Chromatography Applications HPLC - Supelco

Chromatography
Applications
Searchable by technique, industry,
and analyte
Volume 1: HPLC, UHPLC, LC-MS, SFC, SPE
Volume 2: GC, GC-MS, SPME, Air Monitoring
Volume 3: Chiral
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Method development can be a time-consuming part of the This tool demonstrates how Sigma-Aldrich’s analytical
chromatographic process. Finding the best column, sample brands—Supelco, Fluka, Cerilliant, and RTC—are uniquely
prep technique, mobile phase or carrier gas, and detection able to provide customers with a wide spectrum of
method is a challenging process. Just having a starting point chromatographic components across the analytical workflow:
can go a long way in reducing method development time. • Sample collection and sample prep devices
Sigma-Aldrich has developed thousands of applications • UHPLC, HPLC, LC-MS columns
showing commonly analyzed compounds across many fields • GC columns
of research and testing.
• Chiral columns
This tool is interactive, searchable, and organized by research • Mobile phase solvents
market focus within analytical technique, analyte, compound
class, and key word. Each application includes links to
• Buffers and additives
the website which features additional information and • Derivitization reagents
convenient ordering. • Analytical standards
Sigma-Aldrich continually adds new applications to its
portfolio. If you have a suggestion for an application not found
in this notebook, contact techservice@sial.com.
a
Expanded Publication
Learn How to Navigate the New Format
This expanded edition of the Chromatography Applications
Notebook consists of three separate, but linked, volumes.
Note that the application pages are numbered consecutively,
Volume 1 through Volume 3, beginning with 1.
Inside each volume is a Master Table of Contents and a
comprehensive Analyte Index. These pages can be used to
navigate from one volume to another.
When searching in the index, the page number(s) will help
Master Table of Contents
indicate the correct volume to visit. Volume page number Volume 1 Volume 2 Volume 3
keys are found in the index footer. HPLC, UHPLC or LC-MS
Agriculture
Air Monitoring and Industrial Hygiene
1
1
21
GC or GC-MS
Environmental
Air Monitoring and Industrial Hygiene
1429
1429
1572
Chiral
Agriculture
Chemicals and Industrial Polymers
2469
2469
2487
Chemicals and Industrial Polymers 28 Petroleum 1592 Clinical 2619
Clinical 122 Biofuels 1629 Cosmetics, Personal Care, and Cleaning Products 2654
Cosmetics, Personal Care, and Cleaning Products 276 Chemicals and Industrial Polymers 1639 Environmental 2657
Environmental 294 Agriculture 1795 Flavors and Fragrances 2665
Flavors and Fragrances 322 Food and Beverages 1891 Food and Beverages 2691
Food and Beverages 325 Flavors and Fragrances 2082 Forensics and Toxicology 2709
Forensics and Toxicology 407 Cosmetics, Personal Care, and Cleaning Products 2180 Life Science and Biopharma 2726
General Analytical 492 Pharmaceutical (Small Molecule) 2193 Pharmaceutical (Small Molecule) 2845
Life Science and Biopharma 493 Clinical 2229 Vitamins, Nutraceuticals, and Natural Products 3029
Petroleum 841 Forensics and Toxicology 2280
Pharmaceutical (Small Molecule) 843 Life Science and Biopharma 2304
Vitamins, Nutraceuticals, and Natural Products 1232 General Analytical 2308
SFC 1338 Air Monitoring 2318
Chemicals and Industrial Polymers 1338 Environmental 2318
Pharmaceutical (Small Molecule) 1340 Industrial Hygiene 2326
Vitamins, Nutraceuticals, and Natural Products 1346 Agriculture 2348
Food and Beverage 2350
SPE 1347 Flavors and Fragrances 2353
Agriculture 1347
Chemicals and Industrial Polymers 1359 SPME 2354
Clinical 1360 Environmental 2354
Cosmetics, Personal Care, and Cleaning Products 1380 Air Monitoring and Industrial Hygiene 2377
Environmental 1381 Biofuels 2378
Food and Beverages 1394 Chemicals and Industrial Polymers 2379
Forensics and Toxicology 1420 Agriculture 2392
Pharma and Biopharma 1427 Food and Beverages 2403
Cosmetics, Personal Care, and Cleaning Products 2446
Pharmaceutical (Small Molecule) 2448
Clinical 2457
Forensics and Toxicology 2460
b
Master Table of Contents
Applications Notebook Table of Contents
Volume 1 Volume 2 Volume 3

HPLC, UHPLC or LC-MS 1 GC or GC-MS 1429 Chiral 2469
Agriculture 1 Environmental 1429 Agriculture 2469
Air Monitoring and Industrial Hygiene 21 Air Monitoring and Industrial Hygiene 1572 Chemicals and Industrial Polymers 2487
Chemicals and Industrial Polymers 28 Petroleum 1592 Clinical 2619
Clinical 122 Biofuels 1629 Cosmetics, Personal Care, and Cleaning Products 2654
Cosmetics, Personal Care, and Cleaning Products 276 Chemicals and Industrial Polymers 1639 Environmental 2657
Environmental 294 Agriculture 1795 Flavors and Fragrances 2665
Flavors and Fragrances 322 Food and Beverages 1891 Food and Beverages 2691
Food and Beverages 325 Flavors and Fragrances 2082 Forensics and Toxicology 2709
Forensics and Toxicology 407 Cosmetics, Personal Care, and Cleaning Products 2180 Life Science and Biopharma 2726
General Analytical 492 Pharmaceutical (Small Molecule) 2193 Pharmaceutical (Small Molecule) 2845
Life Science and Biopharma 493 Clinical 2229 Vitamins, Nutraceuticals, and Natural Products 3029
Petroleum 841 Forensics and Toxicology 2280
Pharmaceutical (Small Molecule) 843 Life Science and Biopharma 2304
Vitamins, Nutraceuticals, and Natural Products 1232 General Analytical 2308
SFC 1338 Air Monitoring 2318
Chemicals and Industrial Polymers 1338 Environmental 2318
Pharmaceutical (Small Molecule) 1340 Industrial Hygiene 2326
Vitamins, Nutraceuticals, and Natural Products 1346 Agriculture 2348
Food and Beverage 2350
SPE 1347 Flavors and Fragrances 2353
Agriculture 1347
Chemicals and Industrial Polymers 1359 SPME 2354
Clinical 1360 Environmental 2354
Cosmetics, Personal Care, and Cleaning Products 1380 Air Monitoring and Industrial Hygiene 2377
Environmental 1381 Biofuels 2378
Food and Beverages 1394 Chemicals and Industrial Polymers 2379
Forensics and Toxicology 1420 Agriculture 2392
Pharma and Biopharma 1427 Food and Beverages 2403
Cosmetics, Personal Care, and Cleaning Products 2446
Pharmaceutical (Small Molecule) 2448
Clinical 2457
Forensics and Toxicology 2460
c
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d
HPLC, UHPLC or LC-MS Agriculture
1. Oxamyl (10 µg/mL) 8. Fluomethuron (10 µg/mL) 15. Swep (10 µg/mL) Gradient Program
HPLC, UHPLC or LC-MS 2. Methomyl (10 µg/mL)
3. Fenuron (10 µg/mL)
4. Monuron (10 µg/mL)
9. Carbaryl (10 µg/mL)
10. Diuron (10 µg/mL)
11. Propham (10 µg/mL)
16. Mexacarbate (10 µg/mL)
17. Chlorpropham (10 µg/mL)
18. Barban (10 µg/mL)
Time (min) %A %B
0 90 10
13 50 50
5. Aminocarb (10 µg/mL) 12. Siduron (10 µg/mL) 19. Neburon (10 µg/mL) 23 40 60
Agriculture 6. Propoxur (10 µg/mL)
7. Carbofuran (10 µg/mL)
13. Methiocarb (10 µg/mL)
14. Linuron (10 µg/mL)
9 29
34
10
10
90
90
EPA Method 632: HPLC Analysis of Pesticides on 3 4 10 15

17
18
Ascentis® C18 8
13 14
19
column . . . . . . . Ascentis C18, 25 cm x 4.6 mm I.D., 5 μm particles (581325-U) 5 7
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile 1

6 12
11
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0 10 20 30
sample . . . . . . . . . . . . . . . . . . . . . as indicated in 16% acetonitrile in water
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003194
1 Pub Date: August 2014

HPLC Analysis of Acidic Herbicides in Water on a 1. Tetrachloroterephthalic acid

2. Picloram Gradient Program
Polymeric C18 Column after SPE using 3. Dicamba Time (min) % B
4. 2,4-D 2.5 40
Supelclean™ ENVI™-Carb 5. 2,4,5-T 6 5.0 60
6. 2,4,5-TP 13.0 60
using Zymark AutoTrace Extraction WorkStation 1.20 7. Dinoseb 13.5 40
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 8. Dacthal 9
9. Pentachlorophenol 5
sample/matrix . . . Fresh 1 L water samples, dechlorinated with sodium thiosulfate
when necessary, at ambient temperature and pH. 8
1 3
SPE tube/cartridge . . . . . . . . . . . . Supelclean ENVI-Carb, 250 mg/6 mL (57092) 2 7
condition . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mL DI water at 20 mL/min
4
sample addition . . . . . . . . . . . . . . . . . . . . 0.9 L water sample at 20 mL/min Extracted Sample
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 min using clean nitrogen
washing . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mL DI water at 20 mL/min
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mL 0.1% phosphoric acid in
methylene chloride:acetonitrile (80:20) at 5 mL/min Background
column . . polymeric-coated silica-based PAH specialty column, 20 cm × 3 mm I.D.,
5 μm (Supelco equivalent, SUPELCOSIL LC-PAH, available upon request)
mobile phase . . gradient, (A): 0.05% phosphoric acid in DI water; (B): acetonitrile 0 2 4 6 8 10 12 14 16
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min Min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C Figures provided by A. Lichtman, Nassau County Dept. of Health, Hempstead,
detector . . . . . . . . . . . . . . . . . . . . photodiode array- peak width: 0.053 min, New York, USA. Request Application Note No. T196100.
sampling interval: 0.320 sec, monitor 210 nm & 225 nm
injection . . . . . . . . . . . . . . . 10 μL of extract (4-5 ppb each analyte in water)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0150

HPLC Analysis of Ancymidol Enantiomers on

N OH
Astec® CYCLOBOND® I 2000 HP-RSP CH3
* O
column . . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D.,
5 μm particles (24024AST) N
mobile phase . . (A) 5 mM ammonium acetate, pH 6.0; (B) acetonitrile; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004684
0 10 20
Min

HPLC Analysis of Animal Feed Additives on 1 1. 4-hydroxyphenolarsonic acid

2. Roxarsone
Ascentis® Phenyl
2
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 10 mM ammonium formate
(pH 3.0 with formic acid); (B) methanol; (78:22, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in mobile phase A
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003712

HPLC Analysis of Atrazine Herbicides on Ascentis® 1. Desethylatrazine 10. Diuron

Express C18 2. Metoxuron 11. Isoproturon
3. Hexazinone 12. Metobromuron
This application demonstrates the suitability of Ascentis Express C18 for the 4. Simazine 13. Metazachlor
efficient separation of 17 triazine pesticides. 5. Cyanazine 14. Sebuthylazine
column . . Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 μm particles (53814-U) 6. Methabenzthiazuron 15. Terbutylazine
mobile phase . . (A) 20 mM ammonium acetate, pH 6.4 unadjusted; (B) acetonitrile 7. Chlorotoluron 16. Linuron
gradient . . 20 to 28% B in 11 min; 28 to 65% B in 5 min; held at 65% B for 4 min 8. Atrazine 17. Metolachlor
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 9. Monolinuron
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 °C 16
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 3
sample . . . . . . . . . . . . . . . . . . . . . 10-25 μg/mL in 10:90, water: acetonitrile 11
7
1 12 15
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005654
9 10 14
5 6
4 8 17
13
0 10 20
Min

HPLC Analysis of Bromacil Enantiomers on Astec® H

CHIROBIOTIC® T2 H3C N O
C H3
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST)
N *
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (70:30, A:B) Br
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O C H3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004595
0 2 4 6 8 10 12 14 16
Min.

HPLC Analysis of 2(2-Chlorophenoxy) Propionic O
Acid Enantiomers on Astec® CHIROBIOTIC® T H3C *

OH
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST)
mobile phase . . . . . . . . . . . . . . . . . 0.1 wt% ammonium formate in methanol O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm Cl
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004579
0 2 4
Min.

Acid Enantiomers on Astec® CHIROBIOTIC® TAG O *

OH
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST)
mobile phase . . . . . . . . . . . . . . . . . 0.1 wt% ammonium acetate in methanol CH3
Cl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004606
0 2 4
Min.

Acid Enantiomers on Astec® CHIROBIOTIC® TAG O *

OH
CH 3
mobile phase . . . . . . . . . . . . . . 0.1 wt% mM ammonium formate in methanol Cl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004607
0 2 4 6
Min.

HPLC Analysis of Devrinol (Napropamide) CH3

C H3
Enantiomers on Astec® CHIROBIOTIC® V N

column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) O *
mobile phase . . . . . . . . . (A) 0.1% triethylamine, pH 5.0; (B) THF; (80:20, A:B) O CH 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004622
0 2 4 6 8 10 12
Min

HPLC Analysis of Enilconazol (Imazalil) Cl

Enantiomers on Astec® CYCLOBOND® I 2000
column . . . CYCLOBOND I 2000, 25 cm x 4.6 mm I.D., 5 μm particles (20024AST) O *
mobile phase . . (A) 0.1% triethylamine acetate, pH 4.1; (B) acetonitrile; (90:10, A:B) H2 C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min Cl
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C N
N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004690
0 2 4 6 8 10
Min

HPLC Analysis of Fusarium Mycotoxins on 1. Nivalenol

Ascentis® Express Phenyl-Hexyl 2. Deoxynivalenol
3. 15-Acetyldeoxynivalenol
The trichothecene vomitoxins of Fusarium are significant plant pathogens of 4. 3-Acetyldeoxynivalenol
grain and forage crops that can lead to anorexic effects with livestock. This
application demonstrates resolution of deoxynivalenol and three of its 1
3 4
analogs.
column . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 10 cm x 2.1 mm I.D., 2
2.7 μm particles (53336-U)
mobile phase . . . . . . . . . . . . . . (A) water, acetonitrile, 90:10; (B) acetonitrile
gradient . . 0% B held for 0.5 min; to 15% B in 1 min; held at 15% B for 6 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 bar (2780 psi)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 218 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 5 mg/L ea. in 95:5, water:acetonitrile
0 2 4 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005539 Min

HPLC Analysis of Paraquat and Diquat on 1. Diquat

Ascentis® Express HILIC (UV Detection) 2. Paraquat
2
column . . Ascentis Express HILlC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U)
mobile phase . . . . . . (A) 200 mM ammonium TFA; (B) acetonitrile; (20:80, A:B) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . UV, 257 nm (paraquat) and 308 nm (diquat)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg/L in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005718
1.0 2.0
Min

HPLC Analysis of Strigol Enantiomers on Astec® H3 C CH 3 H

CYCLOBOND® I 2000 RSP O O
column . . CYCLOBOND I 2000 RSP, 25 cm x 4.6 mm I.D., 5 μm particles (20324AST) H O

mobile phase . . . . . (A) 0.1% triethylamine, pH 4.1; (B) acetonitrile; (75:25, A:B) O
H
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min OH O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
CH 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004707
0 2 4 6 8
Min

HPLC Analysis of 16 Triazine and Phenyl Urea 1. Atrazine desisopropyl 10. Isoproturon
Compounds on Ascentis® Express RP-Amide 2. Atrazine desethyl 11. Terbumeton
3. Carbendazime 12. Diuron
This application demonstrates the suitability of Ascentis Express RP-Amide 4. Terbumeton desethyl 13. Propazine
for the efficient separation of 16 triazine and phenyl urea compounds. 5. Simazine 14. Terbuthylazine
Lower column temperature can improve resolution of the critical pair. 6. Terbuthylazin desethyl 15. Linuron
column . . Ascentis Express RP-Amide, 15 cm x 3 mm I.D., 2.7 μm particles (53919-U) 7. Cyanazine 16. Terbutryne
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile 8. Atrazine
9. Chlortoluron 10 14
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 to 53% B in 70 min 6 9 16
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 12 15
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 2 7 8 11 13
1 45
sample . . . . . . . . . . . . . . . . . . . . . . 20 mg/L (except terbuthylazin desethyl
and terbuthylazine 60 mg/L) in 60:40, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005651
0 20 40 60
Min

HPLC Analysis of 16 Triazine and Phenyl Urea 1. Atrazine desisopropyl 9. Chlortoluron

Compounds on Ascentis® Express RP-Amide, 2. Atrazine desethyl 10. Isoproturon
3. Carbendazime 11. Terbumeton
Modified Gradient 4. Terbumeton desethyl 12. Diuron
This application demonstrates the suitability of Ascentis Express RP-Amide 5. Simazine 13. Propazine
for the efficient separation of 16 triazine and phenyl urea compounds. 6. Terbuthylazin desethyl 14. Terbuthylazine 14 16
Lower column 7. Cyanazine 15. Linuron
10 15
temperature can improve resolution of the critical pair. 8. Atrazine 16. Terbutryne
9 12
column . . Ascentis Express RP-Amide, 15 cm x 3 mm I.D., 2.7 μm particles (53919-U)
gradient . . 10 to 25% B in 30 min; 25 to 60% B in 15 min; held at 60% B for 5 min 3 11 13
2 8
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 1 4 5 7
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm 0 10 20 30 40
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL Min
sample . . . . . . . . . . . . . . . . . . . 20 mg/L each (except terbuthylazin desethyl
and terbuthylazine 60 mg/L) in 60:40, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005652

HPLC Analysis of B-Trichothecenes on Ascentis® 1. Nivalenol

2 2. Deoxynivalenol
Express C18 3. 15-Acetyldeoxynivalenol
1
Mycotoxins are toxic secondary metabolites produced by fungi. Those 4. 3-Acetyldeoxynivalenol
chemicals exist in food as a result of fungal infection of crops. Mycotoxins
have strong resistance to decomposition and digestion, so they remain in
the food chain in meat and dairy products. They also resist to temperature,
such as cooking and freezing. Their effects on human and animal health 3
4
include death, cancer, weakened immune systems and as allergens or
irritants.
column . . . Ascentis Express C18, 10 cm x 3 mm I.D., 2.7 μm particles (53814-U)
mobile phase . . . . . . . . (A) water:acetonitrile:methanol (92:4:4); (B) acetonitrile
gradient . . 0% B held for 5 minutes, to 8% B in 3 minutes, 8% B held for 2 minutes
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4061 psi (280 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 2 4 6 8 10 12
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 μL
sample . . . . . . . . . . . . . . . 2 ppm each in water:methanol:acetonitrile (92:4:4)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005561

LC-MS Analysis of Ancymidol Enantiomers on N OH CH3

Astec® CYCLOBOND® I 2000 HP-RSP * O
column . . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D., N

5 μm particles (24024AST)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS (m/z = 257)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004683
0 10 20
Min

LC-MS/MS Analysis of Neonicotinoid Pesticides in 1, 2 1. Dinotefuran

Dandelion Blossoms on Ascentis® Express C18 2. Nitenpyram
3. Thiamethoxam
after Dispersive SPE (QuEChERS) using Supel™ QuE 4. Imidacloprid
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . Dispersive (QuEChERS) 6 5. Clothianidin
7
6. Acetamiprid
sample/matrix . . 3 g pulverized dandelion blossoms, homogenized in 10 mL water
7. Thiacloprid
extraction tube . . . . . . . . . . . . . . Supel QuE Acetate extration tube (55234-U)
extraction process . . add 25 mL of acetonitrile; add contents of Supel QuE acetate 3
4
extraction tube; shake immediately for 1 minute; centrifuge at 3400 rpm for 5 minutes
5
clean-up tube . . . . . . . . . . . . . . . . . . . . Supel QuE PSA/C18 tube (55288-U)
clean-up process . . . . . . . . . . . . . Transfer 1 mL of the acetonitrile layer into a
Supel QuE PSA/C18 cleanup tube; shake for 1 minute;
centrifuge at 3500 rpm for 3 minutes; (draw off 700 uL of supernatant,
evaporate to dryness at 50 °C under nitrogen; 0 2 4 6 8 10 12
reconstitute in 200 μL of 50:50 0.1% formic acid:0.1% formic acid in methanol) Min
column . . Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 μm particles (53814-U)
mobile phase . . (A) 0.1% formic acid in water; (B) 0.1% formic acid in methanol
gradient . . 30% B from 0 to 5 min; to 100% B in 0.2 min; held at 100% B for 5.3
min; to 30% B in 0.5 min; held at 30% B for 5 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 μL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3800 psi (262 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . MS, ESI(+), MRM, m/z 203.2/129.2, 271.2/225.0, 292.1/211.0,
256.0/175.2, 250.0/132.0, 223.2/126.0, 253.0/125.8
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006046

UHPLC Analysis of Atrazine on Titan™ C18 1. Atrazine, 8.27 1
column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U)

mobile phase . . (A) 10 mM ammonium acetate, pH 7.0; (B) acetonitrile (70:30, A;B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, XIC m/z 216.10
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . 300 ng/mL each in acetonitrile:water (3:1)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006089
0 2 4 6 8 10
Min

HPLC, UHPLC or LC-MS Air Monitoring and Industrial Hygiene
Carbonyl-DNPH
Air Monitoring and Industrial Hygiene 1.
2.
Formaldehyde
Acetaldehyde
1
3. Acetone
EPA Method TO-11/IP-6A and ASTM® D5197: HPLC 2 3
4 4. Acrolein
5. Propionaldehyde
Analysis of Aldehydes and Ketones on 6. Crotonaldehyde
SUPELCOSIL™ LC-18 after Collection/Desorption 5 7. Butyraldehyde
8. Benzaldehyde
using LpDNPH 9. Isovaleraldehyde
10. Valeraldehyde
sample/matrix . . . . . . . . . . . . . 5 μg each carbonyl-DNPH on LpDNPH cartridge. 11. o-Tolualdehyde
6
Cartridge eluted with 5.0 mL acetonitrile 12. m-Tolualdehyde
adsorbent tube . . . . . . . . . . . . . . . . . . . . . . . . LpDNPH Cartridge (21024-U) 13. p T-olualdehyde
14. Hexaldehyde
column . . . . . SUPELCOSIL LC-18, 25 cm × 4.6 mm I.D., 5 μm particles (58298) 7 15. 2,5-Dimethylbenzaldehyde
mobile phase . . . . . . . . . . . . . (A): acetonitrile:tetrahydrofuran:water, 30:10:60;
8 12
(B): acetonitrile:water, 60:40 gradient 0% B for 1 min,
linear gradient to 100% B over 10 min 9 10
11 13
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
14 15
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIS, 360 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL of extract
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795-0298
Blank Cartridge
0 5 10 15 20 25 30
Min

HPLC Analysis of 13 Aldehyde/Ketone DNPH 1. Formaldehyde-2,4-DNPH

Derivatives on Ascentis® Express C18 2. Acetaldehyde-2,4-DNPH
3. Acetone-2,4-DNPH
column . . Ascentis Express C18, 10 cm x 4.6 mm I.D., 2.7 μm particles (53827-U) 4. Acrolein-2,4-DNPH
mobile phase . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (40:60 v/v, A:B) 5. Propionaldehyde-2,4-DNPH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 1 6. Crotonaldehyde-2,4-DNPH
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4685 psi (323 bar) 7. 2-Butanone-2,4-DNPH
2 8. Butyraldehyde-2,4-DNPH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 360 nm
9. Benzaldehyde-2,4-DNPH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 3
10. Valeraldehyde-2,4-DNPH
sample . . . . . . carb Method 1004 DNPH Mix 2 (47651-U) dilute 3:2 with water 4 11. m-Tolualdehyde 2,4-DNPH
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005635 12. Hexaldehyde-2,4-DNPH
5
67 13. Methacrolein-2,4-DNPH
8
9
10 11
12 13
0 2 4 6
Min

HPLC Analysis of 13 Aldehyde/Ketone DNPH 1. Formaldehyde-2,4-DNPH

2. Acetaldehyde-2,4-DNPH
Derivatives on Ascentis® Express C18 3. Acetone-2,4-DNPH
(High Flow Rate) 4. Acrolein-2,4-DNPH
column . . Ascentis Express C18, 10 cm x 4.6 mm I.D., 2.7 μm particles (53827-U) 5. Propionaldehyde-2,4-DNPH
1 6. Crotonaldehyde-2,4-DNPH
mobile phase . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (40:60, v/v, A:B)
7. 2-Butanone-2,4-DNPH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 mL/min 8. Butyraldehyde-2,4-DNPH
2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8296 psi (572 bar) 9. Benzaldehyde-2,4-DNPH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 360 nm 3 10. Valeraldehyde-2,4-DNPH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4 11. m-Tolualdehyde 2,4-DNPH
sample . . . . . . Carb Method 1004 DNPH Mix 2 (47651-U) dilute 3:2 with water 12. Hexaldehyde-2,4-DNPH
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005636 5 13. Methacrolein-2,4-DNPH
67
8
9
10
11
12 13
0 2 4
Min

HPLC Analysis of Aldehydes and Ketones as DNPH R
Derivatives on Ascentis® Express C18 HN 1. Formaldehyde-2,4-dnph

NH O 2. Acetaldehyde-2,4-dnph
This application demonstrates the suitability of Ascentis Express C18 for the 3. Acetone-2,4-dnph
separation of aldehyde/ketone-DNPH derivatives. 4. Acrolein-2,4-dnph
+ 5. Propionaldehyde-2,4-dnph
N
column . . Ascentis Express C18, 10 cm x 4.6 mm I.D., 2.7 μm particles (53827-U) 1
O
- 6. Crotonaldehyde-2,4-dnph
7. 2-Butanone-2,4-dnph
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (40:60, A:B) 8. Butyraldehyde-2,4-dnph
9. Benzaldehyde-2,4-dnph
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 10. Valeraldehyde-2,4-dnph
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4685 psi (323 bar) 2 11.
12.
m-Tolualdehyde 2,4-dnph
Hexaldehyde-2,4-dnph
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 4 13. Methacrolein-2,4-dnph
+
5 N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 360 nm 6 7 O
-
O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 89
10 11
13
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample in water 12
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005382

1.0 2.0 3.0 4.0 5.0 6.0
Min

HPLC Analysis of Isocyanate Derivatives on 1. N,N'-(2-Methyl-1,3-phenylene)bis(4-(2-pyridinyl)-1-piperazinecarboxamide)

Ascentis® ES-Cyano 2. N,N'-1,6-Hexanediylbis(4-(2-pyridinyl)-1-piperazinecarboxamide)
3. N,N'-(4-Methyl-1,3-phenylene)bis(4-(2-pyridinyl)-1-piperazinecarboxamide)
This application demonstrates the suitability of Ascentis Cyano for the 4. N,N'-(Methylenediphenylene)bis(4-(2-pyridinyl)-1-piperazinecarboxamide)
efficient separation of isocyanate derivatives.
3
column . . . . . . Ascentis Cyano, 15 cm x 4.6 mm I.D., 5 μm particles (577306-U)
1
mobile phase . . . . . . . . . . . . . . . . . . (A) 0.1% ammonium acetate in water;
(B) 0.1% ammonium acetate in acetonitrile; (70:30, A:B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . 20 μg/mL in 80:12:8, water: acetonitrile: dimethylsulfoxide
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005707
0 10 20 30
Min

NIOSH Method 7903: IC Analysis of Inorganic Acids 1

1
after Collection/Desorption using ORBO™-53
1. CO 3-2
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . 1 ppm of each component 2. PO 4-3
adsorbent tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ORBO-53 (20265) 1 ppm Each Anion
3. Br -
column . . . . . . . . . . . . . . . . . Dionex S2 Fast Run Anion, 25 cm × 3 mm I.D. 4. NO -3 3
5
mobile phase . . . . . . . . . . . . . . . . . . . . . . . 3 mM NaHCO 3/2.4 mM Na 2CO 3 4
5. SO 4-2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . conductivity (10μSiemans/ cm)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0873 Blank
0 10 15 20 25
Min
Chromatogram provided by Ms. M. Cassinelli,

NIOSH Inorganic Methods Development Section,
Cincinnati, OH, USA

OSHA Method 42/47 and ASTM® D5836: HPLC 2

Analysis of Isocyanates on SUPELCOSIL™ LC-8 after 3, 4
Collection/Desorption using ORBO™-80
1. Solvent (ACN/DMSO)
sample/matrix . . calibration standard: isocyanate derivatives, 5 μg/mL in acetonitrile 2. 1-2PP
blank: 1-(2-pyridyl)piperazine (1-2PP) coated glass fiber filter 3. 2,4-TDI
desorbed in acetonitrile/DMSO, 9:1 4. 2,6-TDI
5 5. HDI
adsorbent tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ORBO-80 (20811) 6. MDI
column . . . . . . SUPELCOSIL LC-8, 25 cm × 4.6 mm I.D., 5 μm particles (58297)
mobile phase . . . . . . . . . . . . . 0.05 M ammonium acetate in water:acetonitrile, 6
70:30 (pH 6-6.2 with acetic acid)
flow rate . . . . . . . . . . . . . linear gradient, 0.7 mL/min to 2 mL/min in 15 min 1
1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0376
Blank Filter
Calibration Standard
0 5 10 15 20 25
Min

HPLC, UHPLC or LC-MS Chemicals and Industrial Polymers
1 1. Acrylamide
Chemicals and Industrial Polymers 2. Acrylic Acid
HPLC Analysis of Acrylamide and Acrylic Acid on

Discovery® HS F5 2
column . . . . Discovery HS F5, 15 cm × 4.6 mm I.D., 5 μm particles (567516-U)

mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05% formic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 206 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mg/mL each in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002569
0 2 4 6 8
Min

HPLC Analysis of Acrylic Acid Derivatives on 1. Acrylate Gradient Program

2. Methacrylate 4
Discovery® Zr-Carbon 3. Sulfopropylacrylate 2
Time (min) %A %B
4. Sulfopropylmethacrylate
column . . . Discovery Zr-Carbon, 7.5 cm × 2.1 mm I.D., 3 μm particles (65726-U) 0 100
80
0
20
5
mobile phase . . . . . . . . . . (A) 30 mM phosphoric acid, pH 1.9; (B) acetonitrile 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.42 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
detector . . . . . . . . . . . . . . UV, 210 nm (200 nm will enhance sensitivity ~2x) 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . acrylate @ 0.05 g/L, methacrylate @ 0.05 g/L,
sulfopropylacrylate @ 0.02 g/L, sulfopropylmethylacrylate @ 0.02 g/L
in 30 mM H3 PO4, pH 1.9
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002143
1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of Amides on Discovery® 1. Codeine

3 2. Strychnine
RP-Amide C16 3. Quinidine 6
2 4. Quinine
column . . . Discovery RP-Amide C16, 15 cm × 4.6 mm, 5 μm particles (505013) 5. Noscapine
mobile phase . . . . . . . . . . (A) methanol; (B) 25mM monopotassium phosphate, 6. Papaverine
pH 3.0; (20:80, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000691
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of trans-1-Amino-2-Indanol 1. trans-1-Amino-2-indanol enantiomer

Enantiomers on LARIHC™ CF6-P 2. trans-1-Amino-2-indanol enantiomer
column . . LARIHC CF6-P, 25 cm x 4.6 mm I.D., 5 μm particles (AZYP Part No.L1001, 1

OH
available from Supelco/Sigma-Aldrich as a custom item.)
mobile phase . . . (A) methanol; (B) acetonitrile; (C) acetic acid; (D) triethylamine, NH 2
(70:30:0.3.0.2, A:B:C:D) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . Trans-1-Amino-2-indanol, 0.3 mg/mL in ethanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005256
0 2 4 6 8 10 12
Min
Ref: 1. Ping Sun, Chunlei Wang, Zachary S. Breitbach, Ying Zhang, and Daniel W. Armstrong,
Development of New HPLC Chiral Stationary Phases Based on Native and Derivatized
Cyclofructans Anal. Chem. 81, 10215 (2009)
2. Ping Sun and Daniel W. Armstrong, Effective enantiomeric separations of racemic primary
amines by the isopropyl carbamate-cyclofructan6 chiral stationary phase
J. Chromatogr. A. 1217, 4904 (2010)

HPLC Analysis of Aniline Homologs on 3

1. Aniline
Discovery® C18 2. N-Ethylaniline
3. N-Propylaniline
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 4. N,N-Dimethylaniline
mobile phase . . . . . . . . . . . . . . . . . . . (A) methanol; (B) water (60:40, A:B) 1 5. N-Butylaniline
6. N,N-Diethylaniline
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 2 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000295
5
6
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Aromatics on Ascentis® 1. Butylbenzene (2.5 mL/mL) 4

2. o-terphenyl (150 mg/mL )
RP-Amide 3. Amylbenzene (2.5 mL/mL) 3
4. Triphenylene (50 mg/mL) (2.5 mL/mL)
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 2
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (30:70, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in acetonitrile 0 2 4 6 8 10 12 14 16
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002607

HPLC Analysis of Atrolactic Acid Enantiomers on

Astec® CHIROBIOTIC® R
column . . . . . . CHIROBIOTIC R, 25 cm x 4.6 mm I.D., 5 μm particles (13024AST) O
mobile phase . . . . . . . . . . . . . . . . . 0.1 wt% ammonium acetate in methanol H3C
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min OH
OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004594
0 2 4 6 8
Min.

HPLC Analysis of Atrolactic Acid Enantiomers on

Astec® CHIROBIOTIC® TAG
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) O
mobile phase . . . . . . . . . . . . . . . . . 0.1 wt% ammonium acetate in methanol *
H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C OH OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004604
0 2 4 6
Min.

HPLC Analysis of Benzene and Deuterated 5. Toluene = 101,281 plates

4. Benzene = 104,463 plates D
Benzene on Ascentis® Express C18 D D
3. D6 Benzene = 101,852 plates
column . . . . . . . . Ascentis Express C18, 15 cm × 4.6 mm I.D., 2.7 μm particles 2. Acetophenone = 120,045 plates
mobile phase . . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) water (55:45, A:B) Pressure = 480 bar (7,000 psi) D D H
D H
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
H H
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm H
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004046
0 2 4 6 8 10 12 14 16 18
Min

3 1. 4-hydroxybenzoic acid (50 mg/mL)

HPLC Analysis of Benzoic Acid Deriviatives on 2. Acetylsalicylic acid (50 mg/mL)
Ascentis® RP-Amide 1 3. Benzoic acid (50 mg/mL)
4. 2-hydroxybenzoic acid (50 mg/mL)
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 5. Ethyl paraben (50 mg/mL)
mobile phase . . (A) 0.1% TFA in water; (B) 0.1% TFA in acetonitrile; (70:30, A:B) 2
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0 10 20
sample . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in 0.1% TFA in water Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002627

HPLC Analysis of Benzoin Enantiomers on Astec® O

Cellulose DMP *
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST)

OH
mobile phase . . . . . . . . . . . . (A) heptane; (B) IPA; (C) TFA; (90:10:0.1, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C (+)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm (-)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg/mL in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004986
0 10 20
Min

HPLC Analysis of 4-Benzyl-2-oxazolidinone

Enantiomers on Astec® CHIROBIOTIC® TAG
H
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) * N
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004605
0 2 4 6
Min.

HPLC Analysis of Binaphthol Enantiomers on Peak 1:

Astec® CYCLOBOND® I 2000 DNP 13.95 min Peak 2:
HO
15.43 min
column . . CYCLOBOND I 2000 DNP, 25 cm × 4.6 mm I.D., 5 μm particles (25024AST)
mobile phase . . (A) 0.1% triethyl ammonium acetate, pH 4.1; (B) acetonitrile; (75:25, 200
OH
A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
mAU
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004028
0 5 10 15 20
Min

HPLC Analysis of 1,1′-Binaphthyl-2,2′-diyl

Hydrogenphosphate Enantiomers on
Astec® (R,R) P-CAP™ O
OH
column . . . . Astec (R,R) P-CAP, 25 cm x 4.6 mm I.D., 5 μm particles (31024AST) P
mobile phase . . . . . . . . . . . . . . 20 mM ammonium acetate in: (A) acetonitrile; * O
O
(B) methanol; (70:30, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004718
0 2 4 6 8 10 12
Min

HPLC Analysis of 2,2′-Bis[(S)-(4-methylphenyl) CH3
sulfinyl]-1,1′-binapthaIene Diastereomers on
Ascentis® Express C18 S
R
O
CH3
This application demonstrates the suitability of the Ascentis Express C18 for O
S
the separation of the diastereomers of by HPLC in reversed phase mode.
column . . Ascentis Express C18, 10 cm x 4.6 mm I.D., 2.7 μm particles (53827-U) S
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (25:75, A:B) CH3 O
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min S
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2450 psi (169 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm S CH3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . (1R)-2,2′-Bis[(S )-(4-methylphenyl)sulfonyl]-1,1′-binapthalene,
25 μg/mL in methanol; (1S)-2,2′-Bis[(S)-(4-methylphenyl)sulfonyl]-
1,1′-binapthalene, 50 μg/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005612 0 2 4 6
Min

HPLC Analysis of 2-Bromo-3-Methylbutyric Acid CH3 O

Enantiomers on Astec® CHIROBIOTIC® R
*
column . . . . . . CHIROBIOTIC R, 25 cm x 4.6 mm I.D., 5 μm particles (13024AST) H3C OH
mobile phase . . . . . . (A) 20 mM ammonium acetate; (B) methanol; (15:85, A:B)
Br
(alternate mobile phase of 0.1% ammonium acetate
in methanol gives 3.5 and 4.3 min retention)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004667 0 2 4 6 8 10 12
Min

HPLC Analysis of Catechols and Resorcinols on 1. Resorcinol (50 mg/mL)

2. Catechol (50 mg/mL)
Ascentis® RP-Amide 3. 2-methyl resorcinol (50 mg/mL)
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 4. 4-methyl catechol (50 mg/mL)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . (A) 20 mM phosphoric acid 2 5. 2,5-dimethyl resorcinol (50 mg/mL)
1
(pH 2.0 unadjusted): (B) acetonitrile; (75:25, A:B) 6. 3-methyl catechol (50 mg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 7. 4-nitro catechol (50 mg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL 3 6
sample . . . . . . . . . . . . . . . . as indicated in 20 mM phosphoric acid (pH 2.0) 5 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002597
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Cis-(+/-)-3,3a,8,8a-Tetrahydro-2H-

indeno[1,2-d]oxazol-2-one Enantiomers on Astec® H
N
O
(R,R) P-CAP™-DP *
O
column . . Astec (R,R) P-CAP-DP, 25 cm x 4.6 mm I.D., 5 μm particles (35024AST)
*
mobile phase . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) methanol; (98:2, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004729
0 2 4 6
Min

HPLC Analysis of Coumachlor Enantiomers on

Astec® CYCLOBOND® I 2000 DM 1
column . . CYCLOBOND I 2000 DM, 25 cm x 4.6 mm I.D., 5 μm particles (20924AST) 2
mobile phase . . (A) acetonitrile; (B) 20 mM ammonium acetate, pH 2.9; (20:80, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min 1. Coumachlor enantiomer
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 2. Coumachlor enantiomer
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
sample . . . . . . . . . . . . . . . coumachlor, 1 mg/mL in acetonitrile:water (50:50)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005160
0 10 20
Min

HPLC Analysis of Coumafuryl Enantiomers on O

Astec® CHIROBIOTIC® V
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) OH CH 3
mobile phase . . . . . (A) 20 mM ammonium nitrate, pH 5.5; (B) THF; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min
*
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm O
O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL O

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004639
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Cresols and Phenol on Astec® OH OH
CYCLOBOND® I 2000 CH3

column . . . CYCLOBOND I 2000, 25 cm x 4.6 mm I.D., 5 μm particles (20024AST)
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C OH OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 11.70
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mg/mL in methanol
10.70
peak 1: o-cresol (10.70 min)
peak 2: phenol (11.70 min) CH3
peak 3: m-cresol (14.20 min) 14.20

CH3
peak 4: p-cresol (20.10 min)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004731
20.10

HPLC Analysis of Crufomate (Ruelene)

CH3
Enantiomers on Astec® CYCLOBOND® I 2000
O
column . . . CYCLOBOND I 2000, 25 cm x 4.6 mm I.D., 5 μm particles (20024AST) O
CH 3
H3C P
mobile phase . . (A) acetonitrile; (B) acetic acid; (C) triethylamine; (100:0.2:0.1, A:B:C) NH *
O CH 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient CH3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
Cl
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004703
0 2 4 6 8
Min

HPLC Analysis of DACH-ACR Enantiomers on (R,R) P-CAP (S,S) P-CAP

Astec® P-CAP™ (S,S) 3.94 min.
(R,R) 4.39 min.

column . . . . . . . . . . . Astec (R,R) P-CAP or (S,S) P-CAP, 25 cm x 4.6 mm I.D.,
5 μm particles (31024AST, 33024AST)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
(S,S) 6.12 min.
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
CH2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL (R,R) 4.94 min.
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (R,R)- and (S,S)-DACH-ACR O
NH
(N-(2-acryloylamino-(1R,2R)-cyclohexyl)-acrylamide)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004401 NH O
CH2

HPLC Analysis of (+/-)-N,N-Dibenzyltartaric

Diamide Enantiomers on Astec® (R,R) P-CAP™-DP
column . . Astec (R,R) P-CAP-DP, 25 cm x 4.6 mm I.D., 5 μm particles (35024AST) OH O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min HN
*
* NH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm O OH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004727
0 2 4 6 8
Min

HPLC Analysis of 2,3-Dihydroxy-3-Phenylpropionic HO

H HO
H
HO HO
H
H
Acid Enantiomers on CHIROBIOTIC R OH OH
B
O O
column . . . . . . CHIROBIOTIC R, 25 cm x 4.6 mm I.D., 5 μm particles (13024AST)
mobile phase . . . . . . . . . . . . . . (A) 0.1% ammonia, pH 4.1 with formic acid; HO H H H
H OH HO OH
(B) methanol; (50:50, A:B) OH OH
B
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 258 nm A
racemate A racemate B
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . 2,3-dihydroxy-3-phenyl-propionic acid isomers: A
(A) racemate A (4.85 and 6.95 min)
(B) racemate B (5.33 and 6.29 min)
Figure courtesy DSM Fine Chemicals
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004409

HPLC Analysis of Di-6,6′-Methoxy-bi(2-Naphthol)

O
Enantiomers on Astec® (R,R) P-CAP™ H3C
column . . . . Astec (R,R) P-CAP, 25 cm x 4.6 mm I.D., 5 μm particles (31024AST)

mobile phase . . . . . . . . . . . . . . . . . . (A) ethanol; (B): heptane; (50:50, A:B) OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL H3C
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg/mL in methanol O
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004724
0 2 4 6 8 10 12 14
Min

HPLC Analysis of 5,5-Dimethyl-4-phenyl-2-

oxazolidinone Enantiomers on Astec®
CHIROBIOTIC® T CH3
H3 C
*
mobile phase . . (A) 0.5% triethylamine acetate, pH 4.1; (B) methanol; (80:20, A:B) O NH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004547
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Epoxy resin, Epikote® 1001 on

TSKgel® G3000HXL
column . . . . . TSKgel G3000HXL, 30 cm × 7.8 mm I.D., 6 μm particles (816136)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . tetrahydrofuran
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0783
5 10 15 20
Min

HPLC Analysis of Fatty Acid Methyl Esters (FAMES) 3

1. C8
on SUPELCOSIL™ LC-18 2. C10
3. 12:0 + 18:3
column . . . . . SUPELCOSIL LC-18, 25 cm × 4.6 mm I.D., 5 μm particles (58298) 4. 18:2
mobile phase . . . . . . . . . . . . (A) acetonitrile; (B) acetone; (59.0:41.0, v/v, A:B) 5. 14:0
6. 18:1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 7. 16:0
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI 8. 18:0
injection . . 10 μL of 9% C8 to C18 saturated and unsaturated FAMEs in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797-0501
5
4
2
1 7
0 2 4 6
Min

HPLC Analysis of Fipronil Enantiomers on Astec® N Cl
(R,R) P-CAP™ N
F
column . . . . Astec (R,R) P-CAP, 25 cm x 4.6 mm I.D., 5 μm particles (31024AST) N F

mobile phase . . . . . . . . . . . . . . . . . . . (A) heptane; (B) ethanol; (80:20, A:B) O *
S F
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
NH 2 Cl
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C F F
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL F

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004720
0 2 4 6 8 10
Min

HPLC Analysis of p-Fluorophenyl Methyl Sulfoxide 5.9

Details: A. Berthod et al, J. Chromatogr A, 955 (2002) 53-69. O
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) CH 3
S
mobile phase . . . . . . . . . . . . . . . . . . . (A) hexane; (B) ethanol; (50:50, A:B) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
detector . . . . . . . . . . . . . . . . . . . . . UV, polarimetry to confirm elution order
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004735 F
9.0

HPLC Analysis of Furans on Ascentis® Express C18 1. 5-Hydroxymethyl-2-furaldehyde

This application demonstrates the suitability of Ascentis Express C18 for the 2. Furfuryl alcohol
efficient separation of furans as listed in ASTM method 05837-99. 3. 2-Furaldehyde
4. 2-Acetylfuran
5. 5-Methyl-2-furaldehyde
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 2 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 5
sample . . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL in 80:20, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005700
1.0 2.0 3.0

Min

HPLC Analysis of Furans on Ascentis® Express 1. 5-Hydroxymethyl-2-furaldehyde

Phenyl-Hexyl 2. Furfuryl alcohol
3. 2-Furaldehyde
This application demonstrates the suitability of Ascentis Express 4. 2-Acetylfuran
Hexyl-Phenyl for the efficient separation of furans as listed in ASTM method 5. 5-Methyl-2-furaldehyde
2
D5837-99. 3
1
column . . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 5 cm x 3.0 mm I.D.,
2.7 μm particles (53342-U) 4
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (80:20, A:B) 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL in 80:20, water: acetonitrile 1.0 2.0 3.0

HPLC Analysis of Furoin Enantiomers on OH O

Astec® P-CAP™-DP
column . . . . . (R,R) P-CAP-DP, 25 cm x 4.6 mm I.D., 5 μm particles (35024AST) *
mobile phase . . . . . . . . . . . . . . . . . . . (A) hexane; (B) ethanol; (80:20, A:B)
O O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004587
0 2 4 6 8 10 12 14 16
Min.

HPLC Analysis of Furoin on Astec® (R,R) P-CAP™-DP 8 µg injection Peak 1:

10.48 min
column . . . . . (R,R) P-CAP-DP, 25 cm × 4.6 mm I.D., 5 μm particles (25024AST)
60 OH O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min Peak 2:
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C 13.20 min
40 O O
mAU
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL
20
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004031
0
0 5 10 15
Min

HPLC Analysis of Heterocyclic Amines (HCAs) on 1. 2-Amino-9H-pyrido[2,3-b]indole (AC)

2. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PHIP)
Discovery® HS F5 3. 2-Amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ)
column . . . . Discovery HS F5, 15 cm × 4.6 mm I.D., 5 μm particles (567516-U) 4. 2-Amino-3,4-dimethyl-3H-imidazo [4,5-f]quinoline (MeIQ)
5. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)
mobile phase . . . . . . . . . . . . . . . . . . (A) water (10 mM ammonium acetate,
pH 4.0 with acetic acid); (B) methanol (v/v); (15:85, A:B) 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μg/mL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in mobile phase 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002333
5
0 2 4 6 8 10
Min

HPLC Analysis of Hydrobenzoin Enantiomers on

Astec® CYCLOBOND® I 2000 RSP
1
column . . CYCLOBOND I 2000 RSP, 25 cm x 4.6 mm I.D., 5 μm particles (20324AST)
mobile phase . . (A) acetonitrile; (B) 10 mM ammonium acetate, pH 4.0; (25:75, A:B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 1. Hydrobenzoin enantiomer
2. Hydrobenzoin enantiomer
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . hydrobenzoin, 1 mg/mL in acetonitrile:water (50:50)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005163
0 2 4 6 8
Min

HPLC Analysis of Hydrobenzoin Enantiomers on

Astec® CYCLOBOND® I 2000 OH
column . . . CYCLOBOND I 2000, 25 cm x 4.6 mm I.D., 5 μm particles (20024AST) *

mobile phase . . (A) 20 mM ammonium acetate, pH 4.1; (B) acetonitrile; (80:20, A:B) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min OH

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004692
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Hydrolyzed β-Cyclodextrin on DP=1

TSKgel® G-Oligo-PW
column . . . . TSKgel G-Oligo-PW, 30 cm × 7.8 mm I.D., 6 μm particles (808031)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . distilled water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
DP=4 DP=2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI DP=7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0917 DP=3
DP=5
DP=6
15 20
Min

3 1. Uracil
HPLC Analysis of Hydrophobic Bases on Ascentis® 2. Hydroquinidine
RP-Amide 3. Quinidine
1 4. Diphenhydramine
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 5. Fluoxetine
mobile phase . . (A) 25 mM ammonium phosphate, pH 7; (B) methanol; (35:65, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N05158
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002612 0 2 4 6 8 10
Min

HPLC Analysis of α-Hydroxy Aliphatic Acids on 1. Tartaric acid 1

2. Malic acid 2
Discovery® RP-Amide C16 3. Lactic acid
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 4. Citric acid 4
mobile phase . . . . . . . . . . . . . . . . . . . 25 mM potassium phosphate, pH 3.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000624
0 2 4
Min

HPLC Analysis of Jacobsen Catalyst Enantiomers

on Astec® CYCLOBOND® I 2000 HP-RSP * *
(Ammonium Hydroxide Mobile Phase) N N
CH3 Mn CH 3
5 μm particles (24024AST) H3C O O CH3
Cl
mobile phase . . . . . . . . . . . . . . (A) acetonitrile; (B) methanol; (C) acetic acid; CH3 CH3
CH3 H3C
(D) ammonium hydroxide; (85:15:0.4:0.1, A:B:C:D)
H3C CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min CH3 H3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004714
0 2 4 6 8 10
Min

HPLC Analysis of Jacobsen Catalyst Enantiomers

on Astec® CYCLOBOND® I 2000 HP-RSP (TEA
mobile phase) * *
column . . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D., N N

5 μm particles (24024AST) C H3 Mn CH3
mobile phase . . . . . . . . . . . . . . (A) acetonitrile; (B) methanol; (C) acetic acid;
H3C O O CH 3
(D) triethylamine; (95:5:0.2:0.1, A:B:C:D) Cl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min CH3 CH 3
CH3 H3 C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm H3 C CH3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL CH3 H3C

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004715
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Lactic Acid Enantiomers on CLC-L CLC-D

Astec® CLC-L and CLC-D (D)-Lactic acid, (L)-Lactic acid,
9.24 min 9.27 min
column . . . . . . . . . . . . . . . . . Astec CLC-L and CLC-D, 15 cm x 4.6 mm I.D.,
5 μm particles (53023AST, 53123AST) (L)-Lactic acid, (D)-Lactic acid,
12.58 min 12.18 min
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mM copper sulfate
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm H3C

OH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
OH
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004399

HPLC Analysis of Lactic Acid Enantiomers on OH

Astec® CLC-L, Trace Level Detection (L)-Lactic acid,
12.58 min.
column . . . . . . . . Astec CLC-L, 15 cm x 4.6 mm I.D., 5 μm particles (53123AST) HO
*
CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 2% (D)-Lactic acid,
9.24 min.
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg/mL in water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004663

HPLC Analysis of Luciferin Enantiomers on Astec®

N S
CHIROBIOTIC® V
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) S N OH
HO *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004641
0 2 4 6 8 10
Min

HPLC Analysis of Luciferin Enantiomers on 2.85 min

CHIRALPAK® AGP
column . . . . . . CHIRALPAK AGP, 10 cm x 4 mm I.D., 5 μm particles (58150AST)
mobile phase . . . . . . . . . . . . . . . . . . . . . 10 mM sodium phosphate, pH 6.0 N S
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
S N OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm HO
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . luciferin
O
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004398
7.68 min

HPLC Analysis of α-Methylbenzylamine NH2

Enantiomers on Astec® CHIROBIOTIC® V
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) * CH3
mobile phase . . . . . . . . . . . . . . . . . (A) hexane; (B) isopropanol; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004642
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of α-Methylbenzylamine CH 3

Enantiomers on Astec® CYCLOBOND® I 2000 DMP CH3 O
N
column . . CYCLOBOND I 2000 DMP, 25 cm x 4.6 mm I.D., 5 μm particles (20724AST) S CH 3
* NH
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ethanol O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004698
0 2 4 6 8
Min

HPLC Analysis of 5-Methyl-5-Phenylhydantoin

column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) *
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ethanol H
H3 C N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient O O
N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm H
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004643
0 2 4
Min


(Fast Flow)
H
H3C
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) *
N

O O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min N
H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004613
0 2 4 6 8 10 12
Min.

O
HPLC Analysis of 4-Methyl-5-Phenyl-2-
Oxazolidone Enantiomers on Astec®
HN O
CHIROBIOTIC® TAG
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) H3C
* *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004612
0 2 4 6 8 10
Min.

HPLC Analysis of α-Methyl-α-Phenylsuccinimide

mobile phase . . . . . . . . . . . . . . . . . . . (A) hexane; (B) ethanol; (80:20, A:B) H 3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C O
N
O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm H
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004611
0 10 20
Min.

HPLC Analysis of Methyl Phenyl Sulfoxide

O
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . methanol S
CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.48 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004407
0 2 4 6 8 10 12 14
Min

HPLC Analysis of 1-(1-Naphthyl)ethylamine

NH 2
Enantiomers on Astec® CHIROBIOTIC® V2 H3C
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST)

mobile phase . . . . . . (A) methanol; (B) 20 mM ammonium acetate; (90:10, A:B) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mgl/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004458
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Nonionic Surfactants, 9-10 9-10 Ethoxy Groups

Ethoxy Groups (Octylphenol) on SUPELCOSIL™ (Octylphenol)
LC-Diol
column . . . . . SUPELCOSIL LC-Diol, 25 cm × 4.6 mm I.D., 5 μm particles (58201)
Gradient Program
mobile phase . . . . . . . . . . . . . . . . . . . (A) hexane:methylene chloride (95:5); Time (min) %B
(B) hexane:methylene chloride:methanol (50:40:10)
0 30
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 8 50
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 15 60
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 25 80
35 100
injection . . . . . . . . . . . . 10 μL methylene chloride containing 20 μg surfactant
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0782
0 4 8 12 16 20 24
Min

HPLC Analysis of Octopamine Enantiomers on 1. Octopamine enantiomer 1

LARIHC™ CF6-P 2. Octopamine enantiomer
2
column . . . . . . . . . . . . . . . . . . . . . . . LARIHC CF6-P, 25 cm x 4.6 mm I.D.,
5 μm particles (AZYP Part No.L1001, HO
NH 2
available from Supelco/Sigma-Aldrich as a custom item.)
mobile phase . . . . . . . . . . . . . . (A) methanol: (B) acetonitrile: (C) acetic acid: H Cl
(D) triethylamine, (70:30:0.3.0.2, A:B:C:D)

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . Octopamine, 0.3 mg/mL in ethanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005258
0 2 4 6 8 10 12
Min
Development of New HPLC Chiral Stationary Phases Based on Native and Derivatized
Cyclofructans Anal. Chem. 81, 10215 (2009)
amines by the isopropyl carbamate-cyclofructan6 chiral stationary phase J. Chromatogr. A. 1217, 4904 (2010)

HPLC Analysis of Organic Acids on Ascentis® 1. L-Tartaric acid

Express RP-Amide 2. Lactic acid
3. Citric acid
This application demonstrates the suitability of Ascentis Express RP-Amide 4. Acrylic acid
for the analysis of organic acids under 100% aqueous conditions. 5. Fumaric acid
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 10 cm x 2.1 mm I.D., 4
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% (v/v) TFA in (B) water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 - 4 g/L in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005714
1.0 2.0 3.0 4.0 5.0
Min

HPLC Analysis of Organic Acids on Ascentis® RP- 1. L-Tartaric acid

Amide 2. Lactic acid
3. Citric acid
This application demonstrates the suitability of Ascentis RP-Amide for the 4. Acrylic acid
analysis of organic acids under 100% aqueous conditions. 5. Fumaric acid 4
column . . . Ascentis RP-Amide, 10 cm x 2.1 mm I.D., 3 μm particles (565301-U)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2 5
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005715
1.0 2.0
Min

HPLC Analysis of Organic Acids on Discovery® C18 1. Homovanillic acid

4
2. Sorbic acid
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 2 3. Salicylic acid
mobile phase . . . . . . . . . . . . . (A) methanol; (B) water, 0.1% TFA (40:60, A:B) 1 4. p-Toluic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL each analyte
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797-0657
0 2 4 6
Min

HPLC Analysis of Organic Acids on Ascentis® C8 1 1. Syringic acid (50 µg/mL)

2. Homoveratric acid (50 µg/mL)
column . . . . . . . . Ascentis C8, 15 cm x 4.6 mm I.D., 5 μm particles (581424-U)
mobile phase . . . . . (A) 10 mM ammonium phosphate monobasic (pH 2.50 with
phosphoric acid): (B) acetonitrile; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003160 0 2 4 6 8 10 12 14
Min

HPLC Analysis of Organic Acids on Discovery® C18 2

column . . . . . . . . Discovery C18, 5 cm × 4.6 mm I.D., 5 μm particles (504947) 1. Homovanillic acid (0.0625 µg/mL)
2. Sorbic acid (0.00625 µg/mL)
mobile phase . . . . . . . . . . . (A) 0.1% TFA in (B) water:methanol; (60:40, A:B) 3. Salicylic acid (0.0625 µg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min 4. p-Toluic acid (0.00625 µg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001431 1 3
4
1.0 2.0 3.0

Min

HPLC Analysis of Phenols on SUPELCOSIL™ LC-8 6 1. Phenol

column . . . . . SUPELCOSIL LC-8, 15 cm × 4.6 mm I.D., 5 μm particles (58220-U) 2. 4-Nitrophenol
3. 2,4-Dinitrophenol
mobile phase . . . . . . . . (A) 1% acetic acid/methanol: (B) 1% acetic acid/water 4. 2-Nitrophenol
gradient . . . . . . . . . . . . . . . 35:65 to 100:0 (20 min), return to 35:65 (5 min) 5. o-Chlorophenol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 6. 4,6-Dinitrophenol
7. 2,4-Dimethylphenol
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 8. 4-Chloro-3-methylphenol
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 23 9. 2,4-Dichlorophenol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1031 10. 2,4,6-Trichlorophenol
11. Pentachlorophenol
4
8
1 10 11
5 7 9
0 2 4 6 8 10 12 14 16 18 20
Min

HPLC Analysis of Phenylacetic Acid on Ascentis® 1. Phenylacetic acid

C18 1
This application demonstrates the suitability of Ascentis C18 for the analysis
of phenylacetic acid.
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U)
mobile phase . . . . . . . (A) 20 mM phosphoric acid; (B) acetonitrile; (75:25, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 0 2 4 6 8 10 12 14
sample . . . . . . . . . . . . . 50 μg/mL in water: acetonitrile: methanol (75: 20: 5)
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005720

HPLC Analysis of N-(1-Phenylethyl)maleimide

CH 3 O
Enantiomers on Astec® CHIROBIOTIC® R
column . . . . . . CHIROBIOTIC R, 25 cm x 4.6 mm I.D., 5 μm particles (13024AST) * N
mobile phase . . (A) 0.1% triethylamine acetate, pH 4.1; (B) methanol; (70:30, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004668
5.0 6.0
Min

HPLC Analysis of 3-Phenyllactic Acid Enantiomers

O
on Astec® CHIROBIOTIC® R
column . . . . . . CHIROBIOTIC R, 25 cm x 4.6 mm I.D., 5 μm particles (13024AST) *
OH
mobile phase . . . . . . . . . . . . . . . . . . . 0.1% ammonium acetate in methanol
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004680
0 2 4 6 8 10 12 14
Min

HPLC Analysis of 4-Phenyl-2-Oxazolidinone

Enantiomers on Astec® CHIROBIOTIC® T2
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
HN O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL O
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004599
0 2 4
Min.

HPLC Analysis of 4-Phenyl-2-oxazolidinone

Enantiomers on Astec® CHIROBIOTIC® T
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) *
mobile phase . . (A) 0.5% triethylamine acetate, pH 4.1; (B) methanol; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min HN O

detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004548
0 10 20
Min

HPLC Analysis of Phenylsuccinic Anhydride

Enantiomers on Astec® (R,R) P-CAP™-DP
column . . Astec (R,R) P-CAP-DP, 25 cm x 4.6 mm I.D., 5 μm particles (35024AST)
mobile phase . . (A) acetonitrile; (B) methanol; (C) trifluoroacetic acid; (99:1/0.1 (A:B) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004728
0 10 20
Min

HPLC Analysis of Polyethylene Glycol 200 on n=4

TSKgel® G-OLIGO-PW
column . . . TSKgel G-OLIGO-PW, 30 cm × 7.8 mm I.D., 6 μm particles (808031)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . distilled water n=5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI n=3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0778
n=6
n=7
n=8 n=2
n=9
15 20
Min

HPLC Analysis of Positional Isomers (Xylenes) on 1. m-Xylene 1 3

Astec® CYCLOBOND® I 2000 2. o-Xylene
2
3. p-Xylene
mobile phase . . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) water; (15:85, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . xylene isomers, each compound,
0.1 mg/mL in acetonitrile:water (50:50) ((elution order: m, o, p))
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005166
0 2 4 6 8 10 12
Min

HPLC Analysis of Quaternary amines on 1 1. Paraquat

Discovery® Zr-PS 2. Diquat
column . . . . . Discovery Zr-PS, 7.5 cm × 4.6 mm I.D., 3 μm particles (65741-U)

mobile phase . . (A) 25 mM phosphoric acid, 25 mM ammonium fluoride (pH with 2
ammonium hydroxide): (B) acetonitrile, (50:50, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10
Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paraquat, diquat (50 μg/mL)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001853

HPLC Analysis of Ruelene (Cruformate) CH 3

Enantiomers on Astec® CYCLOBOND® I 2000 O
O
CH 3
column . . . CYCLOBOND I 2000, 25 cm x 4.6 mm I.D., 5 μm particles (20024AST) H3C P
mobile phase . . . . . (A) acetonitrile; (B) acetic acid; (C) TEA; (100:0.3:0.2, A:B:C) NH * O
CH 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C CH 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm CI
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . Ruelene (cruformate), 1 mg/mL in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005158
0 2 4 6 8 10 12 14
Min

HPLC Analysis of trans-Stilbene Oxide Enantiomers (-)

on Astec® Cellulose DMP
O
mobile phase . . . . . . . . . . . . . . . . . . . . . (A) heptane; (B) IPA; (90:10, A:B) (+)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004995
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Structural Isomers on Discovery® 1. Ethylbenzene

2
Zr-Carbon 2. p-Xylene
1
column . . . Discovery Zr-Carbon, 15 cm × 4.6 mm I.D., 3 μm particles (65730-U)
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (20:80, A:B) Isomers resolved on
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min Discovery Zr-Carbon
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001874
1.0 2.0 3.0 4.0 5.0
Min

HPLC Analysis of Sulfonamide Positional Isomers 1. Isomer 1 Gradient

2. Isomer 2 Time (min) %A %B
on Discovery® Zr-Carbon C18 3. Parent drug 0.0 55 45
4. Isomer 3
column . . Discovery Zr-Carbon C18, 15 cm × 4.6 mm I.D., 3 μm particles (65706-U) 5.0 55 45
mobile phase . . . . . . . . . . . . (A) 10mM diethylamine, pH 10.8; (B) acetonitrile; 7.5 25 75
10.0 25 75
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 °C 250
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm 200
mAu
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 150 1 2
100
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001866 4
50
0
0 2 4 6 8 10
Min

HPLC Analysis of Tartaric Acid Enantiomers on

OH O
Astec® CLC-D, Trace Level Detection
column . . . . . . . Astec CLC-D, 15 cm x 4.6 mm I.D., 5 μm particles (53023AST) HO *
*
OH
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . 3 mM copper sulfate, pH 3.2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL (D)-Tartaric acid,
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg/mL in water 5.06 min.
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004665
0.5% (L)-Tartaric acid,

7.37 min.

Normal Phase Mode

HPLC Analysis of 3,5-tBu-MeOBIPHEP Enantiomers (n-Hexane / Ethanol)
Polar Ionic Mode
(Method / Acetic Acid / TEA)
on Astec® CHIROBIOTIC® V in Normal Phase and
8.31 min.
Polar Ionic Modes 25.8 min.
t-Bu t-Bu 27.5 min.

Demonstration of CHIROBIOTIC® column utility in both normal phase and
polar ionic modes. The same CHIROBIOTIC® column can be used in all four t-Bu
N
modes, from aqueous to organic, without memory effects or loss of H3CO
P
performance. t-Bu 9.12 min. O

Column: CHIROBIOTIC® V, 25 cm x 4.6 mm, 5 μm particles (11024AST). t-Bu
F
H3CO
Left: Normal phase mode. 3,5-tBu-MeOBIPHEP enantiomers. n-Hexane:10% P
ethanol in n-hexane (75:25), 1.5 mL/min. t-Bu H3C N HBr
Right: Polar ionic mode. Citalopram enantiomers. Methanol:acetic acid:TEA t-Bu t-Bu CH3
(99.8:0.1:0.1), 0.5 mL/min.
column . . . . . . . . CHIROBIOTIC® V, 25 cm x 4.6 mm, 5 μm particles (11024AST)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004430
Application courtesy of Dr. M. Althous,
Hoffman la Roche, Basel, Switzerland

HPLC Analysis of 3,5-tBu-MeOBIPHEP Enantiomers t-Bu t-Bu 8.31 min.

on Astec® CHIROBIOTIC® V
t-Bu
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST)
P
mobile phase . . . . . . . (A) n-hexane; (B) 10% ethanol in n-hexane; (75:25, A:B) H3CO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min t-Bu
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 °C t-Bu
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm H3CO 9.12 min.
sample . . . . . . . . . . . . . . . . . . 3,5-tBu-MeOBIPHEP ((6,6′-dimethoxybiphenyl- P
2,2′-diyl)bis[bis(3,5-di-tert-butylphenyl)phosphine]), t-Bu
1 mg/mL in ethanol with 5 drops of H2O 2 added for oxidation
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004402 t-Bu t-Bu
Application courtesy of
Dr. M. Althous, Hoffman la Roche,
Basel, Switzerland

HPLC Analysis of 3a,4,5,6-Tetrahydrosuccinimido O

[3,4-b]acenaphthen-10-one Enantiomers on O
*
Astec® CHIROBIOTIC® V NH
mobile phase . . . . . . . . . . . . . . . . . . . (A) hexane; (B) ethanol; (75:25, A:B) *
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004648 0 10 20
Min

3
HPLC Analysis of Toluidine Isomers on Ascentis® Si 2
(Hexane: 2-Propanol) 1. o-Toluidine

2. m-Toluidine
1
column . . . . . . . . Ascentis Si, 15 cm × 4.6 mm I.D., 5 μm particles (581512-U) 3. p-Toluidine
mobile phase . . . . . . . . . . . . . . . . . . (A) hexane; (B) 2-propanol; (96:4, A:B)

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 245 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003799 0 2 4 6 8 10 12
Min

HPLC Analysis of Toluidine Isomers on Ascentis® Si 2 3

(Silica), (80:20 Hexane:Ethyl Acetate) 1. o-Toluidine
2. m-Toluidine
column . . . . . . . . Ascentis Si, 15 cm × 4.6 mm I.D., 5 μm particles (581512-U) 3. p-Toluidine
1
mobile phase . . . . . . . . . . . . . . . . (A) hexane; (B) ethyl acetate; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 245 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003697
0 2 4 6 8 10 12
Min

HPLC Analysis of 16 Triazine and Phenyl Urea 1. Atrazine desisopropyl 10. Isoproturon
Compounds on Ascentis® Express RP-Amide 2. Atrazine desethyl 11. Terbumeton
3. Carbendazime 12. Diuron
This application demonstrates the suitability of Ascentis Express RP-Amide 4. Terbumeton desethyl 13. Propazine
for the efficient separation of 16 triazine and phenyl urea compounds. 5. Simazine 14. Terbuthylazine
Lower column temperature can improve resolution of the critical pair. 6. Terbuthylazin desethyl 15. Linuron
column . . Ascentis Express RP-Amide, 15 cm x 3 mm I.D., 2.7 μm particles (53919-U) 7. Cyanazine 16. Terbutryne
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile 8. Atrazine
9. Chlortoluron 10 14
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 to 53% B in 70 min 6 9 16
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 12 15
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 2 7 8 11 13
1 45
sample . . 20 mg/L (except terbuthylazin desethyl and terbuthylazine 60 mg/L) in
60:40, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005651
0 20 40 60
Min

HPLC Analysis of 16 Triazine and Phenyl Urea 1. Atrazine desisopropyl 9. Chlortoluron

Compounds on Ascentis® Express RP-Amide, 2. Atrazine desethyl 10. Isoproturon
3. Carbendazime 11. Terbumeton
Modified Gradient 4. Terbumeton desethyl 12. Diuron
This application demonstrates the suitability of Ascentis Express RP-Amide 5. Simazine 13. Propazine
for the efficient separation of 16 triazine and phenyl urea compounds. 6. Terbuthylazin desethyl 14. Terbuthylazine 14 16
Lower column 7. Cyanazine 15. Linuron
10 15
temperature can improve resolution of the critical pair. 8. Atrazine 16. Terbutryne
9 12
gradient . . 10 to 25% B in 30 min; 25 to 60% B in 15 min; held at 60% B for 5 min 3 11 13
2 8
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 1 4 5 7
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm 0 10 20 30 40
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL Min
sample . . 20 mg/L each (except terbuthylazin desethyl and terbuthylazine 60 mg/L)
in 60:40, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005652

HPLC Analysis of Triazole Antifungal Agent 1. Enilconazole 5. Miconazole

2. Enilconazole 6. Miconazole
Enantiomers on Astec® CYCLOBOND® I 2000 3. Eberconazole 7. Ketoconazole
HP-RSP mAU 4. Eberconazole 8. Ketoconazole
17.5
column . . . . . . . . . . . Astec CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D., 15 5
5 μm particles (24024AST) 12.5
2,3 6
mobile phase . . . . . . . . . . . (A) 0.1% formic acid; (B) acetonitrile; (70:30, A:B) 10
4 7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 7.5 8
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 5 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 2.5
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 mg/mL in methanol 0
peaks 1&2: enilconazole 0 2 4 6 8 10 12 14 16 18
peaks 2&3: eberconazole Min
peaks 4&5: miconazole
peaks 6&7: ketoconazole
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004737

HPLC Analysis of Tröger′s Base Enantiomers on

(+)
Astec® Cellulose DMP H 3C
N
Tröger′s base is an organic compound that exhibits chirality due to the (-)
presence of two bridgehead stereogenic nitrogen atoms in its structure.
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) N
mobile phase . . . . . . . . . . . . (A) heptane; (B) IPA; (C) DEA; (90:10:0.1, A:B:C)
C H3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004996
0 2 4 6 8 10
Min


1. Tröger’s Base enantiomer
Astec® Cellulose DMP (LC/MS Mobile Phase) 2. Tröger’s Base enantiomer
H 3C
Tröger′s base is an organic compound that exhibits chirality due to the N
presence of two bridgehead stereogenic nitrogen atoms in its structure.

column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) 1 2
N
mobile phase . . . . . . . . . (A) methanol; (B) ammonium formate; (100:0.1, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min C H3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6 8 10


Astec® Cellulose DMP (Polar Organic Mode) 1. Tröger’s Base enantiomer
H 3C 2. Tröger’s Base enantiomer
N
Tröger′s base is an organic compound that exhibits chirality due to the
presence of two bridgehead stereogenic nitrogen atoms in its structure. 1 2
mobile phase . . . . . . . . . . . . . . . . . . . (A) methanol; (B) DEA; (100:0.1, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min C H3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6 8 10
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg/mL in mobile phase Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005001

HPLC Analysis of Xylene Isomers on 1. m-Xylene

2
Discovery® Zr-Carbon 2. p-Xylene 1
3. o-Xylene
column . . . Discovery Zr-Carbon, 15 cm × 4.6 mm I.D., 3 μm particles (65730-U)
3
mobile phase . . . . . . . . . . . . . . . . . . (A) water: (B) acetonitrile; (50:50, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . o-xylene, m-xylene, p-xylene (290 μg/mL)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001880
0 2 4 6 8
Min

LC-MS Analysis of Cyanuric Acid on Ascentis® 1 1. Cyanuric acid

Express HILIC 2. Ammelide
column . . Ascentis Express HILIC, 5 cm x 2.1 mm I.D., 2.7 μm particles (53934-U)

mobile phase . . . . . . . 5 mM ammonium formate in 95:5 (v/v) acetonitrile:water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 psi (22.6 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(-), SIR mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6 8 10 12
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005690

LC-MS Analysis of L-Methionine Sulfoximine and 2 1. L-Methionine sulfone

1 2. L-Methionine sulfoximine
Sulfone on Ascentis® Express OH5
L-methionine sulfoximine and L-buthionine sulfoximine are used in are used
to prevent additional enzyme activity in Chinese hampster ovary (CHO) cell
lines with supplemental Glutamine Synthase (GS). It is of interest to be able
to monitor both purity of such molecules in order to control addition to cell
media as well as for the assay of parent molecules during use. The analytes
are highly polar and thus should be amenable to HILIC-LC-MS analysis. This
first installment investigates the ability of Ascentis Express HILIC and
Ascentis Express OH5 stationary phases for the separation of the methionine
analog and related compounds as well as methionine and buthionine
separations.
column . . Ascentis Express OH5, 10 cm x 3.0 mm I.D., 2.7 μm particles (53769-U)
mobile phase . . . . . . . . . . . . (A) acetonitrile; (B) water with 0.1% formic acid, 0 2 4 6 8 10
pH 3.5 with ammonium formate; (75:25, A:B) Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1350 ppi (93 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI (+), TIC, m/z 150-300
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005791

LC-MS Analysis of Quarternary Ammonium 1. Propantheline (1 µg/mL) (M =368.22)

Compounds on Ascentis® Si (Silica) 2. Oxyphenonium (1 µg/mL) (M = 348.25)
3. Mepenzolate (1 µg/mL) (M= 340.19)
column . . Ascentis Si, 15 cm x 4.6 mm I.D., 5 μm particles (581512-U) (65741-U) 4. Scopolamine (10 µg/mL) (M+H)+ = 304.14
mobile phase . . . . . . . . (A) 0.1% ammonium acetate in water (pH unadjusted): 5. Ipratropium (1 µg/mL) (M = 332.22)
(B) 0.1% ammonium acetate in acetontrile; (10:90, A:B) 6. Methscopolamine (1 µg/mL) (M= 318.16)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min. split to the MS
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . MS, ESI (+) in selected ion recording (SIR) mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 5
sample . . . . . as indicated in 0.1% ammonium acetate in 10:90:water:acetonitrile 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003724
3
4 6
0 2 4 6 8 10 12 14 16 18
Min

LC-MS Analysis of Sudan Red Dyes on Ascentis® 1. Sudan Orange G (M+H)+= 215.07
2. Sudan Red G (M+H)+= 279.10
RP-Amide 3. Sudan I (M+H)+= 249.09
Gradient Program
4. Sudan II (M+H)+= 277.12
column . . . Ascentis RP-Amide, 15 cm × 4.6 mm I.D., 5 μm particles (565324-U) 5. Sudan III (M+H)+= 353.13 Time (min) %A %B
6. Sudan Red 7B (M+H)+= 380.17 0 5 85
mobile phase . . . . . . . . . . . . . (A) 0.1% formic acid in water (pH unadjusted): 7. Sudan IV (M+H)+= 381.16 8 15 85
(B) acetonitrile; (15:85, A:B) 1
10 0 100
15 0 100
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min, split to the MS 16 15 85
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 20 15 85
detector . . . . . . . . . . . . . . MS, APCI (+) in selected ion recording (SIR) mode 2

3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL 6
4
sample . . 1 μg/mL in 30:70, 0.1% formic acid in water (pH unadjusted):acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003860
5 7
0 2 4 6 8 10 12 14
Min

LC-MS Extracted Ion Chromatogram of MSX, 2 1. BSX (log P = log D = 0.52)

Sulfone, and Sulfoxide on Ascentis® Express OH5 1 2. MSX (log P = log D = -1.01)
(HILIC Mode)
This figure shows the extracted ion currents for methionine sulfoximine-
related compounds on Ascentis Express OH5. The OH5 shows excellent peak
shape compared to traditional HILIC on bare silica.
column . . . Ascentis Express OH5, 10 cm x 3 mm I.D., 2.7 μm particles (53769-U)
mobile phase . . . . . . . . (A) 0.1% formic acid, pH 3.5 with ammonium formate;
(B) acetonitrile; (25:75, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1350 psi (93 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+), scan m/z 150-300 0 2 4 6 8 10
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL in 90% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005803

HPLC, UHPLC or LC-MS Clinical
1. Niacinamide
Clinical 2. Pyridoxine
3. Ascorbic Acid
HILIC Analysis of Polar Vitamins on Ascentis® 2 4. Thiamine
Express 2.7 μm OH5
This application demonstrates good retention and selectivity for polar 1 4
vitamins on Ascentis® Express OH5 in HILIC mode.
column . . Ascentis Express OH5 10 cm x 3.0 mm I.D. x 2.7 μm particles (53769-U)
mobile phase . . . . . . . . . 100 mM ammonium formate:Acetonitrile (15:85, v/v),
pH adjusted to 5.0 with formic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min 3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 850 psi (58.6 bar)
column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 276 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . 100 ug/mL in 50:50 methanol:acetonitrile 0 2 4 6 8 10

HPLC Analysis of ACE Inhibitors on Ascentis® 1. Captopril (1 μg/mL) (M+H)+ =218.07

2
Phenyl 2. Enalapril ( 0.1 μg/mL) (M+H)+ =377.19
3. Quinapril (1 μg/mL) (M+H)+=439.21
(pH 2.0 with concentrated formic acid): (B) methanol; (40:60, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min, split to the MS
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . as indicated in 40:60, 13 mM ammonium formate (pH 2.0 with 1
concentrated formic acid):methanol 3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003702
0 2 4 6 8 10 12
Min

1 1. Normorphine
HPLC Analysis of Analgesics, Narcotics on 2. Morphine
Ascentis® Phenyl 2 3. Codeine
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 3

(pH 3.0 with formic acid); (B) acetonitrile; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
Min
sample . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 90:10, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003709

1. Lidocaine 3
HPLC Analysis of Anesthetics, Local on 2. Prilocaine
Ascentis® Phenyl 3. Benzocaine

mobile phase . . . . . . (A) 10 mM ammonium formate (pH 3.0 with formic acid); 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12
sample . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 85:15, water:methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003708

HPLC Analysis of Angiotensins on Discovery® C18 1 1. Angiotensin II

2. Angiotensin III
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 2 3. Angiotensin I
mobile phase . . . . . . . . . . . . . . . . . . (A) 5 mM (NH4)H2 PO4/NH4 OH, pH 7.0; 3
(B) 5 mM (NH4 )H2 PO4/NH 4OH, pH 7.0: CH3 CN (50:50, A:B)
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30-60% B in 15 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001085
0 10 20
Min

HPLC Analysis of Antibacterials/Antifungals on 1 1. Furaltadone

2. Nitrofurantoin
Ascentis® RP-Amide (Formate Mobile Phase) 3. Chloramphenicol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in mobile phase
3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002602
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Anticonvulsant Drugs on 1. Clonazepam

1 2. Clorazepate
Discovery® RP-AmideC16 3. Diazepam
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955)
mobile phase . . (A) acetonitrile; (B) 25 mM potassium phosphate, pH 7.0 (40:60, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000188
0 2 4 6 8 10
Min

HPLC Analysis of Antihypertensive ACE Inhibitors 1. Enalapril 2

2. Lisinopril
on Discovery® RP-AmideC16 3. Captopril
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013)
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000279
1.0 2.0 3.0 4.0 5.0
Min

HPLC Analysis of Antihypertensive and Diuretic 3 1. Atenolol

Drugs on Discovery® RP-Amide C16 2. Amiloride
2 3. Hydrochlorothiazide
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 4. Caffeine
mobile phase . . . . . . . . . . . . . (A) methanol; (B) 25 mM potassium phosphate, 4
dibasic, pH 7.0 (22:78, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000280
0 2 4 6 8 10
Min

HPLC Analysis of Antihypertensive Drugs on Gradient Program 1 1. Pindolol

Ascentis® C18 Time (min) %A %B 2. Acebutolol
0 90 10 3. Labetalol
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 2 90 10
4. Alprenolol
mobile phase . . . . . . . . . . . . . . . (A) 10 mM monobasic potassium phosphate 8 40 60
(pH 7.0 with potassium hydroxide); (B):acetonitrile 12 40 60
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002383
0 2 4 6 8 10 12
Min

HPLC Analysis of Antipsychotic Drugs on Ascentis® 1. Desmethyl venlafaxine

Express RP-Amide 2. Hydroxybupropion
3. Hydroxyrisperidon
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 10 cm x 2.1 mm I.D., 4. Bupropion
2.7 μm particles (53913-U) 5. Zuclopehthixol
mobile phase . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate (in water); 6. Fluphenazine
(B) 5 mM ammonium formate (in 95:5 methanol:water) 6
1 3 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), TIC 100 - 1000 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 μg/L in water 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005373
0 2 4 6 8
Min

HPLC Analysis of Antiviral Drugs on Ascentis® C8 1. Valacyclovir (50 µg/mL) Gradient Program 2
2. Imiquimod (50 µg/mL) Time (min) %A %B
column . . . . . . . . Ascentis C8, 15 cm x 2.1 mm I.D., 3 μm particles (581402-U) 0 95 5
mobile phase . . (A) 10 mM ammonium phosphate (pH 2.5 with phosphoric acid); 10 20 80
(B) acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4
Min
6 8 10
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003201

HPLC Analysis of Barbituates in Serum on *IS

Efficiency of Recovery
Concentration
Discovery® C18 after SPE using 1 Compound
1. Phenobarbital
(μg/mL)
0.5
%Recovery
96.2
%RSD (n=6)
±1.6
Discovery® DSC-18Lt 2. Aprobarbital
1.0
0.5
94.9
98.5
±1.7
±2.1
2 1.0 100.8 ±0.8
A Zymark® RapidTrace® SPE Workstation was used for this analysis.
3. Butabarbital 0.5 97.2 ±1.9
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 1.0 98.7 ±1.8
3
sample/matrix . . 0.5 mL porcine serum spiked with 0.5 μg/mL or 1.0 μg/mL each 4 4. Mephobarbital 0.5
1.0
99.7
101.0
±2.4
±2.0
analyte then diluted with 0.5 mL water 5. Pentobarbital 0.5 96.4 ±1.7
1.0 96.4 ±1.9
SPE tube/cartridge . . . . . . . . . . . Discovery DSC-18Lt, 500 mg/3 mL (52613-U) 5
6 6. Secobarbital 0.5 98.2 ±1.7
condition . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL methanol; 2 mL DI water 1.0 97.7 ±1.8
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL at 0.75 mL/min SPE Method For RapidTrace SPE Workstation Application
washing . . . . . . . . . . 2 mL 5% methanol, then vacuum or air dry for 5-10 min 0 10 20 30 1. Condition & equilibrate each tube/well with 2 mL MeOH &
Min 2 mL DI Water
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 mL methanol 2. Load sample
eluate post-treatment . . . . . dry eluate with nitrogen purge (40 °C; 15-20 min), 3. Wash each tube/well with 2 mL 5% MeOH
Barbiturates from serum using 500 mg/3 mL Discovery DSC-18Lt SPE 4. Vacuum or air dry for 5–10 min
reconstitute in 20 μL mobile phase tubes and Zymark’s RapidTrace SPE Workstation.
*IS = Barbital (internal standard).
This removes any excess water from the sorbent.
The presence of water in the final eluent may prolong
column . . . . . . . . . . . . Discovery C18, 15 cm × 4.6 mm, 5 μm preceded by a Sample Info: 0.5 mL porcine serum spike with 0.5 μg/mL or 1.0 μg/mL eluent evaporation.
2 cm C18 guard column and 0.5 μm frit filter (504955) each analyte then diluted with 0.5 mL water. 5. Elute with 1–2 mL MeOH
6. Dry eluate with nitrogen purge (40 °C; 15–20 min)
mobile phase . . . . . . . . . . . . . . . . . . . (A) methanol; (B) water (40:60, A:B) 7. Reconstitute with 200 μL mobile phase
8. Quantify against internal or external standards via
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min HPLC analyses
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . 30 μL, diluted porcine serum extract
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001056

3 1. Nitrazepam (M+H)+= 282.08

HPLC Analysis of Benzodiazepines on Ascentis® C8 2. Clonazepam (M+H)+= 316.04
4 3. Desmethyldiazepam (M+H)+= 271.05
column . . . . . . . . Ascentis C8, 15 cm x 2.1 mm I.D., 3 μm particles (581402-U) 1 4. Diazepam (M+H)+= 285.07
mobile phase . . . . . . . . . (A) 10 mM ammonium acetate (pH 6.98 unadjusted);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
2
detector . . . . . . . . . . . . . . . . . . . MS, ESI (+), Selected Ion Recording Mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . 1 μg/mL each in 10 mM ammonium acetate (pH 6.98 unadjusted)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003309
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Benzodiazepines on Ascentis® 1. Oxazepam

Express C18, Acetonitrile Mobile Phase 2. Clonazepam
3. Alprazolam
column . . . Ascentis Express C18, 10 cm x 3 mm I.D., 2.7 μm particles (53814-U) 1 4. N-Desmethyldiazepam
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (66:34, A:B) 5. Diazepam
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
2 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 5
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g/L each in 20% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005673
0 2 4 6 8
Min

HPLC Analysis of Benzodiazepines on Ascentis® 1. Clonazepam

2. Oxazepam
Express Phenyl-Hexyl, Methanol Mobile Phase 3. Alprazolam
The Phenyl-Hexyl phase of the Ascentis Express product line displays the 4. N-Desmethyldiazepam
best selectivity of this sample; selectivity is markedly affected by choice of 5. Diazepam
organic modifier. The application also demonstrates feasibility of fast LC with 2
Ascentis Express by virtue of low backpressure and high efficiency. 1 4
2.7 μm particles (53348-U) 3 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3600 psi (248 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 1.0 2.0 3.0
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g/L in 20% methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005674


Express Phenyl-Hexyl with Acetonitrile Mobile 2. Alprazolam
3. Clonazepam
Phase 4. N-Desmethyldiazepam
The Phenyl-Hexyl phase of the Ascentis Express product line displays the 5. Diazepam
best selectivity of this sample; selectivity is markedly affected by choice of 1
organic modifier. The application also demonstrates feasibility of fast LC with
Ascentis Express by virtue of low backpressure and high efficiency. 4
column . . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 5 cm x 4.6 mm I.D., 2
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2600 psi (179 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm 1.0 2.0 3.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL Min
sample . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g/mL of each in 20% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005675

3 1. Propranolol (50 µg/ml)

HPLC Analysis of Beta Blockers on Ascentis® 2. Pindolol (50 µg/ml)
Si (Silica) 1 3. Acebutolol (50 µg/ml)
4. Atenolol (50 µg/ml)
column . . . . . . . . Ascentis Si, 15 cm x 4.6 mm I.D., 5 μm particles (581512-U) 2
mobile phase . . . . . . . . (A) 0.1% ammonium acetate in water (pH unadjusted); 4
(B) 0.1% ammonium acetate in acetonitrile; (15:85, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
sample . . . . . as indicated in 0.1% ammonium acetate in 10:90:water:acetonitrile Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003735

HPLC Analysis of Beta-Receptor Agonist

column . . . . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm, 5 μm particles (12024AST)
mobile phase . . . . . . . . . . . . . . . . . 15 mM ammonium formate in methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004337

HPLC Analysis of Bile Acids on Ascentis® 1. Dehydrocholic Acid

Express C18 2. Cholic Acid
5
3. Chenodeoxycholic Acid
column . . Ascentis Express C18, 5 cm x 4.6 mm I.D., 2.7 μm particles (53826-U) 4. Deoxycholic Acid 3 4
mobile phase . . (A) 10 mM ammonium acetate, pH 4 (adjusted with acetic acid); 5. Lithocholic Acid
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 to 100% B in 10 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(-) in SIR mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
0 2 4 6 8
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL

HPLC Analysis of Bilirubin and Biliverdin on 1. Biliverdin contaminant

2. Biliverdin (0.25 g/L)
Discovery® C18 3. Biliverdin contaminant
column . . . . . . Discovery C18, 10 cm × 2.1 mm I.D., 5 μm particles (569220-U) 4. Bilirubin contaminant
mobile phase . . . . . . . . . . . . . (A) 10 mM ammonium acetate (pH unadjusted) 0.12 5 5. Bilirubin (0.25 g/L)
(B) 90:10, methanol:(100 mM ammonium acetate) 6. Bilirubin contaminant
0.10
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient Gradient Program
0.08 Time (min) %A %B
Volts
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 405 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 0.06 0 40 60
20 20 80
sample . . . . . . . . . . . . . . . . . . . . . as indicated (in 50% MeOH, 1% NH4 OH)
0.04
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002490
0.02
1 3 4 6
0.00
0 10 20 30
Min

HPLC Analysis of Biogenic Amines on Ascentis® 1. Histamine

RP-Amide 2. Spermine
6
3. Spermidine
Following pre-column derivatization of primary amino groups using
4. Tyramine
o-phthaldialdehyde/thiol reagent, the Ascentis RP-Amide was used for the
5. Putrescine
analysis of biogenic amines commonly found in wines. These include 5
6. Cadaverine
histamine, spermine, spermidine, tyramine, putrescine and cadaverine.
Structures along with an optimized chromatogram are presented.
column . . . Ascentis RP-Amide, 15 cm x 2.1 mm I.D., 5 μm particles (565305-U) 2 3
1 4
mobile phase . . . . . . . . (A) 0.1% ammonium acetate in water (pH unadjusted);
(B) acetonitrile
gradient . . . . . . . . . . . . . . 25 to 75% B in 10 min; held at 75% B for 5 min 0 2 4 6 8 10 12 14
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 340 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005680

1. Dopamine 2
HPLC Analysis of Biogenic Amines on Ascentis® Si, 2. Epinephrine
1
HILIC Mode, (90 % Acetonitrile) 3. Norepinephrine
column . . . . . . . . Ascentis Si, 15 cm x 4.6 mm I.D., 5 μm particles (581512-U)

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 10 20
sample . . . 50 ug/mL each in 0.1% ammonium acetate in 10:90:water:acetonitrile Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003717

HPLC Analysis of Biogenic Amines on Ascentis® Si, 1 1. Tyramine

2 3 2. Dopamine
HILIC Mode (85 % Acetonitrile) 3. Epinephrine
column . . . . . . . . Ascentis Si, 15 cm x 4.6 mm I.D., 5 μm particles (581512-U) 4. Levodopa
mobile phase . . . . . . . . (A) 0.1% ammonium acetate in water (pH unadjusted);
(B) 0.1% ammonium acetate in acetonitrile; (15:85, A:B) 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . 50 μg/mL in 0.1% ammonium acetate in 10:90:water:acetonitrile 0 2 4 6 8 10 12 14

HPLC Analysis of β-Blockers and β-Agonists in Gradient Program

Urine and Wastewater on a C18 Column after SPE 16000
Time (min)
0
%A
95
%B
5
Analyte
Atenolol
Rt (min.)
3.0
Q1/Q3
267.2/145
2 90 10
using SupelMIP® SPE-Beta-Receptor 5 50 50
Carazolol
Metoprolol
6.2
5.6
299.1/194.2
268.2/133
6 50 50
Propranolol 6.5 260.2/154.9
Sample Pre-treatment: 7 95 5
Timolol 5.5 317.2/188.1
Clenbuterol 5.6 277.1/202.9
Horse urine was centrifuged at 3000 g for 10 min, diluted 1:1 (v/v) with DI Ritodrine 3.9 288.2/121
water, adjusted to pH 7. Wastewater was filtered with 1 μm filter paper and 12000 Salbutamol 2.6 240.2/147.9
Terbutaline 2.6 226.2/152
adjusted to pH 6-7. Samples were spiked with 10 beta-agonists and beta- Tulobuterol 5.6 228.2/154.1
blockers at the level of 1 ng/mL.
cps
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) Lower Limit of Quantitation
8000
sample/matrix . . . . . . . . . . . . . . . . . . horse urine or waste water spiked with (ng/mL, ppb, or µg/kg)
Analyte 1 mL Horse Urine 10 mL Wastewater
10 beta-agonists and beta-blockers at 1 ng/mL Atenolol 0.1 0.01
SPE tube/cartridge . . SupelMIP SPE - Beta-Receptor, 25 mg/10 mL (LRC) (53223-U) Carazolol
Metoprolol
0.1
0.1
0.01
0.01
condition . . . . . . . . . . . . . . . . . . . . . . . . 1 mL acetonitrile; 1 mL DI water Propranolol
Timolol
0.1
0.1
0.01
0.01
4000
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL Clenbuterol 0.02 0.002
Ritodrine 0.05 0.005
washing . . . 3 x 1 mL DI water; 1 mL acetonitrile; 1 mL 60% acetonitrile/40% DI Salbutamol 0.1 0.01
Terbutaline 0.2 0.02
water; apply 2 min of full vacuum to dry the tube after each step Tulobuterol 0.005 0.0005
elution . . . . . . . . . . . . . . . . . . . . . . 2 x 1 mL 1% formic acid in acetonitrile
0
eluate post-treatment . . . . . . . . evaporate under nitrogen and reconstitute with 0 2 4 6
150 μL 5% acetonitrile in 10 mM ammonium acetate, Rt (min)
pH 4.6 prior to LC-MS-MS analysis Note: Clenbuterol and Tulobuterol were spiked at the levels of 0.1 ng/mL.
column . . . . . . . . . . . . . . . . . . . . . . C18, 5 cm x 3 mm I.D., 3 μm particles

mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 10 mM ammonium acetate,
pH 4.6 (adjusted with acetic acid); and (B) acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS/MS, ESI(+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004059

HPLC Analysis of Bovine Serum Albumin (BSA) on

Zenix® SEC-300 versus SRT® SEC-300, Effect of
Particle Size on Resolution
column . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm, 300 Å particles (Z777051)
column . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm, 300 Å particles (Z777033) SRT SEC-300
mobile phase . . . . . . . . . . . . . . . . . . . . . . 150 mM sodium phosphate, pH 7

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient Zenix SEC-300
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 6 7 8 9 10 11
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg/mL, BSA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006119 SRT SEC-300
Zenix SEC-300
0 1 2 3 4 5 6 7 8 9 10 11
Min

HPLC Analysis of Bronchodilators Theophylline Efficiency of Recovery

Concentration
and Other Caffeine Metabolites from Serum on Compound (μg/mL) % Recovery % RSD (n=6)
Discovery® RP-AmideC16 after SPE using 1. Theobromine 0.1 97.4 ±6.8
0.5 96.4 ±8.5
Discovery® DSC-18 1.0 96.1 ±5.0
2. Paraxanthine 0.1 96.2 ±8.4
SPE tube/cartridge . . . . . . . . . . . . Discovery® DSC-18, 500 mg/3 mL (52603-U) 1.0 95.0 ±8.7
condition . . . . . . . . . . . . . . . . . . . . . . . . 2 mL methanol, then 2 mL water 3. Theophylline 0.1 97.8 ±8.5
0.5 97.8 ±8.8
sample preparation . . . . . . . . . . . . 1 mL porcine serum spiked with 0.1 μg/mL, 1.0 98.5 ±5.7
0.50 μg/mL, or 1.0 μg/mL each analyte 4. Caffeine 0.1 98.8 ±3.9
sample preparation . . . . . . . . . . . . . 1 mL methanol; evaporate to dryness with 0.5 95.6 ±6.7
1.0 97.6 ±5.8
nitrogen stream at room temperature; reconstitute in 200 μL mobile
phase containing 0.2 μg/mL or 7-methylxanthine (IS)
1. Theobromine
sample preparation . . . . . . . . . . . . . . . . . . . . . 2 mL 5% methanol in water; 2. Paraxanthine
dry tube 10 min with nitrogen stream 3. Theophylline
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm, 5 μm particles, preceded by 4. Caffeine
a 2 cm RP-AmideC16 guard column and 0.5 μm frit filter (505013) IS 7-Methylxanthine
mobile phase . . . . . . . . . . . . . . (A) Methanol; (B) 1% acetic acid (17:38, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 272 nm Serum Blank
injection . . . . . . . . . . . . . . . . . . . . 20 μL reconstituted porcine serum extract
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000594 3
IS 2
4
Spiked Serum
(0.50 mg/mL)
0 2 4 6 8 10 12
Min

HPLC Analysis of Caffeine and Metabolites on 1. Uric acid (50 mg/mL) Gradient Program
2. Xanthine (30 mg/mL) Time (min) %A %B
Ascentis® RP-Amide 3. 7-methylxanthine (20 mg/mL) 0 98 2
4. 1-methyluric acid (40 mg/mL) 3 92 8
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 5. 1-methylxanthine (20 mg/mL) 12 80 20
mobile phase . . (A) 0.1% ammonium acetate in water (pH 4.5 with acetic acid ); 6. Theobromine (30 mg/mL) 14 80 20
(B) acetonitrile 7. 1,7-dimethyluric acid (40mg/mL) 14.1 98 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 8. 1,7-dimethylxanthine (50 mg/mL) 15 98 2
9. Theophylline (50 mg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 10. beta-(hydroxyethyl) theophylline (50 mg/mL)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 275 nm 11. Caffeine (50 mg/mL)
4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 9
1 7 10 11
sample . . as indicated in 0.1% ammonium acetate in water (pH 4.5 with acetic acid) 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003750 2
8
3
5
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Caffeine Metabolites and 1. Unknown 6. 1,3-Dimethyluric acid

Analogs on Discovery® C18 2. 7 -Methylxanthine 7. 1,3-Dimethylxanthine
3. 1-Methyluric acid 8. 1,7-Dimethylxanthine
column . . . . . . . . Discovery C18, 15 cm x 4.6 mm I.D., 5 μm particles (504955) 4. 1-Methylxanthine 9. 3,7-Dimethylxanthine
mobile phase . . . . . . . . (A) 1% acetic acid in water; (B) methanol; (92:8, A:B) 5. 3-Methylxanthine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005684
7
3 6 8
5 9
1
0 10 20
Min

HPLC Analysis of Calphostin C on Discovery® 1 1. Calphostin C

HS C18
Gradient Program
column . . . . Discovery HS C18, 25 cm × 2.1 mm I.D., 5 μm particles (568503-U) Time (min) %A % B
mobile phase . . . (A) (0.1% phosphorus acid in water):(0.1% phosphorus acid in 0 70 30
acetonitrile); (B) 0.1% phosphorus acid in acetonitrile; (50:50, A:B) 25 20 80
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 30 20 80
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
sample . . . . . . . . . . . . . . Calphostin C (0.25 g/L) in 40% MeOH, 0.025% TFA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002574
0 10 20 30
Min

HPLC Analysis of Catechols and Resorcinols on 1. Resorcinol (50 mg/mL)

2. Catechol (50 mg/mL)
Ascentis® RP-Amide 3. 2-methyl resorcinol (50 mg/mL)
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 4. 4-methyl catechol (50 mg/mL)
mobile phase . . . . . . . . . . . . (A) 20 mM phosphoric acid (pH 2.0 unadjusted): 2 5. 2,5-dimethyl resorcinol (50 mg/mL)
1
(B) acetonitrile; (75:25, A:B) 6. 3-methyl catechol (50 mg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 7. 4-nitro catechol (50 mg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL 3 6
sample . . . . . . . . . . . . . . . . as indicated in 20 mM phosphoric acid (pH 2.0) 5 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002597
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Codeine and Metabolite on 1. Codeine β-D-glucuronide 2

2. Codeine
Ascentis® C18 1
mobile phase . . . . . . . . . . . . . . . (A) 10 mM monobasic potassium phosphate
(pH 3.0 with phosphoric acid); (B) acetonitrile; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002417
0 2 4 6 8
Min


2. Codeine 1
Ascentis® RP-Amide
column . . . . Ascentis RP-Amide 15 cm x 4.6 mm I.D., 5 μm particles (565324-U)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002599
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Min

HPLC Analysis of Conjugated Estrogens on 1. Estriol 3-(β-D-glucuronide)

2. β-estradiol 3-(β-D-glucuronide) 17-sulfate
Discovery® HS C18 3. Estriol 3-sulfate
4. β-estradiol 3,17-disulfate
column . . . . Discovery HS C18, 15 cm × 4.6 mm I.D., 5 μm particles (568522-U) 5. β-estradiol 3-(β-D-glucuronide)
mobile phase . . . . . . . . (A) 10 mM potassium phosphate (pH 7.01 unadjusted); 6. β-estradiol 17-(β-D-glucuronide)
(B) acetonitrile; (C) methanol 7. Estrone 3-(β-D-glucuronide)
8. Estriol Gradient Program
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 9. β-estradiol 3-sulfate Time (min) %A %B %C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 10. Estrone 3-sulfate
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 11. β-estradiol 0 80 15 5
1
12. α-estradiol 5 80 15 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 13. Estrone 25 40 45 15
sample . . . . . . . . . . . . . . . . . . . . 10 μg/mL each in 60/40, water:acetonitrile 30 40 45 15
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002155 8
7
4 56
10 11
2
3
13
12
9
0 10 20 30
Min

HPLC Analysis of Cortocisteroids in Urine Efficiency of Recovery

on Discovery® HS F5 after SPE using %Recovery ± RSD (n=3)
Compound 0.5 µg/mL spike level 1.0 µg/mL spike level
Discovery® DSC-CN 1. Hydrocortisone 123.3±1.4% 95.9±1.7%
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 2. Prednisilone 107.2±1.1% 91.9±1.1%
sample/matrix . . . . . . . . . . . . . . . . human urine spiked with corticosteroids at 3. Prednisone 103.2±1.0% 88.4±1.8%
0.5 and 1.0 μg/mL and diluted in DI water (1:1, v/v)
SPE well plate . . . . . . . Discovery DSC-CN 96-well SPE, 100 mg/well (575636-U) 1
condition . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol; 1 mL DI water. 1 µg/mL spiked urine extract
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL spiked urine on Discovery DSC-CN
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 20% methanol
3
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol 2
eluate post-treatment . . . . . . . . . . . . Evaporate eluate with nitrogen at 30 °C.
Reconstitute in 200 μL mobile phase. 4
column . . . . . . Discovery HS F5, 5 cm x 4.6 mm I.D., 3 μm particles (567504-U)
mobile phase . . . . . . . . . . . . . . . . . (A) methanol; (B) DI water; (40:60, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm 0 2 4 6 8 10 12 14
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003763
Blank urine extract
on Discovery DSC-CN
0 2 4 6 8 10 12
Min

HPLC Analysis of CYP3A Inhibitors on Ascentis® 1. Fluvoxamine

Express RP-Amide 2. Fluoxetine
3. Ketoconozole
This application demonstrates the suitability of the Ascentis Express RP 3 4. Sertraline
5
Amide for the analysis of CYP3A Inhibitors. 5. Nefazodone
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 10 cm x 2.1 mm I.D., 6. Lovastatin
mobile phase . . . (A) 20 mM ammonium formate in 50:50 (v/v) water:acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1 2 4 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 100-600
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005750 1.0 2.0 3.0 4.0
Min

HPLC Analysis of Diazepam and Metabolites in Efficiency of Recovery

Serum on Discovery® C18 after SPE using %Recovery ± RSD (n=3)
Compound Systematically Generic Method On
Discovery® DSC-8 Developed Method on Conventional C18
Discovery DSC-8
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction)
sample/matrix . . goat serum spiked with diazepam and metabolites at 0.5 μg/mL and 1. Oxazepam 94.7 ± 1.2% 82.8 ± 4.0%
diluted 1:1 in 25 mM ammonium formate, pH 7.1 2. Temazepam 99.9 ± 1.1% 89.1 ± 4.0%
SPE well plate . . . . Discovery DSC-8 96-well SPE Plate, 100 mg/well (575627-U) 3. Desmethyl diazepam 94.2 ± 1.8% 82.4 ± 5.0%
condition . . . . . . . . . . . . . . . . . . 1 mL methanol followed by 1 mL DI water. 4. Diazepam 90.0 ± 3.4% 68.5 ± 9.1%
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL spiked goat serum
washing . . . . . . . . 1 mL 25% methanol in 25 mM ammonium formate, pH 2.75
External
elution . . . . . . . . . 1 mL 60% methanol in 25 mM ammonium formate, pH 2.75 1
Standard
elution . . . . . . . . . . . Evaporate eluate with nitrogen purge (30 °C; ~15 min.).
3
column . . . . . . . . . Discovery C18, 5 cm x 4.6 mm I.D., 5 μm particles (504947) 4
mobile phase . . (A) Methanol; (B)10 mM ammonium acetate, pH 4.5;(45:55, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 0.5 µg/mL
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C Spiked Serum
um
Extract on
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm DSC-8
Excellent
E Peak Shape
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
Application No.Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . G003766
Blank Serumm Low

L background/
Extract on minimal
m interferences
DSC-8 for
f optimal sensitivity
and
a resolution at
reduced
r run times
0 2 4 6
Min

HPLC Analysis of 25-Dihydroxyvitamin D2, 1. 25-Dihydroxyvitamin D2

CH3 2. 25-Hydroxyvitamin D3
CH3
25-Hydroxyvitamin D3 and 3-epi-25- H3 C
H3C
Hydroxyvitamin D3 on Ascentis® Express F5, 20 °C CH3
H3C
OH H3C OH
CH3 H
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) CH3
mobile phase . . . . . . (A) 5 mM ammonium formate; (B) methanol:water (75:25)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min H
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3553 psi (245 bar) H
H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C CH2 H
detector . . . . . . . . . . . . . . . . . . . . . (UV, 265 nm, ESI(+), m/z 100 - 1000) CH2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
HO OH
sample . . . . . . . . . . . . . . . . . . . . . . . . 500 mg/L in 25:75, water:methanol
HO
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005375
3. epi-25-Hydroxyvitamin D3
HO
H3C CH3
3
CH3 H 2
CH3
1
H
H
H
CH2
HO
0 2 4 6
Min


CH3 H3C
25-Hydroxyvitamin D3 and 3-epi-25- H3C CH3 H3C OH
Hydroxyvitamin D3 on Ascentis® Express F5, 30 °C CH3 OH CH3 H
H3C CH3
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U)
mobile phase . . (A) 5 mM ammonium formate in methanol; (B) water (25:75, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min H H
H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C CH2
CH2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 265 nm
ESI(+), m/z 100 - 1000 2. 25-Hydroxyvitamin D3
HO
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL O OH
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 μg/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005376
HO
H3C CH3 3
CH3 H 2
CH3
1
H
H
H
CH2
HO
0 2 4
Min


CH3
CH3 OH H3C
Hydroxyvitamin D3 on Ascentis® Express F5, 40 °C H3C H3C OH
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) CH3 H
CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C CH2 H
H
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 265 nm
CH2
ESI(+), m/z 100 - 1000 HO OH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 300 mg/L in 25:75, water:methanol HO
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005377 3. epi-25-Hydroxyvitamin D3
HO
H3 C CH3
3
CH3 H
CH3 2
H
H 1
H
CH2
HO
0 2 4
Min

HPLC Analysis of Dopa and Carbidopa HO O OH

column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HO * NH2
mobile phase . . (A) 20 mM ammonium acetate, pH 4; (B) methanol; (30:70, A:B)
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm H3C NH NH2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL HO
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL in methanol OH
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004529
0 2 4 6 8
Min

HPLC Analysis of Dopa and Carbidopa HO O OH
Enantiomers on Astec® CHIROBIOTIC® T2 HO * NH2

column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) Dopa (D, L)
mobile phase . . (A) 20 mM ammonium acetate, pH 4; (B) methanol; (10:90, A:B) O

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C H3C NH NH2
HO
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm OH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL S(-) Carbidopa

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004481
0 2 4 6 8 10 12 14
Time (min)

HPLC Analysis of Dopamine and Related Molecular weights calculated

2
Compounds on Discovery® HS F5 by deconvolution of spectra:
column . . . . Discovery HS F5, 15 cm × 4.6 mm I.D., 5 μm particles (567516-U) 1. 3204
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 10 mM ammonium formate 2. 3601
(pH 3.0 with concentrated formic acid), (B) acetonitrile 3. 3615
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min, split to the MS 4. 3583
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . MS in SIR (Selected Ion Recording) mode using (+) or (-) ESI mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL 3
sample . . . . . 50 μg/mL in 10 mM ammonium formate (pH 3.0 with formic acid)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002482 1 4
0 10 20 30
Min

2 1. Mesoridazine
HPLC Analysis of Dopamine Receptor Antagonists 2. Promazine
on Ascentis® Phenyl 3. Thiothixene
4. Chlorpromazine
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 4 5. Trifluoperazine
1
mobile phase . . . . . . (A) 50 mM ammonium formate (pH 3.0 with formic acid); 6. Thioridazine
5 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

HPLC Analysis of Enkephalin Derivative (DAMME) 1. PEG contaminants (m/z 520.1, 564.1, 608.2, 652.2, 696.2)
2. DAMME (m/z 604.2)
on Discovery® HS F5 2
Gradient Program
mobile phase . . . . (A) 25 mM HCO 2H in water; (B) 25 mM HCO 3H in acetonitrile Time (min) %A %B
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
0 95 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C 30 35 65
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+), full scan
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L in 0.1% HCO 2H
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002572
1
0 10 20 30
Min

HPLC Analysis of Fluorinated Corticosteroids on 1. Flumethasone (50 µg/mL) Gradient Program

2. Triamcinolone acetonide (50 µg/mL) Time (min) %B
Ascentis® C18 3. Clobetasol propionate (50 µg/mL) 0 35
4. Fluticasone propionate (50 µg/mL) 10 65
column . . . . . . . Ascentis C18, 5 cm × 4.6 mm I.D., 5 μm particles (581323-U) 1 11 35
2 4
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003939
0 2 4 6 8 10 12
Min

HPLC Analysis of Fluorinated Pyrimidine 1. 5-Fluorocytosine (75 µg/mL) Gradient Program

Nucleosides on Ascentis® RP-Amide 2. 5-Fluorouracil (50 µg/mL) Time (min) %B
2 3. Floxuridine (50 µg/mL) 0 0
column . . . . Ascentis RP-Amide, 5 cm × 4.6 mm I.D., 5 μm particles (565323-U) 1
4. Fudarabine (50 µg/mL) 8 8
5. Trifluridine (60 µg/mL) 10 8
mobile phase . . . . . . . . . . . . . . . . . (A) water with 0.1% ammonium formate
11 0
(pH 3.04 with formic acid), (B) acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 3 4
5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in mobile phase A 0 2 4 6 8 10
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003940

HPLC Analysis of Glucocorticoids on Ascentis® C8 1 1. Prednisolone (50 µg/mL)

2. Betamethasone (50 µg/mL)
column . . . . . . . . Ascentis C8, 15 cm x 4.6 mm I.D., 5 μm particles (581424-U) 3. Dexamethasone (50 µg/mL)
4. Corticosterone (50 µg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 3
2 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003148
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Hippuric and Methylhippuric

1. Hippuric acid
Acids in Urine on SUPELCOSIL™ LC-18 2. o-Methylhippuric acid
3. p-Methylhippuric acid impurity
Urinary excretion of hippuric acid and m-or p-methylhippuric acid in the 4. p-Methylhippuric acid
1
urine is a test of exposure to toluene and m-or p-xylene vapors. 5. m-Methylhippuric acid
column . . . . . SUPELCOSIL LC-18, 7.5 cm × 4.6 mm I.D., 3 μm particles (58984)
mobile phase . . . . . . . . . . . . (A) tetrahydrofuran:10 mM potassium phosphate,
pH 3.0 (B) with phosphoric acid (3:97, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm 4
injection . . 10 μL spiked urine (300 μg/mL HA, o-MHA; 150 μg/mL m-MHA, p-MHA)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0961A
2
5
3
0 2 4 6 8 10
Min

HPLC Analysis of 25-Hydroxyvitamin D3 and 1. 25-Hydroxyvitamin D2
25-Hydroxyvitamin D2 on Ascentis® Express F5 H3C

H 3C OH
mobile phase . . . . . . (A) 7 mM ammonium formate; (B) methanol; (25:75, A:B) CH 3 H
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min CH3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
2. 25-Hydroxyvitamin D3
ESI(+), TIC m/z 100 - 1000 H
H
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL H CH3
sample . . . . . . . . . . . . . . . . . . . . . . . . 300 mg/L in 25:75, water:methanol CH3
H3C
CH2
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) CH3 OH
H3 C
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005374
HO
H
1
CH2 2
HO
0 1.0 2.0 3.0 4.0

Min

HPLC Analysis of 25-Hydroxyvitamin D3 and 1. 25-Hydroxyvitamin D3 2. epi-25-Hydroxyvitamin D3

epi-25-Hydroxyvitamin D3 on Ascentis® Express F5 H3C H3 C
HO
CH3
H3C OH
CH3 H
mobile phase . . . . . . (A) 7 mM ammonium formate; (B) methanol; (25:75, A:B) CH3 CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 265 nm H H
H H
ESI(+), m/z 100 - 1000 H
H
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL CH2
CH2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005378 HO 2
HO
1
0 2 4
Min

HPLC Analysis of Ketoprofen Enantiomers on 3.46

Astec® CHIROBIOTIC® R (MS Detection)
4.31 CH3
column . . . . . . . . CHIROBIOTIC R, 15 cm x 2.1 mm, 5 μm particles (13019AST) O
mobile phase . . (A) 20 mM ammonium acetate, pH 5.6 (B) methanol; (70:30, A:B) 36 O

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
Response
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(-) 24
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004331
12
0
0.98 1.00
0 5 10
Min

HPLC Analysis of Levothyroxine and Liothyronine 1 1. Liothyronine

on Ascentis® Express ES-Cyano 2. Levothyroxine
This application demonstrates the suitabilty of Ascentis Express ES-Cyano for 2
the analysis of Levothyroxine according to USP Monograph 3301 with
Liothyronine as a reference standard.
column . . . . . . . . . . . . . . . . Ascentis Express ES-Cyano, 10 cm x 2.1 mm I.D.,
mobile phase . . . . . . . . . . . . . . . . . . (A) water with 0.05% phosphoric acid;
(B) acetonitrile with 0.05% phophoric acid; (60:40, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3270 psi (225 bar) 0 2 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005601

HPLC Analysis of Metabolites of 1. 2-OH-DMBA(t-diol) 11. 7-OHM-12-MBA-4-ol

2. 7-OHM-12-MBA-(t-10,11-diol) 12. 7-M-12-OHMBA-(3-ol)
7,12-Dimethylbenz[a]anthracene on 3. 7-M-12-OHMBA-(t-8,9-diol) 13. 7-M-12-OHMBA-2-ol
SUPELCOSIL™ LC-18 4. 7-OHM-12-MBA-(t-8,9-diol) 14. 7-OHM-12-MBA
5. 7-OHM-(t-3,4-diol) 15. 7-M-12-OHMBA
column . . . . . SUPELCOSIL LC-18, 25 cm × 4.6 mm I.D., 5 μm particles (58298) 6. Mixed diols, incl. DMBA- 16. DMBA-2-ol
(t-8,9-diol & t-10, 11-diol) 17. DMBA-3-ol
mobile phase . . (A) methanol:(B) water, (50:50, A:B), 10 min to 100:0 at 2.5% /min 7. Position of 7,12-bis-OHMBA 18. DMBA-4-ol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min 8. 7-OHM-12-MBA-2-ol 19. DMBA
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . fluorescence 9. 7-OHM-12-MBA-(3-ol) (parentheses = tentative identifications)
10. 7-M-12-OHMNA-4-ol
sample . . . . rat liver, 9000 × g supernatant fraction from Aroclor-treated animals
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1043 6 11 14 16 18 19
1
8 10
5 9
12 17
4 13
2 15
3
7
0 4 8 12 16 20 24 28 32 36 40 44 48
Min
Figure provided by Drs. J. Milner and J. Grunau, University of Illinois,
Urbana, Illinois, USA.

HPLC Analysis of Methadone Enantiomers on

Astec® CHIROBIOTIC® V2 O
CH3
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) H3C
mobile phase . . . . . . (A) methanol; (B) 20 mM ammonium formate; (95:5, A:B) CH3 HCI
N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
H3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . methadone, 1 mg/mL in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004403
0 10 20 30
Min

HPLC Analysis of Methylphenidate (Ritalin)

Enantiomers on Astec® CHIROBIOTIC® T2 O
CH3
mobile phase . . . . . . . . . . . . . . . . . 15 mM ammonium formate in methanol N O
H
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
HCI
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . methylphenidate, 1 mg/mL in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004405
0 2 4 6 8 10 12
Min

HPLC Analysis of Morphine and Glucuronide 1 1. Morphine 3-β-D-glucuronide

Metabolites on Discovery® HS F5 2. Morphine 6-β-D-glucuronide
3. Morphine
mobile phase . . (A) 10 mM ammonium acetate, pH 6.8 (unadjusted); (B) acetonitrile
gradient . . . . . . . . . . . . . . . 0% B for 1 min, to 30% B in 4 min, held 3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in final mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006123
0 2 4 6 8
Min

HPLC Analysis of NNAL in Urine on Ascentis® Gradient Program

Time (min.) %A %B
Express C18 after SPE using SupelMIP® SPE-NNAL 0.0
1.5
90
70
10
30
2.5 70 30
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 2.6 90 10
6.0 90 10
sample/matrix . . . . . . . . . . . human urine spiked with 60 or 100 pg/mL NNAL
SPE tube/cartridge . . . . . . SupelMIP SPE - NNAL, 25 mg/10 mL (LRC) (53206-U) SupelMIP NNAL Blank Urine; Mixed-Mode Polymer Blank Urine;
MRM 210.2/180.2 MRM 210.2/180.2
condition . . . . . . . . . . . 1 mL dichloromethane; 1 mL methanol; 1 mL DI water
sample addition . . . . . . . . 2 mL (60 pg/mL sample), 5 mL (100 pg/mL sample)
washing . . . . . . 2 x 1 mL DI water, 10 min vacuum; mL toluene; 1 mL toluene:
dichloromethane (9:1, v/v); 1 mL toluene:dichloromethane (4:1, v/v), 2 min. vacuum 0 2
Min
4 6 0 2
Min
4 6
elution . . . . . . . . . . . . . . . . . . 2 x 1 mL 10% methanol in dichloromethane,

apply gentle vacuum between each fraction SupelMIP NNAL Blank Urine; Mixed-Mode Polymer Blank Urine;
MRM 210.2/93.2 MRM 210.2/93.2
eluate post-treatment . . . . . . . . . . . . evaporate under nitrogen and reconstitute
6000
6000
in 0.15 - 0.25 mL mobile phase High
column . . Ascentis Express C18, 5 cm x 2.1 mm I.D., 2.7 μm particles (581307-U) Background
3000
3000
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 10 mM ammonium acetate;
(B) 10 mM ammonium acetate in acetonitrile
0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 0 2 4 6 0 2 4 6
Min Min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . MS/MS, MRM Transitions, ESI(+) (210.2/180 and 210.2/93.2 m/z) NNAL Spiked Urine Extracted with NNAL Spiked Urine Extracted with
SupelMIP; MRM 210.2/180.2 Mixed-Mode Polymer; MRM 210.2/180.2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004434
80
80
40
40
0
0
0 2 4 6 0 2 4 6
Min Min
% Recovery
50 pg/mL 60 pg/mL 100 pg/mL
SPE Procedure Spike Spike Spike
SupelMIP SPE – NNAL * 67% 87%
Mixed-Mode Polymer SPE 47% * *
* Not analyzed

HPLC Analysis of Norepinephrine and 1. Norepinephrine (NE) 2

2. 3,4-Dihydroxyphenyl glycol (DHPG)
3,4-Dihydroxyphenylglycol on Discovery® HS F5
1
mobile phase . . . . . . . . 50 mM ammonium formate, pH to 3.0 with formic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 266 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . 50 μg/mL each (NE and DHPG) in 5% methanol in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002096
0 2 4 6
Min

HPLC Analysis of Norgestrel Enantiomers on

Astec® CYCLOBOND® II 1
2
column . . . . . . CYCLOBOND II, 25 cm x 4.6 mm I.D., 5 μm particles (41020AST)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C 1. Norgestrel enantiomer
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 2. Norgestrel enantiomer
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . norgestrel, 1 mg/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005165
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Oenothein B on Ascentis® 2

1. Unknown impurity ([M-2H]2- = 823.4 Da)
Express RP-Amide, UV and MS Detection 2. Oenothein B ([M-2H]2- = 782.9 Da)
3. Isomer ([M-2H]2- = 782.9 Da)
Optimization of a published method showing much better retention, peak 4. Impurity, oxidized oenothein B ([M-2H]2- = 791.9 Da)
shape and resolution on Supelco Ascentis Express RP-Amide 2.7 μm 5. Impurity, oxidized oenothein B ([M-2H]2- = 791.9 Da)
compared to a C18 phase. Isomers and small amounts of homologues are 6. Impurity, fragment oenothein B ([M-H]- = 784.9 Da)
resolved and detectable with higher sensitivity.
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 10 cm x 4.6 mm I.D.,
2.7 μm particles (53929-U) ESI(-) MS detection (TIC)
mobile phase . . . . . . . . . . . . . . . . . . . . (A) 0.2% formic acid; (B) methanol 3
gradient . . . 5% B for 2 min; to 90% B over 18 min; held at 90% B for 10 min 1 4
5 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . MS (ion trap), m/z 100-1500
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006164 10× sensitivity
UV detection (252–256 nm)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Min

HPLC Analysis of Pain Relievers on Ascentis® Elution:

Express C18 5μm 1. acetaminophen 100 μg/mL
2 2. caffeine 100 μg/mL
This application shows the improvements in efficiency that may be obtained 3. benzoic acid
for USP methods using modern Fused-Core technology. (internal standard) 360 μg/mL
column . . . Ascentis Express C18, 10 cm x 4.6 mm I.D., 5 μm particles (50536-U) 4. acetylsalicylic acid 100 μg/mL
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) water with 3% acetic acid;
(B) methanol with 3% acetic acid; (79:21, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3290 psi (227 bar) 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV,275 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 3
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 100 - 360 μg/mLin mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005894 0 2 4
Min

HPLC Analysis of Penicillin G and 2 1. Penicillin G

1 2. Phenoxymethylpenicillinic acid
Phenoxymethylpenicillinic Acid on Ascentis® C18
mobile phase . . . . . . . . . . (A) 10 mM ammonium acetate (pH 4.5 acetic acid):
(B) acetonitrile, (75:25, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002427
0 2 4 6 8
Min

HPLC Analysis of Pipecolic Acid Enantiomers on

O
Astec® CHIROBIOTIC® T H
N *
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004554
0 1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of Piroxicam and 2-Aminopyridine Efficiency of Recovery

in Urine on Discovery® HS F5 after SPE using %Recovery ± RSD (n=4)
Discovery® DSC-MCAX 2-Aminopyridine Piroxicam
Discovery DSC-MCAX 102 ± 3.5% 101 ± 1.2%
Sample Pre-Treatment:
Piroxicam and 2-aminopyridine (piroxicam′s polar metabolite) was spiked Leading Competitor A30 ± 52.5% 98 ± 3.2%
into human urine at the levels of 4 and 10 μg/mL, respectively. The urine Leading Competitor B36 ± 24.2% 83 ± 4.3%
sample was diluted 1:1 with 10 mM potassium phosphate, pH 3.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 1. 2-Aminopyridine (4 µg/mL spike)
1 2. Piroxicam (10 µg/mL spike)
sample/matrix . . . . . . . . . . . . . human urine spiked with piroxicam (4 μg/mL)
and 2-aminopyridine (10 μg/mL)
SPE tube/cartridge . . . . . . . . . . Discovery DSC-MCAX, 100 mg/3 mL (52783-U) Spiked Urine Samples
condition . . . . . . . . . 1 mL methanol; 1 mL 10 mM potassium phosphate, pH 3 Before DSC-MCAX 2
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL
washing . . . . . . . . . . 1 mL 10 mM potassium phosphate, pH 3; 1 mL methanol
elution . . . . . . . . . . . . . . . . . . . 1 mL 5% ammonium hydroxide in methanol
eluate post-treatment . . evaporate to dryness with nitrogen at room temperature, 1.0 2.0 3.0 4.0 5.0
reconstitute in 1 mL mobile phase Min
column . . . . . Discovery HS F5, 15 cm x 4.6 mm I.D., 5 μm particles (567516-U)
mobile phase . . (A) 10 mM potassium phosphate, pH 6: (B) acetonitrile (85:15, A:B) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
Spiked Urine Samples
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 2
After DSC-MCAX
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003762
1.0 2.0 3.0 4.0 5.0
Min

HPLC Analysis of a Protein Mixture on Zenix® 1
SEC-150 versus Zenix® SEC-300, Effect of Pore Size

on Resolution
column . . Zenix SEC-150, 30 cm x 7.8 mm I.D., 3 μm, 150 Å particles (Z777018)
column . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm, 300 Å particles (Z777033) 4
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2
5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
1. Thyroglobulin, 670 kD
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . protein mixture 2. γ-Globulin, 158 kD 3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006118 3. Ovalbumin, 44 kD
4. Ribonuclease A, 13.7 kD
5. p-Aminobenzoic acid, 137 D
2
1
5
Zenix SEC-300
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

HPLC Analysis of Rapamycin and 32- 1. Rapamycin

Desmethoxyrapamycin on Discovery® HS C18 2. 32-Desmethoxyrapamycin
column . . . . . Discovery HS C18, 15 cm × 4.6 mm ID, 3μm particles (568522-U) 1 2
mobile phase . . . . . . . . . . . . . (A) 10 mM ammonium acetate, pH unadjusted:
(B) acetonitrile (25:75, v/v, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 268 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002015
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Min

HPLC Analysis of Selegiline and Amphetamine

1. Selegiline 1
Metabolites on Discovery® HS F5 2. Methamphetamine 2
column . . . . . Discovery HS F5, 5 cm × 4.6 mm I.D., 5 μm particles (567513-U) 3. Amphetamine

mobile phase . . . (A) water (10 mM ammonium acetate, pH 4.0 with acetic acid:
(B) acetonitrile (v/v), (25:75, A:B)
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002136
1.0 2.0 3.0 4.0 5.0 6.0

Min

1 2 3 1. 5-hydroxytryptophan,
HPLC Analysis of Serotonin Metabolites on (m/z 221)
Ascentis® C18 2. Tryptophan,
(m/z 205)
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 3. 5-hydroxyindoleacetic acid,
(m/z 192)
mobile phase . . (A) 10 mM formic acid/acetate (pH 3.0): (B) methanol; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . ESI (+); overlay of extracted ion chromatograms
of individual (M+H)+ species
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . 25 mg/L in 90:10, 10 mM formic acid/acetate (pH 3.0):methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002449
0 2 4 6 8 10 12
Min

1
HPLC Analysis of Serotonin Synthetic 2 1. 5-hydroxytryptophan
Intermediates and Metabolites on Discovery® C18 2. Serotonin
3. Tryptophan
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 4. 5-hydroxyindoleacetic acid
mobile phase . . (A) water (10 mM ammonium formate, pH 3.0 with formic acid): 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002533
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Serotonin Synthetic 1

Intermediates and Metabolites on
Discovery® HS F5 1. 5-hydroxytryptophan
2. Tryptophan
column . . . . Discovery HS F5, 15 cm × 4.6 mm A.D., 5 μm particles (567516-U) 2 3. 5-hydroxyindoleacetic acid
mobile phase . . . (A) water (10 mM ammonium sulfate, pH 3.0 with formic acid): 4. Serotonin
3
(B)acetonitrile; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002561
0 2 4 6 8 10
Min

HPLC Analysis of Statins in Rat Plasma on Total Ion Chromatogram (MRM, 4 pairs: 557.3/397.2)
Rat Plasma spiked with 5 ng/mL Statins Extracted by Supel-Select HLB SPE
Ascentis® Express C18 after SPE using Supel™- HO O
Select HLB Prasvastatin OH
Atorvastatin
O OH OH OH
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) O
HO
NH N O
sample/matrix . . rat plasma spiked with provastin and atorvastin at 5 and 100 ng/mL
O
SPE tube/cartridge . . . . . . . . . . . Supel-Select HLB SPE, 30 mg/1 mL (54181-U) H
condition . . . . . . . . . . 0.5 mL methanol:acetonitrile (1:1, v/v); 0.5 mL DI water

F
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL HO
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL 5% methanol

Supel-Select HLB SPE
elution . . . . . . . . . . . . . . . . . . . . . . 0.5 mL methanol:acetonitrile (1:1, v/v)
eluate post-treatment . . . . . . . . . . evaporate to dryness with nitrogen at 37 °C,
reconstitute in 0.5 mL DI water
column . . . . . Ascentis Express C18, 5 cm x 2.1 mm, 2.7 μm particles (53822-U)
mobile phase . . . . . . . . . . . . . . . . 0.1% acetic acid diluted in 60% methanol Atorvastatin
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min Pravastatin
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . ABI 3200 QT; ESI(+), MRM (423.3/321.3, 423.3/101.0,
557.3/397.2 and 557.3/453.4)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004432
0 1 2 3 4 5 6
Min
Absolute Recovery ± RSD (n=3)

5 ng/mL spike 100 ng/mL spike
Pravastatin Atorvastatin Pravastatin Atorvastatin
Supel-Select HLB 84 ± 8% 92 ± 5% 103 ± 4.2% 89 ± 3.9%
Competitor W 83 ± 17% 92 ± 2% 104 ± 2.2% 87 ± 1.1%
Competitor P 77 ± 5% 93 ± 2% 102 ± 3.0% 91 ± 1.3%

1 1. Pravastatin
HPLC Analysis of Statins on Ascentis® RP-Amide 2. Atorvastatin
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 3. Fluvastatin
4. Lovastatin
mobile phase . . (A) water with 0.1% formic acid (34673): (B) methanol; (25:75, A:B) 2 3 5. Simvastatin
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003270
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Steroids, Anabolic, on 1. Nandrolone (50 µg/mL)

12 2. Methandienone (50 µg/mL)
Ascentis® C8 3 3. Testosterone (50 µg/mL)
4. Norethandrolone (50 µg/mL)
column . . . . . . . . Ascentis C8, 15 cm x 4.6 mm I.D., 5 μm particles (581424-U) 5. Oxymetholone (50 µg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in mobile phase 0 2 4 6 8 10 12 14
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003238

1 1. Estriol (50 µg/mL)

HPLC Analysis of Steroids on Ascentis® C18 2. β-estradiol (50 µg/mL)
(Mobile phase: 55:45) 3. α-estradiol (50 µg/mL)
4. Estrone (50 µg/mL)
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (55,45:A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . as indicated in 55:45, water:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002330
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Steroids on Ascentis® Express 2

1. Nandrolone
C18 (10 cm x 2.1 mm I.D., 2.7 μm) 2. Methandienone
3. Testosterone
This application demonstrates the suitability of Ascentis Express C18 for the 4. Epitestosterone
1
efficient separation of 5 steroids. 5. Norethandrolone
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
4
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2944 psi (203 bar) 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6 8 10
sample . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg/L in 75:25, water:methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005401

HPLC Analysis of Steroids on Ascentis® Express 2

1. Nandrolone
C18 with Methanolic Mobile Phase 2. Methandienone
3. Testosterone
column . . Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.7 μm particles (53823-U) 1 4. Epitestosterone
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (60:40, A:B) 5. Norethandrolone
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 5
sample . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg/L in 75:25, water:methanol

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005404
0 2 4 6 8 10 12
Min

HPLC Analysis of Steroids on Ascentis® Express F5 1. Hydrocortisone 1 2 3

Sample concentrations at 10 ug/mL in 90:10 water:methanol. Acetonitrile 2. Prednisolone
does not provide adequate selectivity on this phase. Mass spec was used for 3. Prednisone
peak identification.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2393 psi (165 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 2 4 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . 10 μg/mL of each in 90: 10, water: methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005743

HPLC Analysis of Steroids on Ascentis® Express F5, 3

1. Nandrolone
1
Acetonitrile Mobile Phase 2. Testosterone
3. Methandienone
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) 4. Epitestosterone
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (70:30, A:B) 5. Norethandrolone
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005409
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Steroids on Ascentis® Express F5 1 4 1. Nandrolone

2. Testosterone
with Methanolic Mobile Phase 3. Epitestosterone
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) 4. Methandienone
5. Norethandrolone
mobile phase . . . . . . . . . . . . . . . . . . . (A) water (B) methanol; (40:60, A:B)
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005410
0 2 4 6 8 10 12
Min

HPLC Analysis of Steroids on Ascentis® RP-Amide 1 1. Estriol (50 µg/mL)

2. β-estradiol (50 µg/mL)
(Mobile phase: 55:45) 3. 17 α-estradiol (50 µg/mL)
4. Estrone (50 µg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
2 3 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . as indicated in 55:45, water:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002672
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Steroids on Ascentis® RP-Amide 1 1. Prednisone

2. Hydrocortisone 21-acetate
(Mobile phase: 60:40) 3. Cortisone acetate

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002674
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Steroids on Ascentis® Si 1. Cortisone 2 3

2. Prednisone 1
column . . . . . . . . Ascentis Si, 15 cm x 4.6 mm I.D., 5 μm particles (581512-U) 3. Prednisolone
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 245 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 85:15, hexane:2-propanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003728
0 2 4 6 8 10 12
Min

HPLC Analysis of Steroids on Ascentis® Phenyl 1. Prednisolone 1

2. Hydrocortisone 2
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 3 μm particles (581610-U) 3. Prednisone 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003698
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Testosterone and 1-Testosterone 1
on Ascentis® Express C18 1. Testosterone

2. 1-Testosterone
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 10:90 water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005413
0 2 4
Min

HPLC Analysis of Thyroglobulin, gamma-Globulins, 1. Thyroglobulin

Ovalbumin, Myoglobin, and Poly-DL-Alanine on 2. Gamma-globulins
3. Ovalbumin
Zenix® SEC-150, 30 cm x 7.8 mm I.D., 3 μm 4. Myoglobin
particles 5. Poly-DL-alanine
6. Uracil 1
This application demonstrates the suitability of Discovery BIO GFC 150 for 4
the separation of proteins. Separation is by inclusion into the pore volume
with larger molecules being excluded and therefore eluting earlier.
column . . . . . . Zenix® SEC-150, 30 cm x 7.8 mm I.D., 3 μm particles (Z777018)
3
mobile phase . . . . . . . . . . . . . . . . . 150 mM potassium phosphate monobasic,
pH 7.0 (adjusted with potassium hydroxide) 2 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min 5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890 psi (61.4 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
0 10 20
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . 1 g/L each (except uracil, 0.1 g/L ) in mobile phase A
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005727

1
HPLC Analysis of Thyroxine Enantiomers on Astec®
CHIROBIOTIC® TAG 1. L-Thyroxine
2. D-Thyroxine
Baseline resolution of the chiral enantiomers of DL-Thyroxine is achieved by
polar ionic mode on the Astec CHIROBIOTIC TAG with the use of an LC/MS-
compatible mobile phase.
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) 2
mobile phase . . . . . . . . . . . . . . . . . . . . 1% ammonium formate in methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826 psi (57 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . each enantiomer 0.1 mg/L in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005515 0 10 20 30
Min

HPLC Analysis of Tricyclic Antidepressants in 1. Nortriptyline

Serum on Discovery® C18 after SPE using 2. Doxepin
3. Imipramine
Discovery® DSC-18 4. Amitriptyline
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 1 5. Trimipramine (I.S.)
SPE tube/cartridge . . . . . . . . . . . . . Discovery DSC-18, 100 mg/1 mL (52602-U) 2 3
column . . . . . . . . . . . . Discovery C18, 15 cm × 4.6 mm, 5 μm preceded by a 4
2 cm C18 guard column and 0.5 μm frit filter (504955)
mobile phase . . . (A) acetonitrile: (B) methanol: (C) 25 mM potassium phosphate, 5
monobasic (pH 7 with triethlylamine) (45:25:30, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 0 2 4 6 8 10 12 14
injection . . . . . . . . . . . . . . . . . . . . . . . 50 μL, diluted porcine serum extract Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000595
SPE Procedure, Using Zymark RapidTrace SPE Workstation
Efficiency of Recovery Solvent/ Volume Flow Rate
Step Solution (mL) (mL/min) Comments
Concentration % RSD
Compound (μg/mL) % Recovery (n=6) 1. Condition MeOH 2.0 5.0 conditions sorbent
1. Nortriptyline 0.10 103.6 ±4.5 2. Condition H2O 2.0 5.0 conditions sorbent
0.50 97.5 ±4.5 3. Load spiked porcine 2.0A 0.75 applies serum sample
2. Doxepin 0.10 102.2 ±3.0 serum
0.50 100.8 ±1.8 4. Rinse 20% MeOH 2.0 5.0 washes sorbent
3. Imipramine 0.10 92.0 ±1.5 in H2O
0.50 97.5 ±1.7 5. Purge-Cannula H2O 4.0 30.0 cleans sample cannula
4. Amitriptyline 0.10 93.6 ±1.2 6. Rinse vent 0.1 2.0 positions SPE tube over waste port
0.50 95.7 ±1.4
7. Dry N2 Time = dries sorbent
10 min
8. Purge-Cannula MeOH 4.0 30.0 cleans sample cannula
9. Collect MeOH 1.0 1.0 elutes analytes into collection vessel
10. Collect vent 6.0 3.0 pushes residual eluent into vesselB
11. Purge-Cannula H2O 4.0 30.0 cleans sample cannula
A
1 mL porcine serum spiked with 0.1 μg/mL each analyte basified with 3 μL
10 N KOH, then diluted with 1 mL water
B
350 μL water added per mL methanolic eluent before analysis

1. Nordoxepin (50 µg/mL) 5. Doxepin (50 µg/mL)

HPLC Analysis of Tricyclic Antidepressants on 2. Desipramine (50 µg/mL) 6. Imipramine (50 µg/mL)
Ascentis® C8 3. Nortriptyline (50 µg/mL) 7. Amitriptyline (50 µg/mL)
4. Norclomipramine (50 µg/mL) 8. Clomipramine (50 µg/mL)
column . . . . . . . . Ascentis C8, 15 cm x 2.1 mm I.D., 3 μm particles (581402-U) 2
mobile phase . . . . . . . . . . . . . . . . . (A) 25 mM ammonium phosphate dibasic 1
(pH 7.0 with phosphoric acid): (B) methanol; (25:75, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4 5 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 7
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 8
sample . . . . . . . . . . . . . . . . . . . . . . . as indicated in 60:40 water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003156
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Tricyclic Antidepressants on 1. Nordoxepin

3
Ascentis® Express C18 2. Desipramine

3. Nortiptyline
1
column . . Ascentis Express C18, 10 cm × 2.1 mm I.D., 2.7 μm particles (53823-U) 4. Doxepin
5. Norclomipramine
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 100 mM ammonium acetate 4
6. Imipramin
(pH 7.0; titrated with ammonium hydroxide),
(B) water, (C) methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C
detector . . . . . . . . . . . . . . . . . Thermo LCQ Advantage; ESI(+), m/z 250-320 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 2 5
instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jasco X-LC
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004062
1.0 2.0 3.0

Min

HPLC Analysis of Tricyclic Antidepressants on 1. Desmethyl doxepin

2 2. Protriptyline
Discovery® RP-AmideC16 3. Desipramine
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 4. Nortriptyline
1 5. Doxepin
mobile phase . . (A) water/acetonitrile (40:60 v/v); (B) 10 mM potassium phosphate 6. Imipramine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 7. Amitriptyline
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 3 8. Trimipramine
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000163
5
6
8
0 2 4 6 8 10 12
Min

HPLC Analysis of Tryptophan Hydroxylase 1 2 3 4 5 1. 6-fluorotryptophan, (m/z 223);

(10 µg/mL)
Inhibitors on Ascentis® C18 2. p-chlorophenylalanine, (m/z 200);
(10 µg/mL)
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 3. p-chloroamphetamine, (m/z 170);
(10 µg/mL)
mobile phase . . . (A) 40 mM formic acid, (pH ~2.5): (B) methanol; (65:35, A:B) 4. α-propyldopacetamide, (m/z 210);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min (50 µg/mL)
5. Fenfluramine, (m/z 232);
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C (10 µg/mL)
detector . . . . . . . . . . . . . full scan MS; overlay of extracted ion chromatograms
of individual (M+H)+ species
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . as indicated in 90:10, 10 mM formic acid:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002453
0 10 20
Min

HPLC Analysis of Verapamil and Metabolites in Rat 40000 1. Normethyl verapamil (m/z 441.28 m/z)
2. Verapamil (455.305 m/z)
Plasma on Ascentis® Express C18 after 3. Methoxy verapamil (485.323)
Phospholipid Removal using HybridSPE®-
Removal of phospholipids
Phospholipid using HybridSPE-PPT allows
for short 90 sec. run time and
SPE well plate . . . HybridSPE-Precipitation 96-well Plate, 50 mg/well (575656-U) isocratic conditions
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 20000

sample/matrix . . . Rat plasma spiked with verapamil and metabolites at 10 ng/mL
mobile phase . . . . . . . . . . . . . (A) water; (B) acetonitrile, pH 2.55 adjust with
formic acid (30:70, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
0
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS 0 0.5
Min
1.0 1.5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample addition . . . . . . . . . . . . . . . . . 100 μL spiked rat plasma followed by
300 μL 1% formic acid in acetonitrile
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . apply vacuum
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004366

HPLC Analysis of Veterinary Antibiotics on 1 1. Lasalocid A (1 µg/mL); m/z 589.5

2. Monensin (10 µg/mL); m/z 669.6
Ascentis® C18 3. Salinomycin SV (10 µg/mL); m/z 749.5
4. Narasin (3 µg/mL); m/z 763.5
3
2
mobile phase . . (A) 50 mM ammonium acetate (pH 6.0): (B) methanol; (20:80, A:B)
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(-), full scan
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . as indicated in 50:50, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002459
0 10 20
Min

HPLC Analysis of Vitamin D2 and D3 on Ascentis® 1. Vitamin D2 2

Express C18 2. Vitamin D3 1

mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.15 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . 10 mg/L each (vitamins D2 and D3) in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005615
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Vitamin D2 and D3 on Ascentis® 2

Elution Order:
Express C18 5 μm 1. Ergocalciferol (Vitamin D2)
This application demonstrates the suitability of the Ascentis Express C18, 2. Cholecalciferol (Vitamin D3)
5 μm, for the routine analysis of the fat-soluble vitamins D2 and D3 .
Structures along with the optimized chromatogram obtained on the
Ascentis Express C18, 5 μm are presented.
column . . Ascentis® Express C18, 25 cm x 4.6 mm I.D., 5 μm particles (50538-U)
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1726 psi (119 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 265 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
sample . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 90:10, acetonitrile:methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005891

HPLC Analysis of Vitamin D2 and Vitamin D3 on 1 2

1. Ergocalciferol
Ascentis® C18 2. Cholecalciferol
This application demonstrates the suitability of the Ascentis C18 for the
analysis of the fat-soluble vitamins D2 and D3 . Structures along with the
optimized chromatogram obtained on the Ascentis C18 are presented.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005614
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Vitamins, Fat Soluble (D2 and

1. Ergocalciferol (Vitamin D2) 2
D3), on Discovery® HS C18 2. Cholecalciferol (Vitamin D3)
column . . . . Discovery HS C18, 15 cm × 4.6 mm I.D., 3 μm particles (569252-U) 1
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100% acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each analyte
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001419 0 10 20
Min

1 1. Warfarin
HPLC Analysis of Warfarin™ Anticoagulant on
Ascentis® C18
mobile phase . . . . . . . . . . . . (A) 20 mM phosphoric acid (pH 2.0, unadjusted):
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in mobile phase 0 2 4 6 8 10 12 14

HPLC Analysis of Warfarin™ Anticoagulant on 1 1. Warfarin (50 µg/mL)
Ascentis® RP-Amide
mobile phase . . . . . . . . . . . . (A) 20 mM phosphoric acid (pH 2.0 unadjusted):
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0 2 4 6 8 10 12 14
sample . . . . . . . . . . . . . . . . as indicated in 20 mM phosphoric acid (pH 2.0) Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002638

HPLC Analysis of Warfarin™ Enantiomers on Astec® 1. (+)-Warfarin

O O
Cellulose DMP 2. (-)-Warfarin
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) * C H3

(+) 1
mobile phase . . . . . . (A) methanol; (B) acetic acid; (C) TEA; (100:0.2:0.1, A:B:C) OH O
(-) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 278 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg/mL in mobile phase 0 2 4 6 8

HPLC Analysis of Warfarin™ Enantiomers on Astec®

CHIROBIOTIC® V
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 4.65
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
5.22
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004349
0 4 8
Min

HPLC Analysis of Warfarin™ Enantiomers on Astec® O O

CHIROBIOTIC® V (Method 1)
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) * C H3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 4.65
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm

5.22
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004472
Response
0 4 8
Min


column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) OH O
mobile phase . . . . (A) 0.1% triethylamine acetate, pH 5.0; (B) THF; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min * CH3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm O O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004649
0 2 4 6 8 10 12
Min


S-form
CYCLOBOND® I 2000 (UV at 254 nm) O O
6.89 min R-form
column . . CYCLOBOND I 2000, 25 cm × 4.6 mm I.D., 5 μm particles (20024AST) 7.90 min
1000
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile: (B) acetic acid: OH O
(C) triethylamine; (100:0.3:0.25, A:B:C)

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
mAU
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004027
0
0 2 4 6 8
Min

CHIROBIOTIC® V2
* C H3
mobile phase . . (A) 10 mM ammonium acetate, pH 4.1; (B) acetonitrile; (70:30, A:B) OH O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004475
0 10 20
Min.

LC-MS Analysis of 2C Amines Drugs of Abuse on 1. R-(-)-Bromo-DragonFLY HCl

Ascentis® Express HILIC 2. (±)-2,5-Dimethoxy-4-Iodoamphetamine HCl (DOI HCl)
3. (±)-2,5-Dimethoxy-4-Bromoamphetamine HCl (DOB HCl)
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 4. 4-Bromo-2,5-dimethoxyphenethylamine HCl (2C-B)
mobile phase . . . . . . 5 mM ammonium formate in acetonitrile:water (90:10, v/v) 5. 2C-B-FLY HCl
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 ng/mL in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006166 3
1 5
4
0 2 4
Min

LC-MS Analysis of Antiarrhythmic Drugs and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction)
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . rabbit plasma, unfiltered K2-EDTA, spiked with each compound
Metabolites in Plasma on Ascentis® Express HILIC at 100 ng/mL (3:1, plasma:1% formic acid in acetonitrile)
following Sample Prep using HybridSPE®- SPE tube/cartridge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HybridSPE-Phospholipid, 96-well plate (575656-U)
Phospholipid sample addition . . . . . . . . To each well add 100 μL plasma, followed by a 300 μL of 1% formic acid in acetonitrile,
agitate on orbital shaker for 4 minutes. Collect filtrate and analyze directly.
For efficient therapeutic drug monitoring, it is important for clinicians to
have access to fast and robust analytical methods for accurate assessment of column . . . . . . . . . . . . . . . . . . . . . . . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U)
drug efficacy. Industrial trends toward highly specific LC/MS applications mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate; (B) 5 mM ammonium formate in
over traditional ELISA type immunoassay have resulted in the need for high- acetonitrile, 5:95 (A:B), adjusted to pH 7.0 with formic acid
speed chromatographic assays along with simplified sample preparation flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
methods. Often the limitation of a bioanalytical technique is based upon the pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305 psi (90 bar)
effectiveness of the sample preparation technique. Plasma and serum column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
samples are often susceptible to assay irregularities due to matrix-induced detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, 100-1000 m/z
interferences. In this study the impact of matrix interference is investigated injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
with respect to precision and accuracy of antiarrhythmic cardiac drugs from
plasma samples. A robust bioanalytical method was developed using a
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005854
combination of fast HILIC chromatography on Ascentis Express along with a
selective sample preparation using HybridSPE-Phospholipid. Solvents were x105
+ESI EIC(235.20000, 415.1700, 618.0300, 646.0600) Scan Frag=175.0V 081712020.d
LC-MS Ultra CHROMASOLV® and Certified Reference Material grade
8
standards were from Cerilliant. 1. Lidocaine 1 gray: Analytes
7 2. Amiodarone HCl
Endogenous phospholipids can dramatically impact the precision and 3. Desethylamiodarone pink: Phospholipids
accuracy of a bioanalytical method. During development, the sample matrix 6 4. (±)-Flecainide
impact on analyte detection should be priority in method validation. Failure 5
to do so can result in inaccuracies in reported levels, thus impacting the 4
2 4
assessment of patient health. Eliminating matrix effects in LC/MS is
3
imperative to producing reliable and accurate bioanalytical methods. The
2 3
targeted phospholipid selectivity of the HybridSPE-Phospholipid technique
enables simplified sample processing with no detected phospholipid matrix 1
interference, while exhibiting excellent recovery from plasma and serum 0
samples. This application demonstrates how combining selectivity in both 0.1 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4
Counts vs. Acquisition Time (min)
sample preparation and chromatographic separation allows for a simplified
and efficient bioanalytical method resulting in a high precision and accurate Min
assay.

LC-MS Analysis of Antiarrhythmic Drugs and 1. Lidocaine

Metabolites on Ascentis® Express HILIC 1
2. Amiodarone HCl
3. N-Desethylamiodarone HCl
The basic nature of these antiarrhythmic agents makes them ideal 4. (±)-Flecainide
candidates for HILIC (hydrophilic interaction) chromatographic separation.
The benefit of HILIC over traditional reversed-phase chromatography is two-
2
fold for both sample introduction and analyte detection. First, the high
acetonitrile concentration of HILIC mobile phases allows for direct analysis of 4
precipitated plasma samples without the need for additional sample solvent
exchange. Second, the high acetonitrile content provides increased analyte
response in positive ESI MS detection. Of the various HILIC-mode columns
tested, method development for this assay determined the Ascentis Express 3
HILIC, 2.7 μm particles, provided the best chromatographic resolution of the
antiarrhythmic drugs while maintaining high peak efficiency for enhanced
detection levels.
mobile phase . . (A) 5 mM ammonium formate; (B) 5 mM ammonium formate in
acetonitrile, 5:95 (A:B), adjusted to pH 7.0 with formic acid 0 1 2 3 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
Min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305 psi (90 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, 100-1000 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . each compound, 300 ng/mL in 75:25 (1% formic acid acetonitrile:water)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005855

LC-MS Analysis of Antipsychotic Drugs on 1. Desmethyl venlafaxine

Ascentis® Express RP Amide 2. Hydroxybupropion
column . . . . . . . . . . . . . . . Ascentis Express RP Amide, 10 cm x 2.1 mm I.D., 4. Bupropion
2.7 μm particles (53913-U) 5. Zuclopenthixol
mobile phase . . . . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate water; 6. Fluphenazine
(B) 5mM ammonium formate in 95:5 (v/v) methanol:water
gradient . . . . . . . . . . . . held at 35% B for 0.5 min; 35 to 85% B in 5.5 min; 6
held at 85% B for 1 min 1
3 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4641 psi (320 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 100-1000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 ng/mL in waler 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005668
0 2 4 6 8 10
Min

LC-MS Analysis of Benzodiazepines on Ascentis® 1. Demoxepam (1 µg/mL) (M+H)+ = 287.05

Phenyl (Analyte Mix 1) 2. Oxazepam (1 µg/mL) (M+H)+ = 287.05
3. Temazepam (1 µg/mL) (M+H)+ = 301.06
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 4. Clobazam (1 µg/mL) (M+H)+ = 301.06
mobile phase . . . . . . . . (A) 0.1% ammonium acetate in water (pH unadjusted), 5. Diazepam (1 µg/mL) (M+H)+ = 285.07
(B): 0.1% ammonium acetate in acetonitrile, (50:50, A:B)
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min., split to the MS
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003705 2
0 2 4 6 8 10
Min

LC-MS Analysis of Benzodiazepines on Ascentis® 1. Fenoldopam (10 µg/mL) (M+H)+ = 306.08

Phenyl (Analyte Mix 2) 2. Lorazepam (10 µg/mL) (M+H)+ = 321.01
3. Nitrazepam (10 µg/mL) (M+H)+ = 282.08
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 4. Clonazepam (10 µg/mL) (M+H)+ = 316.04
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min., split to the MS 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . MS, ESI (+) in selected ion recording (SIR) mode 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003706
0 2 4 6 8 10 12
Min

1. Taurocholic acid (TUDC) 10. D4- Glycodeoxycholic acid (D4-GDC)

LC-MS Analysis of Bile Acids and Their Conjugates 2. Glycoursodeoxycholic acid (GUDC) 11. Glycodeoxycholic acid (GDC)
on Ascentis® Express C18 3. Taurocholic acid (TCA)
4. Glycocholic acid (GCA)
12. Cholic acid (CA)
13. Taurolithocholic acid (TLC)
D4-DC
A fast robust LC-MS/MS method was developed on a C18 Ascentis Express 5. Chenodeoxycholic acid (TCDC) 14. Glycolithocholic acid (GLC)
6. D4-Taurodeoxycholic acid (D4-TDC) 15. Chenodeoxycholic acid (CDC)
Fused-CoreR Particle Column allowing the 15 bile acid species to be 7. Taurodeoxycholic acid (TDC) 16. D4-Deoxycholic acid (D4-DC)
measured individually rather than the alternative measurement of a total 8. Ursodeoxycholic acid (UDC) 17. Deoxycholic acid (DC)
concentration by colourimetric kinetic enzyme assays. 9. Glycochenodeoxycholic acid (GCDC) 18. Lithocholic acid (LC)
sample/matrix . . . . . . Plasma proteins were removed with addition of 900 μL of TUDC critical pairs with same mass
acetonitrile, containing deuterated internal standards, to 250 μL of human EDTA
plasma. The mixture was vortexed, (centrifuged and the supermatant evaporated
before being reconstituted in a 50:50 solution of methanol and water. 10 μL
(corresponding to 8.57 μL of plasma) was injected into the HPLC.)
UDC
gradient . . . . . . . . . . . . . . . . . . 70 to 95% B in 10 minutes held 4 minutes GCDC DC
11.63
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min TDC
D4-GDC CDC
D4-TDC
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 11.30
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(-), MRM Mode TCDC TLC
7.62
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005599
TCA
CA
GUDC GDC GLC LC
GCA
12.48
1 2 3 4 5 6 7 8 9 10 11 12 13
Min

LC-MS Analysis of Clenbuterol Enantiomers in OH

Clenbuterol
Plasma on Astec® CHIROBIOTIC® T after SPE using 700
CI
HybridSPE®-Phospholipid
600 CH3
column . . . . . . CHIROBIOTIC T, 10 cm x 2.1 mm I.D., 5 μm particles (12018AST) HN
H2N
mobile phase . . . . . . . . . . . . . . . . . 10 mM ammonium formate in methanol 500 CH3
CI H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
400
cps
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+)
300
sample . . clenbuterol, 10 ng/mL in rat plasma (phospholipids removed by extraction
with HybridSPE-Phospholipid) Clenbuterol
200
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004245 Phospholipids
100
0
0 1 2 3 4 5
Min

LC-MS Analysis of Clenbuterol In Plasma on Astec® Max 767 CPS

HybridSPE-PPT
CHIROBIOTIC® T with Phospholipid Removal Using 700 3-step procedure
Minimal signal suppression
HybridSPE® Phospholipid 95% absolute recovery
600
The HybridSPE method provides significant improvement in LC-MS baseline.
sample/matrix . . . . rat plasma spiked with clenbuterol enantiomers at 10 ng/mL 400 Clenbuterol
CPS
SPE well plate . . . HybridSPE-Precipitation 96-well Plate, 50 mg/well (575656-U) Phospholipids
sample addition . . 100 μL spiked rat plasma followed by 300 μL 1% formic acid in 300
acetonitrile. Mix by vortexing the HybridSPE-PPT plate briefly. 200

column . . . . . . CHIROBIOTIC T, 10 cm x 2.1 mm I.D., 5 μm particles (12018AST) 100

0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 0 1 2
Min
3 4 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C Max 775 CPS

detector . . . . . . . . . . ABI 3200 QT; ESI(+), MRM: 184/104 m/z (phospholipids); 6000
Standard Protein PPT
277.2/203.1 m/z (clenbuterol) 3-step procedure
25-70% signal suppression
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 5000 No phospholipid removal
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004431
4000
Phospholipids
CPS 3000
Clenbuterol
2000
1000
0
0 1 2 3 4 5
Min

LC-MS Analysis of Conjugated Bile Acids on 1. Taurocholic acid

Ascentis® Express C18 2. Glycocholic acid
3. Taurochenodeoxycholic acid
column . . Ascentis Express C18, 5 cm x 4.6 mm I.D., 2.7 μm particles (53826-U) 4. Taurodeoxycholic acid
mobile phase . . (A) 10 mM ammonium acetate, pH 4 with acetic acid; (B) acetonitrile 5. Sodium gycochenodeoxycholate
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 to 40% B in 10 min
flow rate . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min (postcolumn split to MS) 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(-), SIR 4
3 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L each in mobile phase A 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005688
0 2 4 6 8
Min

LC-MS Analysis of Conjugated Bile Acids on 1. Taurocholic acid

Ascentis® Express RP-Amide 2. Glycocholic acid
3. Taurochenodeoxycholic acid
column . . Ascentis Express RP-Amide, 5 cm x 4.6 mm I.D., 2.7 μm particles (53922-U) 4. Taurodeoxycholic acid
mobile phase . . (A) 10 mM ammonium acetate, pH 3 with acetic acid; (B) acetonitrile 5. Sodium glycochenodeoxycholate
flow rate . . . . . . . . . . . . . . . . . . . 1.0 mL/min (postcolumn split to the MS) 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(-), SIR
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 3 5
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L in mobile phase A 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005689 1
0 2 4 6 8
Min

LC-MS Analysis of CYP2D6 Inhibitors on Ascentis® 1. Amitriptyline 1

Express HILIC 2. Imipramine
3. Fluoxetine
This application demonstrates the suitability of the Ascentis Express HILIC for 4. Paroxetine 2
the analysis of CYP2D6 Inhibitors.
4
column . . Ascentis Express HILlC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 3
mobile phase . . . . . . . 5 mM ammonium formate in 2:98 (v/v) water:acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 100-400 1.0 2.0 3.0 4.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005691

LC-MS Analysis of DL-α-Hydroxyglutaric acid on 1. L-α-Hydroxyglutaric acid 1

Astec® CHIROBIOTIC® R 2. D-α-Hydroxyglutaric acid 2
Chiral 2-hydroxyglutarates (2-OHG) are important molecular signatures of
both healthy and diseased biological cells, their specific biochemical
pathways and inborn errors of metabolism. The chiral differentiation and
quantification of D-2-OHG and L-2-OHG is key for characterizing neuro-
metabolic disorders like the 2-hydroxyglutaric acidurias that cause neuro-
logical impairment early in life. In this application, an Astec® CHIROBIOTIC® R
column run under polar ionic mobile phase conditions achieved a rapid
separation of the D and L enantiomers of 2-OHG under conditions
amenable for sensitive LC-MS/MS detection.
0 2 4
column . . Astec CHIROBIOTIC® R, 10 cm x 4.6 mm I.D., 5μm particles (13022AST)
Min
mobile phase . . . . . . . . . . . . . . 5:95, (water:methanol) with 0.3% acetic acid
and 0.1% ammonium hydroxide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1700 psi (117 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . MS, ESI(-) TOF, SIM, m/z 147.03, split 100:1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . DL-a-Hydroxyglutaric acid, 1 mg/mL in 50:50, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005887

LC-MS Analysis of 25-Hydroxyvitam D3 and 1. 25-Hydroxy vitamin D3

2. 25-Hydroxy vitamin D2 1
25-Hydroxyvitamin D2 on Ascentis® Express C18
mobile phase . . . . . . . . . . . . . . . . . (A) 10mM ammonium formate in water;
(B) 10mM ammonium formate in acetonitrile; (32:68, A:B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1711 psi (118 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 1.0 2.0 3.0 4.0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005658

LC-MS Analysis of 25-Hydroxyvitamin D2 and 1. 25-Hydroxyvitamin D3 1

25-Hydroxyvitamin D3 on Ascentis® Express F5 2. 25-Hydroxyvitamin D2 2
The 25-OH forms of the secosteroids vitamin D2 and D3 are indicators of the
vitamin D status of an individual. However, because they have different
biological activity and clinical interest, they should be distinguishable. The
F5 phase was able to resolve the two compounds under highly LC-MS/MS
compatible conditions.
mobile phase . . (A) 5 mM ammonium formate in (B) 75:25 (v/v) methanol:water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 0 2 4
detector . . . . . . . . . . . . . . . . . . . . UV, 265 nm (MS, ESI(+), m/z 100-1000)
Min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005858

LC-MS Analysis of Illicit Bath Salts in Urine on injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL

sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 μg/L ea. in acetonitrile
Ascentis® Express HILIC after Solid Phase Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005796
Extraction (SPE) on Supel™-Select SCX
The designer drugs known as "bath salts" are synthetic stimulants and illegal
1. MDPV
in most states as of 2011. This application demonstrates the analysis of bath
2. Buphedrone
salts extracted from human urine samples using polymeric solid phase
1 3. 3-Fluoromethcathinone
extraction (SPE) sample preparation followed by hydrophilic interaction
4. Butylone
liquid chromatography (HILIC) analysis with TOF-MS detection. HILIC
5. Ethylone
conditions on the Ascentis Express HILIC (Si) phase are used for fast, high-
2 4 6. 4-Fluoromethcathinone
resolution separation of nine synthetic bath salts. Recoveries greater than 5
7. Mephedrone
65% were observed for all analytes except MDPV (43.7%). The Figure
8. Methylone
illustrates the detection of bath salts in the spiked urine sample after SPE
9. Methedrone
sample cleanup. Notice there are no interfering peaks in the chromatogram,
demonstrating the effectiveness of the SPE sample cleanup.
7
sample/matrix . . Urine samples were spiked to a level of 100 ng/mL with each target 8 9
analyte. (To ensure full ionization of the analytes, spiked samples were 6
treated with formic acid to a final concentration of 0.1% formic acid.) 3
SPE tube/cartridge . . . . . . . . . . . . . . Supel-Select SCX, 30 mg/1 mL (54240-U)
condition . . . . . . . . . . . . . . 1 mL 1% formic acid acetonitrile then 1 mL water
sample addition . . . . . . . . . . . . . . . . . . . . 1 mL spiked water blank or urine
washing . . . . . . . . . 1 mL water, 1 mL 1% formic acid acetonitrile, 1 mL water
1.0 2.0 3.0 4.0 5.0 6.0
elution . . . . . . . . . . . . . . . . . 2 mL 10% ammonium hydroxide in acetonitrile
Min
eluate post-treatment . . thoroughly mix via vortex agitation, evaporate 1 mL aliquot
to dryness, reconstitute in 100 μL water:methanol
mobile phase . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate acetonitrile;
(B) 5 mM ammonium formate water; (98:2, A:B); premixed
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1842 psi (127 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), TIC, m/z 100-1000

LC-MS Analysis of Lactic Acid Enantiomers in Urine
% Intensity
100 m/z 89.1/43.3
on Astec® CHIROBIOTIC® R 75
L-lactic acid
Chromatogram supplied courtesy of H. Henry and associates at the Clinical 50
Chemistry Laboratory, Centre Hospitalier Universitaire Vaudois, University of
Lausanne, CH-1011, Switzerland. View the related full-length contributed 25
0 D-lactic acid
article in Reporter 30.1.
100
Reference: Biomed. Chromatogr. 2011 "Sensitive determination of D-lactic 13
C-L-lactic acid m/z 90.1/44.3
acid and L-lactic acid in urine by high-performance liquid chromatography– 75
tandem mass spectrometry" H. Henry, N. Marmy Conus, P. Steenhout, A. 50
Béguin and O. Boulat, Published online in Wiley Online Library,
wileyonlinelibrary.com/journal/bmc. 25
0
column . . Astec CHIROBIOTIC R, 15 cm x 2.1 mm I.D., 5 μm particles (13019AST) 100
mobile phase . . . . (A) 33.3 mM ammonium acetate; (B) acetonitrile; (15:85, A:B) m/z 93.1/ 46.3
d4-L-lactic acid
75
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min d4-D-lactic acid
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 °C 50
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (-)ESI 25
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 0
sample . . . . . . . . . . . . . . . . . . . . . . . Urine extracts in 5% (v/v) acetic acid, 0 2 4 6 8 10
Min
10% (v/v) H 2O and 85% (v/v) acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005482

LC-MS Analysis of Nicotine and Metabolites on 1. Anabasine

2. Norcotinine
Kromasil® Eternity™ C18 (Analyte Set 1) 3. Nicotine
column . . Kromasil Eternity C18, 10 cm x 3.0 mm I.D., 2.5 μm particles (K0897111) 4. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) isomer 1
mobile phase . . . . . . . (A) 10 mM ammonium acetate in water:methanol (95:5); 5. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) isomer 2
6. (S)-N-Nitrosoanabasine (NAB)
(B) 10 mM ammonium acetate in water:methanol (10:90)
1
gradient . . . 0% B held for 1 min; to 100% B in 8.5 min;100% B held for 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 4 6
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 psi (23 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+) TOF, EIC
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ng/mL in mobile phase A
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005924
5
0 2 4 6 8
Min

LC-MS Analysis of Nicotine and Metabolites on 1. Nornicotine

2. trans-3'-Hydroxycotinine
Kromasil® Eternity™ C18 (Analyte Set 2) 3. Cotinine
column . . Kromasil Eternity C18, 10 cm x 3.0 mm I.D., 2.5 μm particles (K08971111) 4. (±)-N'-Nitrosonornicotine (NNN)
5. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) isomer 1
mobile phase . . . . . . . (A) 10 mM ammonium acetate in water:methanol (95:5);
6. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) isomer 2
(B) 10 mM ammonium acetate in water:methanol (10:90)
gradient . . . 0% B held for 1 min; to 100% B in 8.5 min;100% B held for 1 min 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 psi (23 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+) TOF, EIC 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
4
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ng/mL in mobile phase A 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005925 2
0 2 4 6 8
Min

LC-MS Analysis of Omeprazole and Metabolites in 1. 5-Hydroxy omeprazole

Plasma on Ascentis® Express RP Amide after 2. Phenacetin
2 3. Omeprazole
Sample Prep using HybridSPE®-Phospholipid 4. Omeprazole sulfone
This application demonstrates the suitability of HybridSPE and the Ascentis
Express RP Amide for the analysis of omeprazole metabolites.
Sample was vorlexed and centrifuged for 2 minutes at 15000 rpm. 4
Supemate was then passed through HybridSPE 96 well plate. Elutent was
collected, evaporated and reconstituted in mobile phase.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 1 3
sample/matrix . . . . . . . . . . . . . . . . . . . . rat plasma spiked with omeprazole,
phenacetin and metabolites at 2 μg/mL 1.0 2.0 3.0 4.0 5.0
SPE well plate . . HybridSPE-Phospholipid, 96-well plate, 15 mg/0.8 mL (575656-U) Min
sample addition . . . . . . . . . . . . . . . 100 μL plasma to each well, followed by
300 mL of 1% formic acid in acetonitrile
eluate post-treatment . . . . . . . . . . . . . . . . . eluate was collected, evaporated,
and reconstituted in mobile phase.
column . . Ascentis Express RP-Amide, 5 cm x 2.1 mm I.D., 2.7 μm particles (53911-U)
mobile phase . . . . . . . . . . . . . (A) water, 10 mM ammonium formate, pH 3.4;
(B) methanol, 10 mM ammonium formate, pH 3.4; (65:35, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z = 50-400
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005711

LC-MS Analysis of Omeprazole and Metabolites on 1. Phenacetin (I.S.)

Ascentis® Express HILIC 3 2. Omeprazole sulfone
1 3. Omeprazole
This application demonstrates the suitability of the Ascentis Express HILIC for 2
4. 5-Hydroxy omeprazole
the analysis of omeprazole metabolites.
mobile phase . . . . . . . . . . . . (A) 5 mM ammonium formate in water, pH 6.5; 4
(B) 5 mM ammonium formate in acetonitrile, pH 6.5; (2:98, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 50-400 1.0 2.0 3.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 μL Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/L each in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005710

LC-MS Analysis of Omeprazole and Metabolites on 1. 5-Hydroxy omeprazole

2. Omeprazole
Ascentis® Express RP Amide (pH 2.7) 3. Phenacetin (I.S.)
This application demonstrates the suitability of the Ascentis Express RP 3 4. Omeprazole sulfone
Amide for the analysis of omeprazole metabolites.
column . . Ascentis Express RP-Amide, 5 cm x 2.1 mm I.D., 2.7 μm particles (53911-U) 2
4
mobile phase . . . . . . . . . . . . (A) 10 mM ammonium formate in water, pH 2.7;
(B) 10 mM ammonium formate in methanol, pH 2.7; (65:35, A:B) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 50-400
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/L in mobile phase 1.0 2.0 3.0

LC-MS Analysis of Omeprazole and Metabolites on 1. 5-Hydroxy omeprazole

Ascentis® Express RP Amide (pH 3.4) 2. Phenacetin (I.S.)
2
3. Omeprazole
This application demonstrates the suitability of the Ascentis Express RP 4. Omeprazole sulfone
Amide for the analysis of omeprazole metabolites.
mobile phase . . . . . . . . . . . . (A) 10 mM ammonium formate in water, pH 3.4; 3
(B) 10 mM ammonium formate in methanol, pH 3.4; (65:35, A:B) 4
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 50-400
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 1.0 2.0 3.0 4.0
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/L in mobile phase Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005712

LC-MS Analysis of Plasma Samples on Ascentis®

Express C18 Showing Baseline Improvements after
Phospholipid
HybridSPE-Precipitation:
No phospholipid removal after
Precipitate proteins by first adding 100 μL rat plasma to the HybridSPE-PPT standard protein PPT
plate followed by 300 μL 1% formic acid in acetonitrile. Do not apply
vacuum.
Mix by vortexing the HybridSPE-PPT plate briefly.
Apply vacuum using a 96-well vacuum manifold and collect the resulting
0 10 20
eluate for analysis. Min
Standard Protein Precipitation:
Complete removal of phospholipid
Combine 100 μL rat plasma with 300 μL 1% formic acid in acetonitrile. after HybridSPE-PPT
Mix by vortexing and centrifuge.
Isolate resulting supernatant for analysis.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 0 10 20
Min
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . rat plasma
SPE well plate . . . HybridSPE-Precipitation 96-well Plate, 50 mg/well (575656-U)
sample addition . . . . . . . . . . . . . . . . . . . . . . 100 μL rat plasma followed by
gradient . . . . . . . . . 0 min - 95% (A); 10 min - 50% (A); 18 min - 50% (A);
18.1 min - 95% (A); 22 min - 95% (A)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . ABI 3200QT; ESI (+), MRM (184/103 m/z)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004243

LC-MS Analysis of Sertraline and Norsertraline on 1. Sertraline

1
Ascentis® Express RP-Amide 2. Norsertraline
column . . . . . . . . . . . . . . . Ascentis Express RP Amide, 10 cm x 3.0 mm I.D.,
mobile phase . . . . . . . . . . . . . (A) 10 mM ammonium formate, water, pH 3.6;
(B) 10 mM ammonium formate, acetonitrile, pH 3.6; (65:35, A:B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 100-1000 m/z 0 2 4 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Min
sample . . . . . . . . . . . . . . . . . . . . . . 200 ng/mL in 25:75, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005736

LC-MS Analysis of Vitamin D2, D3, Gamma- and 2

1. Vitamin D2
Alpha-Tocopherol on Ascentis® Express C18 2. Vitamin D3
3. Gamma Tocopherol
Analysis was conducted using standard solution, detection on LC/MS
4. Alpha Tocopherol
system.
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (5:95, A:B) 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI+
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μg/mL in acetonitrile 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005806
1
0 2 4 6 8 10
Min

LC-MS/MS Analysis of Epinephrine, Metanephrine 1. Metanephrine 3

and Metabolites on Ascentis® Express OH5 2. Normetanephrine
3. Epinephrine
HILIC mode on Ascentis Express OH5 provided rapid separation of 4. Norepinephrine
neurotransmitters in highly MS-compatible mobile phases.
mobile phase . . . . . . . . . (A) 5 mM ammonium formate; (B) 5 mM ammonium
formate in acetonitrile; (15:85, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189 psi (82 bar)
4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS ESI(+), 100-1000 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 1
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 ng/mL each in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005879
1.0 2.0
Min

LC-MS/MS Analysis of 2-Hydroxyglutaric Acid column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CHIROBIOTIC R, 25 cm x 4.6 mm I.D., 5 μm particles (13024AST)
mobile phase . . (A) ethanol:methanol (3:1, v:v); (B) 0.1% v/v TEA in water, adjusted to pH 4.5 with acetic acid; 75:25 (A:B)
Enantiomers on Astec® CHIROBIOTIC® R flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
Chiral 2-hydroxyglutarates (2-OHG) are important molecular signatures of column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
both healthy and diseased biological cells, their specific biochemical detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(-), 100-1000 m/z
pathways and inborn errors of metabolism. The chiral differentiation and injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
quantification of D-2-OHG (R(-)-2-OHG) and L-2-OHG (S(+)-2-OHG) is key for
characterizing neurometabolic disorders like the 2-hydroxyglutaric acidurias
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . each enantiomer, 100 μg/mL in water
that cause neurological impairment early in life. The ability to distinguish Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005848
and quantify the enantiomers of 2-OHG is therefore important to be able to
track its disposition throughout the metabolic pathway of normal vs. Intens.
mutated cells. We show here a rapid and sensitive method to separate and x105 HGA TEST 003 ETOH1.D: EIC 129; 147- All MS, Smoothed (3.0,1, GA)
1
measure D-2-OHG and L-2-OHG using LC-MS/MS detection. An Astec® 2
8
CHIROBIOTIC R (ristocetin chiral selector) column was run under polar ionic D
mobile phase conditions. Using tandem MS eliminated two fundamental 6 L
OH
analytical challenges with these compounds: the lack of a UV-absorbing HO O
chromophore in the 2-OHG molecule, and the abundance of competing 4
low molecular weight acids in urine. The upper panel in the chromatogram O OH
2
shows the separation of D-2-OHG and L-2-OHG obtained during method
development. The lower panel shows the pure L-2-hydroxyglutaric acid. 0
Besides providing the necessary enantioselectivity, Astec® CHIROBIOTIC x107 LHGA TEST 001 ETOH.D: EIC 129; 147 - All MS, Smoothed (3.0,1, GA)
columns have the advantage of operating in aqueous and polar organic 1.25 1
mobile phases that are amenable to polar drugs and metabolites. Their ionic L
1.00
interactions promote analyte ionization which enhances sensitivity in ESI
detection. In addition to the CHIROBIOTIC columns, Sigma-Aldrich also 0.75
provides the high purity mobile phase agents, racemic and pure chiral 2-
0.50
OHG enantiomers and other compounds in the isocitrate dehydrogenase
(IDH) pathway. 0.25
2
0.00
2 4 6 8 10 12 14 16 18
Min

LC-MS/MS Analysis of Interacting Cardiac Drugs 1. Quinidine

2. Verapamil
Digoxin, Quinidine, Amiodarone and Verapamil on 2 3. Digoxin
Titan™ C18 4. Amiodarone
20000
Sample pretreatment: Sample spiked into rat plasma
Intensity/cps
Detector:
MRM transitions:
4
Peak MRM
1. Quinidine 325.2/81.1, 307.1
1 3
2. Verapamil 455.3/165.1
3. Digoxin 798.5/651.5
4. Amiodarone 646.1/58.1, 100.1
0
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . Sample spiked into rat plasma
SPE well plate . . . . . . . . . . . . . . . HybridSPE®-PLus 96-Well Plate (575659-U) 0 2 4 6
condition . . 100 uL of spiked rat plasma was added to the HybridSPE plate well, Min
followed by 300 uL of protein precipitation solvent, acetonitrile with
1% formic acid. (The plate, sealed, secured on a vibrater, agitated
by vibration at 1000 rpm for 2 min. The plate was transfered
to a vacuum manifold. Vacuum was applied at 10 in Hg for 4 min.)
column . . . . . . . . . Titan C18, 5 cm x 2.1 mm I.D., 1.9 μm particles (577122-U)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) Water; (B) Methanol,
both with 0.1% formic acid/10 mM ammonium formate
gradient . . . . . . . . . . . . . 0-1 min, 45%B; 1.5-4 min 90%B, 4.5-8 min 45%B
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2900 psi
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), MRM mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006304

LC-MS/MS Analysis of Steroid Hormones in Plasma MRM Transition

on Ascentis® Express C18 after Sample Prep using 1. Aldosterone 361.0/343.1
HybridSPE®-Phospholipid 2. Corticosterone 347.6/109.0
3. 11-Deoxycorticosterone 331.1/109.0
Immunoassay approaches for steroid determination are typically hindered
4. Testosterone 289.0/109.0
due to the lack of specificity of antibodies for the measurement of the
5. 17α-Methyltestosterone 303.1/97.0
particular steroids. With the wide acceptance of LC-MS/MS in the clinical
6. Progesterone 315.0/109.1
setting, there is a growing trend toward converting traditional IA techniques
toward more specific and robust LC-MS/MS approaches. Though LC-MS/MS
improves assay specificity and allows of multiplexed analyte assays to be 6
conducted simultaneously, it is not without limitations specifically toward 4
interferences form endogenous sample matrix. Often LC-MS/MS assays can 5
be hindered due to ionization effects due to endogenous matrix from
biological samples; this can result in random and arbitrary discrimination in 3
analyte response. The use of HybridSPE-Phospholipid prior to LC-MS/MS
analysis removes the endogenous phospholipids and precipitated proteins
in one step, enabling high analyte recovery, negligable matrix background
and long column lifetime.
SPE tube/cartridge . . . . . . . . . . . . . . . HybridSPE®-Phospholipid, 96-well plate,
50 mg/well (575656-U) 1 2
sample addition . . . . To each well add 100 μL of plasma followed by 300 μL of
precipitation solvent (1% formic acid or 0.5% citric acid in acetonitrile)0.
(Agitate via vortex for 4 minute, place on vacuum manifold and 0 2 4 6 8
apply 10" Hg vacuum for 4 minutes. Collect filtrate and analyze directly.)
column . . Ascentis® Express C18, 10 cm x 2.1 mm I.D., 2.7 μm particles (53823-U) Min
mobile phase . . (A) 5 mM ammonium formate pH 4.0 with formic acid; (B) methanol
gradient . . 60% B for 3 min, 60% B to 95% B in 5 min, held at 95% B for 2 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, MRM
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005895

LC-MS/MS Analysis of Testosterone and 1. Testosterone glucuronide

Metabolites on Ascentis® Express C18 2. 5α-Dihydrotestosterone glucuronide
3. Testosterone
4. 17α-Methyltestosterone
mobile phase . . . . . . . (A) 5 mM ammonium formate water pH 4.0 formic acid; 5. 5α-Dihydrotestosterone
(B) 0.1% formic acid acetonitrile 6. 6β-Hydroxytestosterone
gradient . . . . . . . . . . . . . . . 10 to 70% B in 5 min; held at 70% B for 3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
3 4
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3698 psi (255 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . 1000 ng/mL in 40:60 (water:methanol)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005882
1 6
5
2
0 2 4 6
Min

LC-MS/MS Analysis of Warfarin™ in Plasma on

Ascentis® Express C18 after SPE using HybridSPE®- pink: Phospholipids
gray: Analytes
Phospholipid
Warfarin is a widely prescribed oral anticoagulant for treatment and
prevention of thrombosis and thromboembolism. The pharmacological
effect of warfarin is derived from its ability to inhibit the enzyme vitamin K Warfarin
epoxide reductase, thereby reducing circulating levels of vitamin K which is

required in the clotting process. In spite of its popularity, there are some
downsides to warfarin, including a relatively narrow therapeutic index,
interactions with other clinically important drugs, side effects, genetic
variation in warfarin metabolism, frequent migration of blood levels outside
the therapeutic range, bleeding events, and variability in time to reach Min
therapeutic levels. These, and the fact that warfarin is the second leading
cause of drug-related emergency room visits make it a commonly analyzed
compound in the clinical lab. The clinical implications and importance of
therapeutic drug monitoring of warfarin necessitate reliable and sensitive
analytical methods, like UHPLC-MS/MS, to detect and quantify warfarin and
its enantiomers in serum.
sample/matrix . . . . . . rabbit plasma, unfiltered K2-EDTA, spiked with warfarin at
100 ng/mL (3:1, plasma:1% formic acid in acetonitrile)
SPE tube/cartridge . . . . . . . . HybridSPE®-Phospholipid, 96-well plate (575656-U)
sample addition . . . . . to each well add 100 μL plasma, followed by a 300 μL of
1% formic acid in acetonitrile, agitate on orbital shaker for 4 minutes
elution . . to each well add 100 μL plasma, followed by a 300 μL of 1% formic acid in
acetonitrile, agitate on orbital shaker for 4 minutes
column . . Ascentis® Express C18, 10 cm x 2.1 mm I.D., 2.7 μm particles (53823-U)
mobile phase . . . . . . . . (A) 5mM ammonium formate, pH 4.2 with formic acid;
(B) 5mM ammonium formate in 95:5 acetonitrile:water, 50:50 (A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, 100-1000 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005850

LC-MS/MS Analysis of Vitamin D Metabolites on 6

Ascentis® Express F5 1. 1α,25-Dihydroxyvitamin D3
column . . Ascentis Express F5, 15 cm x 2.1 mm I.D., 2.7 μm particles (53571-U) 4,5 2. 1α,25-Dihydroxyvitamin D2
mobile phase . . (A) 5 mM ammonium formate water; (B) methanol; (25:75, A:B) 3. 25-Hydroxyvitamin D3
3 4. 25-Hydroxyvitamin D2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
5. 3-epi-25-Hydroxyvitamin D3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5178 psi (357 bar) 6. 3-epi-25 hydroxyvitamin D2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 7. 1α-Hydroxyvitamin D3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 100-1000 m/z
injection . . . . . . . . 2 μL, each compound 300 ng/mL in 40:60 (water:methanol) 2 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005878 1
0 2 4 6
Min

LC-MS/MS Analysis of Vitamin D Metabolites on 1. 1α,25-Dihydroxyvitamin D3

Ascentis® Express F5 (Monitored Ions) 2. 1α,25-Dihydroxyvitamin D2
3
column . . Ascentis Express F5, 15 cm x 2.1 mm I.D., 2.7 μm particles (53571-U) 4. 25-Hydroxyvitamin D2
mobile phase . . (A) 5 mM ammonium formate water; (B) methanol; (25:75, A:B) 5. 3-epi-25-Hydroxyvitamin D3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 6. 3-epi-25 hydroxyvitamin D2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5178 psi (357 bar) 7. 1α-Hydroxyvitamin D3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 100-1000 m/z 4
injection . . . . . . . . 2 μL, each compound 300 ng/mL in 40:60 (water:methanol) 6 m/z 401.39
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005876 7
1 m/z 413.39
m/z 434.365
m/z 446.41
0 2 4 6
Min

LC-MS/MS Analysis of Warfarin™ Enantiomers in detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, 100-1000 m/z

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Plasma on Astec® CHIROBIOTIC® V after SPE using Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005849
prevention of thrombosis and thromboembolism. The pharmacological pink: Phospholipids
gray: Analytes
effect of warfarin is derived from its ability to inhibit the enzyme vitamin K
the therapeutic range, bleeding events, and variability in time to reach
compound in the clinical lab. Warfarin is a chiral compound comprising an
equal mixture of (R) and (S) enantiomers. (S)-Warfarin is considerably more
potent and pharmacologically active than (R)-warfarin. Astec CHIROBIOTIC V
columns resolve warfarin enantiomers under LC-MS conditions and hold up
well to biological samples. The clinical implications and importance of
SPE tube/cartridge . . . . . . . . . HybridSPE-Phospholipid, 96-well plate (575656-U)
sample addition . . to each well add 100 μL plasma, followed by a 300 μL of 1%
formic acid in acetonitrile, agitate on orbital shaker for 4 minutes
elution . . . to each well add 100 μL plasma, followed by a 300 μL of 1% formic
acid in acetonitrile, agitate on orbital shaker for 4 minutes
column . . Astec CHIROBIOTIC® V, 10 cm x 4.6 mm I.D., 5 μm particles (11022AST)
mobile phase . . (A) 0.1% formic acid (pH unadjusted); (B) acetonitrile, 75:25 (A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C

LC/MS/MS of Digoxin and Digitoxin in Serum or

Peak MRM (m/z)
Plasma on Titan™ C18 after SPE with HybridSPE®- 1. Digoxin 798.5/651.5
Phospholipid, Minimization of Matrix Effects 2. Digitoxin 782.5/635.5 or 243.2
The cardiac glycosides digitoxin and digoxin are widely prescribed for
treating congestive heart failure. Digitoxin differs from digoxin only in that
digoxin possesses an additional OH group. In fact, digoxin is one of the OH- 2
1
metabolites of digitoxin. Therefore, there is a need for simultaneous and
Intensity/cps
sensitive determination of digitoxin and digoxin in biological fluids using
LC-MS. This chromatogram shows the compounds as standards at very low
level of 0.05 ng/mL.
mobile phase . . . . . . . . . 10 mM ammonium formate in methanol:water (80:20)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4550 psi (314 bar) 0
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI-MS/MS
0 2
detector . . . . . . MRM Transitions: Peak MRM(m/z) (1.) Digoxin 798.5/651.5 (2.) Min
Digitoxin 782.5/635.5 or 243.2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 ng/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006306

LC-MS (TOF) Analysis of Opioids and Opiate- 1. Oxycodone

Dependence Management Drugs on Ascentis® 2. 6-Acetylcodeine
3. 6-Acetylmorphine
Express HILIC 4. Codeine
5
column . . Ascentis Express HILIC, 5 cm x 2.1 mm I.D., 2.7 μm particles (53934-U) 2 5. Hydrocodone
mobile phase . . . . . . 2 mM ammonium formate in 90:10 (v:v) acetonitrile:water 6. Morphine
7. Hydromorphone
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C 1 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS 7
4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006153
6
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Min

LC-MS (TOF) Analysis of Warfarin™ Enantiomers in condition . . Apply 100 μL of plasma to plate, followed by 300 μL of 1% formic acid acetonitrile. Agitate via vortex for 4 min.
elution . . . . . Place on vacuum manifold and apply 10” Hg vacuum for 4 minutes. Collect filtrate and analyze directly.
Plasma on Astec® CHIROBIOTIC® V after SPE Using column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CHIROBIOTIC V,10 cm x 4.6 mm I.D., 5 μm particles (11022AST)
HybridSPE®-Phospholipid mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% formic acid water; (B) acetonitrile, (75:25, A:B)
This application demonstrates the benefit of using Sigma-Aldrich Analytical flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
(Supelco, Fluka, and Cerilliant) consumables for rapid, accurate, sensitive and column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
reliable MS detection in a clinical or bioanalytical setting. detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, 100-1000 m/z
Warfarin was originally developed as a rodenticide, but soon found medical injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
application as an anticoagulant to prevent the formation of blood clots,
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005827
especially after heart attacks. At the time of this writing, it is the most widely
prescribed oral anticoagulant and is sold under Coumadin and various other
brand names. Warfarin is a chiral molecule. The R- and S- forms are cleared Black: Sample prep using
by the body via different pathways and have different pharmacokinetic and HybridSPE®-Phospholipid
pharmacodynamic effects. S-warfarin is more potent than the R-warfarin in
producing the anticoagulant response.
The Astec® CHIROBIOTIC V column provided rapid resolution of the
enantiomers under conditions that are compatible with mass spectrometric
(MS) detection. Considering the rise of MS in the clinical laboratory, this is an
important consideration. The CHIROBIOTIC family of chiral columns are Red: Sample prep using
based on the macrocyclic glycopeptide molecule and affect enantioreso- protein precipitation only
lution under mobile conditions that are compatible with reversed-phase
and MS conditions. Their durability allows them to stand up to repeated
injections of biological extracts.
By using HybridSPE-Phospholipid for the sample prep, ion-suppressing
phospholipids were removed for improved sensitivity and longer column
lifetime. LC-MS Ultra CHROMASOLV solvents were used to supply low
background interference and particulate contaminants. Cerilliant reference
material grade standards provided reliable quantitation.
system: Agilent 1290, 6210 TOF 1.0 2.0 3.0 4.0
sample/matrix . . . . . . . . . Rat plasma stabilized with K2EDTA was acquired from Min
Lampire Biological Laboratories, (Pipersville PA). (Plasma was spiked
directly from stock standard to a level of 100 ng/mL in 3:1
(plasma:1% formic acid acetonitrile).)
SPE tube/cartridge . . Pk1 50 mg/96-Well HybridSPE-Phospholipid Plate (575656-U)

x105 +ESI EIC(235.2000, 415.1700, 618.0300, 646.0600) Scan Frag=175.0V 081712020.d

LC-TOF Analysis of Antiarrhythmic Drugs and 8 1
7.5 1 Recovery from plasma (average) by sample Desmethyl-
1
Peak Cerilliant Part No. prep technique (calculated, ng/mL) Lidocaine Amiodarone amiodarone Flecainide
Metabolites in Plasma on Ascentis® Express HILIC 6.5
7 1. Amiodarone
2. Desmethylamiodarone
A-060
D-055 HybridSPE-Phospholipid 108.4 109.8 104.8 119.0
3. Lidocaine L-018 Protein Precipitation 118.5 105.5 52.9 44.7
after Sample Prep using HybridSPE®-Phospholipid 5.5
6 4. Flecainide F-017
5
The basic characteristics of antiarrhythmic cardiac drugs and associated 4.5 2
4
metabolites make them targets for HILIC chromatographic separation. 4 red: phospholipids
Because HILIC mobile phases consist of a high composition of acetonitrile, 3.5
3
this can also facilitate the direct analysis of precipitated plasma samples 2.5
without the need for additional sample solvent exchange. In most cases, the 2 3
1.5
high organic mobile phase also facilitates increased analyte response in ESI 1
(+) MS detection. This application used HybridSPE-Phospholipid sample 0.5
prep to remove phospholipids and precipitated proteins prior to LC-MS 0
1 2 3 4
analysis on Ascentis Express HILIC. The resulting method was robust and Counts vs. Acquisition Time (min)
sensitive.
sample/matrix . . . Rat plasma stabilized with K2EDTA was acquired from Lampire
Biological Laboratories, (Pipersville PA). Plasma was spiked
directly from stock standard to a level of 400 ng/mL
SPE tube/cartridge . . HybridSPE-Phospholipid 96-Well Plate, 50 mg/2 mL (575656-U)
sample addition . . Apply 100 μL of plasma to plate, followed by 300 μL of 1% formic
acid acetonitrile. Agitate via vortex for 4 min
elution . . . . Place on vacuum manifold and apply 10” Hg vacuum for 4 minutes.
Collect filtrate and analyze directly.
mobile phase . . (A) 5 mM ammonium formate; (B) 5 mM ammonium formate in
acetonitrile; (5:95, A:B, pH 7.0 with formic acid) (LC-MS CHROMASOLV Ultra grade)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305 psi (90 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), full scan, m/z 100-1000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . plasma extract, analyte concentration of final sample
work up is equivalent to 100 ng/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005818

LC-TOF Analysis of Antiarrhythmic Drugs and 1. Amiodarone

Metabolites on Ascentis® Express HILIC 1 2. Desmethylamiodarone
3. Lidocaine
The basic characteristics of these compounds make them targets for HILIC
4. Flecainide
chromatographic separation. Because HILIC mobile phases consist of a high
composition of acetonitrile, this can also facilitate the direct analysis of 2
precipitated plasma samples without the need for additional sample solvent 4
exchange. In most cases, the high organic mobile phase also facilitates
increased analyte response in ESI+ MS detections.
mobile phase . . (A) 5 mM ammonium formate; (B) 5 mM ammonium formate in 3
acetonitrile; (5:95, A:B, pH 7.0 with formic acid) (LC-MS CHROMASOLV Ultra grade)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
0 2 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . each compound, 300 ng/mL in 1% formic acid acetonitrile:water, 75:25 Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005817

LC-TOF Analysis of Antiarrhythmic Drugs and sample . . . . . . . . . . . . . . plasma extract, analyte concentration of final sample work up is equivalent to 100 ng/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005819
Metabolites on Ascentis® Express HILIC:
Improvement in Analyte Response after Sample x105
8 1 +ESI EIC(235.2000, 415.1700, 618.0300, 646.0600) Scan Frag=175.0V 081712020.d Sample Prep Using HybridSPE®-Phospholipid 1
7.5
Prep using HybridSPE®-Phospholipid 7
1
Cerilliant Part No.
Calculated Concentration (ng/mL)
HybridSPE-Phospholipid Protein Precipitation
1. Amiodarone A-060 108.4 118.5
6.5
2. Desmethylamiodarone D-055 109.8 105.5
The basic characteristics of antiarrhythmic cardiac drugs and associated 6 3. Lidocaine L-018 104.8 52.9
4. Flecainide F-017 119 44.7
metabolites make them targets for HILIC chromatographic separation. 5.5
5
Because HILIC mobile phases consist of a high composition of acetonitrile, 4.5 2 4
this can also facilitate the direct analysis of precipitated plasma samples 4 phospholipids
3.5
without the need for additional sample solvent exchange. In most cases, the 3
high organic mobile phase also facilitates increased analyte response in ESI 2.5
(+) MS detection. This application used HybridSPE-Phospholipid sample 2 3

1.5
analysis on Ascentis Express HILIC. These chromatograms compare the 0.5
0
response with HybridSPE-Phospholipid vs. standard protein precipitation 1 2 3 4
sample prep methods. Counts vs. Acquisition Time (min)
sample . . . . . . . . Rat plasma stabilized with K2EDTA was acquired from Lampire x105
Biological Laboratories, (Pipersville PA). Plasma was spiked directly 9.5 1 +ESI EIC(235.2000, 415.1700, 618.0300, 646.0600) Scan Frag=175.0V 081612052.d Sample Prep Using Protein Precipitation Only 1
9
from stock standard to a level of 400 ng/mL 8.5
SPE tube/cartridge . . HybridSPE-Phospholipid 96-Well Plate, 50 mg/2 mL (575656-U) 7.5
8
sample addition . . Apply 100 μL of plasma to plate, followed by 300 μL of 1% formic 7

6.5
acid acetonitrile. Agitate via vortex for 4 min. 6
1
5.5
elution . . . . Place on vacuum manifold and apply 10" Hg vacuum for 4 minutes. 5 phospholipids
Collect filtrate and analyze directly. 4.5
4
2
3.5
3
mobile phase . . . . (A) 5 mM ammonium formate; (B) 5 mM ammonium formate 2.5
in acetonitrile; (5:95, A:B, pH 7.0 with formic acid) 2 4
1.5
(LC-MS CHROMASOLV Ultra grade) 1 3
0.5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 0
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305 psi (90 bar) 1 2 3 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL

UHPLC Analysis of Barbituates on Titan™ C18 3

1. Uracil
Analysis of a Barbituate Mix using a MS compatible mobile phase. 2. Barbital
3. Phenobarbital
column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U) 1 2 4. Cyclobarbital
5. Butalbital
mobile phase . . . . . . . . . . . . . . . (A) 0.1% ammonium acetate (not adjusted); 6. Amobarbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 4
5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7540 psi (520 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006073
1.0 2.0 3.0 4.0

Min

UHPLC Analysis of Barbituates on Titan™ C18 with 1 1. Barbital

MS Detection 2. Phenbarbital
3. Cyclobarbital
column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U) 2 5
4. Butalbital
mobile phase . . . . . . . . . . . . . . . (A) 0.1% ammonium acetate (not adjusted); 4 5. Amobarbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6796 psi (468 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (-) MS TIC MRM
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ng/ml in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006137
1 2 3 4 5
Min

UHPLC Analysis of Catecholamines on Ascentis® 1.

2.
Norepinephrine
Epinephrine
Express F5 3. Dopamine
4. Normetanephrine
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53823-U) 5. Metanephrine
mobile phase . . (A) water; (B) 100 mM ammonium acetate (pH 4.1 with acetic acid);
(C) acetonitrile; (88:2:10, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 1 3 4
2 5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1880 psi (130 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
injection . . . . . . . . . . . . . . Catecholamines 25 μg/mL in 90:10, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006281
1.0 2.0 3.0

Min

2
UHPLC Analysis of Corticosteroids on Titan™ C18 1 1. Hydrocortisone
3 2. Cortisone
Analysis of Corticosteroids on a Titan C18 UPLC column with MS detection
3. Corticosterone
in ESI (+) Mode
4. Cortisone Acetate
gradient . . . . . . . . . . . . . . . . . 30% to 60% B in 3 minutes, hold for 0.2 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7423 psi (512 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+) MS TIC SIR
1.0 2.0 3.0 4.0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006147
Min

UHPLC Analysis of Diazepam and Metabolites on 1. Uracil 3. Nordiazepam 5. Methyl Benzoate

2. Oxazepam 4. Temazepam 6. Diazepam
Titan™ C18, Competitive Comparison
5
This application shows a comparison between Titan™ C18 and a competitive
column for diazepam and its metabolites using a MS compatible mobile
3.303
Titan C18 1.9 mm
phase.
445 bar // 6,450 psi*
Used 75 μm I.D. x 250 mm L tubing in system.
3 6
1
2.265
mobile phase . . . . . . . . . . . (A) 0.1% formic acid; (B) acetonitrile; (60:40, A:B)
4.299
3.080
1.846
0.447
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6450 psi (445 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 1 2 3 4
*system pressure includes instrument blank pressure
1.970
Competitor C18 1.7 μm
1.466
2.545
1.265
0.445
1 2 3 4
Min

UHPLC Analysis of Riboflavin and Impurities on pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5000 psi (345 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
Titan™ C18 with MS Detection detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+), MRM and TIC
Riboflavin (vitamin B2) is a water-soluble vitamin. It is synthesized by all detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diode Array at 276 nm
plants and many microorganisms, but it is not produced by higher animals. injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
It is a precursor of coenzymes that are required for the enzymatic oxidation Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006138
of carbohydrates, so it is essential to basic metabolism. Riboflavin is used as
an additive to food and feed and is also used in fortification of baby food
and cereal. The current USP method for impurity analysis is non-quantitative,
and uses an ion pair reagent in the mobile phase. An alternative to USP and
EP methods using LC-MS/MS to identify and quantify the riboflavin and
Riboflavin
impurities is presented. This new proposed method is also compatible with
UV-Vis detection.
A riboflavin certified reference material (CRM) was used in this study. MRM
transitions for the determination of ions for the parent and four impurites Lumiflavine (7,8,10-trimethylbenzo[g]pteri-
dine-2,4(3H,10H)-dione),
were made. Three transitions were used for each ion. Structures for each of Compound A
these major impurities were confirmed by MS. In addition to improving the
specificity of the method with MS/MS detection, two additional goals were
to decrease analysis time and eliminate the need for ion pairing reagents 7,8-Dimethylbenzo[g]pteri-
that are not compatible with MS. dine-2,4(1H,3H)-dione,
Compound B
Other MS Conditions
Capillary (kV) 2.29
Cone (V) 54.95
Extractor (V) 5.13 6,7-Dimethyl-8-[(2S,3S,4R)-2,3,4,5-tetra-
Source Temperature (°C) 151 hydroxypentyl]pteridine-2,4(3H,8H)-dione,
Desolvation Temperature (°C) 349 Compound C
Cone Gas Flow (L/Hr) 2
Desolvation Gas Flow (L/Hr) 646
Collision Gas Flow (mL/Min) ON 8-(Hydroxymethyl)-7-methyl-10-[(2S,3S,4R)-2,3,4,5-tetra-
column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U) hydroxypentyl]benzo[g]pteridine-2,4(3H,10H)-dione,
Compound D
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% formic acid in water;
(B) 0.1% formic acid in acetonitrile (95:5) 0 2 4 6 8 10
gradient . . . . . . . . . . . . . . 5% to 25% in 6 min, return to 5% b in 0.1 min, Min
Compounds B, C, and D are impurities
and re-equilibrate for 4 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 μL/min

UHPLC-MS Analysis of Vitamin K1 and Related 1. Vitamin K1 2,3 epoxide (m/z 467.37)
Compound on Ascentis® Express 2.7 μm C18 2. Vitamin K1 (m/z 451.37)
column . . . Ascentis Express C18 5 cm x 2.1 mm I.D., 2.7 μm particles (53822-U)

2
mobile phase . . . . . . . . . . . . . . . . (A) methanol; (B) 2-propanol; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1870 psi (129 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . MS-APCI+, full scan 425-525 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 50 ng/mL 90:10, methanol:2-propanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006309
1.0 2.0 3.0

Min

HPLC, UHPLC or LC-MS Cosmetics, Personal Care, and Cleaning Prod-
Gradient Program 1. Propyl gallate

Cosmetics, Personal Care, and Cleaning Time (min) %A %B 2. THBP
0 70 30 3. TBHQ
Products 10 0 100
4. NDGA
5. BHA
20 70 30 6. Ionox 100
HPLC Analysis of Antioxidants on 7. Octyl gallate
2
SUPELCOSIL™ LC-18 7 8. BHT
9. Dodecyl gallate
column . . . . SUPELCOSIL LC-18, 15 cm × 4.6 mm I.D., 5 μm particles (58230-U)
mobile phase . . (A) 5% acetic acid in deionized water; (B) acetonitrile:methanol (1:1)
70% A/30% B to 100% B, linear gradient over 10 min, hold 10min 9
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
5
injection . . . . . . . . . . . . . . . . . . . . . . . . . 10μL, 20 μg/mL each antioxidant
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795-0438 3 4
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Benzalkonium Chloride in 2

Commercial Disinfecting Wipes on Ascentis® 1-4. Benzalkonium chloride Analogs 3
Express HILIC
mobile phase . . . . . (A) 20 mM ammonium acetate; (B) acetonitrile; (10:90, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), TIC 150 - 500 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . Single wipe sheet, extracted with 10 mL methanol 0 2 4 6 8 10 12

HPLC Analysis of Benzalkonium Chlorides on +
Discovery® Cyano CH3

CH2 N R
column . . . . . Discovery Cyano, 15 cm × 4.6 mm I.D., 5 μm particles (59356-U) CH3
mobile phase . . . . . . . . (A) acetonitrile; (B) acetate buffer, pH 4.5, (60:40, A:B) Cl-
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
sample . . . . . . . . . . . . . . . . . . . undefined C8 to C18 Benzalkonium Chlorides
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000726
0 2 4 6
Min

HPLC Analysis of Benzoic Acid and Related 1. 4-Hydroxybenzoic Acid

2. Acetylsalicylic Acid
Compounds on Ascentis® RP-Amide 3. Benzoic Acid
3
4. 2-Hydroxybenzoic Acid
column . . . Ascentis RP-Amide, 15 cm × 4.6 mm I.D., 5 μm particles (565324-U) 5. Ethyl paraben
mobile phase . . . . . . . . (A) water with 0.1% TFA; (B) acetonitrile; (70:30, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 4
1
2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004094
0 10 20
Min

HPLC Analysis of Benzoic Acid and Sorbic Acid on 1. Sorbic Acid 2

Ascentis® Express RP-Amide ("Green" Mobile 2. Benzoic Acid
Phase Conditions)
mobile phase . . . . . . . . . . . . . . (A) 10 mM ammonium phosphate monobasic,
pH 2.5 with phosphoric acid; (B) ethanol; (75:25, A:B) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . 138 bar (2000 psi) column, 248 bar (3600 psi) total
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005588
1.0 2.0
Min

1. Chlorhexidine
HPLC Analysis of Chlorhexidine and Robenidine Gradient Program 1
2. Robenidine
Time (min) %A %B %C
on Ascentis® Phenyl 0 65 25 10 2
7 0 90 10
column . . . . . . Ascentis Phenyl 15 cm x 4.6 mm I.D., 5 μm particles (581616-U)
9 0 90 10
mobile phase . . . . . . . . . . . (A) water; (B) acetonitrile; (C) 500 mM ammonium 9.5 65 25 10
formate, pH 3.0, with formic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 273 nm 0 2 4 6 8 10
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . 100 ug/mL each in 70:30, 25 mM ammonium formate pH 3.0:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003711

HPLC Analysis of Cosmetic Preservatives on 1. 2-Phenoxyethanol

3. 1-Phenoxy-2-propanol
O
4. Methylparaben 5. Ethylparaben
HO
Ascentis® Express C18 (0.6 mL/min) O O CH3
O
OH CH3 O
column . . Ascentis Express C18, 7.5 cm x 3 mm I.D., 2.7 μm particles (53812-U) O CH3
2. Contaminant HO
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (55:45, A:B) HO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 6. Propylparaben O
O 7. Butylparaben
2 8. Benzylparaben
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C O
CH3 O
O CH3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm 5 O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 1
HO
HO
HO
sample . . . . . . . . . . . methyl-, benzylparaben, 50 mg/L; ethylparaben, 30 mg/L; 4 6
propyl-, butylparaben, 100 mg/L; 2-phenoxyethanol, 1.5 g/L;
1-phenoxy-2-propanol, 3 g/L; all in 40% methanol 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005386 3
8
0 2 4 6 8 10 12 14
Min


O
HO
Ascentis® Express C18 (1.2 mL/min) O O CH3
O
OH CH3 O
column . . Ascentis Express C18, 7.5 cm x 3 mm I.D., 2.7 μm particles (53812-U) O CH3
2. Contaminant HO
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (55:45, A:B) HO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min 2
6. Propylparaben O
O 7. Butylparaben
8. Benzylparaben
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C O
CH3 O
O CH3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm 5
O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 1 HO
HO
4 6 HO
sample . . . . . . . . . . . methyl-, benzylparaben, 50 mg/L; ethylparaben, 30 mg/L;
3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005385 8
1.0 2.0 3.0 4.0 5.0 6.0 7.0

Min


Ascentis® Express RP-Amide 2. Unknown
With the publication Borremans, M. et.al. 2004, Chromatographia 59: 47-53 4. Methylparaben
as a reference, this method is 10% faster, but with superior resolution of 5. Ethylparaben
critical pairs; in this case, the minimum USP resolution is 2.4. 4 6. Propylparaben
column . . Ascentis Express RP-Amide, 10 cm x 3 mm I.D., 2.7 μm particles (53918-U) 13 7. Butylparaben
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (60:40, A:B) 5 8. Benzylparaben
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2900 psi (200 bar) 6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm 7
8
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . methyl-, benzylparaben (50 mg/L), ethylparaben (30 mg/L),
propyl-, butylparaben (100 mg/L), 2-phenoxyethanol (1.5 g/L), 0 10 20 30
1-phenoxy-2-propanol (3 g/L), all in 40% methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005738


O
HO
Ascentis® Express RP-Amide at Elevated Flow and O O O
CH3
O
OH CH3
Temperature (1.2 mL/min) O CH3
2. Contaminant HO
column . . Ascentis Express RP-Amide, 10 cm x 3 mm I.D., 2.7 μm particles (53918-U) HO
O
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (60:40, A:B) 1
4 6. Propylparaben O 7. Butylparaben
8. Benzylparaben
CH3 O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min 2
3 O
O CH3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C 5 O
HO
HO
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm HO
6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005384

0 2 4 6 8 10 12
Min


O
HO
Ascentis® Express RP-Amide at Elevated O
OH O O
CH3
O
CH3
Temperature (0.6 mL/min) O CH3
2. Contaminant HO
column . . Ascentis Express RP-Amide, 10 cm x 3 mm I.D., 2.7 μm particles (53918-U) HO
O 7. Butylparaben
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (60:40, A:B) 4
6. Propylparaben O 8. Benzylparaben
CH3 O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 1 O
O CH3
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C 5
HO
O
HO
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm HO
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 6

propyl-, butylparaben, 100 mg/L; 2-phenoxyethanol, 1.5 g/L; 2
7
8
1-phenoxy-2-propanol, 3 g/L; all in 40% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005383
0 10 20
Min

HPLC Analysis of Hydrocortisone in Topical Cream Efficiency of Recovery

Actual
on Discovery® HS C18 after SPE using Discovery® Theoretical Hydrocortisone Actual %
Sample Sample Hydrocortisone weight recovered Absolute Hydrocortisone
DSC-Si No. Weight (g) Weight (g)1 (g) Recovery (%) in Cream
1 1.0258 0.0103 0.0092 89.32 0.9003
Sample Pre-treatment: 2 1.1078 0.0118 0.0111 94.07 0.9975
Dissove 1 g 1% hydrocortisone topical hand cream in 10 mL ethyl acetate. 3 1.1151 0.0112 0.0119 106.25 1.0424
Dilute 2 mL of ethyl acetate-cream sample with 8 mL hexane:ethyl 4 1.1369 0.0114 0.0119 104.39 1.0442
acetate (2:1, v/v). 5 1.1390 0.0114 0.0113 99.12 1.0785
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 6 1.1634 0.0116 0.0123 106.03 1.0567
Average: 99.86 1.02
sample addition . . . . . . hydrocortisone topical hand cream in hexane:ethylacetate
RSD: 6.99 0.06
SPE tube/cartridge . . . . . . . . . . . . . Discovery DSC-Si, 500 mg/3 mL (52695-U) 1
Based on manufacturer's label description of 1% hydrocortisone (w/w)
condition . . . . . . . . . . . . . . . . . . . . . . 1 mL hexane:ethyl acetate (2:1, v/v)
washing . . . . . . . . . . . . . . . . . . . . . . . 1 mL hexane:ethyl acetate (2:1, v/v)
elution . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol:DI water (1:1, v/v)
eluate post-treatment . . . dilute eluate to 2 mL with methanol:DI water (1:1, v/v)
column . . . . Discovery HS C18, 15 cm x 4.6 mm I.D., 5 μm particles (568520-U)
mobile phase . . . . . . . . . . . . . . . . . . . (A) methanol; (B) DI water (1:1, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
Hydrocortisone
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm Ext. Standard
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Hydrocortisone
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003765 Sample
1.0 2.0 3.0
Min

HPLC Analysis of Mandelic Acid Enantiomers on O

*
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) OH
mobile phase . . . . . . . . . . . . . . . . . 0.1 wt% ammonium formate in methanol OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004610
0 2 4 6 8 10 12
Min.

HPLC Analysis of Paraben Preservatives on 1. Methyl paraben

Discovery® C18 1 2. Ethyl paraben
3. Propyl paraben
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 4. Butyl paraben
mobile phase . . . . . . . . . . . . . . . . . . (A) acetonitrile: (B) water, (30:70, A:B)
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 2 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001234
0 2 4 6 8 10 12
Min


2. Ethyl paraben
Discovery® C8 3. Propyl paraben
1
column . . . . . . . Discovery C8, 15 cm × 4.6 mm I.D., 5 μm particles (59353-U) 4. Butyl paraben
mobile phase . . . . . . . . . . . . . . . . . . (A) acetonitrile: (B) water, (30:70, A:B) 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001236
0 2 4 6 8 10
Min


1 2. Ethyl paraben
Discovery® RP-AmideC16 3. Propyl paraben
column . . Discovery RP-Amide C16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 4. Butyl paraben
mobile phase . . . . . . . . . . . . . . . . . . (A) acetonitrile: (B) water, (40:60, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 2 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001235
0 10 20
Min

1. Propyl gallate (10 µg/mL)

HPLC Analysis of Phenolic Antioxidants on 2. Ethoxyquin (10 µg/mL)
Gradient Program
Time (min) %A %B
Ascentis® C8 3. 2,4,5-trihydroxybutyrophenone (10 µg/mL)
4. tert-butylhydroquinone (10 µg/mL) 0 70 30
10 0 100
column . . . . . . . . Ascentis C8, 15 cm x 4.6 mm I.D., 5 μm particles (581424-U) 5. Nordihydroguaiaretic acid (10 µg/mL)
6. 3-tert-butyl-4-hydroxyanisole (10 µg/mL) 15 0 100
mobile phase . . . . . . . . . . (A) 5% acetic acid; (B) (50:50) methanol:acetonitrile 7. 2,6-di-tert-butyl-4-hydroxymethylphenol (10 µg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min 8. Octyl gallate (10 µg/mL)
9. Lauryl gallate (10 µg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 10. 3,5-di-tert-butyl-4-hydroxytoluene (10 µg/mL)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 3 8
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 1 9
sample . . . . . . . . . . . . . . . . . . . as indicated in 50:50 acetonitrile:2-propanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003054
5
6 10
4
7
2
0 2 4 6 8 10 12 14
Min

UHPLC Analysis of Kathon on Titan™ C18 Using MS 1 1. 2-methyl-4-isothiazolin-3-one

2. Methyl chloro isothiazolinone
Detection
column . . . . . . . . Titan C18 50 mm x 2.1 mm, I.D, 1.9 μm particles (577122-U) 2
mobile phase . . . . . . . . . . . . . . . (A) 0.1% formic acid in water (B):methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 μL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI (+) MRM TIC
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diode Array at 275 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . Diluted Kathon at 1 ug/ml in 95:5 0.1% formic acid in water::methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006148
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Min

HPLC, UHPLC or LC-MS Environmental
Gradient Program 1. Oxamyl (10 µg/mL) 8. Fluomethuron (10 µg/mL) 15. Swep (10 µg/mL)
2. Methomyl (10 µg/mL) 9. Carbaryl (10 µg/mL) 16. Mexacarbate (10 µg/mL)
Environmental Time (min) %A %B
0
4
85
75
15
25
3. Fenuron (10 µg/mL)
4. Monuron (10 µg/mL)
10. Diuron (10 µg/mL)
17. Chlorpropham (10 µg/mL)
18. Barban (10 µg/mL) 17
44 55 45 5. Aminocarb (10 µg/mL) 12. Siduron (10 µg/mL) 19. Neburon (10 µg/mL)
EPA Method 632: HPLC Analysis of Pesticides on 50
65
40
40
60
60
6. Propoxur (10 µg/mL)
18
15
Ascentis® C8 3

14
19
8
4
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . (A) DI water; (B) acetonitrile 1 9 13 16
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 5 7 12
2 11
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . as indicated in 16% acetonitrile in DI water 0 10 20 30 40 50 60

1. Oxamyl (10 µg/mL) 8. Fluomethuron (10 µg/mL) 15. Swep (10 µg/mL)
EPA Method 632: HPLC Analysis of Pesticides on 2. Methomyl (10 µg/mL) 9. Carbaryl (10 µg/mL) 16. Mexacarbate (10 µg/mL)
Gradient Program
Time (min) %A %B
3. Fenuron (10 µg/mL) 10. Diuron (10 µg/mL) 17. Chlorpropham (10 µg/mL) 0 90 10
Ascentis® C18 4. Monuron (10 µg/mL)
5. Aminocarb (10 µg/mL)
12. Siduron (10 µg/mL)
18. Barban (10 µg/mL)
19. Neburon (10 µg/mL)
13 50 50
23 40 60
column . . . . . . . Ascentis C18, 25 cm x 4.6 mm I.D., 5 μm particles (581325-U) 6. Propoxur (10 µg/mL)
9 29
34
10
10
90
90
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile
17
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 4 10 15
18
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 8
13 14
19
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm 5 7

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 1
6 12
sample . . . . . . . . . . . . . . . . . . . . . as indicated in 16% acetonitrile in water 11
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003194 2
0 10 20 30
Min

1. HMX (100 mg/L) 4 5. Nitrobenzene (100 mg/L)

EPA Method 8330: HPLC Analysis of 2. RDX (100 mg/L) 6. 2,4,6-Trinitrotoluene (100 mg/L)
Nitroaromatics and Nitramines, Mix A on 3. 1,3,5-Trinitrobenzene (100 mg/L) 3
4. 1,3-Dinitrobenzene (100 mg/L)
7. 2,4-Dinitrotoluene (100 mg/L)
8. 2-Amino-4,6-dinitrotoluene (100 mg/L)
Ascentis® C18 7
5 6
8
mobile phase . . . . . . . . . . . . . . . . . . . (A) water: (B) methanol; (50:50, A:B) 1 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
0 2 4 6 8 10 12 14 16 18
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . acetonitrile Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003278

EPA Method 8330: HPLC Analysis of 1. Tetryl (100 mg/L) 1

2. 2,6-Dinitrotoluene (100 mg/L)
Nitroaromatics and Nitroamines, Mix B on 3. 4-Amino-2,6-dinitrotoluene (100 mg/L)
4. 2-Nitrotoluene (100 mg/L)
2
3
Ascentis® C18 5. 4-Nitrotoluene (100 mg/L) 4 6
6. 3-Nitrotoluene (100 mg/L)
column . . . . . . . Ascentis C18, 25 cm x 2.1 mm I.D., 5 μm particles (581305-U) 5
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water;(B) methanol;(50:50, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
0 10 20
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in acetonitrile Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003285


4. 2,4-D 2.5 40
6. 2,4,5-TP 13.0 60
1 3
4
elution . . . . . . . . . . . . . . . . . . . . 10 mL 0.1% phosphoric acid in methylene
chloride:acetonitrile (80:20) at 5 mL/min Background
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0150

HPLC Analysis of Bisphenol A, Dimethylbisphenol 1

1. Bisphenol A
2. Dimethyl Bisphenol A
A and Nonylphenol on Ascentis® Express RP- 3. Nonylphenol
Amide (UV Detection)
2
This application demonstrates the suitability of Ascentis Express C18 for the
efficient separation of milk thistle related compounds 3
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 to 100% B in 10 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 0 2 4 6 8 10 12 14
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . 50 mg/L of each in 50:50, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005681

HPLC Analysis of Bisphenol A in Water on 1. Bisphenol A enantiomer 1

Ascentis® Express C18 after SPE using Supelclean™
ENVI-18
Bisphenol A is a contaminant that can leach into aqueous solutions from
polycarbonate plastics or metal cans; it may impact human development
and reproduction. This application demonstrates the extraction and analysis
of bisphenol A from water.
SPE tube/cartridge . . Supelclean ENVI-18, 500 mg/6 mL glass SPE tube (54331-U)
condition . . . . . . . . . . . 1 mL 1% formic acid in acetonitrile, then 1 mL water
sample . . . . . . . . . . . . . 5 mL spiked water blank, concentration of 0.2 μg/mL
elution . . . . . . . . . . . . . . . . . . . . . . . . 2 mL 1% formic acid in acetonitrile
eluate post-treatment . . . . . . 1 mL aliquot of eluent, and evaporate/reconstitute
to 0.5 mL in acetonitrile
1.0 2.0 3.0
Min
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3270 psi (256 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005530


Time (min)
0
%A
95
%B
5
Analyte
Atenolol
Rt (min.)
3.0
Q1/Q3
267.2/145
2 90 10
Carazolol
Metoprolol
6.2
5.6
299.1/194.2
268.2/133
6 50 50
Timolol 5.5 317.2/188.1
cps
8000
Metoprolol
0.1
0.1
0.01
0.01
Timolol
0.1
0.1
0.01
0.01
4000
washing . . . 3 x 1 mL DI water; 1 mL acetonitrile; 1 mL 60% acetonitrile/40% DI Salbutamol 0.1 0.01
water; apply 2 min of full vacuum to dry the tube after each step Tulobuterol 0.005 0.0005
elution . . . . . . . . . . . . . . . . . . . . . . 2 x 1 mL 1% formic acid in acetonitrile
column . . . . . . . . . . . . . . . . . . . . . . C18, 5 cm x 3 mm I.D., 3 μm particles 0
0 2 4 6
Rt (min)
eluate post-treatment . . . . . . . . evaporate under nitrogen and reconstitute with
150 μL 5% acetonitrile in 10 mM ammonium acetate, Note: Clenbuterol and Tulobuterol were spiked at the levels of 0.1 ng/mL.
pH 4.6 prior to LC-MS-MS analysis

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004059

HPLC Analysis of Carbamate and Urea Pesticides 1. Methomyl (80 ng) 9. Diuron (80 ng)
2. Oxamyl (80 ng) 10. Propham (80 ng)
on SUPELCOSIL™ LC-8 3. Fenuron (80 ng) 11. Siduron (80 ng)
column . . . . . SUPELCOSIL LC-8, 15 cm × 4.6 mm I.D., 5 μm particles (58220-U) 4. Monuron (80 ng) 12. Linuron (80 ng)
5. Carbofuran (1000 ng) 13. Chlorpropham (80 ng)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) water 6. Propoxur (800 ng) 14. Barban (80 ng)
gradient . . . . . . . . . . . . . . . . . . . . . . 18:82 to 65:35 in 9 min, hold 3 min 7. Carbaryl (200 ng) 15. Neburon (80 ng)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 8. Fluometuron (80 ng)
12
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
5 13
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 2 3 14
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0803 7
6 9 11
1 8 15
10
0 2 4 6 8 10 12
Min

HPLC Analysis of Carbamate and Urea Pesticides

4
on SUPELCOSIL™ LC-8 after SPME using 60 μm 5
PDMS/DVB Fiber 6
3
sample/matrix . . . . 3 mL water containing 8ng/mL of each analyte in 10% NaCl 1 1. Propham
2
SPME fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . PDMS/DVB, 60 μm (57317) 2. Siduron
3. Linuron
extraction . . . . . . . . . . . . . . . . . . . . . . . . immersion, 40 min, rapid stirring 4. Chlorpropham
desorption process . . . . . . . . . . . . . static, 5 min in acetonitrile:water (65:35); 5. Barban
dynamic, valve open during run 6. Neburon
column . . . . . SUPELCOSIL LC-8, 15 cm × 4.6 mm I.D., 5 μm particles (58220-U)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) water
gradient . . . . . . . . . . . . . . . . . . . . . . 18:82 to 65:35 in 9 min, hold 3 min 2 4 6 8
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797-0049

HPLC Analysis of Metabolites of 1. 2-OH-DMBA(t-diol) 11. 7-OHM-12-MBA-4-ol

7,12-Dimethylbenz[a]anthracene on 3. 7-M-12-OHMBA-(t-8,9-diol) 13. 7-M-12-OHMBA-2-ol
5. 7-OHM-(t-3,4-diol) 15. 7-M-12-OHMBA
10. 7-M-12-OHMNA-4-ol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1043 6 11 14 16 18 19
1
8 10
5 9
12 17
4 13
2 15
3
7
0 4 8 12 16 20 24 28 32 36 40 44 48
Min

6 1011 12 1314
HPLC Analysis of Nonvolatile Pesticides in Water 1
2
5 7 15
on SUPELCOSIL™ LC-18-DB after SPE using

Supelclean™ ENVI-Carb™ 8
sample/matrix . . . . . . . . . . . . . . . water spiked with pesticides at 10-50 μg/L

3
SPE tube/cartridge . . . . . . . . . . . . Supelclean ENVI-Carb, 250 mg/3 mL (57088)
9
condition . . . . . . . . 5 mL methylene chloride:methanol (80:20); 1 mL methanol;
10 mL 2% acetic acid in water (keep bed moist until sample addition)
sample addition . . . . . . . . . . . . . . . . . . . . . . . 100 mL sample at 5 mL/min Standard
Analyte % Recovery (± RSD, n = 5)
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 minute with vacuum
1. Oxamyl 111 ± 9.6
elution . . . . . 0.8 mL methanol; 2 x 35 mL methylene chloride:methanol (80:20) 2. Methomyl 105 ± 5.0
3. N-1-Naphthylthiourea —
eluate post-treatment . . . . . . dry eluate to 500 μL under gentle nitrogen stream, 4. Aldicarb 92 ± 0.8
reconstitute to 1 mL with methanol 5. Simazine 91 ± 6.5
6. Monuron 99 ± 3.2
column . . SUPELCOSIL LC-18-DB, 25 cm × 4.6 mm I.D., 5 μm particles (58355-U) 7. Cyanazine 90 ± 5.4
8. Metribuzin 97 ± 3.9
mobile phase . . . . . . . . . . . . . . (A) water:acetonitrile (90:10), (B) acetonitrile; 9. Carbofuran 106 ± 6.2
10. Atrazine 89 ± 5.7
gradient 80% A for 5 min then to 30% A over 30 min 11. Carbaryl 97 ± 3.5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 12. Diuron 88 ± 5.7
13. Propham 95 ± 3.2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 14. Propachlor 96 ± 3.8
15. Linuron 88 ± 5.4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 0 4 8 12 16 20 24 28
Blank
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85-438 Min

HPLC Analysis of Organo-acid Pesticides on 2 1. Solvent

2. Dalapon
Discovery® C18 3. 2,4-D
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 4. 2,4-DB
5. 2,4,5-T
mobile phase . . . . . . . . . . . . . . . . (A) 25 mM potassium phosphate, (pH 2.3): 1 6. 2,4-D Methyl ester
(B) acetonitrile; (60:40, A:B) 3 4 7. 2,4,5-T Methyl ester
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 8. 2,4-DB Methyl ester
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 6 7
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 8
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001042
0 10 20
Min

HPLC Analysis of Organo-acid Pesticides on 2 1. Solvent

2. Dalapon
Discovery® C8 3. 2,4-D
4. 2,4-DB
column . . . . . . . Discovery C8, 15 cm × 4.6 mm I.D., 5 μm particles (59353-U) 5. 2,4,5-T
mobile phase . . . . . . . . . . . . . . . . (A) 25 mM potassium phosphate, (pH 2.3): 4 6. 2,4-D Methyl ester
(B) acetonitrile; (60:40, A:B) 3 7. 2,4,5-T Methyl ester
8. 2,4-DB Methyl ester
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 7
6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 1 5 8
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001041
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Organophosphorus Pesticides on 1 1. Dichlorvos

Discovery® Cyano 2. Ethoprophos
3. Guthion
column . . . . . Discovery Cyano, 15 cm × 4.6 mm I.D., 5 μm particles (59356-U) 4. Methyl parathion
mobile phase . . . . . . . . . . . . . . . . . . (A) water: (B) acetonitrile, (75:25, A:B) 5. Disulfoton
6. Fenchlorvos
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 6 7. Chlorpyrifos
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 8. Prothiophos
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001030 3
5
2 4
7 8
0 10 20 30 40
Min

HPLC Analysis of Organophosphorus Pesticides on 6

1. Dichlorvos
Discovery® RP-Amide C16 2. Guthion
3. Methyl parathion
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 4. Ethoprophos
5. Disulfoton
mobile phase . . . . . . . . . . . . . . . . . . (A) acetonitrile: (B) water; (60:40, A:B) 6. Fenchlorvos
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 7. Chlorpyrifos
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 8. Prothiophos
1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001031 2
5
3
4
8
7
0 10 20
Min

HPLC Analysis of PAHs in Water on SUPELCOSIL™ 1. Naphthalene 1,000 ng/mL Gradient Program
LC-PAH after SPME using 100 μm PDMS Fiber 2.
3.
Acenaphthylene 2,000 ng/mL
Acenaphthene 1,000 ng/mL
Time (min) % ACN
4. Fluorene 200 ng/mL 0 50
sample/matrix . . . . . . . . . . . . 5 μL PAH mix (Cat. No. 4-8743) in 5 mL water 5 50
5. Phenanthrene 100 ng/mL
SPME fiber . . . . . . . . . . . . . . . . . . . . polydimethylsiloxane, 100 μm (57301) 6. Anthracene 100 ng/mL 30 100
extraction . . . . . . . . . . . . . . . . . . . . . . . immersion, 30 min (rapid stirring) 7. Fluoranthene 200 ng/mL 45 100
8. Pyrene 100 ng/mL Flow increased at 2.0 min
desorption process . . . . . . . . . . . static, 200 μL acetonitrile:water, 40:60, 2 min
9. Benzo(a)anthracene 100 ng/mL
column . . . . SUPELCOSIL LC-PAH, 15 cm × 4.6 mm I.D., 5 μm particles (58318) 10. Chrysene 100 ng/mL
gradient . . 0 min - 50% A; 5 min - 50% A; 30 min - 100% A; 45 min - 100% A 11. Benzo(b)fluoranthene 200 ng/mL
12. Benzo(k)fluoranthene 100 ng/mL
flow rate . . . . . . . . . . . . . . . . . 0-2 min: 0.2 mL/min 2-45 min: 1.0 mL/min
13. Benzo(a)pyrene 100 ng/mL
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile:(B) water 14. Dibenzo(a,h)anthracene 200 ng/mL
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 15. Benzo(ghi)perylene 200 ng/mL
16. Indeno(1,2,3 cd)pyrene 100 ng/mL
Indeno(1,2,3-cd)pyrene
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0086
6 11
5
4
2 10
7
9 13
1 3 8 12
16
15
14
0 10 20 30 40
Min

HPLC Analysis of PAHs on SUPELCOSIL™ LC-PAH 1. Naphthalene 7. Fluoranthene

PAHs are traditionally evaluated using an acetonitrile/water gradient. 2. Acenaphthalene 8. Pyrene
Recently customers are looking for alternatives to this method using a 3. Acenaphthene 9. Benzo(a)anthracene
methanol/water gradient due to the risk of an another acetonitrile shortage, 4. Fluorene 10. Chrysene
solvent costs, and toxicity to the environment. This analysis demostrates the 5. Phenanthrene 11. Benzo(b)fluoranthene
separation using a methanol gradient. 6. Anthracene 12. Benzo(k)fluoranthene
13. Benzo(a)pryrene
column . . . . . SUPELCOSIL LC-PAH, 15 cm x 4.6 mm I.D., 5 μm particles (58318) 2 14. Benzo(g,h,i)perylene
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water, (B) methanol 15. Dibenzo(a,h)anthracene
gradient . . . . . . . . . . . . . 55 to 100% B in 5 min; held at 100%B for 10 min 16. Indeno(1,2,3-cd)pyrene
56
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 10 11
7
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EPA 610 mix 9
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005559 3
8
12 13
14 15 16
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Paraquat and Diquat on 1

SUPELCOSIL™ LC-18 after SPE using ENVI™-8 DSK
2
Refer to US EPA Method 549.1 for full details
Sample Pre-treatment: 1. Diquat
250 mL drinking water. Adjust water sample to pH to 10.5 ± 0.2 with 10% 2. Paraquat
sodium hydroxide or 10% hydrochloric acid
sample/matrix . . . . . . . . . . . . . . . . . . . drinking water adjusted to pH to 10.
sample preparation . . . . . . . . . . . . . . . . . . . . . ENVI-8 DSK, 47 mm (57172)
SPE tube/cartridge . . . . . . . . . . . . . . . . ENVI-8 DSK SPE Disk, 47 mm (57172)
condition . . . . 10 mL acetonitrile; 2 × 10 mL reagent water; 10 mL conditioning
solvent A (5 g cetyl trimethyl ammonium bromide and
5 mL conc. ammonium hydroxide in 500 mL DI water, dilute to 1 L);
( 2 × 10 mL reagent water; 10 mL conditioning
solvent B (10 g hexanesulfonic acid, sodium salt and
10 mL conc. ammonium hydroxide in 250 mL DI water, dilute to 500 mL))
sample addition . . . . . . . . . . . . . . . . . . . . . . . . 250 mL water 100 mL/min
elution . . 0.5-1.0 mL acetonitrile (to cover/solvate disk); 2 × 4 mL eluting solution
(13.5 mL orthophosphoric acid and 10.3 mL diethylamine
in 500 mL DI water, dilute to 1 L)
mobile phase . . 3.5 mL triethylamine and 1.0 g 1-hexane-sulfonic acid, sodium salt to
800 mL DI H 2O add orthophosphoric acid to pH 2.5, dilute to 1 L
0 2 4 6 8
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . photodiode array, diquat - 308 nm, paraquat - 257 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0715

2 A. Acetone
HPLC Analysis of Pesticides on Ascentis® C8 Gradient Program
1. Vacor (10 µg/mL)
Time (min) %A %B
(15 cm x 4.6 mm x 5 μm) A
0 65 35 2. Propanil (10 µg/mL)
13 25 75 3. Napropamide (10 µg/mL)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . (A) DI water; (B) acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL 0 2 4 6 8 10 12 14 16 18
Min
sample . . . . . . . . . . as indicated in 2.2% 90:10 acetonitrile:acetone in DI water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003121

HPLC Analysis of Phenyl Urea Pesticides on 1. Fenuron

Ascentis® Express C18 Phase 2. Monuron
3. Fluomethuron
This separation illustrates the use of the highly efficient 2.7 um Ascentis 4. Isoproturon
Express Fused-Core C18 stationary phase in the analysis of some popular 5 5. Diuron
herbicides. The short run times allow analyses using isocratic conditions so 6. Siduron A
that column equilibration time is not required between runs. 3
7. Siduron B
Response Time: 0.02 sec. 2 8. Linuron
Flow Cell: 2.5 μL semi-micro 4 9. Neburon
8
column . . Ascentis® Express C18, 10 cm x 4.6 mm I.D., 2.7 μm particles (53827-U) 1
mobile phase . . . . . (A) 0.025 M potassium phosphate buffer, adj. to pH = 2.5, 6 9
7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 bar
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV 245 nm, VWD Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006189

HPLC Analysis of Triazine Herbicides in Grass on Recovery (± SD, n = 3)

SUPELCOSIL™ LC-8-DB after SPE using 102.0 ± 2.9
102.2 ± 1.6
Supelclean™ LC-SCX 100.5 ± 1.8
Sample Pre-treatment:
5 g fresh grass clippings spiked with 2 ppm each herbicide. Add 4 g 1. Simazine
anhydrous sodium sulfate and 20 mL methylene chloride:acetone (80:20). 1 2. Atrazine
Shake 20 min and allow mixture to stand 1 min. 2 3. Propazine
3
sample/matrix . . . . . . . . . fresh grass clippings spiked with herbicides at 2 ppm
SPE tube/cartridge . . . . . . . . . . . . . Supelclean LC-SCX, 500 mg/3 mL (57018)
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methylene chloride
sample addition . . . . . . . . 2 mL grass extract. Wash with 2 x 2 mL acetonitrile.
0 2 4 6
drying . . . . . . . . . . . . . . . . . . . Dry packing for 5 min. under nitrogen purge
Min
washing . . Dry packing for 5 min under nitrogen purge; wash with 2 x 2 mL DI water
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL methanol
eluate post-treatment . . . . . . . . . . . . . . . . . . . dilute to 2 mL with DI water
column . . . . . . . . . . . . . . . . . . . SUPELCOSIL LC-8-DB, 15 cm × 4.6 mm I.D.,
5 μm with guard column (58347)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84-109

HPLC Analysis of Urea, Carbamate, Triazine, Gradient Program 1. Acetone

Time (min) %B 2. Oxamyl
Aniline Pesticides on Discovery® C18 15 3. Methomyl
0 85 4. Fenuron
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 2 85 5. Aldicarb
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) water 15 42.5 6. Simazine
11
17 42.5 7. Monuron
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 8. Cyanazin
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 9. Carbofuran
12 10. Atrazine
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm 11. Carbaryl
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 14 12. Diuron
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000948 13. Propachlor
6 16 14. Linuron
3 10 15. Chlorpropham
7 16. Barban
2
5 9
4 13
1 8
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Urea Pesticides on Discovery® 3

1. Fenuron
Cyano 2. Monuron
3. Diuron
column . . . . . Discovery Cyano, 15 cm × 4.6 mm I.D., 5 μm particles (59356-U) 4. Linuron
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001067 1
1.0 2.0 3.0

Min

LC-MS Analysis of Cyanuric Acid on Ascentis® 1 1. Cyanuric acid

Express HILIC 2. Ammelide

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 psi (22.6 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(-), SIR mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6 8 10 12
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005690

LC-MS Analysis of Pesticides on Ascentis® C18 Gradient Program 3 1. Thiamethoxam (M+H)+= 292.01
Time (min) %A %B 2. Imidacloprid (M+H)+=256.05
(5 cm x 2.1 mm x 3 μm) 0 90 10 3. Metalaxyl (M+H)+=280.14
10 20 80
mobile phase . . . . . (A) 0.1% ammonium acetate, pH unadjusted; (B) acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . MS, (+) ESI, Selected Ion Recording Mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . 1 μg/mL each in 90:10 water:acetonitrile
2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003130 1
0 2 4 6 8 10 12 14
Min

LC-MS Anaysis of Chloramphenicol in Shrimp on Chloramphenicol in

Shrimp Extract (0.3 ng/g)
Ascentis® Express C18 Using Z-Sep+ Cleanup
Internal Standard in
This application demonstrates the superior cleanup associated with Z-Sep+
using QuEChERS approach.
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 g shrimp
extraction . . . . . . . . . . . . . . . . . 9 mL acetonitrile, then 90 μL of formic acid
extraction process . . post treatment: 8 mL supernatant evaporated to dryness and
reconstituted with 1 mL water
clean-up tube . . . . . . . . . Supel™ Que Z-Sep+ tube, 12 mL, 500 mg (55296-U)
clean-up process . . . . . . . . . . . . . . . . . . . . . . . . . . clean-up volume: 9 mL 0 2 4
Min
column . . . . . . Ascentis Express C18, 5 cm X 2.1mm, 2.7 um particles (53822-U)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) Water; (B) Acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . 15% B for 0.1 min, 15-80% in 1.9 min,
held at 80% for 1.5 min, 80-15% in 0.5 min, at 15% for 3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ambient
detector . . . . . . . . . . . . . . . MS, ESI(+), MRM, m/z 320.9/151.9, 325.9/157.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006249


Detection
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 μL/min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006148
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Min

HPLC, UHPLC or LC-MS Flavors and Fragrances
1. Methyl lactate
Flavors and Fragrances 2. β-Butyrolactone
3. α-Phenylethanol
4. Carvone
GC Analysis of Volatile Flavor Compound 5. α-Ionone 4
Enantiomers on Astec CHIRALDEX® G-TA 6.
7.
Methyl mandelate
δ-Octanolactone
column . . . . Astec CHIRALDEX G-TA, 30 m x 0.25 mm I.D., 0.12 μm (73033AST)
oven . . . . . . . . . . . . . . . . . . . . . . 60 °C, 2 °C/ min to 160 °C, hold 20 min 3
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 250 °C 5
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . helium, 30 cm/sec, set at 75 °C 7
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL, split 100:1
6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006117
2
0 10 20 30 40
Min

HPLC Analysis of 2-Phenylpropionic Acid

Enantiomers on Astec® CYCLOBOND® I 2000 HO O
HP-RSP
column . . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D., * CH 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004717
0 2 4 6 8 10
Min

HPLC Analysis of Triacylglycerols (TAG) of Borago

Officinalis Oil on Ascentis® Express C18
OLO + C22:1LγLn
mobile phase . . . . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) isopropyl alcohol
gradient . . . . . . . 0% B held for 50 min; to 70% B in 5 min; to 0% B in 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min SLO + C24:1LγLn
detector . . . . . . . . . . . . . . . MS, APCI(+); mass spectral range, 250-1200 m/z;
interval, 0.5 s; scan speed, 2143 amu/s; nebulizing gas (N2) flow, 4.0 L/min;
interface temperature, 400 °C; (heat block, 230 °C;
desolvation line (DL) temperature, 250 °C; DL voltage, 34 V;
probe voltage, +4.5 kV; Qarray voltage, 1.0 V;
RF voltage, 90 V; detection gain, 1.05 kV)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
30 40 50
sample . . . . . . . . . . . . . Borago Officinalis, 40 mg diluted in 10 mL of hexane

HPLC, UHPLC or LC-MS Food and Beverages
Food and Beverages Efficiency of Recovery

Concentration (µg/mL) %Recovery ± RSD (n=3)
HPLC Analysis of Acrylamide in Fried Potato Chips 10 78 ± 7%
on Discovery® HS F5 after SPE using Discovery® 55 93 ± 5%
DSC-MCAX and DSC-18
sample/matrix . . Finely grind 2.0 g of potato chips, place in 20 mL vial and add
10 mL water. Mix using vortex until mixture forms a thick paste. Spiked Sample (38µg/kg) H2C
(Place sample in centrifuge tubes and centrifuge at 16.1 rcf for 5 min.
Extract aqueous portion of sample leaving oil layer and solids.)
SPE tube/cartridge . . . . . . . . . . Discovery DSC-MCAX, 300 mg/3 mL (52784-U)
SPE tube/cartridge . . . . . . . . . . . . . . . . Discovery DSC-18, 1g/6mL (52606-U)
condition . . connect a Discovery DSC-MCAX SPE tube to the top of a Discovery DSC-18 O NH2
SPE tube using an SPE tube adapter, pass 1 mL methanol,
then 1 mL DI water through both SPE tubes until the tubes are dry
Acrylamide
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL of aqueous extract
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL DI water
elution . . . . . . . . . . . . . removing upper MCAX SPE tube, pass 2 mL methanol
through lower DSC-18 SPE tube
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100% ultra-pure water Control Standard (55µg/kg)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003759 0 2 4 6 8 10 12 14
Min

HPLC Analysis of Aflatoxin Analogs on 80
Ascentis® C18 B1
column . . . . . . Ascentis C18, 15 cm × 4.6 mm I.D., 3 μm particles (581322-U) 60 B2
FLD Response (LU)

G1
mobile phase . . . . . . (A) acetonitrile: (B) methanol: (C) water; (20:20:60, A:B:C)
G2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
40
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C or 50 °C
detector . . . . . . . . . . . . . . . fluorescence, 360 nm excitation, 455 nm emission
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 20
sample . . . . . . . . . . . . . . . . . Aflatoxin Standard Mix, diluted 1:1 with water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004166
0
0 10 20
Min

HPLC Analysis of Aflatoxins B1, B2, G1 and G2 in 1. Aflatoxin G1

Corn on Ascentis® Express C18 after SPE using 2. Aflatoxin B1
2 3. Aflatoxin G2
Supel™ Tox AflaZea and Fluorescence Detection 4. Aflatoxin B2
Aflatoxins are closely related compounds produced as secondary metablo-
lites of fungi. They are found in grains and nuts like corn, wheat or peanuts.
Shown here is a rapid, sensitive method to extract these compounds from
corn using SPE cartridges specifically designed for aflatoxins, Supel Tox
1 4
AflaZea (aflatoxin zearalenone), which removes interferences from grains,
feeds, TMR samples, peanuts, peanut products and aqueous solutions. 3
Analysis was via HPLC on Ascentis Express Fused-Core C18 colums.
sample/matrix . . . . . . . . . 25 g corn meal in 100 mL acetonitrile:water (84:16) 0 2 4 6 8 10 12
SPE tube/cartridge . . . . . . . . . . . . . . . . Supel™ Tox AflaZea, 6 mL (55314-U) Min
elution . . . . . . . . . . . . . . . . . . . . collect sample in 5 mL silanized glass vial
eluate post-treatment . . . . transfer 200 μL sample, add 300 μL TFA derivatization
solution (70:20:10 water:TFA:acetic acid), heat in microreactor
20 min at 65 °C, cool, add 580 μL deionized water, vortex
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.350 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 psi (22.4 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . FL, Ex 360 nm, Em 440 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005922

HPLC Analysis of Aflatoxins in Wheat on Ascentis® 1. Aflatoxin G2

Express C18 after SPE using Supel™ Tox AflaZea 2. Aflatoxin G1
3. Aflatoxin B2 4
Sample/matrix: 4. Aflatoxin B1
Weigh 25 grams of wheat meal into a 500 mL Erlenmeyer flask. Add 100 mL
of 84:16 acetonitrile:water, seal, and place on an orbital shaker at med-high 2
speed for 90 minutes. Filter the sample through a #2 qualitative filter. Spike
sample at 2 mg/mL using analytical standards. 3
sample/matrix . . . . . . . . . . . . 25 g wheat meal spiked with pesticide standards
SPE tube/cartridge . . . . . . . . . . . . . . . . . Supel™Tox AflaZea, 6 mL (55314-U)
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL 0 2 4 6 8 10 12 14
elution . . . . . . . . . . . . . . . . . . collect eluate in 5 mL silanized culture tubes
Min
eluate post-treatment . . . . . . . combine 200 μL of sample eluate and 880 μL of
DI water in a 2 mL autosampler vial
mobile phase . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (C) acetonitrile;
(720:120:120, A:B:C) containing 0.780 g/L potassium bromide,
230 μL/L nitric acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.400 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4568 psi (315 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FLD 360 ex; 440 em
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
derivatization . . . . . . . . . . . . . . . . . . . via KOBRA electrochemical cell, 50 μA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005907

HPLC Analysis of Aflatoxins on Ascentis® 1. Aflatoxin G2 4

Express C18 2. Aflatoxin G1
3. Aflatoxin B2
Aflatoxins are a complex of closely related toxins produced as secondary 4. Aflatoxin B1
metablolites of fungi. They are found in grains and nuts such as corn, wheat, 2
or peanuts. They are produced by aspergillus flavus and a. Parasiticus.
Members of the group include aflatoxin b1, aflatoxin b2, aflatoxin g1, 3
aflatoxin g2, aflatoxin m1, and aflatoxin m2.
mobile phase . . . . (A) water; (B) methanol; (C) acetonitrile; (720:120:120, A:B:C)
containing 0.780 g/L potassium bromide, 230 μL/L nitric acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.400 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4568 psi (315 bar) 0 2 4 6 8 10 12 14
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C Min
detector . . . . . . . . . . . . . . . . . FLD excitation = 360 nm, emission = 440 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . 200 μL spiked 84:16 acetonitrile:di water in 880 μL di water,
Spike concentrations: B1 = 16 ppb; B2 = 4 ppb;
G1 = 16 ppb; B2 = 4 ppb
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005908

HPLC Analysis of Aflatoxins on Ascentis® Express 1. Aflatoxin G1

C18 with Fluorescence Detection 2. Aflatoxin B1
3. Aflatoxin G2
column . . Ascentis Express C18, 15 cm x 4.6 mm I.D., 2.7 μm particles (53829-U) 4. Aflatoxin B2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6381 psi (440 bar) 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3 4
detector . . . . . . . . . . . . . . . . . . . . . . Fluorescence, Ex 360 nm, Em 440 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μL 0 2 4 6 8
sample . . . . . . . 200 μL aflatoxin standard in acetonitrile; 0.5 μg/mL B2 and G2;
Min
2 μg/mL B1 and G1; 800 μL delivatization solution: 70% water,
20% TFA and 10% acetic acid
heat for 9 min at 65 °C
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005662

4 1. Deoxynivalenol
HPLC Analysis of Aflatoxins on Ascentis® Express 6 2. Aflatoxin M1
C18 3. Aflatoxin G2
4. Aflatoxin G1
This application demonstrates the suitability of Ascentis Express C18 for the 5. Aflatoxin B2
2 6. Aflatoxin B1
efficient separation of various toxins. 7. Fumonisin B1
column . . Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.7 μm particles (53823-U) 8. Fumonisin B2
9. Zearalenone
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate, 10. Ochratoxin A
pH 2.5 (titrated with formic acid); (B) methanol
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 to 100% B in 10 min 10
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 3 5 8
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4714 psi (325 bar) 1 9
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 7
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . APCI (+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 0 2 4 6 8 10 12
sample . . . . . . . . . . . . . . . . . . . . 0.15 - 30 mg/L in 90:10, water:acetonitrile Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005441

HPLC Analysis of an Energy Drink on Ascentis® Caffeine

O
CH3
Niacinamide (Vitamin B3) Pyridoxine hydrochloride (Vitamin B6) Benzoic acid
O
N H3C N
Express HILIC, 10 cm (UV Detection) H3C
N
N
NH2 OH
column . . . Ascentis Express HILIC, 5 cm x 3 mm I.D., 2.7 μm particles (53967-U) O N N HO
mobile phase . . (A) 100 mM ammonium acetate, pH 5 (titrated with acetic acid); CH3
O OH
OH
(B) water; (C) acetonitrile; (9:1:90, A:B:C:) 8
1. Unknown
Asorbic acid O OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 2. Unknown
3. Unknown
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C H3C OH HO
OH 4. Caffeine
5. Niacinamide (Vitamin B3)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm Riboflavin (Vitamin B2) 6. Pyridoxine hydrochloride (Vitamin B6)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL OH 7. Benzoic acid
H3C N N O 8. Asorbic acid
sample . . . . . . . . . . . . . . . . . . . . . . Energy Drink Diluted 1:9 in acetonitrile 4 9. Riboflavin (Vitamin B2)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005387 N

NH
H3C
5
123 67 9 O
0 2 4 6 8 10
Min

HPLC Analysis of an Energy Drink on Ascentis® HO

1. Fructose
OH
2. Glucose
OH
3. Sucrose
Express HILIC (ELSD Detection) O OH HO OH

H HO H O H O
column . . . Ascentis Express HILIC, 5 cm x 3 mm I.D., 2.7 μm particles (53967-U) H OH
H O H H
H HO
2 H OH H
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 100 mM ammonium acetate, OH H
1 OH H
HO O OH
pH 5 (titrated with acetic acid) (B) water (C) acetonitrile; HO OH
OH H
H OH
(9:1:90, A:B:C:) H OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 4. Taurine

O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C S
OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD, 55° C, 3.5 bar nitrogen H2N

O
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 4
sample . . . . . . . . . . . . . . . . . . . . . . Energy Drink Diluted 1:9 in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005388 0 2 4 6 8 10
Min

HPLC Analysis of an Energy Drink on Ascentis® 1. Caffeine

Express HILIC (UV Detection) 2. Niacinamide (Vitamin B3)
3. Pyridoxine hydrochloride (Vitamin B6)
This application demonstrates the suitability of Ascentis Express HILIC for the 4. Benzoic acid
efficient separation of common drink additives used in caffeinated energy 5. Sorbic acid
drinks. The analysis was performed with UVand ELSD detectors in series, this 6. Riboflavin (Vitamin B2)
chromatogram being from the UV.
mobile phase . . . . . . . (A) 100 mM ammonium acetate, pH 5.0 with acetic acid; 1 5
(B) water; (C) acetonitrile; (09:01:90, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812 psi (56 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 2
4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
3 6
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dilute 1:9 in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005753
1.0 2.0
Min

OH
HPLC Analysis of an Energy Drink on Ascentis® 1. Niacinamide (Vitamin B3) 2. Pyridoxine hydrochloride
(Vitamin B6)
3. Caffeine 4. Riboflavin (Vitamin B2)
O
Express RP-Amide (UV detection) N
H3C N
H3C
CH3 HO
OH
N
N
This application demonstrates the suitability of Ascentis Express RP-Amide NH2 5 OH
for the efficient separation of common drink additives used in caffeinated HO
O N N H3C N N O
O
energy drinks. OH
OH CH3 NH
column . . Ascentis Express RP-Amide, 5 cm x 3 mm I.D., 2.7 μm particles (53916-U) H3C N
mobile phase . . . . . . . (A) water with 0.1% TFA; (B) acetonitrile with 0.1% TFA O
5. Asorbic acid O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 3
6. Benzoic acid
O
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146 psi (79 bar) H3C OH

1 OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4 6
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6
Min
sample . . . . . . . . . . . . . . . . . . . . . . Energy Drink Diluted 1:9 in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005389

HPLC Analysis of Anthocyanins in Red Wine on 180

mAU
518nm, 4nm (1.00)
5
Ascentis® C18 170
160
Gradient Program
column . . . . . . Ascentis C18, 25 cm × 4.6 mm I.D., 5 μm particles (581325-U) 150 1. Delphinidin 3-glucoside Time (min) %B
2. Cyanidin 3-glucoside 0-4 2
mobile phase . . (A) water:formic acid (9:1), (B) acetonitrile:water:formic acid (5:4:1) 140 3. Petunidin 3-glucoside 5-6 2-12
4. Peonidin 3-glucoside 7-20 12-15
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 130 5. Malvidin-3-glucoside 21-25 26-90
6. Delphinidin 3-(6”-acetyl)-glucoside 26-30 90
120
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 7. Cyanidin 3-(6”-acetyl)-glucoside 31-35 2
110 8. Petunidin 3-(6”-acetyl)-glucoside
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIS, 518 nm 9. Peonidin 3-(6”-acetyl)-glucoside
100 10. Malvidin 3-(6”-acetyl)-glucoside
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 90
11. Malvidin 3-(6”-caffeoyl)-glucoside
12. Petunidin 3-(6”-cumaryl)-glucoside
sample . . . . . . . . . . . . . . . . . . . . . red wine diluted with water, centrifuged 80 13. Peonidin 3-(6”-cumaryl)-glucoside
and passed through a 0.45 μm filter 70

14. Malvidin 3-(6”- cumaryl)-glucoside
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003922 60

10
50
40
30
3
1
20 4 14
7
10 2 6 8 9
11 12 13
0
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5
Min

HPLC Analysis of Antioxidants on SUPELCOSIL™ Gradient Program 1. Propyl gallate

Time (min) %A %B 2. THBP
LC-18 3. TBHQ
0 70 30
4. NDGA
column . . . . SUPELCOSIL LC-18, 15 cm × 4.6 mm I.D., 5 μm particles (58230-U) 10 0 100 5. BHA
mobile phase . . (A) 5% acetic acid in deionized water; (B) acetonitrile:methanol (1:1) 20 70 30 6. Ionox 100
70% A/30% B to 100% B, linear gradient over 10 min, hold 10min 2 7. Octyl gallate
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 7 8. BHT
9. Dodecyl gallate
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . 10μL, 20 μg/mL each antioxidant
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795-0438 9
1
5
3 4
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Benzoate and Sorbate on 1. Sorbate 2

Ascentis® Express RP-Amide 2. Benzoate
This application demonstrates the suitability of Ascentis Express RP-Amide
for the efficient separation of sorbate and benzoate. 1
mobile phase . . . . . . . . . . . . . . (A) 10 mM ammonium phosphate monobasic,
pH 2.5 with phosphoric acid; (B) ethanol; (75:25, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1.0 2.0 3.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg/L each
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005672

HPLC Analysis of Beverage Additives in Diet Cola 1. Acesulfame

on Ascentis® Express C18 2. Caffeine
3. Benzoic acid
This application demonstrates the suitability of Ascentis Express C18 for the 2 4. Aspartame
efficient separation of common drink additives used in diet soda. 1
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 20 mM ammonium acetate, 3 4
pH 4.7 with acetic acid: (B) acetonitrile; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . 100 - 500 μg/mL in 95:5, buffer: acetonitrile 0 2 4 6

HPLC Analysis of Beverage Additives in Diet Cola 1. Acesulfame

2. Caffeine
on Ascentis® Express RP-Amide 3. Benzoic acid
This application demonstrates the suitability of Ascentis Express RP-Amide 2 4. Aspartame
1
for the efficient separation of common drink additives used in diet cola.
mobile phase . . . . . . . . (A) 20 mM ammonium acetate, pH 4.7 with acetic acid;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 1.0 2.0 3.0 4.0 5.0
sample . . . . . . . . . . . . . . . . . . 500 - 1000 μg/mL in 95:5, buffer: acetonitrile Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005678

HPLC Analysis of Bisphenol A, Dimethylbisphenol 1

1. Bisphenol A
2. Dimethyl Bisphenol A
A and Nonylphenol on Ascentis® Express 3. Nonylphenol
RP-Amide (UV Detection)
2
efficient separation of milk thistle related compounds 3
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 to 100% B in 10 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 0 2 4 6 8 10 12 14
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . 50 mg/L of each in 50:50, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005681

HPLC Analysis of Caffeine and Taurine on 1 1. Caffeine

Ascentis® Express HILIC Column using Evaporative 2. Taurine
Light Scattering Detection (ELSD)
This application demonstrates the suitability of Ascentis Express HILIC for the
efficient separation of caffeine and taurine. Elution is by aqueous normal
phase (ANP) as an increase in organic will increase retention.
mobile phase . . . . . . (A) 100 mM ammonium formate, pH 3.0 with formic acid;
(B) acetonitrile; (10:90, A:B) 1.0 2.0 3.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
Min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD, 55 °C, 3.5 bar nitrogen
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . 1000 μg/mL in 10:90, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005682

HPLC Analysis of Caffeine and Taurine on 1 1. Caffeine

2. Taurine
Ascentis® Express HILIC Column using Evaporative
Light Scattering Detection (ELSD, Fast Analysis)
efficient separation of caffeine and taurine. Elution is by aqueous normal
phase (ANP) as an increase in organic will increase retention.
2
mobile phase . . . . . . . (A) 100 mM ammoniumformate, pH 3.0 with formic acid;
(B) acetonitrile; (15:85, A:B) 1.0 2.0 3.0 4.0
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . 1000 μg/mL in 10:90, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005683

HPLC Analysis of Carotene Compounds on 1. Astaxanthin (M+H)+ = 597.38

Ascentis® RP-Amide 2. Xanthophyll (M+H)+ = 569.42
2
3. Zeaxanthin (M+H)+ = 569.42
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 1 4. Lycopene (M+H)+ = 537.43
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100% acetonitrile 5. α-carotene (M+H)+ = 537.43
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 6. β-carotene (M+H)+ = 537.43
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . MS, ESI(+) in selective ion monitoring (SIR) mode 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 6
sample . . . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL each in 100% acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003138 4
0 10 20
Min

100 3.82
HPLC Analysis of Chloramphenicol in Milk on 95
RT: 0.00 - 5.99
90 SupelMIP SPE Extract of

Ascentis® C18 after SPE using SupelMIP® SPE- 85 Chloramphenicol Spiked Milk
3.78
Chloramphenicol 80
• Average recovery of 84%
Relative Abundance
75
• Increased sample cleanliness
70
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 65 • Reduced background and
improved signal-to-noise
sample addition . . . . . . . . . whole pasteurized milk spiked with chloramphenicol 60
55
at 15 ng/mL and 38 ng/mL 50
SPE tube/cartridge . . SupelMIP SPE - Chloramphenicol, 25 mg/10mL (LRC) (53210-U) 45

40
condition . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol;1 mL DI water 1.03
35
30
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 25
washing . . . 2 x 1 mL MS-grade water, 1 mL 5% acetonitrile in 0.5% acetic acid, 20
15 3.67
2 x 1 mL MS-grade water, 1 mL 20% acetonitrile in 1% ammonium 10 1.68
hydroxide. (dry SPE tubes for 15 min. under gentle vacuum; 5 0.22 0.65 0.79 1.44 1.58 1.84
2.05
2.29 2.51 2.90 3.14 3.35 4.47 4.53 4.79 5.04 5.38 5.50 5.57
3 x 1 mL dichloromethane; dry SPE tubes for 1 min. under gentle vacuum) 0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
elution . . . . elute chloramphenicol with 2 x 1 mL methanol:acetic acid:MS-grade 100
RT: 0.00 - 5.99 1.99 3.81
water (89:1:10, v/v/v) 95
Conventional SPE Extract of
eluate post-treatment . . . . evaporate combined eluate to dryness at 50 °C under 90
Chloramphenicol Spiked Milk
85
nitrogen. reconstitute in 150 mL LC mobile phase prior to LC-MS analysis 80 • Average recovery of 79%
column . . . . . . . Ascentis C18, 2.1 mm x 10 cm I.D., 3 μm particles (581301-U) 75 1.97
3.75 • Higher background and
Relative Abundance
70 reduced signal-to-noise
mobile phase . . . . . . . . . (A) 100 mM ammonium acetate: (B) MS-grade water: 65 1.94 • Complete sample prep
(C) acetonitrile (10:60:30, A:B:C) 60 procedure involved protein
0.99
55 precipitation followed by SPE
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 50 cleanup and three LLE Steps
45
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 40
1.09 3.71
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(-), 320-323 m/z range 35
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
30 2.07
25 1.91
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004433 20 1.81
1.32 2.19 2.77
15 1.77 4.64 4.68
1.38 2.43 2.67 5.00 5.12
10
0.16 2.91 3.31 3.59 4.23 4.36 5.28 5.81
5 0.44 0.79 3.47
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
Min

HPLC Analysis of Diet Soda Additives on Ascentis® 5
Express C18
1. Acesulfame
2. Benzoic acid 6
This application demonstrates the suitability of Ascentis Express C18 for the 4
3. Sorbic acid
efficient separation of common drink additives used in diet cola. 4. Caffeine
5. Aspartame
column . . Ascentis Express C18, 3 cm x 4.6 mm I.D., 2.7 μm particles (53818-U) 6. Neotame
mobile phase . . . . . . . . . 20% A constant; 75 to 20% B, 5 to 60% C in 1 min;
held at 20% B, 60% C for 0.1 min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3191 psi (220 bar)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min 1 2
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . (Diet Soda 100 - 500 μg/mL in buffer)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005392
0.2 0.4 0.6 0.8 1.0
Min

HPLC Analysis of Diet Soda Additives on Ascentis® 1. Acesulfame

6
2. Benzoic acid
Express RP-Amide 3. Sorbic acid
5
4. Caffeine 4
This application demonstrates the suitability of Ascentis Express RP-Amide 5. Aspartame
6. Neotame
for the efficient separation of common drink additives used in diet cola.
mobile phase . . . . . . . . (A) 20 mM ammonium acetate, pH 5.6 with acetic acid; 2
1
(B) water; (C) acetonitrile 3
gradient . . . . . . . . . . . . 20% A constant; 75 to 20% B, 5 to 60% C in 1 min;
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3336 psi (230 bar)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 0.2 0.4 0.6 0.8 1.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . Diet Soda 100 - 500 μg/mL in buffer
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005393

HPLC Analysis of Diet Soda Spiked with Neotame 2. Caffeine

2
3
3. Neotame
on Ascentis® Express C18 1. Benzoic acid O CH3 O

O H3C N
This application demonstrates the suitability of Ascentis Express C18 for the N HO
NH
O
CH3
4. Aspartame
efficient separation of common drink additives used in diet soda. OH N O NH O CH3

O N
column . . Ascentis Express C18, 3 cm x 4.6 mm I.D., 2.7 μm particles (53818-U) CH3 H3C O
O O
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 100 mM ammonium acetate, 1

HO
H3C NH
pH 5.6 (titrated with acetic acid); (B) water; (C) acetonitrile CH3
O NH2
held at 20% B, 60% C for 0.1 min 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min

pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3191 psi (220 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm 0.2 0.4 0.6 0.8 1.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . neotame 350 μg/mL in diet cola

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005390

HPLC Analysis of Diet Soda Spiked with Neotame 2. Caffeine 2

3. Aspartame
on Ascentis® Express RP-Amide 1. Benzoic acid O CH3 3 O

4. Neotame
O H3C N HO O
This application demonstrates the suitability of Ascentis Express RP-Amide N NH CH3 CH3
for the efficient separation of common drink additives used in diet cola. OH 1 N O NH O O O
O N O
column . . Ascentis Express RP-Amide, 3 cm x 4.6 mm I.D., 2.7 μm particles (53921-U) CH3
H3C
HO
NH
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 100 mM ammonium acetate, H3C
CH3 O NH2
pH 5.6 with acetic acid; (B) water; (C) acetonitrile
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3336 psi (230 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
0.2 0.4 0.6 0.8 1.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Min
sample . . . . . . . . . . . . . . . . . . . . diet soda spiked with neotame 350 μg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005391

HPLC Analysis of Fatty Acid Methyl Esters (FAMES) 3

1. C8
on SUPELCOSIL™ LC-18 2. C10
3. 12:0 + 18:3
column . . . . . SUPELCOSIL LC-18, 25 cm × 4.6 mm I.D., 5 μm particles (58298) 4. 18:2
mobile phase . . . . . . . . . . . . (A) acetonitrile; (B) acetone; (59.0:41.0, v/v, A:B) 5. 14:0
6. 18:1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 7. 16:0
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI 8. 18:0
injection . . 10 μL of 9% C8 to C18 saturated and unsaturated FAMEs in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797-0501
5
4
2
1 7
0 2 4 6
Min

HPLC Analysis of Fatty Acids on Ascentis® 1 1. Oleic acid (m/z 281)

2. Palmitic acid (m/z 255)
RP-Amide 3. Stearic acid (m/z 283)

mobile phase . . . . . . (A) 0.1% formic acid in water: (B) acetonitrile; (5:95, A;B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min., split to detector
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(-)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002905
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Flavones on Discovery® HS PEG 1 1. Flavone

2. 7-Hydroxyflavone
column . . . . Discovery HS PEG, 15 cm × 4.6 mm I.D., 5 μm particles (567416-U) 3. Baicalein
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% formic acid in water; 4. 5-Hydroxyflavone
(B) 0.1% formic acid in methanol; (45:55, A:B) 5. Chrysin
2 6. Luteolin
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 7. Myricetin
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 8. Quercetin
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL, each compound 50 μg/mL 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001665
4
6
3
7 8
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Flavonoids in Lime Juice on 1. Apigenin 6,8-di-C-glucoside

Ascentis® Express C18 2. Diosmetin 6,8-di-C-glucoside
3. Eriocitrin 5
Flavonoids are polyphenolic compounds ubiquitous in nature and classified, 4. Narirutin
according to their chemical structure, into flavonols, flavones, flavanones, 5. Hesperidin 3
isoflavones, catechins, anthocyanidins and chalcones. They are widely
present in fruits, vegetables, leaves, nuts, seeds, bark and beverages (tea,
coffee, beer, wine and fruit drinks).
sample/matrix . . . lime juice hand-squeezed in laboratory, centrifuged and filtered 2
through a 0.45 μm nylon filter disc (Acrodisc) 1
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) water/formic acid (99.9:0.1);
(B) water/acetonitrile/isopropanol/formic acid (39.9:20:40:0.1) 4
gradient . . . 10% B for 3 min; 10 to 34% B in 30 min; 34 to 100% B in 1 min;
held at 100% B for 6 min; 100 to10% B in 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min 0 5 10 15 20 25
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient Min
detector . . . . . . . . . . . PDA: 190–370 nm; chromatogram extracted at 283 nm
detector . . . . . . . . . . ESI-MS: mass spectral range, 250-700 m/z; interval, 0.5 s;
scan speed, 938 amu/s; nebulizing gas (N2) flow, 1.5 L/min;
(interface temperature, 350 °C; heat block, 300 °C;
probe voltage, +4.5 kV; Qarray voltage, 1.0 V; RF voltage, 90 V;
detection gain, 1.05 kV)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005593

mAU
HPLC Analysis of Flavonoids in Orange Juice on 600
283 nm, 2 nm (1.00)
Ascentis® C18 550
1. Narirutin
column . . . . . . Ascentis C18, 25 cm × 4.6 mm I.D., 5 μm particles (581325-U) 500
2. Hesperidin
3. Didymin
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) formic acid: (B) 2-propanol: 450
(C) acetonitrile: (D) water (0.1:10:12:78, A:B:C:D:)
400
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 350
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PDA, 283 nm 300
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 250
sample . . . . . . . . . . . . . . . . . . orange juice, passed through a 0.45 μm filter 200

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003936
150
1 3
100
50
0
2.5 5.0 7.5 10.0 12.5 15.0
Min

HPLC Analysis of Fluoroquinolone Antibiotics on 1. Ofloxacin

Ascentis® Express C18 2. Ciprofloxacin
3. Lomefloxacin
This application demonstrates the suitability of the Ascentis Express C18 for
4. Danofloxacin
the analysis of fluoroquinolones.
2 5. Enrofloxacin
column . . Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 μm particles (53814-U) 6. Sarafloxacin
1
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% formic acid in water; 5
(B) 0.1% formic acid in acetonitrile; (85:15, A:B)
3 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min
6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005699 1.0 2.0 3.0
Min

3 1. Norfloxacin (1 µg/mL) (M+H)+ =320.13

HPLC Analysis of Fluoroquinolone Antibiotics on 2. Ciprofloxacin (1 µg/mL) (M+H)+ =332.13
Ascentis® Phenyl 3. Lomefloxacin (1 µg/mL) (M+H)+ =352.13
4. Enrofloxacin (1 µg/mL) (M+H)+ =360.16
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 5. Sarafloxacin (1 µg/mL) (M+H)+ =386.12
mobile phase . . . . . . (A) 13 mM ammonium formate (pH 3.0 with concentrated 1 6. Cinoxacin (1 µg/mL) (M+H)+ =263.05
formic acid): (B) acetonitrile; (80:20, A:B)
2 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . as indicated in 80:20, 13 mM ammonium formate
(pH 3.0 with concentrated formic acid):acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003713
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Free Fatty Acids on 5

SUPELCOSIL™ LC-8 1. 18:3c (2.65 μg)
2. 16:1c (0.53 μg)
column . . . . . . SUPELCOSIL LC-8, 15 cm × 4.6 mm I.D., 3 μm particles (58983) 3. 16:1t (10.6 μg)
1 4. 18:2c (0.53 μg)
mobile phase . . . acetonitrile:tetrahydrofuran: 0.1% phosphoric acid (50.4:21.6:28)
5. 18:2t (2.65 μg)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 6. 16:0 (15.8 μg)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4 7. 18:1c (10.6 μg)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm 8. 18:1t (10.6 μg)
9. 18:0 (10.6 μg)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0969
3 Ox Oxidation products
Ox 2
8
6 7
0 2 4 6 8 10 12
Min

HPLC Analysis of Fusarium Mycotoxins on 1. Nivalenol

Ascentis® Express Phenyl-Hexyl 2. Deoxynivalenol
3. 15-Acetyldeoxynivalenol
The trichothecene vomitoxins of Fusarium are significant plant pathogens of 4. 3-Acetyldeoxynivalenol
grain and forage crops that can lead to anorexic effects with livestock. This
application demonstrates resolution of deoxynivalenol and three of its 1
3 4
analogs.
mobile phase . . . . . . . . . . . . . . (A) water, acetonitrile, 90:10; (B) acetonitrile
gradient . . 0% B held for 0.5 min; to 15% B in 1 min; held at 15% B for 6 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 bar (2780 psi)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 218 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 5 mg/L ea. in 95:5, water:acetonitrile
0 2 4 6

HPLC Analysis of Grapefruit Juice on Ascentis® 1. Apigenin 7-(malonyl apiosyl)-glucoside

Express C18 Column 2. Apigenin 6,8-di-C-glucoside
3. Eriocitrin 5
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 4. Narirutin
sample/matrix . . . . . . . grapefruit juice hand-squeezed in laboratory, centrifuged 5. Hesperidin
and filtered through a 0.45 μm nylon filter disc (Acrodisc) 6. Naringin
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) water/formic acid (99.9:0.1);
(B) water/acetonitrile/isopropanol/formic acid (39.9:20:40:0.1) 4 6
gradient . . . 10% B for 3 min; 10 to 34% B in 30 min; 34 to 100% B in 1 min;
held at 100% B for 6 min; 100 to10% B in 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min 2 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 1
detector . . . . . . . . . . . PDA: 190–370 nm; chromatogram extracted at 283 nm
detector . . . . . . . . . . ESI-MS: mass spectral range, 250-700 m/z; interval, 0.5 s;
scan speed, 938 amu/s; nebulizing gas (N2) flow, 1.5 L/min; 0 5 10 15 20 25
interface temperature, 350 °C; (heat block, 300 °C; Min
probe voltage, +4.5 kV; Qarray voltage, 1.0 V; RF voltage, 90 V;
detection gain, 1.05 kV.)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005592

HPLC Analysis of Hydrolyzed β-Cyclodextrin on DP=1

TSKgel® G-Oligo-PW
column . . . . TSKgel G-Oligo-PW, 30 cm × 7.8 mm I.D., 6 μm particles (808031)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . distilled water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
DP=4 DP=2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI DP=7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0917 DP=3
DP=5
DP=6
15 20
Min

HPLC Analysis of Isoflavones in Soy Milk on 7

Ascentis® Express RP Amide (Milk Brand 1) 1.0 μg/mL standard
This application demonstrates the separation of seven isoflavones on
Ascentis Express RP Amide
4 5
column . . Ascentis Express RP-Amide, 10 cm x 3 mm I.D., 2.7 μm particles (53918-U) 3
gradient . . . . . . . . . . . . . . . held at 15% B for 1 min; 15 to 100% in 5 min; 1 2
held at 100% B for 1 min; 100-15% B in 0.1 min; 6
held at 15% B for 2.9 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2900 psi (200 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm 0 2 4 6 8 10
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL Min
sample . . soy milk, methanol extract (1. Measure 2 mL of each brand of soy milk
3 1. Daidzin
(6 samples) 2. Add 8 mL of methanol and shake for 1 hour 3.
2. Glycitin
Centrifuge tubes at 10000 rpm for 30 minutes 4.
3. Genistin
Transfer aliquot to autosampler vial) 4. Glycitein
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005918 5. Daidzein
6. Equol
Silk® Soy Milk 7. Genistein
1
2 7
0 2 4 6 8 10
Min


Ascentis® Express RP Amide (Milk Brand 2)
This application demonstrates the separation of seven isoflavones on 1.0 μg/mL standard
4 5
gradient . . . . . . . . . . . . . . . held at 15% B for 1 min; 15 to 100% in 5 min; 1 2
held at 100% B for 1 min; 100-15% B in 0.1 min; 6
held at 15% B for 2.9 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2900 psi (200 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm 0 2 4 6 8 10
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . soy milk, methanol extract (1. Measure 2 mL of each brand of soy 1. Daidzin
milk (6 samples) 2. Add 8 mL of methanol and shake for 1 hour 3. 2. Glycitin 3
Centrifuge tubes at 10000 rpm for 30 minutes 4. 3. Genistin
Transfer aliquot to autosampler vial) 4. Glycitein
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005917 5. Daidzein
6. Equol
7. Genistein
8th Continent® Soy Milk
1 5
2 4
0 2 4 6 8 10
Min


12.58 min.
*
CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
9.24 min.
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004663

HPLC Analysis of Mannose and Maltose Sugars on 1. Mannose

Ascentis® Express HILIC 2. Maltose
3. Maltotriose
This application demonstrates the suitability of the Ascentis Express HILIC for 4. Maltotetraose
the analysis of sugars. Structures along with the optimized chromatogram
5. Maltopentaose
obtained on the Ascentis Express HILIC are presented. Separation is by
6. Maltohexaose
normal-phase mechanism as a decrease in mobile phase water content
7. Maltoheptaose 2
results in increased retention. 5
column . . Ascentis Express HILlC, 15 cm x 4.6 mm I.D., 2.7 μm particles (53981-U) 1 6
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 7
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005745
0 2 4 6
Min

HPLC Analysis of Methylimidazoles in Diet Cola on 1. 4-Methylimidazole 1

Ascentis® Express HILIC 2. 2-Methylimidazole
This method features fast analysis of methylimidazole derivatives used in Diet cola sample
caramel-colored carbonated beverages.
2
The purpose of this study was to develop a fast analytical method for
analyzing for 2-methylimidazole and 4-methylimidazole in caramel colored
carbonated beverages. The FDA has recently increased the awareness of the 1
potential hazards of methylimidazole. These compounds can be found in
caramel additives used in a broad range of food stuff. In this study the focus
is the levels of methylimidazole in various colas. Methylimidazoles are a
difficult group of compounds to analyze due their polar nature and low
molecular weight. Traditional reversed phase separation are unsuccessful in
retaining these small polar compounds, with most methods utilizing ion-
exchange resins for analysis. Other common methods are GC analysis with
sample derivitization.
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) Standards
mobile phase . . . . . . . . . . 2 mM ammonium formate in 5:95, water:acetonitrile,
to pH 4.4 with formic acid
0 2 4 6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 50-800 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 mg/L in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005469

HPLC Analysis of Milk Proteins on HiTrap® SP Fract. 2

A280nm
column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HiTrap SP, 1 mL (54817) 100% B
mobile phase . . . . . . . . . . . . . . . . . . . . (A) 50 mM sodium acetate, pH 6.0;
0.5
(B) 50 mM sodium acetate/1.0 M sodium chloride, pH 6.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 0.4
injection . . . . . . . . . . . . . . . . . 0.5 mL filtered casein-precipitated human milk
(buffer exchanged to mobile phase A)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795-0493 0.3
0.2
40% B
Fract. 24
0.1
Fract. 16
Fract. 17
Fract. 9
0
10% B
5 10 15 20 25 30
Volume, mL
Reproduced with permission of GE Healthcare.

HPLC Analysis of Oligosaccharides from Dark Corn 1

1. DP6
Syrup on SUPELCOGEL™ Ag 2. DP5
3. DP4
column . . . . . . . . . . . . . . . . SUPELCOGEL Ag, 30 cm × 7.8 mm I.D. (59315) 6 4. DP3
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . water 5. DP2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min 5 6. Glucose
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI
3
injection . . . . . . . . . 10 μL of 1g syrup/10 mL DI water, filtered (0.20 μm filter) 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0029
0 5 10 15
Min


3. Citric acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005714
1.0 2.0 3.0 4.0 5.0
Min

HPLC Analysis of Organic Acids on Ascentis® RP- 1. L-Tartaric acid

Amide 2. Lactic acid
3. Citric acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2 5
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005715
1.0 2.0
Min


4
2. Sorbic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797-0657
0 2 4 6
Min


mobile phase . . . . . . . . . . . . . . (A) 10 mM ammonium phosphate monobasic
(pH 2.50 with phosphoric acid): (B) acetonitrile; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003160 0 2 4 6 8 10 12 14
Min


column . . . . . . . . Discovery C18, 5 cm × 4.6 mm I.D., 5 μm particles (504947) 1. Homovanillic acid (0.0625 μg/mL)
2. Sorbic acid (0.00625 μg/mL)
mobile phase . . . . . . . . . . . (A) 0.1% TFA in (B) water:methanol; (60:40, A:B) 3. Salicylic acid (0.0625 μg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min 4. p-Toluic acid (0.00625 μg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001431 1 3
4
1.0 2.0 3.0

Min


O
OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004680
0 2 4 6 8 10 12 14
Min


Enantiomers on Astec® CYCLOBOND® I 2000 HO O
HP-RSP
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004717
0 2 4 6 8 10
Min

HPLC Analysis of Phenyl Urea Pesticides on 1. Fenuron

Ascentis® Express C18 Phase 2. Monuron
3. Fluomethuron
This separation illustrates the use of the highly efficient 2.7 um Ascentis 4. Isoproturon
Express Fused-Core C18 stationary phase in the analysis of some popular 5 5. Diuron
herbicides. The short run times allow analyses using isocratic conditions so 6. Siduron A
that column equilibration time is not required between runs. 3
7. Siduron B
Response Time: 0.02 sec. 2 8. Linuron
Flow Cell: 2.5 μL semi-micro 4 9. Neburon
8
column . . Ascentis® Express C18, 10 cm x 4.6 mm I.D., 2.7 μm particles (53827-U) 1
mobile phase . . . . . . . . . . . . . . . . . (A) 0.025 M potassium phosphate buffer, 6 9
adj. to pH = 2.5, (B) acetonitrile; (50:50, A:B)
7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 bar
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV 245 nm, VWD Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006189

HPLC Analysis of Polyphenols in Nero d’Avola Red 1. Gallic acid

Wine on Discovery® HS C18 (UV 280 nm) 2. Caftaric acid
3. Catechin
sample/matrix . . . . . . . . . . . . Nero d’Avola wine sample filtered prior injection 4. Procyanidin
through a 0.45 μm nylon membrane 5. Caffeic acid
column . . . . Discovery HS C18, 15 cm x 2.1 mm I.D., 3 μm particles (569255-U) 6. Epicatechin
mobile phase . . . . . . . . . . . . . . . . . . . . . (A) water, pH 3 with formic acid; 7. Ethylgallate
(B) acetonitrile, pH 3 with formic acid 8. Rutin
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 9. Isoquercitrin
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 10. p-Coumaric acid 8+9
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005595
6
1 45 7
2 10
3
0 20 40 60
Min

HPLC Analysis of Polyphenols in Nero d’Avola Red 2+3

1. Caftaric acid
Wine on Discovery® HS C18 (UV 354 nm) 2. Rutin 1
sample/matrix . . . . . . . . . . . . Nero d’Avola wine sample filtered prior injection 3. Isoquercitrin
through a 0.45 μm nylon membrane
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) water, pH 3 with fomic acid;
(B) Acetonitrile, pH 3 with fomic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 354 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005596
0 20 40 60
Min

HPLC Analysis of Pullulan on an SRT® SEC-300 Gel Calibration Curve
Filtration Column Pullulan Standards

1. MW 342 11.67
6
2. MW 1,320 11.34
Pullulan is a water-soluble polysaccharide polymer consisting of glucan 3. MW 6,000 10.39
Log MW
units. Pullulan is used in the manufacture of edible films that are used in 4. MW 10,000 9.94
5. MW 21,700 9.00
breath fresheners and mints and is also used as a food additive. Another use 4 6. MW 48,800 7.75
of pullulan is as calibration standards in gel filtration chromatography. 7. MW 113,000 6.55
8. MW 210,000 6.03
Gel filtration chromatography (GFC) is used to determine the molecular 9. MW 366,000 5.93
mass composition of pullulan and many other water-soluble polymers using 10. MW 805,000 5.84
2
silica- or polymer-based particles that have been modified with a chemically 6 8 10 12
80 Elution volume (mL)
bonded hydrophilic coating. 1
70
In this particular example, a SRT SEC-300 column from Sepax Technologies
60
was used to analyze several pullulan standards. In the accompanying table
50
MV
shows retention volumes for the pullulan standards and the molecular mass
40 9 2
of each; this data was then used to construct the calibration curve which 8
30 10 3
allows the calculation of the molecular mass and distribution of an unknown 5 4
20 7 6
pullulan polymer or other polymers of similar geometrical shape.
10
column . . . . . . . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm, 300 Å (Z777051) 0
mobile phase . . . . . . . . . . . . . . . . . . . . . 150 mM phosphate Buffer, pH 7.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
Min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600 psi (41.4 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI (30° C)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . 10 mg/mL in mobile phase 0.342 kD, 1.32 kD, 6 kD, 10 kD, 21.7 kD,
48.8 kD 113 kD, 210 kD, 366 kD and 805 kD
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006202

HPLC Analysis of Simple Underivatized Sugars on 1. D-(-)-arabinose 5

apHera™ NH2 2. D-(+)-xylose
3. D-(+)-mannose
column . . . . . . . apHera NH2, 15 cm × 4.6 mm I.D., 5 μm particles (56401AST) 2
4. D-(+)-galactose
mobile phase . . . . . . . . . . . . . . . . . . (A) water: (B) acetonitrile; (20:80, A:B) 5. D-(+)-glucose
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD, 45 °C, 3.5 psi nitrogen
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . 500 μg/mL in 30:70, water: acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003996 3
1 4
0 10 20
Min

HPLC Analysis of Sugars and Sugar Enantiomers 1. D-Fructose

1
on Astec® CYCLOBOND® I 2000 2. D-Glucose
3. Sucrose
column . . . CYCLOBOND I 2000, 25 cm x 4.6 mm I.D., 5 μm particles (20024AST) 4. D-Maltose
2
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (15:85, A:B) 5. Lactose 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . ELSD at 35°C and 3.2 bar nitrogen 5
4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . 0.5 mg/mL each in water:acetonitrile, 25:75
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004682
0 10 20
Min

HPLC Analysis of Sugars and Taurine in an Energy 2 1. Fructose

1
Drink on Ascentis® Express HILIC using UV and 2. Glucose
3. Sucrose
ELSD Detection in Series 4. Taurine
efficient separation of common drink additives used in caffeinated energy
drinks. The analysis was performed with UV and ELSD detectors in series, this
chromatogram being from the ELSD. 3
4
mobile phase . . . . . . . (A) 100 mM ammonium acetate, pH 5.0 with acetic acid; 0 2 4 6 8 10
Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812 psi (56 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . dilute1:9 in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005751

HPLC Analysis of Sugars by HILIC Chromatography 1. dextrose 2 3

using Ascentis® Express OH-5 Column with ELSD 2. sucrose
3. maltitol 1
Detection
This application shows the analysis of sugars by HILIC Chromatography with
ELSD detection. The purpose of the work shown here was to develop a
HILIC method that could quickly separate mono- and di-saccharides with
good sensitivity and reproducibility utilizing the Ascentis Express OH-5
column and an Evaporative Light Scattering Detecctor (ELSD) detector.
column . . Ascentis Express OH-5, 15 cm x 4.6 mm, I.D., 2.7 μm particles (53778-U) 0 2 4 6 8 10
Min
mobile phase . . . . . . . . . . . . . (A) 0.005 M ammonium formate, pH 3.15 with
concentrated TFA (B) acetonitrile (30:70, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 841 psi (58 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . ELSD (detector other: Nebulizer 60º C, Evaporator 80º C,
Gas flow 1.60 SLM (Standard Liters/ Minute), LED 100%,
Smoothing 5.0 S, PMT ( Photomultiplier) Gain 1.0)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . 0.5 mg/mL each prepared in 50:50 Water:Acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005902

HPLC Analysis of Sugars on Ascentis® Express 1. Arabinose peak 1

HILIC (Mobile Phase 5:95) 2. Xylose
3. Arabinose peak 2
This application demonstrates the suitability of the Ascentis Express HILIC for 4. Arabinose peak 3
the analysis of sugars. Structures along with the optimized chromatogram 5. Mannose
obtained on the Ascentis Express HILIC are presented. Separation is by 6. Glucose
2 6
normal-phase mechanism as a decrease in mobile phase water content 7. Galactose peak 1
results in increased retention 8. Galactose peak 2
column . . Ascentis Express HILlC, 15 cm x 4.6 mm I.D., 2.7 μm particles (53981-U) 9. Sucrose
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (5:95, A:B) 5 10. Cellobiose
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4
1 3 7 8
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD, 55 °C, 3.5 bar nitrogen 9
10
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005744 0 2 4 6 8 10 12
Min

HPLC Analysis of Sugars on Ascentis® Express 1. Xylose

HILIC (Mobile Phase 15:85) 2. Glucose
2 3. Sucrose
This application demonstrates the suitability of the Ascentis Express HILIC for 3 4. Maltose
the analysis of sugars. Structures along with the optimized chromatogram 1 4
5 5. alpha-Lactose
obtained on the Ascentis Express HILIC are presented. Separation is by 6. Maltotriose
normal-phase mechanism as a decrease in mobile phase water content
results in increased retention.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD, 55 °C, 3.5 bar nitrogen 0 2 4 6 8
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005746

HPLC Analysis of Sugars on Ascentis® Si 1. Xylose

This application demonstrates the suitability of the Ascentis Si for the 2. Glucose
analysis of sugars. Structures along with the optimized chromatogram 3. Sucrose
2 3 4. Maltose
obtained on the Ascentis Si are presented. Separation is by normal-phase
1 5. alpha-Lactose
mechanism as a decrease in mobile phase water content results in increased
retention. 4 6. Maltotriose
5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005747
Min

HPLC Analysis of Sugars on SUPELCOGEL™ Ca 2 1. Maltotriose

column . . . . . SUPELCOGEL Ca, 30 cm × 7.8 mm I.D., 9 μm particles (59305-U) 2. Maltose
1 5 3. Glucose
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . water 3 7
4 4. Xylose
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 8
5. Arabinose
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 °C 6. Ribitol
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI 7. Arabitol
injection . . . . . . . . . . . . . . . . . . . . 10 μL, 2.5 mg/mL each analyte in water 6 8. Xylitol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795-0016
0 5 10 15 20 25
Min

HPLC Analysis of Sugars on SUPELCOSIL™ LC-NH2

1. Fructose
column . . . . . SUPELCOSIL LC-NH2, 25 cm x 4.6 mm I.D., 5 μm particles (58338) 2. Glucose
3. Sucrose
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (25:75, A:B) 4. Maltose
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 5. Lactose
temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . refractive index (RI)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/mL in water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006195 1
3
2
4
5
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5

Min

HPLC Analysis of Synthetic Food Dyes on 1. Tartrazine 7. Fast green

2. Amaranth 8. Erioglaucine
Ascentis® Express C18 3. Indigo carmine 9. Erythrosin B
column . . Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.7 μm particles (53823-U) 4. New coccine (Acid Red 18) 10. Acid red
mobile phase . . . . . (A) 100 mM ammonium phosphate monobasic, pH 7.0 with 5. Sunset yellow FCF 11. Phloxine B
ammonium hydroxide; (B) water; (C) acetonitrile 6. Allura red 12. Rose bengal
25% B, 65% C held for 5 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 1 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 6
3 4 11
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
2 9 10
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
12
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL each in water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005650
7
8
0 2 4 6
Min


OH O
*
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004665

7.37 min.

HPLC Analysis of Triacylglycerols in Olive Oil on 1. LLL 9. PLO

2. OLLn 10. PoPO
Discovery® HS C18 after Sample Prep using 3. PoOLn 11. PLP 8 9 12 13
4. OLL 12. OOO
Discovery® DSC-Si (Acetone-Acetonitrile Mobile 5. OOLn 13. SOL+OOP
15
Phase, ELSD Detection) 6. PLL

7. POLn
14. POP
15. SOO 4
Olive oil was pre-cleaned using Silica SPE, analyzed as 5% sample in 8. OOL+OOPo 16. SOP 10
acetone (w/v). 14
SPE tube/cartridge . . . . . . . . . . . . . . Discovery DSC-Si, 1 gm/6 mL (52656-U) 56 11 16
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 mL hexane 1 23 7
sample addition . . . . . . . . . . . . . . . . . . . . . . . 0.12 g oil in 0.5 mL hexane

10 20 30 40 50
elution . . . . . . . . . . . . . . . . . . . . . . 10 mL hexane-diethyl ether 87-13 (v/v)
Min
eluate post-treatment . . . evaporation to dryness, reconstituted into 2 mL acetone
mobile phase . . . . . . . . . . . . . . . . . (A) acetone; (B) acetonitrile; (60:40, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914 psi (63 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005863

1. LLL 10. PLO+SLL

HPLC Analysis of Triacylglycerols in Olive Oil on 2. OLLn 11. SOLn
Discovery® HS C18 after Sample Prep using 3. PoOLn
4. OLL
12. PLP
13. OOO
Discovery® DSC-Si (Propionitrile Mobile Phase, 5. OOLn 14. SOL+OOP 8 10 13 14 16
6. PLL 15. POP
ELSD Detection) 7. POLn 16. SOO
8. OOL+LnPP 17. SOP 4
Olive oil was pre-cleaned using Silica SPE, analyzed as 5% sample in 9. PoOO
15
acetone (w/v).
9
SPE tube/cartridge . . . . . . . . . . . . . . Discovery DSC-Si, 1 gm/6 mL (52656-U) 5
2 6 7 11 12 17
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 mL hexane 1 3
sample addition . . . . . . . . . . . . . . . . . . . . . . . 0.12 g oil in 0.5 mL hexane
10 20 30
elution . . . . . . . . . . . . . . . . . . . . . . 10 mL hexane-diethyl ether 87-13 (v/v) Min
eluate post-treatment . . . evaporation to dryness, reconstituted into 2 mL acetone
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . propionitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117 psi (77 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005864

HPLC Analysis of Triacylglycerols in Soybean Oil on 1. LLnLn 8. OOL

Discovery® HS C18 (Acetone-Acetonitrile Mobile 2. LLLn 9. POL+SLL
3. LLL 10. PPL
Phase, ELSD Detection) 4. OLLn 11. OOO
Soybean oil was analyzed as 5% diluted sample in acetone (w/v) 5. PLLn 12. SOL+POO
column . . . . Discovery HS C18, 25 cm x 4.6 mm I.D., 5 μm particles (568523-U) 6. OLL 13. POP
mobile phase . . . . . . . . . . . . . . . . . (A) acetone; (B) acetonitrile; (60:40, A:B) 7. PLL
2 3 6 7 9
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914 psi (63 bar) 4 8
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD
1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 10 11
12
13
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 5% soybean oil in acetone (w/v)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005877 10 20 30 40
Min

HPLC Analysis of Triacylglycerols in Soybean Oil on 1. LLnLn

2. LLLn
Discovery® HS C18 after Sample Prep using 3. LLL
4. OLLn
Discovery® DSC-Si (Propionitrile Mobile Phase, 5. PLLn 2
3 4
5 6 7 8
9
10
6. OLL
ELSD Detection) 7. PLL
8. OOL
Soybean oil was cleaned using Silica SPE and analyzed as 5% sample in 9. POL
acetone (w/v). 10. SLL
11. PPL 1
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 mL hexane 12. OOO 13
11
SPE tube/cartridge . . . . . . . . . . . . . . Discovery DSC-Si, 1 gm/6 mL (52656-U) 13. SOL 12
14
14. POO
sample addition . . . . . . . . . . . . . . . . . . . . . . . 0.12 g oil in 0.5 mL hexane 15. PLS 15
elution . . . . . . . . . . . . . . . . . . . . . . 10 mL hexane-diethyl ether 87-13 (v/v)
eluate post-treatment . . . . vaporation to dryness, reconstituted into 2 mL acetone
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . propionitrile 10 20
Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117 psi (77 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005865

HPLC Analysis of B-Trichothecenes on Ascentis® 1. Nivalenol

2 2. Deoxynivalenol
Express C18 3. 15-Acetyldeoxynivalenol
1
Mycotoxins are toxic secondary metabolites produced by fungi. Those 4. 3-Acetyldeoxynivalenol
chemicals exist in food as a result of fungal infection of crops. Mycotoxins
have strong resistance to decomposition and digestion, so they remain in
the food chain in meat and dairy products. They also resist to temperature,
such as cooking and freezing. Their effects on human and animal health 3
4
include death, cancer, weakened immune systems and as allergens or
irritants.
column . . . Ascentis Express C18, 10 cm x 3 mm I.D., 2.7 μm particles (53814-U)
mobile phase . . . . . . . . (A) water:acetonitrile:methanol (92:4:4); (B) acetonitrile
gradient . . 0% B held for 5 minutes, to 8% B in 3 minutes, 8% B held for 2 minutes
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4061 psi (280 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 2 4 6 8 10 12
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 μL
sample . . . . . . . . . . . . . . . 2 ppm each in water:methanol:acetonitrile (92:4:4)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005561

HPLC Analysis of Vitamins, Fat Soluble (A and E) 1. Retinol acetate (Vitamin A acetate), 50 μg/mL
2. δ-Tocopherol, 165 μg/mL
on Discovery® C18 3. γ-Tocopherol, 200 μg/mL
4. α-Tocopherol (Vitamin E), 292 μg/mL
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 5. α-Tocopherol acetate (Vitamin E acetate), 405 μg/mL
mobile phase . . . . . . . . . . . . . . . . . . . . (A) methanol: (B) water (95:5, A:B)
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 3 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000911A
5
0 2 4 6 8 10
Min

LC-MS Analysis of Aflatoxin M1 in Milk on detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS/MS (MRM transitions)

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Ascentis® Express HILIC after SPE using Supel™ Tox Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006300
AflaZea, Quantifying Picogram Concentrations
Aflatoxin M1 is the metabolite of Aflatoxin B1 and can be found in milk
1. Aflatoxin M1
products when animals have been fed alatoxin-contminated feeds.
300
Aflatoxins are positively associated with hepataocellular carcinomas.
Aflattoxin M1 was detected in milk and quantified at concentrations fo 25
1
pg/mL. Sample preparation included a protein precipitation with solvent
partitionng and sample clean-up using Supel Tox AflaZea SPE tubes. Analysis
was perfomed via LC-MS using an Ascentis Express C18, 5 cm x 2.1 mm I.D.,
2.7 μm HPLC column for the separation. Detection and quanitification of
200
aflatoxin M1 at 25 pg/mL satisfies the current regulatory requirements for
aflatoxin M1 for EU treaty participants as stated in EC 1881/2006.
Sample pretreatment: centrifuge 45 g liquid milk for 60 min at 5000 rpm
(RCF 4863 xg), cool at 0 °C for 30 minutes, remove fat layer, transfer middle
layer to collection flask, add 20 mL acetonitrile to 50 mL centrifuge tube, add
100
20 mL of sample to acetonitrile, add contents of 1 Supel Q non-buffered
tube, immedately disperse salt, quicky place on shaker for 5 minutes,
centrifuge at 5000 rpm for 20 min, remove top layer (loading solution)
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cow milk
0
SPE tube/cartridge . . . . . . . . . . . . . . . . Supel™ Tox AflaZea, 6 mL (55314-U) 0.0 1.0 2.0 3.0 4.0 5.0
sample addition . . . . . . . . . . . . . . . . . . 10 mL, 2 drop/second using vacuum AFM1 in Milk 25 pg/mL
washing . . . . . . . . . . . . . . . . . . . 2 x 2 mL acetonitrile (collect with sample)
eluate post-treatment . . . . . . . . . . evaporate to dryness at 70 °C using nitrogen
sample preparation . . . . . . . . . . . reconstitut in 500 μL 20:80 acetonitrile:water,
filter into 750 μL poly vial using PVDF filter, 0.2 μm
mobile phase . . . . . . . . . . . . . . . . (A) 0.1% (v/v) formic acid; (B) acetonitrile
gradient . . . . 90% A for 0.5 min; to 10% A in 0.5 min; held at 10% for 3 min;
to 90% A in 0.10 min; held at 90% A for 3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min

LC-MS Analysis of Melamine and Hydrolysis 1 1. Melamine

Products on Ascentis® Express HILIC 2. Ammelide
3. Ammeline
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), full scan
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/L in mobile phase 0 2 4 6 8 10 12
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005708
Min

LC-MS Analysis of Melamine and Metformin on 1 1. Melamine

Ascentis® Express HILIC 2. Metformin
column . . . Ascentis Express HILIC, 5 cm x 3 mm I.D., 2.7 μm particles (53967-U)
mobile phase . . (A) 10 mM ammonium formate in water; (B) acetonitrile; (5:95, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551 psi (38 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), SIR, m/z 127, 130
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6 8 10 12 14
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/L
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005754

LC-MS Analysis of Phospholipids in Milk on detector . . . . . . . . . . . . . ELSD. nebulizing gas (nitrogen) flow: 2 mL/min (180 KPa); drift tube temperature: 50 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Ascentis® Express HILIC after SPE using Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005600
Supelclean™ Si, Shotgun HPLC Analysis
Extraction of the lipid fraction was carried out from 10 mL of the cow’s milk 1. Phosphatidylinositol
sample, according to the Folch method to ensure the exhaustive extraction 2. Phosphatidylserine
of the whole lipid content. The total extract was evaporated under vacuum, 3. Phosphatidylethanolamine
3
and the final dry residue (400 mg) was re-dissolved in chloroform/methanol 4. Phosphatidylcholine
2:1 (v/v). Lipid extract (100 mg) was afterward dissolved in 1 mL mixture of 5. Sphingomyelin
chloroform/methanol (2:1, v/v). After the cartridge has been conditioned
with hexane, the non-polar lipids were eluted with 3 mL of hexane/diethyl- 2
ether (8:2, v/v) and 3 mL of hexane/diethyl-ether (1:1, v/v). Recovery of PLs
from the cartridge was obtained by two-step elution, using 4 mL of
methanol as first extraction solvent, and subsequently 2 mL of methanol
followed by 2 mL of chloroform/methanol/water (3:5:2, v/v/v). The
recovered fraction was dried under a gentle stream of nitrogen, yielding
49.7 mg dry residue. The residue was finally re-dissolved in chloroform/ 5
methanol (2:1, v/v). 4
sample/matrix . . . Phospholipids from 10 mL cow’s milk (Folch method extraction) 1
SPE tube/cartridge . . . . . . . . . . . . . . . . . Supelclean LC-Si, 1 g/6 mL (57051)
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hexane 0 20
sample addition . . . . . . . . . . . . 1 mL extract in chloroform/methanol (2:1, v/v) Min
washing . . . . . . . . . . . . . 3 mL of hexane/diethylether (8:2, v/v) and 3 mL of
hexane/diethylether (1:1, v/v) to remove nonpolar lipids
elution . . . . . . . . 1 x 4 mL methanol, followed by 1 x 2 mL methanol, followed
by 1 x 2 mL chloroform/methanol/water (3:5:2, v/v/v).
eluate post-treatment . . dry under a gentle stream of nitrogen, redissolved residue in
chloroform/methanol (2:1, v/v)
mobile phase . . . . . . . . . . . . . . . . (A) acetonitrile; (B) acetonitrile:water (2:1)
gradient . . . . . 0% B held for 5 min; to 30% B in 5 min; to 100% B in 20 min;
held at 100% B for 10 min; to 0% B in 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min

LC-MS Analysis of Sudan Red Dyes on Ascentis® 1. Sudan Orange G (M+H)+= 215.07
2. Sudan Red G (M+H)+= 279.10
RP-Amide 3. Sudan I (M+H)+= 249.09
Gradient Program
4. Sudan II (M+H)+= 277.12
column . . . Ascentis RP-Amide, 15 cm × 4.6 mm I.D., 5 μm particles (565324-U) 5. Sudan III (M+H)+= 353.13 Time (min) %A %B
6. Sudan Red 7B (M+H)+= 380.17 0 5 85
mobile phase . . . . . . . . . . . . . (A) 0.1% formic acid in water (pH unadjusted): 7. Sudan IV (M+H)+= 381.16 8 15 85
10 0 100
15 0 100
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min, split to the MS 16 15 85
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 20 15 85
detector . . . . . . . . . . . . . . MS, APCI (+) in selected ion recording (SIR) mode 2

3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL 6
4
sample . . 1 μg/mL in 30:70, 0.1% formic acid in water (pH unadjusted):acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003860
5 7
0 2 4 6 8 10 12 14
Min

(x10,000,000)
LC-MS Analysis of Triglycerides in Corn Oil on TIC 1. LLLn
2. LLL
Ascentis® C18 1.25 3. PLLn
2 4
4. OLL
5. PLL
column . . . . . . . . . . . . . . . . . . . . two Ascentis C18, 15 cm × 2.1 mm I.D., 6. OOL 5
3 μm particles connected in series (581302-U) 1.00
7.
8.
PLO
PLP
mobile phase . . . . . . . . . . . . . . . (A) 2-propanol: (B) acetonitrile; (60:40, A:B) 9.
10.
OOO
POO 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 0.75
7
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . APCI/MS positive ion mode
0.50
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . corn oil weighed and diluted in acetone (10 mg in 1 ml w/v) 9
0.25 10
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003921 1
8
3
0.00
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0
Min

LC-MS Analysis of Triglycerides in Soybean Oil on (x10,000,000)

3
Ascentis® C18 TIC 6
1.
2.
LLnLn
LLLn
9.
10.
PLO+SLL
PLP
1.50 3. LLL 1 1. OOO
column . . . . . . . . . . . . . . . . . . . . two Ascentis C18, 15 cm × 2.1 mm I.D., 4.
5.
OLLn
PLLn
12.
13.
SLO
POO
7
3 μm particles connected in series (581302-U) 1.25 6.
7.
LLO
PLL
14.
15.
PLS
POP
mobile phase . . . . . . . . . . . . . . . (A) 2-propanol: (B) acetonitrile; (60:40, A:B) 8. OOL 16. SOO
1.00
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 9
2 8
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 0.75
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS APCI mode 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0.50
5
sample . . . . . soybean oil weighed and diluted in acetone (10 mg in 1 ml w/v) 10
12
13
11
1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003923 0.25 14
15 16
0.00
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0
Min

LC-MS Anaysis of Chloramphenicol in Shrimp on Chloramphenicol in

Ascentis® Express C18 Using Z-Sep+ Cleanup
Internal Standard in
This application demonstrates the superior cleanup associated with Z-Sep+
using QuEChERS approach.
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 g shrimp
extraction . . . . . . . . . . . . . . . . . 9 mL acetonitrile, then 90 μL of formic acid
extraction process . . . . . . . . . . . . post treatment: 8 mL supernatant evaporated
to dryness and reconstituted with 1 mL water
clean-up tube . . . . . . . . . Supel™ Que Z-Sep+ tube, 12 mL, 500 mg (55296-U)
clean-up process . . . . . . . . . . . . . . . . . . . . . . . . . . clean-up volume: 9 mL 0 2 4
Min
column . . . . . . Ascentis Express C18, 5 cm X 2.1mm, 2.7 um particles (53822-U)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) Water; (B) Acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . 15% B for 0.1 min, 15-80% in 1.9 min,
held at 80% for 1.5 min, 80-15% in 0.5 min, at 15% for 3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
detector . . . . . . . . . . . . . . . MS, ESI(+), MRM, m/z 320.9/151.9, 325.9/157.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006249

LC-MS/MS Analysis of Carbendazim in Orange 1 1. Carbendazim

Juice (Spiked) on Ascentis® Express C18 after
Supel™ QuE PSA/C18 QuEChERS Cleanup
Simple and fast extraction and analytical method were developed for
analysis of fungicide carbendazim in orange juice samples. QuEChERS
extraction method was used with PSA/C18 cleanup. Ascentis Express C18
column chromatography was coupled to MS/MS detection with limits of
detection at 0,1 ppb.
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . Dispersive (QuEChERS)
sample/matrix . . . . . . . . . . . . . orange juice spiked with carbendazim at 1 ppb
extraction tube . . . . . . . . . . . . . . . . Supel™ QuE Citrate (EN) Tube (55227-U)
extraction process . . . . . add 10 mL of orange juice with pulp to a 50 mL empty
extraction tube (55248-U); add carbendazim at 1 ppb; 0 1 2 3
add 10 mL acetonitrile; (shake for 1 minute; add contents of Min
citrate extraction tube (55227-U); shake for 1 min;
centrifuge at 3200 rpm for 5 minutes)
clean-up tube . . . . . . . . . . . . . . . . . . . . . . . . Supel-Q™ PSA/C18 (55288-U)
clean-up process . . transfer 0.7 mL of the acetonitrile layer to the PSA/C18 cleanup
tube (55288-U); shake 1 minute; (centrifuge for 3 minutes at 3400 rpm;
remove 0.25 mL of the supernatant;
mix with 0.25 mL water for LC-MS analysis)
mobile phase . . . . . . . . . . . . . . . . . . A) 10 mM ammonium acetate in water;
(B) 10 mM ammonium acetate in methanol
gradient . . . . . . . . . 0-1 min: 30% B; 1.5-3.5 min: 100% B; 3.5-7 min: 30% B
flow rate . . . . . . . . . . . . . . . . . 0-1 min: 0.3 mL/min; 1.5-7 min: 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . MS, ESI(+), MRM, m/z 192/160, 192/132
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005619

LC-MS/MS Analysis of Melamine Extracted from Melamine

127/85 transition
Milk on Ascentis® Express HILIC
mobile phase . . (A) 10 mM ammonium formate in 10:90 (v/v) water:acetonitrile;
(B) 10 mM ammonium formate in 30:70 (v/v) water:acetonitrile
gradient . . held at 0% B for 5 min; 0 to 100% B in 0.1 min; held at 100% B for
4.9 min; 100 to 0% B in 0.1 min; held at 0% B for 4.9 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ABI 3200 Q-Trap
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
127/68 transition
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . SPE extract from spiked milk
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005607
0 2 4
Min

LC-MS/MS of Pesticides in Oranges on Ascentis® tR(min) tR(min)

1. Methomyl 0.60 20. Quinalphos 7.23
Express C18 after Sample Prep using Supel™ Que 2. Trichlorfon 1.22 21. Edifenphos 7.28
Z-Sep/C18 QuEChERS sorbent 3. Carbendazim
4. Aldicarb
1.94
2.70
22. Etrimfos
23. Fenthion
7.33
7.34
Sample pretreatment: 10 g of pureed oranges homogenized with rind; 5. Parathion-methyl 5.87 24. Fenitrothion 7.36
6. Methabenzthiazuron 6.16 25. Diazinon 7.37
spike at 50 ppb by adding 16.75 μL of a pesticide mix containing each 7. Naled 6.37 26. Tolclofos-methyl 7.52
analyte at 30 μg/mL 8. Methidathion 6.38 27. Phorate 7.53
This demonstrates the application of the Z-Sep/C18 sorbent to the cleanup 9. Clethodim 6.42 28. Chlorpyrifos-methyl 7.67
10. Phosmet 6.50 29. EPN 7.68
of fruits in the LC-MS-MS analysis of polar pesticides including organo-
11. Ametryn 6.58 30. Terbufos 8.14
phosphates, carbamates and benzoylureas 12. Sethoxydim 6.61 31. Ethion 8.36
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . Dispersive (QuEChERS) 13. Anilazine 6.78 32. Lufenuron 8.40
14. Fenxehamid 6.94 33. Spiromesifen 8.74
sample/matrix . . 10 g of pureed oranges (homogenized with rind); spike at 50 ppb 15. Mecarbam 7.00 34. Octhilinone 8.74
(add 16.75 μL of a custom made pesticide mix, each analyte at 30 μg/mL) 16. Oryzalin 7.06 35. Pyraclostrobin 8.75
17. Diflubenzuron 7.18 36. Carbophenothion 8.83
extraction tube . . . . . . . . . . . . . . . . Supel™ QuE Citrate (EN) Tube (55227-U) 18. Fenoxycarb 7.19 37. Flufenoxuron 9.02
extraction process . . add 10 mL acetonitrile; shake for 1 minute; add contents of a p
19. Iprobenfos 7.21 py
38. Fenpyroximate 9.10
Supel QuE citrate extraction tube (55227-U); shake immediately
for 1 minute; centrifuge at 3200 rpm for 5 minutes
clean-up process . . transfer 0.7 mL of the acetonitrile layer into a Supel QuE Z-Sep/
C18 cleanup tube (55284-U); shake for 1 minute; centrifuge
at 5000 rpm for 5 minutes; (transfer 0.2 mL of the supernatant into
an empty 1.5 mL centrifuge tube; add 0.2 mL of water;
centrifuge at 5000 rpm for 2 minutes.)
mobile phase . . . . . . . . . . . . . . . . . (A) 10 mM ammonium acetate in water;
gradient . . Hold at 30% B for 1 min; 30% to 80% B in 2 min; hold at 80% B for 4
min; hold at 100% B for 3 min; hold at 30% B for 3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2730 psi (188 bar) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS/MS, ESI positive
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005645

HPLC, UHPLC or LC-MS Forensics and Toxicology
1 1. Codeine
Forensics and Toxicology 2. Strychnine
3. Quinidine
HPLC Analysis of Alkaloids on Discovery® 4. Quinine
5. Noscapine
RP-AmideC16 6. Papaverine
4
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 2 6
mobile phase . . . . . . . . . . . . . (A) methanol; (B) 25 mM potassium phosphate,
dibasic, pH 3.0; (20:80, A:B) 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000235 0 2 4 6 8 10 12
Min

HPLC Analysis of 1-Aminoindan Enantiomers on

LARIHC™ CF6-P
*
column . . LARIHC CF6-P, 25 cm x 4.6 mm I.D., 5 μm particles (AZYP Part No.L1001,
available from Supelco/Sigma-Aldrich as a custom item) NH2
mobile phase . . . . . . . . . . . . . . (A) methanol: (B) acetonitrile: (C) acetic acid:
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 1-Aminoindan, 0.3 mg/mL in ethanol 0 2 4 6 8 10 12
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005255

HPLC Analysis of Amphetamine and CH3 NH

* *
Methamphetamine Enantiomers on Astec® CH3
CHIROBIOTIC® V2 NH2 CH3
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) Amphetamine Methamphetamine
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . (A) methanol; (B) acetic acid;

(C) ammonium hydroxide; (99.88:0.1:0.01, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004467
10 12 14 16 18 20 22

HPLC Analysis of Amphetamines in Urine on 1. Amphetamine
10
2. Methylamphetamine
Discovery® HS F5 after SPE using Discovery® DSC-
DSC-MCAX SPE
8
MCAX and Standard C18
6
mAU
1 mL human urine was spiked with 2 mg/mL amphetamine and
methylamphetamine. The spiked sample was diluted 1:1 with 1:1 with
4
50 mM ammonium acetate, pH 6.0. 1
2
2
sample/matrix . . . . . . . . . . . . . . . . . . human urine spiked amphetamine and
0
methylamphetamine at with 2 mg/mL
0 2 4 6 8
SPE tube/cartridge . . . . . . . . . . . Discovery DSC-MCAX, 100 mg/3mL (52783-U) Min
condition . . DSC-MCAX SPE tube: 1 mL methanol; 1 mL 50 mM ammonium acetate,
200
pH 6.0; C-18 SPE tube: 1 mL methanol; 1 mL DI water
washing . . . . . . DSC-MCAX SPE tube: 1 mL 50 mM ammonium acetate, pH 6.0; Standard C18
1 mL 1M acetic acid; 1mL methanol; C-18 SPE tube: 1 mL DI water;
mAU
100
1 mL 20% methanol
elution . . . . . . . . . . . . . DSC-MCAX SPE tube: 1 mL 5% ammonium hydroxide
in methanol; C-18 SPE tube: 1 mL methanol
eluate post-treatment . . . . . . . . . . evaporate to dryness with nitrogen at room
temperature, reconstitute in mobile phase
0
column . . . . . Discovery HS F5, 15 cm x 4.6 mm I.D., 5 μm particles (567516-U) 0 2 4 6 8
mobile phase . . (A) 10 mM ammonium acetate, pH 4.5: (B) methanol; (35:65, A:B) Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min Discussion:
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C Note the Y-axis scale difference between DSC-MCAX and C18 SPE.
DSC-MCAX SPE offered a maximum background height of ~9 mAU.
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm In contrast, standard C18 background levels were 20 times greater
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL than DSC-MCAX.
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003761 Also, on DSC-MCAX absolute recovery averaged at 100.3 and
101.7%, for amphetamine and methylamphetamine, respectively.
On standard C18, absolute recovery averaged at 48 and 79% for
the two compounds.

HPLC Analysis of Amphetamines on Amphetamine

(1 μg/mL) m/z 136.1 (M+H)+
Methamphetamine
(1 μg/mL) m/z 150.1(M+H)+
Discovery® HS F5
mobile phase . . (A) 0.1% ammonium acetate in water; (B) 0.1% ammonium acetate
in acetonitrile; (C) acetonitrile; (10:30:60, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . MS, ESI (+) in Single Ion Recording (SIR) Mode 0 2 4 6 8 10 0 2 4 6 8 10
Min Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ug/mL in acetonitrile MDA MDMA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003731 (1 μg/mL) m/z 180.1 (M+H)+ (1 μg/mL) m/z 194.1 (M+H)+
0 2 4 6 8 10 0 2 4 6 8 10
Min Min

HPLC Analysis of Amphetine, Methamphetamine, 1. MDA

MDA, and MDMA on Discovery® HS F5 2. Amphetamine
3. MDMA
column . . . . Discovery HS F5, 15 cm × 4.6 mm I.D., 5 μm particles (567516-U) 4. Methamphetamine
mobile phase . . . (A) 10 mM ammonium acetate in (B) water: acetonitrile (10:90) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 3 4
sample . . . . . . . . . . . . . . . 50 μg/mL each (amphetamine, methamphetamine,
MDA and MDMA) in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002081
0 2 4 6 8
Min

HPLC Analysis of AQC-Amphetamine Enantiomers * CH 3
on Astec® CYCLOBOND® I 2000 DMP

HN
column . . CYCLOBOND I 2000 DMP, 25 cm x 4.6 mm I.D., 5 μm particles (20724AST) O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL N

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004696
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Barbiturates on Ascentis® C8 1 2 1. Barbital (M-H)-= 183.08

2. Phenobarbital (M-H)-= 231.08
column . . . . . . . . Ascentis C8, 15 cm x 4.6 mm I.D., 5 μm particles (581424-U) 3. Aprobarbital (M-H)-= 209.10
4. Butabarbital (M-H)-= 211.11
mobile phase . . . . . . . . . . . . . . . . . . . (A) methanol; (B) water (45:55, A:B)
5. Mephobarbital (M-H)-= 245.10
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 6. Pentobarbital (M-H)-= 225.13
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3 7. Secobarbital (M-H)-= 237.13
4 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 6
7
sample . . . . . . . . . . . . . . . . . . . . . 10 μg/mL each in 50:50 water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003307
0 2 4 6 8 10 12
Min

HPLC Analysis of Barbiturates on Discovery® C18 1. Methylprylon (2 μg/mL)

2. Butabarbital (50 μg/mL)
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 3. Butalbital (50 μg/mL)
mobile phase . . . (A) acetonitrile; (B) 25 mM potassium phosphate buffer, pH 6.9 4. Hexobarbital (50 μg/mL)
(25:75, A:B) 5. Mephobarbital (25 μg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 3
1 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0141
5
0 2 4 6 8 10
Min

HPLC Analysis of Barbiturates on Discovery® C18, 2

1. Barbital
Unbuffered Mobile Phase 2. Aprobarbital
3. Phenobarbital
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 4. Butabarbital
mobile phase . . . . . . . . . . . . . . . . . . . (A) methanol; (B) water (45:55, A:B) 5. Mephobarbital
1 6. Pentobarbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 7. Secobarbital
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000190 5
6 7
0 10 20
Min

HPLC Analysis of Barbiturates on 3

1 1. Barbital
Discovery® RP-AmideC16 2. Phenobarbital
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 3. Aprobarbital
4. Butabarbital
mobile phase . . . . . . . . (A) methanol:acetonitrile, 60:40; (B) water; (25:75, A:B) 5. Mephobarbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 6. Pentobarbital
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 7. Secobarbital
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000193
4
6 7
0 10 20
Min

HPLC Analysis of Barbiturates on Ascentis® C18 1 1. Barbital (50 µg/mL)

2. Phenobarbital (50 µg/mL)
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 2 3. Aprobarbital (50 µg/mL)
4. Butabarbital (50 µg/mL)
5. Mephobarbital (50 µg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 3 6. Pentobarbital (50 µg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 7. Secobarbital (50 µg/mL)
4 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 6
sample . . . . . . . . . . . . . . . . . . . . . . . as indicated in 65:35 water:methanol 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002877
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Beta-Receptor Agonist

column . . . . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm, 5 μm particles (12024AST)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004337

HPLC Analysis of Bufalin Toxin on Discovery® BIO Gradient Program

Time (min) %B
Wide Pore C18
0 35
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 25 cm × 4.6 mm I.D., 15 80
5 μm particles (568223-U) 17 90
19 90
mobile phase . . (A) 0.1% TFA in water; (B) 0.1% TFA in acetonitrile:water (3:1, A:B) 21 35
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 mL/min 29 35
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 298 nm
injection . . . . . . . . . . . . . . . . . . . . 10 μL bufalin, 0.5 mg/mL in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001722

2 1. Cannabidiol
HPLC Analysis of Cannabinoids on Ascentis® C18 2. Cannabinol
column . . . . . . . Ascentis C18, 10 cm x 4.6 mm I.D., 3 μm particles (581321-U) 1 3. Δ-9-tetrahydrocannabinol
mobile phase . . . . . . . (A) 10 mM ammonium acetate (pH 4.5 with acetic acid);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002326
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Clenbuterol Enantiomers on OH

Astec® CHIROBIOTIC® T2 Cl
*
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) CH 3
mobile phase . . . . . . . . . . 0.1 wt% ammonium trifluoroacetic acid in methanol HN
H2N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min CH 3
Cl H3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004596
0 2 4 6 8 10
Min.


2. Codeine
Ascentis® C18 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002417
0 2 4 6 8
Min


2. Codeine 1
Ascentis® RP-Amide
column . . . . Ascentis RP-Amide 15 cm x 4.6 mm I.D., 5 μm particles (565324-U)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002599
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Min

HPLC Analysis of Dermorphin in Horse Plasma on Dermorphin

Ascentis® Express F5 after SPE with HybridSPE®
Dermorphin is a natural heptapeptide opioid. Because of poor recovery from
horse plasma following protein precipitation, alternate methods of
extraction are sought. While the HybridSPE protocol successfully removes
proteins and phospholipids, dermorphin solubility in plasma remains a
limitation. Dermorphin is effectively retained on the pentafluorophenyl
phase under aqueous normal phase conditions.
sample/matrix . . . . . . . . . . . . . . . . . . . . . horse plasma spiked at 10 μg/mL
SPE tube/cartridge . . [TM"HybridSPE"]-Phospholipid Plate, 50mg/96-Well (575656-U)
sample addition . . . . . . . . . . . . . 3:1, (1% formic acid in acetonitrile); plasma
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) 1.0 2.0
mobile phase . . . . . (A) acetonitrile (B) 20 mM ammonium formate; (90:10, A:B); Min
pH 4.5 with formic acid, after mixing A & B
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305 psi (90 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005540


Discovery DSC-8
External
Standard
3
um
Extract on
Excellent
E Peak Shape
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
Application No.Application No. . . . . . . . . . . . . . . . . . . . . . . . . . G003766
Blank Serumm Low

L background/
Extract on minimal
m interferences
DSC-8 for
and
a resolution at
reduced
r run times
0 2 4 6
Min

HPLC Analysis of Drugs of Abuse on Ascentis® 1. Benzoylecgonine

Express F5 2. Morphine
3. Codeine
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) 4. Oxycodone
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate; 5. Normeperidine
(B) 5 mM ammonium formate in 95:5 (v/v) acetonitrile:water 6. Hydrocodone 9
gradient . . . . . . . . . . . . held at 20% B for 0.5 min; 20 to 100% B in 5.5 min; 7. MDMA
8. Methamphetamine 10
held at 100% B for 4 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 9. Meperidine
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 bar 10. Fentanyl
11. Propoxyphene
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 100-1000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 1 μg/mL in 50:50, water: methanol 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005634 3 11
1 2 4 6
7
8
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Drugs of Abuse on Ascentis® Si 3 1. Heroin (50 µg/ml)

2. Oxycodone (50 µg/ml)
(HILIC Mode) 3. Codeine (50 µg/ml)
2 4 4. Morphine (50 µg/ml)
column . . . . . . . . . Ascentis Si, 5 cm x 4.6 mm I.D., 5 μm particles (581511-U)
mobile phase . . . . . . . . (A) 0.1% ammonium acetate in water (pH unadjusted): 1
(B) 0.1% ammonium acetate in acetontrile; (10:90, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003720

1. Morphine (50 mg/mL)

HPLC Analysis of Drugs of Abuse on Ascentis® Gradient Program
2. Hydromorphone (50 mg/mL)
Time (min) %B 1
Phenyl 0 25 3 3. Codeine (50 mg/mL)
7 40 2 4. Hydrocodone (50 mg/mL)
8 25 4
mobile phase . . . . (A) water with 0.1% ammonium acetate (pH 6.6, unadjusted);
(B) acetonitrile with 0.1% ammonium acetate
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003701

HPLC Analysis of Furosemide (Lasix) in Horse Efficiency of Recovery

Serum on Discovery® C18 after SPE using Concentration Avg. Response %Recovery
Sample (μg/mL) Factor ± RSD (n=3)
Discovery® DSC-18 A 10.00 2.307 93.1 ± 3.1
Sample Pre-treatment: B 5.00 1.168 100.8 ± 1.4
C 1.00 0.107 97.4 ± 2.8
Neat horse serum was acidified with 10 mL 6 M HCl/mL serum. Internal
D 0.50 0.065 120.7 ± 1.3
standard (I.S.) blank was prepared by spiking acidified horse serum with E 0.10 0.009 132.8 ± 8.3
indapamide at 10 mg/mL. Sample A = 10 mg/mL furosemide in I.S. blank.
Sample B = 5 mg/mL furosemide in I.S. blank. Sample C = 0.5 mg/mL
furosemide in I.S. blank. Sample D = 0.1 mg/mL furosemide in I.S. blank.
Sample E = 0.05 mg/mL furosemide in I.S. blank.
sample/matrix . . . . . acidified horse serum spiked with indapamide at 10 mg/mL
SPE tube/cartridge . . . . . . . . . . . . . Discovery DSC-18, 50 mg/1 mL (52601-U) 1. Furosemide (3.683)
condition . . . . . . . . . 1 mL methanol; 1 mL 10 mM potassium phosphate, pH 3 2. Indapamide (4.586)
washing . . . . . . . . . . . . . . . . . . . . . . . . 10 mM potassium phosphate, pH 3
1
elution . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 60% methanol in DI water. serum blank
column . . . . . . . . Discovery C18, 15 cm x 4.6 mm I.D., 5 μm particles (504955)
mobile phase . . . . . . . . . . . . (A) 10 mM KH2PO 4, pH 3 (adjusted with H3 PO 4): 2 0.05 µg/mL spike
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
0.5 µg/mL spike
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 234 nm
5 µg/mL spike
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003764
5 µg/mL ext. std.
0 2 4
Min

HPLC Analysis of Glutethimide Enantiomers on 1. Glutethimide enantiomer

1
Astec® Cellulose DMP 2. Glutethimide enantiomer
2
Glutethimide enantiomers are resolved by normal phase chromatography.
Glutethimide is used to treat insomnia.
mobile phase . . . . . . . . . . . . . . . . (A) heptane; (B) isopropanol; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
sample . . . . . . . . . . . . . . . glutethimide, 1 g/L in heptane:isopropanol (50:50)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005514
0 10 20
Min

HPLC Analysis of Heroin, Cocaine, Amphetamine 1. Heroin

2. Cocaine
and MDMA on Ascentis® Express HILIC 3. Amphetamine
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 4. MDMA (3,4-Methylenedioxymethamphetamine hydrochloride)
mobile phase . . . . . . . . . . . . (A) 10 mM ammonium acetate in water, pH 3.5; 2
3
(B) 10 mM ammonium acetate in acetonitrile; (2.98, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 100-400
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005513
1 4
0 2 4 6 8
Min

HPLC Analysis of Hippuric and Methylhippuric

1. Hippuric acid
Acids in Urine on SUPELCOSIL™ LC-18 2. o-Methylhippuric acid
3. p-Methylhippuric acid impurity
Urinary excretion of hippuric acid and m-or p-methylhippuric acid in the 4. p-Methylhippuric acid
1
urine is a test of exposure to toluene and m-or p-xylene vapors. 5. m-Methylhippuric acid
column . . . . . SUPELCOSIL LC-18, 7.5 cm × 4.6 mm I.D., 3 μm particles (58984)
mobile phase . . . . . . . . . . . . (A) tetrahydrofuran:10 mM potassium phosphate,
pH 3.0 (B) with phosphoric acid (3:97, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm 4
injection . . 10 μL spiked urine (300 μg/mL HA, o-MHA; 150 μg/mL m-MHA, p-MHA)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0961A
2
5
3
0 2 4 6 8 10
Min

HPLC Analysis of Illicit Bath Salts on Ascentis® detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), TIC, m/z 100-1000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Express HILIC sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 μg/L ea. in acetonitrile
With the rapid development of unregulated designer and synthetic Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005537
compounds, the field of illicit drug testing has recently been met with a
changing environment. Of most concern has been the development of a
1. 3,4-Methylenedioxypyrovalerone
class of phenethylamine and cathinone compounds being marketed as (MDPV)
“bath salts,” “jewelry cleaner,” or “plant food.” Though sold as “not for human 1 2. Buphedrone
consumption,” these compounds are reported to generate stimulating 3. 3-Fluoromethcathinone
4. Butylone
affects similar to that of methamphetamine, heroin, and 3,4- methylene- 5. Ethylone
dioxypyrovalerone (MDMA). For a period of time, these compounds could 6. 4-Fluoromethcathinone
be acquired legally through the internet and head shops due to no direct 7. Mephedrone
8. Methylone
legal control. In the US, both state and local governments have instituted 9. Methedrone
bans on the sale of these bath salt compounds. Forensic testing facilities 5
2 4
often experience difficulty in testing these compounds due to the fact that
they are not detected under normal ELISA testing methods; additional more
specific LC-MS methods are necessary. The challenge for LC-MS detection of 3 6 7
these particular bath salts resides in three sets of isobaric compounds that 8 9
require chromatographic resolution for positive confirmation. For example,
both butylone and ethylone have the same mono isotopic mass, making
these compounds indistinguishable, even when using accurate mass time of
flight TOF-MS. Efficient chromatographic separation is necessary for accurate 0 2 4 6
quantitation of these compounds. The polar basic nature of the bath salts Min
makes these compounds difficult to retain on traditional reversed-phase C18
and even polar embedded stationary phases, making them prime subjects
for HILIC chromatographic separation. Shown here is the fast, high-
resolution separation of nine synthetic bath salts on Ascentis Express HILIC.
mobile phase . . . . . . . . . . . . . (A) acetonitrile; (B) 0.25 M ammonium formate;
(98:2, A:B); premixed
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1842 psi (127 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C

HPLC Analysis of Illicit Bath Salts on Ascentis® 2 1. Butyolne

2. Ethylone
Express HILIC, Fast Analysis 3. Mephedrone
1
With the rapid development of unregulated designer and synthetic 4. Methedrone
compounds, the field of illicit drug testing has recently been met with a 5. Methylone
changing environment. Of most concern has been the development of a
3 4
class of phenethylamine and cathinone compounds being marketed as 5
“bath salts,” “jewelry cleaner,” or “plant food.” Though sold as “not for human
consumption,” these compounds are reported to generate stimulating
affects similar to that of methamphetamine, heroin, and 3,4- methylene-
dioxypyrovalerone (MDMA). For a period of time, these compounds could
be acquired legally through the internet and head shops due to no direct
legal control. In the US, both state and local governments have instituted
bans on the sale of these bath salt compounds. Forensic testing facilities
often experience difficulty in testing these compounds due to the fact that 1.0 2.0 3.0
they are not detected under normal ELISA testing methods; additional more Min
specific LC-MS methods are necessary. The challenge for LC-MS detection of
these particular bath salts resides in three sets of isobaric compounds that
require chromatographic resolution for positive confirmation. For example,
both butylone and ethylone have the same mono isotopic mass, making
these compounds indistinguishable, even when using accurate mass time of
flight TOF-MS. Efficient chromatographic separation is necessary for accurate
quantitation of these compounds. The polar basic nature of the bath salts
makes these compounds difficult to retain on traditional reversed-phase C18
and even polar embedded stationary phases, making them prime subjects
for HILIC chromatographic separation. Shown here is the fast, high-
resolution separation of nine synthetic bath salts on Ascentis Express HILIC.
mobile phase . . . . . . . . . A: 5 mM ammonium formate (95:5 acetonitrile:water)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005444

HPLC Analysis of Ketamine Enantiomers on Astec® O
(+)
Cellulose DMP *
NH
CH 3
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) Cl (-)

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004990
0 2 4 6 8 10 12
Min

HPLC Analysis of Ketoprofen Enantiomers on 3.46

Astec® CHIROBIOTIC® R (MS Detection)
4.31 CH3
column . . . . . . . . CHIROBIOTIC R, 15 cm x 2.1 mm, 5 μm particles (13019AST) O
mobile phase . . (A) 20 mM ammonium acetate, pH 5.6 (B) methanol; (70:30, A:B) 36 O

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
Response
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(-) 24
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004331
12
0
0.98 1.00
0 5 10
Min

HPLC Analysis of Metabolites of 7,12- 1. 2-OH-DMBA(t-diol) 11. 7-OHM-12-MBA-4-ol

Dimethylbenz[a]anthracene on 3. 7-M-12-OHMBA-(t-8,9-diol) 13. 7-M-12-OHMBA-2-ol
5. 7-OHM-(t-3,4-diol) 15. 7-M-12-OHMBA
10. 7-M-12-OHMNA-4-ol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1043 6 11 14 16 18 19
1
8 10
5 9
12 17
4 13
2 15
3
7
0 4 8 12 16 20 24 28 32 36 40 44 48
Min

HPLC Analysis of Methadone Enantiomers and Peak

1
Rt (min)
8.5
Peak ID
R-Methadone
Methadone Metabolite EDDP on Astec® 2 9.7 S-Methadone
3 11.0 R,S-EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine)
CYCLOBOND® I 2000 HP-RSP
column . . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D., 1
2
mobile phase . . . . . . . . . . . (A) 0.1% acetic acid; (B) acetonitrile; (80:20, A:B)
* CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 292 nm 3
N
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004734 CH3
H3C
EDDP

HPLC Analysis of Methadone Enantiomers on

Astec® CHIROBIOTIC® V2 O
CH3
mobile phase . . . . . . (A) methanol; (B) 20 mM ammonium formate; (95:5, A:B) CH3 HCI
N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
H3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . methadone, 1 mg/mL in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004403
0 10 20 30
Min

HPLC Analysis of Methcathinone and Ephedrine 1. Methcathinone (100 μg/mL)

2. (+/-) Ephedrine (200 μg/mL)
on Discovery® HS F5
1
mobile phase . . . . . . . . . . . . . . . . (A) 10 mM ammonium acetate (pH 6.98);
(B) acetonitrile (30:70, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . photodiode array
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001673
0 1 2 3 4 5 6 7 8
Min

HPLC Analysis of Methylphenidate Enantiomers O O

on Astec® CHIROBIOTIC® V H3C
H
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) N *
*
mobile phase . . . . . . . (A) methanol; (B) 1% triethylamine, pH 4/1; (95:5, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004505
0 2 4 6 8 10
Min.

HPLC Analysis of Methylphenidate (Ritalin) O O

H 3C
Enantiomers on Astec® CHIROBIOTIC® V2 H
N *
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) *
mobile phase . . . . . . . . . . . . . . . . . . 15 mM ammonium acetate in methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004504
0 2 4 6 8 10 12
Min.

HPLC Analysis of 3-Methylpyrazole and Efficiency of Recovery

4-Methylpyrazole in Urine on Discovery® C18 after Discovery Leading Competitor
Compound (mg/mL) DSC-SCX (n=3) SCX (n=2)
SPE using Discovery® DSC-SCX % Recovery %RSD %Recovery %RSD
1. 3-methylpyrazole (1.0) 89.4 ±10.2% 67.1 ±20%
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 2. 4-methylpyrazole (1.0) 79.4 ±6.8% 50.5 ±30%
sample/matrix . . . . . . . . urine spiked with 3- and 4-methylpyrazole at 1 mg/mL
SPE tube/cartridge . . . . . . . . . . . . Discovery DSC-SCX, 500 mg/3 mL (52686-U)
SPE Procedure
condition . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL methanol; 2 mL DI water
1. Condition and equilibrate with
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 2 mL MeOH & 2 mL DI water.
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL DI water 2 2. Load 1 mL urine sample spiked with
elution . . . . 2 mL 5% methanol in 250 mM potassium phosphate buffer, pH 7.4 1 mg/mL of each analyte.
column . . . . . . . . . . . . Discovery C18, 15 cm × 4.6 mm, 5 μm preceded by a 1 3. Wash with 2 mL DI water.
2 cm guard column and 0.5 μm frit filter (504955) 4. Elute with 2 mL 5% MeOH in
mobile phase . . . . . . . . . . . . . . . MeOH:5 mM phosphate buffer, pH 6 (20:80) 250 mM phosphate buffer, pH 7.4.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 5. Quantify against external standards
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C via HPLC analyses.
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL, dilute urine extract
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001635
0 2 4 6 8 10 12 14
Min

1. Propoxyphene 2
HPLC Analysis of Propoxyphene and Methadone 2. Methadone 1
on Ascentis® Phenyl
mobile phase . . (A) water with 0.1% ammonium acetate: (B) methanol; (20:80, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 20:80, water:methanol 0 2 4 6 8 10
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003699

HPLC Analysis of Ritalinic Acid and Ritalin HO O

(Methylphenidate) Enantiomers on Astec®
CHIROBIOTIC® V2 with T2 Guard Column H
N *
column . . . . . . . . . . . . . . . CHIROBIOTIC T2 guard column, 2 cm x 4 mm I.D., *
5 μm particles (16100AST (requires holder 21150AST))
O O
mobile phase . . (A) 20 mM ammonium acetate, pH 4.1; (B) methanol; (7:93, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Ritalinic acid H
N *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C *
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 3.9
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Ritalin
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004733
12.7
15.5
5.5

HPLC Analysis of Ritalinic Acid Enantiomers on

Astec® CHIROBIOTIC® T2
*
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) * N
mobile phase . . (A) 10 mM ammonium acetate, pH 5.5; (B) acetonitrile; (70:30, A:B) H
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min HO O

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004600
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Selegiline and Amphetamine

1. Selegiline 1
Metabolites on Discovery® HS F5 2. Methamphetamine 2
column . . . . . Discovery HS F5, 5 cm × 4.6 mm I.D., 5 μm particles (567513-U) 3. Amphetamine

mobile phase . . . (A) water (10 mM ammonium acetate, pH 4.0 with acetic acid:
(B) acetonitrile (v/v), (25:75, A:B)
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002136
1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of Spice Cannabinoids on Ascentis® 1. JWH-073 metabolite 5. JWH-073

Express C18 2. JWH-250 6. JWH-018
3. CP-47-497 7. JWH-200
Synthetic cannabinoids (Spice) are a relatively new type of designer drug 4. CP-47 -497 C8 8. HU-210, HU-211
used as a pseudo-legal means to get a cannabis-type high. New synthetic
cannabinoids are continually being introduced as suppliers tweak the
1
molecular structures. The ability to rapidly and reliably identify the 2
continually changing population of these compounds in the blood or urine 4 5
suspected users is a significant analytical challenge facing forensic chemists. 3 8
7
mobile phase . . . . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate water;
(B) 5 mM ammonium formate (95:5, acetonitrile:water)
gradient . . . . . . . . . . . . . 68 to 100% B in 8 min; held at 100% B for 2 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4351 psi (300 bar) 0 2 4 6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 265 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005740

HPLC Analysis of Spice Cannabinoids JWH-018, 1. JWH-073 4-butanoic acid metabolite

5
2. JWH-018 5-pentanoic acid metabolite
JWH-073 and Metabolites on Ascentis® Express 6
3. JWH-073 3-hydroxybutyl metabolite
C18, 2.7 μm particles 4. JWH-018 4-hydroxypentyl metabolite
column . . Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 μm particles (53814-U) 5. JWH-073
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% formic acid in water; 6. JWH-018
1
(B) 0.1% formic acid in water:acetonitrile (10:90) 2 3
gradient . . 20% B held for 3 min; 20 to 100% B in 0.1 min; 100% B held for 2 min 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2150 psi (148 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 315 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 μg/mL each in acetonitrile 0 2 4 6 8

HPLC Analysis of Spice Cannabinoids JWH-018, 1. JWH-073 4-butanoic acid metabolite 5

2. JWH-018 5-pentanoic acid metabolite
JWH-073 and Metabolites on Ascentis® Express 6
3. JWH-073 3-hydroxybutyl metabolite
C18, 5 μm particles 4. JWH-018 4-hydroxybutyl metabolite
column . . . Ascentis Express C18, 10 cm x 3.0 mm I.D., 5 μm particles (50526-U) 5. JWH-073
6. JWH-018
1 3
(B) 0.1% formic acid in water:acetonitrile (10:90)
gradient . . 20% B held for 3 min; 20 to 100% B in 0.1 min; 100% B held for 2 min 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1037 psi (71.5 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 315 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005834 0 2 4 6 8
Min

HPLC Analysis of Spice Cannabinoids on Ascentis® 1. JWH-073 metabolite 5. HU-210, HU-211

2. JWH-200 6. JWH-250
Express F5 3. CP-47, 497 7. JWH-073
Synthetic cannabinoids (Spice) are a relatively new type of designer drug 4. CP-47, 497 8. JWH-018
used as a pseudo-legal means to get a cannabis-type high. New synthetic 3
cannabinoids are continually being introduced as suppliers tweak the 5
1 4
molecular structures. The ability to rapidly and reliably identify the
2
continually changing population of these compounds is a significant 6
analytical challenge facing forensic chemists. A rapid separation of nine of 7
these compounds on Ascentis Express F5 column is shown here. 8
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 50 mM ammonium formate;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 1.0 2.0 3.0
Min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4075 psi (281 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 200 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 100 μg/mL in 45:55 water:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005446

HPLC Analysis of Steroids on Ascentis® C18 1 1. Prednisone

2. Hydrocortisone 21-acetate
(Mobile phase: 60:40) 3. Cortisone acetate

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002432
0 2 4 6 8 10
Min

HPLC Analysis of Tetramisole Enantiomers and 1. Aminorex

Related Compounds on Astec® Cyclobond® I 2. Dexamisole 2
3. Levamisole
2000 DMP 4. Cocaine 3
Cocaine is often found laced with levamisole (one of the tetramisole
enantiomers) and aminorex is a metabolite of levamisole known to cause
disorders.
column . . . . . . . . . . . . . . . . Astec CYCLOBOND I 2000 DMP 10 cm X 2.1 mm
1
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 100 mM ammonium acetate,
pH 5 with acetic acid (B) acetonitrile, 90:10, v/v 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 psi
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL 0 2 4 6 8 10
sample . . . . . . . . . . . . . . . . . . . . 100 ug/mL tetramisole and 50 ug/mL each Min
cocaine and aminorex in water:methanol, 80:20, v/v
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006305

HPLC Analysis of Tetramisole Enantiomers on 1. Dexamisole

1
2. Levamisole
Astec® Cyclobond® I 2000 DMP 2
Cyclobond I 2000 DMP is effective at chiral recognition of tetramsiole
enantiomers. This method appears to be a suitable starting point for further
development towards the LC/MS analysis of tetramisole in various matrices.
The method is also a suitable replacement for published methods utilizing
Cyclobond I 2000 SN (no longer available) for the analysis of Levamisole.
column . . . . . . . . . . . . . ASTEC CYCLOBOND I 2000 DMP 25 cm x 4.6 mm I.D.,
pH 5 with acetic acid (B) acetonitrile, 80:20, v/v
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
0 2 4 6 8 10
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Ref: 1. D. W. Armstrong, C. D. Chang, S. H Lee, J. Chromatogr. A 539, 83-90 (1991)
sample . . . . . . . 200 ug/mL tetramisole in mobile phase A:methanol, 80:20, v/v 2. M. Dolezalova, M. Tkaczykova, J. Pharm. Biomed. Anal. 25, 407-415 (2001)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006293

HPLC Chiral Analysis of Levamisole on Astec® 2

1. Dexamisole
Cyclobond® I 2000 DMP 2. Levamisole
Cyclobond I 2000 DMP is effective at chiral recognition of dexamisole from
levamisole formulations. This method appears to be a suitable starting point
for further development towards the analysis of levamisole chiral purity. The
method is also a suitable replacement for published methods utilizing
Cyclobond I 2000 SN which is no longer available.
column . . . . . . . . . . . . . ASTEC CYCLOBOND I 2000 DMP 25 cm x 4.6 mm I.D.,
5μm particles (20724AST) 1
pH 5 with acetic acid (B) acetonitrile, 80:20, v/v
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1600 psi (110 bar) 0 2 4 6 8 10
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
Ref: 1. D. W. Armstrong, C. D. Chang, S. H Lee, J. Chromatogr. A 539, 83-90 (1991)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 2. M. Dolezalova, M. Tkaczykova, J. Pharm. Biomed. Anal. 25, 407-415 (2001)
sample . . . . . . . 200 ug/mL tetramisole in mobile phase A:methanol, 80:20, v/v
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006294

LC-MS Analysis of 2C Amines Drugs of Abuse in Peak Compound Rentention m/z

Urine on Ascentis® Express HILIC following Sample 1 Bromo Dragonfly 2.03 294
2 DOI 2.18 322
Prep using Supel™-Select SCX SPE 3 2C-T-7 2.27 256.1
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 4 DOB 2.29 274.1
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . spiked urine, 1 mL 5 2C-B 2.54 260
SPE tube/cartridge . . Supel-Select SCX SPE 96-well Plate, 30 mg/well (575664-U) 6 2C-B-Fly 2.74 284
condition . . . . . . . . . . . 1 mL methanol, then 1 mL 0.1% formic acid in water 3, 4
washing . . 2 x 1 mL 10% methanol in water, 2 x 1 mL 1% formic acid in methanol 2
elution . . . . . . . . . . . . . . . . . . 2 mL 5% ammonium hydroxide in acetonitrile
eluate post-treatment . . evaporate to 500 μL with nitrogen at room temperature,
reconstitute in 250 μL acetonitrile
mobile phase . . . . . . 5 mM ammonium formate in acetonitrile:water (90:10, v/v) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1570 psi (108 bar) 6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL 0 2

LC-MS Analysis of Amphetamines on Ascentis® 1. 4-Fluoroamphetamine 1

Express HILIC 2. Amphetamine 2 3
3. Methamphetamine
column . . Ascentis Express HILIC, 10 cm x 3 mm I.D., 2.7 μm particles (53970-U)
mobile phase . . (A) 1 mM ammonium acetate in (B) 10:90 (v/v) water:acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189 psi (82 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
0 2 4 6 8 10 12 14 16
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L each in acetonitrile

LC-MS Analysis of Cannabinoids on Ascentis® 1 1. Cannabidiol m/z, 315.5

2. Cannabinol m/z, 311.4
RP-Amide 3. Δ9-tetrahydrocannabinol m/z, 315.5
4. Δ8-tetrahydrocannabinol m/z, 315.5
mobile phase . . . . . . . (A) 10 mM ammonium acetate (pH 5.0 with acetic acid): 2
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min, split to the MS 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS ESI (+), SIR Mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . 1 μg/mL in water:acetonitrile (1:1)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002681 0 10 20
Min


Clenbuterol
CI
600 CH3
H2N
CI H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
400
cps
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+)
300
sample . . . . . . . . . . . . . . . . . . . . . . . . clenbuterol, 10 ng/mL in rat plasma
(phospholipids removed by extraction with HybridSPE-Phospholipid) Clenbuterol
200
100
0
0 1 2 3 4 5
Min

LC-MS Analysis of Clenbuterol In Plasma on Astec® HybridSPE-PPT Max 767 CPS

HybridSPE® Phospholipid 600
95% absolute recovery
The HybridSPE method provides significant improvement in LC-MS baseline. 500

400 Clenbuterol
CPS
sample/matrix . . . . rat plasma spiked with clenbuterol enantiomers at 10 ng/mL Phospholipids
SPE well plate . . . HybridSPE-Precipitation 96-well Plate, 50 mg/well (575656-U) 300
acetonitrile. Mix by vortexing the HybridSPE-PPT plate briefly.
100
0 1 2 3 4 5
mobile phase . . . . . . . . . . . . . . . . . 10 mM ammonium formate in methanol Min
Max 775 CPS
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min Standard Protein PPT
6000
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 3-step procedure
detector . . . . . . . . . . ABI 3200 QT; ESI(+), MRM: 184/104 m/z (phospholipids); 5000 No phospholipid removal
277.2/203.1 m/z (clenbuterol)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4000
CPS
3000
Clenbuterol
2000
1000
0
0 1 2 3 4 5
Min

LC-MS Analysis of Cocaine, Cocaethylene, 1. Oxycodone

2. Noroxycodone 3 4
Oxycodone, and Noroxycodone on
3. Cocaine
Discovery® HS F5 4. Cocaethylene
mobile phase . . . . . . . . . . . . (A) 10 mM ammonium formate in water, pH 6.8;
(B) 10mM ammonium formate in acetonitrile, pH 6.8; (10:90, A:B) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+)
1.0 2.0 3.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Min
sample . . . . . . . . . . . . . . . . . . . . . . 200 ng/mL in 25:75, water:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005687

LC-MS Analysis of Dermorphin in Horse Plasma on

Ascentis® Express F5
Dermorphin is a heptapeptide derived from frogs. It is a painkiller, and used
illicitly in racehorses so they continue to run through the pain.
sample . . . . . . . . . . . . . . 2 mM ammonium formate (90:10 acetonitrile:water)
Dermorphin
adjusted to pH 4.5 with formic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . ESI(+), TIC 100 – 1000 m/z; APCI (+), XIC m/z = 150.1, 203.7, 378.2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL in 75:25 acetonitrile:water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005826
0 2 4
Min

LC-MS Analysis of Drugs of Abuse on Ascentis® 1. Norfentanyl

Express RP-Amide 2. Benzoylecgonine
4 3. Cocaine
This applic.ation demonstrates the suitability of Ascentis Express RP-Amide 4. Cocaethylene
for the efficient separation of common drink additives used in diet cola 5. EDDP
3
column . . . . . . . . . . . . . . . Ascentis Express RP Amide, 10 cm x 2.1 mm I.D., 6. Methadone
mobile phase . . . . . . . . . . . . . . . . . (A) 10 mM ammonium formate in water;
1 2 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+) 0 2 4 6 8
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 ng/mL in water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005695

LC-MS Analysis of Explosives on Ascentis® 1. HMX 5

Express C18 2. RDX
3. 1,3,5-Trinitrobenzene
column . . Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 μm particles (53814-U) 4. 1,3-Dinitrobenzene
mobile phase . . . . . . . . . . . . . . . . . . . (A) methanol; (B) water; (50:50, A:B) 5. 2,4,6-Trinitrotoluene
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min 6. 2,4-Dinitrotoluene
4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS: APCI(-) 3
6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 100 ng/mL in 50:50, methanol:water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005608
1
2
1.0 2.0
Min

LC-MS Analysis of Fluoroamphetamine Drugs of 1. 2-Fluoroamphetamine 3

Abuse on Ascentis® Express HILIC 2. 4-Fluoroamphetamine
4
3. 2-Fluoromethamphetamine
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 4. 4-Fluoromethamphetamine
gradient . . . . . . . . . 5 mM ammonium formate in acetonitrile:water (90:10, v/v)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006165
0 1 2 3 4
Min

1
LC-MS Analysis of GABA and AICAR on Ascentis® 1.
2.
AICAR (5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside)
Ethyl-β-D-glucuronide
MS Polarity Switch
(2.27 min)
Express HILIC 3. 5-Hydroxytryptophan
4. GABA (γ-Aminobutyric acid) 3
This study focuses on the analysis of possible dopants used in equine racing,
polar compounds requiring HILIC chromatographic separation.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) ESI (+) ESI (–)
sample/matrix . . . . . . . . . . . . . . . . . . . rat plasma or human serum spiked at
0.05 ng/mL with digitoxin and digoxin
SPE well plate . . . . . . . . HybridSPE-PLus 96-well Plate, 50 mg/well (575659-U) 2
sample addition . . . . . . . . . . . . . . . . . 100 μL spiked rat plasma followed by 4

elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . apply vacuum 0 2
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) Min
mobile phase . . . . . . (A) 5 mM ammonium formate in acetonitrile:water (95:5);
(B) 5 mM ammonium formate in acetonitrile:water (70:30)
gradient . . . . . . . . . . . . . . . . . . . . . 0% to 70%B in 3 min, held for 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI+/ESI-
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μg/mL in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006301

LC-MS Analysis of Illicit Bath Salts in Urine on detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), TIC, m/z 100-1000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Ascentis® Express HILIC after Solid Phase sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 μg/L ea. in acetonitrile
Extraction (SPE) on Supel™-Select SCX Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005796
The designer drugs known as "bath salts" are synthetic stimulants and illegal
in most states as of 2011. This application demonstrates the analysis of bath
salts extracted from human urine samples using polymeric solid phase 1. MDPV
extraction (SPE) sample preparation followed by hydrophilic interaction 2. Buphedrone
liquid chromatography (HILIC) analysis with TOF-MS detection. HILIC 1 3. 3-Fluoromethcathinone
conditions on the Ascentis Express HILIC (Si) phase are used for fast, high- 4. Butylone
resolution separation of nine synthetic bath salts. Recoveries greater than 5. Ethylone
65% were observed for all analytes except MDPV (43.7%). The Figure 2 4 6. 4-Fluoromethcathinone
5
illustrates the detection of bath salts in the spiked urine sample after SPE 7. Mephedrone
sample cleanup. Notice there are no interfering peaks in the chromatogram, 8. Methylone
demonstrating the effectiveness of the SPE sample cleanup. 9. Methedrone
sample/matrix . . Urine samples were spiked to a level of 100 ng/mL with each target
7
analyte. (To ensure full ionization of the analytes, spiked samples were 8 9
treated with formic acid to a final concentration of 0.1% formic acid.) 6
SPE tube/cartridge . . . . . . . . . . . . . . Supel-Select SCX, 30 mg/1 mL (54240-U) 3
condition . . . . . . . . . . . . . . 1 mL 1% formic acid acetonitrile then 1 mL water
sample addition . . . . . . . . . . . . . . . . . . . . 1 mL spiked water blank or urine
washing . . . . . . . . . 1 mL water, 1 mL 1% formic acid acetonitrile, 1 mL water
elution . . . . . . . . . . . . . . . . . 2 mL 10% ammonium hydroxide in acetonitrile
1.0 2.0 3.0 4.0 5.0 6.0
eluate post-treatment . . thoroughly mix via vortex agitation, evaporate 1 mL aliquot
to dryness, reconstitute in 100 μL water:methanol Min
mobile phase . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate acetonitrile;
(B) 5 mM ammonium formate water; (98:2, A:B); premixed
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1842 psi (127 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C

LC-MS Analysis of Illicit Bath Salts on Ascentis® Compound m/z

1. Naphyrone 282
Express HILIC, Set 1 1 2. Pentylone 236
3. MDPBP 262
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 4. 3-Methoxymethcathinone 194
mobile phase . . 5 mM ammonium formate in (A) acetonitrile; (B) water; (98:2, A,B) 5. Methedrone 194
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 psi (18.4 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . MS, ESI(+) TOF, Summed Ion Chromatogram 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 4
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 ng/mL in acetonitrile 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005910
0 2 4 6
Min


1. α-Pyrrolidinovalerophenone 232
Express HILIC, Set 2 1 2. 3,4-Methylenedioxypyrovalerone 276
3. Butylone 222
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 2 4. Ethylone 222
mobile phase . . (A) 5 mM ammonium formate in acetonitrile: (B) water; (98:2, A;B) 5. Buphedrone Ephedrine Metabolite 180
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 6. (±)-4-Methylephedrine 180
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 psi (18.4 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
3 4
detector . . . . . . . . . . . . . . . . . . MS, ESI(+) TOF, Summed Ion Chromatogram
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005911 5 6
0 2 4 6 8 10 12 14
Min


1. Pentedrone 192
Express HILIC, Set 3 2. 3-Fluoromethcathinone 182
3. 4-Methylethcathinone 192
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 4. 4-Fluoromethcathinone 182
mobile phase . . (A) 5 mM ammonium formate in acetonitrile; (B) water; (98:2, A:B) 1 5. 3,4-Dimethylmethcathinone 182
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 psi (18.4 bar) 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 ng/mL in acetonitrile 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005912
4
0 2 4 6
Min

LC-MS Analysis of Illicit Bath Salts on Ascentis® 1

Compound m/z
1. Pyrovalerone 246
Express HILIC, Set 4 2. Buphedrone 178
3. N-Ethylcathinone 178
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 4. Mephedrone 178
mobile phase . . 5 mM ammonium formate in (A) acetonitrile; (B) water; (98:2, A:B) 5. Methylone 208
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 6. MDAI 178
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 psi (18.4 bar) 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 4 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005913 6
0 10 20
Min

LC-MS Analysis of Isobaric Bath Salts 3- and 2 1. 3-Fluoromethcathinone

4-Fluoromethcathinone on Ascentis® 2. 4-Fluoromethcathinone
Express HILIC
mobile phase . . . . . . . . . . . 5 mM ammonium formate (98:2 acetonitrile:water)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 psi (87 bar) 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 182 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ng/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006224
0 2 4 6 8
Min

LC-MS Analysis of Isobaric Bath Salts Buphedrone 1. Buphedrone ephedrine

2. 4-Methylephedrine 1
Ephedrine and 4-Methylephedrine on Ascentis®
Express HILIC
mobile phase . . . . . . . . . . . 5 mM ammonium formate (98:2 acetonitrile:water) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 psi (87 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 180 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ng/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006226
0 2 4 6 8
Min

LC-MS Analysis of Isobaric Bath Salts Ethylone and 1 2

1. Butylone
Butylone on Ascentis® Express HILIC 2. Ethylone

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 psi (87 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 222 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ng/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006228
0 2 4 6 8
Min

LC-MS Analysis of Isobaric Bath Salts Mephedrone, 1. Buphedrone

2. N-Ethylcathinone
Buphedrone, N-Ethylcathinone and MDAI on 2 3. Mephedrone
1
Ascentis® Express HILIC 4. MDAI (5,6-Methylenedioxy-2-aminoindane)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 psi (87 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 178 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ng/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006227 4
0 2 4 6 8
Min

LC-MS Analysis of Isobaric Bath Salts 1 1. 3-Methoxymethcathinone

3-Methoxymethcathinone and 2. Methedrone
Methedrone on Ascentis® Express HILIC
column . . Ascentis Express HILIC, 5 cm x 2.1 mm I.D., 2.7 μm particles (53934-U) 2
mobile phase . . . . . . . . . . (5 mM ammonium formate (98:2 acetonitrile:water))
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 psi (87 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 194 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ng/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006225
0 2 4 6 8
Min

LC-MS Analysis of Isobaric Bath Salts 1 1. Pentedrone

Pentedrone, 4-Methylethcathinone and 2. 4-Methylethcathinone
3. 3,4-Dimethylmethcathinone
3,4-Dimethylmethcathinone on Ascentis® Express 2
HILIC
mobile phase . . . . . . . . . . . 5 mM ammonium formate (98:2 acetonitrile:water) 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 psi (87 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 192 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ng/mL 0 2 4 6 8

LC-MS Analysis of Methadone and Metabolites 1. EDDP (2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine)

EDDP and EMDP on Ascentis® Express RP Amide 2. Methadone
3. EMDP (2-Ethyl-5-methyl-3,3-diphenylpyrroline)
mobile phase . . . . . . . . . . . . (A) 10 mM ammonium formate in water, pH 3.6; 2 3
(B) 10 mM ammonium formate in acetonitrile, pH 3.6; (65:35, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 100-1000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 200 μg/L in 25:75, water: acetonitrile 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005709
1.0 2.0 3.0
Min

LC-MS Analysis of Selegiline and Amphetamines 1. Selegiline 3

on Ascentis® Express F5 2. Amphetamine
3. Methamphetamine
column . . . Ascentis Express F5, 10 cm x 4.6 mm I.D., 2.7 μm particles (53590-U) 2
mobile phase . . . . . . . . (A) 10 mM ammonium acetate, pH 4.0 with acetic acid;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
1
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2320 psi (160 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), SIR, m/z 136, 150, 188
0 10 20
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005734

LC-MS/MS Analysis of Drugs of Abuse: Generic 1. Benzoylecgonine

9
Screening on Ascentis® Express F5 2. Morphine
3. Codeine 10
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) 4. Oxycodone
mobile phase . . . . . . . (A) 5 mM ammonium formate water pH 4.0 formic acid; 5. Normeperidine
(B) 5 mM ammonium formate (95:5 acetonitrile:water) 6. Hydrocodone
gradient . . . . . 20%B for 0.5 min; to % B in 5.5 min; held at 100%B for 3 min 7. MDMA
8. Methamphetamine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 9. Meperidine
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2538 psi (175 bar) 10. Fentanyl
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 11. Propoxyphene 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), TIC, m/z 100-1000 3 11
4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 1 2 6
7
sample . . . . . . . . . . . . each compound 1000 ng/mL in 40:60 (water:methanol) 8
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005881
0 2 4 6 8 10
Min

LC-MS/MS Analysis of Spice Compounds from detector . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), MRM, m/z 344/155 (JWH-073 metabolite), 385/155 (JWH-200),
336/121 (JWH-250), 328/155 (JWH-073), and 342/155 (JWH-018)
Plasma on Ascentis® Express F5 after SPE using injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
HybridSPE®-Phospholipid Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005786
Synthetic cannabinoids (Spice) are a relatively new type of designer drug
used as a pseudo-legal means to get a cannabis-type high. New synthetic 1. JWH-073 3-hydroxybutyl metabolite
cannabinoids are continually being introduced as suppliers tweak the 2. JWH-200
molecular structures. The ability to rapidly and reliably identify the ((1-(2-Morpholin-4-ylethyl)indol-3-yl)-naphthalen-1-ylmethanone)
continually changing population of these compounds in the blood or urine 3. JWH-250
suspected users is a significant analytical challenge facing forensic chemists. (2-(2-Methoxyphenyl)-1-(1-penylindol-3-yl)ethanone)
A four prong approach using column selectivity, high purity solvents, 4. JWH-073
(Naphthalen-1-yl-(1-butylindol-3-yl)methanone)
effective sample prep, and reference standards, was used to develop a 5. JWH-018
method to rapidly isolate and identify Spice cannabinoids from plasma. The (Naphthalen-1-yl-(1-pentylindol-3-3yl)methanone)
Ascentis Express F5 column gave the necessary resolution, and the LC-MS
CHROMASOLV solvents and additives gave adduct-free response for
maximum sensitivity. Sample prep employing the HybridSPE-Phospholipid 2
was rapid and effective and the Cerilliant reference standards enabled 1
confident identification. 3
sample/matrix . . . . . . . . . . . . . . rabbit plasma, unfiltered K2-EDTA spiked with
Spice cannabinoids (5 ng/mL each)
SPE tube/cartridge . . . . . . . . . . . . . . . . HybridSPE-Phospholipid, 96-well plate,
50 mg bed wt., 2 mL well vol (575656-U) 4
1% formic acid in acetonitrile, agitate on orbital shaker for 2 minutes 5
elution . . . . . attach collection plate and apply vacuum at 10” Hg for 4 minutes
mobile phase . . . . . . . . . . . . . . . . . (A) 10 mM ammonium formate in water,
pH 6.8 (unadjusted); (B) acetonitrile; (50:50; A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 0 2 4 6
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296 psi (89 bar) Min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C

LC-MS (TOF) Analysis of 2C Amine Drugs of Abuse 1. Bromo DragonFly

2
on Ascentis® Express HILIC 2. 2C-T-7
3. 2C-B
column . . Ascentis Express HILIC, 5 cm x 2.1 mm I.D., 2.7 μm particles (53934-U) 4. 2C-B-Fly
mobile phase . . . . . . 2 mM ammonium formate in 90:10 (v:v) acetonitrile:water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 943 psi (65 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1 4
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006152
0 2
Min

LC-MS (TOF) Analysis of Opioids and Opiate- 1. Oxycodone

Dependence Management Drugs on Ascentis® 2. 6-Acetylcodeine
3. 6-Acetylmorphine
Express HILIC 4. Codeine
5
column . . Ascentis Express HILIC, 5 cm x 2.1 mm I.D., 2.7 μm particles (53934-U) 2 5. Hydrocodone
mobile phase . . . . . . 2 mM ammonium formate in 90:10 (v:v) acetonitrile:water 6. Morphine
7. Hydromorphone
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C 1 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS 7
4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006153
6
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Min


1. Uracil
3. Phenobarbital
5. Butalbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 4
5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7540 psi (520 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006073
1.0 2.0 3.0 4.0

Min


3. Cyclobarbital
4. Butalbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6796 psi (468 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006137
1 2 3 4 5
Min

UHPLC Analysis of Cannabinoids Using Ascentis® 1. Cannabidiol

1
2. Cannabinol
Express 5μm RP-Amide 3. (-)-Δ9-THC
2
3
4. (-)-Δ8-THC
column . . Ascentis Express RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (50774-U) 4
mobile phase . . . . . . . . . . 5 mM ammonium acetate (pH 4.5 with acetic acid)
in 20:80, acetonitrile:water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 μL
sample . . . . . . . . . . . . . . . . 4 cannabinoids 50 μg/mL 50:50, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006303
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

Min

UHPLC Analysis of Cocaine and Cocaethylene on 1. Cocaine, 2.18 1

Titan™ C18 2. Cocaethylene, 3.12
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, XIC m/z 304.15 and 318.17
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006091
0 2 4
Min


5
3.303
Titan C18 1.9 mm
phase.
445 bar // 6,450 psi*
3 6
1
2.265
4.299
3.080
1.846
0.447
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6450 psi (445 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 1 2 3 4
1.970
Competitor C18 1.7 µm
1.466
2.545
1.265
0.445
1 2 3 4
Min

UHPLC Analysis of Heroin and Related 1. Morphine (m/z 286) 7. Papaverine (m/z 340)
Compounds on Titan™ C18 2. Procaine (m/z 237) 8. Flunitrazepam (m/z 314)
3. Codeine (m/z 300) 9. Diazepam (m/z 285)
column . . . . . . . . . Titan C18 5 cm x 2.1 mm I.D., 1.9 μm particles (577122-U) 4. 3-Monoacetylmorphine (m/z 328) 10. Noscapine (m/z 414)
mobile phase . . . . . . . (A) 5 mM ammonium acetate in 95:5, water:acetonitrile; 5. Heroin (m/z 370)
(B) 5 mM ammonium acetate in 5:95, water:acetonitrile 6. Alprazolam (m/z 309) 7
gradient . . . . . . . . . . . . . . 0 to 100% B in 3 min, held at 100% B for 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6770 psi (467 bar) 9
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 5 10
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS-TOF, XIC 3
2 4 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μg/mL in 95:5, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006116 8
0 2
Min

UHPLC Analysis of Morphine and Metabolites on 1. Morphine-3-β-D-glucuronide

Titan™ C18 using MS Detection 2 2. Normorphine
3. Morphine
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) water with 0.1% formic acid;
3
(B) acetonitrile with 0.1% formic acid ; (95:5, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4960 psi (342 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS-TOF, XIC
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL 1
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μg/mL in 99:1, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006115
1 2
Min

HPLC, UHPLC or LC-MS General Analytical
1. Uracil 4. Cinnamyl Alcohol

General Analytical 1 2. Benzamide 5. Dimethyl Phthalate
3. Aniline 6. 2-Nitroaniline
HPLC Analysis of Column Test Mixture on 7. 4’-Bromoacetanilide
Ascentis® Express 5μm RP-Amide 8. 2,2’-Biphenol
3
This application shows a test mixture of compounds of widely varying 2 9. 4,4’-Biphenol
polarity on the 5μm RPA Ascentis Express column.
column . . . . . . Ascentis Express RPA 15 cm x 4.6 mm, 5 μm particles (50773-U) 5 6 8
4 7
mobile phase . . (A) 20 mM potassium phosphate, pH 7; (B) acetonitrile; (70:30, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.0 mL/min 9
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4467 psi (308 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 μL
sample . . . . . . . . . . . . 1. Uracil (0.016 mg/mL), 2. Benzamide (0.161 mg/mL), 1.0 2.0
3. Aniline (0.161 mg/mL), 4. Cinnamyl Alcohol (0.016 mg/mL), Min
5. Dimethyl Phthalate (0.323 mg/mL), (6. 2-Nitroaniline (0.081 mg/mL),
7. 4′-Bromoacetanilide (0.040 mg/mL), 8. 2,2′-Biphenol (0.161 mg/mL),
9. 4,4′-Biphenol (0.040 mg/mL), in 50/50 Water/Acetonitrile )
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006279

HPLC, UHPLC or LC-MS Life Science and Biopharma
Life Science and Biopharma

Calibration Curves for TSKgel® SW mAb Columns
In gel fitration chromatography, the calibration curve of a column shows the
relationship between retention time or retention volume of a set of water-
soluble polymer or protein standards vs. the logarithm of their molecular
masses. The linear portion of a calibration curve is used to estimate the
molecular mass of an unknown protein. An expanded linear range is
desirable as it allows for a wider variety of molecular masses to be analyzed
with that column. This example shows the calibratuion curves for TSKgel®
SuperSW mAb HTP, TSKgel SuperSW mAb HR, TSKgel UltraSW Aggregate
and the industry standard TSKgel G3000SWxl column using several
commonly available globular proteins and p-aminobenzoic acid as a small
MW marker to indicate the void volume of the column. The exclusion
volume of the column is indicated by a trimer of thyroglobulin. [Note that
the calibration curve of the 15 cm x 4.6 mm ID TSKgel SuperSW mAb HTP
column is not included in the Figure as the column volume is every small
and the packing material in the column is the same as the TSKgel SuperSW
mAb HR column].
column . . TSKgel SuperSW mAb HR, 30 cm x 7.8 mm I.D., 4 μm particles (822854)
column . . TSKgel UltraSW Aggregate, 30 cm x 7.8 mm I.D., 3 μm particles (822856)
column . . . . . TSKgel G3000SWxl, 30 cm x 7.8 mm I.D., 5 μm particles (808541)
mobile phase . . 0.2 M sodium phosphate buffer, pH 6.7 and 0.05% sodium azide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006277

Desalting of Immunoglobulins (IgG, Human) on A280

2.0
HiTrap® Desalting Cartridge
column . . . . . . . . . . . . . . . . . . . . . HiTrap Desalting Cartridge, 5 mL (54822)
N-Hydroxysuccinimide
mobile phase . . . . . . . . . . . . . . . . . . 0.2 M sodium carbonate buffer, pH 8.5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 1.5
injection . . . 0.5 mL unbound human IgG in 0.2 M sodium carbonate buffer after
coupling to a HiTrap NHS 1 mL column. Fraction volume 0.5 mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0403
1.0
0.5
h-lgG
5 10 15 20
Fraction number

GC Analysis of Fuel Oil #6 on Equity®-1 (15 m x Fuel Oil #6 pattern

0.10 mm I.D., 0.10 μm), Fast GC Analysis
column . . . . . . . . . . . . . . Equity-1, 15 m x 0.10 mm I.D., 0.10 μm (28039-U)
oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 °C, 50 °C/min to 325 °C
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 350 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . hydrogen, 45 cm/sec constant
injection . . . . . . . . . . . . . 0.3 μL, 100:1 split, 0.1 min pre-injection dwell time
liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mm I.D., straight
sample . . . . . No.6 Fuel Oil standard, 20 mg/mL in hexane:chloroform (47536-U)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003908
1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis Carbohydrates on Proteomix®

SAX-NP5
column . . . . Proteomix SAX-NP5, 15 cm x 4.6 mm I.D., 5 μm particles (Z777228)
mobile phase . . . . . . . . . (A) 0.05% (25% ammonium hydroxide) in acetonitrile;
(B) 0.05% (25% ammonium hydroxide) in water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min
temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C Starch hydrolysate (5 μL)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD
sample . . . . . . . . . . . . . . Corn starch hydrolysate; glucose oligomer (G1 to G6)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005958
Glucose oligomer (G1-G6)
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25

Min
Courtesy of Miyako Kawakatsu, M&S Instruments, Inc.

HPLC Analysis MAb Variants on Proteomix® 0.8 mL/min, 105 bar Time (min) %B
SCX-NP5, Fast Analysis 0 5
5 5
Due to the low pressure of Proteomix SCX, faster analysis and high
40 29.5
resolution can be achieved with a higher flow rate.
column . . . . Proteomix SCX-NP5, 25 cm x 4.6 mm I.D., 5 μm particles (Z777164)
mobile phase . . (A) 2.4 mM Tris, 1.5 mM imidazole, 11.6 mM piperazine, pH 6.0;
(B) A + 0.5 M NaCl, pH 10.5
gradient . . . . . . top (0.8 mL/min): 5% B held for 5 min; to 29.5% B in 35 min;
bottom (1.5 mL/min): 5% B held for 2.7 min; to 29.5% B in 18.6 min 0 5 10 15 20 25 30 35 40
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min or 1.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV 280 nm 1.5 mL/min, 180 bar Time (min) %B
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 0 5
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb 321, 5 mg/mL 2.7 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006032 21.3 29.5
0 5 10 15 20
Min

HPLC Analysis of 15N-Acetylcysteine Enantiomers O
on Astec® (R,R) P-CAP™

*
OH
column . . . . Astec (R,R) P-CAP, 25 cm x 4.6 mm I.D., 5 μm particles (31024AST) HS
mobile phase . . . . . . . . . . . . . . 20 mM ammonium acetate in: (A) acetonitrile; 15

H N O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
CH3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004719
0 2 4 6 8 10
Min

HPLC Analysis of N-Acetylmethionine Enantiomers O

on Astec® (R,R) P-CAP™
S *
column . . . . Astec (R,R) P-CAP, 25 cm x 4.6 mm I.D., 5 μm particles (31024AST) H3 C OH
mobile phase . . . . . . . . . . . . . . 20 mM ammonium acetate in: (A) acetonitrile;
HN
(B) methanol; (70:30, A:B) O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C CH3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004722
0 2 4 6 8 10
Min

HPLC Analysis of N-Acetylphenylalanine

OH
*
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) O
mobile phase . . (A) 0.1% triethylamine acetate, pH 3.8; (B) ethanol; (20:80, A:B) NH O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min CH3

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004556
0 2 4 6 8
Min

O
HPLC Analysis of N-Acetyltryptophan Enantiomers
*
on Astec® CHIROBIOTIC® T OH
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) N

HN O
mobile phase . . . . . . (A) 20 mM ammonium acetate; (B) methanol; (70:30, A:B) H
CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004555
0 2 4 6 8 10 12
Min

HPLC Analysis of Aggregated Antibodies on

Zenix® SEC-300, Fast Analysis
Antibody aggregated
column . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm particles, 300 Å (Z777033) sample
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 μL After sample dilution,
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . antibody sample (17 mg/mL) the aggregation of
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006174 MAb is reversed.
Fresh antibody sample
0 2 4 6 8 10 12
Min

HPLC Analysis of Alanine Enantiomers on Astec® 1. L-Alanine 1 NH 2
2. D-Alanine 2 O
CHIROBIOTIC® T (Formic Acid Mobile Phase H 3C
Modifier) OH

mobile phase . . . . . (A) water; (B) methanol; (C) formic acid; (30:70:0.02, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 300 Μg /mL in 30:70 water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005330
0 2 4 6 8
Min

HPLC Analysis of Alanine Enantiomers on Astec®

O
CHIROBIOTIC® T (No Mobile Phase Modifier)
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H3C *
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (50:50, A:B) OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

NH 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004485
0 2 4 6
Min.

HPLC Analysis of Alanine Enantiomers on Astec® O

CHIROBIOTIC® TAG
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) H3C *
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min NH2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004603
0 2 4 6
Min.

HPLC Analysis of Alanylvaline Enantiomers on CH 3 O

Astec® CHIROBIOTIC® T
NH *
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H 2N * OH
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min H 3C C H3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004582
0 2 4 6
Min.

HPLC Analysis of Alloisoleucine Enantiomers on

Astec® CHIROBIOTIC® R CH3 O
column . . . . . . CHIROBIOTIC R, 25 cm x 4.6 mm I.D., 5 μm particles (13024AST) H3C *

* OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004676
0 2 4 6 8
Min

HPLC Analysis of Amino Acids on Ascentis® Si 3

Gradient Program
(Silica), HILIC Mode Time (min) %A %B %C
0 5 10 85 1
column . . . . . . . . Ascentis Si, 15 cm × 2.1 mm I.D., 5 μm particles (581509-U) 40 5 18 77
mobile phase . . . . . (A) 100 mM ammonium formate (pH 3.0, with formic acid),
(B) water, (C) acetonitrile 1. Threonine
2. Serine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 3. Asparagine 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4. Glutamine
5. Glutamatic acid
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . in 10:90, (50 mM ammonium formate/formic acid, pH 3.0):acetonitrile 2
4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004088
0 10 20
Min

HPLC Analysis of Amino Acids, Polar Neutral and 1. Threonine (0.1 g/L) 3 Gradient Program
2. Serine (0.1 g/L) Time (min) %A %B %C
Acidic on Ascentis® Si 3. Asparagine (0.1 g/L) 0 5 10 85
4. Glutamine (0.1 g/L) 20 5 16 79
column . . . . . . . . Ascentis Si, 10 cm x 2.1 mm I.D., 5 μm particles (581508-U) 5. Glutamatic acid (0.1 g/L) 1
mobile phase . . . . . (A) 100 mM ammonium formate (pH 3.0, with formic acid); 6. Aspartatic acid (0.1 g/L) 5
(B) water; (C) acetonitrile 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 2
sample . . . . . . . . . . . . . . . . . . . . as indicated, in 10:90, (50 mM ammonium 6
formate/formic acid, (pH 3.0):acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003722
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of α-Amylase (Crude) on TSKgel®

Ether-5PW
column . . . . TSKgel Ether-5PW, 7.5 cm × 7.5 mm I.D., 10 μm particles (808641)
mobile phase . . . . . (A) 0.1 M phosphate buffer + 1.1 M sodium sulfate, pH 7.0
(B) 0.1 M phosphate buffer A to B over 60 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7130961B
0 10 20
Min

HPLC Analysis of Amyloid β-Protein Fragment 200

10-35 on Discovery® BIO Wide Pore C18
150
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 25 cm × 4.6 mm I.D., 100
5 μm particles (568223-U)
–p
+p
–p
mobile phase . . . . . . . . . . . . . . . . . (A) 0.1% TFA in water; (B) 0.1% TFA in 50
water:0.1% TFA in acetonitrile; (25:75) 0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 mL/min 0 10 20 30
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL, 1 mg/mL in water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001589

HPLC Analysis of Angiotensins on Discovery® BIO Peptide Sequence

Wide Pore C8 1. Angiotensin II (1.67 g/L) DRVYIHPF
2. Angiotensin III (1.67 g/L) RVYIHPF
1 3. Angiotensin I (1.67 g/L) DRVYIHPFHL
column . . . . . . . . . . . . . . Discovery BIO Wide Pore C8, 15 cm × 4.6 mm I.D.,
mobile phase . . . . . . . . . . . . . (A) 10 mM NH4H2PO 4/ NH4 OH, pH 7; (B) 50:50, 3
2
(20 mM NH4H2PO 4/ NH4 OH, pH 7):CH3CN
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30-60% B in 15 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 μL in water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001503B
0 10 20
Min

HPLC Analysis of Angiotensins on Ascentis® 1 1. Angiotensin II (100 µg/mL)

2. Angiotensin III (100 µg/mL)
RP-Amide 3. Angiotensin I (100 µg/mL)
mobile phase . . . . . . (A) 5 mM ammonium phosphate (pH 7.0 with ammonium
hydroxide); (B) acetonitrile; (77.5:22.5, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002914
0 2 4 6 8 10 12
Min

HPLC Analysis of an mPEG-peptide on Zenix® SEC Zenix SEC-300

140
Phases
120
column . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm particles, 300 Å (Z777033) 100
column . . Zenix C SEC-300, 30 cm x 7.8mm I.D., 3 μm particles, 300 Å (Z777092) Zenix-C SEC-300
80
mAU
mobile phase . . . . . . . . . . . . . . . . 150 mM Sodium Phosphate Buffer, pH 7.0
60
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
40
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm 20
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 0
sample . . . . . . . . . . . . . 20kD mPEG-MAL (methoxy-PEG-Maleimide) (1mg/mL) 0 2.5 5 7.5 10 12.5 15 17.5

HPLC Analysis of Antibody Isotypes on SRT® Gel

Filtration Columns
A change in the relative concentration of IgG subclasses (IgG1, IgG2, IgG3
and IgG4) in tissue indicates a change in immunological response. These SRT® SEC-150 (5 μm, 150 , 7.8 × 300 mm)
chromatograms show the elution profiles of several IgG subclasses,
including lambda and Kappa light chains and biotinylated IgG1. Note that
Lambda
sme of the aggregate peaks in front of the main component are partially
excluded on the smaller (SEC-150) pore size column, but not on the 300 Kappa
Angstrom pore size column. IgA
column . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm particles, 300 Å (Z777051)
IgG2
mobile phase . . . . . . . . . . . . . . . . . . . . . 150 mM phosphate buffer, pH 7.0 IgG4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
IgG1-Biotin
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
SRT® SEC-300 (5 μm, 300 , 7.8 × 300 mm)
Lambda
IgA
IgG2
IgG4
IgG1-Biotin
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

HPLC Analysis of Apotransferrin Tryptic Digest on A. 2 mL/min

5–50% B in 30 min
BIOshell™ A160 Peptide C18, Comparison of 126 bar
Different Flow Rates and Pressures
BIOshell columns packed with five micron Fused-Core particles feature
modest back pressures, which allows the use of very high flow rates as is
demonstrated in the two chromatograms, one of which was obtained at
2mL/min, the other at 4mL/min. Note that at 4mL/min the gradient time
was reduced in half to keep the gradient volume constant. At a flow rate of
4mL/min the back pressure for this 15cm x 4.6mm column is 250 bar
(approx. 3500 psi), which illustrates that these high efficiency columns can
0 5 10 15 20
be operated on standard HPLC systems.
column . . . . . . . . . . . . . . . BIOSHELL A160 Peptide C18, 15 cm x 4.6 mm I.D.,
5 μm particles (67015-U) B. 4 mL/min
mobile phase . . . . . . . . . . . . . (A): water/0.1% TFA (B): acetonitrile/0.1% TFA 5–50% B in 15 min
250 bar
gradient . . . . . . . . . . . . . . . . 5-50%B in 30 minutes; 5-50%B in 15 minutes;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min or 4/mL minute
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 bar, 250 bar
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL in water/0.1% TFA
0 2 4 6 8 10 12
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006220
mL/min

O
HPLC Analysis of AQC-Alanine Enantiomers on
H3C *
Astec® CHIROBIOTIC® T2 OH
O NH
mobile phase . . (A) 20 mM ammonium acetate, pH 4.1; (B) methanol; (50:50, A:B) NH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004602
0 2 4 6 8 10 12 14 16 18
Min.

O
HPLC Analysis of AQC-Phenylalanine Enantiomers
*
HN O
mobile phase . . (A) 10 mM ammonium acetate, pH 4.1; (B) methanol; (50:50, A:B) HN
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004583
0 2 4 6 8 10 12 14
Min.

O
HPLC Analysis of AQC-Serine Enantiomers on
*
Astec® CHIROBIOTIC® T HO OH
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) O NH
NH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004576
0 10 20
Min.

HPLC Analysis of AQC-Serine Enantiomers on O

*
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) HO OH
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm NH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004739 N
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of AQC-Threonine Enantiomers on CH3 O
Astec® CHIROBIOTIC® T *
HO * OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min NH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm N
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004578
0 2 4 6 8 10 12 14 16 18
Min.

HPLC Analysis of AQC-Valine Enantiomers on CH 3 O
H3C OH
O NH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min NH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL N

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004577
0 10 20
Min.

HPLC Analysis of Arginine Enantiomers on Astec® NH 2
CHIROBIOTIC® T (23 °C) H2N NH O

*
NH OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004487
0 10 20 30
Min.

HPLC Analysis of Arginine Enantiomers on Astec® NH 2 1 1. L-Arginine

2. D-Arginine
CHIROBIOTIC® T (25 °C) H 2N NH O 2
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) NH OH
mobile phase . . . . . (A) water, (B) methanol; (C) formic acid; (30:70:0.02, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 300 μg /mL in 30:70 water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005331
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Asparaginase on Zenix® SEC-150
10.895
column . . . . . . . Zenix SEC-150, 30 cm x 7.8 mm I.D., 3 μm particles (Z777018)
mobile phase . . . . . . . . . . . . . . . . . . . . 150 mM sodium phosphate, pH 7.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1015 psi (70 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . asparaginase (2.0 mg/mL in water), RT 10.895 minute,
MW 5000-6000, pI 4.85
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005973
12.831
9.682
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0
Min

HPLC Analysis of Asparagine Enantiomers on O

Astec® CHIROBIOTIC® T (23 °C) OH
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H 2N
*
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (50:50, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
H 2N
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004488
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Min

HPLC Analysis of Asparagine Enantiomers on O 1 1. L-Asparagine

Astec® CHIROBIOTIC® T (25 °C) OH 2. D-Asparagine
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H 2N

2
mobile phase . . . . (A) water); (B) methanol; (C) formic acid; (30:70:0.02, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min H 2N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005332
0 2 4 6 8 10
Min

HPLC Analysis of Aspartic Acid Enantiomers on 1

1. L-Aspartic Acid
Astec® CHIROBIOTIC® T (Methanol Mobile Phase) 2. D-Aspartic Acid 2
mobile phase . . . . . (A) water; (B) methanol; (C) formic acid; (30:70:0.02, A:B:C) O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min HO
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm O NH 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005333
0 2 4 6
Min

HPLC Analysis of Aspartic Acid Enantiomers on

Astec® CLC-L, Trace Level Detection O (L)-Aspartic acid,
11.14 min.
column . . . . . . . . Astec CLC-L, 15 cm x 4.6 mm I.D., 5 μm particles (53123AST) HO *
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mM copper sulfate OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
NH2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004664
3% (D)-Aspartic acid,
9.17 min.

HPLC Analysis of 7-Azatryptophan Enantiomers on

Astec® CHIROBIOTIC® T O
OH
N N
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient H
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . 5 mg/mL in methanol/water/ammonium hydroxide
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004538
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Basic Peptides (Angiotensin 1. IVYIHPR

2. KVYIHPI
Analogs) on Ascentis® Express Peptide ES-C18 3. RVYIHPI
4 4. ac- KVYIHPI
column . . . . . . . . . . . . . Ascentis Express Peptide ES-C18, 10 cm x 3 mm I.D., 1 3 5 5. ac- RVYIHPI
6. ac- IVYIHPR
mobile phase . . . . . (A) 0.1% (v/v) formic acid, pH 4.0 (titrated with ammonium
hydroxide); (B) 50:50, (0.175% formic acid in water,
pH 4.0) : acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3002 psi (207 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL 0 2 4 6 8 10 12 14 16 18
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g/L ea. peptide Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005379

HPLC Analysis of Basic Peptides on Discovery® BIO Peptide 1: ac-GGGLGGAGGLK-amide

Gradient Program
Wide Pore C18, No TFA Peptide 2: ac-KYGLGGAGGLK-amide Time (min) %B
Peptide 3: ac-GGALKALKGLK-amide
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 2.1 mm I.D., Peptide 4: ac-KYALKALKGLK-amide 0 15
45 60
mobile phase . . . . . . (A) 25 mM formic acid in water; (B) 50:50 (25 mM formic
acid in water):(20 mM formic acid in acetonitrile)
40
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.20 mL/min
mAU 215 nm
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL (~0.25 μg each peptide)
20
0
0 10 20 30
Min

HPLC Analysis of t-BOC-Alanine Enantiomers on O
Astec® CHIROBIOTIC® T H3C *

mobile phase . . (A) 20 mM ammonium acetate, pH 6; (B) methanol; (90:10, A:B) H3C
O NH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C H3C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm C H3 O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004573
0 2 4 6
Min.

HPLC Analysis of t-BOC-Methionine Enantiomers O
on Astec® (R,R) P-CAP™ S *

H3C OH
column . . . . Astec (R,R) P-CAP, 25 cm x 4.6 mm I.D., 5 μm particles (31024AST)
mobile phase . . . . . . . . . . . . . . 20 mM ammonium acetate in: (A) acetonitrile; HN O
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm CH3
H3C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004723
0 2 4 6 8
Min

HPLC Analysis of t-BOC-Phenylalanine O

Enantiomers on Astec® CHIROBIOTIC® T *
OH
HN O
mobile phase . . (A) 20 mM ammonium acetate, pH 6; (B) methanol; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min CH 3
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm CH 3
H 3C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004585
0 2 4 6 8 10 12
Min.

HPLC Analysis of t-BOC-Tyrosine Enantiomers on O
OH
HN O
mobile phase . . (A) 20 mM ammonium acetate, pH 6; (B) methanol; (90:10, A:B) HO
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm CH3
H3C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004572
0 2 4 6 8 10
Min.

HPLC Analysis of Bovine Liver Extract on 0.5 1.0

Mono Q® 5/50 GL
column . . . . . . . Mono Q 5/50 GL, 5 cm x 5 mm I.D., 10 μm particles (5-4807)
NaCl concentration M (-----)

0.4
mobile phase . . . . . . . . . . . . . . . . . . . . (A) 50 mM diethanolamine, pH 8.8;
(B) 50 mM diethanolamine + 1.0M NaCl, pH 8.8
)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
0.3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
A280 (
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL 1.6 mg protein
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0376
0.2
0.25
0.1
0 5 10 15 20
Volume (mL)

HPLC Analysis of α-Bromopalmitic Acid *

O
H3C OH
Enantiomers on Astec® CHIROBIOTIC® T2 Br
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004601
0 2 4 6
Min.

HPLC Analysis of Calphostin C on 1 1. Calphostin C

Discovery® HS C18
Gradient Program
column . . . . Discovery HS C18, 25 cm × 2.1 mm I.D., 5 μm particles (568503-U) Time (min) %A % B
mobile phase . . . (A) (0.1% phosphorus acid in water):(0.1% phosphorus acid in 0 70 30
acetonitrile); (B) 0.1% phosphorus acid in acetonitrile; (50:50, A:B) 25 20 80
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 30 20 80
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
sample . . . . . . . . . . . . . . Calphostin C (0.25 g/L) in 40% MeOH, 0.025% TFA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002574
0 10 20 30
Min

HPLC Analysis of Carboxyamido Methylated BSA

Tryptic Digest on Discovery® RP-Amide C16
mobile phase . . . . . . . . . . . . . . . . . . (A) 0.1% TFA, 1% acetonitrile in water;
(B) 0.1% TFA, 40% acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 130 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001087
0 20 40 60 80 100 120
Min

HPLC Analysis of Carboxyamido-Methylated BSA

Tryptic Digest on Discovery® C18
mobile phase . . . . . . . . . . . . . . . . . . (A) 0.1% TFA, 1% acetonitrile in water;
(B): 0.1% TFA, 40% acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 130 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001086
0 20 40 60 80 100 120
Min

HPLC Analysis of Carboxymethylated Gradient Program

Time (min) %A %B
Apohemoglobin (Horse) Tryptic Digest on
0 100 0
Discovery® BIO Wide Pore C18 50 0 100
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 2.1 mm I.D.,
mobile phase . . . . . . . . . . . . . . . . . (A) 0.1% TFA in water; (B) 0.1% TFA in
water:0.1% TFA in acetonitrile, 50:50;
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 50 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35°C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . 5 μL, each at 0.8 mg/mL in 0.1% TFA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001719
0 20 40 60
Min

HPLC Analysis of Carboxymethylated Discovery BIO

Wide Pore C18
Apohemoglobin Tryptic Digest on Discovery® BIO
Wide Pore C18
mobile phase . . (A) (0.1% TFA in water):(0.1% TFA in acetonitrile) 95:5; (B) (0.1%
TFA in water):(0.1% TFA in acetonitrile):50:50;
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 65 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μL
sample . . . . . . . . . . . . . . . . . carboxymethylated apohemoglobin tryptic digest
in 50 mM ammonium bicarbonate
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001505A 0 10
Min

HPLC Analysis of Discovery BIO

Wide Pore C5
Carboxymethylated Apohemoglobin Tryptic
Digest on Discovery® BIO Wide Pore C5
mobile phase . . . . . . . . . (A) 0.1% TFA in water:0.1% TFA in acetonitrile, 95:5;
(B) 0.1% TFA in water:0.1% TFA in acetonitrile; 50:50
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 65 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μL
Min

HPLC Analysis of Carboxymethylated Discovery BIO

Wide Pore C8
Apohemoglobin Tryptic Digest on Discovery® BIO
Wide Pore C8
mobile phase . . (A) 0.1% TFA in water:0.1% TFA in acetonitrile, 95:5; (B) 0.1% TFA
in water::0.1% TFA in acetonitrile, 50:50.
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 65 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μL
Min

HPLC Analysis of Carboxymethylated Cytochrome Gradient Program

c (Chicken) Tryptic Digest on Discovery® BIO Wide Time (min) %A %B Undigested protein
Pore C18 0 100 0
50 0 100
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water/0.1% TFA;
(B) (water/0.1%TFA):(acetonitrile/0.1% TFA); 50:50
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.20 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001721
0 20 40 60
Min

HPLC Analysis of Carnitine Enantiomers on Astec®

CHIROBIOTIC® TAG O
-
CH3
H3C
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) N
+
mobile phase . . . (A) 5 mM ammonium acetate, pH 6; (B) methanol; (25:75, A:B) * CH 3

O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min HO
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+)-MS (m/z = 162)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004524
0 10 20
Min

HPLC Analysis of α-Casein Tryptic Digest on detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Ascentis® Express Peptide ES-C18 Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005597
LC-MS-based shotgun proteomics relies both on the power of the
separation techniques, and the sensitivity of detection methods. As a viable
alternative to classical approaches in this field, we developed a fully
automated, comprehensive 2D LC system, in which RP LC x RP LC was
coupled to MS detection, for the first time, and applied for the analysis of
tryptic digests obtained from a-casein and dephosphorylated a–casein. The
use of a significantly different pH in the two dimensions allowed to attain
high peak capacity, despite the employment of novel identical stationary
phases. Furthermore, such a combination addresses compatibility issues,
thus allowing straightforward interfacing in on-line 2D LC configuration, as
well as direct linkage to mass spectrometer. A theoretical peak capacity of
approximately 8500 was calculated for the set-up employing four serially
coupled C18 columns in the first dimension (4 columns, each 15 cm x
2.1 mm I.D., 2.7 μm particles), operated under basic conditions, and 3 cm
length of the same stationary phase (3 cm x 4.6 mm I.D., 2.7 μm particles),
under acidic conditions for fast second dimension analysis.
Sample Prep: one-tenth gram of α-casein was dissolved in 10 mL of 0.01 M 0.2 0.4 0.6 0.8 1.0
ammonium formate buffer, and the pH was adjusted to 8.0 with ammonium Min
hydroxide; the solution was heated in a boiling water bath for 6 min. After
the solution cooled, 2.0 mg of trypsin from bovine pancreas was added, and
the mixture was allowed to react for 4 h at 37 °C; the reaction was quenched
by adding 0.1% trifluoroacetic acid to pH 2. The digest were stored at 4°C
and filtered prior to injection through a 0.45 μm nylon membrane.
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . tryptic digested α-casein
column . . . . . . . . . . . . . Ascentis Express Peptide ES-C18, 3 cm x 4.6 mm I.D.,
mobile phase . . . . . . . . . . . . . . . . . (A) 0.1% TFA in water; (B) 0.1% TFA in
water:acetonitilre (10:90, A:B)
gradient . . 0-0.05 min, 0-20% B; 0.05-0.40 min, to 40% B; 0.40-0.50 min, to 50% B;
0.50-0.69 min, to 90% B; 0.69-0.70 min, to 0% B; held for 0.3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C

HPLC Analysis of Cetuximab Antibody Fragments Fab

from Papain Digestion on Zenix® SEC-150 +
Papain digestion conditions: Buffer: 5.0 mM L-Cysteine, 2.0 mM EDTA and L-cysteine
300 mM NaCl; Enzyme: 80 μg/mL papain; Temperature: 37 °C; Time: 30 min,

60 min, 120 min, 240 min, and 360 min.
mobile phase . . . . . . . . . . . . 20 mM sodium phosphate, pH 7.0, 150 mM NaCl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min ZENIX-150-E-360MIN
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm ZENIX-150-E-240MIN
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . Cetuximab ERBITUX®, papain digest ZENIX-150-E-120MIN
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006172

ZENIX-150-E-60MIN
ZENIX-150-E-30MIN
ZENIX-150-E-0MIN
ZENIX-150-BUFFER
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Min

HPLC Analysis of Chito-Oligosaccharides on 1. Chitohexose

3 2. Chitopentose
TSKgel® G-Oligo-PW 4
5 3. Chitotetrose
2
column . . . . TSKgel G-Oligo-PW, 30 cm × 7.8 mm I.D., 6 μm particles (808031) 4. Chitotriose
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 5. Chitobiose
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI 1
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . distilled water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0325
15 20
Min

HPLC Analysis of o-Chlorophenylalanine OH

*
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (70:30, A:B) NH 2

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Cl
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004550
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of p-Chlorophenylalanine

Enantiomers on Astec® CHIROBIOTIC® T OH

O
NH2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004546
0 2 4 6 8 10
Min

HPLC Analysis of Citrulline Enantiomers on Astec®

CHIROBIOTIC® T
O
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (50:50, A:B) *

H2N NH OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C NH2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004531
0 1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of Column Test Mix on Discovery® 0.32 mm I.D.

BIO Wide Pore C18, Effect of HPLC Column I.D. on 5 μL/min
0.05 μL injection
Sensitivity
column . . . . . Discovery BIO Wide Pore C18, 10 cm x various I.D., 3 μm particles
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated 0 2 4 6 8 10 12 14 16
Min
sample . . . . . . . . . . . . acetophenone (0.1 μg), benzene (1 μg), toluene (1 μg)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004416 0.50 mm I.D.
12.2 μL/min
0.12 μL injection
0 2 4 6 8 10 12 14 16
Min
1.0 mm I.D.
48.8 μL/min
0.49 μL injection
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Conjugated Estrogens on 1. Estriol 3-(β-D-glucuronide)

2. β-estradiol 3-(β-D-glucuronide) 17-sulfate
Discovery® HS C18 3. Estriol 3-sulfate
4. β-estradiol 3,17-disulfate
column . . . . Discovery HS C18, 15 cm × 4.6 mm I.D., 5 μm particles (568522-U) 5. β-estradiol 3-(β-D-glucuronide)
mobile phase . . . . . . . . (A) 10 mM potassium phosphate (pH 7.01 unadjusted); 6. β-estradiol 17-(β-D-glucuronide)
(B) acetonitrile; (C) methanol 7. Estrone 3-(β-D-glucuronide)
8. Estriol Gradient Program
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 9. β-estradiol 3-sulfate Time (min) %A %B %C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 10. Estrone 3-sulfate
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 11. β-estradiol 0 80 15 5
1
12. α-estradiol 5 80 15 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 13. Estrone 25 40 45 15
sample . . . . . . . . . . . . . . . . . . . . 10 μg/mL each in 60/40, water:acetonitrile 30 40 45 15
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002155 8
7
4 56
10 11
2
3
13
12
9
0 10 20 30
Min

HPLC Analysis of Creatine Kinase on Discovery® Gradient Profile

BIO PolyMA-WAX Min %A %B
0 100 0
Discovery BIO PolyMA-SCX and PolyMA-WAX columns give sharp, efficient
30 90 10
peaks for a wide variety of proteins.
column . . Discovery BIO PolyMA-SCX, 5 cm × 4.6 mm, 5 μm particles (59601-U)
mobile phase . . . . . . . . . . . . . . . . . . (A) 20 mM sodium phosphate, pH 7.0;
(B) 20 mM sodium phosphate, 0.5M NaCl, pH 7.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 0 5 10 15 20 25 30
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001836

HPLC Analysis of Cysteine Enantiomers on Astec® 1. L-Cysteine

CHIROBIOTIC® T (Formic Acid Mobile Phase 2. D-Cysteine
Additive) H 2N O
column . . Astec CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST)
HS OH 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . (200 μg /mL in 50:50 water:methanol)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005334
0 2 4 6
Min

HPLC Analysis of Cysteine Enantiomers on Astec® H 2N O

CHIROBIOTIC® T
*
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HS OH
extraction tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004495
0 2 4 6 8
Min.

HPLC Analysis of Cytochrome c Species Variants Cytochrome c Source 3

on Discovery® BIO PolyMA-SCX 1. Bovine
2. Horse
column . . Discovery BIO PolyMA-SCX, 5 cm × 4.6 mm I.D., 5 μm particles (56901-U) 3. Rabbit
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 20 mM Bis-Tris HCl, pH 7.0; 2
(B) A + 0.5 M NaCl 24-69% B over 20 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μg each variant
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0477B
0 5 10 15 20
Min
Figure used with permission of Mitsubishi Chemical Co.

HPLC Analysis of Cytochrome c Species Variants 1. Cow cytochrome c 3

on Discovery® BIO PolyMA-SCX (Ion Exchange) 2. Horse cytochrome c
3. Rabbit cytochrome c
This Figure shows the separation of three cytochrome c variants on
Discovery BIO PolyMA-SCX. Note the difference in elution order compared
to the reversed-phase separation on Discovery BIO Wide Pore C18 shown in 2
product G001581.
column . . . Discovery BIO PolyMA-SCX, 5 cm x 4.6 mm, 5 μm particles (59601-U)
1
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 20 mM Bis-Tris HCl, pH 7.0;
(B) 20 mM Bis-Tris HCl, 0.5M NaCl, pH 7.0;
24 to 69% B in 20 min. (linear)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μg each variant
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0477
0 5 10 15 20
Min

HPLC Analysis of Cytochrome c Species Variants 1. Horse cytochrome c

2. Rabbit cytochrome c
on Discovery® BIO Wide Pore C18 3. Cow cytochrome c
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 4.6 mm I.D., 4. Pigeon cytochrome c
5. Chicken cytochrome c
5 μm particles (568222-U) 6. Dog cytochrome c
mobile phase . . . . . (A) (0.1% TFA in water):(0.1% TFA in acetonitrile); (70:30),
(B) (0.1% TFA in water):(0.1% TFA in acetonitrile), 64:36
1 4
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 30 min 2 5
3 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . 12 μL each at 0.8 mg/mL in 0.1%TFA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001581
0 10 20
Min

HPLC Analysis of Cytochrome c Tryptic Digest on

Discovery® C18
column . . . . . . . . . Discovery C18, 15cm × 4.6mm I.D., 5μm particles (504955)
mobile phase . . . . . . . . . . . . . . . . . . (A) 0.1% TFA, 3% n-propanol in water;
(B) 0.1% TFA, 3% n-propanol in acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-37% B in 72 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000307
0 10 20 30 40 50
Min

HPLC Analysis of Dansyl-Glutamic Acid O O
Enantiomers on Astec® CHIROBIOTIC® R HO

*
OH
NH
mobile phase . . . . . . . . . . 0.1 wt% ammonium trifluoroacetic acid in methanol S
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
H3C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL N
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg/mL in methanol CH3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004669
0 2 4 6 8 10 12
Min

HPLC Analysis of Dansyl-Leucine Enantiomers on O
Astec® CHIROBIOTIC® R H3C *

OH
CH3 HN
mobile phase . . . . . . . . . . 0.1 wt% ammonium trifluoroacetic acid in methanol S
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL CH3
N
0 2 4 6 8
Min

HPLC Analysis of Dansyl-Methionine Enantiomers O
on Astec® CHIROBIOTIC® R S *
H3C OH
S
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
N
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004671
CH3
0 2 4 6 8 10 12
Min

HPLC Analysis of Dansyl-Methionine Enantiomers

O OH
on Astec® CHIROBIOTIC® T
CH3
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HN * S
mobile phase . . . . . . . . . . . . . . . . . 0.1 wt% ammonium acetate in methanol O S O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL N
H3C CH3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004558
0 1.0 2.0 3.0 4.0

Min

HPLC Analysis of Dansyl-Norleucine Enantiomers O

on Astec® CHIROBIOTIC® R *
H3C OH
O
mobile phase . . (A) 20 mM ammonium acetate, pH 4.1; (B) methanol; (70:30. A:B) HN
S
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
N
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004672
CH3
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Dansyl-Phenylalanine O
Enantiomers on Astec® CHIROBIOTIC® R *

OH
HN
mobile phase . . . . . . (A) 20 mM ammonium acetate; (B) methanol; (70:30, A:B)
S
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
CH3
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL in methanol N
0 20 40
Min

HPLC Analysis of Dansyl-Valine Enantiomers on CH3 O
*
column . . . . . . CHIROBIOTIC R, 25 cm x 4.6 mm I.D., 5 μm particles (13024AST) H3C OH
mobile phase . . . . . . . . . . 0.1 wt% ammonium trifluoroacetic acid in methanol O

HN
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min S
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004675 CH 3
N
CH3
0 2 4 6 8
Time (min)

HPLC Analysis of Dehydroproline Enantiomers on

Astec® CHIROBIOTIC® T OH
*
H
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . water O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 200 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004528
0 2 4
Min

HPLC Analysis of Deoxyribonucleosides and dC

Gradient Program
Ribonucleosides on SUPELCOSIL™ LC-18-S hm5U
Time (min) %A
C m5dC
column . . . . SUPELCOSIL LC-18-S, 15 cm × 4.6 mm I.D., 5 μm particles (58931) U dA 0 100
mobile phase . . 0.05 M K2HPO 4/KH2PO4, pH 4.0:methanol (A) 97.5:2.5; (B) 80:20 G dG 3 100
dU dT 12 0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min A
I 15 0
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C dI 19.2 40
m3dC
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . nucleoside standards in water m1dA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0955
m6 dA
5'-dA
0 5 10 15
Min
Figure provided by Dr. C. W. Gehrke and Mr. K. C. Kuo, University of Missouri-

Columbia, Experimental Station Chemical Laboratories, Columbia, MO USA.

HPLC Analysis of Deoxyribonucleosides on 1 1. Thymidine (0.1 g/L)

2 2. Deoxyadenosine (0.1 g/L)
Ascentis® Si 3. Deoxyguanosine (0.1 g/L)
4. Deoxycytidine (0.1 g/L)
mobile phase . . . . . (A) 150 mM ammonium formate (pH 3.0, with formic acid);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 3
4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated, in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003719
0 2 4 6 8 10
Min

HPLC Analysis of 3,5-Dibromo-p-Tyrosine OH
Enantiomers on Astec® CHIROBIOTIC® T Br *

O
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) NH2
HO
Br
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004552
0 2 4 6 8
Min

HPLC Analysis of 2,3-Dimethyltyrosine

Enantiomers on Astec® CHIROBIOTIC® T CH3 O
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (70:30, A:B) OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min NH2

HO
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004532
0 2 4 6 8
Min

HPLC Analysis of DMB-Labeled Sialic Acids on 1 1. DMB-NGNA

Ascentis® Express RP-Amide: Comparison of 2. DMB-NANA
Biosimilars to Reference Materials

2
Sialic acids affect the bioavailability, function, stability, and metabolism of
glycoproteins. Two forms of sialic acid are commonly present in therapeutic
glycoproteins: N-acetylneuraminic acid (NANA) and N-glycolylneuraminic Reaction
acid (NGNA). One of the most common quantification methods involves by-products
releasing sialic acids from the glycoprotein, derivatizing NANA and NGNA
with 1,2-diamino-4, 5-methylenedioxybenzene (DMB), and analyzing by C18-
HPLC with fluorescence detection. This procedure is subject to interference
from peaks originating from excess reagent and other derivatized impurities, Unknown
limiting sensitivity and reproducibility. The objectives of this study were to
develop a significantly improved HPLC-fluorescence method for DMB-NANA
and DMB-NGNA, and to apply this method to compare two candidate 0 2 4 6 8 10 12 14
biosimilar therapeutic proteins to their respective reference materials. Min
column . . . . . . . . . . . . . . . Ascentis® Express RP-Amide, 10 cm x 2.1 mm I,D.,
mobile phase . . . . . . . (A) Water, 0.1% formic acid, (B) ACN, 0.1% formic acid
gradient . . . . . . . . . . . . . . . . . . . . . . . 0-1 min 6% B; 1.01-4 min 20% B;
4.01-12 min 6% B, total run time 15 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300 psi (89.6 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . fluorescence, λ excitation = 373 nm, λ emission = 448 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . Mix of DMB-labeled NGNA and NANAsialic acid, 5 mg/ml each
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006199

HPLC Analysis of DNA on Proteomix® WAX-NP5

column . . . Proteomix WAX-NP5, 15 cm x 4.6 mm I.D., 5 μm particles (Z777262)
mobile phase . . . . . . . . . . . . . (A) 20 mmol Tris, pH 6.0; (B) A + 1.0 M NaCl
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 to 100% B in 40 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1015 psi (70 bar)
temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 15 16 17 18 19 20 21 22 23 24
Zoomed section
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005963
0 5 10 15 20 25 30 35 40
Min

HPLC Analysis of 3,5-DNB-Tryptophan Methyl Ester O
Enantiomers on Astec® CYCLOBOND® I 2000 SN CH3

*
O
column . . CYCLOBOND I 2000 SN, 25 cm x 4.6 mm I.D., 5 μm particles (20524AST)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ethanol HN O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min N
H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL in methanol O- O
+ +
N N
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004709
O O-
0 2 4
Min

2 1. Mesoridazine
HPLC Analysis of Dopamine Receptor Antagonists 2. Promazine
on Ascentis® Phenyl 3. Thiothixene
4. Chlorpromazine
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 4 5. Trifluoperazine
1
mobile phase . . . . . . (A) 50 mM ammonium formate (pH 3.0 with formic acid); 6. Thioridazine
5 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

HPLC Analysis of E. coli Lysate on Proteomix® SAX Proteomix SAX-NP10

Columns, Particle Size Comparison Peak Capacity: >40
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
mobile phase . . . . . . . . . . . . . . (A) 20 mM Tris, pH 9.0; (B) A + 0.5 M NaCl Proteomix SAX-NP5
Peak Capacity: >60
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
column . . . . . Proteomix SAX-NP5, 5 cm x 4.6mm I.D., 5 μm particles (Z777227) 10 15 20
column . . . Proteomix SAX-NP10, 5 cm x 4.6 mm I.D., 10 μm particles (Z777203)

gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 - 100%B in 30 min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 mg/mL
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006129
0 5 10 15 20 25 30 35
Min

HPLC Analysis of E. coli Lysate on Proteomix® SAX-

NP3, Demonstration of High Loading Capacity
column . . . . . Proteomix SAX-NP3, 5 cm x 4.6mm I.D., 3 μm particles (Z777219)
mobile phase . . . . . . . . . . . . . . (A) 20 mM Tris, pH 9.0; (B) A + 0.5 M NaCl
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 - 100% B in 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
18 19 20 21 22
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in figure Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006130
Injection 50 μL
Injection 20 μL
Injection 10 μL
0 5 10 15 20 25 30
Min

HPLC Analysis of E. coli lysate with various pore

size Zenix® columns
An cell lysate, produced by destroying the membranes of E. Coli cells, is
analyzed on two Zenix gel filtration columns packed with 3 micron particles.
The top chromatogram shows the peak profile on a 150 Å pore size Zenix
SEC-150 gel filtration column, while the bottom chromatogram shows the
same sample on a 300 Å pore size Zenix SEC-300 column. As expected, the
peaks shift to longer retention times on the SEC-300 column. Although
roughly the same number of components are visible in each chromatogram,
the SEC-150 column does a better job of pulling the peaks apart, which
helps when fractions need to be collected for further analysis. Zenix SEC-150
mobile phase . . . . . . . . . . . . . . . . . . . . . . 150 mM Phosphate Buffer, pH 7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ambient Zenix SEC-300
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. coli lysate, 2.5 mg/mL 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

HPLC Analysis of Elastase on Discovery® BIO

PolyMA-SCX, Ion Exchange Gradient Profile
Min %A %B
Discovery BIO PolyMA-SCX and PolyMA-WAX columns give sharp, efficient
0 100 0
peaks for a wide variety of proteins. 30 0 100
column . . . . Discovery BIO PolyMA-SCX, 5cm × 4.6mm, 5μm particles (59601-U)
(B) 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0
gradient . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 100% B in 30 min. (linear)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001833
0 5 10 15 20 25 30
Min

HPLC Analysis of Enkephalin Derivative (DAMME) 1. PEG contaminants (m/z 520.1, 564.1, 608.2, 652.2, 696.2)
2. DAMME (m/z 604.2)
on Discovery® HS F5 2
Gradient Program
mobile phase . . . . (A) 25 mM HCO 2H in water; (B) 25 mM HCO 3H in acetonitrile Time (min) %A %B
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
0 95 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C 30 35 65
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L in 0.1% HCO 2H
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002572
1
0 10 20 30
Min

HPLC Analysis of Erythropoietin on SRT® SEC-300 1. Erythropoietin, main peak 1

Gel Filtration Column 2. Impurity
Erythropoietin (EPO) is a glycoprotein hormone that controls red blood cell
production. EPO was one of the first genetically engineered therapeutic
proteins. It is available under such brand names as Epogen and Aranesp for
the treatment of anemia. Human EPO has a molecular mass of 34 kDa. The 5
micron SRT SEC-300 column is operated at a flow rate of 0.7 mL/min. Note
that after the main peak, an impurity elutes at about 16 minutes.
column . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm partices, 300 Å, (Z777051)
2
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Na2 HPO4-KH 2PO4-NaCl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 μL
sample . . . . . . . . . . . . . . . . . . . . . EPO (Recombinant Human Erythropoietin) Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006182

HPLC Analysis of Exenatide Acetate on Proteomix® Sequence: HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS
SCX-NP5 pI: 4.7
column . . . . Proteomix SCX NP5, 25 cm x 4.6mm I.D., 5 μm particles (Z777161) MW: 4186.6
mobile phase . . . . . (A) 10 mM monosodium phosphate:acetonitrile (56:44, v/v),
adjusted to pH to 2.0 with phosphoric acid;
((B) 0.5 M NaCl/10 mM monosodium phosphate:acetonitrile (56:44, v/v),
adjusted to pH to 2.0 with phosphoric acid)
gradient . . . . . . . . 0 to 65% B in 35 min; held for 5 min; to 0% B in 10 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653 psi (114 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . . 0.325 mg/mL exenatide acetate in mobile phase A
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006163 10 15 20 25 30 35 40
Min

HPLC Analysis of Ferredoxin NADP Reductase on

Ferredoxin
TSKgel® Phenyl-5PW NADP reductase
column . . . . . . . . . . . . . . TSKgel Phenyl-5PW column, 7.5 cm × 7.5 mm I.D.,
10 μm particles (807573) Crude Sample
mobile phase . . . 60 min decreasing linear gradient from 1.5M to 0M ammonium
sulfate in 0.1M sodium phosphate buffer, pH7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C Standard
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . 3mg (crude) or 0.2mg (standard)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0922
0 30 60 90
Min

HPLC Analysis of Fluorinated Pyrimidine 1. 5-Fluorocytosine (75 µg/mL) Gradient Program

Nucleosides on Ascentis® RP-Amide 2. 5-Fluorouracil (50 µg/mL) Time (min) %B
2 3. Floxuridine (50 µg/mL) 0 0
column . . . . Ascentis RP-Amide, 5 cm × 4.6 mm I.D., 5 μm particles (565323-U) 1
4. Fudarabine (50 µg/mL) 8 8
5. Trifluridine (60 µg/mL) 10 8
mobile phase . . . . . . . . . . . . . . . . . (A) water with 0.1% ammonium formate
11 0
(pH 3.04 with formic acid), (B) acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 3 4
5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in mobile phase A 0 2 4 6 8 10
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003940

HPLC Analysis of FMOC-Alanine Enantiomers on OH

Astec® CHIROBIOTIC® T H3C *
O
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HN O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004559
0 2 4 6 8 10 12
Min

HPLC Analysis of FMOC-Arginine Enantiomers on

mobile phase . . . . . . . . . . . . 0.1% ammonium trifluoroacetic acid in methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm O NH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL H2N NH OH
*
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004678 NH O
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of FMOC-Asparagine Enantiomers O

on Astec® CHIROBIOTIC® T H2N *
OH
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) O HN O
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004562
0 2 4 6 8
Min

HPLC Analysis of FMOC-Aspartic Acid Enantiomers O
on Astec® CHIROBIOTIC® R HO *
OH
mobile phase . . (A) 10 mM ammonium acetate, pH 5.0; (B) methanol; (60:40, A:B) O HN O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004677
0 10 20
Min

HPLC Analysis of FMOC-Aspartic Acid Enantiomers

O
HO *
mobile phase . . (A) 20 mM ammonium acetate, pH 4.1; (B) methanol; (60:40, A:B) O HN O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004560
0 1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of FMOC-Citrulline Enantiomers on O

O O
O NH NH OH
mobile phase . . (A) 20 mM ammonium acetate, pH 4.1; (B) methanol; (60:40, A:B) O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004563
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of FMOC-Glutamic Acid O O

Enantiomers on Astec® CHIROBIOTIC® T *
HO OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004561
0 2 4 6 8 10
Min

HPLC Analysis of FMOC-Glutamine Enantiomers O O

on Astec® CHIROBIOTIC® T H2N
*
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004565
0 2 4 6 8 10 12 14
Min

CH3 O
HPLC Analysis of FMOC-Isoleucine Enantiomers on
H3C *
Astec® CHIROBIOTIC® T * OH
HN O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004575
0 2 4 6 8 10
Min.

H3C CH3
HPLC Analysis of FMOC-Leucine Enantiomers on O
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004564
0 2 4 6 8 10 12 14
Min

HPLC Analysis of FMOC-Lysine Enantiomers on

O
mobile phase . . . . . . . . . . . . 0.1% ammonium trifluoroacetic acid in methanol O NH *
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O HN
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm

O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004679
0 2 4 6 8
Min

HPLC Analysis of FMOC-Methionine Enantiomers OH

on Astec® CHIROBIOTIC® T H3C
S *
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004566
0 2 4 6 8 10 12 14
Min

HPLC Analysis of FMOC-Norleucine Enantiomers OH
on Astec® CHIROBIOTIC® T H3C

*
O
O
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) NH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004567
0 10 20
Min

HPLC Analysis of FMOC-Norvaline Enantiomers on O

H 3C *
HN O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004568
0 2 4 6 8 10 12 14
Min.

O
HPLC Analysis of FMOC-Phenylalanine *
OH
Enantiomers on Astec® CHIROBIOTIC® T HN O

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004569
0 10 20
Min.

HPLC Analysis of FMOC-Proline Enantiomers on

Astec® CYCLOBOND® I 2000 SN OH
N *
column . . CYCLOBOND I 2000 SN, 25 cm x 4.6 mm I.D., 5 μm particles (20524AST)
mobile phase . . . . . . . . . . . . . . (A) acetonitrile; (B) methanol; (C) acetic acid; O
(D) ammonium hydroxide; (85:15:0.4:0.1, A:B:C:D) O O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004710
0 10 20
Min

HPLC Analysis of FMOC-Serine Enantiomers on O

Astec® (R,R) P-CAP™
*
column . . . . Astec (R,R) P-CAP, 25 cm x 4.6 mm I.D., 5 μm particles (31024AST) HO OH
mobile phase . . . . . . . . . . . . . . 20 mM ammonium acetate in: (A) acetonitrile;
HN O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004725
0 2 4 6 8 10 12
Min

HPLC Analysis of FMOC-Threonine Enantiomers on CH3 O
Astec® CHIROBIOTIC® T HO *
*
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004571
0 2 4 6
Min.

O
HPLC Analysis of FMOC-Tryptophan Enantiomers
*
N HN O
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004570
0 10 20
Min.

HPLC Analysis of FMOC-Valine Enantiomers on CH3 O
Astec® CHIROBIOTIC® T H3C

*
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004589
0 2 4 6 8 10
Min.

HPLC Analysis of Fusion Protein on 7.087

7.087
Zenix®-C SEC-300
column . . . . . . Zenix-C SEC-300, 30 cm x 7.8 mm I.D., 3 μm particles (Z777092)
7.955
mobile phase . . . . . . 150 mM sodium phosphate buffer, pH 7.0 + 200 mM NaCl 6.283
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1102 psi (76 bar)
column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
5 6 7 8 9
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL fusion protein
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005953
6.283 7.955
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

HPLC Analysis of Fusion Protein on Zenix®-C Zenix-C Sec-300

SEC-300 vs. Zenix® SEC-300
column . . . . . . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm particles (Z777033) Zenix Sec-300
mobile phase . . . . . . 150 mM sodium phosphate buffer, pH 7.0 + 200 mM NaCl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . 1 mg/mL fusion protein, MW 170 kD, pl 6.8-7.0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005951
0 5 10 15 20 25
Min

HPLC Analysis of Fusion Protein on Zenix®-C SEC- 1. 150 mM sodium phosphate buffer,
pH 7.0 + 20 mM NaCl
300, Effect of Mobile Phase Composition 2. 150 mM sodium phosphate buffer,
column . . . . . . Zenix-C SEC-300, 30 cm x 7.8 mm I.D., 3 μm particles (Z777092) pH 7.0 + 10% acetonitrile
gradient . . . . . . . . . . . . . (A) lower, 150 mM sodium phosphate buffer, pH 7.0 3. 150 mM sodium phosphate
(B) middle, 150 mM sodium phosphate buffer, pH 7.0 + 10% acetonitrile buffer, pH 7.0
(C) top, 150 mM sodium phosphate buffer, pH 7.0 + 20 mM NaCl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . 1 mg/mL fusion protein, MW 170 kD, pl 6.8-7.0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005952 1
2
3
0 5 10 15 20 25 30 35
Min
Note: 10% retention time offset for presentation purpose

HPLC Analysis of Glutamic Acid Enantiomers on 1 1. L-Glutamic acid

2. D-Glutamic acid
Astec® CHIROBIOTIC® T (23 °C) OH
2
mobile phase . . . . . (A) water; (B) methanol; (C) formic acid; (20:80:0.02, A:B:C) NH2
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C HO
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004496
0 2 4 6 8
Min

HPLC Analysis of Glutamic Acid Enantiomers on 1. L-Glutamic acid 1

Astec® CHIROBIOTIC® T (25 °C) 2. D-Glutamic acid
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) O OH 2

HO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min NH 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005335
0 2 4 6
Min

HPLC Analysis of Glutamine Enantiomers on 1 1. L-Glutamine

2. D-Glutamine
Astec® CHIROBIOTIC® T (23 °C) NH2 O
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (50:50, A:B) O OH 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004497
0 2 4 6
Min

HPLC Analysis of Glutamine Enantiomers on 1. L-Glutamine 1 NH 2 O
Astec® CHIROBIOTIC® T 2. D-Glutamine O OH

2
NH 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005336
0 2 4 6 8
Min

HPLC Analysis of Glutathione S-Transferase on Active fraction

TSKgel® G3000SWXL (recovered activity = 89%)
column . . . . TSKgel G3000SWXL, 30 cm × 7.8 mm I.D., 5 μm particles (808541)
mobile phase . . 0.3M sodium chloride/0.05M sodium dihydrogen phosphate, pH 7.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Standard
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220nm and enzyme assay
injection . . . . . . . . . . . . . . . 100μL crude glutathione S-transferase preparation
from guinea pig liver (0.7mg)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0939
0 5 10 15
Min

HPLC Analysis of Glycan and Isomers on G0F: asialo, agalacto, core-fucosylated

Proteomix® SAX-NP5, High Resolution Separation biantennary glycan
G1F: asialo, mono-galacto, core-fucosylated
biantennary glycan
mobile phase . . . . . . . . . . . . . (A) 2.5% (v/v) acetic acid, 0.5% TEA in water;
G2F: asialo, di-galacto, core-fucosylated
0.5% acetic acid in acetonitrile
G1F biantennary glycan
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 60 min
SA1F: mono-sialylated, galactosylated,
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
G0F core-fucosylated biantennary glycan
detector . . . . . . . . . . . . . . . . . . . . . . . . . Fluorescence, Ex/Em=360/425 nm (from NGNA, N-glycolylneuraminic acid)
sample . . . . . . . . . . . . . . . . . . . . . . 2-AA (anthranilic acid) labeled N-linked
SA2F: di-sialylated, galactosylated,
oligosaccharide profiling of an IgG1 sample
core-fucosylated biantennary glycan
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005959 (from NGNA, N-glycolylneuraminic acid)
G2F
SA1F
SA2F
0 10 20 30 40 50 60 70
Min
Courtesy of Miyako Kawakatsu, M&S Instruments Inc

HPLC Analysis of Hemoglobin HbA1C on A405nm

100
Mono S® HR5/5
column . . . . . . . . . . . . . . Mono S HR5/5, 5 cm x 5 mm I.D., 10 μm (54808)
mobile phase . . (A) 0.01M malonate-NaOH, pH 5.7; (B) 0.3M LiCl in mobile phase A
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min
% full scale
HbA1c
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIS, 405 nm
%B
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μL hemolysate, dilued 1:5 100
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0412
0
0 5 10
Min

HPLC Analysis of Hemoglobin S on Discovery® BIO Gradient Profile

PolyMA-SCX, Ion Exchange Min %A %B
Discovery BIO PolyMA-SCX and PolyMA-WAX columns give sharp, efficient 0 100 0
30 0 100
peaks for a wide variety of proteins.
column . . . . Discovery BIO PolyMA-SCX, 5cm × 4.6mm, 5μm particles (59601-U)
(B) 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0
gradient . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 100% B in 30 min. (linear)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001834
0 5 10 15 20 25 30
Min

HPLC Analysis of Hemoglobin Variants on 2 1. Hemoglobin A2

Discovery® BIO PolyMA-WAX 1 2. Hemoglobin S
3. Hemoglobin A0
column . . Discovery BIO PolyMA-WAX, 5 cm x 4.6 mm, 5 μm particles (59602-U)
mobile phase . . . . . . . . . . . . . . . . . (A) 10 mM Tris, pH 8.0 with acetic acid; 3
(B) 10 mM Tris, 0.25 M KCl, pH 8.0 with acetic acid
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 32% B in 20 min. (linear)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . 50 μg each Hb variants (A2, S and A0)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001830
0 10 20 30
Min

HPLC Analysis of Histidine Enantiomers on Astec® NH2 1. L-Histidine

2
CHIROBIOTIC® T (15 °C) O 2. D-Histidine 1
HN
mobile phase . . . . (A) water; (B) methanol; (C) formic acid; (30:70:0.006, A:B:C)
N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005337
0 20 40
Min

HPLC Analysis of Histidine Enantiomers on Astec® 1 1. L-Histidine

NH2 2. D-Histidine
CHIROBIOTIC® T (23 °C)
O
2
mobile phase . . (A) 160 mM sodium phosphate, pH 4.5; (B) ethanol; (40:60, A:B) OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004498
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Homophenylalanine Enantiomers

NH2
OH
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (90:10, A:B) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004533
0 2 4 6 8
Min

HPLC Analysis of Horse Serum Proteins in

Acetonitrile as a Function of Ammonium Formate
Concentration
Although horse serum proteins can be analyzed under standard SEC
conditions, the resolution is poor. However, very high efficiency separations
can be achieved when using pure acetonitrile to which ammonium formate
has been added. About 200 mM ammonium formate is required for all
proteins to elute within the pore volume of SRT SEC-150 column. The
position of the components can be modulated by decreasing the
ammonium formate concentration.
Ref.: V. McKay; X. Huang, LC-GC North America;Sep2006 Supplement, Vol. 24, p25, 2D Chromatography
of Proteins: Horse Serum Analysis
Courtesy of Miyako Kawakatsu, M&S Instruments Inc. 200 mM
mobile phase . . . . . . . . . . . . . . . . 100–200 mM CH3COONH4/CH3CN, pH 6.3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 mL/min 150 mM
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SofTA ELSD
sample . . . . . . . . . . . . . . . . . Molecular weight marker proteins, horse serum
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006212 100 mM
0 5 10 15 20
Min

HPLC Analysis of HSA and HSA Fusion Protein on

Zenix®-C SEC-300
mobile phase . . . . . . . . . . . . . (A) 150 mM sodium phosphate buffer, pH 7.0;
(B) IPA; (95:5, v/v, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . HSA fusion peptide 5 mg/mL (MW 75 kD, pl 5.0,
HSA conjugated peptide in diabetes treatment)
HSA fusion protein
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005956
HSA
4 6 8 10 12
Min

HPLC Analysis of HSA Fusion Protein on

Proteomix® SAX-NP5
column . . . . . Proteomix SAX-NP5 25 cm x 4.6mm I.D., 5 μm particles (Z777229)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm HSA, 2 mg/mL, 10 μL
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C E2HSA, 5 mg/mL, 10 μL
mobile phase . . Piperazine, Triethanolamine, bis-tris propane and N-methylpiperazine,
20 mM each,(A) pH 9.7; (B) pH 3.0 ( adjust by HCl )
HSA+E2HSA (1:1 v/v), 30 μL
sample . . . . . . . . . . . . . . . . HSA fusion peptide 5 mg/mL, 10 μL (MW 75 kD,
pI 5.0, HSA conjugated peptide in diabetes treatment)
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2016 psi (139 bar) 0 5 10 15 20 25 30

HPLC Analysis of HSA Fusion Protein on Zenix®-C

SEC-300, Effect of Mobile Phase Composition
mobile phase . . 150 mM sodium phosphate buffer, pH 7.0 or (A) 150 mM sodium
phosphate buffer, pH 7.0; (B) IPA; (95:5, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
HSA conjugated peptide in diabetes treatment)
B. R=1.58
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005955
A. R=1.09
2 4 6 8 10 12
Min

HPLC Analysis of HSA Fusion Protein on Zenix®-C

SEC-300 vs. Zenix® SEC-300
mobile phase . . . . . . . . . . . . . . . . 150 mM sodium phosphate buffer, pH 7.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
HSA conjugated peptide in diabetes treatment) Zenix™-C SEC-300
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005957
Zenix™ SEC-300
2 4 6 8 10 12 14
Min

HPLC Analysis of Human Growth Hormone (hGH) hGH

on Discovery® BIO PolyMA-WAX
Untreated hGH
This Figure shows the degradation products of human growth hormone
(hGH) well-separated on a Discovery BIO PolyMA-WAX anion-exchange
column. The difference between the resolved compounds is the conversion
of protein amide(s) to carboxylate(s), demonstrating both the power of the
ion-exchange technique, and the efficiency of the Discovery BIO PolyMA-
WAX column.
column . . Discovery BIO PolyMA-WAX, 5 cm × 4.6 mm I.D., 5 μm particles (59602-U) Desamido hGH derivative
Heated hGH: 60 °C, 5 hr.
mobile phase . . (A) 20 mM Tris-HCl, pH 8.0; (B) 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg hGH Heated hGH: 70 °C, 8 hr. Desamido hGH derivatives
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0659
0 5 10 15 20 25 30
Min

HPLC Analysis of Human Serum Albumin Tryptic

Digest on Ascentis® Express C18
Sample Prep: 0.1 gram of albumin was dissolved in 10 mL of 0.01 M
ammonium formate buffer, and the pH was adjusted to 8.0 with ammonium
hydroxide; the solution was heated in a boiling water bath for 6 min. After
the solution cooled, 2.0 mg of trypsin from bovine pancreas was added, and
the mixture was allowed to react for 4 h at 37 °C; the reaction was quenched
by adding 0.1% trifluoroacetic acid to pH 2. The digest was stored at 4 °C
and filtered prior to injection through a 0.45 ìm nylon membrane
sample/matrix . . . . . . . . . . . . . . . . . . . human serum albumin, tryptic digest 0.2 0.4 0.6 0.8 1.0
column . . Ascentis Express C18, 3 cm x 4.6 mm I.D., 2.7 μm particles (53818-U) Min
mobile phase . . . . . . . . . . . . . (A) 10 mM ammonium acetate in water, pH 9;
(B) 10 mM ammonium acetate in methanol/water, pH 9; (90:10, A:B)
gradient . . . . 0% B in 0.50 min; 55% B in 0.25 min; 90% B for 0.50-0.69 min;
0% B for 0.70-1.00 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005598

HPLC Analysis of Immunoglobulin G (IgG) on

Zenix® SEC-150 versus Zenix® SEC-300, Effect of
Pore Size on Resolution
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL, IgG
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006120 Zenix SEC-150
Zenix SEC-300
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Immunoglobulins (IgM) on A280nm

Q Sepharose® Fast Flow 5.0 100
media . . . . . . . . . . . . . . . . . . . . . . . Q Sepharose Fast Flow (Q1126-100ML)

column . . . . . . . . . . . . . . . . . . . . . . . 30 cm × 50 mm I.D. (Q1126-100ML) 4.0 80
mobile phase . . . . . . (A) 0.02 M NaH2PO4 , pH 6.5; (B) A + 0.5 M NaCl, pH 6.5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 cm/hr
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 3.0 60
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cell culture supernatant
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0370
2.0 40
IgM
containing
1.0 Start of fraction 20
elution
100 200 300 400

Volume, mL

HPLC Analysis of Inhibin α-Subunit Fragment 1-32 2
on Discovery® HS C18
Molecular weights calculated by
column . . . . Discovery HS C18, 10 cm × 2.1 mm ID, 5 μm particles (568501-U) deconvolution of spectra:
mobile phase . . (A) 20 mM formic acid in water; (B) (20 mM formic acid in water): 1. 3673
2. 3601
(20 mM formic acid in acetonitrile), 50:50 3. 3715
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 4. 3204
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg/L in 0.1 % formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002581
1 4
3
0 10 20
Min

HPLC Analysis of Inhibin α-Subunit Fragment 1-32 Molecular weights calculated

2
on Discovery® HS F5 by deconvolution of spectra:
column . . . . . Discovery HS F5, 10 cm × 2.1 mm ID, 5 μm particles (567510-U) 1. 3204
mobile phase . . (A) 80 mM formic acid, 40 mM ammonium hydrate (pH ~3.5) in 2. 3601
water; (B) 80 mM formic acid, 40 mM ammonium 3. 3615
hydrate in 80% acetonitrile 4. 3583
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+), full scan 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg/L in 0.1 % formic acid 1 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002580
0 10 20 30
Min

HPLC Analysis of Insulin, Bovine on Discovery® C8 2 1. Subunit A oxidized

column . . . . . . . . Discovery C8, 5 cm × 4.6 mm I.D., 5 μm particles (59353-U) 2. Holoenzyme
3 3. Subunit B oxidized
mobile phase . . . . 50 mM sodium phosphate, monobasic, 50 mM sodium sulfate,
pH 3.0 (w/phosphoric acid), (A) +5% acetonitrile,
(B) +50% acetonitrile Gradient Program
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Time (min) %B
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 33.3
1 18 73.3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000765
0 10 20
Min

HPLC Analysis of Insulin on SRT®-C SEC-300 1. Insulin 1
column . . . . . . SRT-C SEC-300, 30 cm x 7.8 mm I.D., 5 μm particles (Z777110)

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . insulin
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006167
Min

HPLC Analysis of Insulin (Species Variants) on 2 1. Insulin, bovine

Ascentis® Express Peptide ES C18 3 2. Insulin, human
3. Insulin, porcine
These three insulins have more than 76% identical positions in a primary 1
structure alignment. The human and porcine proteins are most similar with
85% identity. Baseline resolution of these insulins in achieved on Ascentis
Express Peptide ES-C18, with relative band spacing of these three peaks
approximating their relative sequence similarities. Ascentis Express Peptide
ES-C18 is ideally suited for RP chromatography of small proteins or large
peptides.
column . . . . . . . . . . . . Ascentis Express Peptide ES C18, 15 cm x 2.1 mm I.D.,
mobile phase . . . . (A) 70:30, water with 0.1% TFA:acetonitrile with 0.09% TFA;
(B) 55:45, water with 0.1% TFA:acetonitrile with 0.09% TFA 6.0 7.0
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 100% B in 7.5 min Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4040 psi (278 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . 200 μg/mL insulin (bovine, human, porcine) in 0.1% formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005794

HPLC Analysis of Insulin (Species Variants) on 1 1. Bovine insulin

Gradient 2. Human insulin
Discovery® BIO Wide Pore C5 Time (min) %A %B 2 3. Porcine insulin
column . . . . . . . . . . . . . . Discovery BIO Wide Pore C5, 15 cm × 4.6 mm I.D., 0 100 0 3
5 μm particles (568422-U) 30 0 100
mobile phase . . . . . (A): 71:29, (0.1% TFA in water):(0.1% TFA in acetonitrile);
(B): 68:32, (0.1% TFA in water):(0.1% TFA in acetonitrile)
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . 5 μL 5 μL in 0.1% TFA containing 5ug each of three insulin variants
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001580
0 2 4 6 8 10
Min

HPLC Analysis of Insulin (Species Variants) on Gradient 1. Bovine insulin (5 μg)

Time (min) %A %B 1 2. Human insulin (5 μg)
Discovery® BIO Wide Pore C8 3. Porcine insulin (5 μg)
0 100 0 2
mobile phase . . . . . . . . . . (A) (0.1% TFA in water):(0.1% TFA in acetonitrile),
71:29 (B): (0.1% TFA in water):(0.1% TFA in acetonitrile), 68:32,
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . 5 μL in 0.1% TFA containing 5ug each of three insulin variants
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001507
2 4 6 8 10
Min

HPLC Analysis of Insulin (Specirs Variants) on 1. Insulin, bovine

2
Ascentis® Express Phenyl-Hexyl 1 3 2. Insulin, human
3. Insulin, porcine
These three insulins have more than 76% identical positions in a primary
85% identity. The Phenyl-Hexyl phase provides an alternate retention and
selectivity to a C18 phase, particularly the C18 phase on a larger pore silica,
Ascentis Express ES-C18
column . . . . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 15 cm x 2.1 mm,
(B) 55:45, water with 0.1% TFA:acetonitrile with 0.09% TFA
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 100% B in 7.5 min 0 2 4 6
Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3960 psi (273 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 μg/mL in 0.1% formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005795

HPLC Analysis of Isoleucine Enantiomers on Astec® CH 3 OH

CHIROBIOTIC® T (23 °C ) H 3C *
O
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (70:30, A:B) NH 2

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004500
0 2 4 6
Min.

HPLC Analysis of Isoleucine Enantiomers on Astec®

1. L-Isoleucine 1 O
CHIROBIOTIC® T (25 °C ) 2. D-Isoleucine
OH
column . . Astec CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) 2
H2N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C H3C CH3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005338
0 2 4 6 8 10
Min

HPLC Analysis of β-Lactoglobulin, Gradient Program

Carboxymethylated, Tryptic Digest on Discovery® Time (min) %A %B
BIO Wide Pore C18 0 100 0
50 0 100
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water/0.1% TFA;
(B) (water/0.1% TFA):(acetonitrile/0.1% TFA), 50:50
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.20 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001720
0 20 40 60
Min

HPLC Analysis of Leucine Enantiomers on Astec® CH3 NH 2

CHIROBIOTIC® T (23 °C) O

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004501
0 2 4 6
Min.

HPLC Analysis of Leucine Enantiomers on Astec® 1

O
CHIROBIOTIC® T (25 °C) 1. L-Leucine OH
2. D-Leucine
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) 2 H2N
mobile phase . . . . . (A) water; (B) methanol; (C) formic acid; (30:70:0.02, A:B:C) H3C CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005339
0 2 4 6 8
Min

HPLC Analysis of Light Scattering Detection of BSA 1.15 BSA monomer

on Zenix® SEC-300
Intensity (rel.)
The chromatogram shows an overlay of the ultraviolet (UV), differential 0.75
refractive index (RI) and multi angle light scattering data (MALS) for a sample
BSA dimer
of bovine serum albumin; showing the prsenece of dimer and trimer as well
LS
as the monomer. The chromatogram illustrates the power of light scattering 0.35 RI BSA trimer
used with Sepax’s Zenix® SEC in protein characterization. The light scattering
UV
data makes it possible to determine the molecular mass for the monomer,
dimer and trimer. -0.05
Acknowledgements and thanks to Matthew Thompson for this data. 0 2 4 6 8 10
Postdoctoral Researcher - Gianneschi Lab, Department of Chemistry & Min
Biochemistry University of California, San Diego
column . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm particles, 300 Å (Z777033)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1M phosphate buffer
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . Hitachi-Elite LaChromL-2130 pump equipped with amulti-angle light
scattering detector (DAWN-HELIOS: Wyatt Technology),
a refractive index detector (Hitachi L-2490) and a
UV-Vis detector (Hitachi 2420)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BSA (20mg/mL)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006198

HPLC Analysis of Lipoxidase on TSKgel®

Phenyl-5PW
column . . . TSKgel Phenyl-5PW, 7.5 cm × 7.5 mm I.D., 10 μm particles (807573)
mobile phase . . . . . . . . (A) 0.1M phosphate buffer, pH 7.0 + 1.5M (NH4)2 SO 4;
(B) 0.1M phosphate buffer, pH 7.0;
gradient . . . . . . . . . . . . . . . . . . . . . . linear, 0% B to 100% B over 60 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0320
0 30 60
Min

HPLC Analysis of Luciferin Enantiomers on Astec®

N S
CHIROBIOTIC® V
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) S N OH
HO *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004641
0 2 4 6 8 10
Min

HPLC Analysis of Lysine Enantiomers on Astec® 1. L-Lysine O
CHIROBIOTIC® T 2. D-Lysine H2N

OH
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) NH2
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005340
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Lysine Enantiomers on Astec® O
CHIROBIOTIC® T (Ethanol Mobile Phase) H2N *

OH
NH 2
mobile phase . . (A) 100 mM sodium phosphate, pH 4.5; (B) ethanol; (70:30, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004502
0 2 4 6
Min.

HPLC Analysis of Lysozyme, Myoglobin and Discovery BIO Wide Pore C5

Impurities on Discovery® BIO Wide Pore C5
600 1. Impurity in Lysozyme
column . . Discovery BIO Wide Pore C5, 15cm × 4.6mm I.D., 5μm particles (568422-U) 2. Impurity in Myoglobin
mobile phase . . . . . . . . (A) (75:25) 0.1% TFA in water:0.1% TFA in acetonitrile;
mAU 220 nm
(B) (25:75) 0.1% TFA in water:0.1% TFA in acetonitrile 400
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 25 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 1 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 μL in 0.1% TFA 0
0 10 20
Min

HPLC Analysis of Lysozyme on SRT® SEC-300, 10.5
30 cm x 7.8 mm I.D., 5 μm particles

The positively-charged protein, lyzoyme (pI=11), exhibits excellent peak
shape and recovery on Discovery BIO GFC columns due to its highly-inert
surface. Low protein recovery is usually attributed to 1) hydrophobic
interactions with the stationary phase and 2) electrostatic interactions with
the silica suface. Discovery BIO GFC surface is hydrophilic and densely
bonded to silica, therefore providing high mass recovery and good peak
shape.
column . . . . . . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm particles (Z777051)
mobile phase . . . . . . 50 mM phosphate buffered saline, 200 mM Na2SO4, pH 7.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . 1 mg/mL in 50 mM phosphate buffered saline
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004736

HPLC Analysis of mAb 016 on Proteomix® SCX-NP5

Column at Different Flow Rates Time Flow Rate Time Flow Rate
(min) (mL/Min) %B (min) (mL/Min) %B
This figure shows the how flow rate (F) affects analysis time and resolution 0 0.8 0 0 1.5 0
during gradient analysis. When comparing the performance of different 30 0.8 6 16 1.5 6
column formats or flow rates or gradient times, the rule of thumb is to keep
the gradient volume, i.e. the product of gradient time and flow rate,
constant. When gradient volume is kept constant, the compounds elute
from the column in the same eluent composition.
Note that in this example the gradient time (tg) was reduced by the same
factor as the increase in flow rate, thereby proportionaly increasing the
gradient steepness, since gradient steepness is equal to the change in
composition per unit time; in this case 6%B/16 vs. 6%B/30. It is now easy to
see that the gradient volume was kept constant, since 30 min x 0.8 mL/min
= 15 min x 1.6 mL/min = 24 mL. Clearly, the separation deteriorated
substantially at the higher flow rate.
column . . Proteomix SCX-NP5, 25 cm x 4.6mm I.D., 5 μm particles, PEEK (Z777164)
mobile phase . . . . . (A) 20 mM phosphate buffer, pH 7.5 and (B) A + 1 M NaCl 0.8 mL/min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 1.5 mL/min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . mAb 016 (11.8 mg/mL) 4 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006173 0 5 10 15 20 25 30 35
Min

HPLC Analysis of MAb 016 on Proteomix® SCX NP5

versus Competitive SCX using a Salt Gradient
column . . . . . . . . . Proteomix SCX-NP5, 25 cm x 4.6 mm I.D. (PEEK hardware),
5 μm particles (Z777164)
mobile phase . . . . . . . (A) 20 mM phosphate buffer, pH 7.5; (B) A + 1 M NaCl
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 6% B in 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb 016 (11.8 mg/mL)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006107
Competitive SCX
Proteomix SCX
0 5 10 15 20 25 30
Min


versus Competitive SCX using pH and Salt Note improved resolution
Gradients on the Proteomix SCX
compared to competitive
column . . . . . . . . . Proteomix SCX-NP5, 25 cm x 4.6 mm I.D. (PEEK hardware), column.
mobile phase . . . . . . . . . . . . . . . . . . . . (A) 2.4 mM Tris, 1.5 mM imidazole,
11.6 mM piperazine, pH 6.0; (B) A + 0.5 M NaCl, pH 10.5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb 321 (5 mg/mL)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006098
Competitor SCX
Proteomix SCX
17.5 20.0 22.5 25.0 27.5 30.0 32.5

Min

HPLC Analysis of mAb Charge Variants on TSKgel® 40

CM-STAT
A TSKgel CM-STAT weak cation exchange (WCX) column was applied to 30
separate charge variants of several monoclonal antibodies. The typical
analysis time on conventional 25 cm long WCX columns of about eighty
mV
20 E
minutes could be significantly reduced when separation was performed on
a 10 cm TSKgel CM-STAT column, filled with 7 μm particles. The analysis D
profiles for five antibodies show that high resolution analysis can be 10 C
obtained in about 20 minutes analysis time.
B
From "Ion Exchange Chromatography for the Characterization of Biother-
apeutics" in The Supelco Reporter, Vol. 29.3, page 20. 0 A
column . . . . . . TSKgel CM-STAT, 10 cm x 4.6 mm I.D., 7 μm particles (821966) 0 10 20 30 40
mobile phase . . (A) 20 mM MES buffer, pH 6.0; (B) 0.5 M NaCl in buffer A, pH 6.0 Min
gradient . . . . . . . . . . . . . 0% B (0 min), 30% B (15 min), 100% B (15 min),
0% B (17 min), 10% B (17 min), 10% B (21 min)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . . . . . . . monoclonal antibodies (mAb A through E)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005458

HPLC Analysis of mAb on Antibodix® WCX, Effect

of Particle Size (5 cm columns)
column . . Antibodix WCX-NP3 PEEK, 5 cm x 4.6 mm I.D., 3 μm particles (Z777288)
N = 7260
column . . . . . Z777280 (Antibodix WCX-NP1.7, 5 cm x 4.6 mm, 1.7 μm particles)
mobile phase . . (A) 10 mM sodium phosphate buffer, pH 7.5; (B) A + 100 mM NaCl
gradient . . . . . . . . . . . NP3 column: 15 to 55% B in 30 min, held for 3 min;
NP1.7 column: 15 to 100% B in 60 min, held for 1.7 min N = 15680
Gray: Antibodix WCX-NP3
flow rate . . . . . . . . . . . . . . . . 0.5 mL/min for 3 min; 0.3 mL/min for 1.7 min (5 cm × 4.6 mm, 3 μm non-porous particles)
pressure . . . . . . . . . . . . . . . . . . . . . . 2180 psi (3 min), 3425 psi (1.7 min) Pressure: 2,180 psi

detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm Black: Antibodix WCX-NP1.7
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL (5 cm × 4.6 mm, 1.7 μm non-porous particles)
Pressure: 3,425 psi
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . Monoclonal antibody, 5 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005941
0 5 10 15 20 25 30 35 40
Min

HPLC Analysis of mAb on Antibodix® WCX-NP5, N = 12,170

25 cm Columns
column . . . . Antibodix WCX-NP5, 25 cm x 4.6 mm I.D., 5 μm particles (Z777294)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . monoclonal antibody
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005942
0 5 10 15 20 25 30
Min

HPLC Analysis of MAb on Proteomix® SCX-NP5

Comparing Different Buffer Systems and pH
Gradients
column . . . . Proteomix SCX-NP5, 25 cm x 4.6 mm I.D., 5 μm particles (Z777161)
mobile phase . . . . . . . . . . . . . . . . Mobile phase A: (A) 20 mM MES, pH 6.0;
(B) A + 1 M NaCl, pH 6.0Mobile phase B: (A) 2.4 mM Tris,
1.5 mM imidazole, 11.6 mM piperazine, pH 6.0; (B) A, pH 10.5
gradient . . . . . . . . . . . Gradient A: 7% B held for 2 min; to 14% B in 20 min,
Gradient B: 0 to 30% B in 2 min; to 55% B in 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm A. MES buffer
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006141 B. pH gradient
0 5 10 15 20 25 30
Min

HPLC Analysis of mAb F on Zenix®-300 and

MAb MW 145 kD
Zenix®-C 300 pl = 8.4
Zenix-C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073 psi (74 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . 1.23 mg/mL mAb F in 10 mM sodium succinate, pH 5.0 Zenix-300
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005950
5 6 7 8 9 10 11 12 13
Min

HPLC Analysis of mAb-X22 on Antibodix® A: 15–75% B in 30 min

WCX-NP10, Effect of Gradient Profile B: 15–65% B in 30 min
C: 15–55% B in 30 min
column . . Antibodix WCX-NP10, 25 cm x 4.6 mm I.D., 10 μm particles (Z777272) D: 15–47.5% B in 30 min
mobile phase . . (A) 10 mM sodium phosphate buffer, pH 7.5; (B), A + 100 mM NaCl E: 5–40% B in 30 min and 40% B after 30 min
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) 15-75% B in 30 min
(B) 15-65% B in 30 min
(C) 15-55% B in 30 min
(D) 15-47.5% B in 30 min
(E) 15-40% B in 30 min and 40% B after 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
A
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb-X22, 1.5 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005943 B
0 5 10 15 20 25 30 35 40
Min

HPLC Analysis of mAb-X22 on Antibodix®

WCX-NP10, Impact of Initial Salt Concentration
column . . Antibodix WCX-NP10, 25 cm x 4.6 mm I.D., 10 μm particles (Z777272)
mobile phase . . (A) 10 mM sodium phosphate buffer, pH 7.5; (B), A + 100 mM NaCl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb-X22, 1.5 mg/mL A
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005944
B
0 5 10 15 20 25 30 35 40 45 50
Min


WCX-NP10, Impact of Mobile Phase pH
mobile phase . . . . . . . . . . . . . (A) 10 mM sodium phosphate buffer at 7.5 pH;
(B), A + 100 mM NaCl at 7.0 pH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL A. pH 7.5
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb-X22, 1.5 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005945
B. pH 7.0
0 10 20 30 40 50 60
Min

Aqpz is 23 kD
HPLC Analysis of Membrane Protein Aquaporin-Z BOG Detergent is nonionic
(AqpZ) on SRT® SEC-300, Octyl Glucoside Mobile
Phase Additive Nanofilm SEC-250
SRT SEC-300 gave the best separation of the three columns tested. It
exhibited fewer secondary interactions and provided baseline separation of
the high molecular weight proteins.
Zenix™ SEC-300
column . . . . . . . . SRT-SEC 300, 30 cm x 7.8mm I. D., 5 μm particles (Z777110)
mobile phase . . . . . . . . . . . . . . . . . . 20 mM tris-HCl pH 7.0, 190 mM NaCl,
SRT®-C SEC-300
10 mM KCl, 40 mM octyl glucoside
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
4 5 6 7 8 9 10 11 12 13 14
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . aqpZ, 6 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006169

HPLC Analysis of Methionine Enantiomers on OH

Astec® CHIROBIOTIC® T (No Additives)
S *
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H 3C O
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (30:70, A:B) NH 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004503
0 2 4 6 8 10
Min.

HPLC Analysis of Methionine Enantiomers on 1. L-Methionine S NH2

H3C
Astec® CHIROBIOTIC® T 2. D-Methionine
1
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HO O
mobile phase . . . . . (A) water; (B) methanol; (C) formic acid; (30:70:0.02, A:B:C) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005341
0 2 4 6 8
Min

HPLC Analysis of Methionine Enantiomers on O

S *
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) H 3C OH
NH2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004598
0 2 4 6 8 10
Min.

HPLC Analysis of 3-Methylaspartic Acid

Enantiomers on Astec® CHIROBIOTIC® T CH3 O
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HO *
* OH
mobile phase . . . . . (A) methanol; (B) water; (C) formic acid; (80:20:0.02, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min O NH2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004553
0 2 4 6 8
Min


column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) H
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . methanol * N
O
H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.48 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C N
O H
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004525
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of α-Methyl-α-Phenylsuccinimide

mobile phase . . . . . . . . . . . . . . . . . . . (A) ethanol; (B) hexane; (20:80, A:B) H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C O
N
O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm H
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004544
0 1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of α-Methyl-m-Tyrosine

Enantiomers on Astec® CHIROBIOTIC® T H3C
O
*
HO
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . methanol NH2
extraction tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004557
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Milk Proteins on HiTrap® SP Fract. 2

A280nm
column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HiTrap SP, 1 mL (54817) 100% B
mobile phase . . . . . . . . . . . . . . . . . . . . (A) 50 mM sodium acetate, pH 6.0;
0.5
(B) 50 mM sodium acetate/1.0 M sodium chloride, pH 6.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 0.4
injection . . . . . . . . . . . . . . . . . 0.5 mL filtered casein-precipitated human milk
(buffer exchanged to mobile phase A)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795-0493 0.3
0.2
40% B
Fract. 24
0.1
Fract. 16
Fract. 17
Fract. 9
0
10% B
5 10 15 20 25 30
Volume, mL

HPLC Analysis of Monoclonal Antibodies (mAb) Antibodix WCX-NP10

with Charge Variance on Antibodix® WCX-NP10 25 cm × 4.6 mm, 10 μm (non-porous particles)
Pressure: 840 psi
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840 psi (58 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm MAb+ 1 lysine
MAb Acidic isomer
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL MAb+ 2 lysine
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005940
0 5 10 15 20 25
Min

HPLC Analysis of Monoclonal Antibody Fragments 1. scFC 2

2. F(ab´)2
on BIOshell™ A400 Protein C4
Digesting an antibody into a few large fragments facilitates a middle-down
approach for subsequent structural characterization. The IdeS is a
Streptococcal protease (commercialized as FabRICATOR) that cleaves IgG in 1
the hinge region resulting in two fragments: F(ab′)2 and scFc.
Monoclonal antibody SO57 (0.5 mg) was digested by IdeS protease
according to Genovis (www.genovis.com) FragIT kit protocol. The digest was
then diluted 1:5 with mobile phase A, before injection; final protein conc.
<0.3 g/L.
Genovis FragIT kit is available from Sigma, outside the USA.
column . . . . . . . . . . . . . . . . BIOshell A400 Protein C4, 10 cm x 2.1 mm I.D.,
0 2 4 6 8 10
mobile phase . . . . . . (A) 80:20, [0.1% TFA in water]:[0.1% TFA in acetonitrile] ;
(B) 50:50, [0.1% TFA in water]:[0.1% TFA in acetonitrile] Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 psi initial (69 bar initial)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006204

HPLC Analysis of Monoclonal Antibody Heavy and Heavy Chain

Light Chains on Zenix® SEC-300
Light Chain
mobile phase . . . . . . . . . . . . . . 0.1% TFA, 0.1% formic acid, 20% acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min Reduced MAb
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . Monoclonal antibody (1 mg/mL) reduced with 20 mM DTT
Intact MAb
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006171
20 mM DTT
10 15 20 25
Min

HPLC Analysis of a Monoclonal Antibody on 1. Aggregate 3

Discovery® BIO GFC 150 after Partial Proteolysis 2. Intact IgG
3. F(ab’)2
with IdeS Protease 4. scFc 2
Proteolysis of antibodies can be useful for simplifying further analysis. This 4
application demonstrates separation of the two fragments, F(ab′)2 and scFc
from the intact IgG, after partial digestion with IdeS protease.
column . . Discovery® BIO GFC 150, 30 cm X 4.6 mm I.D., 3 μm particles (567329-U)
mobile phase . . . . . . . . . . . . . . . . . 0.15 M potassium phosphate monobasic,
pH 7.0 with potassium hydroxide (567329-U)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.35 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar)
1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 0.0 2.5 5.0 7.5 10.0 12.5 15.0
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . digested SO57 mAb, ~0.5 g/L Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005896

HPLC Analysis of a Monoclonal Antibody on 1. aggregate 3

Discovery® BIO GFC 300 after Partial Proteolysis 2. intact IgG 2
3. F(ab’)2
with IdeS Protease 4. scFc 4
Proteolysis of antibodies can be useful for simplifying further analysis. This
application demonstrates separation of the two fragments, F(ab′)2 and scFc
from the intact IgG, after partial digestion with IdeS protease.
column . . Discovery® BIO GFC 300, 30 cm X 4.6 mm I.D., 3 μm particles (567335-U)
pH 7.0 with potassium hydroxide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.35 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1350 psi (93 bar)
1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 0 2 4 6 8 10 12 14
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005899

HPLC Analysis of a Monoclonal Antibody on SRT®

0.02
SEC-300
column . . . . . . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm particles (Z777051) Buffer Peak
Zoom-in
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb 10, 2.5, 0.5 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006170
0.0 2.5 5.0 7.5 10.0 12.5 15.0
1.8
1.6
1.4
1.2
1.0
AU
0.8
0.6
0.4
0.2
0.0
00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

HPLC Analysis of a Monoclonal Antibody on 1. intact IgG 2

Zenix® SEC-150, 30 cm X 4.6 mm I.D., 3 μm 2. F(ab')2
3. scFc
particles after Proteolysis with IdeS Protease 3
Proteolysis of antibodies can be useful for simplifying further analysis. This
application demonstrates separation of the two fragments, F(ab′)2 and scFc,
after digestion with IdeS protease
column . . . . . . Zenix® SEC-150, 30 cm X 4.6 mm I.D., 3 μm particles (Z777018)
pH 7.0 with potassium hydroxide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.35 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar) 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 0.0 2.5 5.0 7.5 10.0 12.5 15.0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005889

HPLC Analysis of mPEG and mPEG-Peptide

(Methoxy-PEG-Maleimide) on Zenix® and
Zenix®-C SEC
column . . . . . Zenix-C SEC-300, 30 cm x 7.8 mm, I.D., 3 μm particles (Z777092)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL Zenix SEC-300
sample . . . . . . . . 20kD peptide mPEG-MAL (methoxy-PEG-Maleimide), 1 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005948 Zenix-C SEC-300
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Min

HPLC Analysis of mPEG and mPEG-Peptide on Zenix SEC-300

Zenix® and Zenix®-C SEC
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 2 3 4 5 6 7 8 9 10 11 12 13 14
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . 4kD peptide and 20kD mPEG-MAL(methoxy-PEG-Maleimide) (6 mg/mL)
mPEG-peptide
2xmPEG-peptide
2 3 4 5 6 7 8 9 10 11 12 13 14
Min

HPLC Analysis of Norleucine Enantiomers on

*
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (80:20, A:B) H3C OH
flow velocity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C NH2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004534
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Min

HPLC Analysis of Norphenylephrine Enantiomers

OH
on Astec® CYCLOBOND® I 2000 AC (pH 4.1)
column . . CYCLOBOND I 2000 AC, 25 cm x 4.6 mm I.D., 5 μm particles (20124AST) HO
*
NH2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004693
0 2 4 6 8
Min

HPLC Analysis of Norvaline Enantiomers on Astec®

CHIROBIOTIC® T
NH2
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min H3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004535
0 1.0 2.0 3.0 4.0 5.0 6.0

Min

1 1. Thymine (0.1 g/L)

HPLC Analysis of Nucleic Acid Bases on 2. Uracil (0.1 g/L)
Ascentis® Si 3. Adenine (0.1 g/L)
4. Guanine (0.1 g/L)
column . . . . . . . . Ascentis Si, 10 cm x 2.1 mm I.D., 5 μm particles (581508-U) 5. Cytosine (0.1 g/L)
mobile phase . . . . . (A) 200 mM ammonium formate (pH 3.0, with formic acid): 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003723
0 2 4 6 8 10
Min

HPLC Analysis of Nucleosides on Ascentis® 1. Acenaphthene, 80 μg/mL in mobile phase

2. Adenosine, 35 μg/mL in mobile phase
Express HILIC 3. Cytosine, 75 μg/mL in mobile phase
column . . Ascentis Express HILIC, 10 cm × 2.1 mm I.D., 2.7 μm particles (53939-U)
2
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 100 mM ammonium formate,
pH 3.0 with concentrated formic acid: 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004183
0 1 2 3 4 5
Min

HPLC Analysis of Nucleosides on Ascentis® Express 1. Ribothymidine 10. 3-Methylcytidine

OH5 (HILIC Mode) 2. Uridine 11. 1-Methyladenosine
3. 2-Thiocytidine 12. 7-Methylguanosine
Nucleosides are involved in many aspects of metabolism. Analogs of 4. 2’-O-Methylcytidine
nucleosides have medical application in antivirals or anticancer treatments. 5. Pseudouridine
This application demonstrates the utility of hydrophilic interaction 6. Inosine 7
chromatography (HILIC) for the retention and resolution of nucleosides and 7. 5-Methylcytidine
analogs thereof. Elution is not solely explained by logD but by other ionic 8. Cytidine
and solvophobic effects. 9. Guanosine
mobile phase . . . . . . . . . . . . . . . . (A) 5 mM ammonium acetate, pH 5.0 with 1 8
acetic acid in 95:5, acetonitrile:water; 9
(B) 5 mM ammonium acetate, pH 5.0 with acetic 6 10
acid in 80:20, acetonitrile:water 2
11
gradient . . 0% B held for 1 min; to 100% B in 10 min; held at 100% B for 1 min 3 5
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 12
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
0 2 4 6 8 10 12
sample . . 10 - 100 μg/mL in 95:5, acetonitrile:water (pH of buffer stock (in water)
Min
was adjusted before further dilution with water and/or acentonitrile)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005644

1 1. Thymidine (0.1 g/L) 5. Deoxyguanosine (0.1 g/L)

HPLC Analysis of Nucleosides on Ascentis® Si 2 2. Uridine (0.1 g/L) 6. Deoxycytidine (0.1 g/L)
34
3. Deoxydenosine (0.1 g/L) 7. Guanosine (0.1 g/L)
(HILIC Mode) 4. Adenosine (0.1 g/L) 8. Cytidine (0.1 g/L)
mobile phase . . . . . (A) 150 mM ammonium formate (pH 3.0, with formic acid): 6
5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 7
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003721
0 2 4 6 8 10
Min

(×10,000,000)
HPLC Analysis of Nucleosides on Ascentis® TIC 13
1.75
Si (Silica) 1.
2.
LLL
OLLn
10.
11.
PLO
PoPO
14
3. PLLn 12. PLP

column . . . . . . . . Ascentis Si, 10 cm × 2.1 mm I.D., 5 μm particles (581508-U) 1.50 4. OLL 13. OOO
5. OOLn 14. POO
mobile phase . . . . . (A) 150 mM ammonium formate (pH 3.0, with formic acid): 6. PLL 15. POP
125
(B) acetonitrile; (10:90, A:B) 7. PLnO 16. SOO
8. OOL 17. SOP 8
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 1.00
9. OOPo
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 10
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm 0.75
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 15
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μg/mL in mobile phase 0.50

4
9 16
11
5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003680 0.25
1 2
3
67 12 17
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0
Min

HPLC Analysis of Nucleosides on Ascentis® Phenyl 1. Cytidine (0.1 g/L)

column . . . . . Ascentis Phenyl, 15 cm x 2.1 mm I.D., 5 μm particles (581613-U) 2. Contaminants from Deoxyadenosine,
Deoxyguanosine, and Guanosine Standards
mobile phase . . . . . . . . . 10 mM ammonium formate (pH 3.0 with formic acid)
3. Deoxycytidine (0.1 g/L)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 1 4. Uridine (0.1 g/L)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 5. Guanosine (0.1 g/L)
detector . . . . UV, 270 nm; 750 psi back pressure regulator on outlet of flow cell 3
6. Adenosine (0.1 g/L)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 7. Deoxyguanosine (0.1 g/L)
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated, in mobile phase 4 8. Deoxyadenosine (0.1 g/L)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003710 9. Thymidine (0.1 g/L)
6
5 7 8 9
2
0 10 20
Min

HPLC Analysis of Nucleotides on SUPELCOSIL™ Gradient Program 1. HX

Time (min) %B 5 2. XAN
LC-18-T 3. INO
0 0 4. IMP
column . . . SUPELCOSIL LC-18-T, 15 cm × 4.6 mm I.D., 3 μm particles (58970-U) 2.5 0 5. NAD +
mobile phase . . (A) 0.1M potassium phosphate buffer/4mM tertrabutylammonium 5 30 6. ADO
10 60 7. AMP
hydrogen sulfate, pH6.0; (B) A:methanol (70:30), pH 7.2 13 100
11
8. GDP
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 17 100 14 9. NADP +
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 18 0 9 10. ADP-rib
injection . . . . . . . . . . . . . . . 20 μL deionized water (25-530 μM each analyte) 6 13
11. GTP
12. ADP
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0951 1 13. NADH
3 7 15
12 14. ATP
2
8 15. NADPH
4
10
0 4 8 12 16 20
Min

HPLC Analysis of Oligonucleotides on 9

10
1. A10
7 8
Proteomix® SAX 4
6 2. A11
3. A12
column . . . . Proteomix SAX-NP5, 25 cm x 4.6 mm I.D., 5 μm particles (Z777229) 5 4. A13
5. A14
mobile phase . . . . . . . . . . . . . . . . A: 20 mM Tris, pH 8.0 B: A + 0.5 M NaCl 3 6. A15
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 - 100% B in 30 minutes 7. A16
8. A17
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min 1 2 9. A18
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073 psi (74 bar) 10. A20
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . (10 mM each oligonucleotide in water)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005994
0 5 10 15 20 25 30 35
Min

HPLC Analysis of Opioid Glucuronide Metabolites 1. Buprenorphine 8. Morphine

2. Norbuprenorphine 9. Buprenorphine gluc
on Ascentis® Express HILIC Ascentis Express HILIC 3. Oxycodone 10. Norbuprenorphine gluc
4. Hydrocodone 11. Codeine gluc
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) x106 5. Codeine 12. Hydromorphone gluc
6. Oxymorphone 13. Oxymorphone gluc
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . urine 1 1
7. Hydromorphone 14. Morphine gluc
SPE well plate . . . . . . . . . . Supel™l-Select HLB 96-Well 60mg/Well (575662-U) 0.8
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol, 1 mL water 0.6

5,6
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL urine 0.4
7
washing . . . . . . . . . . . . . . . . . . . . . . . . 1 mL water, 1 mL 25% methanol 0.2 4
2 3 8 Opioid Drugs
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL acetonitrile 0
column . . . Ascentis Express HILIC, 10 cm x 2.1 mm x 2.7 μm particles (53939-U) 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5
mobile phase . . . . . . . . . . . . . . . . . . (A): 5 mM ammonium formate pH 4.2 x10 4
(B): 5 mM ammonium formate (95:5 acetonitrile:water) pH 5.2 7

11
gradient . . 100% B for 3 minutes, 80% B in 7 minutes, hold 80%B for 2 minutes 6
5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 4 9 13 12
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 3
10 14
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF MS 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 μL 1 Opioid Glucuronides

0
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 ng/mL 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006290 Counts vs. Acquisition Time (min)

HPLC Analysis of Ornithine Enantiomers on Astec® NH2

CHIROBIOTIC® T H2N O
*
mobile phase . . (A) 20 mM ammonium acetate, pH 4.1; (B) methanol; (50:50, A:B) OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004506
0 10 20
Min.

HPLC Analysis of Ovalbumin and BSA Charge Ovalbumin
Variants on Proteomix® SAX-NP3

mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mM Tris buffer, pH 8.0
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 - 0.3 M NaCl in 15 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
BSA
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1600 psi (110 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006132
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

HPLC Analysis of pBr322 DNA-Hae III Digest on

TSKgel® DEAE-NPR Gradient Program
540
column . . . . . . . . . . TSKgel DEAE-NPR, 3.5 cm × 4.6 mm I.D., 2.5 μm particles Time (min) %A 504
(with 5 mm DEAE-NPR guard column) (813075) 0 25 587
mobile phase . . (A) 1 M NaCl/25 mm Tris-HCl, pH 9.0; (B) 25 mM Tris-HCl, pH 9.0 0.5 45
5 50 458
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 20 62 434
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 21 100
24 100
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm 25 25
injection . . . . . . . . . . . . . . . 20 μL of 50 μg/mL Hae III digest of pBR322 DNA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0036 234 267
213
184 192
104123
89 124
57 80
64
51
21
18
0 2 4 6 8 10 12 14 16
Min
Ref: 1. Elena D. Katz, Lawrence A. Haff, Roy Eksteen, Rapid separation, quantitation, and purification
of products of polymerase chain reaction by liquid chromatography J. Chromatogr. A. 512, 433-444 (1990)

HPLC Analysis of PEG separation on two Zenix® 1. PEG 81,400 Da

SEC-300 columns in Tandem 1 2. PEG 50,630 Da
Pegylation of biotherapeutic drugs improves solubility, increases half life in 2 3. PEG 40,000 Da
the blood stream and reduces immunogenicity. Size exclusion chromatog- 4. PEG 26,100 Da
raphy (SEC) is often applied to characterize protein–polyethylene glycol 5. PEG 8,730 Da
3 5
(PEG) conjugates and establish the number of attached PEG chains, since
SEC is a precise and robust technique that combines ease of set up and
strainghtforward method develpopment. 4
This chromatogram shows the separation of four polyethyleneglycol
polymers varying in molecular mass 8730 to 81,400 Da on two Zenix® SEC-
300 columns in series.
column . . . . . . . . . . . . . . . . . . . Zenix SEC-300 (2 ea), 30 cm x 7.8 mm I.D.,
3 μm partcles, 300 Å, connected in tandem (Z777033)
mobile phase . . . . . . . . . . . . . . . . 150 mM Sodium Phosphate Buffer pH=7.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 15 20 25 30 35 40 45 50
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Refractive Index at 30 °C
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.PEG 81,400 Da (2mg/mL)
2.PEG 50,630 Da (2mg/mL)
3.PEG 40,000 Da (2mg/mL)
4.PEG 26,100 Da (2mg/mL)
5.PEG 8,730 Da (2mg/mL)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006200

HPLC Analysis of Pegylated Exenatide on Zenix®

SEC-300 and Zenix®-C SEC-300
mobile phase . . (A) 50 mM Ammonium acetate: (B) acetonitrile; (90:10, A:B, v/v)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609 psi (42 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C Zenix-C SEC-300 Tailing 1.07
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
sample . . . . . . . . . . . . . . . . . 3.3 mg/ml PEG-Exanatide in water (PEG 23 kD) Zenix SEC-300 Tailing 2.37
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005949
10 12 14 16 18 20
Min

HPLC Analysis of Pepides, Alberta Mix, on Gradient Program Peptide 1: ac-RGAGGLGLGK-amide

Peptide 2: ac-RGGGGLGLGK-amide
Discovery® BIO Wide Pore C18, No TFA Time (min) %A %B
Peptide 3: ac-RGAGGLGLGK-amide
0 90 10 Peptide 4: ac-RGVGGLGLGK-amide
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 2.1 mm I.D., 20 50 50 Peptide 5: ac-RGVVGLGLGK-amide
mobile phase . . (A) 25 mM formic acid in water, (B) (25 mM formic acid in water):
(20 mM formic acid in acetonitrile, (50:50) 0% TFA (25 mM formic acid), 1 μL injection
5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.20 mL/min 100
4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +ES
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001638B 2
% 1
0
0 10 20
Min

HPLC Analysis of Pepides, Alberta Mix on Gradient Peptide 1: ac-GGGLGGAGGLK -amide

Discovery® BIO Wide Pore C5 Min %B Peptide 2: ac-KYGLGGAGGLK -amide
0 15 Peptide 3: ac-GGALKALKGLK -amide
column . . . . . . . . . . . . . . . Discovery BIO Wide Pore C5, 15cm × 2.1mm I.D., 35 50 Peptide 4: ac-KYALKALKGLK -amide
1
mobile phase . . (A) 25mM formic acid in water; (B) (50:50) 25mM formic acid in 2
water, 20mM formic acid in acetonitrile 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.20 mL/min 4

injection . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL (~0.25 μg each peptide)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
sample . . . . . . . . . . . . . . . . . . . . . . RP Peptide Ionic Interactions Standard,
p/n RPS-I0020 (Alberta Peptide Institute) 0 10 20 30

HPLC Analysis of Pepides, Synthetic, on Discovery® Peptide 1: ac-GGGLGGAGGLK-amide Gradient Program

BIO Wide Pore C18 Peptide 2: ac-KYGLGGAGGLK-amide Time (min) %B
Peptide 3: ac-GGALKALKGLK-amide 0 15
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 2.1 mm I.D., Peptide 4: ac-KYALKALKGLK-amide 45 60
mobile phase . . (A) 25 mM formic acid in water; (B) (50:50) 25 mM formic acid in
water, 20 mM formic acid in acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.20 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL (~0.25 μg ea peptide)
p/n RPS-I0020 (Alberta Peptide Institute)
0 10 20 30
Min

HPLC Analysis of Peptide Mix, Complex, on Gradient Program

Time (min) %A %B
Ascentis® C18 0
90
90
36
10
64
mobile phase . . (A) 25 mM formic acid in water; (B): 50:50, 25 mM formic acid in
water:25 mM formic acid in acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . tryptic digest of carboxymethylated protein
mixture 2.4 g/L in 0.1% formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002441
0 10 20 30 40 50 60 70 80 90
Min

HPLC Analysis of Peptide Mix, Complex on Gradient Program

Time (min) %A %B
Ascentis® C18 0 90 10
column . . . . . . . Ascentis C18, 25 cm x 2.1 mm I.D., 5 μm particles (581305-U) 80 50 50
mobile phase . . (A) (50:50), 50 mM formic acid titrated with acetate (pH 3.0):water;
(B) (50:50), 50 mM formic acid titrated with acetate (pH 3.0): acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL 0 20 40 60 80
sample . . . . . . . . . . . . . . . . . . . . . . . . . tryptic digest of carboxymethylated Min
protein mixture 1.5 g/L in 50 mM formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002669

HPLC Analysis of Peptide Mixture on Zenix® SEC- 6.936
100 8.089
Zenix® SEC columns can be operated in aqueous-organic mobile phases, as 5.931
is shown ion this example where hydrophobic peptides and proteins are
7.252
eluted with excellent peak shape in 65% acetonitrile 35% water containing
0.1% TFA.
column . . . . . Zenix-100, 30 cm x 7.8 mm I.D., 3 μm particles, 100 Å (Z777008)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . ACN:H2O:TFA=65:35:0.1(v/v)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . ribonuclease A (5.931 min), human insulin (6.936 min),
thymosin a1 (7.252 min), somatostatin (8.089 min)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006196
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Min

HPLC Analysis of Peptides and Proteins on 1. Ribonuclease A

3
Proteomix® SCX-NP1.7, 3 cm x 4.6 mm I.D. Column 2. Cytochrome c

3. Lysozyme
column . . Proteomix SCX-NP1.7, 3 cm x 4.6 mm I.D., 1.7 μm particles (Z777145)
mobile phase . . . . . (A) 20 mM sodium phosphate, pH 6.0; (B) A + 1.0 M NaCl
gradient . . . . . . . . . . . . . . . . . . . . . 0 to 50% B in 5 min; 15 min prewash 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 2

detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006144
0.0 1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of Peptides and Proteins on 1. Ribonuclease A 3

Proteomix® SCX-NP1.7, 5 cm x 4.6 mm I.D. Column 2. Cytochrome c
1
3. Lysozyme
gradient . . . . . . . . . . . . . . . . . . . . 0 to 75% B in 25 min; 15 min prewash
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006145
0 2 4 6 8 10 12 14 16 18 20
Min

HPLC Analysis of Peptides from BSA on Gray: BSA tryptic digest, 10 µg on-column
Proteomix® SCX-NP3, Volatile Buffer Black: BSA tryptic digest, 5 µg on-column
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.35 mL/min

(B) A + 1 M NaCl; (C) acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . . 90% A, 0% B, 10% C held for 1.5 min;
to 40% A, 50% B, 10% C in 18.5 min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5511 psi (380 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
sample . . . . . . . . . . . . . . . . . . trypsin digested bovine serum albumin (BSA)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006022
0 5 10 15 20
Min

HPLC Analysis of Peptides on Discovery® BIO 1 1. ac-GGGLGGAGGLK-amide

PolyMA-SCX (Ion Exchange) 2. ac-KYGLGGAGGLK-amide
3. ac-GGALKALKGLK-amide
Polymer particles are frequently used in bioseparations. Their advantages 4. ac-KYALKALKGLK-amide
over inorganic particles, however, are often offset by generally lower
efficiency. Discovery BIO PolyMA-SCX and PolyMA-WAX particles offer the
benefits of polymeric particles, but have higher efficiency and better
2
resolution than competitive particles. An example of the high efficiency of
Discovery BIO PolyMA-SCX is shown in this Figure. The Discovery BIO 3
PolyMA-SCX column gives efficient, well-resolved separation of a four- 4
component peptide mixture.
column . . Discovery BIO PolyMA-SCX, 5 cm x 4.6 mm I.D., 5 μm particles (59601-U)
mobile phase . . ((A) 5% acetonitrile in 20 mM ammonium carbonate, pH 3.5 with
phosphoric acid; (B) 5% acetonitrile in 20 mM ammonium carbonate,
480 mM ammonium phosphate, pH 3.5 with phosphoric acid) 0 10 20 30
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 100% B in 24 min (linear) Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
p/n RPS-10020 (Alberta Peptide Institute)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001824

HPLC Analysis of Peptides on Discovery® BIO Wide 1. Gly-Tyr

1 2. Val-Tyr-Val
Pore C18 (5 cm Column) 3. Met-Enkephalin
column . . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 5 cm × 4.6 mm I.D., 2 4. Leu-Enkephalin
5. Angiotensin II
mobile phase . . (A) (5:95), 0.5% TFA in water:0.5% TFA in acetonitrile; (B) (25:75),
0.5% TFA in water:0.5% TFA in acetonitrile 3
gradient . . . . . . . . . . . . . . . . . . . . . 2-24% B in 22 min, 8 min. at 100%A 4 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001562
0 10 20
Min

HPLC Analysis of Peptides on Discovery® BIO Wide Gradient Program

Time (min) %A %B
Pore C18 (Gradient #1) 0 100 0
column . . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15cm × 4.6mm I.D., 1 100 0
5μm particles (568222-U) 15 0 100
mobile phase . . . . . . . (A) (80:20), 0.1% TFA in water:0.1% TFA in acetonitrile;

(B) (66:34), 0.1% TFA in water:0.1% TFA in acetonitrile
gradient . . . . . . . . . . . . . . . . . . . 0-100% B in 14 min after 1 minute delay
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
sample . . . . . . . . . . . . . . . . . . . . . . . . Sigma Peptide Mix (Cat. No. P2693) Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001512

HPLC Analysis of Peptides on Discovery® BIO Wide Gradient

Pore C18 (Gradient #2) Column
Volumes %A %B
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 4.6 mm I.D., 0 100 0
5 μm particles (568222-U) 2 100 0
mobile phase . . . . . . . . (A) (80:20), 0.1%TFA in water:0.1% TFA in acetonitrile; 9 0 100
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . 5 μL Sigma Peptide Mix P2693 (~25 μg ea. Peptide) (~25 μg ea. peptide)
sample . . . . . . . . . . . . . . . . . . . . . . . . Sigma Peptide Mix (Cat. No. P2693)
0 2 4 6 8 10 12 14 16

HPLC Analysis of Peptides on Discovery® BIO Wide 0–16 min window

Pore C18, 0-16 Min. Window
0.1%TFA in water:0.1% TFA in acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 65 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . 50μL carboxymethylated apohemoglobin 0 2 4 6 8 10 12 14 16
tryptic digest in 50 mM ammonium bicarbonate Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001730


column . . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15cm × 4.6mm I.D.,
0.1% TFA in water:0.1% TFA in acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 65 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . 50μL carboxymethylated apohemoglobin 28 30 32 34 36 38
trypic digest in 50 mM ammonium bicarbonate Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001727

HPLC Analysis of Peptides on Discovery® BIO Wide Peptide Amino Acid Sequence
Pore C18 (PFPA Mobile Phase Additive) 1. Arg8-vassopressin CYFQNCPRG-amide; disulfide
2. Bradykinin, fragment 1-5 RPPGF
Sigma peptide mix (5 μm) 3. Oxytocin CYIQNCPLG-amide; disulfide
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 4.6 mm I.D., 4. Met-Enkephalin YGGFM
5 μm particles (568222-U) 5. LHRH* pEHWSYGLRPG-amide**
6. Leu-Enkephalin YGGFL
mobile phase . . . . . . . . . (A) 81:19, (water/0.1% PFPA):(acetonitrile/0.1%PFPA); 7. Bradykinin RPPGFSPFR
(B) 62:38, (water/0.1%PFPA):(acetonitrile/0.1%PFPA) 8. Bombesin pEQRLGNQWAVGHLM-amide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 9. Substance P RPKPQQFFGLM-amide**
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL (~25 μg each peptide) * Luteinizing Hormone Releasing
Hormone 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C ** pE is pyroglutamate
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
sample . . . . . . . . . . . . . . . . . . . . . . . . Sigma Peptide Mix (Cat. No. P2693)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001594
4
6 7
1
2 3 8
9
0 2 4 6 8 10 12 14 16 18
Min


mobile phase . . (A) (95:5), 0.1% TFA in water:0.1% TFA acetonitrile; (B) (50:50),
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 65 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm 0 2 4 6 8 10 12 14 16
injection . . . . . . . . . . . 50μL carboxymethylated apohemoglobin tryptic digest in Min
50 mM ammonium bicarbonate (568422-U)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001732


0.1%TFA in water:0.1% TFA in acetonitirle
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 65 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 28 30 32 34 36 38
injection . . . . . . . . . . . . . . . . . . . . 50 μL carboxymethylated apohemoglobin
tryptic digest in 50 mM ammonium bicarbonate
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001729


gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 65 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1ml/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm 0 2 4 6 8 10 12 14 16
injection . . . . . . . . . . . . . . . . . . . . . 50μL carboxymethylated apohemoglobin Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001731


0.1% TFA in water:0.1%TFA in acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 65 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm 28 30 32 34 36 38
Min
injection . . . . . . . . . . . . . . . . . . . . . 50μL carboxymethylated apohemoglobin
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001728

C1 C3
HPLC Analysis of Peptides on Proteomix®
SCX-NP3, Volatile Buffer, ELSD and UV Detection
column . . . . . Proteomix SCX-NP3, 5 cm x 4.6 mm I.D., 3 μm particles (Z777151)
Pressure
(B) 50 mM ammonium acetate:acetonitrile (4:1, v:v) C4
C2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min SofTA
detector . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD (SofTA) and UV, 280 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006021
@280nm
0.0 2.5 5.0 7.5 10.0 12.5 15.0

Min
Courtesy of Miyako Kawakatsu, M&S Instruments Inc.

HPLC Analysis of Peptides on Ascentis® RP-Amide Gradient Program 1. Gly-Phe (62.5 mg/mL)
Time (min) %B 2. Val-Tyr-Val (250 mg/mL)
2
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 5 9-21 3. Methionine Enkephalin (250 mg/mL)
15 21-27 4. Angiotensin II (250 mg/mL)
mobile phase . . . . . . . . . . (A) 0.1% TFA in water; (B) 0.1% TFA in acetonitrile
5. Leucine Enkephalin (250 mg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 1 4 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002613
0 10 20
Min

HPLC Analysis of Peptides on Discovery® BIO Wide Gradient

Pore C18, Preparative Scale Column
Volumes %A %B
column . . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 10 mm I.D., 0 100 0
10 μm particles (567208-U) 2 100 0
9 0 100
mobile phase . . . . . . . (A) (80:20), 0.1% TFA in water:0.1% TFA in acetonitrile;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cat. No. P2693
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001511 0 2 4 6 8 10 12 14 16
Min

5
HPLC Analysis of Peptides on Discovery® BIO Wide 1.
Peptide
Arg8-vasopressin
Sequence
CFQNCPRG-amide; disulfide
Pore C5 2.
3.
Bradykinin, fragment 1–5
Oxytocin
RPPGF
CYQNCPLG-amide; disulfide
4. Met-enkephalin YGGFM
column . . . . . . . . . . . . . . Discovery BIO Wide Pore C5, 15 cm × 4.6 mm I.D., 5. Luteinizing hormone
5 μm particles (568422-U) 4 67 8 releasing hormone PEHWSYGLRPG-amide
1 3 9 6. Leu-enkephalin YGGFL
mobile phase . . . (A): 0.1% PFPA (pentafluoropropionic acid) in water:acetonitrile 7. Bradykinin RPPGFSPFR
2 8. Bombesin PEQRLGNQWAVGHLM-amide
(81:19); (B): 0.1% PFPA in water:acetonitrile (62:38) 9. Substance P RPKPQQFFGLM-amide
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 19 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
0 2 4 6 8 10 12 14 16 18
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL ~0.25 μg each peptide
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cat. No. P2693
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001492

HPLC Analysis of Peroxidase on TSKgel®

G3000SWXL
column . . . . TSKgel G3000SWXL, 30 cm × 7.8 mm I.D., 5 μm particles (808541) Recovered Activity = 98%
mobile phase . . . . . . . . . . . . . . . . 0.3M NaCl/0.05M phosphate buffer, pH 7.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . 100 μL crude peroxidase preparation from Japanese horseradish (0.15mg)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0940
0 5 10 15
Min

HPLC Analysis of Phenylalanine Enantiomers on 1. L-Phenylalanine NH2

1
Astec® CHIROBIOTIC® T (Formic Acid Mobile Phase 2. D-Phenylalanine O
Additive) OH

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005342
0 2 4 6 8 10
Min

HPLC Analysis of Phenylalanine Enantiomers on

NH 2
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004508
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Phenylalanine Methyl Ester 1. Phenylalanine methyl ester enantiomer

Enantiomers on LARIHC™ CF6-P 2. Phenylalanine methyl ester enantiomer
available from Supelco/Sigma-Aldrich as a custom item)
O 1
C H3 2
mobile phase . . . . . . . . . . . . . . (A) methanol: (B) acetonitrile: (C) acetic acid: O
H Cl
(D) triethylamine, (70:30:0.3:0.2. A:B:C:D) NH 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . Phenylalanine methyl ester, 0.3 mg/mL in ethanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005259
0 20 40 60 80
Min
Development of New HPLC Chiral Stationary Phases Based on Native and Derivatized Cyclofructans
Anal. Chem. 81, 10215 (2009)
amines by the isopropyl carbamate-cyclofructan6 chiral stationary phase J. Chromatogr. A.
1217, 4904 (2010)

HPLC Analysis of Pipecolic Acid Enantiomers on

O
Astec® CHIROBIOTIC® T H
N *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004554
0 1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of Polar Neutral and Acidic Amino Gradient Program

Min %A %B %C
Acids on Ascentis® Si (Silica) 0 5 10 85 3
column . . . . . . . . Ascentis Si, 10 cm × 2.1 mm I.D., 5 μm particles (581508-U) 20 5 16 79
mobile phase . . . . . (A) 100 mM ammonium formate (pH 3.0, with formic acid),
(B) water, (C) acetonitrile 1 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 1. Threonine
2. Serine 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
3. Asparagine
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+), full scan 4. Glutamine
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 5. Glutamatic acid
sample . . . . . . . . . . . . . . . . . . . . . 100 μg/mL in 10:90, (50 mM ammonium 6. Aspartatic acid 2
formate / formic acid, pH 3.0):acetonitrile 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003688
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Poly(ethyleneglycol) polymers on

SRT® SEC-150 and SRT® SEC-300
SEC-150 SEC-300
Although proteins are the most common applications of Sepax SRT and
Zenix gel filtration columns, they can also be used to other water-soluble
polymers such as the poly(ethyleneglycol) samples shown in these
chromatograms. The conditions are very similar to those used for protein
analysis, namely 150mM phosphate buffer at pH 7.0. Judging by the narrow
peak widths all PEG polymers shwon here have rather narrow size PEG 3
distributions.
column . . . . . . SRT-150, 30 cm x 7.8 mm I.D., 5 μm particles, 150 Å (Z777045) PEG 2
column . . . . . . SRT-300, 30 cm x 7.8 mm I.D., 5 μm particles, 150 Å (Z777051)
mobile phase . . . . . . . . . . . . . . . . . . . . . 150 mM phosphate Buffer, pH 7.0 PEG 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 0.0 2.5 5.0 7.5 10.0 12.5 15.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 720 psi (49.6 bar) Min Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI(30 C°) No Molecular Wt. Log Mw Elution Vol (mL) No Molecular Wt. Log Mw Elution Vol (mL)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 1 932000 5.969416 – 1 932000 5.969416 5.853
sample . . 1.5 mg/mL in mobile phase PEG 1. MW 636, 6,550, 118,000, 932,000; PEG 2 496000 5.695482 5.517 2 496000 5.695482 5.875
2. MW 2010, 42,700, 496,000 ; PEG 3. MW 232, 17,900, 271,000 3 271000 5.432969 5.559 3 271000 5.432969 5.91
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006206 4 118000 5.071882 5.622 4 118000 5.071882 6.426
5 42700 4.630428 5.923 5 42700 4.630428 7.568
6 17900 4.252853 6.803 6 17900 4.252853 8.927
7 6550 3.816241 8.201 7 6550 3.816241 10.201
8 2010 3.303196 9.603 8 2010 3.303196 11.137
9 628 2.79796 10.465 9 628 2.79796 11.604
10 232 2.365488 10.983 10 232 2.365488 11.89

HPLC Analysis of Proline Enantiomers on Astec® O

1 1. L-Proline
H
CHIROBIOTIC® T (Formic Acid Mobile Phase N 2. D-Proline
OH
Additive)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005343
2
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Proline Enantiomers on Astec®

CHIROBIOTIC® T O
H
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) N *
OH
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (95:5, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004526
0 1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of Proline Enantiomers on Astec®

CHIROBIOTIC® T, Fast Analysis
OH
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) N *
H
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . water O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.65 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004527
0 1.0 2.0 3.0 4.0

Min

HPLC Analysis of Proteins and Peptides on 1. Ribonuclease A 1

Proteomix® WCX, Comparison of Different 2. Cytochrome c 3
3. Lysozyme 2
Particle Sizes
column . . . . . Proteomix WCX-NP10, 5 cm x 4.6 mm, 10 μm particles (Z777169)
column . . . . . . Proteomix WCX-NP5, 5 cm x 4.6 mm x 5 μm particles (Z777193)
column . . Proteomix WCX-NP1.7, 5 cm x 4.6 mm I.D., 1.7 μm particles (Z777180)
column . . . . Proteomix WCX NP3, 5 cm x 4.6 mm I.D., 3 μm particles (Z777185)
mobile phase . . . . . (A) 20 mM sodium phosphate, pH 6.5, (B) A + 1.0 M NaCl
gradient . . . . . . . . . . . . . . . . . . . . 0 to 100% B in 25 min, 15 min prewash
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 1.7 μm
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 3 μm
sample . . . . . . . . . Ribonuclease A, Cytochrome C and Lysozyme (1 mg/mL) min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006143 5 μm
10 μm
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Min

HPLC Analysis of Proteins and Peptides on 1. Ribonuclease A

Proteomix® WCX-NP1.7 Demonstrating Lot-to-Lot 2. Cytochrome c 1 3
3. Lysozyme
Reproducibility
column . . Proteomix WCX-NP1.7, 5 cm x 4.6 mm I.D., 1.7 μm particles (Z777180) 2
gradient . . . . . . . . . . . . . . . . . . . . 0 to 100% B in 25 min, 15 min prewash
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL Lot 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006142
Lot 2
Lot 3
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Min

HPLC Analysis of Proteins by Size Exclusion on 1. Thyroglobulin, 670 kDa

Zenix® and SRT® SEC-300, Comparison of Particle 2. BSA dimer, 132 kDa
Size and Column I.D. 3. Bovine serum albumin (BSA), 66 kDa
4. Ribonuclease A, 13.7 kDa
column . . . . . Zenix 300, 30 cm x 4.6 mm I.D., 3 μm particles, 300 Å (Z777028) 5. Uracil, 120 Da
column . . . . Zenix 300, 30 cm x 7.8 mm I. D., 3 μm particles, 300 Å (Z777033)
column . . . . . . SRT SEC-300, 30 cm x 7.8 mm 5 μm particles, 300 Å (Z777051) Zenix SEC-300 3
30 cm x 4.6 mm I.D., 3 μm particles 4
0.35 mL/min, 3 μL injection
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214nm 5
2
sample . . . . . . . . . . . . . . . each compound 1 mg/mL except uracil, 2.5 μg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006179
Zenix SEC-300
1 mL/min, 10 μL injection
SRT SEC-300
1 mL/min, 10 μL injection
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Proteins by Size Exclusion on 1. Thyroglobulin

Zenix® Columns, Effect of Pore Size 2. Bovine serum albumin (BSA) dimer
3. Bovine serum albumin (BSA)
column . . Zenix SEC-150, 30 cm x 7.8 mm I.D., 3 μm, 150 Å particles (Z777018) 4. Ribonuclease A
column . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm, 300 Å particles (Z777033) 5. Uracil 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
3 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . each 1 mg/mL except uracil, 2.5 mg/mL
5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006125 Zenix SEC-150 2
3
4
1
5
2
Zenix SEC-300
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Proteins by Size Exclusion on 1. Thyroglobulin 7 min assay

2. Bovine serum albumin 1.0 mL/min
Zenix® SEC-300 Columns, Effect of Flow Rate 3. Ribonuclease A 3 Plate count: 17617
(15 cm column) 4. Methacrifos 2 Back pressure: 850 psi
5. Uracil
column . . Zenix SEC-300, 15 cm x 7.8 mm I.D., 3 μm, 300 Å particles (Z777030) 1 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated on figure
2 4.5 min assay
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm 1.5 mL/min
4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 1 Plate count: 16215
sample . . . . . . . . . . . . . . . . . . . . . each 1 mg/mL except uracil, 2.5 mg/mL Back pressure: 1350 psi
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006127
3
2 3.5 min assay
4 2.0 mL/min
1
Plate count: 13541
Back pressure: 1600 psi
0 1 2 3 4 5 6 7 8
Min

HPLC Analysis of Proteins by Size Exclusion on 1. Thyroglobulin

2. Glucose-6-phosphate dehydrogenase
Zenix® SEC-300 Columns, Effect of Flow Rate 3. Bovine serum albumin
(30 cm column) 4. Ribonuclease 3 4
5. Uracil
column . . Zenix SEC-300, 30 cm x 4.6 mm I.D., 3 μm, 300 Å particles (Z777028) 3 4
mobile phase . . . . . . . . . . . . . . . . . . . 150 mM potassium phosphate, pH 6.8
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated on figure
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 2 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 2
5
sample . . . . . . . . . . . . . . . . . . . . . each 1 mg/mL except uracil, 2.5 mg/mL 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006128
0.5 mL/min 1
0.2 mL/min
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0
Min

HPLC Analysis of Proteins on Ascentis® Express 1. Ribonuclease, 13.7 kDa

Peptide ES-C18 2. Adrenomedullin, 5.73 kDa
3. Cytochrome c, 12.2 kDa
column . . . . . . . . . . . . Ascentis Express Peptide ES-C18, 15 cm x 2.1 mm I.D., 4. Lysozyme, 14.3 kDa
2.7 μm particles (53307-U) 5. Superoxide dismutase, 32.5 kDa
mobile phase . . . . . . . (A) 75:25, 0.1% TFA in water: 0.08% TFA in acetonitrile; 6. Soybean trypsin inhibitor, 20.1 kDa
(B) 25:75, 0.1% TFA in water: 0.08% TFA in acetonitrile 7. Carbonic anhydrase, 29.0 kDa
gradient . . . . . . . . . . . . . . held at 0% B for 0.5 min; 0 to 100% B in 7 min 8. Ovalbumin, 44.3 kDa
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 4
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4743 psi (327 bar) 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 5
1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 7 8
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 - 0.6 g/L in mobile phase A
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005724
0 2 4 6 8
Min

HPLC Analysis of Proteins on Discovery® BIO

3
PolyMA-SCX (Analyte Set #2) 1. Ribonuclease A
2 2. α-Chymotrypsinogen A
Absorbance (280 nm)

Proteins, mixture
The large pore diameter of Discovery BIO PolyMA-SCX and PolyMA-WAX 3. Cytochrome c
particles allows full access to small peptides and very large proteins and
protein aggregates. This Figure shows the power of ion-exchange to resolve
proteins with very little difference in molecular weight, an advantage over
size-exclusion separations.
1
column . . . Discovery BIO PolyMA-SCX, 5 cm x 4.6 mm, 5 μm particles (59601-U)
(B) 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0;
5 to 100% B in 15 min. (linear)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
injection . . . . . . . . . . . 10 μg trypsin inhibitor, 5 μg conalbumin, myoglobin or 0 5 10 15 20
10 μg ribonuclease A, 5 μg cytochrome c, a-chymotrypsinogen A Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm Figure used with permission of Mitsubishi Chemical Co.
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0658

HPLC Analysis of Proteins on Discovery® BIO 1

1. Myoglobin
PolyMA-WAX (Analyte Set #1) 2. Conalbumin
3. Trypsin inhibitor
Absorbance (280 nm)

The large pore diameter of Discovery BIO PolyMA-SCX and PolyMA-WAX
particles allows full access to small peptides and very large proteins and
protein aggregates. This Figure shows the separation of proteins that vary
from 18 to 80 kDA. 2
column . . Discovery BIO PolyMA-WAX, 5 cm x 4.6 mm, 5 μm particles (59602-U)
mobile phase . . . . . . . . . . . (A) 10 mM Tris, pH 8.0 with HCl; (B) 10 mM Tris, 3
0.5 M NaCl, pH 8.0 with HCl; 5 to 100% B in 15 min. (linear)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . 10 μg trypsin inhibitor, 5 μg conalbumin, myoglobin or
10 μg ribonuclease A, 5 μg cytochrome c, a-chymotrypsinogen A 0 5 10 15
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0657 Min

HPLC Analysis of Proteins on Mono Q HR 5/5 A280nm

0.1 Gradient Program
column . . . . . . . . . . . . . . . . . . . . Mono Q HR 5/5, 10 μm particles (54807) Time (min) % B
mobile phase . . . . . . . . . . . . . (A): 20 mM bis-Tris, pH 6.0; (B): A + 1M NaCl 0 0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 10 0
hPL 25 10
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 26 40
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 mL
sample . . . . . . . . human placental lactogen fraction from size exclusion medium
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0421
NaCl
0 20
Min

HPLC Analysis of Proteins on SRT® SEC-1000, 1. Thyroglobulin, 669,000 Da

30 cm x 7.8 mm I.D., 5 μm particles 2. Ferritin, 440,000 Da 5
3. Carbonic anhydrase, 29,000 Da 4
column . . . . . . SRT® SEC-1000, 30 cm x 7.8 mm I.D., 5 μm particles (Z777063) 4. Aprolinin, 6,500 Da 3
mobile phase . . . . . . . . . . . . 150 mM potassium phosphate monobasic, pH 7.0 5. Cyanocobalamin, 1,355 Da
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
1 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10 g/L in buffer
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005726 0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Proteins on Antibodix® WCX- A. Antibodix WCX-NP10

NP10, Comparison of WCX Columns 25 cm × 4.6 mm I.D., 10 μm (non-porous)
column . . Antibodix WCX-NP10, 25 cm x 4.6 mm I.D., 10 μm particles (Z777272) B. Vendor D WCX
mobile phase . . (A) 10 mM sodium phosphate buffer, pH 6.0; (B) A + 1.0 M NaCl 25 cm × 4.0 mm I.D., 10 μm (non-porous)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 1. Cytochrome c (12.2 kDa)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 2. Lysozyme (14.3 kDa)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm 3. Ribonuclease A (13.7 kDa)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 1 2 3
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL each
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005946
A
3
2
B 1
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Min

HPLC Analysis of Proteins (2) on BIOshell™ A400 1. Superoxide Dismutase 6. Carbonic Anydrase
2. Serum albumin, bovine 7. β-Amylase
Protein C4 3. α-Lactalbumin 8. Glucose-6-phosphate dehydrogenase
4. β-Lactoglobulin A 9. Thioredoxin
This application demonstrates the utility of the BIOshell A400 Protein C4 5. Catalase 10. Phosphorylase b
column for the reversed-phase chromatography of proteins from a variety of
3
sources and of different molecular weights.
sample . . . . . . protein mixture, g/L ea: superoxide dismutase, 0.3; bovine serum
albumin, 0.3; alpha-lactalbumin, 0.2; beta-lactoglobulin A, 0.3; 10
catalase, 0.3; (carbonic anhydrase, 0.3; beta-Amylase, 1.0;
glucose-6-phosphate dehydrogenase, 1.0; thioredoxin, 0.3; 9
phosphorylase b, 2.5.) 6
8
column . . BIOshell A400 Protein C4, 10 cm X 2.1 mm I.D., 3.4 μm particles (66825-U) 1
4
(B) 25:75, [0.1% TFA in water]:[0.1% TFA in acetonitrile] 7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2410 psi (166 bar) 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006236
0 2 4 6 8 10
Min

HPLC Analysis of Proteins on Discovery® BIO Wide 1. RNase (13.7 kDa, 1 mg/mL)
Pore C5 2. Cytochrome c (12.4 kDa, 1 mg/mL)
3. Lysozyme (14.3 kDa, 1 mg/mL)
column . . . . . . . . . . . . . . Discovery BIO Wide Pore C5, 15 cm × 4.6 mm I.D., 4. BSA (67.0 kDa, 2.5 mg/mL)
5 μm particles (568422-U) 5. Myoglobin (18.8 kDa, 1 mg/mL)
mobile phase . . (A) (0.1% TFA in water): (B) (0.1% TFA in CH3CN); (75:25, A:B) 6. Ovalbumin (45.3 kDa, 3.5 mg/mL)
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 25 min 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 600
2 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm Impurity in 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 μL in 0.1% TFA 400 Lysozyme 1 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001488B Impurity in
Myoglobin
200
0 10 20
Min

HPLC Analysis of Proteins on Proteomix® SCX 1. Ribonuclease A 3

1
Columns, Effect of Particle Size of Resolution 2. Cytochrome c
3. Lysozyme
column . . Proteomix SCX-NP1.7, 5 cm x 4.6 mm I.D., 1.7 μm particles (Z777146) 2
mobile phase . . (A) 20 mM sodium phosphate buffer, pH 6.0, (B) A + 1.0 M NaCl
gradient . . . . . . . . . . . . . . . . . . . 0-25 min from 0-75% B, 15 min prewash
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 mL/min 1.7 μm
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 3 μm
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . each 1 mg/mL
10 μm
0 2 4 6 8 10 12 14 16 18 20
Min

HPLC Analysis of Proteins on Proteomix® 1. Ribonuclease A 3

SCX-NP1.7, Demonstration of Lot-to-Lot 2. Cytochrome c
3. Lysozyme 1
Reproducibility
gradient . . . . . . . . . . . . . . . . . . . . . 0 - 75% B in 25 min, 15 min prewash
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 mL/min
Lot A
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL each compound Lot B
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006136
Lot C
0 2 4 6 8 10 12 14 16 18 20
Min


30 cm x 7.8 mm I.D., 5 μm particles 2. Alcohol dehydrogenase, 150,000 Da
3. Albumin, 66,000 Da
column . . . . . . . SRT® SEC-150, 30 cm x 7.8 mm I.D., 5 μm particles (Z777045) 4. Carbonic anhydrase, 29,000 Da
mobile phase . . . . . . . . . . . . . . . . . . . 150 mM potassium phosphate, pH 7.0 5. Aprotinin, 6,500 Da
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 6. Cyanocobalamin, 1,355 Da
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 7. p-Aminobenzoic acid, 137 Da
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005728
3 7
2 4 5
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Proteins on SRT® SEC-2000, 1. Hemocyanin, 8,500,000 Da

30 cm x 7.8 mm I.D. 2. Thyroglobulin, 669,000 Da
3. Carbonic anhydrase, 29,000 Da 3
This application demonstrates the suitability of Discovery BIO GFC100 for the and Aprotinin, 6,500 Da
separation of proteins. Separation is by inclusion into the pore volume with 4. Uracil, 112 Da
larger molecules being excluded and therefore eluting earlier. 1 4
column . . . . . . SRT® SEC-2000, 30 cm x 7.8 mm I.D., 5 μm particles (Z777069)
mobile phase . . . . . . . . . . . . 150 mM potassium phosphate monobasic, pH 7.0 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14 16
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1- 10 g/L in buffer Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005729


30 cm x 7.8 mm I.D., 5 μm 2. Ferritin, 440,000 Da
3. Alcohol dehydrogenase, 150,000 Da
column . . . . . . . SRT® SEC-300, 30 cm x 7.8 mm I.D., 5 μm particles (Z777059) 4. Albumin, 66,000 Da
mobile phase . . . . . . . . . . . . 150 mM potassium phosphate monobasic, pH 7.0 5. Carbonic anhydrase, 29,000 Da
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 6. Aprotinin, 6,500 Da 7
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 7. p-Aminobenzoic acid, 137 Da 3 4
5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10 g/L in buffer 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005730 1
0 2 4 6 8 10 12 14 16
Min


30 cm x 7.8 mm I.D., 5 μm particles 2. Carbonic anhydrase, 29,000 Da
3. Aprotinin, 6,500 Da
column . . . . . . . SRT® SEC-300, 30 cm x 7.8 mm I.D., 5 μm particles (Z777051) 4. Cyanocobalamin, 1,355 Da
mobile phase . . . . . . . . . . . . . . . . . . . 150 mM potassium phosphate, pH 7.0 5. p-Aminobenzoic acid, 137 Da
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 2
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10 g/L in buffer 3 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005725
0 2 4 6 8 10 12 14 16
Min


2. Ferritin, 440,000 Da
30 cm x 7.8 mm I.D. 3. Albumin, 66,000 Da
column . . . . . . . SRT® SEC-500, 30 cm x 7.8 mm I.D., 5 μm particles (Z777057) 4. Carbonic anhydrase, 29,000 Da 3
mobile phase . . . . . . . . . . . . 150 mM potassium phosphate monobasic, pH 7.0 5. Aprotinin, 6,500 Da 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 6. p-Aminobenzoic acid, 137 Da 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 2 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005733
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Proteins on Superdex® Gel A280nm
Filtration column
0.1
column . . . . . . . . . . . . . . . HiLoad 16/60 Superdex® 75 prep grade (54802-U)
mobile phase . . . . . . . . . . . . . . . . . . . . . 0.15M ammonium acetate, pH 6.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
0.05
sample . . . . . . . . . . . . . . . . . . . . . . . IGF-1, ZZ fusion protein and impurties IGF-1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0420
VO VC
Fraction 1 2 3 4
0 1.0 2.0
Time (h)

HPLC Analysis of Proteins on TSKgel® Butyl-NPR 1. Myoglobin

2. Ribonuclease
column . . . . TSKgel Butyl-NPR, 3.5 cm × 4.6 mm I.D., 2.5 μm particles (814947) 3. Lysozyme
mobile phase . . . . . (A) 0.1 M phosphate buffer, pH 7.0 plus ammonium sulfate, 3 5 4. α-Chymotrypsin
5. β-Chymotrypsinogen
2.3 M to 0 M in12 min (B & C) = 0.1 M phosphate buffer,
pH 7.0 plus ammonium sulfate, 1.8 M to 0 M in 60 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1025 4
2
0 2 4 6 8 10
Min

HPLC Analysis of Proteins on TSKgel® Ether-5PW

Proteins, mixture 1 1. Myoglobin
column . . . . TSKgel Ether-5PW, 7.5 cm × 7.5 mm I.D., 10 μm particles (808641) 2. Ribonuclease
3. Lysozyme
mobile phase . . . . . . . . 0.1 M phosphate buffer, pH 7.0 plus ammonium sulfate, 4. α-Chymotrypsin
1.8 M to 0 M in 60 min 5. β-Chymotrypsinogen
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1027
5
2
4
0 15 30 45
Min

HPLC Analysis of Proteins on TSKgel® Phenyl-5PW 1. Myoglobin

2. Ribonuclease
Proteins, mixture 3. Lysozyme
column . . . TSKgel Phenyl-5PW, 7.5 cm × 7.5 mm I.D., 10 μm particles (807573) 1 5
4. α-Chymotrypsin
mobile phase . . . . . . . . 0.1 M phosphate buffer, pH 7.0 plus ammonium sulfate, 5. β-Chymotrypsinogen
1.8 M to 0 M in 60 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
2 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1026
0 30 60
Min

HPLC Analysis of Proteins on Zenix® SEC-300, 1. Thyroglobulin, 669,000 Da

30 cm x 4.6 mm I.D., 3 μm 2. γ-Globulins, ~300,000 Da
3. Ovalbumin, 44,281 Da
This application demonstrates the suitability of Discovery BIO GFC300 for the 4. Myoglobin, 17,699 Da
separation of proteins. Separation is by inclusion into the pore volume with 4
5. Poly-DL-alanine, 1,000-5,000 Da
larger molecules being excluded and therefore eluting earlier. 6. Uracil, 112 Da
column . . . . . . Zenix® SEC-300, 30 cm x 4.6 mm I.D., 3 μm particles (Z777033) 3
mobile phase . . . . . . . . . . . . . . . . . 150 mM potassium phosphate monobasic, 2
pH 7.0 (adjusted with potassium hydroxide)
1 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 mL/min
5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652 psi (45 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
0 10 20
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Min
sample . . . . . . . . . . . . . . 1 g/L each (except uracil, 0.1 g/L) in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005732

HPLC Analysis of Proteins on Zenix® SEC-300, 1. Thyroglobulin, 669,000 Da

30 cm x 7.8 mm I.D., 3 μm 2. BSA (bovine serum albumin), 66,463 Da
3. Ribonuclease A, 13,700 Da
This application demonstrates the suitability of Discovery BIO GFC300 for the
4. Poly-DL-alanine, 1,000-5,000 Da 3
separation of proteins. Separation is by inclusion into the pore volume with 2
5. Uracil, 112 Da
larger molecules being excluded and therefore eluting earlier.
mobile phase . . . . . . . . . . . . . . . . . 150 mM potassium phosphate monobasic,
pH 7.0 (adjusted with potassium hydroxide) 1 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min 4
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812 psi (56 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm 0 10 20
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL Min
sample . . . . . . . . . . . . . . 1 g/L each (except uracil, 0.1 g/L) in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005731

HPLC Analysis of Proteins on TSKgel® CM-5PW 1. Trypsinogen

2. Ribonuclease A 3 4
column . . . . . TSKgel CM-5PW, 7.5 cm × 7.5 mm I.D., 10 μm particles (813068) 3. α-Chymotrypsinogen
mobile phase . . . . . . . . (A) 0.1 M sodium phosphate, monbasic buffer, pH 7.0; 4. Cytochrome c
(B) 0.5 M sodium chloride, A to B in 60 min 5. Lysozyme
5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 1
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1002
0 10 20 30 40
Min

HPLC Analysis of Proteins on TSKgel® DEAE-NP 2

column . . . . TSKgel DEAE-NPR, 3.5 cm × 4.6 mm I.D., 2.5 μm particles (813075) A to B in 10 min 1. Ovalbumin
at 1.5 mL/min 2. Trypsin inhibitor
mobile phase . . (A) 20 mM Tris-HCl buffer, pH 8.0; (B) 0.5 M sodium chloride in A
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1020
1
2 3 4 5 6
Min

HPLC Analysis of Protein Standards (1) on 1. Lysozyme

2. Haptoglobulin, phenotype 1-1
BIOshell™ A400 Protein C4 3. Conalbumin
This application demonstrates the utility of the BIOshell A400 Protein C4 4. Trypsin inhibitor, soybean
column for the reversed-phase chromatography of proteins from a variety of 5. Glutamate dehydrogenase
sources and of different molecular weights. 6. β-Galactosidase
column . . BIOshell A400 Protein C4, 10 cm X 2.1 mm, 3.4 μm particles (66825-U) 7. Serum albumin, chicken
mobile phase . . . . . . (A) 75:25, [0.1% TFA in water]:[0.1% TFA in acetonitrile] ; 8. Lactate dehydrogenase 4
8
(B) 25:75, [0.1% TFA in water]:[0.1% TFA in acetonitrile]
5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 3
1
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1430 psi (99 bar) 7
6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . protein mixture, g/L ea: lysozyme, 0.1; haptoglobulin, 0.3;
conalbumin, 0.3; trypsin inhibitor, 0.3; glutamate dehydrogenase, 0.8;
beta-galactosidase, 0.7; ovalbumin, 0.8; lactate dehydrogenase, 0.4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006201
0 2 4 6 8
Min

HPLC Analysis of Proteins with Different pI Values 1. Ovalbumin

on Proteomix® SCX-NP3 2. Ribonuclease A
3. Cytochrome c
column . . . . . Proteomix SCX-NP3, 5 cm x 4.6 mm I.D., 3 μm particles (Z777151) 2 5
4. Aprotinin
mobile phase . . . . . . (A) 10 mM sodium phosphate, pH 6.0; (B) A + 1 M NaCl 1 5. Lysozyme
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006146
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

HPLC Analysis of Protein Tryptic Digest on

Ascentis® RP-Amide Gradient Program
Time (min) %A %B
column . . . Ascentis RP-Amide, 25 cm x 2.1 mm I.D., 5 μm particles (565306-U) 0 90 10
mobile phase . . (A) (50:50) 50 mM formic acid titrated with acetate (pH 3.0):water; 80 50 50
(B) (50:50) 50 mM formic acid titrated with acetate (pH 3.0):acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL
sample . . . . . . . . . . . . . . . . . . . . tryptic digest of carboxymethylated protein 0 20 40 60 80
mixture 1.5 g/L in 50 mM formic acid Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002670

HPLC Analysis of Pullulan on an SRT® SEC-300 Gel Calibration Curve
Filtration Column Pullulan Standards

1. MW 342 11.67
6
2. MW 1,320 11.34
Pullulan is a water-soluble polysaccharide polymer consisting of glucan 3. MW 6,000 10.39
Log MW
units. Pullulan is used in the manufacture of edible films that are used in 4. MW 10,000 9.94
5. MW 21,700 9.00
breath fresheners and mints and is also used as a food additive. Another use 4 6. MW 48,800 7.75
of pullulan is as calibration standards in gel filtration chromatography. 7. MW 113,000 6.55
8. MW 210,000 6.03
Gel filtration chromatography (GFC) is used to determine the molecular 9. MW 366,000 5.93
mass composition of pullulan and many other water-soluble polymers using 10. MW 805,000 5.84
2
silica- or polymer-based particles that have been modified with a chemically 6 8 10 12
80 Elution volume (mL)
bonded hydrophilic coating. 1
70
In this particular example, a SRT SEC-300 column from Sepax Technologies
60
was used to analyze several pullulan standards. In the accompanying table
50
MV
shows retention volumes for the pullulan standards and the molecular mass
40 9 2
of each; this data was then used to construct the calibration curve which 8
30 10 3
allows the calculation of the molecular mass and distribution of an unknown 5 4
20 7 6
pullulan polymer or other polymers of similar geometrical shape.
10
column . . . . . . . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm, 300 Å (Z777051) 0
mobile phase . . . . . . . . . . . . . . . . . . . . . 150 mM phosphate Buffer, pH 7.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
Min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600 psi (41.4 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RI (30° C)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . 10 mg/mL in mobile phase 0.342 kD, 1.32 kD, 6 kD, 10 kD, 21.7 kD,
48.8 kD 113 kD, 210 kD, 366 kD and 805 kD
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006202

1
HPLC Analysis of Purines and Pyrimidines of
Nucleic Acids on Ascentis® Si (Silica) 1. Thymine
2. Uracil
column . . . . . . . . Ascentis Si, 10 cm × 2.1 mm I.D., 5 μm particles (581508-U) 3. Adenine
4. Guanine
mobile phase . . . . . (A) 200 mM ammonium formate (pH 3.0, with formic acid): 3 5. Cytosine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 4
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μg/mL in mobile phase

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003683
0 2 4 6 8 10
Min

HPLC Analysis of Pyroglutamic Acid Enantiomers H O

N
on Astec® CHIROBIOTIC® T O *
OH
mobile phase . . . . . . . . . . . . . . (A) acetonitrile; (B) methanol; (C) acetic acid;
(D) triethylamine; (54.5:45:0.3:0.2, A:B:C:D)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004511
0 10 20
Min.

HPLC Analysis of Recombinant Tumor Necrosis

Factor Receptor-Fc Fusion Protein (rhTNFR-Fc) on
column . . . . . . . . . . . . . . . . . . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm,
300 Å particles (Z777051)
column . . . . . . . . . . . . . . . . . . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm,
300 Å particles (Z777051) (Z777033)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . Recombinant tumor necrosis factor receptor-Fc fusion
protein (rhTNFR-Fc), including oligomers (440 kD and 360 kD),
monomer (150 kD), and impurity (120 kD)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006121
Zenix SEC-300
SRT SEC-300
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

HPLC Analysis of Reduced Monoclonal Antibody 1. scFc

3
2. LC
Fragments on BIOshell™ A400 Protein C4 3. Fd’
2
Digesting an antibody into a few large fragments facilitates a middle-down
approach for subsequent structural characterization. The IdeS enzyme, from
Streptococcus pyogenes and commercialized as FabRICATOR, cleaves IgG in 1
the hinge region resulting in two fragments: F(ab′)2 and scFc. Reduction of F
(ab′)2 dissociates it into its component polypeptides, Fd′ and LC. This
application demonstrates the baseline resolution of the three fragments
scFC, LC, and Fd′, in reasonable time with excellent peak shape.
Monoclonal antibody SO57 (0.5 mg) was digested by IdeS protease
according to Genovis (www.genovis.com) FragIT kit protocol. The digest was
then exposed to 10 mM dithiobutylamine for one hour, at pH 7, before
being diluted 1:5 with mobile phase A prior to injection; final protein conc.
<0.3 g/L. 0 2 4 6 8
Genovis FragIT kit is available from Sigma, outside the USA. Min
column . . BIOshell A400 Protein C4, 10 cm x 2.1 mm I.D., 3.4 μm particles (66825-U)
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 to 70%B in 12 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 psi initial (69 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
(B) 50:50, [0.1% TFA in water]:[0.1% TFA in acetonitrile]
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006235

HPLC Analysis of Ribonucleosides on Ascentis® Si 1 1. Uridine (0.1 g/L)

2 2. Adenosine (0.1 g/L)
column . . . . . . . . Ascentis Si, 10 cm x 2.1 mm I.D., 5 μm particles (581508-U) 3. Guanosine (0.1 g/L)
mobile phase . . . . . (A) 150 mM ammonium formate (pH 3.0, with formic acid): 4. Cytidine (0.1 g/L)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm 4
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003725
0 2 4 6 8 10
Min

HPLC Analysis of RNA on Proteomix® WAX-NP5

column . . . Proteomix WAX-NP5, 15 cm x 4.6 mm I.D., 5 μm particles (Z777262)
mobile phase . . . . . . . . . . . . (A) 20 mmol Tris, pH 6.0; (B): A + 1.0 M NaCl
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20%-100%B (40 min)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1015 psi (70 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RNA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005967
0 5 10 15 20 25 30 35 40
Min

HPLC Analysis of RP Peptide Performance Peptide 1: -RGAGGLGLGK-amide Gradient

Peptide 2: ac-RGGGGLGLGK-amide
Standard on Discovery® BIO Wide Pore C18, Peptide 3: ac-RGAGGLGLGK-amide
Min %A %B
0 90 10
No TFA Peptide 4: ac-RGVGGLGLGK-amide
20 50 50
Peptide 5: ac-RGVVGLGLGK-amide
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 2.1 mm I.D., 0% TFA (25 mM formic acid), 1 μL injection
mobile phase . . (A) 25 mM formic acid in water; (B) (50:50) 25 mM formic acid in 100 5
water:20 mM formic acid in acetonitrile 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.20 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 2 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +ESI % 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . RP Peptide Performance Standard,
p/n RPS-P0010 (Alberta Peptide Institute)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001638A 0
0 10 20
Min

HPLC Analysis of Separation of Low MW

Polysaccharides on SRT®
Achyranthes bidentata polysaccharides (ABPS) is extracted from the root of a
Chinese medicinal herb. ABPS acts as an immune modulator. It is tested for
use as a replacement of antibiotics added to feedstock to protect piglets
from microorganisms. ABPS contains mainly fructose and glucose units. The
chromatograms show that the smallest pore size column, SRT SEC-100,
provides the largest number of peaks, which is not unexpected given the
low molecular mass (< 10,000 Da) of the sample.
Note that since ABPS does not carry an electrical charge, a 15 mM buffer
concentration is sufficient to prevent interaction of the sample with the
underlying silica surface.
column . . . . SRT SEC-100 30 cm x 4.6 mm I.D., 5 μm particles, 100 Å (Z777037)
column . . . . SRT SEC-150 30 cm x 4.6 mm I.D., 5 μm particles, 150 Å (Z777043)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . 0.15 M phosphate buffer, pH 7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.35 mL/min SRT SEC-100 (100 Å)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . Achyranthes bidentata polysaccharides (ABPS)
SRT SEC-150 (150 Å)
from plant root extract (MW < 10,000)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006207
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Min

HPLC Analysis of Serine Enantiomers on Astec® 1. L-Serine

1 O
CHIROBIOTIC® T (Formic Acid Mobile Phase 2. D-Serine HO OH
Additive) NH 2
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005344
0 2 4 6
Min

HPLC Analysis of Serine Enantiomers on Astec® O

CHIROBIOTIC® T
*
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HO OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min NH 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004513
0 2 4 6
Min.

HPLC Analysis of Serine Enantiomers on Astec® O

CHIROBIOTIC® TAG
*
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) HO OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004614
0 2 4 6 8 10
Min.

HPLC Analysis of Six Enkephalin Peptides (Chiral

and Achiral) on Astec® CHIROBIOTIC® T2
mobile phase . . (A) 5 mM ammonium formate, pH 3.3; (B) acetonitrile; (25:75, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004738
10 20 30 40
Min

HPLC Analysis of Size Exclusion Separation of 1. 70 kDa protein (undisclosed drug candidate)
Proteins and PEGylated Proteins 2. 40 kDa PEG molecule attached to protein identified as peak 1
3. Two PEG molecules attached to protein identified as peak 1
Pegylation of biotherapeutic drugs improves solubility, increases half life in
the blood stream and reduces immunogenicity. Size exclusion chromatog- 1
raphy (SEC) is often applied to characterize protein–polyethylene glycol
(PEG) conjugates and establish the number of attached PEG chains, since
SEC is a precise and robust technique that combines ease of set up and
strainghtforward method develpopment.
column . . Zenix SEC-150, 30 cm x 7.8 mm I.D., 3 μm particles, 150 Å, (Z777018)
mobile phase . . . . . . . . . . . . . . . . . . . . . 150 mM phosphate buffer, pH 7.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL, 2.0 mg/mL
sample . . . . . 1. Protein with MW 70KD (from an undisclosed drug development 3 2
company) 2. Protein 1 attached with 1 PEG (MW 40KD) molecule
3, Protein 1 attached with 2 PEG (MW 40KD) molecules 0 1 2 3 4 5 6 7 8 9 10 11 12

HPLC Analysis of Substance P and Its Fragments 1. Fragment 1-4 Gradient Program
on Discovery® C18 2. Fragment 1-7 Time (min) %B
3. Fragment 1-9 0 5
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 4. Fragment 8-11 6 29
mobile phase . . . . . . . . . . (A) water + 0.1% TFA; (B) acetonitrile + 0.1% TFA 5. Holopeptide 11 34
3
6. Fragment 2-11
gradient . . . . . . . . . . . . . . . . . . . . linear; 5-29%B (6 min); 29-34B (5 min) 7. Fragment 7-11 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 6
1
sample . . . . . . . . . . . . . . . . . . . . . . . . Substance P in initial mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000322
7
0 2 4 6 8 10 12
Min

HPLC Analysis of Sulfa Drugs on Titan™ C18 1 1. Sulfanilamide

2. Sulfacetamide
column . . . . . . . . . Titan C18 5 cm x 2.1 mm I.D., 1.9 μm particle (577122-U)
3. Sulfadiazine
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) water with 0.1% acetic acid; 4. Sulfathiazole
(B) acetonitrile with 0.1% acetic acid ; (90:10, A:B) 5. Sulfamerazine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 6. Sulfamethizole
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2670 psi (184 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 3
2 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm 5
6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006114
0 2 4
Min

HPLC Analysis of Superoxide Dismutase on

Discovery® BIO PolyMA-WAX
Gradient Profile
Discovery BIO PolyMA-SCX and PolyMA-WAX columns give sharp, efficient Min %A %B
peaks for a wide variety of proteins. 0 100 0
column . . Discovery BIO PolyMA-WAX, 5 cm × 4.6 mm, 5 μm particles (59602-U) 30 90 10
mobile phase . . . . . . . . . . . . . ((A) 20 mM Tris, pH 8.0 with hydrocloric acid;
(B) 20 mM Tris, 0.5 M sodium chloride,
pH 8.0 with hydrochloric acid)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001835
0 5 10 15 20 25 30
Min

HPLC Analysis of Synephrine Enantiomers on

Astec® CYCLOBOND® I 2000 AC HO
column . . CYCLOBOND I 2000 AC, 25 cm x 4.6 mm I.D., 5 μm particles (20124AST)

* CH 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 °C OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004695
0 2 4 6 8 10
Min

HPLC Analysis of the Monoclonal Antibody (mAb)

Erbitux (Cetuximab) on BIOshell™ A400 Protein C4
Antibodies can exhibit considerable hydrophobicity, and so efficient 0.4 mL/min
reversed-phase elution of this intact mAb requires the use of high
temperature. The top chromatogram illustrates that BIOshell™ Fused-Core®
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
columns can be operated at high flow rate while maintaining good peak
shape at a reasonable back pressure. Min
column . . . . . . . . . . . . . . . BIOshell™ A400 Protein C4, 10 cm X 2.1 mm I.D.,
mobile phase . . . . . . . . (A) 80:20, 0.1% TFA in water: 0.1% TFA in acetonitrile;
(B) 20:80, 0.1% TFA in water: 0.1% TFA in acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . . . . . 20 to 35%B in 10 min (or 5 min) 0.2 mL/min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470 psi (or 940 psi) 0 1 2 3 4 5 6 7 8 9 10
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 °C Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 μL
sample . . . . . . . . . . . . . . . . . . . . . 1 g/L mAb in 20 mM Tricine/KOH, pH 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006254

HPLC Analysis of the Peptide Glycine-Leucine- CH3

Alanine Enantiomers on Astec® CHIROBIOTIC® TAG
O CH3 O
H2N NH *
mobile phase . . (A) 5 mM ammonium acetate, pH 4.1; (B) acetonitrile; (35:65, A:B) NH * OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O CH3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004617
0 10 20
Min.

HPLC Analysis of Threonine Enantiomers on OH O 1 1. L-Threonine

Astec® CHIROBIOTIC® T (Formic Acid Mobile 2. D-Threonine
H3C OH 2
Phase Additive) NH2

mobile phase . . . . . (A); water; (B) methanol; (C) formic acid; (30:70:0.02, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005345
0 2 4 6
Min

HPLC Analysis of Threonine Enantiomers on OH O
*
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H3C OH
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (30:70, A:B) NH2

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004517
0 2 4 6 8
Min.

HPLC Analysis of Thyroglobulin on a SRT® 1. Thyroglobulin aggregate

3
SEC-1000 Gel Filtration Column 2. Thyroglobulin
3. Uracil
Thyroglobulin is a very large protein, with a molecular mass of 670 kDa.
Thus,a large pore size gel filtration columns is required to prevent partial
exclusion of not just the monomer, but also of the dimer and higher order
aggregates. This was accomplished using the Sepax SRT SEC-1000 column,
which has a nominal pore size of 1000 Angstrom. The nucleic acid base
Uracil was added to the sample as a marker to indicate the inclusion volume
since a small molecule like Uracil is not restricted from entering any pores in
the column.
column . . SRT SEC-1000, 30 cm x 4.6 mm I.D., 5 μm particles, 1000 Å (Z777061)
mobile phase . . . . . . . . . . . . . . . . . . . . . 150 mM phosphate buffer, pH 7.0 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.35 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 1. Thyroglobulin (670kD) 1 mg/mL;
2. Uracil (120D), 0.1 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006181 1
8 9 10 11 12 13 14 15
Min

1
HPLC Analysis of Thyroxine Enantiomers on Astec®
CHIROBIOTIC® TAG 1. L-Thyroxine
2. D-Thyroxine
Baseline resolution of the chiral enantiomers of DL-Thyroxine is achieved by
polar ionic mode on the Astec CHIROBIOTIC TAG with the use of an LC/MS-
compatible mobile phase.
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) 2
mobile phase . . . . . . . . . . . . . . . . . . . . 1% ammonium formate in methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826 psi (57 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . each enantiomer 0.1 mg/L in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005515 0 10 20 30
Min

HPLC Analysis of BIOshell™ A160 Peptide C18 and 3 1. Asp-Phe 6. Angiotensin II
CN 5 μm Columns, Comparison of Different Phases

2. Angiotensin (1-7) amide 7. Neurotensin
3. Tyr-Tyr-Tyr 8. β-endorphin
3 4. Bradykinin 9. Sauvagine
The availability of two distinct chemistries (C18 alkyl vs. CN) is helpful to 6 5. Leu-Enk 10. Mellitin
check changes in elution order as we see for peaks # 5 and # 6 on the C18 5
and CN columns. As expected, peptide retention is shorter on the CN 2
6
column, while peak shape is symmetrical and efficiency very high on both 1 4
5
2 7 9 10
column types. 8
column . . BIOshell A160 Peptide C18, 15 cm x 4.6 mm I.D., 5 μm particles (67015-U) BIOshell A160
Peptide CN, 5 µm
4
column . . BIOshell A160 Peptide CN, 15 cm x 4.6 mm I.D., 5 μm particles (67081-U) 1
7
10
8
mobile phase . . . . . . . . . . . . . (A): water/0.1% TFA (B): acetonitrile/0.1% TFA 9
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-50%B in 30 minutes
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min BIOshell A160
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 bar, 250 bar Peptide C18, 5 µm
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm 0 5 10 15 20 25 30
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01 mg/mL in water/0.1% TFA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006219

HPLC Analysis of tRNA Hydrolysate on Gradient Program

SUPELCOSIL™ LC-18-S Time (min) %A %B %C
0 100 — —
column . . . . SUPELCOSIL LC-18-S, 15 cm × 4.6 mm I.D., 5 μm particles (58931) 7.2 100 — —
Selective,
mobile phase . . (A) 0.1M potassium phosphate, dibasic (pH 5.3):methanol, 97.5:2.5; 12 90 10 —
Quantitative Analysis
(B) 0.01M potassium phosphate, dibasic (pH 5.1):methanol, 80:20; (C) 0.01M 15 75 25 —
19.2 40 60 —
potassium phosphate, dibasic (pH 4.9):acetonitrile, 65:35 21.6 38 62 —
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 27 — 100 —
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 °C 48 — — 100
55 — — 100 Detection at
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm, 254 nm, 280 nm 210 nm, 250 mAU
injection . . . . . . . . . . . . . . . . . . 50 μL hydrolysate buffer (pH 7.8) containing ψC U G
ψ C U
A
5 μg bovine tRNA Leu hydrolysate m2G
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0501
m1G
mC3 Um m22G hU
m1A
m5C T 0 Min 10
ψm Detection at
254 nm, 50 mAU
Detection at
280 nm, 50 mAU
0 10 20 30
Min
Figure provided by Dr. C.W. Gehrke and Mr. K.C. Kuo of the Interdisciplinary
Chromatography-Mass Spectrometry Laboratories, University of Missouri
(Columbia, MO, USA).

HPLC Analysis of Trypsin on TSKgel® Butyl-NPR α-Trypsin

column . . . . TSKgel Butyl-NPR, 3.5 cm × 4.6 mm I.D., 2.5 μm particles (814947)
mobile phase . . . . . . . 2 M to 0 M ammonium sulfate in 20 mM Tris-HCI buffer,
pH 7.5 in 10 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . commercial trypsin sample
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1204
β-Trypsin
0 2 4 6 8 10
Min

HPLC Analysis of Tryptic Digest on Ascentis®

Express Peptide ES-C18
column . . . . . . . . . . . . Ascentis Express Peptide ES-C18, 15 cm x 2.1 mm I.D.,
(B) 25:75, (0.4% formic acid in water):acetonitrile
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 70% B in 40 min.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
0 10 20 30 40
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), TOF
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 pg/L in 0.1% formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005381

HPLC Analysis of Tryptic Digest of Protein Mixture

on Ascentis® Express Peptide ES-C18
column . . . . . . . . . . . . Ascentis Express Peptide ES-C18, 15 cm x 21 mm I.D.,
(B) 0.4% formic acid in 25:75 (v/v) water:acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), TOF
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 pmol/L in 0.1% formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005629 0 20 40
Min

HPLC Analysis of Tryptophan Enantiomers on 1. L-Tryptophan NH 2
2. D-Tryptophan 1
Astec® CHIROBIOTIC® T (Formic Acid Mobile Phase
O
Additive) N
H
HO

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005346
0 2 4 6 8 10
Min

HPLC Analysis of Tryptophan Enantiomers on

Astec® CHIROBIOTIC® T * NH 2

O
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (20:80, A:B) N HO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004521
0 2 4 6 8 10 12 14
Min

NH2
HPLC Analysis of Tyrosine Enantiomers on Astec® 1. L-Tyrosine L
CHIROBIOTIC® T 2. D-Tyrosine D O
HO
mobile phase . . . . . (A) water; (B) methanol; (C) formic acid; (30:70:0.02, A:B:C) HO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005347
0 2 4 6 8
Min

HPLC Analysis of o-Tyrosine Enantiomers on

Astec® CHIROBIOTIC® T OH O

OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004537
0 2 4 6 8
Min

HPLC Analysis of m-Tyrosine Enantiomers on

column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HO *

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004536
0 2 4 6 8
Min

HPLC Analysis of p-Tyrosine Enantiomers on NH2

*
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (50:50, A:B) HO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
HO
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004522
0 2 4 6
Min

HPLC Analysis of Valine Enantiomers on Astec® 1 NH2

1. L-Valine
CHIROBIOTIC® T (Formic Acid Mobile Phase 2. D-Valine
H3C O
Additive) 2 CH3 OH

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005348
0 2 4 6 8 10
Min

HPLC Analysis of Valine Enantiomers on Astec® NH 2

CHIROBIOTIC® T
H 3C O
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (70:30, A:B) CH3 OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004523
1.0 2.0 3.0 4.0 5.0

Min

HPLC Analysis of White-Faced Hornet Venom on A280

Phospholipase A %B
Mono S® HR5/5 100
Phospholipase B
column . . . . . . . . . . . . . . . . . . . . . Mono S HR5/5, 10 μm particles (54808)
mobile phase . . . . . (A) 50 mM BICINE, pH 8.4; (B) A + 0.35 M sodium chloride Hyaluronidase
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Acid phosophatase
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . 7 mg venom in mobile phase A
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0411
50
0 10 20 30 40
Min

HPLC Analysis of Xanthines on Ascentis® C18 1 1. 7-methylxanthine

2. 1-methylxanthine
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 3. 1,7-dimethylxanthine
mobile phase . . . . . . . . . . . . . . . (A) 10 mM monobasic potassium phosphate 4. β-hydroxytheophylline
(pH 3.0 with phosphoric acid); (B) methanol; (94:6, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 3
2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002465
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Xylo-oligosaccharide on

Proteomix® SAX-NP5
mobile phase . . . . . . . . . . . . . (A) 0.05% ammonium hydroxide in acetonitrile
(B) 0.05% ammonium hydroxide in water
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15-60% B (20 min)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1015 psi (70 bar)
temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ELSD
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xylose oligosaccharides
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005960
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5
Min

HPLC Analysis of Yeast Lysate on Proteomix®

SAX-NP3 Demonstrating High Separation
Efficency
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
mobile phase . . . . . . . . . . . . . . (A) 20 mM Tris, pH 9.0; (B) A + 0.5 M NaCl
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 - 100% B in 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006131
0 5 10 15 20 25 30 35 40 45
Min

3
HPLC Analysis Proteins on Proteomix® SCX-NP1.7, 1. Ribonuclease A
Demonstration of Column Stability with Repeated 2. Cytochrome c
1
3. Lysozyme
Injections
column . . . . . . . . . . . . . . . Proteomix SCX-NP1.7 guard cartridge with holder,
1 cm x 4 mm I.D. (Z777144) 1st Injection
gradient . . . . . . . . . . . . . . . . . . . . 0 - 100% B in 25 min, 15 min prewash 125th Injection
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 250th Injection
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 0 5 10 15 20
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . each compound 1 mg/mL Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006135

HPLC Separation of an Oligonucleotide and

Impurities on Proteomix® SAX-NP5 versus
Porous SAX
mobile phase . . . . . . . . . . . . (A) 0.025 M Tris buffer pH9; (B) A + 1.0 M LiCi
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 to 75% B in 45 min Proteomix SAX-NP5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μL SAX Column A
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50-mer Oligo
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005961
0 5 10 15 20 25 30 35 40 45
Min
Proteomix SAX-NP5
SAX Column A
20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0

Zoomed section Min

HPLC Separation of an Oligonucleotide and Its

Degradation Fragments on Proteomix® SAX-NP5
mobile phase . . . . . . . . (A) 25 mM Tris, 1 mM EDTA 10% acetonitrile (8.0 pH);
(B) A + 1.0 M NaCl
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 75% B in 50 min 16 20 25 30 35 40
Zoomed section Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MW 12196
sample . . . . . . . . . . Oligonucleotide (MW 12196) and its degradation fragments
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005962
0 5 10 15 20 25 30 35 40 45
Min

HPLC (Size Exclusion) Analysis of Proteins on R

U
Zenix®-C SEC-300, Effect of DTT and EDTA O
mobile phase . . (A) 150 mM sodium phosphate buffer, pH 7.0 (B) 150 mM sodium T G
phosphate buffer, pH 7.0 with 2 mM DTT, 0.1 mM EDTA
(C) 150 mM sodium phosphate buffer, pH 7.0 with 2 mM DTT,
0.1 mM EDTA
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
R
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
B Blue: Protein mix 2, 150 mM sodium
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm phosphate buffer, pH 7.0 with 2 mM DTT,
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL T 0.1 mM EDTA
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . proteins
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005954 Red: Protein mix 1, 150 mM sodium
phosphate buffer, pH 7.0 with 2 mM DTT,
0.1 mM EDTA
Black: Protein mix 1, 150 mM

sodium phosphate buffer, pH 7.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5

Min
Separation efficiency not adversely affected by addition of DTT or EDTA.

Improved HPLC Selectivity of Discovery® BIO Wide Peak Peptide

1. Arg8-vassopressin
Amino Acid Sequence
CYFQNCPRG-amide; disulfide
2. Bradykinin, fragment 1-5 RPPGF
Pore RP Phases 3. Oxytocin CYIQNCPLG-amide; disulfide
4. LHRH* pEHWSYGLRPG-Amide**
column . . Discovery BIO Wide Pore C18, 15 cm x 4.6 mm I.D., 5 μm particles vs. 5. Met-enkephalin
6. Bradykinin
YGGFM
RPPGFSPFR
competitive wide pore C18 column of same dimensions (568222-U) 7. Leu-enkephalin
8. Bombesin
YGGFL
pEQRLGNQWAVGHLM-amide**
mobile phase . . . . . . . . . . . . . . . . . (A) 0.1% TFA in 80:20 water:acetonitrile; 9. Substance P RPKPQQFFGLM-amide
(B) 0.1% TFA in 66:34 water:acetonitrile *Luteinizing Hormone Releasing Hormone

**pE is pyroglutamate
Gradient of 0 to 100% B in 14 min. after 1 min. delay
(A) Discovery Bio Wide Pore C18
gradient . . . . . . . . . . . . . . . . . . . 0 to 100% B in 14 min. after 1 min. delay
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 1
5
7
sample . . . . peptide mixture (Sigma P2693), ~0.25 μg each peptide in 0.1% TFA 2 6 8
3 9
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004415
0 2 4 6 8 10 12 14
Min
(B) Competitor
Discovery column resolves

synthetic peptides better than
the competitive column
0 2 4 6 8 10 12 14
Min

LC-MS Analysis of Amino Acids on Ascentis® 1. Tryptamine

Express HILIC 2. Tyramine 5
3. Tryptophan 6
column . . Ascentis Express HILIC, 15 cm x 4.6 mm I.D., 2.7 μm particles (53981-U) 4. Methionine
mobile phase . . (A) 13 mM ammonium acetate in 10:90 (v/v) water: acetonitrile; 5. β-Alanine
(B) 13 mM ammonium acetate in water 6. Alanine 4
3
gradient . . . . . . . . . . . . . . . . held at 0% B for 1 min; 0 to 90% B in 19 min
1 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L in Mobile Phase A Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005664

LC-MS Analysis of CYP2D6 Inhibitors on Ascentis® 1. Amitriptyline 1

Express HILIC 2. Imipramine
3. Fluoxetine
This application demonstrates the suitability of the Ascentis Express HILIC for 4. Paroxetine 2
the analysis of CYP2D6 Inhibitors.
4
mobile phase . . . . . . . 5 mM ammonium formate in 2:98 (v/v) water:acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 100-400 1.0 2.0 3.0 4.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005691

LC-MS Analysis of β-Lactoglobulin Tryptic Digest 27.66

859.2
on a Discovery® BIO Wide Pore C18 Capillary 100 21.42 22.80
696.2 818.7
Column 21.00
Relative Abundance
419.4
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 0.5 mm I.D., 18.46
5 μm particles (65519-U) 623.4
mobile phase . . . . . . . . . . (A) 0.1% TFA in water; (B) 0.1% TFA in acetonitrile 17.17 15.90
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-40% B in 70 min 50 337.3 458.9 29.17
27.17 555.2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 μL/min 1044.6 30.49
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 12.36 772.1 35.06 36.66
573.3 522.6 322.0
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +ESI mode 24.54 34.37
37.71
injection . . . . . . . . . . . . . . . . . 500 pmol (5μL) β-Lactoglobulin tryptic digest 775.4 522.5
522.6
in 50 mM ammonium bicarbonate 0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001591 0 10 20 30 40
Min

LC-MS Analysis of L-Methionine Sulfoximine, 3 1. L-Methionine sulfone

Sulfone and Sulfoxide on Ascentis® Express HILIC 2. L-Methionine sulfoximine
2 3. L-Methionine sufloxide
L-methionine sulfoximine and L-buthionine sulfoximine are used in are used
to prevent additional enzyme activity in Chinese hampster ovary (CHO) cell
1
lines with supplemental Glutamine Synthase (GS). It is of interest to be able
to monitor both purity of such molecules in order to control addition to cell
media as well as for the assay of parent molecules during use. The analytes
are highly polar and thus should be amenable to HILIC-LC-MS analysis. This
first installment investigates the ability of Ascentis Express HILIC and
Ascentis Express OH5 stationary phases for the separation of the methionine
analog and related compounds as well as methionine and buthionine
separations.
mobile phase . . . . . . . . . . . . (A) acetonitrile; (B) water with 0.1% formic acid, 0 2 4 6 8 10
pH 3.5 with ammonium formate; (75:25, A:B) Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1350 psi 93 bar
temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI (+), TIC, m/z 150-300
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL in 90:10, methanol:water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005790

3 1. Peptide 1 (100 µg/mL)

LC-MS Analysis of Peptides, Basic, on Gradient Program
2. Peptide 2 (100 µg/mL)
Time (min) %A %B
Ascentis® C18 0 80 20 3. Peptide 3 (100 µg/mL)
20 40 60 4. Peptide 4 (100 µg/mL)
mobile phase . . . . . (A) 20 mM formic acid in water; (B) (50:50), 20 mM formic
acid in water:20 mM formic acid in acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002444
1
0 10 20
Min

LC-MS Analysis of Peptides, Hydrophobic, on Gradient Program 5 1. Peptide 1 (20 mg/L)

Time (min) %A %B 2. Peptide 2 (20 mg/L)
Ascentis® C18 0 80 20 4 3. Peptide 3 (20 mg/L)
15 50 50 3 4. Peptide 4 (20 mg/L)
column . . . . . . . Ascentis C18, 10 cm x 2.1 mm I.D., 5 μm particles (581326-U) 5. Peptide 5 (20 mg/L)
mobile phase . . . . . . . . . . . . . . (A) (50:50), 50 mM formic acid titrated with 2
acetate (pH 3.0):water; (B) (50:50), 50 mM formic acid 1
titrated with acetate (pH 3.0):acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in 10 mM formic acid 0 2 4 6 8 10 12 14

LC-MS Analysis of Peptides, Hydrophobic, on Gradient Program 5 1. Peptide 1 (20 mg/L)

Time (min) %A %B 2. Peptide 2 (20 mg/L)
Ascentis® RP-Amide 0 85 15 3 4 3. Peptide 3 (20 mg/L)
15 55 45 4. Peptide 4 (20 mg/L)
column . . . Ascentis RP-Amide, 10 cm x 2.1 mm I.D., 5 μm particles (565304-U) 5. Peptide 5 (20 mg/L)
mobile phase . . (A) (50:50), 50 mM formic acid titrated with acetate (pH 3.0):water;
(B) (50:50), 50 mM formic acid titrated with 1 2
acetate (pH 3.0):acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in 10 mM formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002682
0 2 4 6 8 10 12 14
Min

Peptide 1: ac-RGAGGLGLGK-amide Peptide 3: ac-RGVGGLGLGK-amide

LC-MS Analysis of Peptides on Discovery® BIO Peptide 2: ac-RGGGGLGLGK-amide Peptide 4: ac-RGVVGLGLGK-amide
Wide Pore C18, Effect of TFA Concentration on 100 A1: 0% TFA (25mM formic acid), 1µL injection
4
5
MS Signal Gradient Program

Time (min) %A B%
0 90 10
column . . . . . . . . . . . . . Discovery® BIO Wide Pore C18, 15 cm x 2.1 mm I.D., 20 50 50
2
3
% 1
mobile phase . . . . . . . (A): aqueous component (25 mM formic acid, 0.01% TFA
or 0.1% TFA); (B) (50:50) water:acetonitrile containing
same ionic additives as aq. component
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.20 mL/min
0 10 20
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (+)ESI
100 A2: 0.01% TFA, 3µL injection
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 μL Gradient Program
sample . . . . . . . . . . . . . . . . . . . . . . . . . . RP Peptide Performance Standard Time (min) %A %B
0 80 20
(Alberta Peptide Inst., Cat. No. RPS-P0010) 20 40 60 5
4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004414
2 3
% 1
0
0 10 20
70 A3: 0.1% TFA, 3µL injection
Gradient Program
Time (min) %A %B
0 80 20
20 40 60
1,2: NOTE: Coelution of peptides 1 and 2

3 4 5
0
0 10 20
Min

LC-MS Analysis of Peptides, Serum Derived, on Gradient Program 1. Peptide 1, (M+H)+ = 1142.0,
Time (min) %A %B (M+2H)2+ = 572.6; (10 mg/L)
Ascentis® RP-Amide 0 70 30 2. Peptide 2, (M+2H)2+ = 810.0; (10 mg/L)
25 45 55 3. Peptide 3, (M+2H)2+ = 649.5; (10 mg/L)
column . . . Ascentis RP-Amide, 10 cm x 2.1 mm I.D., 5 μm particles (565304-U) 4. Peptide 4, (M+2H)2+ = 885.5; (10 mg/L)
mobile phase . . (A) (50:50), 50 mM formic acid titrated with acetate (pH 3.0):water; 3
5. Peptide 5, (M+2H)2+ = 977.8,
(B) (50:50), 50 mM formic acid titrated with acetate (pH 3.0):acetonitrile (M+3H)3+ = 652.6; (10 mg/L)
5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+) 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in 10 mM formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002689 4
1
0 10 20
Min

LC-MS Analysis of Polar Amino Acids on Ascentis® 1. Threonine

2. Serine
Si (HILIC Mode) 3. Asparagine 3
column . . . . . . . . Ascentis Si, 10 cm x 2.1 mm I.D., 5 μm particles (56403AST) 4. Glutamine
mobile phase . . . . . . (A) 100 mM ammonium formate, pH 3.0 with formic acid; 5. Glutamic acid 1 5
(B) water; (C) acetonitrile 6. Aspartic acid 4
gradient . . . . . . . . . . . . 5% A constant; 10 to 16% B, 85 to 79% C in 20 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g/L each in mobile phase 0 10 20
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005722

LC-MS Analysis of Polypeptide Drugs on Ascentis® 1. Lypressin

Express RP-Amide 2. Oxytocin 3
3. Eptifibatide
This application demonstrates the suitability of the Ascentis Express RP 4. Desmopressin 4
Amide for the analysis of polypeptide drugs. 5. Bivalirudin
mobile phase . . . . . (A) 10 mM ammonium acetate in water, pH 4.2; (B) 10 mM
ammonium acetate in 90:10 (v/v) acetonitrile:water, pH 4.7
2
gradient . . . . . . . . . . . . held at 20% B for 0.1 min; 20 to 80% B in 2.9 min;
80 to 20% B in 0.01 min; held at 20% B for 0.99 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 1.0 2.0 3.0
Min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF, ESI(+), m/z 100-2500
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
sample . . . . . . . 1 μL/mL in 10mM ammonium formate, 80:20, acetonitrile:water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005723

LC-MS/MS Analysis of 2-Hydroxyglutaric Acid detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(-), 100-1000 m/z

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Enantiomers on Astec® CHIROBIOTIC® R sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . each enantiomer, 100 μg/mL in water
Chiral 2-hydroxyglutarates (2-OHG) are important molecular signatures of Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005848
both healthy and diseased biological cells, their specific biochemical
pathways and inborn errors of metabolism. The chiral differentiation and
Intens.
quantification of D-2-OHG (R(-)-2-OHG) and L-2-OHG (S(+)-2-OHG) is key for x105 HGA TEST 003 ETOH1.D: EIC 129; 147- All MS, Smoothed (3.0,1, GA)
characterizing neurometabolic disorders like the 2-hydroxyglutaric acidurias 1
2
8
that cause neurological impairment early in life. The ability to distinguish
D
and quantify the enantiomers of 2-OHG is therefore important to be able to 6 L
track its disposition throughout the metabolic pathway of normal vs. OH
HO O
mutated cells. We show here a rapid and sensitive method to separate and 4
measure D-2-OHG and L-2-OHG using LC-MS/MS detection. An Astec® O OH
CHIROBIOTIC R (ristocetin chiral selector) column was run under polar ionic 2
mobile phase conditions. Using tandem MS eliminated two fundamental
analytical challenges with these compounds: the lack of a UV-absorbing 0
x107 LHGA TEST 001 ETOH.D: EIC 129; 147 - All MS, Smoothed (3.0,1, GA)
chromophore in the 2-OHG molecule, and the abundance of competing

1.25 1
low molecular weight acids in urine. The upper panel in the chromatogram
L
shows the separation of D-2-OHG and L-2-OHG obtained during method 1.00
development. The lower panel shows the pure L-2-hydroxyglutaric acid.
0.75
Besides providing the necessary enantioselectivity, Astec® CHIROBIOTIC
columns have the advantage of operating in aqueous and polar organic 0.50
mobile phases that are amenable to polar drugs and metabolites. Their ionic 0.25
interactions promote analyte ionization which enhances sensitivity in ESI
2
detection. In addition to the CHIROBIOTIC columns, Sigma-Aldrich also 0.00
2 4 6 8 10 12 14 16 18
provides the high purity mobile phase agents, racemic and pure chiral 2-
OHG enantiomers and other compounds in the isocitrate dehydrogenase Min
(IDH) pathway.
mobile phase . . . . . (A) ethanol:methanol (3:1, v:v); (B) 0.1% v/v TEA in water,
adjusted to pH 4.5 with acetic acid; 75:25 (A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C

LC-MS with Online Solid Phase Extraction and column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
(SPE/LC-MS) of Thyroid Hormones in Biological sample . . . . . . . . . . . . . . . . . . . protein precipitated rabbit plasma spiked with desired amount of T4, T3 and rT3
Fluids on Ascentis® Express Phenyl-Hexyl using RP- Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006223
Amide Traps
An online SPE-LC/MS method was developed for the determination of 1. 3,3',5-Triiodo- L-thyronine (T3) 1
thyroid hormones in biological fluids. The method exploits RP-Amide or C8 2. 3,3',5'-Triiodo- L-thyronine (rT3)
as the trapping column and Phenyl phase as the separation column, 3. L-Thyroxine (T4)
2
respectively. The preliminary experiments demonstrated that, under the
optimized conditions, both RP-Amide and C8 effectively trapped the thyroid
hormones extracted from spiked rabbit plasma sample which had been
protein precipitated. However, the RP-Amide traps are advantageous over
the commonly used C8 traps with higher signals and recoveries. 3
Additionally, RP-Amide traps are more flexible with washing solvent as it is
compatible with 100% aqueous mobile phases and can be used with up to
20% methanol with minimal sample loss.
The online SPE/LC-MS system comprises a trapping column, a switching
valve and an LC-MS instrument. The program consists of three distinct steps.
The valve switches between position 1 for loading/washing (Step 1) and
equilibration (Step 3), and position 2 for LC-MS analysis (Step 2). In Step 1
samples are loaded onto the trap and washed with mobile phase 4 6 8
containing low percent organic and high flow rate to remove salts and Min
other interferences which are directed to the waste. In Step 2 the analytes
are eluted from the trap, separated and detected by the LC-MS at optimum
flow for both chromatographic separation and MS signals. In Step 3, the
system returns back to valve position 1 for re-equilibration under the sample
loading/washing conditions.
mobile phase . . . . . . . . . . (A) water; (B) methanol, both with 0.1% acetic acid
gradient . . . . . . . . . . . . . . . 5% B for 3 min; to 70% B in 2 min, hold 3 min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1800 psi
detector . . MS, ESI(+), MRM mode (777.7/731.8 for T4; 651.8/605.5 for T3 or rT3)

Lot-to-Lot Reproducibility of Monoclonal Antibody

Analysis on Three Lots of Zenix® SEC-300 Columns
Exceptional lot-to-lot reproducibility is demonstrated for three lots of
3 micron Zenix SEC-300 columns for the release test of monoclonal
antibody-based therapeutics. Monomer retention time, peak efficiency and
peak height are shown to be virtually identical on the three lots.
column . . . . . . . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm, 300 Å (Z777033)
mobile phase . . . . . . . . . . . . . . . . . . . . . . 150 mM Phosphate Buffer, pH 7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 mg/ml MAb
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006210 Lot 091109-MGB
Lot 28076-1MG
Lot 28076-2MG
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

Mobile Phase Optimization in the Analysis of

Lysozyme on Zenix® SEC-300
50 mM sodium phosphate, pH 7.0
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated on figure
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.35 mL/min 100 mM sodium phosphate, pH 7.0
temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 50 mM sodium phosphate + 200 mM Na2SO4, pH 7.0
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lysozyme, 1 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005972
150 mM sodium phosphate, pH 7.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0

Min

Protein Molecular Weight Calibration Curve on 7

Zenix® Size Exclusion Columns, Effect of Pore Size Zenix SEC-150
6 Zenix SEC-300
mobile phase . . . . . . . . . . . . . . . . . . . . . . 150 mM sodium phosphate, pH 7 5
LogMW
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 3
sample . . . . . . . . . . . . . . . . . . . . . each 1 mg/mL except uracil, 2.5 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006126 2
1
4.0 6.0 8.0 10.0 12.0 14.0
Elution Volume (mL)

Protein MW Calibration Curves on SRT® SEC-100, 1. Thyroglobulin, 670 kDa 6. Myoglobin, 17.6 kDa
2. γ-Globulin, 158 kDa 7. Ribonuclease A, 13.7 kDa
-150 and -300, 30 cm x 7.8 mm I.D., 5 μm particles 3. Bovine serum albumin dimer, 132 kDa 8. Vitamin B12, 1.35 kDa
4. Bovine serum albumin, 66 kDa 9. Uracil, 120 Da
column . . . . . . . . . . . . . SRT® SEC-100, -150 and -300, 30 cm x 7.8 mm I.D., 5. Ovalbumin, 44 kDa
mobile phase . . . . . . . . . . . . . . . . 150 mM potassium phosphate, monobasic,
pH 7 (adjusted with potassium phosphate) 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004237
Log (MW)
4
Discovery BIO GFC 150

2
1
5 6 7 8 9 10 11 12 13
Elution Volume (mL)

Proteomix WCX-NP1.7, 3 cm x 4.6 mm I.D., 1.7 μm

particles
mobile phase . . . . . . . . (A) 20 mM sodium acetate, pH 5.15; (B) A + 1 M LiCl
gradient . . . . 12% to 40% B in 6.7 min; to 100% B in 0.1 min; held for 2 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1.5 mL/min) Papain digest MAb
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . 3335-3916 psi (230 - 270 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL papain digested MAb 321
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006023
Intact MAb
Papain control
0 1 2 3 4 5 6 7 8 9 10
Min

Size Exclusion Analysis of Avastin and Erbitux on

Zenix® SEC-300
The quality control analysis of recombinant monoclonal antibody
therapeutics by gel filtration chromatography is a standard release test to
determine antibody purity and the level of aggregation. Bevacizumab, sold
under the trade name Avastin, is a humanized monoclonal antibody used in
the treatment of colorectal, breat and lung cancer. Similarly, cetuximab, sold
under the trade name Erbitux, is a chimeric monoclonal antibody used in
the treatment of metastatic colorectal cancer. Both mAbs have molecular
masses of about 150 kD.
column . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm particles, 300 Å (Z777033)
mobile phase . . . . . . . . . . . . . . . . . . . . . 150 mM phosphate buffer, pH 7.0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 0 1 2 3 4 5 6 7 8 9 10 11 12 13
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL Min
sample . . (top) Bevacizumab (trade name Avastin), a humanized monoclonal antibody
(bottom) Cetuximab (trade name Erbitux), a chimeric monoclonal antibody
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006208

Stability and Reproducibility of Zenix® 300 under 2

3
4
1. Thyroglobulin
High Salt and High pH Mobile Phase Conditions 2. Bovine serum albumin (BSA)
column . . . . . . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm particles (Z777018) 3. Ribonuclease A
4. Uracil
mobile phase . . . . . . . . . . . . . 50 mM sodium phosphate + 1 M NaCl, pH 8.5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . each compound 1 mg/mL except uracil, 0.1 mg/mL 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005971 1st
30th
60th
90th
120th
150th
180th
210th
240th
270th
300th
2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

Trace HPLC Analysis of Proline Enantiomers on

(L)-Proline,
Astec® CLC-L 11.87 min.
column . . . . . . . . Astec CLC-L, 15 cm x 4.6 mm I.D., 5 μm particles (53123AST) O
H
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mM copper sulfate N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min *
OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004662
1.4% (D)-Proline,
6.29 min.

4
UHPLC Analysis of Antifungals on Ascentis® 1. Metronidazole
2. Fluconazole
Express 2.7 μm C18 3. Voriconazole
4. Ketoconazole 6
column . . . Ascentis Express C18 5 cm x 3.0 mm I.D., 2.7 μm particles (53811-U) 5. Posaconazole
6. Itraconazole
mobile phase . . . . . . . . . . . . (A) 5 mM ammonium acetate pH 6.8 unadjusted
in 80:20, water:acetonitrile; 5
(B) 5 mM ammonium acetate pH 6.8 unadjusted in 5:95, water:acetonitrile;
gradient . . . . . . . . . . . . 0% B; to 100% B in 3 min; 100% B held for 0.6 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2790 psi (192 bar) 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL 1
sample . . . . . . . . . . . . . . . . 6 antifungal 50 μg/mL in 70:30, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006256
1.0 2.0 3.0

Min

UHPLC Analysis of Antifungals on Titan™ C18 1. Metronidazole 4

2. Fluconazole
column . . . . . . . . . Titan C18, 5 cm x 3.0 mm I.D., 1.9 μm particles (577126-U) 3. Voriconazole
mobile phase . . . . . . . . . . . . (A) 5 mM ammonium acetate pH 6.8 unadjusted 4. Ketoconazole 6
5. Posaconazole
in 80:20, water:acetonitrile; 6. Itraconazole
(B) 5 mM ammonium acetate pH 6.8 unadjusted in 5:95, water:acetonitrile;
gradient . . . . . . . . . . . . 0% B; to 100% B in 3 min; 100% B held for 0.6 min 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4350 psi (300 bar) 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL 2
sample . . . . . . . . . . . . . . . . 6 antifungal 50 μg/mL in 70:30, water:methanol 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006255
1.0 2.0 3.0

Min

250000
UHPLC Analysis of Benzodiazepines using Beta- 240000
1: 463.00>287.05(+)
2: 468.50>246.00(+)
1.
2.
Oxazepam glucuronide
Oxazepam-D5 glucuronide
3: 497.30>321.15(+)
Glucuronidase Enzymatic Digestion on Titan™ C18 230000 4: 477.00>255.00(+)
5: 330.00>289.20(+)
3.
4.
Lorazepam glucuronide
Temazepam glucuronide
220000 6: 289.90>254.15(+)
5. α-hydroxyalprazolam-D5
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 210000 7: 285.90>250.00(+)
8: 291.00>138.10(+) 6. 7-Aminoclonazepam-D4
9: 283.90>135.00(+)
SPE well plate . . . . . . . . . . Supel™-Select HLB 96-Well 60 mg/well (575662-U) 200000
10: 325.00>284.05(+)
7. 7-Aminoclonazepam
190000 11: 358.90>176.00(+) 8. 7-Aminoflunitrazepam-D7
condition . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL acetonitrile, 1 mL water 12: 286.90>240.95(+) 9. 7-Aminoflunitrazepam
180000 13: 292.00>246.05(+) 12. Oxazepam
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 mL digested urine 170000
14: 281.90>236.05(+)
15: 320.90>274.95(+) 13. Oxazepam-D5
16: 315.90>315.80(+)
washing . . . . . . . . . . . . . . . . . . . . . . . . 1 mL water, 1 mL 25% methanol 160000 17: 309.00>280.90(+)
14.
15.
Nitrazepam
Lorazepam
18: 314.00>286.05(+)
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL acetonitrile 150000 19: 388.00>314.90(+)
20: 270.90>208.05(+)
17. Alprazolam
18. Alprazolam-D5
column . . . . . . . . . . . TITAN C18, 5 cm x 2.1 mm, 1.9 μm particles (577122-U) 140000 21: 300.90>254.95(+)
22: 321.00>275.05(+) 19. Flurazepam
130000 23: 314.00>268.00(+) 20. Nordiazepam
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A): 5 mM ammonium formate 120000 24: 275.90>213.00(+) 21. Temazepam
25: 289.90>198.15(+)
(B): 5 mM ammmonium formate (95:5 acetonitrile:water) 110000 26: 284.90>153.90(+) 22. Flunitrazepam-D7
27: 325.90>284.95(+) 23. Flunitrazepm
gradient . . . . . . . 10% B to 90% B in 2.5 minutes, hold 90% B for 0.5 minutes 100000 24. Nordiazepam-D5
25. Diazepam D5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 90000
26. Diazepam
80000
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 27. Midazolam
70000
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS/MS 60000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL 50000
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 ng/mL in water 40000

30000
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006291
20000
10000
0
10000-
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Min

UHPLC Analysis of Haloperidol and Metabolite on 1. Haloperidol metabolite II

2. Haloperidol
Titan™ C18
column . . . . . . . . . . . Titan C18 5 cm x 2.1 mm, 1.9 μm particles (577122-U) 2
mobile phase . . . . . . . . . . A) 10 mM potassium phosphate, monobasic pH 3.0;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
1
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4310 psi (297 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 200 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . 1–10 μg/mL in 70:30, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006282
1.0
Min

UHPLC Analysis of Vitamin K1 and Related 1. Vitamin K1 2,3 epoxide

2
Compound on Ascentis® Express 5 μm C18 2. Vitamin K1
column . . . Ascentis Express C18 15 cm x 4.6 mm I.D., 5 μm particles (50537-U)

mobile phase . . . . . . . . . . . . . . . . (A) methanol; (B) 2-propanol; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1035 psi (71 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . Vitamin K1 and related, 50 μg/mL 90:10, methanol:2-propanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006261
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

Min

2
UHPLC-MS Analysis of Anticonvulsants on 1. Gabapentin
2. Pregabalin
Ascentis® Express 2.7μm HILIC 1 3. Vigabatrin

mobile phase . . . . . . . . . . . . . . (A) 5 mM ammonium formate pH unadjusted
in 95:5, acetonitrile:water;
(B) 5 mM ammonium formate pH unadjusted
in 75:25, acetonitrile:water; (25:75, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1240 psi (85.5 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS-ESI-, full scan 100-500 m/z 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 μL
sample . . . . . . . . . . . 3 anticonvulsants 1 μg/mL in 99:1, acetonitrile:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006302
0 2 4 6 8 10
Min

UHPLC-MS Analysis of Basic Drugs and Peak MRM(M/Z)

1. Quinidine 325.2/81.2
Metabolites with Titan™ C18 2. Propranolol 260.1/116.2
3. Haloperidol 376.1/165.1
1.010
MRM transitions 4. Haloperidol metabolite II 378.1/149.1
20000
1. Quinidine 325.2/81.2 1 5. Haloperidol cholor analog 392.1/181.0
6. Fluoxetine 310.1/44.2
2. Propranolol 260.1/116.2
3. Haloperidol 376.1/165.1
4. Haloperidol metabolite II 378.1/149.1
5. Haloperidol cholor analog 392.1/181.0
6. Fluoxetine 310.1/44.2
Intensity/cps
column . . . . . . . . . . . Titan C18 10 cm x 2.1 mm, 1.9 μm particles (577124-U)
2.938
mobile phase . . . . . . . . (A) Water; (B) Acetonitrile, both with 0.1% Formic acid
10000
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35%B Isocratic 5
1.729
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
0.704
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484 psi (7120)
0.536
3
1.255
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
2.570
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), MRM 2
4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 ng/mL in 50% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006280
0
0.0 1.0 2.0 3.0
Min

UHPLC-MS Analysis of Spice Cannabinoid 1. JWH-250 5-Hydroxypentyl metabolite (352.21 m/z)

13
Metabolites from Urine Using Titan™ C18 2. JWH-250 4-Hydroxypentyl metabolite (352.21 m/z)
3. JWH-073 4-Butanoic acid metabolite (358.16 m/z)
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 4. JWH-073 4-Hydroxybutyl metabolite (344.18 m/z)
5. JWH-018 5-Pentanoic acid metabolite (372.17 m/z) 11
SPE well plate . . . . . . . . SupelSelect HLB 96-Well Plate, 60mg/well (575662-U) 6. JWH-073 3-Hydroxybutyl metabolite (344.18 m/z)
10
condition . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol, then 1 mL water 7. JWH-018 5-Hydroxypentyl metabolite (358.20 m/z)
12
8. AM2201 4-Hydroxypentyl metabolite (376.19 m/z)
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ng/mL in matrix 9. JWH-018 4-Hydroxypentyl metabolite (358.19 m/z)
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . none 10. JWH-122 5-Hydroxypentyl metabolite (372.21 m/z)
11. JWH-122 4-Hydroxypentyl metabolite (372.21 m/z)
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 20% methanol in water 12. JWH-210 5-Hydroxypentyl metabolite (386.23 m/z) 2
1
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL methanol 13. JWH-210 4-Hydroxypentyl metabolite (386.23 m/z)
eluate post-treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . none 8
column . . . . . . . . . . . Titan C18 10 cm x 2.1 mm, 1.9 μm particles (577124-U) 4 9

6
mobile phase . . . . . . . . . . . . . (A) 0.1% formic acid in 95:5, water:acetonitrile;
(B) 0.1% formic acid in 5:95, water:acetonitrile; 7
gradient . . . . . . . . . . . . . . . . . 25% B held for 1 min; to 45% B in 0.5 min; 3

45% B held for 4.5 min; to 80% B in 0.5 min; 5
80% B held for 4 min

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8756 psi (604 bar)
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
Min
detector . . . . . . . . . . . . . . . . . . . . . . . . MS-ESI+,100 - 1000 m/z scan, XIC
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ng/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006307

UHPLC-MS Analysis of Spice Cannabinoids Using 1. AM2233 (459.11 m/z)

2, 3
2. RCS-4 (322.19 m/z)
Titan™ C18 3. JWH-015 (328.18 m/z)
4. JWH-073 (328.18 m/z)
column . . . . . . . . TITAN C18, 100 mm x 2.1 mm x 1.9 μm particles (577124-U) 5. JWH-203 (340.164 m/z)
mobile phase . . . . . . . . . . . . . (A) 0.1% formic acid in 95:5, water:acetonitrile; 6. RCS-8 (376.246 m/z)
7. JWH-210 (370.23 m/z)
(B) 0.1% formic acid in 5:95, water:acetonitrile;
gradient . . 25% B held for 1 min; to 45% B in 0.5 min; 45% B held for 4.5 min;
to 80% B in 0.5 min; 80% B held for 4 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
1
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8756 psi (604 bar) 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 100 ng/mL in 60:40, water:methanol 5 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006308
0 2 4 6 8 10
Min

USP Analysis of Actos (Pioglitazone) using 1. Pioglitazone (Actos)

1
Ascentis® Express C18 5μm 2. Benzophenone
column . . . . . Ascentis® Express C18, 15 cm x 4.6 mm, 5 μm particles (50537-U)

mobile phase . . . . . . . . . . . . (A) 100 mM ammonium acetate; (B) acetonitrile;
(C) acetic acid; (55:45:2, A:B:C) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1160 psi (80 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 269 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . pioglitazone 50 μg/mL, benzophenone 13 μg/mL in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006203
0 2 4 6 8 10 12 14
Min

HPLC, UHPLC or LC-MS Petroleum
5. Toluene = 101,281 plates

Petroleum 4. Benzene = 104,463 plates D
3. D6 Benzene = 101,852 plates D D
HPLC Analysis of Benzene and Deuterated 2. Acetophenone = 120,045 plates
Benzene on Ascentis® Express C18 Pressure = 480 bar (7,000 psi) D D H
D H H
column . . . . . . . . Ascentis Express C18, 15 cm × 4.6 mm I.D., 2.7 μm particles
mobile phase . . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) water (55:45, A:B) H H
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004046
0 2 4 6 8 10 12 14 16 18
Min

HPLC, UHPLC or LC-MS Petroleum
UHPLC Analysis of Deuterated Isomers on 1. Diazepam, 2.523

Titan™ C18 2. N,N-Dimethylaniline, 2.931
3. Toluene D8, 3.349
column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U) 4. Toluene H8, 3.473
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (50:50, A:B) 5. Naphthalene D8, 4.641
1 6. Naphthalene H8, 4.847
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5650 psi (390 bar) 7. p-Xylene D10, 5.306
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2 8. p-Xylene H10, 5.565
9. Biphenyl, 7.788
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 3 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006122
8
4 6 7
0 2 4 6 8
Min

HPLC, UHPLC or LC-MS Pharmaceutical (Small Molecule)
Hexobarbital (+/-)
Pharmaceutical (Small Molecule)
Mephobarbital (+/-)
GC Analysis of Barbiturate Enantiomers on
β-DEX™ 120
column . . . . . . . . . . . . . β-DEX 120, 30 m × 0.25 mm I.D., 0.25 μm (24304)
oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 °C
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 300 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 20 cm/sec @ 275 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100:1 split
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92-0342 13 14 15 16 17 18 19
Min

GC Analysis of Methylphenidate (Ritalin)

Enantiomers (N-Trifluoroacetyl Derivatives) on
Astec® CHIRALDEX® B-DA
column . . . . Astec CHIRALDEX B-DA, 30 m x 0.25 mm I.D., 0.12 μm (72023AST)
oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 °C
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 250 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 35 psi
sample . . . . . . . . peaks 1 & 2: methylphenidate enantiomers (N-TFA derivative)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005070
0 10 20 30
Min

1
High Efficiency Resolution on Ascentis® Express Ascentis Express C18
36,800
Versus 3μm Particles
1. Naphthalene
column . . . Ascentis Express C18, 15 cm x 4.6 mm I.D., 2.7 μm particles and C18, 2. p-Xylene
Nearly
15 cm × 4.6 mm I.D., 3 μm particles (53829-U) Twice The
3. Biphenyl Efficiency
mobile phase . . . . . . . . . . (A) water; (B) acetontrile; (35:65 or 27.5:72.5, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
35,700
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003979 2
37,100
0 2 4
Min
21,700
C18, 3 μm
21,100
2
21,600
0 2 4
Min

Higher Efficiency of Ascentis® Express Compared Ascentis Express C18

1
1. Quinidine
80
to 3 μm Particles Gives Better Sensitivity C18, 3 µm
2. Fluoxetine
Sensitivity 3. Diphenhydramine
column . . . Ascentis Express C18, 5 cm x 2.1 mm I.D., 2.7 μm particles and C18, Gap
5 cm x 2.1 mm I.D., 3 μm particles (53822-U)
60
mobile phase . . . . . . . . . . . (A) 25 mM dibasic ammonium phosphate (pH 7.0): 3
1
(B) water: (C) acetonitrile; (35:0:65 or 35:4:61, A:B:C) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
mV
40
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 2 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
20
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003977
0
0.0 1.0 2.0 3.0 4.0
Min

OH
HPLC Analysis of Acebutolol Enantiomers on
H3C O
Astec® CHIROBIOTIC® T2 *
O O HN CH3
CH3
mobile phase . . (A) methanol; (B) acetic acid; (C) triethylamine; (99.8:0.1:0.1, A:B:C) H3C NH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004482
0 10 20
Min.

HPLC Analysis of Acebutolol Enantiomers on OH

Astec® CHIROBIOTIC® V2 H3C O
*
O HN C H3
mobile phase . . (A) methanol; (B) acetic acid; (C) triethylamine; (100:0.1:0.1, A:B:C) O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min C H3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C H3C NH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004457
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of ACE Inhibitors on Ascentis® 1. Captopril (1 μg/mL) (M+H)+ =218.07

2
Phenyl 2. Enalapril ( 0.1 μg/mL) (M+H)+ =377.19
3. Quinapril (1 μg/mL) (M+H)+=439.21
mobile phase . . . . . . (A) 13 mM ammonium formate (pH 2.0 with concentrated
formic acid): (B) methanol; (40:60, A:B)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . as indicated in 40:60, 13 mM ammonium formate 1
(pH 2.0 with concentrated formic acid):methanol 3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003702
0 2 4 6 8 10 12
Min

HPLC Analysis of Acetaminophen, Caffeine, 1. Maleic acid 5. Caffeine

Chlorpheniramine, Ibuprofen, Naproxen, 2. Phenylephrine 6. Chlorpheniramine
3. Acetaminophen 7. Naproxen
Phenylephrine, and Pseudoephedrine on 4. Pseudoephedrine 8. Ibuprofen
Ascentis® Express C18
This application demonstrates the suitability of Ascentis Express C18 for the 7
5
efficient separation of various over the counter drugs.
mobile phase . . . . . . . (A) 5 mM ammonium phosphate monobasic, pH 2.0 with
phosphoric acid: acetonitrile (98:2); (B) acetonitrile;
gradient . . . . . . . . . . . . . . . . 0 to 68% B in 3 min; held at 68% B for 2 min 8
1 2 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 6
4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 1.0 2.0 3.0 4.0 5.0
Min
sample . . . . . . . . . . . . . . . . . . . . . . . . 100 μg/mL in 95:5, water: methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005716

HPLC Analysis of Acidic and Neutral Drug Panel on 5 1. Hydrochlorothiazide (9)

2 2. Chlorthalidone (2)
Ascentis® Express C18 1 3. Prednisolone (2) 6
4. Pravastatin (4)
column . . Ascentis Express C18, 10 cm × 4.6 mm I.D., 2.7 μm particles (53827-U) 5. Carbamazepine (2)
6. Diclofenac (14)
mobile phase . . . . . . . . . . . . . . . . . . . (A) water with 0.1% phosphoric acid, 7. Ibuprofen (15)
(B) acetonitrile with 0.1% phosphoric acid 8. Progesterone (2)
7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.76 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient Gradient Program
Time (min) %A B%
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm 0 70 30
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL 3 4
2 60 40 8
4 5 95
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004212
0 1 2 3 4 5
Min

HPLC Analysis of Albuterol Enantiomers on Astec® HO

CHIROBIOTIC® T2 H3C
CH3
HO *
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) NH
CH3
mobile phase . . . . . . . . . . 0.1 wt% ammonium trifluoroacetic acid in methanol OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004593
0 2 4 6 8 10 12
Min.

HPLC Analysis of Alkaloids on Discovery® 1 1. Codeine

RP-AmideC16 2. Strychnine
3. Quinidine
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 4. Quinine
5. Noscapine
mobile phase . . . . . . . . . . . . . (A) methanol; (B) 25 mM potassium phosphate, 6. Papaverine
dibasic, pH 3.0; (20:80, A:B) 4
2 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000235
0 2 4 6 8 10 12
Min

HPLC Analysis of Alkaloids on Discovery® Zr-PBD 1. Codeine

column . . . . . Discovery Zr-PBD, 15 cm × 4.6 mm I.D., 5 μm particles (65723-U) 2. Strychnine
1 3. Papaverine
mobile phase . . . . . . . . . . . . . . . . (A) 20 mM potassium phosphate (pH 12): 4. Quinine
(B) acetonitrile; (90:10, A:B) 5. Quinidine
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.35 mL/min 65 °C, 12 min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 °C 4
3 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL each compound 50 μg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001840
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Alprenolol Enantiomers on CH2

CH3
(+)
Astec® Cellulose DMP *
O NH CH3
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min (-)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004984
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Alprenolol Enantiomers on CH 2

CH 3
*
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) O NH CH3
mobile phase . . . . . . . . . . . . . . . . . 15 mM ammonium formate in methanol OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004486
0 2 4 6 8 10 12
Min.

HPLC Analysis of Alprenolol Enantiomers on

CH 2
Astec® CHIROBIOTIC® T2 CH 3
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) *

NH CH 3
O
mobile phase . . . . . . . . . . 0.1 wt% ammonium trifluoroacetic acid in methanol
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004592
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Amines on Discovery® 1. Doxylamine

2. Methapyrilene
Zr-PBD at 20° C 3. Chlorpheniramine
column . . . . Discovery Zr-PBD, 7.5 cm × 4.6 mm I.D., 3 μm particles (65717-U) 4. Triprolidine
mobile phase . . . . . . . . . . (A) 50 mM Triethylammonium Hydroxide (pH 12.6): 3
(B) acetonitrile (75:25, A:B) 1
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001860
0 2 4 6 8 10 12
Min

HPLC Analysis of Amines on Discovery® Zr-PBD 1. Doxylamine

2. Methapyrilene
at 80° C 3 3. Chlorpheniramine
4. Triprolidine
column . . . . Discovery Zr-PBD, 7.5 cm × 4.6 mm I.D., 3 μm particles (65717-U)
mobile phase . . . . . . . . . . (A) 50 mM Triethylammonium Hydroxide (pH 12.6): 4
(B) acetonitrile (75:25, A:B) 1 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001861
1.0 2.0 3.0 4.0 5.0

Min

HPLC Analysis of Amines on Discovery® Zr-PS Gradient

Time (min) %A %B
column . . . . . . Discovery Zr-PS, 5 cm × 4.6 mm I.D., 3 μm particles (65740-U) 0 100 0
mobile phase . . . . . . . . . . . . . . . . . (A) 25 mM HCl, pH 1.8; (B): acetonitrile 19 8 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 1 44 0 60
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 2 1. Tripelennamine
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 2. Triprolidine
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 3. Meclizine
sample . . . . . . . . . . tripelennamine, triprolidine (1mg/mL), meclizine (3mg/mL)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001870
1.0 2.0 3.0 4.0

Min

HPLC Analysis of Aminoglutethimide Enantiomers 1. Aminoglutethimide 1

on Astec® Cellulose DMP enantiomer
2. Aminoglutethimide
Aminoglutethimide enantiomers are resolved by normal phase chroma- enantiomer
tography.
column . . ASTEC Cellulose DMP; 25 cm x 4.6 mm I.D., 5 μm particles (51099AST)
mobile phase . . (A) heptane; (B) isopropanol; (C) diethylamine; (70:30:0.1, A:B:C) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
sample . . . . . . . . . . . . aminoglutethimide, 1 g/L in 50:50, heptane:isopropanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005466
0 20 40
Min

HPLC Analysis of 1-Aminoindan Enantiomers on

LARIHC™ CF6-P
*
available from Supelco/Sigma-Aldrich as a custom item) NH2
mobile phase . . . . . . . . . . . . . . (A) methanol: (B) acetonitrile: (C) acetic acid:
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 1-Aminoindan, 0.3 mg/mL in ethanol 0 2 4 6 8 10 12
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005255

HPLC Analysis of Aminopterin and Related 1. Aminopterin

1 2 2. Folic acid
Compounds on Ascentis® Express C18
3. Methotrexate
analysis of aminopterin, folic acid and methotrexate. Structures along with
the optimized chromatogram obtained on the Ascentis Express C18 are 3
presented
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 2 4 6 8 10
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005665

HPLC Analysis of Aminopterin and Related 1. Aminopterin

Compounds on Ascentis® RP-Amide 2. Folic acid
3. Methotrexate
This application demonstrates the suitability of the Ascentis RP-Amide for 1
the analysis of aminopterin, folic acid and methotrexate. Structures along 2
with the optimized chromatogram obtained on the Ascentis RP-Amide are
presented. 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14 16

HPLC Analysis of Amlodipine Enantiomers on

Astec® CHIROBIOTIC® V2
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) O
Cl
O
mobile phase . . (A) methanol; (B) 20 mM ammonium acetate, pH 4.1; (95:5, A:B)
H C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 3
O * O CH 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm H3C N NH 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL H

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004463
0 10 20 30
Min

HPLC Analysis of 2-AMP and Piroxicam on 1. 2-Aminopyridine

Ascentis® Express F5 2. Piroxicam

2
mobile phase . . (A) 10 mM ammonium formate, pH=3; (B) acetonitrile; (75:25, A:B)
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1635 psi (113 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . 100 μg/mL Piroxicam and 10 μg/mL 2-AMP in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005606
0 2 4 6 8 10 12
Min

HPLC Analysis of Anaesthetic Drugs on Ascentis® 1 1. 4-hydroxyphencyclidine (m/z 260)

2. Phencyclidine (m/z 244)
RP-Amide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μg/mL each in mobile phase 0 2 4 6 8

1 1. Normorphine
HPLC Analysis of Analgesics, Narcotics on 2. Morphine
Ascentis® Phenyl 2 3. Codeine
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 3

mobile phase . . . . . . (A) 10 mM ammonium formate (pH 3.0 with formic acid);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003709

2 1. Acetaminophen
HPLC Analysis of Analgesics on Ascentis® C18 2. Gentisic acid
1
mobile phase . . . . . . . . . . . . (A) 20 mM phosphoric acid (pH 2.0, unadjusted);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002354 0 2 4 6 8 10 12
Min

HPLC Analysis of Analgesics on Discovery® HS F5

Gradient Program 1. p-Aminophenol (100 μg/mL)
column . . . . Discovery HS F5, 15 cm × 4.6 mm I.D., 5 μm particles (567516-U) Time (min) %A %B 2. Acetaminophen (10 μg/mL)
mobile phase . . . (A) 10 mM ammonium acetate, 0.1% formic acid; (B) methanol 0 90 10 3. Acetanilide (10 μg/mL)
3 90 10 4. Phenacetin (10 μg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
10 50 50
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 15 50 50
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL Discovery HS F5:
4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001526 Good Retention
2 3
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Analgesics on Discovery® 4

Gradient Program
RP-Amide C16 Time (min) %B
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 0 90

2 90
mobile phase . . . . . . (A) 25 mM potassium phosphate (pH 2.3); (B) acetonitrile 6 70
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 10 70
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 1. Doxylamine
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm 2. Pseudoephedrine
3. Codeine
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10μL 4. Acetaminophen
2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000785A 5. Chlorpheniramine
3
5
0 2 4 6 8 10
Min

1. Lidocaine 3
HPLC Analysis of Anesthetics, Local on Ascentis® 2. Prilocaine
Phenyl 3. Benzocaine

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003708

HPLC Analysis of Angiotensins on Ascentis® 1 1. Angiotensin II (100 µg/mL)

2. Angiotensin III (100 µg/mL)
RP-Amide 3. Angiotensin I (100 µg/mL)
mobile phase . . . . . . (A) 5 mM ammonium phosphate (pH 7.0 with ammonium
hydroxide); (B) acetonitrile; (77.5:22.5, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002914
0 2 4 6 8 10 12
Min

HPLC Analysis of Angiotensins on Discovery® BIO Peptide Sequence

1. Angiotensin II DRVYIHPF
Wide Pore C18 2. Angiotensin III RVYIHPF
column . . . . . . . . . . . . . Discovery BIO Wide Pore C18, 15 cm × 4.6 mm I.D., 3. Angiotensin I DRVYIHPFHL
5 μm particles (568222-U) 1
mobile phase . . . . . . . . . . . . . . . (A) (10 mM NH4 OAc, pH 7):(50% CH3CN in
20 mM NH4 OAc, pH 7), 65:35 ; 3
2
(B) (10 mM NH4 OAc, pH 7):(50% CH3CN in
20 mM NH4OAc, pH 7), 25:75
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 12.5 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 0 2 4 6 8 10 12
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 μL (10 μg each) in water Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001585

C18
HPLC Analysis of Antianxiety Drugs on Ascentis® 1. Oxazepam:FW = 286
2. Alprazolam: FW = 311 1X
Express C18 vs. Standard C18, Demonstrating High 3. Cloazepam: FW = 315 41.5 Min.
4. N-desmethyldiazepam: FW = 270
Throughput on Ascentis Express 5. Diazepam: FW = 284
column . . . . . . . . . . . . . . . . . C18, 25 cm × 4.6 mm I.D., 5 μm particles and

Ascentis Express C18, 10 cm × 4.6 mm I.D., 2.7 μm particles (53827-U)
0 10 20 30 40
Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
Ascentis Express C18
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004039 1X 2X 3X 4X
0 10 0 10 0 10 0 10

HPLC Analysis of Antianxiety Drugs on Ascentis® 3 1. Oxazepam

2. Alprazolam
Phenyl 2 3. Cloazepam
1 4. n-desmethyldiazepam
column . . . . . Ascentis Phenyl, 15 cm × 4.6 mm I.D., 5 μm particles (581616-U) 5. Diazepam
5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003664 4
0 2 4 6 8 10 12 14 16 18
Min


2. Nitrofurantoin
Ascentis® RP-Amide (Formate Mobile Phase) 3. Chloramphenicol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002602
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Antibacterials/Antifungals on 1 1. Norfloxacin

2. Ciprofloxacin
Ascentis® RP-Amide, Phosphate Mobile Phase 3. Lomefloxacin
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002589
0 2 4 Min 6 8 10


2 2. Nitrofurantoin
Ascentis® C18
3. Chloramphenicol
mobile phase . . . . . . . (A) 10 mM ammonium formate (pH 3.0 with formicacid);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002325
0 2 4 6 8 10
Min

HPLC Analysis of Antibiotics (Fluoroquinolones Gradient Program

1
from Tablets) on Discovery® HS C18 Time (min) %A %B
2 3
0 90 10 5
column . . . . Discovery HS C18, 15 cm × 4.6 mm I.D., 5 μm particles (568522-U) 25 65 35
4
mobile phase . . . . . . (A) 25 mM potassium phosphate (pH 3.0); (B) acetonitrile 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
1. Levofloxacin
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2. Ciprofloxacin
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 3. Lomefloxacin
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4. Sparfloxacin
5. Grepafloxacin
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001425 6. Trovafloxacin
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Antibiotics on Discovery® C18 Gradient Program

Time (min) %A %B
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 1
0 90 10
mobile phase . . . . . . (A) 25 mM potassium phosphate (pH 3.0); (B) acetonitrile 25 65 35 2 3
5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1. Levofloxacin 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 2. Ciprofloxacin
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 3. Lomefloxacin
4. Sparfloxacin
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001424 5. Grepafloxacin
6. Trovafloxacin
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Antibiotics on Ascentis® C18 1 1. Cefsulodin

2 2. Cefadroxil
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 3. Cephapirin
(pH 3.0 with phosphoric acid); (B) acetonitrile (93:7, A:B) 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002407
0 2 4 6 8 10 12
Min

1 1. Sulfanilamide
HPLC Analysis of Antibiotic Sulfa Drugs on 2. Sulfacetamide
Ascentis® C18 3. Sulfadiazine
4. Sulfathiazole
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 5. Sulfamerazine
mobile phase . . . . . . (A) water with 1% acetic acid; (B) methanol; (85:15, A:B) 2 6. Sulfamethizole
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 4
3 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002345 0 2 4 6 8 10 12 14
Min

1 1. Sulfanilamide (50 mg/mL)

HPLC Analysis of Antibiotic Sulfa Drugs on 2. Sulfacetamide (50 mg/mL)
Ascentis® RP-Amide 3. Sulfadiazine (50 mg/mL)
4. Sulfathiazole (50 mg/mL)
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 5. Sulfamerazine (50 mg/mL)
mobile phase . . . . . . . . (A) 1% acetic acid in water; (B) methanol, (85:15, A:B) 6. Sulfamethizole (50 mg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3 4 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . as indicated in 1% acetic acid in water
0 10 20

1 1. Scopolamine
HPLC Analysis of Anticholinergic Drugs on 2. Methyl scopolamine
Ascentis® C18
(pH 3.0 with phosphoric acid); (B) acetonitrile; (85:15, A:B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002351
0 2 4 6 8
Min

HPLC Analysis of Anticholinergic Drugs on 1 1. Scopolamine

2. Methyl scopolamine
Ascentis® RP-Amide
mobile phase . . . . . . . . . . . . . . . (A) 10 mM monobasic potassium phosphate 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in mobile phase 0 2 4 6 8 10 12

HPLC Analysis of Anticonvulsant Drugs on 1. Clonazepam

1 2. Clorazepate
Discovery® RP-AmideC16 3. Diazepam
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000188
0 2 4 6 8 10
Min

HPLC Analysis of Anticonvulsants on 1 1. Clonazepam

Discovery® C18 2. Clorazepate
3. Diazepam
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Anticonvulsants on 1 1. Clonazepam

Discovery® C8 2. Clorazepate
3. Diazepam
column . . . . . . . Discovery C8, 15 cm × 4.6 mm I.D., 5 μm particles (595353-U)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000748
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Antihistamines on Discovery® C8 2 1. Maleic acid

column . . . . . . . Discovery C8, 15 cm × 4.6 mm I.D., 5 μm particles (59353-U) 2. Doxylamine
3. Chlorpheniramine
mobile phase . . (A) acetonitrile; (B) 25 mM potassium phosphate, pH 2.3; (8:92, A:B) 4. Carbinoxamine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 1 5. Brompheniramine
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001278 3
4 5
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Antihistamines on Ascentis® C18 1. Maleic acid 4

2. (+/-)-chlorpheniramine
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 3. (+/-)-brompheniramine
mobile phase . . . . . . . . . . . . . . . (A) 10 mM monobasic potassium phosphate 4. Triprolidine
1
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002365
0 2 4 6 8 10 12
Min

HPLC Analysis of Antihypertensive ACE Inhibitors 1. Enalapril 2

2. Lisinopril
on Discovery® RP-AmideC16 3. Captopril
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000279
1.0 2.0 3.0 4.0 5.0
Min

HPLC Analysis of Antihypertensive and Diuretic 3 1. Atenolol

Drugs on Discovery® RP-Amide C16 2. Amiloride
2 3. Hydrochlorothiazide
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 4. Caffeine
mobile phase . . . . . . . . . . . . . (A) methanol; (B) 25 mM potassium phosphate, 4
dibasic, pH 7.0 (22:78, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000280
0 2 4 6 8 10
Min

HPLC Analysis of Antihypertensive Drugs on 1

Ascentis® RP-Amide (UV, 220 nm) 1. Hydroxymetoprolol
2. Metoprolol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002598
0 2 4 6 8
Min

HPLC Analysis of Antihypertensive Drugs on 2 1. Pindolol

Ascentis® RP-Amide (UV, 254 nm) 2. Acebutolol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002600
1.0 2.0 3.0 4.0 5.0 6.0

Min

HPLC Analysis of Antihypertensive Drugs 1. Captopril

(ß-Calcium Channel Blockers and ACE Inhibitor) on 2. Pindolol
2 3. Desmethyldiltiazem
Discovery® RP-AmideC16 4. Nifedipine
5. Diltiazem
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 1 6. Verapamil
mobile phase . . . . . . . . (A) methanol; (B) 25 mM KH2PO4 , pH 7.0 (40:60, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000112
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Anti-Inflammatory 1. 2-Aminopyridine

2. Piroxicam 2
Drugs (Aminopyridine and Piroxicam) on
Discovery® HS F5
mobile phase . . . . . . . . . . . . . . . . (A) 10 mM potassium phosphate (pH 2.5); 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001657 0 2 4 6
Min

HPLC Analysis of Anti-Inflammatory Drugs on 1. Beclomethasone (50 µg/mL)

Ascentis® C18 1 2. Etodolac (50 µg/mL)
3. Celecoxib (50 µg/mL)
(pH 3.0 with formic acid); (B) acetonitrile; (45:55, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in mobile phase 0 2 4 6 8 10 12 14

HPLC Analysis of Anti-Inflammatory Drugs on 2 1. Piroxicam

2. Sulindac
Discovery® RP-Amide C16 3. Tolmetin
4. Ketoprofen
column . . Discovery RP-Amide C16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 1 5. Naprosyn
mobile phase . . . . . . . (A) acetonitrile; (B) 25 mM KH2PO4 , pH 3.0; (40:60, A:B) 5 6. Diflunisal
6 7. Indomethacin
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 4
8. Ibuprofen
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 9. Diclofenac
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 7 10. Mefanamic acid
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL, 1 μg/mL each analyte
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000061 9
8
10
0 10 20
Min

HPLC Analysis of Anti-Inflammatory Drugs on 1. Piroxicam 1

Ascentis® C8 2. Indomethacin 2

(pH 3.0 with phosphoric acid); (B) acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002391
0 2 4 6 8 10
Min

HPLC Analysis of Anti-Inflammatory Drugs on 1. Piroxicam

2. Indomethacin 1
Ascentis® RP-Amide
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μg/mL each in mobile phase 0 2 4 6 8 10

HPLC Analysis of Anti-Inflammatory Drugs on 1. 2-Aminopyridine

2. Piroxicam 2
Discovery® HS F5
mobile phase . . . . . . . . . . . . . . . . (A) 10 mM potassium phosphate (pH 6.8);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001658
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Antineoplastic Drugs on 1. Methotrexate

1
Ascentis® C18
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in mobile phase 1.0 2.0 3.0 4.0 5.0 6.0

1 1. Methotrexate
HPLC Analysis of Antineoplastic Drugs on
Ascentis® RP-Amide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002396
0 2 4 6 8 10
Min

1. R-(-)-deprenyl hydrochloride 1
HPLC Analysis of Antiparkinsonian Drugs on
Ascentis® C18
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002398
1.0 2.0 3.0 4.0 5.0 6.0
Min

2 1. Dimetridazole
HPLC Analysis of Antiprotozoal Drugs on Ascentis® 2. Furaltadone
Phenyl 3. Furazolidone
4. Sulfanitran
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 4 5. Unknown
mobile phase . . . . . . . . . . . . . . . (A) 10 mM ammonium acetate (pH 5.0 with 3
acetic acid); (B) acetonitrile; (60:40, A:B) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in 85:15, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003703 0 2 4 6 8 10
Min

HPLC Analysis of Antipsychotic Drugs on Ascentis® 1. Desmethyl venlafaxine

Express RP-Amide 2. Hydroxybupropion
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 10 cm x 2.1 mm I.D., 4. Bupropion
2.7 μm particles (53913-U) 5. Zuclopehthixol
mobile phase . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate (in water); 6. Fluphenazine
(B) 5 mM ammonium formate (in 95:5 methanol:water) 6
1 3 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 μg/L in water 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005373
0 2 4 6 8
Min

1 1. Clozapine
HPLC Analysis of Antipsychotic Drugs on 2. Thioridazine
Ascentis® C18
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002400
0 2 4 6 8 10 12
Min

HPLC Analysis of Antipsychotic Drugs on Ascentis® 1 1. Clozapine

2. Thioridazine
RP-Amide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in mobile phase 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002603
0 2 4 6 8
Min

HPLC Analysis of Antipyretics/Analgesics/ 4 1. Paracetamol

2. Aspirin (acetylsalicylic acid)
Antifungals on Discovery® RP-AmideC16 3. Sorbic acid
1 4. Benzoic acid
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 2 5. Salicylic acid
3
mobile phase . . . . . . . . . . . . . . . . . . . 0.1% TFA in (25:75) acetonitrile:water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000088 5
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Antiretroviral Drugs on Ascentis® Gradient Program 1. Lamivudine (50 µg/mL)
Time (min) %A %B 2. Stavudine (50 µg/mL)
RP-Amide 0 95 5 3. Zidovudine (50 µg/mL)
10 15 85 4. Nevirapine (35 µg/mL)
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 5. Saquinavir (100 µg/mL)
12 15 85 4
mobile phase . . . . . . (A) 25 mM ammonium phosphate (pH 5.5); (B) acetonitrile 12.5 95 5 6. Ritonavir (100 µg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 7. Lopinavir (100 µg/mL)
1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 5
2 7
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 3 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . as indicated in 25 mM ammonium phosphate (pH 5.5)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002988
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Antitussive/Antihistamine/ 1. Acetaminophen Gradient Program

Antipyretic Mix on Discovery® RP-Amide C16 2. Doxylamine Time (min) %A %B
3. Pseudoephedrine
0 90 10
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 4. Codeine 1 2 90 10
5. Chlorpheniramine
mobile phase . . . . . . (A) 25 mM potassium phosphate (pH 2.3); (B) acetonitrile 4 70 30
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 8 70 30
10 50 50
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL, each compound 1 μg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000787
3
4
5
0 2 4 6 8 10
Min

HPLC Analysis of Antiviral Drugs on Ascentis® C8 1. Valacyclovir (50 µg/mL) Gradient Program 2
2. Imiquimod (50 µg/mL) Time (min) %A %B
mobile phase . . . . . . . . . . . . . (A) 10 mM ammonium phosphate (pH 2.5 with 10 20 80
phosphoric acid); (B) acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4
Min
6 8 10
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003201

HPLC Analysis of Anxiolytic Drugs on Ascentis® 1
Phenyl 1. Trazadone
2. Buspirone
column . . . . . Ascentis Phenyl, 15 cm × 4.6 mm I.D., 5 μm particles (581616-U)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
0 3 6 9 12 15

HPLC Analysis of Anxiolytic Drugs on Nordoxepin is notoriously 3 1. Nordoxepin

Discovery® C18 difficult to chromatograph, 2. Oxazepam
and the slight tailing is a 3. Chlordiazepoxide
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) good peak shape for this 4. Desmethyldiazepam
mobile phase . . . . . . . . (A) acetonitrile; (B) 25mM KH2PO4 , pH 7.0 (30:70, A:B) drug. 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797-0662
0 10
Min

HPLC Analysis of Anxiolytic Drugs on 1

1. Demoxepam
SUPELCOSIL™ LC-8 2. Nitrazepam (int. std.)
3. N-desmethyl Chlordiazepoxide
column . . . . . SUPELCOSIL LC-8, 15 cm × 4.6 mm I.D., 5 μm particles (58220-U) 4. Oxazepam
mobile phase . . methanol:acetonitrile:0.005 M KH2PO4 and 0.1 M ammonium acetate 3 5. Chlordiazepoxide
buffer (pH 6.0 with glacial acetic acid),26.5:16.5:57 (v/v/v) 6. Temazepam
7. N-desmethyl Diazepam
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 8. Diazepam
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 245 nm 5 7
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 2 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1390
6
8
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Anxiolytic Drugs on 1 1. Chlordiazepoxide
Ascentis® C18
mobile phase . . . . . . . (A) 10 mM monobasic potassium phosphate (pH 7.0 with
potassium hydroxide): (B) acetonitrile; (50:50, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002403
1.0 2.0 3.0 4.0 5.0 6.0
Min

1 1. Trazodone
HPLC Analysis of Anxiolytic Drugs on Ascentis® 2. Buspirone
Phenyl
mobile phase . . . . . . . (A) 10 mM ammonium acetate (pH 5.5 with acetic acid):
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003704

1 1. Chlordiazepoxide
HPLC Analysis of Anxiolytic Drugs on Ascentis®
RP-Amide
potassium hydroxide); (B) acetonitrile; (50:50, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL

HPLC Analysis of Arotinolol Enantiomers on Astec®

H2N
CHIROBIOTIC® T
mobile phase . . . . . . . . . . . . . . (A) acetonitrile; (B) methanol; (C) acetic acid; S
N H3C
(D) triethylamine; (55:45:0.3:0.2, A:B:C:D) CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min *
S S NH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C CH3
OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004539
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Aspartic Acid Enantiomers on O
Astec® CHIROBIOTIC® T, Ethanol Mobile Phase

HO *
mobile phase . . . . . . (A) water; (B) ethanol; (C) formic acid; (20:80:0.02, A:B:C)
O NH 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004489
0 2 4 6 8
Min.

HPLC Analysis of Aspirin and Salicylic Acid on 1. Aspirin

Ascentis® Express RP-Amide 2. Salicylic acid
1
Salicylic acid is the primary metabolite of aspirin, and is likely responsible for 2
its antiinflammatory properties via suppression of cyclooxygenase genes; it
also is thought to suppress colonic carcinogenesis. Ascentis Express RP-
Amide provides good retention and resolution at low pH by a combination
of hydrophobic and H-bonding intereactions.
mobile phase . . . . . . . . . . . . . (A) water with 0.1% TFA; (B) acetonitrile with
0.1% TFA; (65:35, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 0 2 4 6 8
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2200 psi (152 bar)
Min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 75:25, water:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005797

HPLC Analysis of Atenolol Enantiomers on Astec® NH2

CH3
CHIROBIOTIC® T
* O
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H3C NH O

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004490
0 10 20
Min.

HPLC Analysis of Atropine Enantiomers on Astec®

Cellulose DMP N
CH 3
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) (+) (-)
O O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004985
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Barbiturates on Ascentis® C8 1 2 1. Barbital (M-H)-= 183.08

2. Phenobarbital (M-H)-= 231.08
column . . . . . . . . Ascentis C8, 15 cm x 4.6 mm I.D., 5 μm particles (581424-U) 3. Aprobarbital (M-H)-= 209.10
4. Butabarbital (M-H)-= 211.11
5. Mephobarbital (M-H)-= 245.10
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 6. Pentobarbital (M-H)-= 225.13
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3 7. Secobarbital (M-H)-= 237.13
4 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 6
7
sample . . . . . . . . . . . . . . . . . . . . . 10 μg/mL each in 50:50 water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003307
0 2 4 6 8 10 12
Min

HPLC Analysis of Barbiturates on Discovery® C18 1. Methylprylon (2 μg/mL)

2. Butabarbital (50 μg/mL)
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 3. Butalbital (50 μg/mL)
mobile phase . . . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) 25 mM potassium 4. Hexobarbital (50 μg/mL)
phosphate buffer, pH 6.9 (25:75, A:B) 5. Mephobarbital (25 μg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 3
1 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0141
5
0 2 4 6 8 10
Min

HPLC Analysis of Barbiturates on Discovery® C18, 2

1. Barbital
Unbuffered Mobile Phase 2. Aprobarbital
3. Phenobarbital
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 4. Butabarbital
mobile phase . . . . . . . . . . . . . . . . . . . (A) methanol; (B) water (45:55, A:B) 5. Mephobarbital
1 6. Pentobarbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 7. Secobarbital
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000190 5
6 7
0 10 20
Min

HPLC Analysis of Barbiturates on Discovery® C8 2

1. Barbital
2. Aprobarbital
column . . . . . . . Discovery C8, 15 cm × 4.6 mm I.D., 5 μm particles (59353-U) 3. Phenobarbital
mobile phase . . . . . . . . . . . . . . . . . . . (A) methanol; (B) water (45:55, A:B) 4. Butabarbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 5. Mephobarbital
1 6. Pentobarbital
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 7. Secobarbital
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000791 4
5
6 7
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Barbiturates on Discovery® 3

1 1. Barbital
RP-AmideC16 2. Phenobarbital
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 3. Aprobarbital
4. Butabarbital
mobile phase . . . . . . . . (A) methanol:acetonitrile, 60:40; (B) water; (25:75, A:B) 5. Mephobarbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 6. Pentobarbital
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 7. Secobarbital
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000193
4
6 7
0 10 20
Min

HPLC Analysis of Barbiturates on Ascentis® C18 1 1. Barbital (50 µg/mL)

2. Phenobarbital (50 µg/mL)
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 2 3. Aprobarbital (50 µg/mL)
4. Butabarbital (50 µg/mL)
5. Mephobarbital (50 µg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 3 6. Pentobarbital (50 µg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 7. Secobarbital (50 µg/mL)
4 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 6
sample . . . . . . . . . . . . . . . . . . . . . . . as indicated in 65:35 water:methanol 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002877
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Basic Drug Panel on Ascentis® 3

1. Quinidine (5)
2. Dipyridamole (29)
Express C18 3. Propranolol (12)
4. Haloperidol (8)
column . . Ascentis Express C18, 10 cm × 4.6 mm I.D., 2.7 μm particles (53827-U) 5. Amlodipine (29)
mobile phase . . . . . . (A) water with 0.05M potassium phosphate and 0.1% TEA 6. Fluoxetine (3)
and 0.6% OSA-Na at pH = 2.9, (B) acetonitrile 1
Gradient Program
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.76 mL/min Time (min) %A B%
0 63 37
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 2 5 59 41
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004213 6
0 1 2 3 4 5 6 7
Min

HPLC Analysis of Basic Peptides (Angiotensin 1. IVYIHPR

2. KVYIHPI
Analogs) on Ascentis® Express Peptide ES-C18 3. RVYIHPI
4 4. ac- KVYIHPI
column . . . . . . . . . . . . . Ascentis Express Peptide ES-C18, 10 cm x 3 mm I.D., 1 3 5 5. ac- RVYIHPI
6. ac- IVYIHPR
mobile phase . . . . . (A) 0.1% (v/v) formic acid, pH 4.0 (titrated with ammonium
hydroxide); (B) 50:50, (0.175% formic acid in
water, pH 4.0) : acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3002 psi (207 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL 0 2 4 6 8 10 12 14 16 18
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g/L ea. peptide Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005379

HPLC Analysis of Bendroflumethiazide O

NH 2
O O
S
Enantiomers on Astec® CHIROBIOTIC® T S
NH
O
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) F
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (70:30, A:B) N *
F H
F
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004491
0 2 4 6 8 10 12
Min.

HPLC Analysis of Benoxaprofen Enantiomers on

CH3
HO N
Cl
mobile phase . . . . . . . (A) 0.1 wt% triethylamine, pH 7.0; (B) THF; (90:10, A:B) O
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004637
0 2 4 6 8 10 12 14 16 18
Min

3 1. Nitrazepam (M+H)+= 282.08

HPLC Analysis of Benzodiazepines on Ascentis® C8 2. Clonazepam (M+H)+= 316.04
4 3. Desmethyldiazepam (M+H)+= 271.05
column . . . . . . . . Ascentis C8, 15 cm x 2.1 mm I.D., 3 μm particles (581402-U) 1 4. Diazepam (M+H)+= 285.07
mobile phase . . . . . . . . . (A) 10 mM ammonium acetate (pH 6.98 unadjusted);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
2
detector . . . . . . . . . . . . . . . . . . . MS, ESI (+), Selected Ion Recording Mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . 1 μg/mL each in 10 mM ammonium acetate (pH 6.98 unadjusted)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003309
0 2 4 6 8 10 12 14 16
Min


Express C18, Acetonitrile Mobile Phase 2. Clonazepam
3. Alprazolam
column . . . Ascentis Express C18, 10 cm x 3 mm I.D., 2.7 μm particles (53814-U) 1 4. N-Desmethyldiazepam
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (66:34, A:B) 5. Diazepam
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
2 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 5
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g/L each in 20% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005673
0 2 4 6 8
Min

HPLC Analysis of Benzodiazepines on Ascentis® 1. Clonazepam

2. Oxazepam
Express Phenyl-Hexyl, Methanol Mobile Phase 3. Alprazolam
The Phenyl-Hexyl phase of the Ascentis Express product line displays the 4. N-Desmethyldiazepam
best selectivity of this sample; selectivity is markedly affected by choice of 5. Diazepam
organic modifier. The application also demonstrates feasibility of fast LC with 2
Ascentis Express by virtue of low backpressure and high efficiency. 1 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3600 psi (248 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 1.0 2.0 3.0
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g/L in 20% methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005674


Express Phenyl-Hexyl with Acetonitrile Mobile 2. Alprazolam
3. Clonazepam
Phase 4. N-Desmethyldiazepam
The Phenyl-Hexyl phase of the Ascentis Express product line displays the 5. Diazepam
best selectivity of this sample; selectivity is markedly affected by choice of 1
organic modifier. The application also demonstrates feasibility of fast LC with
Ascentis Express by virtue of low backpressure and high efficiency. 4
column . . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 5 cm x 4.6 mm I.D., 2
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2600 psi (179 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm 1.0 2.0 3.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL Min
sample . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g/mL of each in 20% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005675

HPLC Analysis of Benzoin Enantiomers on Astec® O

Cellulose DMP *

OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C (+)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm (-)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004986
0 10 20
Min

HPLC Analysis of Benzoin Enantiomers on Astec®

O
(R,R) P-CAP™-DP
column . . Astec (R,R) P-CAP-DP, 25 cm x 4.6 mm I.D., 5 μm particles (35024AST) *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004726
0 2 4 6 8
Min

HPLC Analysis of Beta Blockers on Ascentis® 2
Express C18 1. Atenolol

2. Pindolol
column . . Ascentis Express C18, 5 cm × 2.1 mm I.D., 2.7 μm particles (53822-U) 3. Timolol
mobile phase . . (A) 0.1% acetic acid in water; (B) 0.1% acetic acid in acetonitrile 4. Metoprolol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . ABI 3200 QT; ESI(+), MS/MS
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agilent 1100 3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004063
4
1
0.2 0.4 0.6 0.8 1.0 1.2 1.4

Min

HPLC Analysis of Beta Blockers on Ascentis® Si 3 1. Propranolol (50 µg/ml)

2. Pindolol (50 µg/ml)
column . . . . . . . . Ascentis Si, 15 cm x 4.6 mm I.D., 5 μm particles (581512-U) 1 3. Acebutolol (50 µg/ml)
mobile phase . . . . . . . . (A) 0.1% ammonium acetate in water (pH unadjusted); 4. Atenolol (50 µg/ml)
2
(B) 0.1% ammonium acetate in acetontrile; (15:85, A:B)
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . as indicated in 0.1% ammonium acetate in 10:90:water:acetonitrile
0 2 4 6 8 10 12 14

3 1. Propranolol (50 µg/ml)

HPLC Analysis of Beta Blockers on Ascentis® 2. Pindolol (50 µg/ml)
Si (Silica) 1 3. Acebutolol (50 µg/ml)
4. Atenolol (50 µg/ml)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003735

HPLC Analysis of Bovine Serum Albumin (BSA) on

column . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm, 300 Å particles (Z777051)
column . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm, 300 Å particles (Z777033) SRT SEC-300

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 6 7 8 9 10 11
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg/mL, BSA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006119 SRT SEC-300
Zenix SEC-300
0 1 2 3 4 5 6 7 8 9 10 11
Min

HPLC Analysis of Bromacil Enantiomers on Astec® H

CHIROBIOTIC® T H3C N O
C H3
N *
mobile phase . . . . . . . . . . . . . . . . . . . . (A) water; (B) ethanol; (60:40, A:B) Br
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
O CH3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004492
0 2 4 6 8 10
Min.


Concentration
0.5 96.4 ±8.5
Discovery® DSC-18 1.0 96.1 ±5.0
0.5 97.8 ±8.8
sample preparation . . 1 mL methanol; evaporate to dryness with nitrogen stream at 0.5 95.6 ±6.7
1.0 97.6 ±5.8
room temperature; reconstitute in 200 μL mobile phase
containing 0.2 μg/mL or 7-methylxanthine (IS)
sample preparation . . . . . . . . . . . . . . . . . . . . . 2 mL 5% methanol in water; 1. Theobromine
dry tube 10 min with nitrogen stream 2. Paraxanthine
column . . . . Discovery RP-AmideC16, 15 cm × 4.6 mm, 5 μm particles, preceded 3. Theophylline
by a 2 cm RP-AmideC16 guard column and 0.5 μm frit filter (505013) 4. Caffeine
mobile phase . . . . . . . . . . . . . . (A) Methanol; (B) 1% acetic acid (17:38, A:B) IS 7-Methylxanthine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000594
3
IS 2
4
Spiked Serum
(0.50 mg/mL)
0 2 4 6 8 10 12
Min

HPLC Analysis of Bupivacaine Enantiomers on

Astec® CHIROBIOTIC® V2, Acetate Mobile Phase C H3
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) N *

NH
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min H3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm C H3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004464
0 2 4 6 8 10 12 14
Min


C H3
Astec® CHIROBIOTIC® V2, Formate Mobile Phase
NH
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) N *
mobile phase . . (A) 20 mM ammonium formate, pH 4.1; (B) methanol; (5:95, A:B)
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
H 3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL C H3

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004619
0 2 4 6 8 10 12 14 16 18
Min


CH3
NH
mobile phase . . . . . . . . (A) 1% triethylamine, pH 4.1; (B) methanol; (5:95, A:B)

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C H 3C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 202 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL CH3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004623
0 2 4 6 8
Min

HPLC Analysis of Butorphanol Enantiomers on OH
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
N *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004574
0 2 4 6 8
Min.

HPLC Analysis of Butorphanol Enantiomers on OH
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) *
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
N *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004591
0 2 4 6 8
Min.

HPLC Analysis of Calcium Channel Blockers on 2 1. (+/-)verapamil

2. Methoxyverapamil
Ascentis® C18 1

(pH 3.0 with phosphoric acid); (B) acetonitrile; ( 60:40, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002437
0 2 4 6 8 10
Min

1. (+/-)verapamil 2
HPLC Analysis of Calcium Channel Blockers on 2. Methoxyverapamil 1
Ascentis® RP-Amide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL

HPLC Analysis of Cardiac Drugs on Ascentis® C8 1. Maleic acid (50 µg/mL) 4. Timolol (50 µg/mL) 6
2. Acebutolol (50 µg/mL) 5. Oxprenolol (50 µg/mL)
6. Propranolol (50 µg/mL)
column . . . . . . . . Ascentis C8, 15 cm x 2.1 mm I.D., 3 μm particles (581402-U) 3. Metoprolol (50 µg/mL)
mobile phase . . . . . . . . . . . . . . . . . 0.1% ammonium acetate:acetonitrile with

0.1% ammonium acetate, (47:53) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 1 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003172 0 2 4 6 8 10
Min

HPLC Analysis of Catecholamines on

1. Norepinephrine
Discovery® C18 1
2. Epinephrine
3. 3,4-Dihydroxybenzylamine
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 2
4. Dopamine
mobile phase . . (A) acetonitrile: (B) 50 mM KH2PO4 , pH 3.0 with phosphoric acid,
100 mg/L EDTA, 200 mg/L 1-octane-sulfonic acid (5:95, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797-0659
0 10
Min

HPLC Analysis of Cephalsporin Antibiotics on 1. Cefadroxil 1

2. Cefsulodin 2 3
Ascentis® RP-Amide 3. Cephapirin
(pH 3.0 with phosphoric acid): (B) acetonitrile; (93:7, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in mobile phase 0 2 4 6 8

1. Chlorhexidine
HPLC Analysis of Chlorhexidine and Robenidine Gradient Program 1
2. Robenidine
Time (min) %A %B %C
on Ascentis® Phenyl 0 65 25 10 2
7 0 90 10
column . . . . . . Ascentis Phenyl 15 cm x 4.6 mm I.D., 5 μm particles (581616-U)
9 0 90 10
mobile phase . . . . . . . . . . . (A) water; (B) acetonitrile; (C) 500 mM ammonium 9.5 65 25 10
formate, pH 3.0, with formic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 273 nm 0 2 4 6 8 10
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . 100 ug/mL each in 70:30, 25 mM ammonium formate pH 3.0:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003711

HPLC Analysis of Chloroquine Enantiomers on

Cl N
mobile phase . . . (A) methanol; (B) acetic acid; (C) triethylamine; (98:1:1, A:B:C) HN *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min N CH3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C C H3
H3C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004620
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Chlorpheniramine Enantiomers

on Astec® CYCLOBOND® I 2000
N
mobile phase . . (A) 1% triethylamine acetate, pH 4.1; (B) acetonitrile; (90:10, A:B)
* N
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm CH3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL Cl
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004688
0 10 20
Min

HPLC Analysis of Chlorthalidone Enantiomers on O
NH O O
S
mobile phase . . (A) 10 mM ammonium acetate, pH 4.1; (B) methanol; (80:20, A:B) NH 2
HO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C Cl
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004584
0 2 4 6 8 10 12 14 16
Min.


Astec® CYCLOBOND® I 2000 HP-RSP
column . . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D., NH O O
5 μm particles (24024AST) *
S
mobile phase . . (A) 10 mM ammonium acetate, pH 4.0; (B) acetonitrile; (85:15, A:B) NH2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min HO
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
Cl
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004712
0 2 4 6 8 10
Min


column . . CYCLOBOND I 2000 RSP, 25 cm x 4.6 mm I.D., 5 μm particles (20324AST) O O
NH
mobile phase . . (A) 1% triethylamine acetate, pH 4.1; (B) acetonitrile; (95:5, A:B) *
S
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C HO
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL Cl

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004704
0 10 20
Min

HPLC Analysis of Chlorzoxazone on 1

2 1. 6-Hydroxychlorzoxazone
Discovery® HS-PEG 2. Chlorzoxazone
column . . . . Discovery HS PEG, 15 cm × 4.6 mm I.D., 5 μm particles (567416-U)

mobile phase . . (A) 20 mM acetic acid in water (pH 4.5 with ammonium hydroxide):
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 285 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001674
0 10 20
Min

HPLC Analysis of Cholesterol-Lowering Drugs 1 1. Pravastatin

2. Fluvastatin
(Statins) on Discovery® Cyano 3. Atorvastatin
column . . . . . . Discovery Cyano, 15 cm × 4.6mm I.D., 5 μm particles (59356-U) 2 4. Lovastatin
5. Simvastatin
mobile phase . . . . . . . . . . . . . . . . . 60:40 water (10 mM ammonium acetate,
pH 4.0 with acetic acid):acetonitrile 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in 70:30 water:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002552
0 2 4 6 8 10
Min

HPLC Analysis of Ciprofibrate Enantiomers on O

H3C
CH 3
(D) triethylamine; (90:10:0.3:0.2, A:B:C:D) *
Cl
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004689
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Citalopram Enantiomers on N

column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) O
mobile phase . . (A) methanol; (B) acetic acid; (C) triethylamine; (99.8:0.1:0.1, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min F
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 213 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL H3C HBr
N
sample . . . . . . . . . . . . . . . . . . . . . . . citalopram, 1 mg/mL in mobile phase
0 10 20 30
Min


Astec® CHIROBIOTIC® T Cl
*
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) CH3
mobile phase . . . . . . . . . . . . . . . . . 15 mM ammonium formate in methanol HN
H2N
Cl H3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004493
0 2 4 6 8 10 12
Min.


Cl
mobile phase . . . . . . . . . . . . . (A) methanol; (B) acetic acid; (C) triethylamine; CH 3

HN
(99.98:0.01:0.01, A:B:C) H2 N
Cl H 3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004621
0 10 20
Min


Astec® CHIROBIOTIC® T2 Cl
*
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) CH 3
H2N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min CH 3
Cl H3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004596
0 2 4 6 8 10
Min.

HPLC Analysis of Clopidogrel Enatiomers on CH3

O O
Astec® CYCLOBOND® I 2000 DNP
column . . CYCLOBOND® I 2000 DNP, 25 cm x 4.6 mm I.D., 5 μm particles (25024AST) N
mobile phase . . (A) 20 mM ammonium acetate, pH 4.0; (B) acetonitrile; (80:20, A:B) S Cl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1100 psi (76 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004774
0 10 20
Min

HPLC Analysis of Cold Remedy Ingredients on 1. Doxylamine (100 µg/mL) Gradient Program
2. Acetaminophen (75 µg/mL) Time (min) %A %B
Ascentis® RP-Amide 3. Chlorpheniramine (125 µg/mL) 0 90 10
4. Dextromethorphan (150 µg/mL) 15 70 30
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 1 17 70 30
mobile phase . . . . . . (A) 5 mM ammonium formate (pH 2.75 with formic acid); 17.1 90 10
(B) acetonitrile
2 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
sample . . . as indicated in 5 mM ammonium formate (pH 2.75 with formic acid)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003006
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Coumachlor Enantiomers on O
OH CH3
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
O O Cl
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004638
0 10 20
Min

HPLC Analysis of Cyclophosphamide Enantiomers O

O
P Cl
on Astec® CHIROBIOTIC® T * N
N
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H
mobile phase . . . . . . . . . . . . . (A) water; (B) ethanol; (C) THF; (90:5:5, A:B:C)
Cl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004494
0 2 4 6 8 10 12
Min.

HPLC Analysis of Cylert (Pemoline) and Ritalin 1 1. Cylert

2. Ritalin
(Methylphenidate) on Discovery® C18
mobile phase . . . . . . . . . . . . . . . . . 80:20 water 10 mM ammonium acetate,
pH 3.0 with formic acid):acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 2
sample . . . . . . Cylert (50 μg/mL) and Ritalin (100 μg/mL) in 85:15 water:CH3OH
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002558
0 2 4 6 8 10
M in

HPLC Analysis of Deprenyl (Selegiline)

* CH3
Enantiomers on Astec® CYCLOBOND® I 2000 DMP
CH
column . . CYCLOBOND I 2000 DMP, 25 cm x 4.6 mm I.D., 5 μm particles (20724AST) N
mobile phase . . (A) 20 mM ammonium acetate, pH 4.0; (B) methanol; (65:35, A:B) H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004681
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of N-Desmethylnefopam

Enantiomers on Astec® CHIROBIOTIC® V2
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . (A) methanol; (B) acetic acid; O
*
(C) ammonium hydroxide; (100:0.1:0.02, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm CH3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004655
0 10 20
Min

HPLC Analysis of Dextromethorphan Enantiomers CH3
on Astec® CHIROBIOTIC® V2 O

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min *
N CH3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004468
0 10 20
Min

HPLC Analysis of Diclofenac and Salicylic Acid on 1 1. Salicylic acid (50 mg/mL)
2. Diclofenac (50 mg/mL)
Ascentis® RP-Amide
2
mobile phase . . . . . . . . . . . . (A) 20 mM phosphoric acid (pH 2.0 unadjusted);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0 2 4 6 8 10 12 14 16 18
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002617

HPLC Analysis of Diclofenac and Salicylic Acid on 1 1. Salicylic acid

2. Diclofenac
Ascentis® C18
mobile phase . . . . . . . . . . . . (A) 20 mM phosphoric acid (pH 2.0 unadjusted);
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002424
0 2 4 6 8 10 12 14
Min

HPLC Analysis of 3,4-Dihydroxyphenylalanine OH

(DOPA) Enantiomers on Astec® CHIROBIOTIC® T
*
O
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (25:75, A:B) NH2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004586
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of 3,4-Dihydroxyphenylalanine

HO O OH
(DOPA) Enantiomers on Astec® CHIROBIOTIC® TAG
HO * NH2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004608
0 2 4 6 8 10 12
Min.

HPLC Analysis of 2,3-Dihydroxy-3-Phenylpropionic HO

H HO
H
HO HO
H
H
Acid Enantiomers on CHIROBIOTIC R OH OH
B
O O
mobile phase . . . . . . . . . . . . . . (A) 0.1% ammonia, pH 4.1 with formic acid; HO H H H
H OH HO OH
(B) methanol; (50:50, A:B) OH OH
B
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 258 nm A
racemate A racemate B
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . 2,3-dihydroxy-3-phenyl-propionic acid isomers: A
(A) racemate A (4.85 and 6.95 min)
(B) racemate B (5.33 and 6.29 min)
Figure courtesy DSM Fine Chemicals
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004409

HPLC Analysis of Diperodon Enantiomers on O NH

Astec® Cellulose DMP, LC/MS Conditions O
O
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) NH O *
mobile phase . . . . . . . . . (A) methanol; (B) ammonium formate; (100:0.1, A:B) N
H2O
(Note: 0.1 w/v ammonium formate, 0.1 g/100 mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004987 0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Diperodon Enantiomers on O NH

Astec® Cellulose DMP (no additives) O
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) NH O *

O
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile; (100, A)
N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min H2O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005004
0 20 40 60
Min

HPLC Analysis of Diuretic Drugs on Ascentis® C18 1 1. Furosemide

2. Indapamide
(pH 3.0 with phosphoric acid); (B) acetonitrile; (A:B, 40:60)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 234 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002423
0 2 4 6 8
Min

HPLC Analysis of Diuretic Drugs on Ascentis® 1 1. Furosemide

2. Indapamide
RP-Amide
(pH 3.0 with phosphoric acid); (B) acetronitrile; (60:40, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 234 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL

HPLC Analysis of DMB-Labeled Sialic Acids on 1 1. DMB-NGNA

Ascentis® Express RP-Amide: Comparison of 2. DMB-NANA
Biosimilars to Reference Materials

2
Sialic acids affect the bioavailability, function, stability, and metabolism of
glycoproteins. Two forms of sialic acid are commonly present in therapeutic
glycoproteins: N-acetylneuraminic acid (NANA) and N-glycolylneuraminic Reaction
acid (NGNA). One of the most common quantification methods involves by-products
releasing sialic acids from the glycoprotein, derivatizing NANA and NGNA
with 1,2-diamino-4, 5-methylenedioxybenzene (DMB), and analyzing by C18-
HPLC with fluorescence detection. This procedure is subject to interference
from peaks originating from excess reagent and other derivatized impurities, Unknown
limiting sensitivity and reproducibility. The objectives of this study were to
develop a significantly improved HPLC-fluorescence method for DMB-NANA
and DMB-NGNA, and to apply this method to compare two candidate 0 2 4 6 8 10 12 14
biosimilar therapeutic proteins to their respective reference materials. Min
column . . . . . . . . . . . . . . . Ascentis® Express RP-Amide, 10 cm x 2.1 mm I,D.,
mobile phase . . . . . . . (A) Water, 0.1% formic acid, (B) ACN, 0.1% formic acid
gradient . . . . . . . . . . . . . . . . . . . . . . . 0-1 min 6% B; 1.01-4 min 20% B;
4.01-12 min 6% B, total run time 15 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300 psi (89.6 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . fluorescence, λ excitation = 373 nm, λ emission = 448 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . Mix of DMB-labeled NGNA and NANAsialic acid, 5 mg/ml each
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006199

HPLC Analysis of Dorzolamide Enantiomers on O O

H3C S O
Astec® CHIROBIOTIC® V2 * S
O
S
NH2
mobile phase . . . . . . . . . . . . . . . (A) 3.81 mM ammonium trifluoroacetic acid;
(B) methanol; (5:95, A:B) HN C H3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004478
0 10 20
Min.

HPLC Analysis of cis/trans-Dothiepin Enantiomers

H3 C
on Astec® CYCLOBOND® I 2000 AC
N
column . . CYCLOBOND I 2000 AC, 25 cm x 4.6 mm I.D., 5 μm particles (20124AST) CH3
mobile phase . . (A) 1.0% triethylamine acetate, pH 4.1; (B) acetonitrile; (85:15, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL *
peak 1: 9.48 min.
peak 2: 12.48 min.
S
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004732

HPLC Analysis of Enilconazole (Imazalil) Peak 1:

Cl 11.86 min
Enantiomers on Astec® CYCLOBOND® I 2000 Peak 2:
HP-RSP 20 O 15.08 min
15 Cl
column . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm × 4.6 mm I.D., N N
mAU
10
mobile phase . . (A) 10 mM ammonium acetate, pH 4.0: (B) acetonitrile; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C 0
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 0 4 8 12 16 20
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004029

HPLC Analysis of Enilconazol (Imazalil) Cl
Enantiomers on Astec® CYCLOBOND® I 2000 O *

HP-RSP H2C
column . . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D., Cl

N
5 μm particles (24024AST) N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004588
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Ephedrine Compounds on 1. Norephedrine 4

Ascentis® Express HILIC 2. Ephedrine 1
3. Pseudoephedrine
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 4. Synephrine
mobile phase . . (A) acetonitrile; (B) 40 mM ammonium acetate in water; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005352
0 2 4 6 8 10
Min

HPLC Analysis of Ephedrine Enantiomers on H3 C

CHIRALPAK® AGP NH
column . . . . . . . . CHIRAL-AGP, 10 cm x 4 mm I.D., 5 μm particles (58170AST) * OH

mobile phase . . . . . . . . . . . 1 mM octanoic acid in 10 mM (NH4)2 HPO4, pH 6.9 H3 C *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004730
0 2 4 6
Min

HPLC Analysis of Epinephrine, Epinastine, 4

1. Epinephrine
2. Benzyalkonium chloride
and Benzalkonium Chlorides on Ascentis® 3. Benzyalkonium chloride
4. Benzyalkonium chloride
Express HILIC 5. Epinastine
3
column . . Ascentis Express HILIC, 15 cm x 4.6 mm I.D., 2.7 μm particles (53981-U) 5

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . ESI(+), XIC m/z 304 & 332 (BAC), 250 (epinastine), 180 (epinephrine)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12
Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L ea. in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005411

HPLC Analysis of Erectile Dysfunction Drugs on 1 1. Tadalafil

Discovery® C18 2. Sildenafil
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 3. Vardenafil
mobile phase . . . . . . (A) water (10 mM NH4 COOH); (B) acetonitrile; (50:50, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 2
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002546
0 2 4 6 8 10
Min

HPLC Analysis of Erectile Dysfunction Drugs on 1
Discovery® HS F5
1. Tadalafil
column . . . . Discovery HS F5, 15 cm × 4.6 mm I.D., 5 μm particles (567516-U) 2. Sildenafil
mobile phase . . . . . . . . . . . . . . . . . . . 50:50 water (10 mM NH4COOH):CH3CN 3. Vardenafil
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002542
0 2 4 6 8 10
Min

CH 3
HPLC Analysis of Etodolac Enantiomers on Astec® H 3C
H OH
Cellulose DMP N
*
O
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) (-)
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
(+)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004988
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Exenatide Acetate on Proteomix® Sequence: HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS
SCX-NP5 pI: 4.7
column . . . . Proteomix SCX NP5, 25 cm x 4.6mm I.D., 5 μm particles (Z777161) MW: 4186.6
mobile phase . . (A) 10 mM monosodium phosphate:acetonitrile (56:44, v/v), adjusted
to pH to 2.0 with phosphoric acid; ((B) 0.5 M NaCl/10 mM monosodium
phosphate:acetonitrile (56:44, v/v), adjusted to pH to 2.0 with
phosphoric acid)
gradient . . . . . . . . 0 to 65% B in 35 min; held for 5 min; to 0% B in 10 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653 psi (114 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . . 0.325 mg/mL exenatide acetate in mobile phase A
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006163 10 15 20 25 30 35 40
Min

HPLC Analysis of Fenofibric Acid Enantiomers on Cl O

CH 3
column . . . CYCLOBOND I 2000, 25 cm x 4.6 mm I.D., 5 μm particles (20024AST) H3 C

*
mobile phase . . (A) 0.1% triethylamine acetate, pH 4.0; (B) acetonitrile; (20:80, A:B) O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004691
0 10 20
Min

HPLC Analysis of Fenoprofen Enantiomers on

O
mobile phase . . . . . . . . . . (A) 1% triethylamine, pH 7.0; (B) THF; (90:10, A:B) O
*
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C CH 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004624
0 2 4 6 8 10 12
Min

HPLC Analysis of Fenoprofen Methyl Ester

O
* CH3
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) O O
mobile phase . . . . . . . (A) 20 mM sodium citrate, pH 6.3; (B) THF; (90:10, A:B)
CH 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004651
0 2 4 6 8 10
Min

HPLC Analysis of Fenoterol Enantiomers on Astec® OH

CHIROBIOTIC® V
OH
chiral CH3
*
mobile phase . . . . . (A) 20 mM ammonium nitrate, pH 5.5; (B) THF; (90:10, A:B) HO NH *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 275 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004625
0 2 4 6 8 10 12 14 16 18
Min

1 1. Fentanyl
HPLC Analysis of Fentanyl Tranquilizer on
Ascentis® C18
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002435
0 2 4 6 8 10
Min

HPLC Analysis of Fluoroquinolone Antibiotics on 1. Ofloxacin

Ascentis® Express C18 2. Ciprofloxacin
3. Lomefloxacin
This application demonstrates the suitability of the Ascentis Express C18 for
4. Danofloxacin
the analysis of fluoroquinolones.
2 5. Enrofloxacin
column . . Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 μm particles (53814-U) 6. Sarafloxacin
1
(B) 0.1% formic acid in acetonitrile; (85:15, A:B)
3 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min
6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005699 1.0 2.0 3.0
Min

3 1. Norfloxacin (1 µg/mL) (M+H)+ =320.13

HPLC Analysis of Fluoroquinolone Antibiotics on 2. Ciprofloxacin (1 µg/mL) (M+H)+ =332.13
Ascentis® Phenyl 3. Lomefloxacin (1 µg/mL) (M+H)+ =352.13
4. Enrofloxacin (1 µg/mL) (M+H)+ =360.16
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 5. Sarafloxacin (1 µg/mL) (M+H)+ =386.12
mobile phase . . . . . . . . . . . . . . (A) 13 mM ammonium formate (pH 3.0 with 1 6. Cinoxacin (1 µg/mL) (M+H)+ =263.05
concentrated formic acid): (B) acetonitrile; (80:20, A:B)
2 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . as indicated in 80:20, 13 mM ammonium formate
(pH 3.0 with concentrated formic acid):acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003713
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Fluoxetine Enantiomers on F

F
Astec® CHIROBIOTIC® V2 HN
CH3
F
O 8.47
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 9.95
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Response
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004465
0 7 14
Min

HPLC Analysis of Fluoxetine Enantiomers on

F
Narrowbore Astec® CHIROBIOTIC® V2 F
CH3
HN
column . . . . . CHIROBIOTIC V2, 10 cm x 2.1 mm I.D., 5 μm particles (15018AST) F
mobile phase . . . . . . . . . . . . . . . . . . 13 mM ammonium acetate in methanol O *

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004476
0 2 4
Min.

HPLC Analysis of Fluoxetine (Prozac®) and 1 1. Norfluoxetine

2. Fluoxetine
Norfluoxetine on Discovery® Zr-PS
column . . . . . Discovery Zr-PS, 7.5 cm × 4.6 mm I.D., 3 μm particles (65741-U) 2
mobile phase . . . . . . . . . . . . . . . . (A) 25 mM potassium phosphate (pH 12):
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0 2 4 6 8 10

HPLC Analysis of Fluoxetine (Prozac®) on

Discovery® C18
mobile phase . . . . . . . (A) acetonitrile; (B) 25 mM KH2PO4 , pH 7.0 (40:60, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000900
0 2 4 6
Min

HPLC Analysis of Fluoxetine (Prozac®) on

Discovery® Cyano
Fluoxetine (Prozac®)
column . . . . . Discovery Cyano, 15 cm × 4.6 mm I.D., 5 μm particles (59356-U)
mobile phase . . . . . . . (A) acetonitrile; (B) 25 mM KH2PO4 , pH 7.0; (40:60, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000899
0 2 4 6
Min

HPLC Analysis of Flurbiprofen Enantiomers on

Astec® Cellulose DMP
mobile phase . . . . . . . . (A) heptane; (B) isopropanol; (C) TFA; (95:5:0.1, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g/L in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005467
0 2 4 6 8 10 12
Min

HPLC Analysis of Flurbiprofen Enantiomers on

Astec® CHIROBIOTIC® V O OH

mobile phase . . . . . (A) 20 mM ammonium nitrate, pH 5.5; (B) THF; (80:20, A:B) * CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL F
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004626
0 2 4 6 8 10 12
Min

HPLC Analysis of Fluroquinolone Antibiotics on 1

1. Ciprofloxacin
Discovery® C18 2. Danofloxacin
3. Enrofloxacin
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 4. Sarafloxacin
3
mobile phase . . . (A) water (10 mM ammonium acetate, pH 3.0 with acetic acid);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002547
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Furosemide and Indapamide on 1
Discovery® C18
column . . . . . . . . Discovery C18, 15 cm × 4.6 mm ID, 5 μm particles (504955) 1. Furosemide
2. Indapamide
mobile phase . . . . . . . . . . . . . . . (A) 10mM monobasic potassium phosphate,
(pH 3.0 with H3PO4 ): (B) acetonitrile; (40:60, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 234 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
1.0 2.0 3.0 4.0 5.0 6.0
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL each in mobile phase Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002044

HPLC Analysis of Glutethimide Enantiomers on 1. Glutethimide enantiomer

1
Astec® Cellulose DMP 2. Glutethimide enantiomer
2
Glutethimide enantiomers are resolved by normal phase chromatography.
Glutethimide is used to treat insomnia.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
sample . . . . . . . . . . . . . . . glutethimide, 1 g/L in heptane:isopropanol (50:50)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005514
0 10 20
Min

HPLC Analysis of Homatropine Enantiomers on 1 & 2: Homatropine enantiomers

1
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004997 2 4 6
Min

HPLC Analysis of Homatropine Enantiomers on HO

HC
Astec® Cellulose DMP, LC/MS Conditions O 3
*
N
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
0 2 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004998


Actual
1 1.0258 0.0103 0.0092 89.32 0.9003
Dilute 2 mL of ethyl acetate-cream sample with 8 mL hexane:ethyl acetate 4 1.1369 0.0114 0.0119 104.39 1.0442
(2:1, v/v). 5 1.1390 0.0114 0.0113 99.12 1.0785
Average: 99.86 1.02
RSD: 6.99 0.06
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
Hydrocortisone
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Hydrocortisone
1.0 2.0 3.0
Min

HPLC Analysis of Hydroxychloroquine

Enantiomers on Astec® CHIROBIOTIC® V Cl N
CH 3
mobile phase . . . (A) methanol; (B) acetic acid; (C) triethylamine; (98:1:1, A:B:C) N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min HN *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm CH3 OH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004627
0 2 4 6 8
Min

HPLC Analysis of Hydroxymetoprolol, Metoprolol 100 1. Hydroxymetoprolol

1
and Alprenolol on Discovery® HS F5 2. Metoprolol

3. Alprenolol
%
column . . . . . Discovery HS F5, 5 cm × 2.1 mm I.D., 3 μm particles (567500-U)
mobile phase . . . . . . . . . . 5 mM ammonium acetate in 10:90 water:acetonitrile 0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 2
100
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (+) ESI %
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated (in mobile phase) 3
100
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002381
%
0
0 2 4 6
Min

Cl
HPLC Analysis of Hydroxyzine Enantiomers on
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) N N
*
mobile phase . . . . . . . . . . . . (A) heptane; (B) IPA; (C) DEA; (90:10:0.1, A:B:C) O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C HO
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm

(+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg/mL in mobile phase (-)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004989
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Ibuprofen Enantiomers on Astec®

CH3
CHIROBIOTIC® V
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) O
CH3 *
mobile phase . . . . . . . . . . . . (A) 20 mM sodium citrate; (B) THF; (90:10, A:B)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C H3 C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004628
0 2 4 6
Min

HPLC Analysis of Ifosfamide Enantiomers on

O
Astec® CHIROBIOTIC® T O NH
*P Cl
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) THF; (90:10, A:B) N
Cl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004499
0 2 4 6 8 10
Min

HPLC Analysis of Indapamide Enantiomers on

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005000
0 2 4 6 8
Min

HPLC Analysis of Indapamide Enantiomers on

Astec® Cellulose DMP, LC/MS 1. Indapamide enantiomer
O 2. Indapamide enantiomer
mobile phase . . . . . . . . . (A) methanol; (B) ammonium formate; (100:0.1, A:B) NH
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min H 3C
1
Cl
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm H 2N S O 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μl/h O

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004999
0 2 4 6 8
Min

HPLC Analysis of Indoprofen Enantiomers on O
Astec® CHIROBIOTIC® V CH 3
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) N

mobile phase . . . . . . . (A) 20 mM sodium citrate, pH 6.3; (B) THF; (90:10, A:B) *
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004652
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Insulin, Bovine on Discovery® C8 2 1. Subunit A oxidized

column . . . . . . . . Discovery C8, 5 cm × 4.6 mm I.D., 5 μm particles (59353-U) 2. Holoenzyme
3 3. Subunit B oxidized
mobile phase . . . . 50 mM sodium phosphate, monobasic, 50 mM sodium sulfate,
pH 3.0 (w/phosphoric acid), (A) +5% acetonitrile,
(B) +50% acetonitrile Gradient Program
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Time (min) %B
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 33.3
1 18 73.3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000765
0 10 20
Min

HPLC Analysis of Insulin (Species Variants) on 2 1. Insulin, bovine

Ascentis® Express Peptide ES C18 3 2. Insulin, human
3. Insulin, porcine
These three insulins have more than 76% identical positions in a primary 1
85% identity. Baseline resolution of these insulins in achieved on Ascentis
Express Peptide ES-C18, with relative band spacing of these three peaks
approximating their relative sequence similarities. Ascentis Express Peptide
ES-C18 is ideally suited for RP chromatography of small proteins or large
peptides.
column . . . . . . . . . . . . Ascentis Express Peptide ES C18, 15 cm x 2.1 mm I.D.,
(B) 55:45, water with 0.1% TFA:acetonitrile with 0.09% TFA 6.0 7.0
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 100% B in 7.5 min Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4040 psi (278 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . 200 μg/mL insulin (bovine, human, porcine) in 0.1% formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005794

HPLC Analysis of Insulin (Species Variants) on 1 1. Bovine insulin

Gradient 2. Human insulin
Discovery® BIO Wide Pore C5 Time (min) %A %B 2 3. Porcine insulin
5 μm particles (568422-U) 30 0 100
mobile phase . . . . . (A): 71:29, (0.1% TFA in water):(0.1% TFA in acetonitrile);
(B): 68:32, (0.1% TFA in water):(0.1% TFA in acetonitrile)
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . 5 μL 5 μL in 0.1% TFA containing 5ug each of three insulin variants
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001580
0 2 4 6 8 10
Min

HPLC Analysis of Insulin (Species Variants) on Gradient 1. Bovine insulin (5 μg)

Time (min) %A %B 1 2. Human insulin (5 μg)
Discovery® BIO Wide Pore C8 3. Porcine insulin (5 μg)
0 100 0 2
mobile phase . . . . . . . (A) (0.1% TFA in water):(0.1% TFA in acetonitrile), 71:29
(B): (0.1% TFA in water):(0.1% TFA in acetonitrile), 68:32,
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . 5 μL in 0.1% TFA containing 5ug each of three insulin variants
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001507
2 4 6 8 10
Min

HPLC Analysis of Insulin (Specirs Variants) on 1. Insulin, bovine

2
Ascentis® Express Phenyl-Hexyl 1 3 2. Insulin, human
3. Insulin, porcine
These three insulins have more than 76% identical positions in a primary
85% identity. The Phenyl-Hexyl phase provides an alternate retention and
selectivity to a C18 phase, particularly the C18 phase on a larger pore silica,
Ascentis Express ES-C18
column . . . . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 15 cm x 2.1 mm,
(B) 55:45, water with 0.1% TFA:acetonitrile with 0.09% TFA
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 100% B in 7.5 min 0 2 4 6
Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3960 psi (273 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 μg/mL in 0.1% formic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005795

HPLC Analysis of Isopromethazine Enantiomers on H3C C H3

Astec® CHIROBIOTIC® V N
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) H3C *

mobile phase . . (A) methanol; (B) acetic acid; (C) triethylamine; (100:0.1:0.1, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
N
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
S
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004640
0 2 4 6 8 10
Min

HPLC Analysis of Isoproterenol Enantiomers on

column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HO NH CH3
mobile phase . . . . . . . . . . . . . . (A) acetonitrile; (B) methanol; (C) acetic acid; *
(D) triethylamine; (55:45:0.3:0.2, A:B:C:D) CH3

HO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004543
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Ketamine Enantiomers on Astec® O
(+)
Cellulose DMP *
NH
CH 3
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) Cl (-)

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004990
0 2 4 6 8 10 12
Min

N
HPLC Analysis of Ketoconazole Enantiomers on
Astec® Cellulose DMP, No Additives N
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) O R

O
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile; (100, A) N N O S O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min H3C H Cl Cl
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 1 & 2: Ketoconazole enantiomers 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005005
0 10 20
Min

HPLC Analysis of Ketoconazole Enantiomers on N
Astec® CYCLOBOND® I 2000 HP-RSP

N
5 μm particles (24024AST) O
*
O
N N O * O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
H3 C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C H
Cl Cl
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004716
0 2 4 6 8 10 12
Min

HPLC Analysis of Ketoprofen Enantiomers on O CH 3

O
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004666
0 2 4 6 8
Min

HPLC Analysis of Ketoprofen Enantiomers on O CH3

O
mobile phase . . . . . . . . . . . . (A) 20 mM sodium citrate; (B) THF; (90:10, A:B)
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004629
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Ketorolac Enantiomers on Astec® O
CHIROBIOTIC® T N
O
mobile phase . . . . . . . . . . . . . . 0.1 wt% mM ammonium acetate in methanol
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004581
0 2 4
Min.

HPLC Analysis of Labetalol Enantiomers on Astec® H2N O

CHIROBIOTIC® V
HO
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) C H3
mobile phase . . . . . . . . . . . . . (A) methanol; (B) acetic acid; (C) triethylamine; *
(99.8:0.15:0.05, A:B:C) NH *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 206 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004630
0 20 40
Min

HPLC Analysis of β-Lactam Antibiotics on Discovery RP-AmideC16

Gradient Program
Discovery® RP-Amide C16 Time (min) %B
0 5
column . . Discovery RP-Amide C16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 2
5 35
mobile phase . . . . . . . . (A) 0.01% TFA in water; (B) 0.01% TFA in acetonitrile 20 35
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 3
1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 1. Amoxicillin
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 2. Ampicillin
4 5 6
3. Piperacillin
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001178 4. Penicillin G
5. Penicillin V
6. Cloxacillin
0 10 20
Min

HPLC Analysis of Lansoprazole (Degradation
Lansoprazole
Profile) on Ascentis® Express C18 0.4
column . . . . Ascentis Express C18, 15 cm × 4.6mm, 2.7 μm particles (53829-U)
mobile phase . . . . . . . . . . . . . . (A) water, (B) acetonitrile:0.5% triethylamine
AU
in water, pH=7.0 (80:20)
0.2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 285 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL 0.0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004160
0 10 20 30 40
Min

HPLC Analysis of Lansoprazole Enantiomers on F

F
Astec® CYCLOBOND® I 2000 DMP

F
column . . CYCLOBOND I 2000 DMP, 25 cm x 4.6 mm I.D., 5 μm particles (20724AST) H
N H3C O
mobile phase . . . . . . . . . . . . . . (A) acetonitrile; (B) methanol; (C) acetic acid; S*
(D) ammonium hydroxide; (95:5:0.4:0.1, A:B:C:D)
N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004687
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Lorazepam Enantiomers on H

O
N
Astec® (R,R) P-CAP™
column . . . . Astec (R,R) P-CAP, 25 cm x 4.6 mm I.D., 5 μm particles (31024AST) * OH
mobile phase . . . . . . . . . . . . . . . . . . . (A) ethanol; (B) heptane; (50:50, A:B) Cl N

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
Cl
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004721
0 10 20
Min

HPLC Analysis of Lorazepam Enantiomers on H

N
O
* OH
Cl
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004551
0 2 4 6 8
Min

HPLC Analysis of Lorazepam Enantiomers on H O

N
* OH
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) Cl N
Cl
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004609
0 2 4 6 8 10
Time (min)


versus Competitive SCX using a Salt Gradient
column . . . . . . . . . Proteomix SCX-NP5, 25 cm x 4.6 mm I.D. (PEEK hardware),
mobile phase . . . . . . . (A) 20 mM phosphate buffer, pH 7.5; (B) A + 1 M NaCl
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 to 6% B in 30 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb 016 (11.8 mg/mL)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006107
Competitive SCX
Proteomix SCX
0 5 10 15 20 25 30
Min


versus Competitive SCX using pH and Salt Note improved resolution
Gradients on the Proteomix SCX
compared to competitive
column . . . . . . . . . Proteomix SCX-NP5, 25 cm x 4.6 mm I.D. (PEEK hardware), column.
mobile phase . . (A) 2.4 mM Tris, 1.5 mM imidazole, 11.6 mM piperazine, pH 6.0;
(B) A + 0.5 M NaCl, pH 10.5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb 321 (5 mg/mL)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006098
Competitor SCX
Proteomix SCX
17.5 20.0 22.5 25.0 27.5 30.0 32.5

Min

HPLC Analysis of mAb on Antibodix® WCX, Effect

of Particle Size (5 cm columns)
column . . Antibodix WCX-NP3 PEEK, 5 cm x 4.6 mm I.D., 3 μm particles (Z777288)
N = 7260
column . . . . . Z777280 (Antibodix WCX-NP1.7, 5 cm x 4.6 mm, 1.7 μm particles)
gradient . . . . . . . . . . . NP3 column: 15 to 55% B in 30 min, held for 3 min;
NP1.7 column: 15 to 100% B in 60 min, held for 1.7 min N = 15680
Gray: Antibodix WCX-NP3
flow rate . . . . . . . . . . . . . . . . 0.5 mL/min for 3 min; 0.3 mL/min for 1.7 min (5 cm × 4.6 mm, 3 μm non-porous particles)
pressure . . . . . . . . . . . . . . . . . . . . . . 2180 psi (3 min), 3425 psi (1.7 min) Pressure: 2,180 psi

detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm Black: Antibodix WCX-NP1.7
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL (5 cm × 4.6 mm, 1.7 μm non-porous particles)
Pressure: 3,425 psi
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . Monoclonal antibody, 5 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005941
0 5 10 15 20 25 30 35 40
Min

HPLC Analysis of mAb on Antibodix® WCX-NP10, Vendor D WCX

Competitive Comparison 25 cm × 4 mm I.D., 10 μm (non-porous)
Pressure: 1,490 psi
mobile phase . . . . (A) 10 mM phosphate buffer, pH 7.5; (B) A + 100 mM NaCl Antibodix WCX-NP10
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 to 55% B in 30 min 25 cm × 4.6 mm I.D., 10 μm (non-porous)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min Pressure: 840 psi
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005939
0 5 10 15 20 25 30
Min

HPLC Analysis of mAb on Antibodix® WCX-NP5, N = 12,170

25 cm Columns
column . . . . Antibodix WCX-NP5, 25 cm x 4.6 mm I.D., 5 μm particles (Z777294)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005942
0 5 10 15 20 25 30
Min


WCX-NP10, Impact of Mobile Phase pH
mobile phase . . . . . . . . . . . . . (A) 10 mM sodium phosphate buffer at 7.5 pH;
(B), A + 100 mM NaCl at 7.0 pH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL A. pH 7.5
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAb-X22, 1.5 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005945
B. pH 7.0
0 10 20 30 40 50 60
Min

HPLC Analysis of Mandelic Acid Enantiomers on O

*
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004610
0 2 4 6 8 10 12
Min.

HPLC Analysis of Mephenytoin Enantiomers on 1

Astec® CHIROBIOTIC® V Mephenytoin
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) H3C H
N 2
mobile phase . . . . . . . . . . . . . . . . . . . . (A) hexane; (B) ethanol; (95:5, A:B) O
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm O C H3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004471
0 10 20
Min

HPLC Analysis of Mephobarbital Enantiomers on 1. Mephobarbital enantiomer 1

Astec® Cellulose DMP 2. Mephobarbital enantiomer
Mephobarbital enantiomers are resolved by normal phase chromatography.

2
Mephobarbital (aka methyphenobarbital) is used as a sedative, anxiolytic
and anticonvulsant. It is the N-methylated analogue of phenobarbital, and
has similiar indications, therapeutic value, and tolerability.
column . . Astec Cellulose DMP; 25 cm x 4.6 mm I.D., 5 μm particles (51099AST)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
sample . . . . . . . . . . . . . . . mephobarbital, 1 g/L in 50:50, heptane:isopropanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005519 0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Mepivacaine Enantiomers on

Astec® CHIROBIOTIC® V2 N
CH3
CH3
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) NH
mobile phase . . (A) methanol; (B) 20 mM ammonium acetate, pH 4.1; (95:5, A:B) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O

H3C
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004466
0 10 20
Min

HPLC Analysis of Meptazinol Enantiomers on

CH3
N
mobile phase . . . . . . . . . . . . . . . . . 0.1 wt% ammonium formate in methanol HO *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
CH3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004660
0 2 4 6 8
Min

HPLC Analysis of Metaproterenol Enantiomers on

HO
NH CH3
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) HO

CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004406
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Methocarbamol (Guaiacol 1. Methocarbamol enantiomer 1

2. Methocarbamol enantiomer 2
Glyceryl Ether Carbamate) Enantiomers on Astec®
Cellulose DMP
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005372

HPLC Analysis of Methorphan Enantiomers on CH3

O
mobile phase . . . . . . . . . 0.05 wt% ammonium trifluoroacetic acid in methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm *
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL N CH3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004659
0 10 20
Min

HPLC Analysis of Methylphenidate Enantiomers O O

on Astec® CHIROBIOTIC® V H3C
H
*
mobile phase . . . . . . . (A) methanol; (B) 1% triethylamine, pH 4/1; (95:5, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004505
0 2 4 6 8 10
Min.

HPLC Analysis of Methylphenidate (Ritalin)

Enantiomers on Astec® CHIROBIOTIC® T2 O
CH3
mobile phase . . . . . . . . . . . . . . . . . 15 mM ammonium formate in methanol N O
H
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
HCI
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . methylphenidate, 1 mg/mL in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004405
0 2 4 6 8 10 12
Min

HPLC Analysis of Methylphenidate (Ritalin) O O

H 3C
Enantiomers on Astec® CHIROBIOTIC® V2 H
N *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004504
0 2 4 6 8 10 12
Min.

HPLC Analysis of Methylphenobarbital CH3

(Mephobarbital) Enantiomers on Astec® O N O
CHIROBIOTIC® V
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) H3C
*
NH
mobile phase . . . . . . . . . . . . . . . . . . . (A) ethanol; (B) hexane; (25:75, A:B)

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004631 0 2 4 6 8
Min

HPLC Analysis of α-Methyl Phenylalanine OH

Enantiomers on Astec® CHIROBIOTIC® T H3 C

*
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004590
0 2 4 6 8 10
Min.

HPLC Analysis of 5-Methyl-5-Phenylhydantoin O
Enantiomers on Astec® CHIROBIOTIC® V2 HN

NH

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.48 mL/min H 3C
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004477
0 2 4 6 8 10 12
Min.

HPLC Analysis of Methyl Phenyl Sulfoxide

O
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . methanol S
CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.48 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004407
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Methylselenocysteine O
Enantiomers on Astec® CHIROBIOTIC® TAG H 3C *

column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) Se OH
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (30:70, A:B) N H2

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV at 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004615
0 2 4 6 8 10 12
Min.

HPLC Analysis of Metoprolol Enantiomers on H3C

O
CH3
Astec® Cellulose DMP *
O NH CH3
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) OH
mobile phase . . . . . . . . . . . . (A) heptane; (B) IPA; (C) DEA; (90:10:0.1, A:B:C) (+)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004991
(-)
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Metoprolol Enantiomers on

Astec® CHIROBIOTIC® V O
H3C CH3
*
mobile phase . . (A) methanol; (B) acetic acid; (C) triethylamine; (99.8:0.1:0.1, A:B:C) O NH CH3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004632
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Metoprolol Enantiomers on O

H3C CH3
Astec® CHIROBIOTIC® T2 *
O NH CH3
OH
mobile phase . . . . . . . . . . 0.1 wt% ammonium trifluoroacetic acid in methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004597
0 2 4 6 8 10
Min.

HPLC Analysis of Metoprolol Enantiomers on O

H3C
Astec® CHIROBIOTIC® T2 (MS Mobile Phase) CH3
*
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) O NH CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004484
0 2 4 6 8 10 12 14
Min.

H3C
HPLC Analysis of Mianserin Enantiomers on Astec® N
Cellulose DMP
N
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) (+) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min (-)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004993
0 2 4 6 8 10 12
Min

HPLC Analysis of Mianserin Enantiomers on Astec® 1. Mianserin enantiomer

Cellulose DMP, LC/MS Conditions 2. Mianserin enantiomer
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) H 3C

1
N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min N
*
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005003 0 2 4 6 8
Min

HPLC Analysis of Mianserin Enantiomers on Astec® 1. Mianserin enantiomer

Cellulose DMP (No Additives) H 3C
N
2. Mianserin enantiomer

N
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile; (100, A) * 1 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6

HPLC Analysis of Mianserin Enantiomers on Astec® H3C

N
Cellulose DMP, POM Conditions
N
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
0 2 4 6 8 10
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004992
Min

HPLC Analysis of Mianserin Enantiomers on Astec® H3 C

CHIROBIOTIC® V N

mobile phase . . (A) methanol; (B) acetic acid; (C) triethylamine; (99.8:0.1:0.1, A:B:C) N
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004633
0 2 4 6 8 10
Min

HPLC Analysis of Mianserin Enantiomers on Astec® H3C

N
CHIROBIOTIC® V2
N

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004469
0 2 4 6 8 10 12
Min

HPLC Analysis of Miconazole Enantiomers on 1. Miconazole enantiomer

Astec® CYCLOBOND® I 2000 HP-RSP 2. Miconazole enantiomer

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . miconazole, 1 mg/mL in acetonitrile:water (50:50)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005164
0 20
Min

HPLC Analysis of Miconazole Enantiomers on N

Cl
column . . CYCLOBOND I 2000 RSP, 25 cm x 4.6 mm I.D., 5 μm particles (20324AST) N
mobile phase . . (A) 1% triethylamine acetate, pH 4.1; (B) acetonitrile; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min Cl *
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm O Cl
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg/mL in methanol Cl
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004706
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Monoclonal Antibodies (mAb) Antibodix WCX-NP10

with Charge Variance on Antibodix® WCX-NP10 25 cm × 4.6 mm, 10 μm (non-porous particles)
Pressure: 840 psi
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840 psi (58 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm MAb+ 1 lysine
MAb Acidic isomer
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL MAb+ 2 lysine
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005940
0 5 10 15 20 25
Min

HPLC Analysis of Morphine and Glucuronide 1 1. Morphine 3-β-D-glucuronide

Metabolites on Discovery® HS F5 2. Morphine 6-β-D-glucuronide
3. Morphine
mobile phase . . (A) 10 mM ammonium acetate, pH 6.8 (unadjusted); (B) acetonitrile
gradient . . . . . . . . . . . . . . . 0% B for 1 min, to 30% B in 4 min, held 3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in final mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006123
0 2 4 6 8
Min

HPLC Analysis of Mosapride Enantiomers on CH3
Astec® CHIROBIOTIC® V H2N O F

O
NH N
mobile phase . . . . . (A) 20 mM ammonium nitrate, pH 5.5; (B) THF; (60:40, A:B) Cl *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004650
0 2 4 6 8 10
Min

HPLC Analysis of mPEG and mPEG-Peptide

(Methoxy-PEG-Maleimide) on Zenix® and
Zenix®-C SEC
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL Zenix SEC-300
sample . . . . . . . . 20kD peptide mPEG-MAL (methoxy-PEG-Maleimide), 1 mg/mL
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Min

HPLC Analysis of mPEG and mPEG-Peptide on Zenix SEC-300

Zenix® and Zenix®-C SEC
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 2 3 4 5 6 7 8 9 10 11 12 13 14
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . 4kD peptide and 20kD mPEG-MAL(methoxy-PEG-Maleimide) (6 mg/mL)
mPEG-peptide
2xmPEG-peptide
2 3 4 5 6 7 8 9 10 11 12 13 14
Min

HPLC Analysis of 1-(1-Naphthyl)ethylamine

NH 2
Enantiomers on Astec® CHIROBIOTIC® V2 H3C

mobile phase . . . . . . (A) methanol; (B) 20 mM ammonium acetate; (90:10, A:B) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mgl/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004458
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Naproxen Enantiomers on Astec® CH 3
CHIROBIOTIC® V OH
*
mobile phase . . . . . . . . . (A) 0.1% triethylamine, pH 7.0; (B) THF; (90:10, A:B) H3C O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004644
0 2 4 6 8 10 12
Min

HPLC Analysis of Nebivolol Enantiomers on Astec® OH OH

CHIROBIOTIC® V2
O * NH * O
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) * *
mobile phase . . . . . . . . . . . . 0.05% ammonium trifluoroacetic acid in methanol

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min F F
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004658
0 10 20
Min

HPLC Analysis of Nicardipine Enantiomers on O

+
Astec® CHIROBIOTIC® V O
- N

mobile phase . . . . . . . . . . . . . (A) methanol; (B) acetic acid; (C) triethylamine; O O CH3
(100:0.02:0.01, A:B:C)
H 3C N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O * O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm H3C N CH3
H
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004654
0 2 4 6 8 10 12
Min

H
HPLC Analysis of Nicardipine Enantiomers on H3C N C H3
O * O
mobile phase . . (A) methanol; (B) 20 mM ammonium acetate, pH 4.1; (95:5, A:B) O O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min H3C N
O-
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C + CH3
N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL O

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004470
0 10 20
Min

HPLC Analysis of Nitrazepam Anticonvulsant 1 1. Nitrazepam
Drugs on Ascentis® C18

(pH 7.0 with potassium hydroxide): (B) acetonitrile; (50:50, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002356
1.0 2.0 3.0 4.0 5.0
Min

HPLC Analysis of Norphenylephrine Enantiomers OH

1
on Astec® CYCLOBOND® I 2000 AC (pH 5.0) HO NH2
*
mobile phase . . (A) methanol; (B) 20 mM ammonium acetate, pH 5.0; (5:95, A:B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min 1. Norphenylephrine enantiomer
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 2. Norphenylephrine enantiomer
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . norphenylephrine, 1 mg/mL in acetonitrile:water (50:50)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005159
0 2 4 6
Min

HPLC Analysis of NSAIDs on Discovery® 1. Ketoprofen 1

Zr-Carbon C18 2. Ibuprofen
3. Naproxen
column . . Discovery Zr-Carbon C18, 15 cm × 4.6 mm I.D., 3 μm particles (65706-U) 3
mobile phase . . (A) 50 mM phosphoric acid (pH 1.75): (B) acetonitrile; (50:50, A:B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . ketoprofen, ibuprofen, naproxen, each 1 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001864
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Min

HPLC Analysis of NSAIDs on Ascentis® RP-Amide 2 1. Ketoprofen (250 µg/mL)

2. Naproxen (50 µg/mL)
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 3. Ibuprofen (250 µg/mL)
mobile phase . . . . . . . . . . . (A) 10 mM dibasic potassium phosphate (pH 3.0):
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min.
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . as indicated in 10 mM dibasic potassium phosphate (pH 3.0)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002621 0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Oenothein B on Ascentis® 2

1. Unknown impurity ([M-2H]2- = 823.4 Da)
Express RP-Amide, UV and MS Detection 2. Oenothein B ([M-2H]2- = 782.9 Da)
3. Isomer ([M-2H]2- = 782.9 Da)
Optimization of a published method showing much better retention, peak 4. Impurity, oxidized oenothein B ([M-2H]2- = 791.9 Da)
shape and resolution on Supelco Ascentis Express RP-Amide 2.7 μm 5. Impurity, oxidized oenothein B ([M-2H]2- = 791.9 Da)
compared to a C18 phase. Isomers and small amounts of homologues are 6. Impurity, fragment oenothein B ([M-H]- = 784.9 Da)
resolved and detectable with higher sensitivity.
2.7 μm particles (53929-U) ESI(-) MS detection (TIC)
mobile phase . . . . . . . . . . . . . . . . . . . . (A) 0.2% formic acid; (B) methanol 3
gradient . . . 5% B for 2 min; to 90% B over 18 min; held at 90% B for 10 min 1 4
5 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . MS (ion trap), m/z 100-1500
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006164 10× sensitivity
UV detection (252–256 nm)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Min

HPLC Analysis of Oligonucleotides on 9

10
1. A10
7 8
Proteomix® SAX 4
6 2. A11
3. A12
column . . . . Proteomix SAX-NP5, 25 cm x 4.6 mm I.D., 5 μm particles (Z777229) 5 4. A13
5. A14
mobile phase . . . . . . . . . . . . . . . . A: 20 mM Tris, pH 8.0 B: A + 0.5 M NaCl 3 6. A15
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 - 100% B in 30 minutes 7. A16
8. A17
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min 1 2 9. A18
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073 psi (74 bar) 10. A20
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . (10 mM each oligonucleotide in water)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005994
0 5 10 15 20 25 30 35
Min

HPLC Analysis of Omeprazole Enantiomers on O
H
Astec® CYCLOBOND® I 2000 DMP N S
column . . CYCLOBOND I 2000 DMP, 25 cm x 4.6 mm I.D., 5 μm particles (20724AST)

H3C N CH3
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile; (B) acetic acid; O N
(C) ammonium hydroxide; (100:0.4:0.1, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 302 nm CH3 CH3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004685
0 10 20
Min

HPLC Analysis of Oxamniquine Enantiomers on

HO
O NH CH3
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) N
+ N *
H
mobile phase . . . . . . . . . . . . . . . . . 0.1 wt% ammonium formate in methanol O- CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004656
0 2 4 6 8 10 12 14
Min

H
HPLC Analysis of Oxazepam Enantiomers on Cl N
O 1. Oxazepam enantiomer
2. Oxazepam enantiomer
Astec® CYCLOBOND® I 2000 DNP * OH
N
column . . CYCLOBOND I 2000 DNP, 25 cm x 4.6 mm I.D., 5 μm particles (25024AST) 1
mobile phase . . . . . . . . . . . (A) acetonitrile; (B) 20 mM ammonium phosphate,
pH 2.9; (20:80, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
sample . . . . . . . . . . . . . . . . oxazepam, 1 mg/mL in acetonitrile:water (50:50)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005162
0 10 20
Min

HPLC Analysis of Oxazepam Enantiomers on H O

N
OH
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) Cl N
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004545
0 2 4 6 8 10
Min

HPLC Analysis of Oxprenolol Enantiomers on 1

1. Oxprenolol enantiomer
Astec® Cellulose DMP 2. Oxprenolol enantiomer
Oxprenolol enantiomers are resolved by normal phase chromatography.
Oxprenolol is a nonselective beta-blocker and is used to treat angina and
hypertension.
mobile phase . . (A) heptane; (B) isopropanol; (C) diethylamine; (80:20:0.1, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
sample . . . . . . . . . . . . . . . . . oxprenolol, 1 g/L in 50:50 heptane:isopropanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005522 0 10 20
Min

HPLC Analysis of Oxprenolol Enantiomers on OH

Astec® CHIROBIOTIC® T O NH C H3
*
C H2 C H3
(D) triethylamine; (54.5:45:0.3:0.2, A:B:C:D)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004507
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Oxprenolol Enantiomers on OH

Astec® CHIROBIOTIC® T2 O NH CH 3
*
mobile phase . . . . . . . . . . . . . . . . . . 5 mM ammonium formate in methanol CH 2 CH 3
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004480
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Paclitaxel and Related 1. Baccatin III

Compounds on Ascentis® Express F5 5 μm 1
2. 10-Deacetylpaclitaxel
3. Cephalomannine
An efficient reverse phase HPLC method for the HPLC analysis of of 2 4. 10-Deacetyl-7-epipaclitaxel
Paclitaxel (Taxol) and related compounds is developed using the Ascentis® 5. Paclitaxel
Express F5 5μm Fused Core HPLC column. Paclitaxel is a natural product 6. 7-Epipaclitaxel
with antitumor activity. TAXOL (paclitaxel) is obtained via a semi-synthetic
3 4 5
process from Taxus baccata.
The developed method can be easily used for the routine quality control of
6
bulk and Parenterals dosage form of Paclitaxel within a short analysis time.
This method offers the advantages of higher efficies, reduced backpressure
and shorter run times over a traditionally run methods over 5 μm totally
porous particle columns.
column . . . . Ascentis Express F5, 15 cm x 4.6 mm I.D., 5 μm particles (50631-U)
0 2 4 6 8 10 12 14
Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2727 psi (188 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . taxols 25 μg/mL in 70:15:15, water:methanol:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005890

HPLC Analysis of Pain Relievers on Ascentis® Elution:

Express C18 5μm 1. acetaminophen 100 μg/mL
2 2. caffeine 100 μg/mL
This application shows the improvements in efficiency that may be obtained 3. benzoic acid
for USP methods using modern Fused-Core technology. (internal standard) 360 μg/mL
column . . . Ascentis Express C18, 10 cm x 4.6 mm I.D., 5 μm particles (50536-U) 4. acetylsalicylic acid 100 μg/mL
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) water with 3% acetic acid;
(B) methanol with 3% acetic acid; (79:21, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3290 psi (227 bar) 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV,275 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 3
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 100 - 360 μg/mLin mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005894 0 2 4
Min


2. Ethyl paraben 4
Discovery® Cyano 3. Propyl paraben 1
column . . . . . Discovery Cyano, 15 cm × 4.6 mm I.D., 5 μm particles (59356-U) 4. Butyl paraben
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001237
0 2 4 6
Min

HPLC Analysis of Pegylated Exenatide on Zenix®

SEC-300 and Zenix®-C SEC-300
mobile phase . . (A) 50 mM Ammonium acetate: (B) acetonitrile; (90:10, A:B, v/v)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609 psi (42 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C Zenix-C SEC-300 Tailing 1.07
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
sample . . . . . . . . . . . . . . . . . 3.3 mg/ml PEG-Exanatide in water (PEG 23 kD) Zenix SEC-300 Tailing 2.37
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005949
10 12 14 16 18 20
Min

HPLC Analysis of Penicillamine Enantiomers on CH 3 O

HS
*
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) H3C
OH
mobile phase . . (A) 0.1% triethylamine acetate, pH 3.8; (B) ethanol; (20:80, A:B) NH 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004549
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Peptide Antibiotics on 1 1. Bacitracin A

2. Virginiamycin M1
Discovery® C8 2
column . . . . . . . . . Discovery C8, 15 cm × 4.6 mm I.D., 5 μm particles (59353)
mobile phase . . (A) 25mM potassium phosphate, pH 3; (B) acetonitrile, (60:40, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 10 μL
0 2 4 6 8 10
Min

HPLC Analysis of a Peptide Drug on Antibodix® Gradient: 0–50% B

WCX-NP10, Gradient Optimization to Separate over 20 min Gradient: 0–20% B
over 20 min, hold
Impurity for 20 min
mobile phase . . . . . . . . . . . . . . . . . (A) 3 mM potassium phosphate, pH 5.0;
(B) 20 mM potassium phosphate, pH 7.5
gradient . . (A) 0% - 50% B (20 min); (B) 0% - 20% B (20 min), 20% B after 20 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . 2 mg/mL, proprietary peptide drug, pI 7-8
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005932
0 5 10 15 20 25 30
Min

HPLC Analysis of Phensuximide Enantiomers on

*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O O
N
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL CH3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004645
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Phensuximide Enantiomers on H3C

Astec® CYCLOBOND® I 2000 DMP O
N
O
mobile phase . . . . . . . . . . . . . . . . . (A) isopropanol; (B) hexane; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004699
0 2 4 6 8 10 12
Min

HPLC Analysis of Phenylephrine Enantiomers on

OH
Astec® CYCLOBOND® I 2000 AC
mobile phase . . (A) 0.5% w/v sodium acetate, pH 5.5; (B) methanol; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min * CH3
NH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm OH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004694
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Pilocarpine, Betaxolol, and 1. Pilocarpine

Benzalkonium Chloride on Ascentis® Express HILIC 1 2. Benzalkonium chloride
3. Benzalkonium chloride
column . . Ascentis Express HILIC, 15 cm x 4.6 mm I.D., 2.7 μm particles (53981-U) 4. Betaxolol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . ESI (+), XIC m/z 304, 332 (BAC), 209 (pilocarpine), 308 (betaxolol)
2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 4
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005412
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Pindolol Enantiomers on Astec® H

N
CHIROBIOTIC® T
mobile phase . . . . . . . . . . . . . . . . . 15 mM ammonium formate in methanol H3C NH O

*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
CH3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004509
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Pindolol Enantiomers on Astec® H

N
CHIROBIOTIC® V
OH
mobile phase . . . . . . . . . . . . . (A) methanol; (B) acetic acid; (C) triethylamine;
(99.98:0.01:0.01, A:B:C) H3C NH O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C CH3

detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004634 0 2 4 6 8 10 12
Min

HPLC Analysis of Polar and Non-Polar Analytes on 1. Uracil

6
2. Phenol
Ascentis® Express C18, Column Test Mixture 3. Acetophenone
4. Benzene
40
column . . Ascentis Express C18, 5 cm × 3.0 mm I.D. 2.7 μm particles (53811-U) 5. Toluene 2
6. Naphthalene 5
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80% acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min 1 4
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3700 psi (255 bar)

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 ° C
mV
20
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004158
3
0
0 10 20 30
Sec

HPLC Analysis of Polar and Non-Polar Analytes on 1. Uracil

2. Acetophenone
Ascentis® Express C8, Column Test Mixture 3. Benzene
4
4. Toluene 3
column . . . Ascentis Express C8, 5 cm × 4.6 mm I.D., 2.7 μm particles (58336-U)
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.76 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 0 0.2 0.4 0.6 0.8 1.0 1.2
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003954

1
HPLC Analysis of Polar Compounds on Ascentis®
Express OH5 (HILIC Mode) 1. Caffeine (netural, pKa = 0.52, log D = -0.63)
2. Fenoprop (2-(2,4,5-trichlorophenoxy)propionic
The impact of partitioning and ion exchange in HILIC separations is acid, pKa = 2.93, log D = -0.05)
demonstrated using a mixture of acidic, neutral, and basic polar compounds 3. Codeine (base, pKa = 8.29, log D = -0.02)
in this Figure. All three analytes have similar log D values, both acid and base
are ionized at mobile phase pH.
column . . . Ascentis Express OH5, 10 cm x 3 mm I.D., 2.7 μm particles (53769-U)
mobile phase . . . . . . 5 mM ammonium formate (95:5 acetonitrile:water) pH 6.8
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 965 psi (66.5 bar) 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 100 μg/mL in 25:75, water:methanol 0 1 2 3 4 5
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005802

HPLC Analysis of Prednisolone (Fluka Secondary Prednisolone

Pharma Standard) on Ascentis® C18
mobile phase . . . . . . . . . . . . (A) water; (B) methanol:acetonitrile (50:50, A:B)
gradient . . . . . 40% B for 14 min; to 80% B in 6 min; held at 80% B for 5 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . 125 μg/mL in water:acetonitrile (60:40)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005589
0 10 20 30
Min

HPLC Analysis of Procainamide and N- 1. 5-Fluorocytosine (to)

2. N-Acetylprocainamide
Acetylprocainamide on Discovery® HS F5 3. Procainamide
1
3
mobile phase . . . . . . . . . . . . . . . . (A) 25 mM ammonium phosphate (pH 7):
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001686
0 2
Min

HPLC Analysis of Proglumide Enantiomers on O OH
Astec® Cellulose DMP O CH3
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) NH *

N
CH3
(+)
mobile phase . . . . . . . . . . . . (A) heptane; (B) IPA; (C) TFA; (90:10:0.1, A:B:C) O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm (-)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004994
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Proglumide Enantiomers on O OH
O CH3
N
mobile phase . . . . . (A) 13 mM ammonium acetate; (B) acetonitrile; (90:10, A:B) NH * CH 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004635
0 2 4 6 8
Min

HPLC Analysis of Promethazine and Degradants 5 1. Degradation product

2. Degradation product
on Ascentis® Phenyl 3. Degradation product
4. Degradation product
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 2 5. Promethazine
mobile phase . . . . . . . (A) 0.1% ammonium acetate: (B) methanol; (25:75, A:B) 6. Degradation product
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 4
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003700 0 2 4 6 8 10 12 14
Min

HPLC Analysis of Promethazine Enantiomers on

S
mobile phase . . (A) methanol; (B) acetic acid; (C) triethylamine; (99.8:0.1:0.1, A:B:C) CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min N
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C N CH3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm H3C

HCI
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004404
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Promethazine Sulfoxide H3C CH3

N
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) * CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min N
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL S
O
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004646
0 10 20
Min

HPLC Analysis of Propranolol Enantiomers on CH3
H3C NH O
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004510
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Propranolol Enantiomers on CH3

Astec® CHIROBIOTIC® TAG *
H3 C NH O
OH
mobile phase . . . . . . . . . . . . . . . . . 0.1 wt% ammonium formate in methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004616
0 20 40
Min.

HPLC Analysis of a Protein Mixture on Zenix® 1
SEC-150 versus Zenix® SEC-300, Effect of Pore Size

on Resolution
column . . Zenix SEC-300, 30 cm x 7.8 mm I.D., 3 μm, 300 Å particles (Z777033) 4
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 2
5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
1. Thyroglobulin, 670 kD
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . protein mixture 2. γ-Globulin, 158 kD 3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006118 3. Ovalbumin, 44 kD
4. Ribonuclease A, 13.7 kD
5. p-Aminobenzoic acid, 137 D
2
1
5
Zenix SEC-300
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

HPLC Analysis of Proteins on Proteomix® 1. Ribonuclease A 3

1
SCX Columns, Effect of Particle Size of Resolution 2. Cytochrome c
3. Lysozyme
gradient . . . . . . . . . . . . . . . . . . . 0-25 min from 0-75% B, 15 min prewash
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 mL/min 1.7 μm
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 3 μm
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . each 1 mg/mL
10 μm
0 2 4 6 8 10 12 14 16 18 20
Min

HPLC Analysis of Pseudoephedrine Enantiomers 1. Pseudoephedrine enantiomer

on Astec® CHIROBIOTIC® TAG 2. Pseudoephedrine enantiomer
H3C
column . . . . CHIROBIOTIC TAG, 25 cm x 4.6 mm I.D., 5 μm particles (14024AST) NH
2
mobile phase . . (A) methanol; (B) acetic acid; (C) triethylamine;(99.8:0.1:0.1, A:B:C) OH 1
H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005350
0 10 20
Min

HPLC Analysis of Pseudoephedrine Enantiomers H3C

NH
* OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004530
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Pyroglutethimide Enantiomers N

on Astec® CHIROBIOTIC® V
mobile phase . . (A) 0.1% triethylamine acetate, pH 7.0; (B) acetonitrile; (90:10, A:B) *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C H3C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm O N O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL H

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004647
0 2 4 6 8 10 12
Min

HPLC Analysis of Quaternary Ammonium Drugs 1. Ipratropium bromide

on Discovery® HS F5 2. Mepenzolate bromide
3. Pipenzolate bromide
column . . . . Discovery HS F5, 15 cm × 4.6 mm I.D., 5 μm particles (567516-U) 4. Isopropamide iodide
mobile phase . . . . . . . . . . . . . 10 mM ammonium acetate in 80% acetonitrile 5. Oxyphenonium bromide
6. Propantheline bromide
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 225 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 2 3 4
6
1 5
sample . . . . . . . . . . . . . . . . . 50 mg/mL in 70:30, 10 mM ammonium acetate
in 80% acetonitrile:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002221
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Quinidine and Trace Impurity on 1 1. Quinidine (50 μg/mL)

Discovery® HS F5 2. Impurity

mobile phase . . . . . . . . . . . . . . . (A) 25 mM ammonium phosphate (pH 7.0):
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001666
0 10 20
Min

HPLC Analysis of Rabeprazole Enantiomers on H

N O H3C O
Astec® CYCLOBOND® I 2000 DMP S

*
column . . CYCLOBOND I 2000 DMP, 25 cm x 4.6 mm I.D., 5 μm particles (20724AST) N
mobile phase . . . . . . . . . . . . . . (A) acetonitrile; (B) methanol; (C) acetic acid; N O

(D) ammonium hydroxide; (95:5:0.4:0.1, A:B:C:D) H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004686
0 10 20
Min

HPLC Analysis of Recombinant Tumor Necrosis

Factor Receptor-Fc Fusion Protein (rhTNFR-Fc) on
column . . . . . . . . . . . . . . . . . . . . SRT SEC-300, 30 cm x 7.8 mm I.D., 5 μm,
300 Å particles (Z777051) (Z777033)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . Recombinant tumor necrosis factor receptor-Fc fusion
protein (rhTNFR-Fc), including oligomers (440 kD and 360 kD),
monomer (150 kD), and impurity (120 kD)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006121
Zenix SEC-300
SRT SEC-300
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Min

HPLC Analysis of Ritalinic Acid and Ritalin HO O

(Methylphenidate) Enantiomers on Astec®
CHIROBIOTIC® V2 with T2 Guard Column H
N
*
column . . . . . . . . . . . . . . . CHIROBIOTIC T2 guard column, 2 cm x 4 mm I.D.,
O O
5 μm particles (16100AST (requires holder 21150AST)) H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Ritalinic acid H
N *
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C *
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 3.9
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Ritalin
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004733
12.7
15.5
5.5

HPLC Analysis of Ritalinic Acid Enantiomers on

*
column . . . . . CHIROBIOTIC T2, 25 cm x 4.6 mm I.D., 5 μm particles (16024AST) * N
mobile phase . . (A) 10 mM ammonium acetate, pH 5.5; (B) acetonitrile; (70:30, A:B) H
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 mL/min HO O

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004600
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Salbutamol Enantiomers on HO

Astec® CHIROBIOTIC® T H 3C
CH 3
HO *
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) NH
CH 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004512
0 2 4 6 8 10 12
Min.

HPLC Analysis of Salicylic Acid and Acetylsalicylic 1. Salicylic acid

Acid (Aspirin) on Ascentis® Express OH5 2. Acetylsalicylic acid
2
mobile phase . . . . . . . . . . 2 mM ammonium acetate in 10:90 water:acetonitrile,
pH to 7.0 with acetic acid, glacial 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 850 psi (59 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . 100 μg/mL salicylic acid, 200 μg/mL acetylsalicylic
acid in methanol:acetonitrile (3:7)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006124
0 2
Min

HPLC Analysis of Sertraline Enantiomers on Astec® CH3

CYCLOBOND® I 2000 RSP HN

*
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Cl
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004705
Cl
0 10 20
Min

HPLC Analysis of Sibutramine Enantiomers on CH 3

Astec® CHIROBIOTIC® V N * CH 3
H3C
Sibutramine, one of a few drugs approved for long-term treatment of
obesity, is a chiral compound. The (R) enantiomer has the desired effect of CH 3
reducing body weight and food intake, but the (S) enantiomer has the
opposite effect: A perfect example of why chiral HPLC is important to Cl
pharmaceutical and clinical analysts. Astec® CHIROBIOTIC® V successfully
separates sibutramine enantiomers, using a simple, MS-friendly mobile
phase.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 0 2 4 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm Min.
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004479

HPLC Analysis of Sibutramine Enantiomers on CH3

N * CH3
Sibutramine, one of a few drugs approved for long-term treatment of H3C
obesity, is a chiral compound. The (R) enantiomer has the desired effect of
CH3
reducing body weight and food intake, but the (S) enantiomer has the
opposite effect: A perfect example of why chiral HPLC is important to
pharmaceutical and clinical analysts. Astec® CHIROBIOTIC® V2 successfully Cl
separates sibutramine enantiomers, using a simple, MS-friendly mobile
phase.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 0 2 4 6 8
Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004661

HPLC Analysis of Sinus Medications on Discovery® 1. Pseudoephedrine

1 2. Paracetamol
RP-AmideC16 3. Chlorpheniramine
mobile phase . . . . . . . (A) acetonitrile: (B) 50 mM KH2PO4 , pH 3.0 (25:75, A:B)
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000087
0 2 4 6
Min

HPLC Analysis of Sotalol Enantiomers on Astec® OH
CHIROBIOTIC® T O *
NH CH3
H3C
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) S CH3
NH
mobile phase . . . . . . . . . . . . . . . . . 15 mM ammonium formate in methanol O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004514
0 2 4 6 8 10 12 14 16
Min.

OH
HPLC Analysis of Sotalol Enantiomers on Astec®
CHIROBIOTIC® V *
NH CH3
O
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) H3C
mobile phase . . . . . . . . . . . . . (A) methanol; (B) acetic acid; (C) triethylamine; S CH 3
NH
(99.98:0.01:0.01, A:B:C) O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004636
0 10 20 30
Min

1 1. Pravastatin
HPLC Analysis of Statins on Ascentis® RP-Amide 2. Atorvastatin
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 3. Fluvastatin
4. Lovastatin
mobile phase . . (A) water with 0.1% formic acid (34673): (B) methanol; (25:75, A:B) 2 3 5. Simvastatin
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003270
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Sulconazole Enantiomers on 1. Sulconazole enantiomer

1
Astec® Cellulose DMP 2. Sulconazole enantiomer 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 1 g/L in 50:50, heptane:isopropanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005468
0 10 20
Min

HPLC Analysis of Sulfa Drugs on Discovery® C18 1 1. Sulfanilamide

column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 2. Sulfathiazole
3. Sulfamerazine
mobile phase . . . . . . . (A) methanol; (B) water with 1% acetic acid (20:80, A:B) 4. Sulfamethazine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797-0655 2
4
0 2 4 6
Min

HPLC Analysis of Sulfa Drugs on Titan™ C18 1 1. Sulfanilamide

2. Sulfacetamide
column . . . . . . . . . Titan C18 5 cm x 2.1 mm I.D., 1.9 μm particle (577122-U)
3. Sulfadiazine
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) water with 0.1% acetic acid; 4. Sulfathiazole
(B) acetonitrile with 0.1% acetic acid ; (90:10, A:B) 5. Sulfamerazine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 6. Sulfamethizole
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2670 psi (184 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 3
2 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm 5
6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006114
0 2 4
Min

HPLC Analysis of Tamoxifen E/Z isomers and 1. Endotoxifen

2. 4-Hydroxytamoxifen
Related Compounds on Ascentis® Express RP- 3. Endotoxifen
Amide 4. 4-Hydroxytamoxifen 5
column . . Ascentis Express RP-Amide, 10 cm x 3 mm I.D., 2.7 μm particles (53918-U) 5. N-Desmethyltamoxifen 4

6. Tamoxifen 6
mobile phase . . (A) 0.1% formic acid in water; (B) 0.1% formic acid in acetonitrile
23
gradient . . . . . . . . . . . . . . held at 25% B for 2 min; 25 to 40% B in 15 min;
1
40 to 25% B in 0.1 min; held at 25% B for 2.9 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2292 psi (158 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm 0 10 20
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL Min
sample . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L in 10:90, water: methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005748

HPLC Analysis of Taxols on Ascentis® Express F5 1. Baccatin III

2. 10-DeacetyIpaclitaxel
This application demonstrates the suitability of Ascentis Express F5 for the
efficient separation of taxols. 3. Cephalomannine
1 4. 10-Deacetyl-7-epipaclitaxel
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) 5. Paclitaxel
mobile phase . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (60:40, A:B) 2 6. 7-Epipaclitaxel
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 5 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005749
0 2 4 6 8 10 12
Min

HPLC Analysis of Taxols on Discovery® HS F5 1. 10-deacetyl baccatin III Gradient Program

column . . . . Discovery HS F5, 15 cm × 2.1 mm I.D., 3 μm particles (567503-U) 2. Baccatin III Time (min) %A %B
3. 10-deacetyl-7-xylosyl-β-paclitaxel
mobile phase . . (A) 10 mM ammonium acetate (pH 6.8 unadjusted); (B) acetonitrile 4. 10-deacetyl-7-β-xylosyl paclitaxel C 0 65 35
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 5. 10-deacetyl paclitaxel 5 65 35
20 55 45
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 6. 7-β-xylosyl paclitaxel
23 55 45
7. Cephalomannine 65 35
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm 25
8. 10-deacetyl-7-epi paclitaxel
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 9. Paclitaxel
sample . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL (in 50:50, water:acetonitrile) 10. 7-epi paclitaxel
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002150 100 10
9
% 8
6
3 7
1 2 4
5
0
0 1 2 3 4 5
Min

HPLC Analysis of Terbutaline Enantiomers on CH3

H3C
Astec® CHIROBIOTIC® T NH
* OH
H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min HO OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004515
0 2 4 6 8 10 12
Min.

HPLC Analysis of Terfenadine Enantiomers on
11.043
12.037
mobile phase . . . . . (A) heptane; (B) ethanol; (C) diethylamine; (98:2:0.1, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 0 2 4 6 8 10 12 14 16 18
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g/L in methanol

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005465

HPLC Analysis of Tetracycline Antibiotics on 1 1. Oxytetracycline

2. Rolitetracycline
Ascentis® Phenyl 3. Demeclocycline
4. Minocycline
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% ammonium acetate; 2 3
(B) acetonitrile with 0.1% ammonium acetate; (80:20, A:B) 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 353 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0 2 4 6 8 10 12 14
sample . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in 80:20, water:methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003715

HPLC Analysis of Thalidomide Enantiomers on O
column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) N O
mobile phase . . (A) 18 mM ammonium nitrate, pH 5.5; (B) acetonitrile; (65:35, A:B) *
N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min H
O O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004653
0 2 4 6
Min

HPLC Analysis of Thalidomide Enantiomers on

Astec® CHIROBIOTIC® V2 Thalidomide O
N * O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL N
H
Response
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm O
O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004473
0 7 14
Min

HPLC Analysis of Allegra® on Discovery® Cyano 2

1. Methanol
2. Pseudoephedrine
column . . . . . Discovery Cyano, 15 cm × 4.6 mm I.D., 5 μm particles (59356-U) 3. Fexofenadine
mobile phase . . . . . . . . . . . . . . . . acetonitrile:25 mM KH2PO 4, pH 7.0 (15:85)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001279
1
0 2 4 6 8 10
Min

HPLC Analysis of Tolperisone Enantiomers on

Astec® CHIROBIOTIC® V2 H3C
CH3
N
mobile phase . . . . . . . . . . . . . . . . . . . 0.1% ammonium formate in methanol *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004657
0 2 4 6 8
Min

HPLC Analysis of Tramadol Enantiomers on Astec® H 3C
CYCLOBOND® I 2000 DMP N

*
column . . CYCLOBOND I 2000 DMP, 25 cm x 4.6 mm I.D., 5 μm particles (20724AST) H3C
mobile phase . . (A) 0.1% triethylamine acetate, pH 5.0; (B) acetonitrile; (80:20, A:B)
*
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C OH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg/mL in methanol O
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004700 CH 3
0 2 4 6 8
Min

HPLC Analysis of Triadimenol Enantiomers on Cl
Astec® CYCLOBOND® I 2000 HP-RSP HO CH3
column . . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D., CH3
5 μm particles (24024AST) O CH3

N
mobile phase . . (A) 20 mM ammonium acetate, pH 4.0; (B) acetonitrile; (70:30, A:B) N N
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004396
0 2 4 6 8 10 12 14 16
Time (min)

1. Nordoxepin (50 µg/mL) 5. Doxepin (50 µg/mL)

HPLC Analysis of Tricyclic Antidepressants on 2. Desipramine (50 µg/mL) 6. Imipramine (50 µg/mL)
Ascentis® C8 3. Nortriptyline (50 µg/mL) 7. Amitriptyline (50 µg/mL)
4. Norclomipramine (50 µg/mL) 8. Clomipramine (50 µg/mL)
mobile phase . . . . . . . . . . . . . . . . . (A) 25 mM ammonium phosphate dibasic 1
(pH 7.0 with phosphoric acid): (B) methanol; (25:75, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4 5 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 7
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 8
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003156
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Tricyclic Antidepressants on 1. Doxepin

Ascentis® ES Cyano 1 2. Trimipramine
3. Amitriptyline
This application demonstrates the suitability of Ascentis ES Cyano for the 4. Imipramine
efficient separation of tricyclic antidepressants. 5. Nordoxepin
column . . . . Ascentis ES Cyano, 15 cm x 4.6 mm I.D., 5 μm particles (577306-U) 3 6. Nortriptyline
mobile phase . . (A) 10 mM potassium phosphate dibasic, pH 7 with phosphoric acid; 7. Desipramine
(B) acetonitrile; (C) methanol; (25:60:15, A:B:C) 8. Protriptyline
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
2 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm 4 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 7 8
sample . . . . . . . . . . . . . . . . . . . . . . 100 μg/mL in water: methanol (70:30)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005630
0 2 4 6 8 10 12 14 16
Min


3
Ascentis® Express C18 2. Desipramine

3. Nortiptyline
1
column . . Ascentis Express C18, 10 cm × 2.1 mm I.D., 2.7 μm particles (53823-U) 4. Doxepin
5. Norclomipramine
mobile phase . . . . . . . . . . . . (A) 100 mM ammonium acetate (pH 7.0; titrated 4
6. Imipramin
with ammonium hydroxide), (B) water, (C) methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C
detector . . . . . . . . . . . . . . . . . Thermo LCQ Advantage; ESI(+), m/z 250-320
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 6
instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jasco X-LC 2 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004062
1.0 2.0 3.0

Min

HPLC Analysis of Tricyclic Antidepressants on 1. Desmethyl doxepin

2 2. Protriptyline
Discovery® RP-AmideC16 3. Desipramine
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 4. Nortriptyline
1 5. Doxepin
mobile phase . . (A) water/acetonitrile (40:60 v/v); (B) 10 mM potassium phosphate 6. Imipramine
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 3 8. Trimipramine
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000163
5
6
8
0 2 4 6 8 10 12
Min


2. Nortriptyline 1 2
Discovery® Zr-PBD 3. Imipramine 3 4
4. Amitriptyline
column . . . . . Discovery Zr-PBD, 15 cm × 4.6 mm I.D., 3 μm particles (65718-U)
mobile phase . . . . . . . . . . . . . . . (A) 20 mM potassium phosphate (pH 12.0):
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001862 1.0 2.0 3.0 4.0 5.0 6.0
Min

HPLC Analysis of Tricyclic Antidepressants on 1 1. Desmethyl doxepin

Discovery® C8 2. Protriptyline
3. Desipramine
3
column . . . . . . . Discovery C8, 15 cm × 4.6 mm I.D., 5 μm particles (59353-U) 4. Nortriptyline
5. Doxepin
mobile phase . . . (A) methanol: (B) 10 mM potassium phosphate, dibasic, pH 7.0 6. Imipramine
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 4 8. Trimipramine
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000541
2 5
6
7
8
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Tricyclic Antidepressants on 1. Trimipramine

1
2. Doxepin 2 4
Discovery® Cyano 3. Amitriptyline
4. Imipramine 3
column . . . . . Discovery Cyano, 15 cm × 4.6 mm I.D., 5 μm particles (59356-U) 5. Desmethyl doxepin
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . (A) acetonitrile: (B) methanol: 6. Nortriptyline
(C) 10 mM potassium phosphate, 7. Desipramine 5 7
dibasic, pH 7.0, (60:20:20, A:B:C) 8. Protriptyline 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000727 8
0 2 4 6
Min

HPLC Analysis of Trimipramine Enantiomers on

N
column . . . . . CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 μm particles (15024AST) H3C *
N
CH3 CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004474
0 2 4 6 8 10 12 14
Min.

HPLC Analysis of Tryptophan Hydroxylase 1 2 3 4 5 1. 6-fluorotryptophan, (m/z 223);

(10 µg/mL)
Inhibitors on Ascentis® C18 2. p-chlorophenylalanine, (m/z 200);
(10 µg/mL)
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 3. p-chloroamphetamine, (m/z 170);
(10 µg/mL)
mobile phase . . . (A) 40 mM formic acid, (pH ~2.5): (B) methanol; (65:35, A:B) 4. α-propyldopacetamide, (m/z 210);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 mL/min (50 µg/mL)
5. Fenfluramine, (m/z 232);
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C (10 µg/mL)
detector . . . . . . . . . . . . . . . . . . . . . . full scan MS; overlay of extracted ion
chromatograms of individual (M+H)+ species
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . as indicated in 90:10, 10 mM formic acid:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002453
0 10 20
Min

HPLC Analysis of Ulcer Treatment Drugs on 1. Ranitidine (40 µg/mL) 5. Pirenzepine (40 µg/mL)
2. Famotidine (40 µg/mL) 6. Omeprazole (40 µg/mL)
Ascentis® RP-Amide 3. Cimetidine (40 µg/mL) 7. Lansoprazole (40 µg/mL)
6
4. Nizatidine (40 µg/mL)
mobile phase . . . . . . . . . . . . . . . . . . . . . (A) 10 mM ammonium phosphate Gradient Program 3
Time (min) %A %B
(pH 6.2 with phosphoric acid); (B) acetonitrile 0 95 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 10 85 15
2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 18 45 55 1
5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 23 45 55 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . as indicated in 95:5 water:methanol
0 10 20

HPLC Analysis of Veterinary Antibiotics on 1. Lasalocid A (1 μg/mL); m/z 589.5

Discovery® HS C18 2. Monensin (10 μg/mL); m/z 669.6
3. Salinomycin SV (10 μg/mL); m/z 749.5
column . . . . Discovery HS C18, 5 cm × 2.1 mm I.D., 3 μm particles (569253-U) 4. Narasin (3 μg/mL); m/z 763.5
mobile phase . . . . . . . . . . . . . . (A) (50 mM acetic acid/ammonium hydroxide,
pH 6.0]: (B) methanol; (20:80, A:B): 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
3 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 °C 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002575
0 2 4 6 8 10 12 14 16
Min

1 1. Warfarin
HPLC Analysis of Warfarin™ Anticoagulant on
Ascentis® C18
mobile phase . . . . . . . . . . . . (A) 20 mM phosphoric acid (pH 2.0, unadjusted):
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each in mobile phase 0 2 4 6 8 10 12 14

HPLC Analysis of Warfarin™ Anticoagulant on 1 1. Warfarin (50 µg/mL)
Ascentis® RP-Amide
mobile phase . . . . . . . . . . . . (A) 20 mM phosphoric acid (pH 2.0 unadjusted):
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0 2 4 6 8 10 12 14
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002638

HPLC Analysis of Warfarin™ Enantiomers on Astec® 1. (+)-Warfarin

O O
Cellulose DMP 2. (-)-Warfarin
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) * C H3

(+) 1
mobile phase . . . . . . (A) methanol; (B) acetic acid; (C) TEA; (100:0.2:0.1, A:B:C) OH O
(-) 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 278 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg/mL in mobile phase 0 2 4 6 8


CHIROBIOTIC® V
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 4.65
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
5.22
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004349
0 4 8
Min


column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) * C H3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 4.65
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm

5.22
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004472
Response
0 4 8
Min


column . . . . . . CHIROBIOTIC V, 25 cm x 4.6 mm I.D., 5 μm particles (11024AST) OH O
mobile phase . . . . (A) 0.1% triethylamine acetate, pH 5.0; (B) THF; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min * CH3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm O O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004649
0 2 4 6 8 10 12
Min


S-form
CYCLOBOND® I 2000 (UV at 254 nm) O O
6.89 min R-form
column . . CYCLOBOND I 2000, 25 cm × 4.6 mm I.D., 5 μm particles (20024AST) 7.90 min
1000
mobile phase . . . . . . . . . . . . (A) acetonitrile: (B) acetic acid: (C) triethylamine; OH O
(100:0.3:0.25, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
mAU
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004027
0
0 2 4 6 8
Min

HPLC Analysis of Warfarin™ Enantiomers on Astec® 1. Warfarin enantiomer

CYCLOBOND® I 2000 (UV at 278 nm) 2. Warfarin enantiomer
O O
* C H3 1
mobile phase . . . . . (A) acetonitrile; (B) acetic acid; (C) TEA; (100:0.3:0.2, A:B:C) 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 278 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005008 0 2 4 6 8
Min

CHIROBIOTIC® V2
* C H3
mobile phase . . (A) 10 mM ammonium acetate, pH 4.1; (B) acetonitrile; (70:30, A:B) OH O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004475
0 10 20
Min.

3
HPLC Analysis Proteins on Proteomix® SCX-NP1.7, 1. Ribonuclease A
Demonstration of Column Stability with Repeated 2. Cytochrome c
1
3. Lysozyme
Injections
column . . . . . . . . . . . . . . . Proteomix SCX-NP1.7 guard cartridge with holder,
1 cm x 4 mm I.D. (Z777144) 1st Injection
gradient . . . . . . . . . . . . . . . . . . . . 0 - 100% B in 25 min, 15 min prewash 125th Injection
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 250th Injection
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 0 5 10 15 20
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . each compound 1 mg/mL Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006135

HPLC (Size Exclusion) Analysis of Proteins on R

U
Zenix®-C SEC-300, Effect of DTT and EDTA O
mobile phase . . . . . . . . . . . . . . (A) 150 mM sodium phosphate buffer, pH 7.0 T G
(B) 150 mM sodium phosphate buffer,
pH 7.0 with 2 mM DTT, 0.1 mM EDTA
(C) 150 mM sodium phosphate buffer, pH 7.0 with 2 mM DTT,
0.1 mM EDTA
R
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
B Blue: Protein mix 2, 150 mM sodium
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C phosphate buffer, pH 7.0 with 2 mM DTT,
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm T 0.1 mM EDTA
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . proteins Red: Protein mix 1, 150 mM sodium
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005954 phosphate buffer, pH 7.0 with 2 mM DTT,
0.1 mM EDTA
Black: Protein mix 1, 150 mM

sodium phosphate buffer, pH 7.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5

Min
Separation efficiency not adversely affected by addition of DTT or EDTA.

Hyper-Fast Separations on Ascentis® Express at Ascentis Express C18 1.

2.
Estradiol
β-Estradiol
10 cm length
Half the Pressures of sub-2 μm Columns 2 3.
4.
Impurity
Estrone
1400 psi 4 5.
column . . . . . Ascentis Express C18, 10 cm × 2.1 mm I.D., 2.7 μm particles and Estrone degradant
sub-2 μm particle column, 5 cm × 2.1 mm I.D. (53823-U)

1
mobile phase . . . . . . . . . . . . (A) water; (B) acetonitrile; (55:45 or 54:46, A:B)
Twice The
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min Resolution
At Equal
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient Pressure
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 200 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003973 5
3
1.0 2.0 3.0 4.0 5.0 6.0

Min
1650 psi
1
2
4
C18 Sub-2 µm
5 cm length
5
3
1.0 2.0 3.0 4.0

Min

LC-MS Analysis of Anesthetics on Ascentis® 1. Lidocaine 1

Express F5 2. Mepivacaine 2
3. Procainamide
mobile phase . . . . . . . . (A) 10 mM ammonium acetate, pH 4.0 with acetic acid; 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2320 psi (160 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(+), SIR, m/z 235, 236, 247 0 10 20
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005661

LC-MS Analysis of Angiotensins on Discovery® BIO 3.34 8.36

649.1 1046.6
1046.6
Wide Pore C18 100 II I
60
524.0
column . . . . . . . . . . . . Discovery BIO Wide Pore C18, 10 cm × 0.32 mm I.D., 40 1047.5

3 μm particles (65527-U) 20 784.4
0.32 mm I.D. capillaries 611.3
mobile phase . . (A) 65:35, (10mM ammonium acetate, pH 7):(50% acetonitrile in can give up to
Relative Abundance
Relative Abundance
20mM ammonium acetate, pH 7); (B) 25:75, (10mM ammonium 207 times the sensitivity
466.5
931.5
4.10 of 4.6 mm I.D. columns. MS confirms
acetate, pH 7):(50% acetonitrile in 20mM ammonium acetate, pH 7) 931.5
40
identity of peaks.
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0-100% B in 12.5 min 50 III 20
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 μL/min 669.4
100 649.1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +ESI mode
injection . . . . . . . . . . . . . . . . . . . . 50 pmol angiotensin I, II and III in water 50
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001592
0
0 400 800 1200
0 10 20
Min m/z

LC-MS Analysis of Antipsychotic Drugs on 1. Desmethyl venlafaxine

Ascentis® Express RP Amide 2. Hydroxybupropion
column . . . . . . . . . . . . . . . Ascentis Express RP Amide, 10 cm x 2.1 mm I.D., 4. Bupropion
2.7 μm particles (53913-U) 5. Zuclopenthixol
mobile phase . . . . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate water; 6. Fluphenazine
(B) 5mM ammonium formate in 95:5 (v/v) methanol:water
gradient . . . . . . . . . . . . held at 35% B for 0.5 min; 35 to 85% B in 5.5 min; 6
held at 85% B for 1 min 1
3 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4641 psi (320 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z 100-1000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 ng/mL in waler 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005668
0 2 4 6 8 10
Min

LC-MS Analysis of Benzodiazepines on Ascentis® 1. Demoxepam (1 µg/mL) (M+H)+ = 287.05

Phenyl (Analyte Mix 1) 2. Oxazepam (1 µg/mL) (M+H)+ = 287.05
3. Temazepam (1 µg/mL) (M+H)+ = 301.06
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 4. Clobazam (1 µg/mL) (M+H)+ = 301.06
mobile phase . . . . . . . . (A) 0.1% ammonium acetate in water (pH unadjusted), 5. Diazepam (1 µg/mL) (M+H)+ = 285.07
(B): 0.1% ammonium acetate in acetonitrile, (50:50, A:B)
4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min., split to the MS
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003705 2
0 2 4 6 8 10
Min

LC-MS Analysis of Benzodiazepines on Ascentis® 1. Fenoldopam (10 µg/mL) (M+H)+ = 306.08

Phenyl (Analyte Mix 2) 2. Lorazepam (10 µg/mL) (M+H)+ = 321.01
3. Nitrazepam (10 µg/mL) (M+H)+ = 282.08
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 4. Clonazepam (10 µg/mL) (M+H)+ = 316.04
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min., split to the MS 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003706
0 2 4 6 8 10 12
Min


Clenbuterol
CI
600 CH3
H2N
CI H3C
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
400
cps
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+)
300
sample . . . . . . . . . . . . . . . clenbuterol, 10 ng/mL in rat plasma (phospholipids
removed by extraction with HybridSPE-Phospholipid) Clenbuterol
200
100
0
0 1 2 3 4 5
Min



400 Clenbuterol
CPS
100
0 1 2 3 4 5
Max 775 CPS
6000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4000
CPS
3000
Clenbuterol
2000
1000
0
0 1 2 3 4 5
Min

LC-MS Analysis of Erythromycin Analogs on 1. Erythromycin ethylsuccinate

Ascentis® Express HILIC 2. Erythromycin estolate
3. Erythromycin
This application shows the suitability of Ascentis Express Silica columns for 4. Erythromycin c 3
the analysis of erythromycin analogs in aqueous normal phase/HILIC mode 1
of chromatography. Other relatively hydrophobic aminoglycosides have also
been shown to separate under these conditions. 2 4
mobile phase . . (A) 100 mM ammonium formate, pH 3.0 (titrated with formic acid);
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1247 psi (86 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 2 4 6 8 10 12 14
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+); SIR Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005696

LC-MS Analysis of Pilocarpine and Betaxolol in the 1. Pilocarpine

Presence of Benzalkonium Chlorides on Ascentis® 2. Benzalkonium chloride
1 3. Benzalkonium chloride
Express HILIC 4. Betaxolol
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . (A) 2 mM arrmonium acetate,
pH 6.7 in (B) 10:90 (v/v) water:acetonitrile 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1160 psi (80 bar) 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), XlC, m/z 304, 332 (BACs),
m/z 209 (pilocarpine), m/z 308 (betaxolol) 0 2 4 6 8 10 12 14
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005721

LC-MS Analysis of Sertraline and Norsertraline on 1. Sertraline

1
Ascentis® Express RP-Amide 2. Norsertraline
mobile phase . . . . . . . . . . . . . (A) 10 mM ammonium formate, water, pH 3.6;
(B) 10 mM ammonium formate, acetonitrile, pH 3.6; (65:35, A:B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 100-1000 m/z 0 2 4 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005736

Proteomix WCX-NP1.7, 3 cm x 4.6 mm I.D.,

1.7 μm particles
mobile phase . . . . . . . . (A) 20 mM sodium acetate, pH 5.15; (B) A + 1 M LiCl
gradient . . . . 12% to 40% B in 6.7 min; to 100% B in 0.1 min; held for 2 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1.5 mL/min) Papain digest MAb
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . 3335-3916 psi (230 - 270 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL papain digested MAb 321
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006023
Intact MAb
Papain control
0 1 2 3 4 5 6 7 8 9 10
Min

UHPLC Analysis of p-Acetophenetide (Phenacetin) 1 1. p-Acetophenetide

Titan™ C18
column . . . . . . . TITAN C18, 10 cm x 2. 1 mm I.D., 1.9 um particles (577124-U)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006101
2 4
Min

UHPLC Analysis of Antibiotics/Antimalarials on 1. Dapsone, 2.05 3

Titan™ C18 2. Cinoxacin, 2.93
3. Pyrimethamine, 3.81
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . MS, XIC m/z 249.09, 249.07, 263.14
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006088
0 2 4
Min


1. Uracil
3. Phenobarbital
5. Butalbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 4
5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7540 psi (520 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006073
1.0 2.0 3.0 4.0

Min


3. Cyclobarbital
4. Butalbital
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6796 psi (468 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006137
1 2 3 4 5
Min

UHPLC Analysis of Buspirone on Titan™ C18 1. Buspirone, 6.11 1

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006090
0 2 4 6 8 10
Min

UHPLC Analysis of Cocaine and Cocaethylene on 1. Cocaine, 2.18 1

Titan™ C18 2. Cocaethylene, 3.12
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006091
0 2 4
Min

2
UHPLC Analysis of Corticosteroids on Titan™ C18 1 1. Hydrocortisone
3 2. Cortisone
Analysis of Corticosteroids on a Titan C18 UPLC column with MS detection
3. Corticosterone
in ESI (+) Mode
4. Cortisone Acetate
gradient . . . . . . . . . . . . . . . . . 30% to 60% B in 3 minutes, hold for 0.2 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7423 psi (512 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+) MS TIC SIR
1.0 2.0 3.0 4.0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006147
Min

UHPLC Analysis of p-Coumaric Acid on Titan™ C18 1 1. p-Coumaric Acid, 2.23

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006103
0 2 4
Min

UHPLC Analysis of Desipramine and Imipramine 1. Desipramine, 7.57 2

on Titan™ C18 2. Imipramine, 10.46

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006105
0 2 4 6 8 10 12 14
Min


5
3.303
Titan C18 1.9 mm
phase.
445 bar // 6,450 psi*
3 6
1
2.265
4.299
3.080
1.846
0.447
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6450 psi (445 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 1 2 3 4
1.970
Competitor C18 1.7 µm
1.466
2.545
1.265
0.445
1 2 3 4
Min

UHPLC Analysis of Formoterol on Titan™ C18 1

1. Formoterol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006092
0 2 4
Min

UHPLC Analysis of Hydroquinidine on Titan™ C18 1

1. Hydroquinidine, 2.39
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006094
0 2 4
Min


Detection
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 μL/min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006148
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Min

UHPLC Analysis of Losartan and Quinapril on 1. Losartan

1
2. Quinipril
Titan™ C18
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, XIC m/z 423.17 and 439.22 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006095
0 2 4 6
Min

UHPLC Analysis of Mesoridazine and Haloperidol 1. Mesoridazine, 3.71 2

on Titan™ C18 2. Haloperidol, 7.14
column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U) 1

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006099
0 2 4 6 8
Min

UHPLC Analysis of Methapyrilene and 1 1. Methapyrilene, 2.91

Tripelennamine on Titan™ C18 2. Tripelennamine, 3.73
column . . . . . . . Titan C18, 10 cm x 2. 1 mm I.D., 1.9 um particles (577124-U)

mobile phase . . (A) 10 mM ammonium acetate, pH 7.0; (B) acetonitrile (70:30, A;B) 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006106
0 2 4 6 8 10
Min

UHPLC Analysis of Nevirapine and Imiquimod on 2 1. Nevirapine, 2.12

2. Imiquimod, 3.46
Titan™ C18
column . . . . . . . TITAN C18, 10 cm x 2. 1 mm I.D., 1.9 um particles (577124-U)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006113
0 2 4 6 8
Min

UHPLC Analysis of Nifedipine on Titan™ C18 1 1. Nifedipine, 2.53

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006100
0 2 4 6 8 10
Min

UHPLC Analysis of Paroxetine Titan™ C18 1. Paroxetine, 7.08 1

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006102
0 2 4 6 8 10
Min

UHPLC Analysis of Phenylbutazone on Titan™ C18 1. Phenylbutazone, 10.21

1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006104
2 4 6 8 10 12
Min

UHPLC Analysis of Quinidine and Metabolite on 1. Quinidine

Titan™ C18 2. Hydroxyquinidine (as impurity)
1
mobile phase . . . . . . . . . . (A) 10 mM potassium phosphate, monobasic pH 3.0;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4310 psi (297 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 200 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
sample . . . . . . . . . . . . . . . . . . . Quinidine 5 μg/mL in 80:20, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006218
1.0 2.0
Min

UHPLC Analysis of Riboflavin and Impurities on pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5000 psi (345 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
Titan™ C18 with MS Detection detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI (+), MRM and TIC
Riboflavin (vitamin B2) is a water-soluble vitamin. It is synthesized by all detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diode Array at 276 nm
plants and many microorganisms, but it is not produced by higher animals. injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
It is a precursor of coenzymes that are required for the enzymatic oxidation Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006138
of carbohydrates, so it is essential to basic metabolism. Riboflavin is used as
an additive to food and feed and is also used in fortification of baby food
and cereal. The current USP method for impurity analysis is non-quantitative,
and uses an ion pair reagent in the mobile phase. An alternative to USP and
EP methods using LC-MS/MS to identify and quantify the riboflavin and
Riboflavin
impurities is presented. This new proposed method is also compatible with
UV-Vis detection.
A riboflavin certified reference material (CRM) was used in this study. MRM
transitions for the determination of ions for the parent and four impurites Lumiflavine (7,8,10-trimethylbenzo[g]pteri-
dine-2,4(3H,10H)-dione),
were made. Three transitions were used for each ion. Structures for each of Compound A
these major impurities were confirmed by MS. In addition to improving the
specificity of the method with MS/MS detection, two additional goals were
to decrease analysis time and eliminate the need for ion pairing reagents 7,8-Dimethylbenzo[g]pteri-
that are not compatible with MS. dine-2,4(1H,3H)-dione,
Compound B
Other MS Conditions
Capillary (kV) 2.29
Cone (V) 54.95
Extractor (V) 5.13 6,7-Dimethyl-8-[(2S,3S,4R)-2,3,4,5-tetra-
Source Temperature (°C) 151 hydroxypentyl]pteridine-2,4(3H,8H)-dione,
Desolvation Temperature (°C) 349 Compound C
Cone Gas Flow (L/Hr) 2
Desolvation Gas Flow (L/Hr) 646
Collision Gas Flow (mL/Min) ON 8-(Hydroxymethyl)-7-methyl-10-[(2S,3S,4R)-2,3,4,5-tetra-
column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U) hydroxypentyl]benzo[g]pteridine-2,4(3H,10H)-dione,
Compound D
(B) 0.1% formic acid in acetonitrile (95:5) 0 2 4 6 8 10
gradient . . . . . . . . . . . . . . 5% to 25% in 6 min, return to 5% b in 0.1 min, Min
Compounds B, C, and D are impurities
and re-equilibrate for 4 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 μL/min

UHPLC analysis of Taxanes on Titan C18 Column 1. baccatin III 4. paclitaxel

column . . . . . . . . . . . . . . . . Titan C18 10 cm x 2.1 mm, 1.9 μm (577124-U) 2. 10-deacetyl paclitaxel 5. 10-deacetyl-7-epipaclitaxel
mobile phase . . . . . . . . . . . . . . . (A) water; (B) (50:50, methanol:acetonitrile) 3. cephalomannine 6. 7-epipaclitaxel
gradient . . . . . . . . . . . . . . 60 to 80% B in 4.5 min; 80% B held for 1.5 min 4
1 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 6
5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6090 psi (420 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006139
0 2 4 6
Min

UHPLC Analysis of Temsirolimus on Titan™ C18 1. Temsirolimus 1

Intravenous drug for the treatment of renal cell carcinoma
column . . . . . . . . Titan C18 10 cm x 2.1 mm I.D., 1.9 um particles (577124-U)
mobile phase . . . . . . . . . . . . . . . (A) 0.1% formic acid in water, (B) methanol
gradient . . . . . . 75% B for 2 minutes, 75% B to 90% B from 2 to 12 minutes,
90% B from 12 to 14 minutes.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3300 psi (228 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 276 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ug/mL in 10% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006283 0 2 4 6 8 10 12 14
Min

UHPLC Analysis of Verapamil and Metabolites on 1. Norverapamil

2. Verapamil
Titan™ C18 3. Methoxyverapamil
2
column . . . . . . . . . . . Titan C18 5 cm x 2.1 mm, 1.9 μm particles (577122-U)
mobile phase . . . . . . . . . . (A) 10 mM potassium phosphate, monobasic pH 3.0;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4290 psi (296 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 200 nm 1 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006284 0 2 4
Min

UHPLC-MS Analysis of Antineoplastic and 1. Fluoxetine 2

Antidepressants in Plasma on Titan™ C18 1.9 μm 2. Tamoxifen 1
after SPE using HybridSPE®-PLus

SPE tube/cartridge . . . . . . . . . . . . . . . . . . . . . HybridSPE-PLus 96-Well Plate,
50 mg/2 mL per well (575659-U)
sample addition . . 400 μL of 1% formic acid acetonitile:spiked plasma (3:1) per well
sample preparation . . . . . . . . . . . . . . . . final analyte concentration 50 ng/mL
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate;
(B) 5 mM ammonium formate in 90:10 (v:v) acetonitrile:water
gradient . . . . . . . . . . . . . 35 to 90% B in 1.5 min, held at 90% B for 0.4 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 1.0 2.0
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006151

6
UHPLC-MS Analysis of Benzodiazepines and 1. Oxazepam glucuronide (463 m/z)
2. Lorazepam glucuronide (497 m/z)
Glucuronide Metabolites on Titan™ C18
3. Temazepam glucuronide (477 m/z)
column . . . . . . . . . . . Titan C18 5 cm x 3.0 mm, 1.9 μm particles (577126-U) 4. Oxazepam (287 m/z) 4
mobile phase . . . . . . . . . . . . . (A) 0.1% formic acid in 95:5, water:acetonitrile; 5. Lorazepam (321 m/z)
(B) 0.1% formic acid in 5:95, water:acetonitrile; 6. Temazepam (301 m/z) 5
gradient . . . . . . . . . . . . . 35% B to 60% B in 1 min; 60% B held for 0.5 min 1 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5800 psi (400 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . MS-TOF, ESI+, XIC, 100 -1000 m/z scan
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0.2 0.4 0.6 0.8 1.0 1.2 1.4
sample . . . . . . . . . . . . . . . . . . . . . . . . 300 ng/mL in 97:3, water:methanol
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006150

UHPLC-MS Analysis of Spice Cannabinoid 1. JWH-250 5-Hydroxypentyl metabolite (352.21 m/z)

13
Metabolites from Urine Using Titan™ C18 2. JWH-250 4-Hydroxypentyl metabolite (352.21 m/z)
3. JWH-073 4-Butanoic acid metabolite (358.16 m/z)
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 4. JWH-073 4-Hydroxybutyl metabolite (344.18 m/z)
5. JWH-018 5-Pentanoic acid metabolite (372.17 m/z) 11
SPE well plate . . . . . . . . SupelSelect HLB 96-Well Plate, 60mg/well (575662-U) 6. JWH-073 3-Hydroxybutyl metabolite (344.18 m/z)
10
condition . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol, then 1 mL water 7. JWH-018 5-Hydroxypentyl metabolite (358.20 m/z)
12
8. AM2201 4-Hydroxypentyl metabolite (376.19 m/z)
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ng/mL in matrix 9. JWH-018 4-Hydroxypentyl metabolite (358.19 m/z)
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . none 10. JWH-122 5-Hydroxypentyl metabolite (372.21 m/z)
11. JWH-122 4-Hydroxypentyl metabolite (372.21 m/z)
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 20% methanol in water 12. JWH-210 5-Hydroxypentyl metabolite (386.23 m/z) 2
1
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL methanol 13. JWH-210 4-Hydroxypentyl metabolite (386.23 m/z)
eluate post-treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . none 8
column . . . . . . . . . . . Titan C18 10 cm x 2.1 mm, 1.9 μm particles (577124-U) 4 9

6
mobile phase . . . . . . . . . . . . . (A) 0.1% formic acid in 95:5, water:acetonitrile;
(B) 0.1% formic acid in 5:95, water:acetonitrile; 7
gradient . . . . . . . . . . . . . . . . . 25% B held for 1 min; to 45% B in 0.5 min; 3

45% B held for 4.5 min; 5

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8756 psi (604 bar)
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ng/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006307

UHPLC-MS Analysis of Spice Cannabinoids and 1. JWH-073 4-Butanoic acid metabolite 9

Metabolites Using Titan™ C18 2.
3.
JWH-073 4-Hydroxybutyl metabolite
JWH-018 5-Pentanoic acid metabolite
column . . . . . . . . . . . Titan C18 10 cm x 2.1 mm, 1.9 μm particles (577124-U) 4.
5.
JWH-073 3-Hydroxybutyl metabolite
JWH-018 5-Hydroxypentyl metabolite
mobile phase . . . . . . . . . . . . . (A) 0.1% formic acid in 95:5, water:acetonitrile; 6. JWH-018 4-Hydroxypentyl metabolite
(B) 0.1% formic acid in 5:95, water:acetonitrile; 7. AM2201
8. JWH-073 8
gradient . . 45% B held for 4.5 min; to 80% B in 1.5 min; 80% B held for 4 min 7
9. XLR-11
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min 10. JWH-018
11. JWH-081
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7208 psi (497 bar) 12. JWH-122 14
11
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 13. JWH-019 10 12
14. UR-144 13
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 2 4
sample . . . . . . . . . 14 Spice cannabinoids 300 ng/mL in 80:20, water:methanol 6
5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006185 1 3
0 2 4 6 8 10
Min

UHPLC-MS Analysis of Spice Cannabinoids Using 1. AM2233 (459.11 m/z)

2, 3
2. RCS-4 (322.19 m/z)
Titan™ C18 3. JWH-015 (328.18 m/z)
4. JWH-073 (328.18 m/z)
column . . . . . . . . TITAN C18, 100 mm x 2.1 mm x 1.9 μm particles (577124-U) 5. JWH-203 (340.164 m/z)
mobile phase . . . . . . . . . . . . . (A) 0.1% formic acid in 95:5, water:acetonitrile; 6. RCS-8 (376.246 m/z)
7. JWH-210 (370.23 m/z)
(B) 0.1% formic acid in 5:95, water:acetonitrile;
gradient . . 25% B held for 1 min; to 45% B in 0.5 min; 45% B held for 4.5 min;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
1
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8756 psi (604 bar) 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 100 ng/mL in 60:40, water:methanol 5 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006308
0 2 4 6 8 10
Min

UHPLC-MS/MS Analysis of Various Drug 3 1. Metoprolol

Substances on Titan™ C18 1.9 μm 2. Popranolol
3. Carbamazepine
column . . . . . . . . . Titan C18, 5 cm x 2.1 mm I.D., 1.9 μm particles (577122-U) 4. Diazepam
(B) 5 mM ammonium formate in 90:10 (v:v) acetonitrile:water 2
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 to 70% B in 3.0 min 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS/MS
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 μL 4
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 ng/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006149
0 2
Min

UHPLC Separation of Temsirolimus and Sirolimus 1. Temsirolimus 2

on Titan™ C18 2. Sirolimus
1
column . . . . . . . . . . . Titan C18 10 cm x 2.1 mm, 1.9 um particles (577124-U)
mobile phase . . . . . . . . . . . . . . . (A) 0.1% formic acid in water, (B) methanol
gradient . . . . . . 75% B for 2 minutes, 75% B to 90% B from 2 to 12 minutes,
90% B from 12 to 14 minutes.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3300 psi (228 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 276 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . 100 ug/mL each in 20% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006270
0 2 4 6 8 10 12 14
Min

USP HPLC Analysis of Ampicillin Sodium on 1. Ampicillin Sodium

1
Ascentis® Express 5μm C18
This application was generated by RTC on an Ascentis Express C18.
column . . Ascentis Express C18, 10 cm × 4.6 mm I.D., 5 μm particles (50536-U)
mobile phase . . . . (A:B:C:D) (A) acetonitrile,(B)water, (C)1M Potassium phosphate
monobasic in water, (D)1N acetic acid in water (80:909:10:1)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006262
1.0 2.0
Min

USP HPLC Analysis of L-Methionine on Ascentis® 1. L-Methionine 1

Express OH5
This USP application was generated by RTC on an Ascentis® Express OH5
column.
column . . . . . . Ascentis Express OH5 15 cm x 4.6 mm, 5 μm particles (50347-U)
mobile phase . . . . . . . . . . . . . . . . . (A)0.005M Ammounium formate in Water
(pH: 3.15 with Trifluoroacetic Acid) ), (B)Acetonitrile (30:70, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914 psi (63 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agilent 380-ELSD
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mg/mL water:acetonitrile (1:1) 0 2 4

HPLC, UHPLC or LC-MS Vitamins, Nutraceuticals, and Natural Products
Vitamins, Nutraceuticals, and Natural 1. Yohimbine
Products
1
HPLC Analysis of Alkaloids from Herbal
Supplements on Ascentis® Express F5, HILIC Mode
column . . . Ascentis® Express F5, 3 cm x 3.0 mm I.D., 2.7 μm particles (53574-U)
pH to 6.0 with acetic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1100 psi (76 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 275 nm 0 2 4 6 8 10
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL Min
sample . . . . . . . . . . . . . . . methanolic extract of Yohimbe Herbal Supplement
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005905

HPLC Analysis of Alkaloids on Discovery® Zr-PBD 1. Codeine

column . . . . . Discovery Zr-PBD, 15 cm × 4.6 mm I.D., 5 μm particles (65723-U) 2. Strychnine
1 3. Papaverine
mobile phase . . . . . . . . . . . . . . . . (A) 20 mM potassium phosphate (pH 12): 4. Quinine
(B) acetonitrile; (90:10, A:B) 5. Quinidine
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.35 mL/min 65 °C, 12 min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 °C 4
3 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL each compound 50 μg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001840
0 2 4 6 8 10 12 14
Min

1. Dehydroascorbic acid (100 µg/mL) 2

HPLC Analysis of Ascorbic Acid on Ascentis® RP- 2. Ascorbic acid (20 µg/mL)
Amide
mobile phase . . . . . . . . . . . . . . . . . . 25 mM monobasic potassium phosphate
(pH 3.0 with phosphoric acid)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 1
sample . . . . . . . . . . . . . . . . . . . . . . . . . as indicated each in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002722 1.0 2.0 3.0 4.0 5.0
Min

HPLC Analysis of Aswagandha Extract on 1. Withanoside IV

Discovery® C18 2. Withanoside V
3. Withaferin A
This application demonstrates the suitability of Discovery C18 for the 4. 12-Deoxywithastramonolide
efficient separation of withania extract by the USP monograph. 5. Withanolide A
column . . . . . . . . Discovery C18, 25 cm x 4.6 mm I.D., 5 μm particles (504971) 2
6. Withanolide B
mobile phase . . . . . . (A) 1 mM potassium phosphate, monobasic; (B) acetonitrile
gradient . . 5 to 45% B in 18 min; 45 to 80% B in 7 min; held at 80% B for 3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 1 34 5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3191 psi (220 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 °C 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL in water: acetonitrile (86:14)
0 10 20
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005669
Min

HPLC Analysis of Benzoic Acid Derivatives on 1. 4-Hydroxybenzoic acid

Ascentis® C18 2. Acetylsalicylic acid
3. Benzoic acid
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 4. 2-Hydroxybenzoic acid (Salicylic acid)
mobile phase . . (A) 10mM TFA in water; (B) 10 mM TFA in acetonitrile; (70:30, A:B) 5. Ethylparaben (Ethyl 4-hydroxybenzoate)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005676 5
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Benzoin Methyl Ether

Enantiomers on Astec® CHIROBIOTIC® V O
*
mobile phase . . . . . . . . . . (A) 1% triethylamine, pH 7.0; (B) THF; (90:10, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
CH
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004618
0 2 4 6 8 10 12
Min

HPLC Analysis of Biotin Enantiomers on Astec® H

N
H
CHIROBIOTIC® T O S
*
H H
mobile phase . . . (A) 0.1% sodium phosphate, pH 4.1; (B) methanol; (60:40, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 °C
O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004540
0 2 4 6 8
Min

HPLC Analysis of Carotene Compounds on 1. Astaxanthin (M+H)+ = 597.38

Ascentis® RP-Amide 2. Xanthophyll (M+H)+ = 569.42
2
3. Zeaxanthin (M+H)+ = 569.42
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 1 4. Lycopene (M+H)+ = 537.43
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100% acetonitrile 5. α-carotene (M+H)+ = 537.43
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 6. β-carotene (M+H)+ = 537.43
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . MS, ESI(+) in selective ion monitoring (SIR) mode 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 6
sample . . . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL each in 100% acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003138 4
0 10 20
Min

HPLC Analysis of Carotenes on Ascentis® Express 2

1. Alpha-carotene
RP-Amide, Methanol Mobile Phase 2. Beta-carotene
Analysis was conducted using juice extract, detection via UV.
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653 psi (114 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV-VIS, 454 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . V8 Splash® fruit juice was extracted with hexane,
the extract dissolved 1:1 into ethanol for HPLC injection 0 2 4 6 8 10 12 14
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005859
Min

HPLC Analysis of Carotenoids by Normal Phase 1. Astaxanthin

Chromatography on Ascentis® Si 2. Xanthophyll 2
Ascentis silica is used in the analysis of naturally occurring carotenoid 1
pigments in the form of alcohols, xanthophyll and astaxanthin.
mobile phase . . . . (A) hexane; (B) ethyl acetate; (C) isopropanol; (85:10:5, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 450 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50-100 μg/mL hexane Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005685

HPLC Analysis of Carotenoids on Ascentis® 1. Astaxanthin (100 μg/mL)

2. Xanthophyll (50 μg/mL
Si (Silica), Normal Phase 2
column . . . . . . . . Ascentis Si, 15 cm × 4.6 mm I.D., 5 μm particles (581512-U) 1
mobile phase . . . . (A) hexane: (B) ethyl acetate: (C) isopropanol; (85:10:5, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in hexane
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003813
0 2 4 6 8 10
Min

HPLC Analysis of Carotenoids on Ascentis® Si 1. Astaxanthin

2. Xanthophyll
2
mobile phase . . . . (A) hexane; (B) ethylacetate; (C) isopropanol; (85:10:5, A:B:C:) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in hexane
0 2 4 6 8 10

HPLC Analysis of Catechins on Ascentis® RP-Amide 1. Gallocatechin, m/z 305 5. Epigallocatechin gallate, m/z 457
2. Epigallocatechin, m/z 305 6. Gallocatechin gallate, m/z 457
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 3. Catechin, m/z 289 7. Epicatechin gallate, m/z 441
mobile phase . . . . . . . . . . . . . . (A) 10 mM ammonium formate (pH 3.0 with 4. Epicatechin, m/z 289 8. Catechin gallate, m/z 441
concentrated formic acid); (B) acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Gradient Program
Time (min) %A %B
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 85 15
detector . . . . . . . . . . . . . . . MS, ESI (-) in selected ion recording (SIR) mode 1 85 15 8
10 55 45
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 12 85 15 7
sample . . . . . . . . . . . . . . . . . . . . . . 10 μg/mL each in water:acetonitrile 1:1 3 4 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002723
5
1 2
0 2 4 6 8 10 12
Min

HPLC Analysis of Chinese Ginseng Extract on

4 1. Ginsenoside Re
This application demonstrates the suitability of Ascentis Express C18 for the 2. Ginsenoside Rg1
3. Ginsenoside Rf
efficient separation of Chinese ginseng root extract. 4. Ginsenoside Rb1
5
2 6
5.
6.
Ginsenoside Rc
Ginsenoside Rb2
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile 7. Ginsenoside Rd
gradient . . held at 25% B for 1.5 min; 25 to 85% B in 12 min; held at 85% for 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3481 psi (240 bar)
7
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
sample . . . . . . . . . . Extracted from dried, ground plant material; 10 mg/mL in
water:acetonitrile (85:15); sonicate 10 min at ambient
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005396
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Conjugated Flavonoids on 1. Maysin 1

Discovery® HS-C18 2. Apimaysin
3. Methoxymaysin
mobile phase . . . . . (A) 10 mM sodium citrate (pH 7.0 with 10 mM citric acid); 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 340 nm 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL Min
sample . . . . . . . . . . . . 50 μg/mL each (Maysin, Apimaysin, Methoxymaysin) in
10 mM sodium citrate (pH 7.0 with 10 mM citric acid)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002048


CH3
H3C
Hydroxyvitamin D3 on Ascentis® Express F5, 20 °C CH3
H3C
OH H3C OH
CH3 H
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) CH3
mobile phase . . . . . . (A) 5 mM ammonium formate; (B) methanol:water (75:25)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min H
H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C CH2 H
detector . . . . . . . . . . . . . . . . . . . . . (UV, 265 nm, ESI(+), m/z 100 - 1000) CH2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
HO OH
HO
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005375
HO
H3C CH3
3
CH3 H 2
CH3
1
H
H
H
CH2
HO
0 2 4 6
Min


CH3 H3C
25-Hydroxyvitamin D3 and 3-epi-25- H3C CH3 H3C OH
Hydroxyvitamin D3 on Ascentis® Express F5, 30 °C CH3 OH CH3 H
H3C CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min H H
H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C CH2
CH2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 265 nm
ESI(+), m/z 100 - 1000 2. 25-Hydroxyvitamin D3
HO
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL O OH
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005376
HO
H3C CH3 3
CH3 H 2
CH3
1
H
H
H
CH2
HO
0 2 4
Min


CH3
CH3 OH H3C
Hydroxyvitamin D3 on Ascentis® Express F5, 40 °C H3C H3C OH
CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C CH2 H
H
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 265 nm
CH2
ESI(+), m/z 100 - 1000 HO OH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 300 mg/L in 25:75, water:methanol HO
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005377 3. epi-25-Hydroxyvitamin D3
HO
H3 C CH3
3
CH3 H
CH3 2
H
H 1
H
CH2
HO
0 2 4
Min

HPLC Analysis of Dioxibrassinin Enantiomers on 5.34

Astec® P-CAP™ CH3
column . . Astec (R,R) P-CAP-DP, 25 cm x 4.6 mm I.D., 5 μm particles (35024AST) 6.55 S
HO
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min S
NH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 255 nm O
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
N
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg/mL in methanol H
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004400

1. α-Tocopherol (100 μg/mL)

HPLC Analysis of Fat Soluble Vitamins by Gradient Program
Time (min) %A %B 2. Menadione (150 μg/mL)
Normal Phase Chromatography on Ascentis® Si, 0
10
90 10
70 30 1
3.
4.
γ-Tocopherol (200 μg/mL)
Chlolecalciferol (100 μg/mL)
4
15 cm Column 12 70 30
13 90 10
column . . . . . . . . Ascentis Si, 15 cm × 4.6 mm I.D., 5 μm particles (581512-U)
3
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . (A) hexane, (B) ethyl acetate
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . as indicated in 96:4 (hexane:isopropanol)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003693
0 2 4 6 8 10 12
Min

HPLC Analysis of Fat Soluble Vitamins by 1. alpha-Tocopherol

Normal Phase Chromatography on Ascentis® Si 2. Menadione
3. gamma-Tocopherol
(25 cm Column) 4. Chlolecalciferol
Ascentis silica is used in the analysis of fat-soluble vitamins using a normal
phase gradient. These include vitamin K 3 (menadione), vitamin E (alpha and 1 4
gamma tocopherol), and vitamin D3 (cholecalciferol).
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . (A) hexane; (B) ethyl acetate 2
gradient . . . . . . . . . . . . . . 10 to 30% B in 10 min; held at 30% B for 2 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
0 2 4 6 8 10 12
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005697
Min

HPLC Analysis of Fat Soluble Vitamins on Ascentis® 1. Ergocalciferol (D2) 5
Express RP-Amide, Methanol Mobile Phase 2. Cholecalciferol (D3)

3. DL-Alpha-tocopherol acetate (E)
Analysis was conducted using standard solution, detection via UV. 4. Alpha-tocopherol (E)
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 15 cm x 4.6 mm I.D., 5. Phylloquinone (K1)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . methanol 4
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653 psi (114 bar)
1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . 0.4 mg/mL tocopherol acetate, alpha-tocopherol 2 4 6
and K1, 0.04 mg/mL D2 and D3, all in ethanol
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005862

HPLC Analysis of Fat Soluble Vitamins on Ascentis® 1. Vitamin D3

2
2. Vitamin E
RP-Amide
The Ascentis RP-Amide is shown to be suitable for the analysis of fat soluble
vitamins, alpha tocopherol (vitamin E) and cholecalciferol (vitamin D3 ). 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 215 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 0 2 4 6 8 10 12 14

HPLC Analysis of Fat-Soluble Vitamins on 1. Retinol acetate (A)

Ascentis® Express C18, Methanol Mobile Phase 2. Delta-tocopherol (E)
3. Ergocalciferol (D2)
Analysis was conducted using standard solution, detection via UV. 4. Cholecalciferol (D3)
column . . Ascentis Express C18, 15 cm x 4.6 mm I.D., 2.7 μm particles (53829-U) 5. Alpha-tocopherol (E)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . methanol 6. DL-alpha-tocopherol acetate (E)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 7. Phylloquinone (K1)
8. Retinol palmitate (A)
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3249 psi (224 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 2 4 7
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . 0.5 mg/mL tocopherol acetate, alpha-tocopherol, 6
3
delta-tocopherol and K1, 0.05 mg/mL D2, D3 and 1 8
retinol acetate, 1.5 mg/mL retinol palmitate, all in ethanol.
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005861 0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Fat Soluble Vitamins on Ascentis® 1. Retinol acetate (A)

Express RP-Amide, Acetonitrile Mobile Phase 2. DL-Alpha-tocopherol acetate (E)
3. Phylloquinone (K1)
Analysis was conducted using standard solution, detection via UV.
4. Ergocalciferol (D2)
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 15 cm x 4.6 mm I.D., 5. Cholecalciferol (D3)
2.7 μm particles (53931-U) 1 6. Alpha-tocopherol (E)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . acetonitrile 7. Delta-tocopherol (E)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 8. Retinol palmitate (A)
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1682 psi (116 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 8
sample . . . . . . . . . . . . . . . . 0.5 mg/mL tocopherol acetate, alpha-tocopherol, 2
delta-tocopherol and K1, 0.05 mg/mL D2, D3 and 5 6
retinol acetate, 1.5 mg/mL retinol palmitate, all in ethanol 4 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005860
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Flavanone Enantiomers on O

Astec® CYCLOBOND® I 2000 DMP, Ethanol:Hexane
Mobile Phase
mobile phase . . . . . . . . . . . . . . . . . . . (A) ethanol; (B) hexane; (20:80, A:B) O *
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004697
0 2 4 6
Min

HPLC Analysis of Flavanone Enantiomers on

Astec® CYCLOBOND® I 2000 DMP, IPA:Heptane
Mobile Phase
1
mobile phase . . . . . . . . . . . . . . . . (A) isopropanol; (B) heptane; (30:70, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 1. Flavanone enantiomer
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 2. Flavanone enantiomer
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . flavanone, 1 mg/mL in heptane
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005161
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Flavonoids on Ascentis® 1. Gallic acid 6. Rutin Gradient Program

2. (+) catechin 7. p-coumaric acid Time (min) %A %B
RP-Amide 3. Syringic acid 8. Quercitrin 0 85 15
4. Vanillic acid 9. Myricetin
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 5. Caffeic acid 10. Quercetin
30 65 35
35 15 85
mobile phase . . . . . . . . . . . . (A) 0.085% orthophosphoric acid; (B) acetonitrile;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 1
7
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 5
10
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . as indicated in mobile phase A 9
2 6 8
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003025
0 10 20 30 40
Min

HPLC Analysis of Folinic Acid Enantiomers on O OH

O
NH
OH
mobile phase . . (A) methanol; (B) acetic acid; (C) triethylamine; (100:1:0.5, A:B:C) N N *
NH
O
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min

H2N N N
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C H H
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004541
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Folinic Acid Enantiomers on

O OH
Astec® CHIROBIOTIC® T (RP mode)
O
column . . . . . . CHIROBIOTIC T, 25 cm x 4.6 mm I.D., 5 μm particles (12024AST) O OH
O NH
mobile phase . . . (A) 1% triethylamine acetate, pH 4.5; (B) ethanol; (50:50, A:B) N * O
N NH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C H2N N
H
N
H
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004542
0 2 4 6 8
Min

HPLC Analysis of Furocoumarins on Ascentis® 1

1. Xanthotoxin
Express RP-Amide 2. Psoralen
3. Angelicin
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 10 cm x 4.6 mm I.D., 4. Bergapten
2.7 μm particles (53929-U) 5. Isobergapten
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2843 psi (196 bar) 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 4
5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 25 μg/mL in water:methanol (90:10)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005394 0 2 4
Min

3
HPLC Analysis of Ginsenosides on Ascentis®
4 1. Ginsenoside Re
Express C18 5 2. Ginsenoside Rg1
7
column . . Ascentis Express C18, 15 cm x 4.6 mm I.D., 2.7 μm particles (53829-U) 3.
4.
Ginsenoside Rf
Ginsenoside Rb1
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile 2 6 5. Ginsenoside Rc
6. Ginsenoside Rb2
gradient . . 25% B for 1.5 min; 25 to 85% B in 12 min; hold at 85% B for 1 min 1
7. Ginsenoside Rd
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005443
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Ginsenosides on Ascentis® 1. Ginsenoside Re 5. Ginsenoside Rc

Express 5 μm 2. Ginsenoside Rg1 6. Ginsenoside Rb2
3. Ginsenoside Rf 7. Ginsenoside Rd
column . . . Ascentis Express C18, 15 cm x 4.6 mm I.D., 5 μm particles (50537-U) 4. Ginsenoside Rb1
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water, (B) acetonitrile 3 7
6
gradient . . . . 25% B held for 3 min; to 85% B in 12 min;85% B held for 1 min 4 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
1 2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1470 psi (101 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 82:18, water:acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005888
0 2 4 6 8 10
Min

3
HPLC Analysis of Ginsenoside Standards on
4 1. Ginsenoside Re
Ascentis® Express C18 5 2. Ginsenoside Rg1
7 3. Ginsenoside Rf
This application demonstrates the suitability of Ascentis Express C18 for the 4. Ginsenoside Rb1
efficient separation of ginseng. 2 6 5. Ginsenoside Rc
6. Ginsenoside Rb2
7. Ginsenoside Rd
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A): water; (B): acetonitrile
gradient . . held at 25% for 1.5 min; 25 to 85% B in 12 min; held at 85% B for 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3481 psi (240 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 205 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
sample . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in water:methanol (82:18)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005397 0 2 4 6 8 10 12 14
Min

HPLC Analysis of Herbal Supplement Containing 2 1. Taxifolin

2. Silychristin
Milk Thistle on Ascentis® Express C18 3. Apigen 7-glucoside
4. Silydianin
This application demonstrates the suitability of Ascentis Express C18 for the 5. Quercitrin
efficient separation of milk thistle containing herbal supplement. 6. Silybin A
7. Silybin B
column . . Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 μm particles (53814-U) 8. Isosilybin A
9. Isosilybin B
(B) methanol
7
gradient . . . . . . . . . . . . . . held at 35% B for 3 min; 35 to 45% B in 10 min;
held at 45% B for 2 min; 45 to 100% B in 5 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 6
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4105 psi (283 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 8
3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 1 4
sample . . . . . . . . . . . 20 mg/mL in water:ethanol (5:95); sonicate 15 minutes; 9
filter 0.45 μm; dilute to water:ethanol (80:20) 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005604
0 10 20
Min

1
HPLC Analysis of Horn Goat Weed Standards 1. Hyperoside
2. Epimedin A
Using Ascentis® Express C18 5 μm 3. Epimedin B
4. Epimedin C
column . . . Ascentis Express C18, 15 cm x 4.6 mm I.D., 5 μm particles (50537-U) 5. Icariin
6. Icaritin
mobile phase . . (A) 0.1% acetic acid in water; (B) 0.1% acetic acid in acetonitrile;
gradient . . . . . . . . . . . . . . . 5% B held for 0.75 min; to 40% B in 3.75 min; 4
to 100% B in 3.5 min; 100% B held for 2 min 3 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 6
2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2120 psi (146 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL 70:30, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006310
0 2 4 6 8 10
Min

HPLC Analysis of 25-Hydroxyvitamin D3 and 1. 25-Hydroxyvitamin D2
25-Hydroxyvitamin D2 on Ascentis® Express F5 H3C

H 3C OH
mobile phase . . . . . . (A) 7 mM ammonium formate; (B) methanol; (25:75, A:B) CH 3 H
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
ESI(+), TIC m/z 100 - 1000 H
H
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL H CH3
sample . . . . . . . . . . . . . . . . . . . . . . . . 300 mg/L in 25:75, water:methanol CH3
H3C
CH2
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) CH3 OH
H3 C
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005374
HO
H
1
CH2 2
HO
0 1.0 2.0 3.0 4.0

Min

HPLC Analysis of 25-Hydroxyvitamin D3 and epi- 1. 25-Hydroxyvitamin D3 2. epi-25-Hydroxyvitamin D3

25-Hydroxyvitamin D3 on Ascentis® Express F5 H3C H3 C
HO
CH3
H3C OH
CH3 H
mobile phase . . . . . . (A) 7 mM ammonium formate; (B) methanol; (25:75, A:B) CH3 CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 265 nm H H
H H
ESI(+), m/z 100 - 1000 H
H
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL CH2
CH2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005378 HO 2
HO
1
0 2 4
Min


Ascentis® Express RP Amide (Milk Brand 1) 1.0 μg/mL standard
This application demonstrates the separation of seven isoflavones on
4 5
gradient . . held at 15% B for 1 min; 15 to 100% in 5 min; held at 100% B for 1 min; 1 2
100-15% B in 0.1 min; held at 15% B for 2.9 min 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2900 psi (200 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10
sample . . . . . . . soy milk, methanol extract (1. Measure 2 mL of each brand of Min
soy milk (6 samples) 2. Add 8 mL of methanol and shake for 1 hour 3.
3 1. Daidzin
Centrifuge tubes at 10000 rpm for 30 minutes 4.
2. Glycitin
Transfer aliquot to autosampler vial)
3. Genistin
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005918 4. Glycitein
5. Daidzein
6. Equol
Silk® Soy Milk 7. Genistein
1
2 7
0 2 4 6 8 10
Min


Ascentis® Express RP Amide (Milk Brand 2)
This application demonstrates the separation of seven isoflavones on 1.0 μg/mL standard
4 5
gradient . . held at 15% B for 1 min; 15 to 100% in 5 min; held at 100% B for 1 min; 1 2
100-15% B in 0.1 min; held at 15% B for 2.9 min 6
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2900 psi (200 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 260 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10
Min
sample . . . . . soy milk, methanol extract (1. Measure 2 mL of each brand of soy
milk (6 samples) 2. Add 8 mL of methanol and shake for 1 hour 3. 1. Daidzin
Centrifuge tubes at 10000 rpm for 30 minutes 4. 2. Glycitin 3
Transfer aliquot to autosampler vial) 3. Genistin
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005917 4. Glycitein
5. Daidzein
6. Equol
7. Genistein
8th Continent® Soy Milk
1 5
2 4
0 2 4 6 8 10
Min

1. Daidzin (M+H)+ = 417.11 5. Glycitein (M+H)+ = 285.06

HPLC Analysis of Isoflavones on Ascentis® Phenyl 2. Glycitin (M+H)+ = 447.12 6. Equol (M+H)+ = 243.09
3. Genistin (M+H)+ = 433.10
column . . . . . Ascentis Phenyl, 15 cm × 4.6 mm I.D., 5 μm particles (581616-U) 4. Daidzein (M+H)+ = 255.05
7. Genistein (M+H)+ = 271.05
mobile phase . . . . . . . . . . . . . . (A) 10 mM ammonium formate (pH 3.0 with

concentrated formic acid), (B) methanol Gradient Program
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min split to the MS Time (min) %A %B 4 5
0 60 40
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 16 0 40
detector . . . . . . . . . . . . . . . . MS, ESI(+) in selected ion recording (SIR) mode 15 0 100
16 60 40
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 20 60 40
sample . . . . . . . glycitein, genistein at 1 μg/mL, equol at 50 μg/mL and daidzin,
glycitin, genistin at 10 μg/mL in 60:40 10 mM ammonium 1
2 7
6
formate (pH 3.0 with concentrated formic acid):methanol
3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003868
0 2 4 6 8 10
Min

HPLC Analysis of Lactic Acid Enantiomers on CLC-L CLC-D

Astec® CLC-L and CLC-D (D)-Lactic acid, (L)-Lactic acid,
9.24 min 9.27 min
column . . . . . . . . . . . . . . . . . Astec CLC-L and CLC-D, 15 cm x 4.6 mm I.D.,
5 μm particles (53023AST, 53123AST) (L)-Lactic acid, (D)-Lactic acid,
12.58 min 12.18 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C O
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm H3C

OH
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
OH
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004399


12.58 min.
*
CH3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min O
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
9.24 min.
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004663

HPLC Analysis of Maté Leaves on Ascentis® * 1. Gentisic acid

2. 3,4-Dihydroxy-phenylacetic acid
14. Feruloylquinic acid (4-FQA)
15. Rutin
25
Express C18 22
23
24
3. Theobromine
4. Neo-chlorogenic acid (3-CQA)
16. Quercetin-3-O-glycoside
17. Kaempferol-3-O-glycoside
5. Dicaffeic acid 18. Dicaffeoylquinic acid (3,4-di-CQA)
sample/matrix . . UAE (Ultrasonic Extraction) maté extract (dried and minced leaves 26 6. Coumaroylquinic acid (CQA) 19. Dicaffeoylquinic acid (3,5-di-CQA)
(7.5 g) from maté were extracted with 225 mL of methanol 7. Chlorogenic acid (5-CQA)
8. Caffeine
20. Dicaffeoylquinic acid (4,5-di-CQA)
21. Caffeoylferuloylquinic acid (3F,4C-QA)
in an ultrasonic bath, thermostated at 75 ± 0.5 °C 9. Dicaffeic acid 22. Dicaffeoylquinic acid (1,5-di-CQA)
10. Crypto-chlorogenic acid (4-CQA) 23. Caffeoylsinapylquinic acid (CSQA)
(potency: 90 W; frequency: 40 kHz)) 11. Caffeoylquinic acid (1-CQA) 24. Caffeoylferuloylquinic acid (3C,4F-QA)
column . . . Ascentis Express C18, 150 x 4.6 mm I.D., 2.7 μm particles (53829-U) 41.0 12. Feruloylquinic acid (3-FQA) 25. Caffeoylferuloylquinic acid (3F,5C-QA)
Min 13. Feruloylquinic acid (5-FQA) 26. Caffeoylferuloylquinic acid (4C,5F-QA)
mobile phase . . . . . . . . . . . . . . . . . . . . . (A) water, pH 3 with formic acid;
(B) acetonitrile, pH 3 with formic acid
gradient . . . . . . . . . . . . . . . . . 0 to 5% B in 20 min; 5 to 40% B in 30 min 4 7 8 10 15 18 19 20
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . PDA: range 215- 420 nm (8 μL detector cell volume,
cell temp. 40 °C, sampling rate 12.5 Hz, time constant 0.025 s)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 3
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005594
17
9
5 13
11
16 21 *
6 12 14
0 20 40
Min

HPLC Analysis of Milk Thistle Standards on 1. Taxifolin

Ascentis® Express C18 2. Silychristin
3 3. Apigen 7 -glucoside
This application demonstrates the suitability of Ascentis Express C18 for the 4. Silydianin
efficient separation of milk thistle related compounds. 5 5. Quercitrin
column . . . Ascentis Express C18, 10 cm x 3 mm I.D., 2.7 μm particles (53814-U) 6. Silybin A
mobile phase . . . . . . . . . . . . . (A) water with 0.1% formic acid; (B) methanol 7. Silybin B
gradient . . . . . . . . . . . . . . held at 35% B for 3 min; 35 to 45% B in 10 min; 8. Isosilybin A
held at 45% B for 2 min; 45 to 100% B in 5 min 9. Isosilybin B
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 1 2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4105 psi (283 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4 6 7 89
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL in 80:20, water:methanol 0 10 20
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005445
Min


3. Citric acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2 5
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005714
1.0 2.0 3.0 4.0 5.0
Min


RP-Amide 2. Lactic acid
3. Citric acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 2 5
3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005715
1.0 2.0
Min


4
2. Sorbic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797-0657
0 2 4 6
Min


mobile phase . . . . . (A) 10 mM ammonium phosphate monobasic (pH 2.50 with
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003160 0 2 4 6 8 10 12 14
Min


column . . . . . . . . Discovery C18, 5 cm × 4.6 mm I.D., 5 μm particles (504947) 1. Homovanillic acid (0.0625 μg/mL)
2. Sorbic acid (0.00625 μg/mL)
mobile phase . . . . . . . . . . . (A) 0.1% TFA in (B) water:methanol; (60:40, A:B) 3. Salicylic acid (0.0625 μg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min 4. p-Toluic acid (0.00625 μg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001431 1 3
4
1.0 2.0 3.0

Min

1. Propyl gallate (10 µg/mL)

HPLC Analysis of Phenolic Antioxidants on 2. Ethoxyquin (10 µg/mL)
Gradient Program
Time (min) %A %B
Ascentis® C8 3. 2,4,5-trihydroxybutyrophenone (10 µg/mL)
4. tert-butylhydroquinone (10 µg/mL) 0 70 30
10 0 100
column . . . . . . . . Ascentis C8, 15 cm x 4.6 mm I.D., 5 μm particles (581424-U) 5. Nordihydroguaiaretic acid (10 µg/mL)
6. 3-tert-butyl-4-hydroxyanisole (10 µg/mL) 15 0 100
mobile phase . . . . . . . . . . (A) 5% acetic acid; (B) (50:50) methanol:acetonitrile 7. 2,6-di-tert-butyl-4-hydroxymethylphenol (10 µg/mL)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min 8. Octyl gallate (10 µg/mL)
9. Lauryl gallate (10 µg/mL)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 10. 3,5-di-tert-butyl-4-hydroxytoluene (10 µg/mL)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm 3 8
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 1 9
sample . . . . . . . . . . . . . . . . . . . as indicated in 50:50 acetonitrile:2-propanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003054
5
6 10
4
7
2
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Phenolic Compounds on 1. Hydroquinone

2. Resorcinol
Ascentis® Express RP-Amide 3. Catechol
Chromatography of Seven Phenol Compounds on Ascentis Express RPA 4. Phenol
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 10 cm x 4.6 mm I.D., 5. m-Cresol, p-Cresol
2.7 μm particles (53929-U) 2 3 6. o-Cresol
mobile phase . . . (A) 20 mM phosphoric acid, pH 2; (B) acetonitrile; (75:25, A:B) 4
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 270 nm 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg/L in mobile phase
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005719
0 2 4 6
Min

HPLC Analysis of Phenolics from Rosemary on 1. Rosmarinic acid

Ascentis® Express C8 2. Carnosol
1 3. Carnosic acid
efficient separation of phenolics from rosemary.
column . . . . Ascentis Express C8, 5 cm x 3 mm I.D., 2.7 μm particles (53848-U) 3
mobile phase . . (A) 0.1% formic acid in water; (B) 0.1% formic acid in acetonitrile 2
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 to 100% B in 8.5 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 0 2 4 6 8
sample . . . . . . . 100 mg/L in 75 μM EDTA, 1.5 mM ascorbate, 15% acetonitrile
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005717


O
OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004680
0 2 4 6 8 10 12 14
Min


Enantiomers on Astec® CYCLOBOND® I HO O
2000 HP-RSP
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 235 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004717
0 2 4 6 8 10
Min

HPLC Analysis of Procyanidins on Ascentis® Phenyl 1
1. Procyanidin B1 (M-H)- = 577.14

column . . . . . Ascentis Phenyl, 15 cm × 4.6 mm I.D., 5 μm particles (581616-U) 2. Procyanidin B3 (M-H)- = 577.14
mobile phase . . . . . . . . . . . . . . . . . . . . . . (A) 10 mM ammonium formate, 3. Procyanidin B2 (M-H)- = 577.14
(pH 3.0 with concentrated formic acid): (B) acetonitrile; (85:15, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min split to the MS
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . MS, ESI (-) in Single Ion Recording (SIR) Mode
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
3
sample . . . . . . . . . . . . . . . . 10 mg/mL in 85:15, 10 mM ammonium formate, 2
(pH 3.0 with concentrated formic acid): acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003879
0 2 4 6
Min

HPLC Analysis of Silymarin on Ascentis® 1. Taxifolin 5. Silybin A

Express C18 2. Silychristin 6. Silybin B
3. Apigen 7 -glucoside 7. Isosilybin A
This application demonstrates the suitability of Ascentis Express C18 for the 4. Silydianin 8. Isosilybin B
efficient separation of silymarin. 2
mobile phase . . . . . . . . . . . . . (A) water with 0.1% formic acid; (B) methanol
gradient . . . . . . . . . . . . . . held at 35% B for 3 min; 35 to 45% B in 10 min; 6
held at 45% B for 2 min; 45 to 100% B in 5 min 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min 1 3 4 7
8
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4105 psi (283 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 0 10 20
sample . . . . . . . . . . . . . . . . . . . . . . . 100 μg/mL in 90:10, water:methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005739

HPLC Analysis of Steroidal Glycosides on Ascentis® Gradient Program 1. Digoxin (100 µg/mL)
Phenyl Time (min) %B 2. Glycyrrhetinic acid (100 µg/mL)
0 70 3. Digitonenin (200 ug/mL)
column . . . . . Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 μm particles (581616-U) 10 90
mobile phase . . . . . (A) 10 mM ammonium formate (pH 3 with formic acid); (B) 10.5 70
methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.25 mL/min 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
2
detector . . . . . . . . . . . . . . . . . . . . . . . . ELSD at 45 °C, gain 6, 3.5 bar N2 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003714
0 2 4 6 8 10
Min

HPLC Analysis of Steroids on Ascentis® Si 1. Cortisone 2 3

2. Prednisone 1
column . . . . . . . . Ascentis Si, 15 cm x 4.6 mm I.D., 5 μm particles (581512-U) 3. Prednisolone
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 245 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 85:15, hexane:2-propanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003728
0 2 4 6 8 10 12
Min


OH O
*
OH
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
O OH
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004665

7.37 min.

HPLC Analysis of Vitamin D2 and D3 on Ascentis® 1. Vitamin D2 2

Express C18 2. Vitamin D3 1

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.15 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . 10 mg/L each (vitamins D2 and D3) in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005615
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Vitamin D2 and D3 on Ascentis® 2

Elution Order:
Express C18 5 μm 1. Ergocalciferol (Vitamin D2)
This application demonstrates the suitability of the Ascentis Express C18, 2. Cholecalciferol (Vitamin D3)
5 μm, for the routine analysis of the fat-soluble vitamins D2 and D3 .
Structures along with the optimized chromatogram obtained on the
Ascentis Express C18, 5 μm are presented.
column . . Ascentis® Express C18, 25 cm x 4.6 mm I.D., 5 μm particles (50538-U)
1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1726 psi (119 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 265 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
sample . . . . . . . . . . . . . . . . . . . . . 50 μg/mL in 90:10, acetonitrile:methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005891

HPLC Analysis of Vitamin D2 and Vitamin D3 on 1 2

1. Ergocalciferol
Ascentis® C18 2. Cholecalciferol
analysis of the fat-soluble vitamins D2 and D3 . Structures along with the
optimized chromatogram obtained on the Ascentis C18 are presented.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005614
0 2 4 6 8 10 12 14 16 18
Min

HPLC Analysis of Vitamins, Fat Soluble (A and E), 1. Retinol acetate (Vitamin A acetate), 50 μg/mL
on Discovery® C8 2. δ-Tocopherol, 165 μg/mL
3. γ-Tocopherol, 200 μg/mL
column . . . . . . . Discovery C8, 15 cm × 4.6 mm I.D., 5 μm particles (59353-U) 4. α-Tocopherol (Vitamin E), 292 μg/mL
mobile phase . . . . . . . . . . . . . . . . (A) acetonitrile: (B) water (90:10 v/v, A:B) 5. α-Tocopherol acetate (Vitamin E acetate), 405 μg/mL
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm 3
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000912
1
0 2 4 6 8
Min

HPLC Analysis of Vitamins, Fat Soluble (A and E), 1. Retinol

on SUPELCOSIL™ LC-18 2. Retinyl acetate
3. δ-Tocopherol
column . . . . . SUPELCOSIL LC-18, 25 cm × 4.6 mm I.D., 5 μm particles (58298) 4. γ-Tocopherol
mobile phase . . . . . . . . . . . . . . (A) methanol: (B) deionized water (98:2, A:B) 5. α-Tocopherol
6. Tocopherol acetate
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
detector . . . . . . . . . . . . . . . . . . . . . UV, 325 nm (retinol, retinyl acetate) or
290 nm (tocopherols, tocopherol acetate) 1
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 2
6
sample . . . . . . . . . . . . . . . . . . . . . . 0.2-1 mg/mL each analyte in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712-0679
3
4 5
0 2 4 6 8 10 12
Min

HPLC Analysis of Vitamins, Fat Soluble (D2 and

1. Ergocalciferol (Vitamin D2) 2
D3), on Discovery® HS C18 2. Cholecalciferol (Vitamin D3)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100% acetonitrile
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL each analyte
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001419 0 10 20
Min

1. alpha Tocopherol
HPLC Analysis of Vitamins, Fat Soluble, on 2. Menadione
Gradient Program
Time (min) %B
Ascentis® Si 3. gamma Tocopherol 0 10
4. Chlolecalciferol 10 30
column . . . . . . . . Ascentis Si, 15 cm x 4.6 mm I.D., 5 μm particles (581512-U) 1 12 30
4
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . (A) hexane; (B) ethylacetate 13 10
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 290 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
sample . . . . . . . . . . . . . . . . . . . . . as indicated in 96:4 (hexane:isopropanol)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003727 0 2 4 6 8 10 12
Min

HPLC Analysis of B-vitamins in vitamin water using 1. Pyridoxine 4

Ascentis® Express RP-Amide and UV detection 2. Niacinamide
3. Pantothenate
The vitamin water drink was filtered using 0.2 um Nylon filter and injected
4. Cyanocobalamin
into UHPLC for B-vitamin analysis
mobile phase . . . . . . . . . (A) 20 mM potassium phosphate, pH 3; (B) methanol
gradient . . . . . . . . . held at 0.5% B for 1.5 minutes, to 25% B in 1.0 minutes,
held at 25% B for 1.7 minute, held at 0.5% B for 1.2 minutes
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 mL/min
1 3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5584 psi (385 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm 0 2 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL Min
sample . . . . . . . . vitamin water was filtered through 0.2um nylon syringe filter
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006214

HPLC Analysis of B-vitamins on Ascentis® Express 1. Thiamine

2. Nicotinic acid
RP-Amide using UV detection 3. Pyridoxine
column . . . . . . . . . . . . . . . Ascentis Express RP-Amide, 10 cm x 3.0 mm I.D., 4. Niacinamide
2.7 μm particles (53918-U) 5. Calcium pantothenate
mobile phase . . . . . . . . . (A) 20 mM potassium phosphate, pH 3; (B) methanol; 6. Cyanocobalamin
2 3 7. Biotin
gradient . . . . . . . . . . . 0.5% B held for 1.5 minutes, to 25% B in 1.0 minutes,
held at 25% B for 1.7 minute, held at 0.5% B for 1.2 minutes 8. Folic acid
9. Riboflavin
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5584 psi (385 bar) 4 6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm 8
5 9
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL 7
sample . . . . . . . . . . . . . . . . . 100 ug/mL of all vitamins except 250 ug/mL of
calcium pantothenate, biotin and riboflavin in water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006215
0 2 4
Min

2 1. Niacin (50 µg/mL)

HPLC Analysis of Vitamins, Water Soluble, on 2. Ascorbic acid (100 µg/mL)
Ascentis® RP-Amide 1 3. Pyridoxine (100 µg/mL)
4. Thiamine (75 µg/mL)
mobile phase . . 25 mM potassium phosphate, dibasic (pH 3.5 with phosphoric acid)
3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 0 2 4 6 8 10 12 14
sample . . . . . . . . . . . . . . . . . . as indicated in 25 mM potassium phosphate, Min
dibasic (pH 3.5 with phosphoric acid)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003035

HPLC Analysis of Vitamins, Water Soluble, on Gradient Program 1. Ascorbic acid

Time (min) %B 2. Nicotinic acid
Discovery® C8 3. Thiamine
0 0 4. Pyridoxine
column . . . . . . . Discovery C8, 15 cm × 4.6 mm I.D., 5 μm particles (59353-U) 3 8 30 5. Nicotinamide
mobile phase . . (A) 50 mM potassium phosphate, monobasic, pH 4.5 to methanol: 6. Folic acid
(B) 50 mM potassium phosphate, monobasic, pH 4.(30:70) in 12 min 7. Riboflavin
1 2 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 7
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000789
0 2 4 6 8 10 12
Min

HPLC Analysis of Vitamins, Water Soluble, on 1. Ascorbic acid Gradient Program

Discovery® RP-Amide C16 2. Nicotinic acid Time (min) %B
3. Thiamine
0 0
column . . Discovery RP-Amide C16, 15 cm × 4.6 mm I.D., 5 μm particles (505013) 3 4. Pyridoxine
8 30
1 5. Nicotinamide
mobile phase . . . . . . . . . . . . . (A) methanol; (B) 50 mM potassium phosphote, 6. Folic acid
monobasic, pH 4.5 (30:70, A:B) 7. Riboflavin
2 7
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 5
4 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001171
0 10 20
Min

HPLC Analysis of Vitamins, Water Soluble on 3

Gradient Program
Discovery® C18 Time (min) %B
column . . . . . . . Discovery C18, 15 cm × 4.6 mm I.D., 5 μm particles (504955) 0 0
8 30
mobile phase . . . . (A) 50 mM potassium phosphate, dibasic, pH 4.5 to methanol:
(B) 50mM potassium phosphate, dibasic, pH 4.0 (30:70, A:B) in 8 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 1. Ascorbic acid
2. Nicotinic acid
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 5 3. Thiamine
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm 1 4. Pyridoxine
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 5. Nicotinamide
6. Folic acid
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000790 6 7. Riboflavin
2
4 7
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Vitexin and Related Compounds 3 1. vitexin 4"-O-glucoside

on Ascentis® Express RP-Amide 2. vitexin 2"-O-rhamnoside
3. vitexin
Vitexin is an apigenin flavone glucoside; a chemical coupound found in the 4. isovitexin
passion flower; Vitex agnus-castus (chaste tree or chasteberry) and in the
Phyllostachys nigra bamboo leaves.[1] It is also found in the pearl millet
(Pennisetum millet).[2] Vitexin is also found in Hawthorn. 4
Improved peak shape for the conjugates was obtained at higher temper-
atures. Note that citric acid may not be necessary as it was added as an 2
attempt to improve peak shape at lower temps. It is expected that 1
phophoric acid or other acids will provide similar results.
2.7 um particles (53918-U)
mobile phase . . . . . . . . . . . . (A) 10 mM citric acid (B) acetontrile (85:15, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min 0 2 4 6 8 10
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 335 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . 100 ug/mL each in 40% methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005903

HPLC Analysis of Water-Soluble B-Vitamins on 1. Nicotinic acid

Ascentis® Express C18 2. Calcium pantothenate
3. Pyridoxine 9
A mix of 9 water-soluble B-vitamins was separated on Ascentis 3 4. Niacinamide
Express C18 column 5. Thiamine
column . . Ascentis Express C18, 5 cm x 3.0 mm I.D., 2.7 μm particles (53811-U) 6. Biotin
mobile phase . . . . (A) 20 mM potassium phosphate, dibasic, pH 7; (B) methanol 7. Folic acid
gradient . . . . . . . . . . . . . . . . . . 0.5% B for 1.3 min, 0.5-30%B over 1.7 min; 8. Cyanocobalamine
4 9. Riboflavin
to 30% B in 1.4 min, 2 min equilibration at 0.5% B
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4130 psi (285 bar) 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm 8
sample . . . . . . . 9 vitamins in water, 10 μg/mL for nicotinic acid and folic acid,
12 μg/mL for thiamine hydrochloride and cyanocobalamin,
(20 μg/mL for pyridoxine, 30 μg/mL for riboflavin, 40 μg/mL for 2 5 7
niacineamide, 100 μg/mL for calcium pantothenate, 120 μg/mL for biotin) 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005625
0 1 2 3 4 5
Min

HPLC Analysis of Water-Soluble B-Vitamins on 1. Calcium pantothenate

Ascentis® Express F5 2. Nicotinic acid
1
3. Pyridoxine
A mix of 9 water-soluble B-vitamins was separated on Ascentis 3 4. Biotin
Express F5 column 5. Niacinamide 8
column . . . Ascentis Express F5, 5 cm x 3.0 mm I.D., 2.7 μm particles (53576-U) 6. Folic acid
mobile phase . . (A) 20 mM potassium phosphate, dibasic, pH 6.2; (B) acetonitrile 7. Thiamine
2
gradient . . . . . . . . . . . . . . . . . . . . 0% B for 1.5 min, 0-10% over 0.5 min; 8. Riboflavin
to 15% B in 1.5 min, 2 min re-equilibration at 0% B 5 9. Cyanocobalamin
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 4 9
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4931 psi (340 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 7
6
sample . . . . . . all vitamins in water, 10 μg/mL for nicotinic acid and folic acid,
12 μg/mL for thiamine hydrochloride and cyanocobalamin,
(20 μg/mL for pyridoxine, 30 μg/mL for riboflavin, 40 μg/mL for niacineamide,
100 μg/mL for calcium pantothenate, 120 μg/mL for biotin) 0.0 1.0 2.0 3.0

HPLC Analysis of Water Soluble B Vitamins on 1. Nicotinic acid 6. Biotin

Ascentis® Express RP-Amide 2. Calcium Pantothenate 7. Folic acid
3. Pyridoxine 8. Cyanocobalamin
A mix of 9 water-soluble B-vitamins was separated on Ascentis 4. Thiamine 9. Riboflavin
Express RPA column 5. Niacinamide
mobile phase . . (A) 20 mM potassium phosphate, dibasic, pH 6.2; (B) acetonitrile 2 3 5 9
gradient . . . . . . . . . . . . . . . . . . . 0% B for 0.5 min, to 15% B over 0.3 min; 1 8
up to 35% B in 1.2 min, 1 min equilibration at 0% B
7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5076 psi (350 bar) 6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . 9 vitamins in water, 10 μg/mL for nicotinic acid and folic acid, 0.0 1.0 2.0
12 μg/mL for thiamine hydrochloride and cyanocobalamin, Min
(20 μg/mL for pyridoxine, 30 μg/mL for riboflavin, 40 μg/mL for niacineamide,
100 μg/mL for calcium pantothenate, 120 μg/mL for biotin)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005623

HPLC Analysis of Water Soluble Vitamins on 2

1. Niacin
Ascentis® RP-Amide 2. Ascorbic acid
1 3. Pyridoxine
column . . . Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U) 4. Thiamine
mobile phase . . . . . . . . . . . . . 25 mM potassium phosphate monobasic, pH 3.5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005735
0 2 4 6 8 10 12 14
Min

6
HPLC Analysis of Withania (Aswagandha) Standard 1.
2.
Withanoside IV
Withanoside V
4
5
3. Withaferin A
on Ascentis® Express F5 4.
5.
12-deoxywithastramonolide
Withanolide A
6. Withanone
This application demonstrates the suitability of Ascentis Express F5 for the 7. Withanolide B 3
efficient separation of withania standards.

1 2
gradient . . . . . . . . . . . . 20 to 100% B in 10 min: held at 100% B for 0.5 min

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2756 psi (190 bar) 1.0 2.0 3.0 4.0 5.0
Min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005447

HPLC Analysis of Withania Compounds on 1. Withanoside IV

Ascentis® Express Phenyl-Hexyl 2. Withanoside V
3. Withaferin A
This application demonstrates the suitability of Ascentis Express Phenyl- 4. 12-Deoxywithastramonolide 5
Hexyl for the efficient separation of withania standards. 5. Withanolide A 4 6
column . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 10 cm x 2.1 mm I.D., 6. Withanone
2.7 μm particles (53336-U) 7. Withanolide B 3
gradient . . . . . . . . . . . . 20 to 100% B in 10 min; held at 100% B for 0.5 min
1 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 7
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2321 psi (160 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm 0 2 4 6 8 10
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Min
sample . . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL in water: methanol (80:20)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005660

HPLC Analysis of Withania Extract on Ascentis® 1. Withanoside IV

Express F5 2. Withanoside V
3. Withaferin A
This application demonstrates the suitability of Ascentis Express F5 for the
4. 12-Deoxywithasramonolide
efficient separation of withania extract.
5. Withanolide A
column . . . Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U) 6. Withanone
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile 7. Withanolide B
gradient . . . . . . . . . . . . 20 to 100% B in 5 min; held at 100% B for 0.5 min 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2750 psi (190 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 4
5
sample . . . . . . 20 mg/mL in water:acetonitrile (50:50); sonicate for 15 minutes; 2
filter 0.45 μm; dilute to water:acetonitrile (80:20) 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005613
6
0 2 4
Min

HPLC Analysis of Withania Extract on Ascentis® 1. Withanoside IV

Express Phenyl-Hexyl 2. Withanoside V
3. Withaferin A
This application demonstrates the suitability of Ascentis Express Phenyl- 4. 12-Deoxywithastramonolide
Hexyl for the efficient separation of withania extract. 5. Withanolide A 3
column . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 10 cm x 2.1 mm I.D., 6. Withanone
2.7 μm particles (53336-U) 7. Withanolide B
gradient . . . . . . . . . . . . 20 to 100% B in 10 min; held at 100% B for 0.5 min 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 4
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 bar (2320 psi) 5
2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 6 7
sample . . . . . . . . . 20 mg/mL in water:acetonitrile (50:50); sonicate 15 minutes;
filter 0.45 μm; dilute to water:acetonitrile (80:20)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005610 0 2 4 6 8 10
Min

HPLC Analysis of Withania Standards on 1. Withanoside IV

Discovery® C18 2. Withanoside V 4
5 6
3. Withaferin A
This application demonstrates the suitability of Discovery C18 for the 2
4. 12-Deoxywithastramonolide
efficient separation of withania standards by the USP monograph.
5. Withanolide A
column . . . . . . . . Discovery C18, 25 cm x 4.6 mm I.D., 5 μm particles (504971) 6. Withanone
mobile phase . . . . . . (A) 1 mM potassium phosphate, monobasic; (B) acetonitrile 7. Withanolide B
gradient . . 5 to 45% B in 18 min; 45 to 80% B in 7 min; held at 80% B for 3 min 7
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 1 3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 bar (3200 psi)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 227 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL in water: acetonitrile (86:14)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005611 0 10 20
Min

HPLC Analysis of Xanthines on Ascentis® C18 1 1. 7-methylxanthine

2. 1-methylxanthine
column . . . . . . . Ascentis C18, 15 cm x 4.6 mm I.D., 5 μm particles (581324-U) 3. 1,7-dimethylxanthine
mobile phase . . . . . . . . . . . . . . . (A) 10 mM monobasic potassium phosphate 4. β-hydroxytheophylline
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 3
2
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G002465
0 2 4 6 8 10 12 14 16
Min

HPLC Analysis of Yohimbe Alkaloids on Ascentis® 1

1. Ajmalicine
Express F5, HILIC Mode 2 2. Yohimbine
pH to 6.0 with acetic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1100 psi (76 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 275 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ug/mL each in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005904
0 2 4 6 8 10
Min

LC-MS Analysis of Carotene Compounds on 1 1. Astaxanthin

Ascentis® RP-Amide 2. Xanthophyll
3. Zeaxanthin
column . . . Ascentis RP-Amide, 15 cm x 2.1 mm I.D., 5 μm particles (565305-U) 4. Lycopene
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . acetonitrile 3 5. α-Carotene
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 6. ß-Carotene
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, APCI(+), SIM
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg/L in acetonitrile 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005609 5 6
4
0 10 20
Min

LC-MS Analysis of 25-Hydroxyvitam D3 and 1. 25-Hydroxy vitamin D3

2. 25-Hydroxy vitamin D2 1
25-Hydroxyvitamin D2 on Ascentis® Express C18
mobile phase . . . . . . . . . . . . . . . . . (A) 10mM ammonium formate in water;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1711 psi (118 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL 1.0 2.0 3.0 4.0
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005658

LC-MS Analysis of 25-Hydroxyvitamin D2 and 1. 25-Hydroxyvitamin D3 1

25-Hydroxyvitamin D3 on Ascentis® Express F5 2. 25-Hydroxyvitamin D2 2
The 25-OH forms of the secosteroids vitamin D2 and D3 are indicators of the
vitamin D status of an individual. However, because they have different
biological activity and clinical interest, they should be distinguishable. The
F5 phase was able to resolve the two compounds under highly LC-MS/MS
compatible conditions.
mobile phase . . (A) 5 mM ammonium formate in (B) 75:25 (v/v) methanol:water
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 0 2 4
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
Min
detector . . . . . . . . . . . . . . . . . . . . UV, 265 nm (MS, ESI(+), m/z 100-1000)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005858

LC-MS Analysis of Vaccinium Anthocyanidins on 1. Delphinidin

2. Cyanidin 3
Ascentis® Express C18 at Elevated Temperature
3. Pelargonidin 4
and Flow 4. Peonidin
Fruits of the genus Vaccinium are popular sources for anthocyanidin 5. Malvidin
antioxidants. This genus includes the commonly known blueberry,
huckleberry, and bilberry. 2
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% TFA in water;
(B) 0.1% TFA in 75:25 (v/v) acetonitrile:water; (78:22, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5440 psi (375 bar) 1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), TOF
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 0 2 4
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL in 0.1% formic acid Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005628

LC-MS Analysis of Vaccinium Antioxidants on 1. Delphinidin 3

2. Cyanidin 4
3. Pelargonidin
Fruits of the genus Vaccinium are popular sources for anthocyanidin 4. Peonidin
antioxidants. This genus includes the commonly known blueberry, 5. Malvidin
huckleberry, and bilberry.
2 5
column . . Ascentis Express C18, 10 cm X 2.1 mm I.D., 2.7 μm particles (53823-U)
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% TFA in water;
(B) 0.1% TFA in 75:25 (v/v) acetonitrile:water; (78:22, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3260 psi (225 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), TOF
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μg/mL in 0.1% formic acid 0 2 4 6 8 10 12

LC-MS Analysis of Vitamin D2, D3, Gamma- and 2

1. Vitamin D2
Alpha-Tocopherol on Ascentis® Express C18 2. Vitamin D3
3. Gamma Tocopherol
Analysis was conducted using standard solution, detection on LC/MS
4. Alpha Tocopherol
system.
mobile phase . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile; (5:95, A:B) 3
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI+
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μg/mL in acetonitrile 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005806
1
0 2 4 6 8 10
Min

LC-MS/MS Analysis of Vitamin D Metabolites on 6

Ascentis® Express F5 1. 1α,25-Dihydroxyvitamin D3
column . . Ascentis Express F5, 15 cm x 2.1 mm I.D., 2.7 μm particles (53571-U) 4,5 2. 1α,25-Dihydroxyvitamin D2
mobile phase . . (A) 5 mM ammonium formate water; (B) methanol; (25:75, A:B) 3. 25-Hydroxyvitamin D3
3 4. 25-Hydroxyvitamin D2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
5. 3-epi-25-Hydroxyvitamin D3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5178 psi (357 bar) 6. 3-epi-25 hydroxyvitamin D2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C 7. 1α-Hydroxyvitamin D3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 100-1000 m/z
injection . . . . . . . . 2 μL, each compound 300 ng/mL in 40:60 (water:methanol) 2 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005878 1
0 2 4 6
Min

LC-MS/MS Analysis of Vitamin D Metabolites on 1. 1α,25-Dihydroxyvitamin D3

2. 1α,25-Dihydroxyvitamin D2
Ascentis® Express F5 (Monitored Ions) 3. 25-Hydroxyvitamin D3
3
column . . Ascentis Express F5, 15 cm x 2.1 mm I.D., 2.7 μm particles (53571-U) 4. 25-Hydroxyvitamin D2
mobile phase . . (A) 5 mM ammonium formate water; (B) methanol; (25:75, A:B) 5. 3-epi-25-Hydroxyvitamin D3
6. 3-epi-25 hydroxyvitamin D2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
7. 1α-Hydroxyvitamin D3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5178 psi (357 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), 100-1000 m/z
6 m/z 401.39
injection . . . . . . . . 2 μL, each compound 300 ng/mL in 40:60 (water:methanol)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005876 7
1 m/z 413.39
m/z 434.365
m/z 446.41
0 2 4 6
Min

UHPLC Analysis of Boswellic Acids on Ascentis® 1. 11-Keto-β-boswellic acid

Express 2.7 μM Phenyl-Hexyl 2. 3-O-Acetyl-11-keto-β-boswellic acid
3. α-Boswellic acid
column . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 10 cm x 3.0 mm I.D., 4. β-Boswellic acid
2.7 μm particles (53345-U) 5. 3-O-Acetyl-α-boswellic acid
mobile phase . . . . . . . . (A) 0.1% phosphoric acid; (B) acetonitrile; (10:90, A:B) 6. 3-O-Acetyl-β-boswellic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 850 psi (59 bar)
1 4 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm 2 6
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . 6 Boswellic Acids 50μg/mL in 70:30, water:methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006257
0 2 4
Min

UHPLC Analysis of Boswellic Acids on Titan™ C18 1. 11-Keto-β-boswellic acid

column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U) 2. 3-O-Acetyl-11-keto-β-boswellic acid
mobile phase . . . . . . . . (A) 0.1% phosphoric acid; (B) acetonitrile; (10:90, A:B) 3. α-Boswellic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 4. β-Boswellic acid
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2450 psi (169 bar) 5. 3-O-Acetyl-α-boswellic acid
6. 3-O-Acetyl-β-boswellic acid
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006258 3
2 4
5
6
0 2 4 6 8 10 12
Min

UHPLC Analysis of Fat Soluble Vitamins on 1. Retinyl acetate

Titan™ C18, Acetonitrile Mobile Phase 1 2. (+)-γ-tocopherol
3. α-tocopherol
column . . . . . . TITAN C18, 50 mm x 2.1mm I.D. x 1.9 μm particles (577122-U) 4. (+)-δ-tocopherol
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100% acetonitrile 5. DL-α-tocopherol acetate
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1755 psi (121 bar) 2
3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . All compounds at 0.5 mg/mL except 5
retinyl acetate at 0.05 mg/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006221
0 2 4 6
Min

UHPLC Analysis of Fat Soluble Vitamins on 1. Retinyl acetate

Titan™ C18, Methanol Mobile Phase 2. (+)-γ-tocopherol
3. α-tocopherol
column . . . . . . . Titan C18, 50 mm x 2.1 mm I.D., 1.9 μm particles (577122-U) 4. (+)-δ-tocopherol
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100% methanol 5. DL-α-tocopherol acetate
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 6. Retinyl palmitate
1
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2741 psi (189 bar)
23 6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 280 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . All compounds at 0.5 mg/mL except
retinyl acetate at 0.05 mg/mL in methanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006222
0 2 4 6
Min

UHPLC Analysis of Galangin on Titan™ C18 1 1. Galangin, 2.05

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006093
0 2 4
Min

UHPLC Analysis of Horn Goat Weed Standards 1 1. Hyperoside

2. Epimedin A
Using Titan™ C18 3. Epimedin B
4. Epimedin C
column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U) 5. Icariin
6. Icaritin
mobile phase . . (A) 0.1% acetic acid in water; (B) 0.1% acetic acid in acetonitrile;
gradient . . . 25% B held for 3 min; to 100% B in 6 min; 100% B held for 2 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5130 psi (354 bar)
6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μg/mL 70:30, water:methanol 2 3 4 5
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006311
0 2 4 6 8 10 12
Min

UHPLC Analysis of Turmeric Curcumin Herbal 1 1. Curcumin

Supplement on Ascentis® Express RP-Amide 5 μm 2. Demethoxycurcumin
3. Bisdemethoxycurcumin
extraction . . . . . . . . . . . . . Weigh approximately 100 mg of herbal supplement
ad add 100 mL ethanol
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sonicate 20 min
sample preparation . . . . . . . . . . . . . . . . . . . . . . . Filter with 0.22 μm PVDF
column . . Ascentis Express RP-Amide, 10 cm x 4.6 mm I.D., 5 μm particles (50774-U)
(B) acetonitrile with 0.1% formic acid;
gradient . . 40% B held for 1 min; 40 to 100% B in (6 min); 100% B held for 5 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 mL/min
2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1270 psi (88 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 3
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 250 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 μL 0 2 4 6 8 10 12
sample . . . . . . . . . . . . . . . . . . . turmeric curcumin hernal supplement extract Min
in ethanol dilute 1:1 with water
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006289

UHPLC Analysis of B-vitamins in vitamin water on 1. Pyridoxine

2. Niacinamide 4
Titan™ C18 using UV detection
3. Pantothenate
The vitamin water drink was filtered using 0.2 um Nylon filter and injected 4. Cyanocobalamin
into UHPLC for B-vitamin analysis
column . . . . . . . TITAN C18, 50 mm x 2.1 mm I.D., 1.9 μm particles (577122-U)
mobile phase . . . . . . . . . (A) 20 mM potassium phosphate, pH 3; (B) methanol
gradient . . . . . . . . . . . 0.5% B held for 0.5 minutes, to 30% B in 1.3 minutes,
held at 30% B for 1 minute, held at 0.5% B for 0.8 minutes
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min 2
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7977 psi (550 bar)
1 3
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL 0 2
sample . . . . . . . . vitamin water was filtered through 0.2um nylon syringe filter Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006216

UHPLC Analysis of B-vitamins on Titan™ C18 using 1. Thiamine

UV detection 2. Nicotinic acid
3. Pyridoxine
column . . . . . TITAN C18, 50 mm x 2.1 mm I.D., x 1.9 μm particles (577122-U) 4. Niacinamide
mobile phase . . . . . . . . . (A) 20 mM potassium phosphate, pH 3; (B) methanol 5. Calcium pantothenate
gradient . . . . . . . . . . . 0.5% B held for 0.5 minutes, to 30% B in 1.3 minutes, 6. Folic acid
2
held at 30% B for 1 minute, held at 0.5% B for 0.8 minutes 3 7. Cyanocobalamin
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min 8. Biotin
9. Riboflavin
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7977 psi (550 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C 6
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm 4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL 7
1 9
sample . . . . . . . . . . . . . . . . . 100 ug/mL of all vitamins except 250 ug/mL of
calcium pantothenate, biotin and riboflavin in water 5
8
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006217
0 2
Min

UHPLC Gradient Analysis of Boswellia 1. 11-Keto-β-boswellic acid

2. 3-O-Acetyl-11-keto-β-boswellic acid
Herbal Supplement on Ascentis® Express 2.7μm 3. α-Boswellic acid
4. β-Boswellic acid
Phenyl-Hexyl 5. 3-O-Acetyl-α-boswellic acid
4 6. 3-O-Acetyl-β-boswellic acid
extraction:
1. Weigh 100 mg Bosewellia Herbal Supplement
2. Add 10 mL ethanol 2
3. Sonicate 30 min at 40°C
4. Dilute 100 μL extract, 400 μL ethanol, 500 μL water 3
mobile phase . . . . . . . . . . . . . . . . . (A) 0.1% phosphoric acid; (B) acetonitrile 1
5
gradient . . 50% B for 0.5 min; to 100% B in 4.5 min; held at 100% B for 5 min
gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2390 psi (165 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 2 4 6 8 10
Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006252

UHPLC Gradient Analysis of Boswellia Herbal 1. 11-Keto-β-boswellic acid

Supplement on Titan™ C18 3. α-Boswellic acid
extraction . . . . . . . . . . . . . . . 1. Weigh 100 mg Bosewellia Herbal Supplement 5. 3-O-Acetyl-α-boswellic acid
2. Add 10 mL ethanol 6. 3-O-Acetyl-β-boswellic acid
3. Sonicate 30 min at 40°C
4
4. Dilute 100 μL extract, 400 μL ethanol, 500 μL water
2
1 3
mobile phase . . . . . . . . . . . . . . . . . (A) 0.1% phosphoric acid; (B) acetonitrile
6
gradient . . 40% B for 0.5 min; to 100% B in 2 min; held at 100% B for 10 min 5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4050 psi (279 bar) 0 2 4 6 8 10 12
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
sample . . . . . . . . . . . . . . . . . . . . Bosewellia Extract in 50:50, water:ethanol
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006260

UHPLC Gradient Analysis of Boswellic Acids on 3 5

1.
2.
11-Keto-β-boswellic acid
3-O-Acetyl-11-keto-β-boswellic acid
Ascentis® Express Phenyl-Hexyl 3. α-Boswellic acid
4
column . . . . . . . . . . . . . Ascentis Express Phenyl-Hexyl, 10 cm x 3.0 mm I.D., 5. 3-O-Acetyl-α-boswellic acid
6. 3-O-Acetyl-β-boswellic acid
mobile phase . . . . . . . . . . . . . . . . . (A) 0.1% phosphoric acid; (B) acetonitrile

gradient . . 50% B for 0.5 min; to 100% B in 4.5 min; held at 100% B for 5 min 1 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2390 psi (165 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006285
0 2 4 6 8 10
Min

UHPLC Gradient Analysis of Boswellic Acids on 1. 11-Keto-β-boswellic acid

Titan™ C18 3. α-Boswellic acid
column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U) 4. β-Boswellic acid
mobile phase . . . . . . . . . . . . . . . . . (A) 0.1% phosphoric acid; (B) acetonitrile 5. 3-O-Acetyl-α-boswellic acid
gradient . . 40% B for 0.5 min; to 100% B in 2 min; held at 100% B for 10 min 6. 3-O-Acetyl-β-boswellic acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 1 3
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4050 psi (279 bar) 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm 5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL 6
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006259
0 2 4 6 8 10 12
Min

SFC Chemicals and Industrial Polymers
SFC
Chemicals and Industrial Polymers
SFC Analysis of Diastereomers on Astec® Cellulose
DMP, Methanol Additive
This application shows the ability of Astec Cellulose DMP to separate a 0 1 2 3 4 5 6
mixture of diastereomers. The analytes are proprietary pharmaceutical-type
Min
compounds.
SFC data provided by Dr. Christina Kraml, Lotus Separations, LLC, Princeton,
NJ. Instrument was a Berger Analytical SFC system equipped with a dual
pump (FCM-1200), autosampler (ALS 3100), column oven (TCM-200) and
diode-array detector (DAD-4100); from Mettler Toledo.
mobile phase . . . . . . . . . . . . . . . . . . . . (A) CO 2; (B) methanol; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005176

SFC Chemicals and Industrial Polymers
SFC Analysis of 1-(1-Naphthyl)ethylamine 1. 1-(1-Naphthyl)ethylamine

Enantiomers on Astec® Cellulose DMP 1 2. 1-(1-Naphthyl)ethylamine
column . . Astec Cellulose DMP, 15 cm x 4.6 mm I.D., 5 μm particles (51098AST) 2

mobile phase . . . . . . . 20% methanol (0.1% MSA, methanesulfonic acid) in CO 2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1740 psi (120 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 1.0 2.0 3.0 4.0 5.0 6.0
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg/mL in methanol Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005490

SFC Pharmaceutical (Small Molecule)
Pharmaceutical (Small Molecule)

SFC Analysis of Aminoglutethimide Enantiomers
on Astec® Cellulose DMP
This application demonstrates the ability of the Astec® Cellulose DMP
column to separate Aminoglutethimide enantiomers via Supercritical Fluid
Chromatography. DMPC-derivatized cellulose is widely used for chiral
separations by SFC, both analytical and prep. The Astec® Cellulose DMP 0 2 4
works well in SFC mode, providing rapid separations with excellent Min
selectivity.
mobile phase . . (A) CO2; (B) methanol containing 0.1% DEA in CO 2; (75:25, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1740 psi (120 bar)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 230 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005454

SFC Analysis of Chlorpheniramine Enantiomers on 1. Chlorpheniramine enantiomer

Astec® Cellulose DMP, Methanol Gradient 2. Chlorpheniramine enantiomer
SFC data provided by Dr. Christina Kraml, Lotus Separations, LLC, 1

Princeton, NJ. N
Instrument: Berger Analytical SFC system equipped with a dual pump (FCM-
CH3
1200), autosampler (ALS 3100), column oven (TCM-200) and diode-array * N
CH3 2
detector (DAD-4100); from Mettler Toledo. Cl

gradient . . . . . . . . . . . . Gradient of 5-65% methanol in 3 min, hold for 1 min
0 1 2 3 4 5
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) CO2 ; (B) methanol;
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mL/min Min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . chlorpheniramine, 2 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005175

SFC Analysis of Propranolol Enantiomers on Astec® 1. Propranolol enantiomer CH 3
Cellulose DMP, Methanol Additive 2. Propranolol enantiomer H3C NH

*
O
SFC data provided by Dr. Christina Kraml, Lotus Separations, LLC, OH

Princeton, NJ.
Instrument: Berger Analytical SFC system equipped with a dual pump (FCM- 1
1200), autosampler (ALS 3100), column oven (TCM-200) and diode-array
detector (DAD-4100); from Mettler Toledo. 2
mobile phase . . . . . . . . . . . . . . . . . . . . . (A)CO 2; (B) methanol; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 1 2 3 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . propranolol, 2 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005172

SFC Analysis of Warfarin™ Enantiomers on Astec® 1 1. Warfarin enantiomer

Cellulose DMP, Ethanol Additive 2. Warfarin enantiomer
SFC data provided by Dr. Christina Kraml, Lotus Separations, LLC,
Princeton, NJ. 2
detector (DAD-4100); from Mettler Toledo.
mobile phase . . . . . . . . . . . . . . . . . . . . . (A) CO2 ; (B) ethanol; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 0 2 4 6 8
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL Min
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . warfarin enantiomers, 2 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005171

SFC Analysis of Warfarin™ Enantiomers on Astec®

1. Warfarin enantiomer
Cellulose DMP, Methanol Additive 2. Warfarin enantiomer
Princeton, NJ. 1
Instrument: Berger Analytical SFCsystem equipped with a dual pump (FCM-
detector (DAD-4100); from Mettler Toledo. 2
mobile phase . . . . . . . . . . . . . . . . . . . . (A) CO 2; (B) methanol; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 1 2 3 4
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . warfarin enantiomers, 2 mg/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005170

SFC Analysis of Warfarin™ Enantiomers on Astec® 1. Warfarin enantiomer

Cellulose DMP, Methanol Gradient 2. Warfarin enantiomer
SFC data provided by Dr. Christina Kraml, Lotus Separations, LLC, 1 2

Princeton, NJ.
mobile phase . . . . . . . . . . . . . . . . . . . . . (A) carbon dioxide; (B) methanol;
Gradient of 5-65% methanol in 3 min., hold for 1 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar) 1 2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . warfarin
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005174

SFC Vitamins, Nutraceuticals, and Natural Products
O
1. Flavanone enantiomer
Vitamins, Nutraceuticals, and Natural 2. Flavanone enantiomer
Products 1
2 *
O
SFC Analysis of Flavanone Enantiomers on Astec®
Cellulose DMP, Methanol Additive
Princeton, NJ.
mobile phase . . . . . . . . . . . . . . . . . . . (A) CO2 ; (B) isopropanol; (80:20, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450 psi (100 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 0 1 2
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . flavanone, 2 mg/mL
Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005173

SPE Agriculture
1. Methamidiphos 17. Tolylfluanid

SPE 2.
3.
Dichlorvos
Acephate
18.
19.
Heptachlor epoxide
Captan
4. Propoxur 20. Thiabendazole
Agriculture 5.
6.
Ethoprophos (I.S.)
Hexachlorobenzene
21.
22.
Folpet
cis-Chlordane
7. γ-BHC 23. Imazalil 23
GC Analysis of Agriculture Pesticides in Orange 8. Diazinon 24. 4,4'-DDE
9. Chlorothalonil 25. Dieldrin 15 17 22
(Spiked) on SLB®-5ms after QuEChERS Cleanup 10. Methyl chlorpyrifos 26. Endosulfan sulfate 16
19, 20 24 25
11. Carbaryl 27. Dicofol 18 21
using Primary-Secondary Amine (PSA) 12. Dichlofluanid 28. cis-Permethrin
13. Chlorpyrifos 29. trans-Permethrin 15.0 16.0
Chromatograms of the spiked orange samples are presented in the Figure. 14. p-Dichlorobenzophenone 30. Coumaphos Min
Several background peaks eluting prior to nine minutes are due to 15. Cyprodinil
impurities in the toluene. Despite extract cleanup, matrix peaks are also 16. Pencanazole
present in the chromatograms. Further sample cleanup may be possible by
increasing SPE sorbent weight. Nevertheless, all pesticides were detected. A 4
first order fit was used for calibration. Linearity for the five-point calibration 14
curves was excellent, with 28 of the 29 pesticides having r2 values >0.995 at
a range of 50-500 ppb. Proper calibration of imazalil was not possible due to 13 28
11 12 29
its presence in the orange blanks. 6
8
5 9 10
12 3 7
sample/matrix . . . 15 g of ground-up orange, 75 μL of the internal standard stock

solution (ethoprophos at 20 ppm in methanol), 10 20
75 μL of the pesticide stock solution (29 pesticides, each at 20 ppm in methanol) Min
column . . . . . . . . . . . . . . SLB-5ms, 30 m x 0.25 mm I.D, 0.25 μm (28471-U)

oven . . . . . . . . . . . . . . . . . . . 100 °C (1 min), 10 °C/min to 300 °C (5 min)
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C
MSD interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 °C
scan range . . . . . . . . . selected ion monitoring (SIM), 7 monitoring groups used
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 1 mL/min constant
injection . . . . . . . . . . 1.0 μL pulsed (20 psi until 0.20 min), splitless (1.0 min)
liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D., single taper
sample . . . . . . . . . . . . . . . . . . . . . . . . . extract of an orange spiked with a
29-component pesticide mix plus 1 internal standard, each at 100 ppb
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003590

SPE Agriculture
GC Analysis of Diphenylamine and Methoxychlor 1

1. Diphenylamine
in Apples on a 14% Cyanopropylphenyl Column Pesticide-containing Sample 2. Methoxychlor
(Relative Abundance Scale 5× Blank)
after SPE using Supelclean ENVI-Carb
Pesticides in fruits and vegetables
sample/matrix . . . . . . . . . . . . homogenize 50 g sample in 100 mL acetonitrile
then add 10 g NaCl and homogenize 5 min
SPE tube/cartridge . . . . . . . . . . . . . . . . . . . . . . . . ENVI-Carb, 500 mg/6 mL
column . . . . . . . . . 14% cyanopropylphenyl polysiloxane, 30 m × 0.25 mm I.D.,
0.15 μm (Supelco equivalent, Equity-1701, 28372-U)
oven . . . . . . . . . . . . 70 °C (2 min), 25 °C/min to 130 °C, 2 °C/min to 220 °C, 2
10 °C/min to 280 °C (4.6 min)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD, 285 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . helium
Blank Extract
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL splitless
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0129 5 10 15 20 25 30 35 40 45 50 55
Min
Chromatograms courtesy of J. Fillion, Agriculture and Agri-Food Canada
(Laboratory Services Division, Pesticide Laboratory), Ottawa, Ontario.

SPE Agriculture
GC Analysis of FAMEs in Popcorn Oil on SP™-2560 carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hydrogen, 40 cm/sec. at 180 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (0.5 μL, 100:1 split)
(75 m x 0.18 mm I.D., 0.14 μm) after SPE liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL, 100:1 split
Fractionation using Discovery® Ag-Ion, Fast GC Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003760
Analysis
16:0 Popcorn Oil FAME Extract
Sample pretreatment: A 1 g sample of popcorn oil was scraped from the 18:0 without SPE
inside of a popped microwave oven popcorn bag and mixed with 8 mL 18:1c
deionized water (DI) and 4 mL petroleum ether, centrifuged to remove 18:1t
particulates. The ether layer (upper layer) was collected. This petroleum
ether extraction step was repeated four times. The upper ether layers were
combined, evaporated, and reconstituted in 16 mL toluene. A 1 mL aliquot
was transferred to conical reaction vial and 2 mL 7% boron triflluoride in 18:2c/c
methanol were added. The reaction was incubated at 80 °C for 15 minutes
using a heating block, then cooled to room temperature. 1 mL DI water was 6 8 10 12 14 16 18 20
Min
added and FAMEs were extracted twice using 1 mL hexane each time. The
upper hexane layers were combined into a fresh vial, evaporated, and 16:0 Fr. 1: hexane:acetone (96:4)
reconstituted in 5 mL hexane and 50 mg anhydrous sodium sulfate. 18:0
saturated fatty acids
trans monoenes
Discovery Ag-Ion operates by argentation chromatography which is a
powerful analytical and sample prep technique for the separation or 18:1t
fractionation of cis and trans geometric and positional fatty acid (FA), fatty
acid methyl ester (FAME), diacylglycerol (DAG) and triacylglycerol (TAG)
isomers. Discovery Ag-Ion SPE is shown in this application to separate
FAMEs from an edible oil sample.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 6 8 10 12 14 16 18 20
Min
sample/matrix . . . . . . . . . . . . . . . . . . . 1 g of popcorn oil FAME′s in Hexane
SPE tube/cartridge . . . . . . . . . . . . . Discovery Ag-Ion, 750 mg/6 mL (54225-U) 18:1c Fr. 2: hexane:acetone (90:10)
cis monoenes
condition . . . . . . . . . . . . . . . . . . . . . . . . . 4 mL acetone and 4 mL hexane
sample addition . . . . . . . . . . . . . . . . . . . . 1 mL of the hexane FAME extract
elution . . . . . . . . . . . . . . . . . . 6 mL hexane:acetone (96:4, v/v) followed by
4 mL hexane:acetone (90:10, v/v)
column . . . . . . . . . . . . . . . SP-2560, 75 m x 0.18mm I.D., 0.14 μm (23348-U) 6 8 10 12 14 16 18 20
Min
oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 °C isothermal
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 220 °C

SPE Agriculture
GC Analysis of Organophosphorus Pesticides injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL, splitless

liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D. dual tapered
(OP-Pest) in Cabbage, Onion, and Mushrooms Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005815
on SLB-5ms after SPE using Supelclean™
ENVI-Carb™ II/PSA 1. Methamidophos 22. Disulfoton 43. Quinalphos and Phenthoate
2. Dichlorvos 23. Isazofos 44. Methidathion
Sample pretreatment: To a 10 g sample of homogenized food (green 3. Phosdrin 24. Kitazine 45. Bromophos-ethyl
4. Acephate 25. Formothion 46. Iodophenfos
onions, apples, Napa cabbage, button mushrooms) was added 10 mL 5. Trichlorphan 26. Dichlofenthion and Phosphamidon isomer #2 47. Profenfos
acetonitrile and 4 g magnesium sulfate (that had been heated at 550 °C) and 6. Omethoate 27. Chloropyrifos-methyl 48. Fensulfothion
1 g sodium chloride. After centrifugation, the supernatant was removed and 7. Ethoprophos 28. Methyl parathion 49. Ethion
8. Dibromfos 29. Ronnel 50. Famphur
mixed with 1 g magnesium sulfate and centrifuged. 5 mL of the supernatant
9. CIPC 30. Paraoxon-ethyl 51. Edinphos
was removed and evaporated to 1 mL for SPE loading. 10. Dicrotophos 31. Pirmiphos-methyl and Fenitrothion 52. Pyridaphenthion
Organophosphorous pesticide use in agriculture is widespread due to the 11. Sulfotepp 32. Malathion 53. Phosmet
12. Cadusafos 33. Chloropyrifos 54. EPN
fact that they are more amenable to environmental degradation in 13. Monocrotophos 34. Fenthion 55. Phosalone
comparison to organochlorine or organonitrogen compounds. There are a 14. Phorate 35. Parathion 56. Azinphos-methyl
large number of pesticides in the organophosphorous group, and because 15. Thiometon 36. Isocarbophos 57. Azinphos-ethyl
16. Dimethoate and Demeton-ethyl 37. Trichloronate 58. Coumaphos
of their potential health effects, they are of particular concern on produce 17. Unknown 38. Fosthiazate isomer #1 59. Temephos
49
imported from areas in which they are commonly used. This chromatogram 18. Terbufos 39. Fosthiazate isomer #2
illustrates the separation of 63 of these pesticides by GC on the SLB-5ms 19. Dyfonate 40. Pirmiphos-ethyl
20. Propetamphos 41. Isophenfos-methyl
using nitrogen phosphorus detection (NPD). 21. Phosphamidon isomer #1 and Diazinon 42. Phosfolan and Isophenfos
10
sample/matrix . . . . . 10 g homogenized food sample (green onions, apples, Napa 42 53 52 55 58
16 31 50
14 26 43
cabbage, button mushrooms) 2 36 51
20 37 48
SPE tube/cartridge . . . . . . . . . Supelclean ENVI-Carb II/PSA SPE Tube (54067-U) 19 21
22
24
29 34
35 40 59
15 28
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mL acetone:toluene (65:35) 1
7 18 23 25 30 33 41 44 45
27 32 38
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL sample extract 4 9 47
3 12
elution . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mL of acetone:toluene (65:35) 46
6 13 39
eluate post-treatment . . Evaporate fraction to 0.5 mL then adjust volume to 1 mL 11
8
17
with ethyl acetate for GC-NPD analysis 5
column . . . . . . . . . . . . . . SLB-5ms, 30 m x 0.25 mm I.D., 0.25 μm (28471-U)
oven . . . . 110 °C (2 min), 2.5 °C/min to 205 °C, 10 °C/min to 310 °C (10 min)
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C 20 40
Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NPD, 310 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . helium, 0.9 mL/min constant flow

SPE Agriculture
GC Analysis of Pesticides in Avocado on SLB®-5ms 1. Trifluralin 10. Metolachlor

11. Chloropyrifos
(20 m x 0.18 mm I.D., 0.36 μm) after QuEChERS
2. α-BHC
3. Hexachlorobenzene 12. Endosulfan I
4. Dimethoate 13. 4,4'-DDT
Cleanup using Supel™ QuE Z-Sep+, Fast GC 5. Simazine 14. Methoxychlor
15. Coumaphos
Analysis 6. Quintozene
7. γ-BHC 16. Cyflurthrin isomer
8. Heptachlor 17. Cypermethrin isomer
About this analysis: This application demonstrates the use of dispersive SPE 9. Malathion 18. Deltamethrin
for the extraction of pesticides from Avocado. Cleanup is achieved using Z-
Sep + for fat removal. FInal analysis of the extracts was done by LVI-GC/MS
SIM on an SLB-5ms column.
sample/matrix . . . . . . . . . . . . . . . . Avocado spiked with pesticides at 20 ng/g
extraction tube . . . . . . . . . . . . . . Supel QuE Acetate extraction tube (55234-U) 2
extraction process . . add 25 mL of acetonitrile; shake for 1 minute; add contents of
Supel QuE acetate extraction tube; shake immediately for 1 minute; 8 10, 11
centrifuge at 3200 rpm for 5 minutes 9
clean-up tube . . . . . . . . . . . . . . . Supel Que Z-Sep+ cleanup tube (55296-U) 3
4 5 67 14 17
clean-up process . . transfer 3 mL of the acetonitrile layer into a Supel Que Z-Sep+ 13 15 18
12
cleanup tube; shake for 1 minute; centrifuge at 3400 rpm for 16
3 minutes; draw off supernatant for LVI-GCMS analysis
column . . . . . . . . . . . . . . SLB-5ms, 20 m x 0.18 mm I.D., 0.36 μm (28576-U) 10 12 14 16 18
Min
oven . . . . . . . . . . . . . . . . . . . . 70 °C (2 min), 15 °C/min to 325 °C (6 min)
inj. temp. . . . . . . . . . . . . . . 60 °C (0.28 min), 600 °C/min to 325 °C (5 min)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD
MSD interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 1 mL/min, constant
injection . . . . . . PTV solvent vent; 10 μL, injection dispense speed 15000 μL/min
liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D. FocusLiner with taper
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pesticides spiked at 20 ng/g
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005805

SPE Agriculture
GC Analysis of Pesticides in Fruits and Vegetables Diphenylamine
on a 14% Cyanopropylphenyl Column after SPE

using Supelclean ENVI-Carb™ Pesticide-containing Sample
(relative abundance scale 5× blank)
Homogenize 50 g chopped fruit/vegetable sample in 100 mL acetonitrile.
Add 10 g sodium chloride and homogenize 5 min. Transfer 13 mL of
acetonitrile layer to clean centrifuge tube. Centrifuge at high speed for
Methoxychlor
5 min. Evaporate 10 mL aliquot of supernatant to 0.5 mL under Nitrogen at
35°C.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 5 10 15 20 25 30 35 40 45 50 55 60 65 70
sample/matrix . . . . . . . extract from 50 g homogenized fruit or vegetable sample Min
SPE tube/cartridge . . . . . . . . . . . . . . . . . . ENVI-Carb, 500 mg/6 mL (57094)
sample addition . . . . . . . . . . . . . . . . . . . 0.5 mL concentrated sample extract
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mL acetonitrile:toluene (3:1) Blank Extract
eluate post-treatment . . Concentrate to 2 mL by rotary evaporation. Add 2 x 10 mL
acetone concentrating to 2 mL after each acetone addition.
(Add 50 μL I.S. (500 ng/μL cis-chlordane in acetone)
then dilute to 2.5 mL with acetone.)
column . . . . . . . . . 14% cyanopropylphenyl polysiloxane, 30m × 0.25 mm I.D.,
0.15 μm (Supelco equivalent, Equity-1701, 28372-U) 5 10 15 20 25 30 35 40 45 50 55
oven . . . . . . . . . . . . 70 °C (hold 2 min) to 130 °C at 25 °C; 130 °C to 220 °C Min
at 2 °C/min; 220 °C to 280 °C at 10 °C/min and hold 4.6 min For extraction recovery data, request Bulletin T196900.
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD, 285 °C Chromatograms provided by J. Fillion, Agriculture and Agri-Food Canada
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL, splitless
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0128

SPE Agriculture
7 1. Methamidophos 11. Chlorothiophos

GC Analysis of Pesticides in Spinach on Equity®-1 2. Dichlorovos 12. Tetrasul
6500 3. Acephate 13. Endosulfan sulfate
after SPE using Supelclean™ ENVI-Carb-II/PSA 4. Quintozene 14. Acrinathrin
5. Methyl parathion 15. Bitertanol
6. Carbaryl 16. cis-Permethrin
Sample Pre-treatment: 7. Methyl chloropyriphos 17. trans-Permethrin
Homogenize 10 g of spinach in 10 mL of acetonitrile. 8. Vinclozolin 18. Cypermethrin isomers
9. Procymidone 19. Deltamethrin
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 5 10. Imazalil
sample/matrix . . . . . . . . 10 g of fresh spinach spiked at 0.2 ppm each pesticide

SPE tube/cartridge . . . Supelclean ENVI-Carb-II/PSA, 500mg/300mg/6mL (55119-U) 6 Peak Compound Pesticide Pesticide
Abundance
ID Class Recovery (%)
condition . . . . . . . . . . . . . . . . . . . . . . . 5 mL acetonitrile:toluene (3:1, v/v) 1 Methamidophos Organophosphorous 80
3500 15
sample addition . . . . . . . . . . . . . . . . . . . . 1 mL concentrated sample extract 2
10
16
2
3
Dichlorovos
Acephate
Organophosphorous
Organophosphorous
70
60
elution . . . . . . . . . . . . . . . . . . . . . . . . 20 mL acetonitrile:toluene (3:1, v/v) 1
4 Quintozene Organochloride 92
5 Methyl parathion Organophosphorous 97
eluate post-treatment . . . . . Evaporate eluate to dryness with nitrogen at 40 °C. 12 6 Carbaryl Carbamate 128
Reconstitute in 1 mL acetone:hexane (1:1, v/v). 3

9 11
7
8
Methyl chloropyriphos
Vinclozolin
Organophosphorous
Organochloride
99
83
column . . . . . . . . . . . . . . Equity-1, 30 m x 0.25 mm I.D., 0.25 μm (28046-U) 9
10
Procymidone
Imazalil
Dicarboximide
Imidazole
84
104
oven . . . . . . . 50 °C (5 min), 25 °C/min to 125 °C, 10 °C/min to 30 °C (8 min) 8 17
11 Chlorothiophos Phophosulfi de 106
12 Tetrasul Organochloride 87
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 °C, aux: 325 °C (8 min) 4 13 13 Endosulfan sulfate Organochloride 124
14 14 Acrinathrin Organophosphorous 118
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD, scan range 45-450 amu 18 15 Bitertanol Biphenol 108
19
carrier gas . . . . . . . . . . . . . . . . . . helium, 0.9 mL/min., constant flow mode 0
16
17
Permethrin cis and trans
Cypermethrin isomers
Pyrithroid
Organochloride
82
74
injection . . . . . . . . . . . . . . . . . . . . . 1 μL, splitless (splitter open at 1 min) 10 16 22
Min
28 34 18 Deltamethrin Organobromine 134

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003758

SPE Agriculture
GC Analysis of Sterols (Silylated Derivatives) in elution . . . . . . . . . . . . . . . . . . . . . . . esters fraction: 2.5 mL of hexane:ethyl acetate (90:10); free sterols fraction;
5 mL of hexane:ethyl acetate (90:10) and 3 mL x 5 of ethanol:diethyl ether:hexane (50:25:25)
Olive Oil (Free Sterol Fraction) on SLB®-5ms after eluate post-treatment . . . . . . . . . . . . Evaporate to dryness and perform silylation as described in the Analysis Note.
SPE using Discovery DSC-Si column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SLB-5ms, 20 m x 0.20 mm I.D., 0.20 μm (28564-U)
Sample pre-treatment oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 °C (1 min), 10 °C/min to 325 °C (10 min)
Weigh 0.25 gm oil into test tube. For spiked sample add 12.5 μL of inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C
65-100 μg/mL mixed sterol std. Add 1 ml of hexane:ethyl acetate (90:10). detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD, rn/z 500 - 600
After SPE fractionation, evaporate solvent from each fraction, add 1 ml of MSD interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 °C
30% methanolic sodium methoxide:MeOH:MTBE solution (13:27:60), mix and carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 0.6 mL/min, constant
let sit at room temp for 30 min. Add 1 ml of water and 2 mL of n-heptane.
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL, splitless
Withdraw aqueous phase (bottom) and replace w/ 1 mL of 1% citric acid
solution. Withdraw organic phase and dry over anhydrous sodium sulfate. liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D. single taper
Silylation: Evaporate organic phase to dryness. Add 125 μL of Sylon BFT and sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Olive oil sample; fractionated, transesterified, and silylated
125 μL of pyridine. Heat at 70°C for 20 min. The sample is then ready for Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005816
GC-MS analysis.
Sterol content is one in a battery of tests used to analyze the composition 1. Cholesterol 5
and determine the grade and authenticity of olive oil. Test results are 2. Brassicasterol
compared against standards such as those established by the International 3. Campesterol
Olive Oil Council (IOC) and US Dept. of Agriculture. In vegetable oils, sterols 4. Stigmasterol
exist in the free form, or esterified to fatty acids. In this application, sterols 5. β-Sitosterol
were analyzed in olive oil separately as free and esterified. This was
accomplished by taking advantage of the polarity difference between these
two groups to fractionate them using silica gel SPE. In place of
saponification, the fractions were transesterified to liberate the esterified
sterols. The fractions were then silylated, and analyzed by GC-MS. The 3
silylated sterols all contained a significant molecular ion in their mass
spectum, which made it possible to perform peak identification free of 4
matrix interference. The sterols isolated in the free fraction are shown here. 1 2
sample/matrix . . . . . . . . olive oil spiked with a mixed sterol standard at 5 μg/g 18 20 22
SPE tube/cartridge . . . . . . . . . . . . . . . . Discovery DSC-Si, 1 g/6 mL (52656-U)
Min
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 x 5 mL hexane

SPE Agriculture

4. 2,4-D 2.5 40
6. 2,4,5-TP 13.0 60
1 3
4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0150

SPE Agriculture

2
condition . . 10 mL acetonitrile; 2 × 10 mL reagent water; 10 mL conditioning solvent
A (5 g cetyl trimethyl ammonium bromide and 5 mL conc. ammonium
hydroxide in 500 mL DI water, dilute to 1 L);
( 2 × 10 mL reagent water; 10 mL conditioning
solvent B (10 g hexanesulfonic acid, sodium salt and 10 mL conc. ammonium
hydroxide in 250 mL DI water, dilute to 500 mL))
elution . . 0.5-1.0 mL acetonitrile (to cover/solvate disk); 2 × 4 mL eluting solution
(13.5 mL orthophosphoric acid and 10.3 mL diethylamine
in 500 mL DI water, dilute to 1 L)
0 2 4 6 8
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0715

SPE Agriculture

102.2 ± 1.6
3
0 2 4 6
Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84-109

SPE Agriculture
LC-MS/MS Analysis of Neonicotinoid Pesticides in 1, 2 1. Dinotefuran

Dandelion Blossoms on Ascentis® Express C18 2. Nitenpyram
3. Thiamethoxam
after Dispersive SPE (QuEChERS) using Supel™ QuE 4. Imidacloprid
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . Dispersive (QuEChERS) 6 5. Clothianidin
7
6. Acetamiprid
sample/matrix . . 3 g pulverized dandelion blossoms, homogenized in 10 mL water
7. Thiacloprid
extraction tube . . . . . . . . . . . . . . Supel QuE Acetate extration tube (55234-U)
extraction process . . add 25 mL of acetonitrile; add contents of Supel QuE acetate 3
4
extraction tube; shake immediately for 1 minute; centrifuge at 3400 rpm for 5 minutes
5
clean-up tube . . . . . . . . . . . . . . . . . . . . Supel QuE PSA/C18 tube (55288-U)
clean-up process . . Transfer 1 mL of the acetonitrile layer into a Supel QuE PSA/C18
cleanup tube; shake for 1 minute; centrifuge at 3500 rpm for 3 minutes;
(draw off 700 uL of supernatant, evaporate to dryness at 50 °C under nitrogen;
reconstitute in 200 μL of 50:50 0.1% formic acid:0.1% formic acid in methanol) 0 2 4 6 8 10 12
column . . Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 μm particles (53814-U) Min
gradient . . 30% B from 0 to 5 min; to 100% B in 0.2 min; held at 100% B for 5.3
min; to 30% B in 0.5 min; held at 30% B for 5 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 μL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3800 psi (262 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C
detector . . . . . . . MS, ESI(+), MRM, m/z 203.2/129.2, 271.2/225.0, 292.1/211.0,
256.0/175.2, 250.0/132.0, 223.2/126.0, 253.0/125.8
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006046

SPE Chemicals and Industrial Polymers
1 3 5 7 9 Analyte %Recovery (± CV)

Chemicals and Industrial Polymers 2
1.
2.
Phenol
2-Chlorophenol
101.8 ± 3.7
104.7 ± 1.7
3. 2-Methylphenol 107.0 ± 1.9
GC Analysis of Phenols in Water on a 8 4. 2-Bromophenol (Int. Std.) —
6 5. 3-Methylphenol 104.9 ± 1.6
5% Phenyl Column after SPE using Supelclean™ 10 6. 2-Nitrophenol 96.3 ± 2.8
7. 2,4-Dimethylphenol 105.3 ± 2.2
ENVI-Chrom P 4
11
12 8. 2,4-Dichlorophenol 106.3 ± 2.2
9. 4-Chloro-3-methylphenol 104.6 ± 2.0
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 10. 2,4,6-Trichlorophenol 103.9 ± 2.1
13 11. 4-Nitrophenol 99.9 ± 4.1
sample/matrix . . . . . . . . . . . . . . . . . . . water spiked with phenol compounds 12. 2,3,4,6-T etrachlorophenol 104.6 ± 1.5
SPE tube/cartridge . . . . . . . . Supelclean ENVI-Chrom P, 250 mg/6 mL (57225-U) 13. Pentachlorophenol 97.7 ± 4.8
0 2 4 6 8 10 12 14
condition . . . . . . . . . . 6 mL methyl t-butyl or ethyl acetate; 6 mL acetonitrile. Min
Equilibrate with 6 mL DI water
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 mL water sample.
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 min using vacuum
elution . . . . . . . . . . . . . . 5 mL methyl t-butyl ether acetate dropwise allowing
initial 2 mL to soak in SPE bed
column . . . . . . . . . . . . . . . . 5% Phenyl Polysiloxane, 15 m × 0.53 mm I.D.,
oven . . . . . . 65 °C, 10 °C/min to 185 °C (1 min), 20 °C/min to 275 °C (5 min)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 300 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL, splitless (45 sec hold)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712-0073

SPE Clinical
Clinical *IS
1 Compound
Concentration
(μg/mL) %Recovery %RSD (n=6)
1. Phenobarbital 0.5 96.2 ±1.6
HPLC Analysis of Barbituates in Serum on 2. Aprobarbital
1.0
0.5
94.9
98.5
±1.7
±2.1
Discovery® C18 after SPE using Discovery® 2 1.0 100.8 ±0.8
3. Butabarbital 0.5 97.2 ±1.9
DSC-18Lt 3
1.0 98.7 ±1.8
4 4. Mephobarbital 0.5 99.7 ±2.4
A Zymark® RapidTrace® SPE Workstation was used for this analysis. 1.0 101.0 ±2.0
5. Pentobarbital 0.5 96.4 ±1.7
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 5 1.0 96.4 ±1.9
sample/matrix . . . . . . . . . . . . 0.5 mL porcine serum spiked with 0.5 μg/mL or 6 6. Secobarbital 0.5 98.2 ±1.7
1.0 μg/mL each analyte then diluted with 0.5 mL water 1.0 97.7 ±1.8
SPE tube/cartridge . . . . . . . . . . . Discovery DSC-18Lt, 500 mg/3 mL (52613-U) SPE Method For RapidTrace SPE Workstation Application
0 10 20 30 1. Condition & equilibrate each tube/well with 2 mL MeOH &
condition . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL methanol; 2 mL DI water Min 2 mL DI Water
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL at 0.75 mL/min 2. Load sample
3. Wash each tube/well with 2 mL 5% MeOH
Barbiturates from serum using 500 mg/3 mL Discovery DSC-18Lt SPE
washing . . . . . . . . . . 2 mL 5% methanol, then vacuum or air dry for 5-10 min tubes and Zymark’s RapidTrace SPE Workstation.
4. Vacuum or air dry for 5–10 min
This removes any excess water from the sorbent.
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 mL methanol *IS = Barbital (internal standard). The presence of water in the final eluent may prolong
Sample Info: 0.5 mL porcine serum spike with 0.5 μg/mL or 1.0 μg/mL eluent evaporation.
eluate post-treatment . . . . . dry eluate with nitrogen purge (40 °C; 15-20 min), each analyte then diluted with 0.5 mL water. 5. Elute with 1–2 mL MeOH
reconstitute in 20 μL mobile phase 6. Dry eluate with nitrogen purge (40 °C; 15–20 min)
7. Reconstitute with 200 μL mobile phase
column . . . . . . . . . . . . Discovery C18, 15 cm × 4.6 mm, 5 μm preceded by a 8. Quantify against internal or external standards via
2 cm C18 guard column and 0.5 μm frit filter (504955) HPLC analyses

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 214 nm
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001056

SPE Clinical

Time (min)
0
%A
95
%B
5
Analyte
Atenolol
Rt (min.)
3.0
Q1/Q3
267.2/145
2 90 10
Carazolol
Metoprolol
6.2
5.6
299.1/194.2
268.2/133
6 50 50
Timolol 5.5 317.2/188.1
cps
8000
Metoprolol
0.1
0.1
0.01
0.01
Timolol
0.1
0.1
0.01
0.01
4000
washing . . . . . . . . . . . . . . . . . . . . . . 3 x 1 mL DI water; 1 mL acetonitrile; Salbutamol 0.1 0.01
1 mL 60% acetonitrile/40% DI water; Tulobuterol 0.005 0.0005
apply 2 min of full vacuum to dry the tube after each step
0
elution . . . . . . . . . . . . . . . . . . . . . . 2 x 1 mL 1% formic acid in acetonitrile 0 2 4 6
column . . . . . . . . . . . . . . . . . . . . . . C18, 5 cm x 3 mm I.D., 3 μm particles Rt (min)
Note: Clenbuterol and Tulobuterol were spiked at the levels of 0.1 ng/mL.
150 μL 5% acetonitrile in 10 mM ammonium acetate,
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004059

SPE Clinical

Concentration
0.5 96.4 ±8.5
Discovery® DSC-18 1.0 96.1 ±5.0
0.5 97.8 ±8.8
sample preparation . . 1 mL methanol; evaporate to dryness with nitrogen stream at 0.5 95.6 ±6.7
1.0 97.6 ±5.8
room temperature; reconstitute in 200 μL mobile phase
containing 0.2 μg/mL or 7-methylxanthine (IS)
sample preparation . . . . . . . . . . . . . . . . . . . . . 2 mL 5% methanol in water; 1. Theobromine
dry tube 10 min with nitrogen stream 2. Paraxanthine
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm, 5 μm particles, preceded by 3. Theophylline
a 2 cm RP-AmideC16 guard column and 0.5 μm frit filter (505013) 4. Caffeine
IS 7-Methylxanthine
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
1
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000594 3
IS 2
4
Spiked Serum
(0.50 mg/mL)
0 2 4 6 8 10 12
Min

SPE Clinical
HPLC Analysis of Cortocisteroids in Urine on Efficiency of Recovery

Discovery® HS F5 after SPE using Discovery® %Recovery ± RSD (n=3)
Compound 0.5 µg/mL spike level 1.0 µg/mL spike level
DSC-CN 1. Hydrocortisone 123.3±1.4% 95.9±1.7%
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 2. Prednisilone 107.2±1.1% 91.9±1.1%
sample/matrix . . . . . . . . . . . . . . . . human urine spiked with corticosteroids at 3. Prednisone 103.2±1.0% 88.4±1.8%
0.5 and 1.0 μg/mL and diluted in DI water (1:1, v/v)
SPE well plate . . . . . . . Discovery DSC-CN 96-well SPE, 100 mg/well (575636-U) 1
condition . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol; 1 mL DI water. 1 µg/mL spiked urine extract
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL spiked urine on Discovery DSC-CN
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 20% methanol
3
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol 2
eluate post-treatment . . . . . . . . . . . . Evaporate eluate with nitrogen at 30 °C.
mobile phase . . . . . . . . . . . . . . . . . (A) methanol; (B) DI water; (40:60, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 240 nm 0 2 4 6 8 10 12 14
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL Min
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003763
Blank urine extract
on Discovery DSC-CN
0 2 4 6 8 10 12
Min

SPE Clinical

Discovery DSC-8
External
Standard
3
um
Extract on
Excellent
E Peak Shape
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
Application No.Application No. . . . . . . . . . . . . . . . . . . . . . . . . G003766
Blank Serumm Low

L background/
Extract on minimal
m interferences
DSC-8 for
and
a resolution at
reduced
r run times
0 2 4 6
Min

SPE Clinical
HPLC Analysis of NNAL in Urine on Ascentis® Gradient Program

Time (min.) %A %B
0.0 90 10
Express C18 after SPE using SupelMIP® SPE-NNAL 1.5 70 30
2.5 70 30
2.6 90 10
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 6.0 90 10
sample/matrix . . . . . . . . . . . human urine spiked with 60 or 100 pg/mL NNAL
SupelMIP NNAL Blank Urine; Mixed-Mode Polymer Blank Urine;
SPE tube/cartridge . . . . . . SupelMIP SPE - NNAL, 25 mg/10 mL (LRC) (53206-U) MRM 210.2/180.2 MRM 210.2/180.2
condition . . . . . . . . . . . 1 mL dichloromethane; 1 mL methanol; 1 mL DI water

sample addition . . . . . . . . 2 mL (60 pg/mL sample), 5 mL (100 pg/mL sample)
washing . . . . . . 2 x 1 mL DI water, 10 min vacuum; mL toluene; 1 mL toluene: 0 2
Min
4 6 0 2
Min
4 6
dichloromethane (9:1, v/v); 1 mL toluene:dichloromethane (4:1, v/v), 2 min. vacuum

SupelMIP NNAL Blank Urine; Mixed-Mode Polymer Blank Urine;
elution . . . . . . . . . . . . . . . . . . 2 x 1 mL 10% methanol in dichloromethane, MRM 210.2/93.2 MRM 210.2/93.2
apply gentle vacuum between each fraction
6000
6000
High
eluate post-treatment . . . . . . . . . . . . evaporate under nitrogen and reconstitute Background
3000
3000
in 0.15 - 0.25 mL mobile phase
0
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 10 mM ammonium acetate; 0 2 4 6 0 2 4 6
Min Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min NNAL Spiked Urine Extracted with NNAL Spiked Urine Extracted with
SupelMIP; MRM 210.2/180.2 Mixed-Mode Polymer; MRM 210.2/180.2
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . MS/MS, MRM Transitions, ESI(+) (210.2/180 and 210.2/93.2 m/z)
80
80
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
40
40
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004434
0
0
0 2 4 6 0 2 4 6
Min Min
% Recovery
50 pg/mL 60 pg/mL 100 pg/mL
SPE Procedure Spike Spike Spike
SupelMIP SPE – NNAL * 67% 87%
Mixed-Mode Polymer SPE 47% * *
* Not analyzed

SPE Clinical
HPLC Analysis of Piroxicam and 2-Aminopyridine Efficiency of Recovery

in Urine on Discovery® HS F5 after SPE using %Recovery ± RSD (n=4)
Discovery® DSC-MCAX 2-Aminopyridine Piroxicam
Discovery DSC-MCAX 102 ± 3.5% 101 ± 1.2%
Piroxicam and 2-aminopyridine (piroxicam′s polar metabolite) was spiked Leading Competitor A30 ± 52.5% 98 ± 3.2%
into human urine at the levels of 4 and 10 μg/mL, respectively. The urine Leading Competitor B36 ± 24.2% 83 ± 4.3%
sample was diluted 1:1 with 10 mM potassium phosphate, pH 3.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 1. 2-Aminopyridine (4 µg/mL spike)
1 2. Piroxicam (10 µg/mL spike)
sample/matrix . . . . . . . . . . . . . human urine spiked with piroxicam (4 μg/mL)
and 2-aminopyridine (10 μg/mL)
SPE tube/cartridge . . . . . . . . . . Discovery DSC-MCAX, 100 mg/3 mL (52783-U) Spiked Urine Samples
condition . . . . . . . . . 1 mL methanol; 1 mL 10 mM potassium phosphate, pH 3 Before DSC-MCAX 2
washing . . . . . . . . . . 1 mL 10 mM potassium phosphate, pH 3; 1 mL methanol
elution . . . . . . . . . . . . . . . . . . . 1 mL 5% ammonium hydroxide in methanol
eluate post-treatment . . evaporate to dryness with nitrogen at room temperature, 1.0 2.0 3.0 4.0 5.0
reconstitute in 1 mL mobile phase Min
mobile phase . . (A) 10 mM potassium phosphate, pH 6: (B) acetonitrile (85:15, A:B) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min
Spiked Urine Samples
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 °C 2
After DSC-MCAX
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003762
1.0 2.0 3.0 4.0 5.0
Min

SPE Clinical
HPLC Analysis of Statins in Rat Plasma on Total Ion Chromatogram (MRM, 4 pairs: 557.3/397.2)
Rat Plasma spiked with 5 ng/mL Statins Extracted by Supel-Select HLB SPE
Ascentis® Express C18 after SPE using Supel™- HO O

Atorvastatin
Select HLB Prasvastatin OH
O OH OH OH
HO
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) O NH N O
sample/matrix . . rat plasma spiked with provastin and atorvastin at 5 and 100 ng/mL O
H
SPE tube/cartridge . . . . . . . . . . . Supel-Select HLB SPE, 30 mg/1 mL (54181-U)
condition . . . . . . . . . . 0.5 mL methanol:acetonitrile (1:1, v/v); 0.5 mL DI water HO
F
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL

Supel-Select HLB SPE
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL 5% methanol
elution . . . . . . . . . . . . . . . . . . . . . . 0.5 mL methanol:acetonitrile (1:1, v/v)
Atorvastatin
column . . . . . Ascentis Express C18, 5 cm x 2.1 mm, 2.7 μm particles (53822-U)
Pravastatin
mobile phase . . . . . . . . . . . . . . . . 0.1% acetic acid diluted in 60% methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . ABI 3200 QT; ESI(+), MRM (423.3/321.3, 423.3/101.0,
557.3/397.2 and 557.3/453.4)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004432
0 1 2 3 4 5 6
Min
Absolute Recovery ± RSD (n=3)

5 ng/mL spike 100 ng/mL spike
Pravastatin Atorvastatin Pravastatin Atorvastatin
Supel-Select HLB 84 ± 8% 92 ± 5% 103 ± 4.2% 89 ± 3.9%
Competitor W 83 ± 17% 92 ± 2% 104 ± 2.2% 87 ± 1.1%
Competitor P 77 ± 5% 93 ± 2% 102 ± 3.0% 91 ± 1.3%

SPE Clinical
HPLC Analysis of Tricyclic Antidepressants in 1. Nortriptyline

2. Doxepin
Serum on Discovery® C18 after SPE using 3. Imipramine
Discovery® DSC-18 4. Amitriptyline
1 5. Trimipramine (I.S.)
2 3
SPE tube/cartridge . . . . . . . . . . . . . Discovery DSC-18, 100 mg/1 mL (52602-U)
4
column . . . . . . . . . . . . Discovery C18, 15 cm × 4.6 mm, 5 μm preceded by a
2 cm C18 guard column and 0.5 μm frit filter (504955) 5
mobile phase . . . (A) acetonitrile: (B) methanol: (C) 25 mM potassium phosphate,
monobasic (pH 7 with triethlylamine) (45:25:30, A:B:C)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ambient 0 2 4 6 8 10 12 14
SPE Procedure, Using Zymark RapidTrace SPE Workstation
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000595
Solvent/ Volume Flow Rate
Step Solution (mL) (mL/min) Comments
Efficiency of Recovery 1. Condition MeOH 2.0 5.0 conditions sorbent
Concentration % RSD 2. Condition H2O 2.0 5.0 conditions sorbent
Compound (μg/mL) % Recovery (n=6)
3. Load spiked porcine 2.0A 0.75 applies serum sample
1. Nortriptyline 0.10 103.6 ±4.5 serum
0.50 97.5 ±4.5
4. Rinse 20% MeOH 2.0 5.0 washes sorbent
2. Doxepin 0.10 102.2 ±3.0 in H2O
0.50 100.8 ±1.8
3. Imipramine 0.10 92.0 ±1.5
0.50 97.5 ±1.7 6. Rinse vent 0.1 2.0 positions SPE tube over waste port
4. Amitriptyline 0.10 93.6 ±1.2 7. Dry N2 Time = dries sorbent
0.50 95.7 ±1.4 10 min
8. Purge-Cannula MeOH 4.0 30.0 cleans sample cannula
9. Collect MeOH 1.0 1.0 elutes analytes into collection vessel
10. Collect vent 6.0 3.0 pushes residual eluent into vesselB
A
1 mL porcine serum spiked with 0.1 μg/mL each analyte basified with 3 μL
10 N KOH, then diluted with 1 mL water
B
350 μL water added per mL methanolic eluent before analysis

SPE Clinical
HPLC Analysis of Verapamil and Metabolites in Rat 40000 1. Normethyl verapamil (m/z 441.28 m/z)
2. Verapamil (455.305 m/z)
Plasma on Ascentis® Express C18 after 3. Methoxy verapamil (485.323)
Removal of phospholipids
Phospholipid using HybridSPE-PPT allows
for short 90 sec. run time and
SPE well plate . . . HybridSPE-Precipitation 96-well Plate, 50 mg/well (575656-U) isocratic conditions

sample/matrix . . . Rat plasma spiked with verapamil and metabolites at 10 ng/mL
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile,
pH 2.55 adjust with formic acid (30:70, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 mL/min
0
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS 0 0.5
Min
1.0 1.5
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
sample addition . . . . . . . . . . . . . . . . . . . 100 μL spiked rat plasma followed
by 300 μL 1% formic acid in acetonitrile
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004366

SPE Clinical
LC-MS Analysis of Antiarrhythmic Drugs and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction)
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . rabbit plasma, unfiltered K2-EDTA,
Metabolites in Plasma on Ascentis® Express HILIC spiked with each compound at 100 ng/mL (3:1, plasma:1% formic acid in acetonitrile)
following Sample Prep using HybridSPE®- SPE tube/cartridge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HybridSPE-Phospholipid, 96-well plate (575656-U)
Phospholipid sample addition . . . . . . . . To each well add 100 μL plasma, followed by a 300 μL of 1% formic acid in acetonitrile,
agitate on orbital shaker for 4 minutes. Collect filtrate and analyze directly.
For efficient therapeutic drug monitoring, it is important for clinicians to
have access to fast and robust analytical methods for accurate assessment of column . . . . . . . . . . . . . . . . . . . . . . . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U)
drug efficacy. Industrial trends toward highly specific LC/MS applications mobile phase . . . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate; (B) 5 mM ammonium formate in acetonitrile,
over traditional ELISA type immunoassay have resulted in the need for high- 5:95 (A:B), adjusted to pH 7.0 with formic acid
speed chromatographic assays along with simplified sample preparation flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
methods. Often the limitation of a bioanalytical technique is based upon the pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305 psi (90 bar)
effectiveness of the sample preparation technique. Plasma and serum column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
samples are often susceptible to assay irregularities due to matrix-induced detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, 100-1000 m/z
interferences. In this study the impact of matrix interference is investigated injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
with respect to precision and accuracy of antiarrhythmic cardiac drugs from
plasma samples. A robust bioanalytical method was developed using a
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005854
combination of fast HILIC chromatography on Ascentis Express along with a
selective sample preparation using HybridSPE-Phospholipid. Solvents were x105
+ESI EIC(235.20000, 415.1700, 618.0300, 646.0600) Scan Frag=175.0V 081712020.d
LC-MS Ultra CHROMASOLV® and Certified Reference Material grade
8
standards were from Cerilliant. 1. Lidocaine 1 gray: Analytes
7 2. Amiodarone HCl
Endogenous phospholipids can dramatically impact the precision and 3. Desethylamiodarone pink: Phospholipids
accuracy of a bioanalytical method. During development, the sample matrix 6 4. (±)-Flecainide
impact on analyte detection should be priority in method validation. Failure 5
to do so can result in inaccuracies in reported levels, thus impacting the 4
2 4
assessment of patient health. Eliminating matrix effects in LC/MS is
3
imperative to producing reliable and accurate bioanalytical methods. The
2 3
targeted phospholipid selectivity of the HybridSPE-Phospholipid technique
enables simplified sample processing with no detected phospholipid matrix 1
interference, while exhibiting excellent recovery from plasma and serum 0
samples. This application demonstrates how combining selectivity in both 0.1 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4
sample preparation and chromatographic separation allows for a simplified
and efficient bioanalytical method resulting in a high precision and accurate Min
assay.

SPE Clinical
LC-MS Analysis of Clenbuterol In Plasma on Astec® Max 767 CPS

HybridSPE-PPT
HybridSPE® Phospholipid 95% absolute recovery
600
The HybridSPE method provides significant improvement in LC-MS baseline.
sample/matrix . . . . rat plasma spiked with clenbuterol enantiomers at 10 ng/mL 400 Clenbuterol
CPS
SPE well plate . . . HybridSPE-Precipitation 96-well Plate, 50 mg/well (575656-U) Phospholipids
acetonitrile. Mix by vortexing the HybridSPE-PPT plate briefly. 200


0
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min 0 1 2
Min
3 4 5
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C Max 775 CPS

detector . . . . . . . . . . ABI 3200 QT; ESI(+), MRM: 184/104 m/z (phospholipids); 6000
Standard Protein PPT
277.2/203.1 m/z (clenbuterol) 3-step procedure
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 5000 No phospholipid removal
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004431
4000
Phospholipids
CPS 3000
Clenbuterol
2000
1000
0
0 1 2 3 4 5
Min

SPE Clinical
LC-MS Analysis of Omeprazole and Metabolites in 1. 5-Hydroxy omeprazole

Plasma on Ascentis® Express RP Amide after 2. Phenacetin
2 3. Omeprazole
Sample Prep using HybridSPE®-Phospholipid 4. Omeprazole sulfone
This application demonstrates the suitability of HybridSPE and the Ascentis
Express RP Amide for the analysis of omeprazole metabolites.
Sample was vorlexed and centrifuged for 2 minutes at 15000 rpm. 4
Supemate was then passed through HybridSPE 96 well plate. Elutent was
collected, evaporated and reconstituted in mobile phase.
sample/matrix . . . . . . . . . . . . . . . . . . . . rat plasma spiked with omeprazole,
phenacetin and metabolites at 2 μg/mL 1.0 2.0 3.0 4.0 5.0
SPE well plate . . HybridSPE-Phospholipid, 96-well plate, 15 mg/0.8 mL (575656-U) Min
sample addition . . . . . . . . . . . . . . . . . . . . . . . 100 μL plasma to each well,
followed by 300 mL of 1% formic acid in acetonitrile
eluate post-treatment . . . . . . . . . . . . . . . . . eluate was collected, evaporated,
and reconstituted in mobile phase.
mobile phase . . . . . . . . . . . . . (A) water, 10 mM ammonium formate, pH 3.4;
(B) methanol, 10 mM ammonium formate, pH 3.4; (65:35, A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI(+), m/z = 50-400
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005711

SPE Clinical
LC-MS Analysis of Plasma Samples on Ascentis®

Express C18 Showing Baseline Improvements after
Phospholipid
HybridSPE-Precipitation:
No phospholipid removal after
Precipitate proteins by first adding 100 μL rat plasma to the HybridSPE-PPT standard protein PPT
plate followed by 300 μL 1% formic acid in acetonitrile. Do not apply
vacuum.
Mix by vortexing the HybridSPE-PPT plate briefly.
Apply vacuum using a 96-well vacuum manifold and collect the resulting
0 10 20
eluate for analysis. Min
Standard Protein Precipitation:
Complete removal of phospholipid
Combine 100 μL rat plasma with 300 μL 1% formic acid in acetonitrile. after HybridSPE-PPT
Mix by vortexing and centrifuge.
Isolate resulting supernatant for analysis.
Min
SPE well plate . . . HybridSPE-Precipitation 96-well Plate, 50 mg/well (575656-U)
sample addition . . . . . . . . . . . . . . . . . . . . . . 100 μL rat plasma followed by
gradient . . . . . . . . . . . . . . . . . . . . . . 0 min - 95% (A); 10 min - 50% (A);
18 min - 50% (A); 18.1 min - 95% (A); 22 min - 95% (A)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . ABI 3200QT; ESI (+), MRM (184/103 m/z)
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004243

SPE Clinical
LC-MS/MS Analysis of Steroid Hormones in Plasma MRM Transition

on Ascentis® Express C18 after Sample Prep using 1. Aldosterone 361.0/343.1
HybridSPE®-Phospholipid 2. Corticosterone 347.6/109.0
3. 11-Deoxycorticosterone 331.1/109.0
Immunoassay approaches for steroid determination are typically hindered
4. Testosterone 289.0/109.0
due to the lack of specificity of antibodies for the measurement of the
5. 17α-Methyltestosterone 303.1/97.0
particular steroids. With the wide acceptance of LC-MS/MS in the clinical
6. Progesterone 315.0/109.1
setting, there is a growing trend toward converting traditional IA techniques
toward more specific and robust LC-MS/MS approaches. Though LC-MS/MS
improves assay specificity and allows of multiplexed analyte assays to be 6
conducted simultaneously, it is not without limitations specifically toward 4
interferences form endogenous sample matrix. Often LC-MS/MS assays can 5
be hindered due to ionization effects due to endogenous matrix from
biological samples; this can result in random and arbitrary discrimination in 3
analyte response. The use of HybridSPE-Phospholipid prior to LC-MS/MS
analysis removes the endogenous phospholipids and precipitated proteins
in one step, enabling high analyte recovery, negligable matrix background
and long column lifetime.
SPE tube/cartridge . . . . . . . . . . . . . . . HybridSPE®-Phospholipid, 96-well plate,
50 mg/well (575656-U) 1 2
sample addition . . . . To each well add 100 μL of plasma followed by 300 μL of
precipitation solvent (1% formic acid or 0.5% citric acid in acetonitrile).
(Agitate via vortex for 4 minute, place on vacuum manifold and 0 2 4 6 8
apply 10" Hg vacuum for 4 minutes. Collect filtrate and analyze directly.)
column . . Ascentis® Express C18, 10 cm x 2.1 mm I.D., 2.7 μm particles (53823-U) Min
gradient . . 60% B for 3 min, 60% B to 95% B in 5 min, held at 95% B for 2 min
mobile phase . . (A) 5 mM ammonium formate pH 4.0 with formic acid; (B) methanol
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, MRM
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005895

SPE Clinical
LC-MS/MS Analysis of Warfarin™ in Plasma on

Ascentis® Express C18 after SPE using HybridSPE®- pink: Phospholipids
gray: Analytes
Phospholipid
prevention of thrombosis and thromboembolism. The pharmacological
effect of warfarin is derived from its ability to inhibit the enzyme vitamin K Warfarin

the therapeutic range, bleeding events, and variability in time to reach Min
compound in the clinical lab. The clinical implications and importance of
SPE tube/cartridge . . . . . . . . HybridSPE®-Phospholipid, 96-well plate (575656-U)
sample addition . . to each well add 100 μL plasma, followed by a 300 μL of 1%
formic acid in acetonitrile, agitate on orbital shaker for 4 minutes
column . . Ascentis® Express C18, 10 cm x 2.1 mm I.D., 2.7 μm particles (53823-U)
mobile phase . . . . . . . . (A) 5mM ammonium formate, pH 4.2 with formic acid;
(B) 5mM ammonium formate in 95:5 acetonitrile:water, 50:50 (A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, 100-1000 m/z
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005850

SPE Clinical
LC-MS/MS Analysis of Warfarin™ Enantiomers in detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, 100-1000 m/z

injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
Plasma on Astec® CHIROBIOTIC® V after SPE using Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005849
prevention of thrombosis and thromboembolism. The pharmacological pink: Phospholipids
gray: Analytes
effect of warfarin is derived from its ability to inhibit the enzyme vitamin K
the therapeutic range, bleeding events, and variability in time to reach
compound in the clinical lab. Warfarin is a chiral compound comprising an
equal mixture of (R) and (S) enantiomers. (S)-Warfarin is considerably more
potent and pharmacologically active than (R)-warfarin. Astec CHIROBIOTIC V
columns resolve warfarin enantiomers under LC-MS conditions and hold up
well to biological samples. The clinical implications and importance of
SPE tube/cartridge . . . . . . . . . HybridSPE-Phospholipid, 96-well plate (575656-U)
1% formic acid in acetonitrile, agitate on orbital shaker for 4 minutes
column . . Astec CHIROBIOTIC® V, 10 cm x 4.6 mm I.D., 5 μm particles (11022AST)
mobile phase . . (A) 0.1% formic acid (pH unadjusted); (B) acetonitrile, 75:25 (A:B)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C

SPE Clinical
LC-MS (TOF) Analysis of Warfarin™ Enantiomers in condition . . Apply 100 μL of plasma to plate, followed by 300 μL of 1% formic acid acetonitrile. Agitate via vortex for 4 min.
elution . . . . . Place on vacuum manifold and apply 10” Hg vacuum for 4 minutes. Collect filtrate and analyze directly.
Plasma on Astec® CHIROBIOTIC® V after SPE Using column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CHIROBIOTIC V,10 cm x 4.6 mm I.D., 5 μm particles (11022AST)
HybridSPE®-Phospholipid mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (A) 0.1% formic acid water; (B) acetonitrile, (75:25, A:B)
This application demonstrates the benefit of using Sigma-Aldrich Analytical flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
(Supelco, Fluka, and Cerilliant) consumables for rapid, accurate, sensitive and column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
reliable MS detection in a clinical or bioanalytical setting. detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+, 100-1000 m/z
Warfarin was originally developed as a rodenticide, but soon found medical injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL
application as an anticoagulant to prevent the formation of blood clots,
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005827
especially after heart attacks. At the time of this writing, it is the most widely
prescribed oral anticoagulant and is sold under Coumadin and various other
brand names. Warfarin is a chiral molecule. The R- and S- forms are cleared Black: Sample prep using
by the body via different pathways and have different pharmacokinetic and HybridSPE®-Phospholipid
pharmacodynamic effects. S-warfarin is more potent than the R-warfarin in
producing the anticoagulant response.
The Astec® CHIROBIOTIC V column provided rapid resolution of the
enantiomers under conditions that are compatible with mass spectrometric
(MS) detection. Considering the rise of MS in the clinical laboratory, this is an
important consideration. The CHIROBIOTIC family of chiral columns are Red: Sample prep using
based on the macrocyclic glycopeptide molecule and affect enantioreso- protein precipitation only
lution under mobile conditions that are compatible with reversed-phase
and MS conditions. Their durability allows them to stand up to repeated
injections of biological extracts.
By using HybridSPE-Phospholipid for the sample prep, ion-suppressing
phospholipids were removed for improved sensitivity and longer column
lifetime. LC-MS Ultra CHROMASOLV solvents were used to supply low
background interference and particulate contaminants. Cerilliant reference
material grade standards provided reliable quantitation.
system: Agilent 1290, 6210 TOF 1.0 2.0 3.0 4.0
sample/matrix . . . . . . . . . Rat plasma stabilized with K2EDTA was acquired from Min
Lampire Biological Laboratories, (Pipersville PA). (Plasma was spiked
directly from stock standard to a level of 100 ng/mL in
3:1 (plasma:1% formic acid acetonitrile).)
SPE tube/cartridge . . Pk1 50 mg/96-Well HybridSPE-Phospholipid Plate (575656-U)

SPE Clinical
x105 +ESI EIC(235.2000, 415.1700, 618.0300, 646.0600) Scan Frag=175.0V 081712020.d

LC-TOF Analysis of Antiarrhythmic Drugs and 8 1
7.5 1 Recovery from plasma (average) by sample Desmethyl-
1
Peak Cerilliant Part No. prep technique (calculated, ng/mL) Lidocaine Amiodarone amiodarone Flecainide
Metabolites in Plasma on Ascentis® Express HILIC 6.5
7 1. Amiodarone
2. Desmethylamiodarone
A-060
D-055 HybridSPE-Phospholipid 108.4 109.8 104.8 119.0
3. Lidocaine L-018 Protein Precipitation 118.5 105.5 52.9 44.7
after Sample Prep using HybridSPE®-Phospholipid 5.5
6 4. Flecainide F-017
5
The basic characteristics of antiarrhythmic cardiac drugs and associated 4.5 2
4
metabolites make them targets for HILIC chromatographic separation. 4 red: phospholipids
Because HILIC mobile phases consist of a high composition of acetonitrile, 3.5
3
this can also facilitate the direct analysis of precipitated plasma samples 2.5
without the need for additional sample solvent exchange. In most cases, the 2 3
1.5
high organic mobile phase also facilitates increased analyte response in ESI 1
1 2 3 4
analysis on Ascentis Express HILIC. The resulting method was robust and Counts vs. Acquisition Time (min)
sensitive.
sample/matrix . . . Rat plasma stabilized with K2EDTA was acquired from Lampire
Biological Laboratories, (Pipersville PA). Plasma was spiked
directly from stock standard to a level of 400 ng/mL
SPE tube/cartridge . . HybridSPE-Phospholipid 96-Well Plate, 50 mg/2 mL (575656-U)
sample addition . . . . . . . . Apply 100 μL of plasma to plate, followed by 300 μL
of 1% formic acid acetonitrile. Agitate via vortex for 4 min
elution . . . . Place on vacuum manifold and apply 10” Hg vacuum for 4 minutes.
Collect filtrate and analyze directly.
(B) 5 mM ammonium formate in acetonitrile; (5:95, A:B, pH 7.0 with formic acid)
(LC-MS CHROMASOLV Ultra grade)
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305 psi (90 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL
sample . . . . . . . . . . . . . . . . . . . plasma extract, analyte concentration of final
sample work up is equivalent to 100 ng/mL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005818

SPE Clinical
LC-TOF Analysis of Antiarrhythmic Drugs and injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL

sample . . . . . . . . . . . . . . plasma extract, analyte concentration of final sample work up is equivalent to 100 ng/mL
Metabolites on Ascentis® Express HILIC: Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005819
Improvement in Analyte Response after Sample
x105
Prep using HybridSPE®-Phospholipid 8
7.5
1 +ESI EIC(235.2000, 415.1700, 618.0300, 646.0600) Scan Frag=175.0V 081712020.d Sample Prep Using HybridSPE®-Phospholipid 1
1 Calculated Concentration (ng/mL)
The basic characteristics of antiarrhythmic cardiac drugs and associated 7

1. Amiodarone
Cerilliant Part No.
A-060
HybridSPE-Phospholipid
108.4
Protein Precipitation
118.5
6.5
metabolites make them targets for HILIC chromatographic separation. 6
2.
3.
Desmethylamiodarone
Lidocaine
D-055
L-018
109.8
104.8
105.5
52.9
Because HILIC mobile phases consist of a high composition of acetonitrile, 5.5 4. Flecainide F-017 119 44.7
5
this can also facilitate the direct analysis of precipitated plasma samples 4.5 2 4
without the need for additional sample solvent exchange. In most cases, the 4 phospholipids
high organic mobile phase also facilitates increased analyte response in ESI 3.5
3
prep to remove phospholipids and precipitated proteins prior to LC-MS 2 3
1.5
analysis on Ascentis Express HILIC. These chromatograms compare the 1
response with HybridSPE-Phospholipid vs. standard protein precipitation 0.5
sample prep methods. 0

1 2 3 4
sample . . . . . . . . . . . . . Rat plasma stabilized with K2EDTA was acquired from Counts vs. Acquisition Time (min)
Lampire Biological Laboratories, (Pipersville PA). Plasma was spiked

x105
directly from stock standard to a level of 400 ng/mL 9.5 1 +ESI EIC(235.2000, 415.1700, 618.0300, 646.0600) Scan Frag=175.0V 081612052.d Sample Prep Using Protein Precipitation Only 1
9
SPE tube/cartridge . . HybridSPE-Phospholipid 96-Well Plate, 50 mg/2 mL (575656-U) 8.5
sample addition . . . . . . . . . . . . Apply 100 μL of plasma to plate, followed by 7.5
8
300 μL of 1% formic acid acetonitrile. Agitate via vortex for 4 min. 7

6.5
elution . . . . . . . . . . . Place on vacuum manifold and apply 10" Hg vacuum for 6
1
4 minutes. Collect filtrate and analyze directly. 5.5
5 phospholipids
column . . Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 μm particles (53939-U) 4.5
4 2
mobile phase . . . . . . . . . . . . . . . . . . . . . . . (A) 5 mM ammonium formate; 3.5
(B) 5 mM ammonium formate in acetonitrile; 2.5
3
(5:95, A:B, pH 7.0 with formic acid) (LC-MS CHROMASOLV Ultra grade) 2 4
1.5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 1 3
0.5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305 psi (90 bar) 0
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1 2 3 4

SPE Cosmetics, Personal Care, and Cleaning Prod-
Cosmetics, Personal Care, and Cleaning Theoretical
Actual
Hydrocortisone Actual %
Products Sample
No.
Sample
Weight (g)
Hydrocortisone
Weight (g)1
weight recovered
(g)
Absolute
Recovery (%)
Hydrocortisone
in Cream
1 1.0258 0.0103 0.0092 89.32 0.9003
HPLC Analysis of Hydrocortisone in Topical Cream 2 1.1078 0.0118 0.0111 94.07 0.9975
on Discovery® HS C18 after SPE using Discovery® 3

4
1.1151
1.1369
0.0112
0.0114
0.0119
0.0119
106.25
104.39
1.0424
1.0442
DSC-Si 5 1.1390 0.0114 0.0113 99.12 1.0785
6 1.1634 0.0116 0.0123 106.03 1.0567
Average: 99.86 1.02
Dissove 1 g 1% hydrocortisone topical hand cream in 10 mL ethyl acetate.
RSD: 6.99 0.06
Dilute 2 mL of ethyl acetate-cream sample with 8 mL hexane:ethyl acetate 1
(2:1, v/v).
SPE tube/cartridge . . . . . . . . . . . . . Discovery DSC-Si, 500 mg/3 mL (52695-U)
Hydrocortisone
Ext. Standard
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Hydrocortisone
Sample
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 1.0 2.0 3.0
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003765

SPE Environmental
Environmental Analyte % Recovery

(± CV)
1. Hexachlorobenzene 87 ± 11
GC Analysis of a 7-Component Organochlorine 2. g-BHC (Lindane) 99 ± 13
Pesticide Mix in Drinking Water on a 5% Phenyl 3. Heptachlor 96 ± 12
4. Aldrin 94 ± 13
Column after SPE using Supelclean™ ENVI™-18 5. Heptachlor epoxide 98 ± 13
6. Endrin 93 ± 11
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 7. Methoxychlor 110 ± 13
sample/matrix . . . . . . . . . . . . . . . . . . . drinking water spiked with pesticides
SPE tube/cartridge . . . . . . . . . . . . . Supelclean ENVI-18, 500 mg/6 mL (57064) Extracted Pesticides
4 5
condition . . . . 2 x 6 mL hexane:ethyl ether (1:1); 6 mL methanol; 6 mL DI water 2 3
sample addition . . . . . . . . . . . . . . . . . . 250 mL water sample at 10 mL/min
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 min with vacuum
elution . . . . . . . . . . . . . . . . . . . . . . . . 2 x 15 mL hexane:ethyl ether (1:1) 6
eluate post-treatment . . . . . . . . . . . concentrate to 2 mL with nitrogen stream
oven . . . . . . . . . . . . . . . . . . . . . . . . 150 °C (2 min), 10 °C/min to 275 °C
1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ECD, 310 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 7
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1192
0 4 8 12 16
Min
Blank
0 4 8 12
Min

SPE Environmental
4
GC Analysis of a 20-Component Organochlorine Standard
3
1.
2.
a-BHC
b-BHC
Pesticide Mix in Water on a 5% Phenyl Column 1
12
3.
4.
g-BHC (Lindane)
d-BHC
after SPE using Supelclean™ ENVI™-8 11
5.
6.
Heptachlor
Aldrin
7. Heptachlor epoxide
Chlorinated pesticides in hazardous waste 8. g-Chlordane
Sample Pre-treatment: 5 9 9. Endosulfan I
19 10. a-Chlordane
Add 5 mL methanol to 100 mL aqueous hazardous waste. Adjust to pH 5-7 if 8 11. Dieldrin
6 14 15
necessary. 10 12. 4,4'-DDE
18 13. Endrin
7 20 14. Endosulfan II
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 13 17 15. 4,4'-DDD
sample/matrix . . . . . . . . . . . . . . . . . . . . waste water spiked with pesticides 16. Endrin aldehyde
17. Endosulfan sulfate
SPE tube/cartridge . . . . . . . . . . . . . . . . . . Supelclean ENVI-8, glass hardware, 18. 4,4'-DDT
2 16 19. Endrin ketone
500 mg/6 mL (57107 (500 mg/6 mL); 57108 (1 g/6 mL)) 20. Methoxychlor
condition . . . . . . . . . . . . . . . 3 mL methanol; h 2 mL 5% methanol in water 21. Decachlorobiphenyl (Surr.)
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . load sample at 5 mL/min 21 Surr.

drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3 min with nitrogen stream
elution . . . . 2 x 4 mL hexane:acetone (90:10), drop wise. allow to soak into bed
0.50 μm (Supelco equivalent, Equity-5, 28252-U) 0 5 10
Min
15 20
oven . . . . . . . . . . . . . . . . . . . . . . . . . 150 °C (0.5 min) to 275 °C (5 min)

inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 °C
Extracted Sample
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ECD, 300 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL split/splitless (45 sec delay)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0399 Surr.
0 5 10 15 20
Min

SPE Environmental
GC Analysis of Glycols in Water on SPB®-1000 after 1. 2-Methoxyethanol 5. Ethylene glycol

SPE using ENVI-Carb™ Plus 2. 2-Ethoxyethanol 6. Diethylene glycol
3. 2-Butoxyethanol 7. Triethylene glycol
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 4. Propylene glycol
sample/matrix . . . . . . . . . . . . . . . . tap water spiked at 2 μg/mL with glycols
SPE tube/cartridge . . ENVI-Carb Plus reversible cartridge, 400 mg/ 1mL (54812-U)
condition . . 1 mL methylene chloride, 2 x 2 mL methanol, 3 mL deionized water
3
(cartridge not allowed to dry during conditioning) 2
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mL spiked tap water
1 6 7
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 min, 10 mm mercury 4
5
elution . . . . . . . . . . . . . . . . . . . . . 2 mL 80:20 methanol:methylene chloride
column . . . . . . . . . . . . . . . SPB-1000, 30 m x 0.25 mm I.D., 0.25 μm (24313) 0 10 20
oven . . . . . . . . . . . . . . . . . . . . 50 °C (1 min), 8 °C/min to 200 °C (7 min) Min
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 220 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 1.5 mL/min. constant
injection . . . . . . . . . . . . . . . . . . . 1 μL, splitless (splitter open at 0.75 min.)
liner . . . . . . . . . . . . . . . . . . . . 4 mm I.D. FocusLiner™ inlet liner with taper
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005284

SPE Environmental
Elution Profile of Oil Interferences and PCB Congeners from Sulfoxide SPE
GC Analysis of PCBs in Transformer Oil on a
0.8
5% Phenyl Column after SPE using Supelclean™ Oil Biphenyl
CB3
CB1
Sulfoxide 0.7 CB10 CB15
CB30 CB37
0.6 CB54 CB77
Commercial insulation oil (Japan Industrial Standard JIS C2320-1999, CB101 CB104
insulating oil, Class 1-2/4, paraffin oil) was spiked with a Kanechlor PCB mix
Recovery (arbitrary)
0.5 CB126 CB155
at the total levels of 3.7 ppm (mg/kg) and diluted with hexane (1:1 v/v). CB169 CB188
SPE tube/cartridge . . . Supelclean Sulfoxide SPE Tube, Glass 6 g/20 mL (55252-U) 0.4 CB189 CB194
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) CB202 CB206
0.3 CB208 CB209

column . . . . . . 5% phenyl polysiloxane (Supelco equivalent, SLB-5ms, 28471-U)
sample addition . . . . . . . . . . . . . . . . . . . . . transformer oil spiked with PCBs
0.2
detector . . . . . . . . . . . . QMS (For full method details, please see reference (1))
extraction process . . . . . . . Sample Pre-treatment: paraffin oil was spiked with a 0.1
Kanechlor PCB mix at the total levels of 3.7 ppm (mg/kg)
and diluted with hexane (1:1 v/v). (Commercial insulation oil (Japan Industrial 0
Standard JIS C2320-1999, insulating oil, Class 1-2/4).) 0 5 10 15 20 25 30 35 40
Elution Volume (mL, hexane)
condition . . . . . . . . . . . . . . . . . . . . . . . . 20 mL acetone; 40 mL of hexane
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL diluted oil sample Observed Concentrations of PCB Homologues of a PCB-fortified Insulation Oil Sample (n = 3).
elution . . . . 12 mL hexane (elutes oil interferences); 25 mL hexane (elutes PCBs) mono- di- tri- tetra- penta- hexa- hepta- octa- nona- deca- total
eluate post-treatment . . . . . . . . . . . . . . . . . . . . . concentrate under nitrogen CBs CBs CBs CBs CBs CBs CBs CBs CBs CBs CBs
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004220
Mean 0.045 0.29 0.80 0.87 0.90 0.35 0.55 0.13 nd nd 3.9
Conc.
mg/kg
SD 0.003 0.05 0.08 0.06 0.08 0.002 0.01 0.004 – – 0.13
Mean 102 91 92 108 106 95 97 97 95 9 –

Recovery
%
SD 5.2 2.6 5.1 5.4 6.8 2.8 2.7 3.3 1.2 3.4 –
Ref: 1. Numata et al. , Anal. Chem. 75, 1450-1457 (2003)

SPE Environmental
GC Analysis of PCBs in Transformer Oil on SPB®-5

after SPE using Supelclean™ LC-Florisil® and LC-Si
Connect Supelclean LC-Si SPE tube (1 g/6 mL) in series to male luer outlet of
Supelclean LC-Florisil SPE tube (1 g/6 mL) using an SPE Tube Adapter
(57020-U). Apply up to 0.2 g transformer oil or 0.1 g waste oil to upper frit of
Florisil SPE tube. Pass 5 x 2 mL isooctane through both Florisil and LC-Si SPE
tubes. Oil interferences will be retained on SPE tubes whereas PCBs will elute
with isooctane fractions. Collect and combine isooctane fractions using 10
mL volumetric flask. Analyze isooctane extract using GC-ECD.
sample/matrix . . . . . . . . . . used transormer oil containing 50 ppb Aroclor 1254.
0 4 20 24 28 32
SPE tube/cartridge . . . . . . . . . . . . . Supelclean LC-Florisil, 1.0 g/6 mL (57057)
Min
SPE tube/cartridge . . . . . . . . . . . . . . . . Supelclean LC-Si, 1.0 g/6 mL (57051)
condition . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL isooctane (Florisil tube)
sample addition . . . . . . . . . . . . . . . . 0.2 g transformer oil or 0.1 g waste oil
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 x 2 mL isooctane
column . . . . . . . . . . . . . SPB-5, 30 m × 0.32 mm I.D., 0.25 μm film (24048)
oven . . . . . . . . . . . . . . . . 40 °C (4 min) to 300 °C at 10 °C/min, hold 5 min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ECD
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . nitrogen
injection . . . . . . . . . . . . . . . . 1 μL, splitless (30 sec delay), then split (50:1)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-0854

SPE Environmental
GC Analysis of Phenols in Water on a 1 3 5 7 9

1.
Analyte
Phenol
%Recovery (± CV)
101.8 ± 3.7
5% Phenyl Column after SPE using Supelclean™ 2 2. 2-Chlorophenol 104.7 ± 1.7
ENVI-Chrom P 8 4. 2-Bromophenol (Int. Std.) —
6 5. 3-Methylphenol 104.9 ± 1.6
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 10 6. 2-Nitrophenol 96.3 ± 2.8
sample/matrix . . . . . . . . . . . . . . . . . . . water spiked with phenol compounds 4
11
12 8. 2,4-Dichlorophenol 106.3 ± 2.2
SPE tube/cartridge . . . . . . . . Supelclean ENVI-Chrom P, 250 mg/6 mL (57225-U) 9. 4-Chloro-3-methylphenol 104.6 ± 2.0
10. 2,4,6-Trichlorophenol 103.9 ± 2.1
condition . . . . . . . . . . 6 mL methyl t-butyl or ethyl acetate; 6 mL acetonitrile. 13 11. 4-Nitrophenol 99.9 ± 4.1
Equilibrate with 6 mL DI water 12. 2,3,4,6-T etrachlorophenol 104.6 ± 1.5
13. Pentachlorophenol 97.7 ± 4.8
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 mL water sample. 0 2 4 6 8 10 12 14
Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 300 °C
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712-0073

SPE Environmental
GC Analysis of Polynuclear Aromatic Analyte % Recovery (± CV)

1. Acenaphthylene 66 ± 28
Hydrocarbons (PAHs) in Water on a 5% Phenyl 2. Fluorene 95 ± 7.5
3. Phenanthrene 95 ± 8
Column after SPE using Supelclean™ ENVI-18 4. Anthracene 95 ± 11
5. Pyrene 97 ± 7.1
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 6. Benzo(a)anthracene 94 ± 8.3
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Chrysene 101 ± 8.8
SPE tube/cartridge . . . . . . . . . . . . . Supelclean ENVI-18, 500 mg/6 mL (57064) 8. Benzo(b)fluoranthene 103 ± 10
9. Benzo(k)fluoranthene 103 ± 9.4
condition . . . . 2 x 6 mL toluene:methanol (10:1); 6 mL methanol; 6 mL DI water 11. Indeno(1,2,3-cd)pyrene 102 ± 10
sample addition . . . . . . . . . . . . . . . . . . 250 mL water sample at 10 mL/min 10. Benzo(a)pyrene 94 ± 11
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 min under vacuum 1 2 12. Dibenzo(a,h)anthracene 104 ± 9.7
13. Benzo(ghi)perylene 101 ± 11
elution . . . . . . . . . . . . . . . . . . . . . . . . . 2 x 1 mL toluene:methanol (10:1) 3,4
column . . . . . . . . . . . . . . . . . 5% phenyl polysiloxane, 30m × 0.25 mm I.D., 5
0.25 μm film (Supelco equivalent, SLB-5ms, 28471-U)
oven . . . . . . . . . . . . . . . . . . . . . . . . 70 °C (2 min) to 280 °C at 8 °C/min
6,7
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 310 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
8,9
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80-207
10
12
11 13
8 12 16 20 24 28
Min

SPE Environmental
GC Analysis of Semivolatiles in Drinking Water on 1. Acenapthene -d 10

mg/L*
5.0 24. Heptachlor epoxide
g/L*
2.0
a 5% Phenyl Column after SPE using ENVI™-18 DSK 2.
3.
Phenanthrene -d10
Chrysene -d 12
5.0
5.0
25.
26.
2,2',3',4,6-Pentachlorobiphenyl
g -Chlordane
2.0
2.0
4. Hexachlorocyclopentadiene 2.0 27. Pyrene 2.0
Sample Pre-treatment: 5. Dimethylphthalate 2.0 28. a -Chlordane 2.0
Adjust 1 L drinking water to pH <2 with 6 N HCl. Add 5 mL methanol and 6. Acenaphthylene 2.0 29. trans-Nonachlor 2.0
7. 2-Chlorobiphenyl 2.0 30. 2,2',4,4',5,6'-Hexachlorobiphenyl 2.0
mix thoroughly 8. Diethyphthalate 2.0 31. Endrin 2.0
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 9. Fluorene 2.0 32. Butylbenzylphthalate 2.0
10. 2,3-Dichlorobiphenyl 2.0 33. di(2-ethylhexyl)Adipate 2.0
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . acidified drinking water 11. Hexachlorobenzene 2.0 34. 2,2'3,3',4,4',6-Heptachlorobiphenyl 2.0
12. Simazine 2.0 35. Methoxychlor 2.0
SPE tube/cartridge . . . . . . . . . . . . . . . ENVI-18 DSK SPE Disk, 47 mm (57171) 13. Atrazine 2.0 36. 2,2'3,3',4,5',6,6'-Octachlorobiphenyl 2.0
condition . . . . . . . . . . . 5 mL dichloromethane; 5 mL methanol; 5 mL DI water 14. Pentachlorophenol 8.0 37. Benz(a)anthracene 2.0
15. g-BHC 2.0 38. Chrysene 2.0
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mL water sample 16. Phenanthrene 2.0 39. Di(2-ethylhexyl)phthalate 2.0
17. Anthracene 2.0 40. Benzo(b)fluoranthene 2.0
elution . . . . . . . . . . . . . . . . . . 2 x 5 mL acetonitrile rinsing internal chamber 18. 2,4,5-Trichlorobiphenyl 2.0 41. Benzo(k)fluoranthene 2.0
of sample reservoir thoroughly 19. Alachlor 2.0 42. Benzo(a)pyrene 2.0
20. Heptachlor 2.0 43. Perylene -d 12 5.0
column . . . . . . . . . . . . . . . . 5% phenyl polysiloxane, 30 m × 0.25 mm I.D., 21. di-n-Butylphthalate 2.0 44. Indeno(1,2,3 -cd)Pyrene 2.0
0.25 μm (Supelco equivalent, SLB-5ms, 28471-U) 22. 2,2',4,4'-Tetrachlorobiphenyl 2.0 45. Dibenz(a,h)anthracene 2.0
23. Aldrin 2.0 46. Benzo(ghi)perylene 2.0
oven . . . . . . . . . 40 °C to 160 °C (3 min), then to 300 °C (3 min) at 6 °C/min 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, scan range m/z = 45-450
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 33 cm/sec
injection . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL split/splitless, 1 min delay 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749-0798 16

7
8
5
6 10 13, 14
9 15 18 21
17 19 22 27,28,29
4
11 20 3
23 25 37
12 1 26 36
24
30 31 34
33 38 39 43
32 35 41 42 44
45 46
40
600 1200 1800 2400

8:00 16:00 Min 24:00 32:00

SPE Environmental

4. 2,4-D 2.5 40
6. 2,4,5-TP 13.0 60
1 3
4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0150

SPE Environmental

Time (min)
0
%A
95
%B
5
Analyte
Atenolol
Rt (min.)
3.0
Q1/Q3
267.2/145
2 90 10
Carazolol
Metoprolol
6.2
5.6
299.1/194.2
268.2/133
6 50 50
Timolol 5.5 317.2/188.1
cps
8000
Metoprolol
0.1
0.1
0.01
0.01
Timolol
0.1
0.1
0.01
0.01
4000
washing . . . . . . . . . . . . . . . . . . . . . . 3 x 1 mL DI water; 1 mL acetonitrile; Salbutamol 0.1 0.01
1 mL 60% acetonitrile/40% DI water; apply 2 min of full Tulobuterol 0.005 0.0005
vacuum to dry the tube after each step
0
elution . . . . . . . . . . . . . . . . . . . . . . 2 x 1 mL 1% formic acid in acetonitrile 0 2 4 6
Rt (min)
150 μL 5% acetonitrile in 10 mM ammonium acetate, Note: Clenbuterol and Tulobuterol were spiked at the levels of 0.1 ng/mL.

column . . . . . . . . . . . . . . . . . . . . . . C18, 5 cm x 3 mm I.D., 3 μm particles
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 mL/min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004059

SPE Environmental
6 1011 12 1314
HPLC Analysis of Nonvolatile Pesticides in Water 1
2
5 7 15
on SUPELCOSIL™ LC-18-DB after SPE using

Supelclean™ ENVI-Carb™ 8
sample/matrix . . . . . . . . . . . . . . . water spiked with pesticides at 10-50 μg/L

3
SPE tube/cartridge . . . . . . . . . . . . Supelclean ENVI-Carb, 250 mg/3 mL (57088)
9
condition . . . . . . . . 5 mL methylene chloride:methanol (80:20); 1 mL methanol;
10 mL 2% acetic acid in water (keep bed moist until sample addition)
sample addition . . . . . . . . . . . . . . . . . . . . . . . 100 mL sample at 5 mL/min Standard
Analyte % Recovery (± RSD, n = 5)
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 minute with vacuum
1. Oxamyl 111 ± 9.6
elution . . . . . 0.8 mL methanol; 2 x 35 mL methylene chloride:methanol (80:20) 2. Methomyl 105 ± 5.0
3. N-1-Naphthylthiourea —
eluate post-treatment . . . . . . dry eluate to 500 μL under gentle nitrogen stream, 4. Aldicarb 92 ± 0.8
reconstitute to 1 mL with methanol 5. Simazine 91 ± 6.5
6. Monuron 99 ± 3.2
column . . SUPELCOSIL LC-18-DB, 25 cm × 4.6 mm I.D., 5 μm particles (58355-U) 7. Cyanazine 90 ± 5.4
8. Metribuzin 97 ± 3.9
mobile phase . . . . . . . . . . . . . . (A) water:acetonitrile (90:10), (B) acetonitrile; 9. Carbofuran 106 ± 6.2
10. Atrazine 89 ± 5.7
gradient 80% A for 5 min then to 30% A over 30 min 11. Carbaryl 97 ± 3.5
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min 12. Diuron 88 ± 5.7
13. Propham 95 ± 3.2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm 14. Propachlor 96 ± 3.8
15. Linuron 88 ± 5.4
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL 0 4 8 12 16 20 24 28
Blank
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85-438 Min

SPE Environmental

2
condition . . 10 mL acetonitrile; 2 × 10 mL reagent water; 10 mL conditioning solvent
A (5 g cetyl trimethyl ammonium bromide and 5 mL conc. ammonium
hydroxide in 500 mL DI water, dilute to 1 L);
( 2 × 10 mL reagent water; 10 mL conditioning solvent B (10 g hexanesulfonic
acid, sodium salt and 10 mL conc. ammonium hydroxide
in 250 mL DI water, dilute to 500 mL))
elution . . . . . . . . . . . . . . . . . . 0.5-1.0 mL acetonitrile (to cover/solvate disk);
2 × 4 mL eluting solution (13.5 mL orthophosphoric acid
and 10.3 mL diethylamine in 500 mL DI water, dilute to 1 L)
0 2 4 6 8
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0715

SPE Environmental

102.2 ± 1.6
3
0 2 4 6
Min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 mL/min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 254 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84-109

SPE Food and Beverages
1. Methamidiphos 17. Tolylfluanid

Food and Beverages 2.
3.
Dichlorvos
Acephate
18.
19.
Heptachlor epoxide
Captan
4. Propoxur 20. Thiabendazole
GC Analysis of Agriculture Pesticides in Orange 5. Ethoprophos (I.S.) 21. Folpet
6. Hexachlorobenzene 22. cis-Chlordane
(Spiked) on SLB®-5ms after QuEChERS Cleanup 7. γ-BHC 23. Imazalil 23
8. Diazinon 24. 4,4'-DDE
using Primary-Secondary Amine (PSA) 9. Chlorothalonil 25. Dieldrin 15 17 22
10. Methyl chlorpyrifos 26. Endosulfan sulfate 19, 20 24 25
16
Chromatograms of the spiked orange samples are presented in the Figure. 11. Carbaryl 27. Dicofol 18 21
Several background peaks eluting prior to nine minutes are due to 12. Dichlofluanid 28. cis-Permethrin
impurities in the toluene. Despite extract cleanup, matrix peaks are also 13. Chlorpyrifos 29. trans-Permethrin 15.0 16.0
14. p-Dichlorobenzophenone 30. Coumaphos Min
present in the chromatograms. Further sample cleanup may be possible by 15. Cyprodinil
increasing SPE sorbent weight. Nevertheless, all pesticides were detected. A 16. Pencanazole
first order fit was used for calibration. Linearity for the five-point calibration
curves was excellent, with 28 of the 29 pesticides having r2 values >0.995 at 4
a range of 50-500 ppb. Proper calibration of imazalil was not possible due to
14
its presence in the orange blanks.
11 12 29
sample/matrix . . . 15 g of ground-up orange, 75 μL of the internal standard stock 5 6
8
9 10
solution (ethoprophos at 20 ppm in methanol), 75 μL of the pesticide 12 3 7
26 27 30
stock solution (29 pesticides, each at 20 ppm in methanol)
column . . . . . . . . . . . . . . SLB-5ms, 30 m x 0.25 mm I.D, 0.25 μm (28471-U) 10 20
oven . . . . . . . . . . . . . . . . . . . 100 °C (1 min), 10 °C/min to 300 °C (5 min) Min
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C

MSD interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 °C
scan range . . . . . . . . . selected ion monitoring (SIM), 7 monitoring groups used
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 1 mL/min constant
injection . . . . . . . . . . 1.0 μL pulsed (20 psi until 0.20 min), splitless (1.0 min)
sample . . . . . . . extract of an orange spiked with a 29-component pesticide mix
plus 1 internal standard, each at 100 ppb
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003590

GC Analysis of a 7-Component Organochlorine Analyte % Recovery

Pesticide Mix in Drinking Water on a 5% Phenyl (± CV)
1. Hexachlorobenzene 87 ± 11
Column after SPE using Supelclean™ ENVI™-18 2. g-BHC (Lindane) 99 ± 13
3. Heptachlor 96 ± 12
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 4. Aldrin 94 ± 13
sample/matrix . . . . . . . . . . . . . . . . . . . drinking water spiked with pesticides 5. Heptachlor epoxide 98 ± 13
SPE tube/cartridge . . . . . . . . . . . . . Supelclean ENVI-18, 500 mg/6 mL (57064) 6. Endrin 93 ± 11
7. Methoxychlor 110 ± 13
condition . . . . 2 x 6 mL hexane:ethyl ether (1:1); 6 mL methanol; 6 mL DI water
sample addition . . . . . . . . . . . . . . . . . . 250 mL water sample at 10 mL/min Extracted Pesticides
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 min with vacuum 4 5
elution . . . . . . . . . . . . . . . . . . . . . . . . 2 x 15 mL hexane:ethyl ether (1:1) 2 3
eluate post-treatment . . . . . . . . . . . concentrate to 2 mL with nitrogen stream

0.25 μm (Supelco equivalent, Equity-5, 28089-U) 6
oven . . . . . . . . . . . . . . . . . . . . . . . . 150 °C (2 min), 10 °C/min to 275 °C

detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ECD, 310 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL 1
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713-1192

7
0 4 8 12 16
Min
Blank
0 4 8 12
Min

4
GC Analysis of a 20-Component Organochlorine Standard
3
1.
2.
a-BHC
b-BHC
Pesticide Mix in Water on a 5% Phenyl Column 1
12
3.
4.
g-BHC (Lindane)
d-BHC
after SPE using Supelclean™ ENVI™-8 11
5.
6.
Heptachlor
Aldrin
7. Heptachlor epoxide
Chlorinated pesticides in hazardous waste 8. g-Chlordane
Sample Pre-treatment: 5 9 9. Endosulfan I
19 10. a-Chlordane
Add 5 mL methanol to 100 mL aqueous hazardous waste. Adjust to pH 5-7 if 8 11. Dieldrin
6 14 15
necessary. 10 12. 4,4'-DDE
18 13. Endrin
7 20 14. Endosulfan II
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 13 17 15. 4,4'-DDD
sample/matrix . . . . . . . . . . . . . . . . . . . . waste water spiked with pesticides 16. Endrin aldehyde
17. Endosulfan sulfate
SPE tube/cartridge . . . . . . . . . . . . . . . . . . Supelclean ENVI-8, glass hardware, 18. 4,4'-DDT
2 16 19. Endrin ketone
500 mg/6 mL (57107 (500 mg/6 mL); 57108 (1 g/6 mL)) 20. Methoxychlor
condition . . . . . . . . . . . . . . . 3 mL methanol; h 2 mL 5% methanol in water 21. Decachlorobiphenyl (Surr.)
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . load sample at 5 mL/min 21 Surr.

drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3 min with nitrogen stream
elution . . . . 2 x 4 mL hexane:acetone (90:10), drop wise. allow to soak into bed
0.50 μm (Supelco equivalent, Equity-5, 28252-U) 0 5 10
Min
15 20
oven . . . . . . . . . . . . . . . . . . . . . . . . . 150 °C (0.5 min) to 275 °C (5 min)

inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 °C
Extracted Sample
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ECD, 300 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL split/splitless (45 sec delay)
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794-0399 Surr.
0 5 10 15 20
Min

GC Analysis of Diphenylamine and Methoxychlor 1

1. Diphenylamine
in Apples on a 14% Cyanopropylphenyl Column Pesticide-containing Sample 2. Methoxychlor
(Relative Abundance Scale 5× Blank)
after SPE using Supelclean ENVI-Carb
Pesticides in fruits and vegetables
sample/matrix . . . . . . . . . . . . homogenize 50 g sample in 100 mL acetonitrile
then add 10 g NaCl and homogenize 5 min
SPE tube/cartridge . . . . . . . . . . . . . . . . . . . . . . . . ENVI-Carb, 500 mg/6 mL
column . . . . . . . . . 14% cyanopropylphenyl polysiloxane, 30 m × 0.25 mm I.D.,
oven . . . . . . . . . . . . 70 °C (2 min), 25 °C/min to 130 °C, 2 °C/min to 220 °C, 2
10 °C/min to 280 °C (4.6 min)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD, 285 °C
Blank Extract
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL splitless
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0129 5 10 15 20 25 30 35 40 45 50 55
Min
Chromatograms courtesy of J. Fillion, Agriculture and Agri-Food Canada

GC Analysis of FAMEs in Cookies on SLB®-IL111 Unfractionated

C16:0 C18:0 C18:2n6c
C18:1 cis & trans
after SPE Fractionation using Discovery® Ag-Ion
A sample of commercially purchased cookies were prepared using an acid
digestions /alkali hydrolysis followed by methylation as described in AOCS C14:0 C20:0
C22:0
Official Method Ce 1k-09 (1). The resulting extracts were then fractionated C20:1n9
using Ag-ion SPE and analyzed by GC/FID on the 100 m SLB-IL111 ionic
10 12 14 16 18 20 22 24
liquid capillary column.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) Fraction 1 t10 t12
t8-9 t11
sample/matrix . . 1 g of commercially purchased cookies was ground and subjected to C16:0 C18:0
t6 t13-14
C18:1 trans isomers
acid digestion and alkali hydrolysis followed t4 t5
t7 t15 t16
by methylation as described in AOCS Official Method Ce 1k-09
14 16
SPE tube/cartridge . . . . . . Discovery Ag-Ion SPE tubes, 750 mg/6 mL (54225-U) C14:0
condition . . 4 mL of acetone; allow solvent to gravity drip completely through tube; C20:0
C22:0
discard eluant; 4 mL of hexane; allow solvent to gravity drip
completely through tube; discard eluant 10 12 14 16 18 20 22 24
sample addition . . . . 1 mL of extract; discard any eluant that drips through tube
elution . . . . (Fraction 1) 6 mL of hexane:acetone (96:4); collect eluant in a fresh Fraction 2
c9 c10c11
container with slight vacuum; ((Fraction 2) 4 mL of hexane: C18:1 trans isomers
acetone (90:10); collect eluant in a fresh container with c8

c12 c14
c6-7 c13
slight vacuum; (Fraction 3) 4 mL of 100% acetone; c4 c5 c15 c16
collect eluant in a fresh container with slight vacuum) 14 16
eluate post-treatment . . evaporate each fraction at room temperature using nitrogen; C16:0
C18:0
C20:1n9
reconstitute each fraction to 1 mL of hexane
column . . . . . . . . . . . . SLB-IL111, 100 m x 0.25 mm I.D., 0.20 μm (29647-U) 10 12 14 16 18 20 22 24
oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 °C
Fraction 3
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C
C18:2n6c
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 250 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL, 10:1 split
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL, 10:1 split
liner . . . . . . 4 mm I.D., split type, single taper wool packed FocusLiner™ design
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005622 10 12 14 16 18 20 22 24
Min

GC Analysis of FAMEs in Cookies on SP™-2560 Unfractionated

C18:1 cis & trans
C18:0
after SPE Fractionation using Discovery® Ag-Ion C16:0 C18:2n6c
A sample of commercially purchased cookies were prepared using an acid

digestions /alkali hydrolysis followed by methylation as described in AOCS
C20:1n9
Official Method Ce 1k-09 (1). The resulting extracts were then fractionated C14:0 C20:0
using Ag-ion SPE and analyzed by GC/FID on the 100 m SP-2560 capillary C22:0
column. 10 20 30

Fraction 1
sample/matrix . . 1 g of commercially purchased cookies was ground and subjected to C16:0 C18:0 t9
t10 t11
acid digestion and alkali hydrolysis followed by methylation as t6-8

t12
t13-14
described in AOCS Official Method Ce 1k-09 C18:1 trans isomers
t15
t4 t5 t16
SPE tube/cartridge . . . . . . Discovery Ag-Ion SPE tubes, 750 mg/6 mL (54225-U)
18 20
condition . . 4 mL of acetone; allow solvent to gravity drip completely through tube; C14:0
C20:0
discard eluant; 4 mL of hexane; allow solvent to gravity C22:0
drip completely through tube; discard eluant

10 20 30
sample addition . . . . 1 mL of extract; discard any eluant that drips through tube
elution . . . . (Fraction 1) 6 mL of hexane:acetone (96:4); collect eluant in a fresh Fraction 2 C18:1 cis isomers c9
c10
container with slight vacuum; ((Fraction 2) 4 mL of hexane: acetone (90:10); c11
c6-8
collect eluant in a fresh container with slight vacuum;(Fraction 3) 4 mL of c4-5
c12
c13 c14 c15 c16
100% acetone; collect eluant in a fresh container with slight vacuum)
18 20
eluate post-treatment . . evaporate each fraction at room temperature using nitrogen;
reconstitute each fraction to 1 mL of hexane C16:0
C18:0 C20:1n9
column . . . . . . . . . . . . . . . SP-2560, 100 m x 0.25 mm I.D., 0.20 μm (24056)

10 20 30
oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 °C
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C Fraction 3 C18:2n6c
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 250 °C

carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hydrogen, 1 mL/min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL, 10:1 split
liner . . . . . . 4 mm I.D., split type, single taper wool packed FocusLiner™ design
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005621
10 20 30
Min

GC Analysis of FAMEs in Popcorn Oil on SP™-2560 carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hydrogen, 40 cm/sec. at 180 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (0.5 μL, 100:1 split)
(75 m x 0.18 mm I.D., 0.14 μm) after SPE liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 μL, 100:1 split
Fractionation using Discovery® Ag-Ion, Fast GC Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003760
Analysis
16:0 Popcorn Oil FAME Extract
Sample pretreatment: A 1 g sample of popcorn oil was scraped from the 18:0 without SPE
inside of a popped microwave oven popcorn bag and mixed with 8 mL 18:1c
deionized water (DI) and 4 mL petroleum ether, centrifuged to remove 18:1t
particulates. The ether layer (upper layer) was collected. This petroleum
ether extraction step was repeated four times. The upper ether layers were
combined, evaporated, and reconstituted in 16 mL toluene. A 1 mL aliquot
was transferred to conical reaction vial and 2 mL 7% boron triflluoride in 18:2c/c
methanol were added. The reaction was incubated at 80 °C for 15 minutes
using a heating block, then cooled to room temperature. 1 mL DI water was 6 8 10 12 14 16 18 20
Min
added and FAMEs were extracted twice using 1 mL hexane each time. The
upper hexane layers were combined into a fresh vial, evaporated, and 16:0 Fr. 1: hexane:acetone (96:4)
reconstituted in 5 mL hexane and 50 mg anhydrous sodium sulfate. 18:0
saturated fatty acids
trans monoenes
Discovery Ag-Ion operates by argentation chromatography which is a
powerful analytical and sample prep technique for the separation or 18:1t
fractionation of cis and trans geometric and positional fatty acid (FA), fatty
acid methyl ester (FAME), diacylglycerol (DAG) and triacylglycerol (TAG)
isomers. Discovery Ag-Ion SPE is shown in this application to separate
FAMEs from an edible oil sample.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 6 8 10 12 14 16 18 20
Min
sample/matrix . . . . . . . . . . . . . . . . . . . 1 g of popcorn oil FAME′s in Hexane
SPE tube/cartridge . . . . . . . . . . . . . Discovery Ag-Ion, 750 mg/6 mL (54225-U) 18:1c Fr. 2: hexane:acetone (90:10)
cis monoenes
condition . . . . . . . . . . . . . . . . . . . . . . . . . 4 mL acetone and 4 mL hexane
sample addition . . . . . . . . . . . . . . . . . . . . 1 mL of the hexane FAME extract
elution . . . . . . . . . . . . . . . . . . 6 mL hexane:acetone (96:4, v/v) followed by
4 mL hexane:acetone (90:10, v/v)
column . . . . . . . . . . . . . . . SP-2560, 75 m x 0.18mm I.D., 0.14 μm (23348-U) 6 8 10 12 14 16 18 20
Min
oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 °C isothermal
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 220 °C

GC Analysis of Organophosphorus Pesticides injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL, splitless

liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D. dual tapered
(OP-Pest) in Cabbage, Onion, and Mushrooms on Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005815
SLB-5ms after SPE using Supelclean™ ENVI-Carb™
II/PSA 1. Methamidophos 22. Disulfoton 43. Quinalphos and Phenthoate
2. Dichlorvos 23. Isazofos 44. Methidathion
Sample pretreatment: To a 10 g sample of homogenized food (green 3. Phosdrin 24. Kitazine 45. Bromophos-ethyl
4. Acephate 25. Formothion 46. Iodophenfos
onions, apples, Napa cabbage, button mushrooms) was added 10 mL 5. Trichlorphan 26. Dichlofenthion and Phosphamidon isomer #2 47. Profenfos
acetonitrile and 4 g magnesium sulfate (that had been heated at 550 °C) and 6. Omethoate 27. Chloropyrifos-methyl 48. Fensulfothion
1 g sodium chloride. After centrifugation, the supernatant was removed and 7. Ethoprophos 28. Methyl parathion 49. Ethion
8. Dibromfos 29. Ronnel 50. Famphur
mixed with 1 g magnesium sulfate and centrifuged. 5 mL of the supernatant
9. CIPC 30. Paraoxon-ethyl 51. Edinphos
was removed and evaporated to 1 mL for SPE loading. 10. Dicrotophos 31. Pirmiphos-methyl and Fenitrothion 52. Pyridaphenthion
Organophosphorous pesticide use in agriculture is widespread due to the 11. Sulfotepp 32. Malathion 53. Phosmet
12. Cadusafos 33. Chloropyrifos 54. EPN
fact that they are more amenable to environmental degradation in 13. Monocrotophos 34. Fenthion 55. Phosalone
comparison to organochlorine or organonitrogen compounds. There are a 14. Phorate 35. Parathion 56. Azinphos-methyl
large number of pesticides in the organophosphorous group, and because 15. Thiometon 36. Isocarbophos 57. Azinphos-ethyl
16. Dimethoate and Demeton-ethyl 37. Trichloronate 58. Coumaphos
of their potential health effects, they are of particular concern on produce 17. Unknown 38. Fosthiazate isomer #1 59. Temephos
49
imported from areas in which they are commonly used. This chromatogram 18. Terbufos 39. Fosthiazate isomer #2
illustrates the separation of 63 of these pesticides by GC on the SLB-5ms 19. Dyfonate 40. Pirmiphos-ethyl
20. Propetamphos 41. Isophenfos-methyl
using nitrogen phosphorus detection (NPD). 21. Phosphamidon isomer #1 and Diazinon 42. Phosfolan and Isophenfos
10
sample/matrix . . . . . 10 g homogenized food sample (green onions, apples, Napa 42 53 52 55 58
16 31 50
14 26 43
cabbage, button mushrooms) 2 36 51
20 37 48
SPE tube/cartridge . . . . . . . . . Supelclean ENVI-Carb II/PSA SPE Tube (54067-U) 19 21
22
24
29 34
35 40 59
15 28
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mL acetone:toluene (65:35) 1
7 18 23 25 30 33 41 44 45
27 32 38
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL sample extract 4 9 47
3 12
elution . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mL of acetone:toluene (65:35) 46
6 13 39
eluate post-treatment . . Evaporate fraction to 0.5 mL then adjust volume to 1 mL 11
8
17
with ethyl acetate for GC-NPD analysis 5
column . . . . . . . . . . . . . . SLB-5ms, 30 m x 0.25 mm I.D., 0.25 μm (28471-U)
oven . . . . 110 °C (2 min), 2.5 °C/min to 205 °C, 10 °C/min to 310 °C (10 min)
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C 20 40
Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NPD, 310 °C

GC Analysis of PCBs in Whole Cow Milk (Spiked) 1.

2.
2,3',6-Trichlorobiphenyl (No. 28)
2,2',5,5'-Tetrachlorobiphenyl (No. 52)
11.
12.
2,2',3,4,4',5'-Hexachlorobiphenyl (No. 138)
3,3',4,4',5-Pentachlorobiphenyl (No. 126)
on SLB®-5ms (20 m x 0.18 mm I.D., 0.18 μm) after 3. 2,2',4,5,5'-Pentachlorobiphenyl (No. 101) 13. 2,3',4,4',5,5'-Hexachlorbiphenyl (No. 167)
4. 3,4,4',5-Tetrachlorobiphenyl (No. 81) 14. 2,3,3',4,4',5-Hexachlorobiphenyl (No. 156)
QuEChERS Cleanup using Supel™ QuE Z-Sep, 5. 3,3',4,4'-Tetrachlorobiphenyl (No. 77) 15. 2,3,3',4,4',5'-Hexachlorobiphenyl (No. 157)
6. 2',3,4,4',5-Pentachlorobiphenyl (No. 123) 16. 2,2',3,4,4',5,5'-Heptachlorobipheny (No. 180)
Fast GC Analysis 7. 2,3',4,4',5-Pentachlorobiphenyl (No. 118) 17. 3,3',4,4',5,5'-Hexachlorobiphenyl (No. 169)
8. 2,3,4,4',5-Pentachlorobiphenyl (No. 114) 18. 2,3,3',4,4',5,5'-Heptachlorobiphenyl (No. 189)
Reference: Stenerson, K.; Ye, M.; Halpenny, M., New Analytical Tools for 9. 2,2',4,4',5,5'-Hexachlorobiphenyl (No. 153) 19. 2,2',3,3',4,4',5'5',6,6'-Decachlorobiphenyl (No. 209)
the Determination of Persistent Organic Pollutants (POPs) in Fatty Food 10. 2,3,3',4,4'-Pentachlorobiphenyl (No. 105)
8
and Beverage Matrices Using QuEChERS Extraction/Cleanup and Gas
14
Chromatography (GC) Analysis. Pittcon 2013. Sigma-Aldrich/Supelco
publication T413034. 7
15
This application demonstrates the use of dispersive SPE or "QuEChERS" for 18
6 10 11
the extraction and cleanup of whole cow′s milk for GC-ECD analysis of 13
16
polychlorinated biphenyl (PCBs). Z-Sep sorbent was used in the cleanup 9
step and found to reduce background and yield recoveries >70% for all PCB 1
3 17
congeners evaluated except no. 209, which was recovered at 68%.
4
Extraction solvent: acetone:hexane (1:1), 20 mL 2 5 19
12
Clean-up volume: 1 mL
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g whole cow′s milk
extraction tube . . . . . . . Supel™ QuE Non-buffered tube (4 g magnesium sulfate,
1 g sodium chloride) (55294-U)
clean-up tube . . . . . . . . . . . . . Supel™ QuE Z-Sep 150 mg, magnesium sulfate
(150 mg, part no. 63135) (55299-U) 10 12 14 16
column . . . . . . . . . . . . . . SLB-5ms, 20 m x 0.18 mm I.D., 0.18 μm (28564-U) Min
oven . . . . . . . . . . . . . . . . . . . 75 °C (1 min), 12 °C/min to 340 °C (10 min)
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . μ-ECD, 340 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . hydrogen, 1.2 mL/min, constant
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL splitless (0.75 min)
liner . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D. FocusLiner™ with taper
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005921

GC Analysis of Pesticides in Avocado on SLB®-5ms 1. Trifluralin 10. Metolachlor

11. Chloropyrifos
(20 m x 0.18 mm I.D., 0.36 μm) after QuEChERS
2. α-BHC
3. Hexachlorobenzene 12. Endosulfan I
4. Dimethoate 13. 4,4'-DDT
Cleanup using Supel™ QuE Z-Sep+, Fast GC 5. Simazine 14. Methoxychlor
15. Coumaphos
Analysis 6. Quintozene
7. γ-BHC 16. Cyflurthrin isomer
8. Heptachlor 17. Cypermethrin isomer
About this analysis: This application demonstrates the use of dispersive 9. Malathion 18. Deltamethrin
SPE for the extraction of pesticides from Avocado. Cleanup is achieved using
Z-Sep + for fat removal. FInal analysis of the extracts was done by LVI-GC/MS
SIM on an SLB-5ms column.
sample/matrix . . . . . . . . . . . . . . . . Avocado spiked with pesticides at 20 ng/g
extraction tube . . . . . . . . . . . . . . Supel QuE Acetate extraction tube (55234-U) 2
extraction process . . . . . . . . . . . add 25 mL of acetonitrile; shake for 1 minute;
add contents of Supel QuE acetate extraction tube; 8 10, 11
shake immediately for 1 minute; centrifuge at 3200 rpm for 5 minutes 9
clean-up tube . . . . . . . . . . . . . . . Supel Que Z-Sep+ cleanup tube (55296-U) 3
4 5 67 14 17
clean-up process . . transfer 3 mL of the acetonitrile layer into a Supel Que Z-Sep+ 13 15 18
12
cleanup tube; shake for 1 minute; centrifuge at 3400 rpm for 3 minutes; 16
draw off supernatant for LVI-GCMS analysis
column . . . . . . . . . . . . . . SLB-5ms, 20 m x 0.18 mm I.D., 0.36 μm (28576-U) 10 12 14 16 18
Min
oven . . . . . . . . . . . . . . . . . . . . 70 °C (2 min), 15 °C/min to 325 °C (6 min)
inj. temp. . . . . . . . . . . . . . . 60 °C (0.28 min), 600 °C/min to 325 °C (5 min)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD
MSD interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 1 mL/min, constant
injection . . . . . . PTV solvent vent; 10 μL, injection dispense speed 15000 μL/min
liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D. FocusLiner with taper
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pesticides spiked at 20 ng/g
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005805

1
GC Analysis of Pesticides in Beef Kidney on
SLB®-5ms (20 m x 0.18 mm I.D., 0.36 μm) after 1. Diphenylamine
2. Hexachlorobenzene
QuEChERS Cleanup using Supel™ Que Z-Sep+, MS 3. γ-BHC
4. Endosulfan I
in SIM Mode, Fast GC Analysis 5. 4,4'-DDE
6. Endosulfan II
Beef kidney was analyzed for pesticides by GC/MS-SIM after extraction and 7. 4,4'-DDD
cleanup. Dispersive SPE was used for both extraction and cleanup. Since 8. Endosulfan sulfate
9. 4,4'-DDT
beef kideny contains approximately 5% fat, Z-Sep + was used for cleanup. 10. Piperonyl Butoxide
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . Dispersive (QuEChERS) 11. Bifenthrin
12. Cyhalothrin
sample/matrix . . 10 gm homogenized beef kidney spiked at 50 ng/g with pesticides 2 7 13. Permethrin isomers
extraction tube . . . . . . . . . . . . . . . . . Supel Que Acetate (AC) Tube (55234-U) 14. Cyfluthrin isomers
extraction process . . . . . . . . . . . add 10 mL of acetonitrile; shake for 1 minute;

add contents of Supel Que acetate extraction tube (55234-U);
shake immediately for 1 minute; centrifuge at 3200 rpm for 5 minutes; 5
(Sample Pretreatment: Homogenize 10 gm beef kidney
and spike with pesticides at 50 ng/g.) 3 11
clean-up tube . . . . . . . . . . . . . . . . . . . . Supel QuE Z-Sep+ tube (55296-U)
clean-up process . . . . . . . . . . . . . transfer 3 mL of the acetonitrile layer into a 9
Supel QuE Z-Sep+ cleanup tube (55296-U); shake for 1 minute; 10
8 13
centrifuge at 3400 rpm for 3 minutes; 4 6 12
14
draw off supernatant for LVI-GCMS analysis
column . . . . . . . . F.S. CAP. SLB-5MS 20 m x 0.18 mm I.D., 0.36 μm (28576-U)
oven . . . . . . . . . . . . . . . . . . . . . . . 70 °C ( 0.5 min), 25 °C/min to 125 °C, 10 20
Min
10 °C/min to 200 °C, 5 °C/min to 300 °C/min (1 min)
inj. temp. . . . . . . programmed, 60 °C (0.28 min), 600 °C/min to 325 °C (5 min)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD
MSD interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 °C
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . helium, 1 mL/min constant flow
injection . . . . . . . . . . . . . . 10 μL LVI, PTV solvent vent, rapid injection speed
liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm ID FocusLiner with taper
sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Beef Kidney extract
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005799

GC Analysis of Pesticides in Fruits and Vegetables Diphenylamine
on a 14% Cyanopropylphenyl Column after SPE

using Supelclean ENVI-Carb™ Pesticide-containing Sample
(relative abundance scale 5× blank)
Homogenize 50 g chopped fruit/vegetable sample in 100 mL acetonitrile.
Add 10 g sodium chloride and homogenize 5 min. Transfer 13 mL of
acetonitrile layer to clean centrifuge tube. Centrifuge at high speed for 5
Methoxychlor
min. Evaporate 10 mL aliquot of supernatant to 0.5 mL under Nitrogen at
35°C.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 5 10 15 20 25 30 35 40 45 50 55 60 65 70
sample/matrix . . . . . . . extract from 50 g homogenized fruit or vegetable sample Min
SPE tube/cartridge . . . . . . . . . . . . . . . . . . ENVI-Carb, 500 mg/6 mL (57094)
sample addition . . . . . . . . . . . . . . . . . . . 0.5 mL concentrated sample extract
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mL acetonitrile:toluene (3:1) Blank Extract
eluate post-treatment . . . . . . . . . . . Concentrate to 2 mL by rotary evaporation.
Add 2 x 10 mL acetone concentrating to 2 mL after each
acetone addition. (Add 50 μL I.S. (500 ng/μL cis-chlordane in acetone)
then dilute to 2.5 mL with acetone.)
column . . . . . . . . . 14% cyanopropylphenyl polysiloxane, 30m × 0.25 mm I.D.,
0.15 μm (Supelco equivalent, Equity-1701, 28372-U) 5 10 15 20 25 30 35 40 45 50 55
oven . . . 70 °C (hold 2 min) to 130 °C at 25 °C; 130 °C to 220 °C at 2 °C/min; Min
220 °C to 280 °C at 10 °C/min and hold 4.6 min For extraction recovery data, request Bulletin T196900.
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD, 285 °C Chromatograms provided by J. Fillion, Agriculture and Agri-Food Canada
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 μL, splitless
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796-0128

7 1. Methamidophos 11. Chlorothiophos

GC Analysis of Pesticides in Spinach on Equity®-1 2. Dichlorovos 12. Tetrasul
6500 3. Acephate 13. Endosulfan sulfate
after SPE using Supelclean™ ENVI-Carb-II/PSA 4. Quintozene 14. Acrinathrin
5. Methyl parathion 15. Bitertanol
6. Carbaryl 16. cis-Permethrin
Sample Pre-treatment: 7. Methyl chloropyriphos 17. trans-Permethrin
Homogenize 10 g of spinach in 10 mL of acetonitrile. 8. Vinclozolin 18. Cypermethrin isomers
9. Procymidone 19. Deltamethrin
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 5 10. Imazalil
sample/matrix . . . . . . . . 10 g of fresh spinach spiked at 0.2 ppm each pesticide

SPE tube/cartridge . . . Supelclean ENVI-Carb-II/PSA, 500mg/300mg/6mL (55119-U) 6 Peak Compound Pesticide Pesticide
Abundance
ID Class Recovery (%)
condition . . . . . . . . . . . . . . . . . . . . . . . 5 mL acetonitrile:toluene (3:1, v/v) 1 Methamidophos Organophosphorous 80
3500 15
sample addition . . . . . . . . . . . . . . . . . . . . 1 mL concentrated sample extract 2
10
16
2
3
Dichlorovos
Acephate
Organophosphorous
Organophosphorous
70
60
elution . . . . . . . . . . . . . . . . . . . . . . . . 20 mL acetonitrile:toluene (3:1, v/v) 1
4 Quintozene Organochloride 92
5 Methyl parathion Organophosphorous 97
eluate post-treatment . . . . . Evaporate eluate to dryness with nitrogen at 40 °C. 12 6 Carbaryl Carbamate 128
Reconstitute in 1 mL acetone:hexane (1:1, v/v). 3

9 11
7
8
Methyl chloropyriphos
Vinclozolin
Organophosphorous
Organochloride
99
83
column . . . . . . . . . . . . . . Equity-1, 30 m x 0.25 mm I.D., 0.25 μm (28046-U) 9
10
Procymidone
Imazalil
Dicarboximide
Imidazole
84
104
oven . . . . . . . 50 °C (5 min), 25 °C/min to 125 °C, 10 °C/min to 30 °C (8 min) 8 17
11 Chlorothiophos Phophosulfi de 106
12 Tetrasul Organochloride 87
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 °C, aux: 325 °C (8 min) 4 13 13 Endosulfan sulfate Organochloride 124
14 14 Acrinathrin Organophosphorous 118
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD, scan range 45-450 amu 18 15 Bitertanol Biphenol 108
19
carrier gas . . . . . . . . . . . . . . . . . . helium, 0.9 mL/min., constant flow mode 0
16
17
Permethrin cis and trans
Cypermethrin isomers
Pyrithroid
Organochloride
82
74
injection . . . . . . . . . . . . . . . . . . . . . 1 μL, splitless (splitter open at 1 min) 10 16 22
Min
28 34 18 Deltamethrin Organobromine 134

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003758

GC Analysis of Phenols in Water on a 1 3 5 7 9

1.
Analyte
Phenol
%Recovery (± CV)
101.8 ± 3.7
5% Phenyl Column after SPE using Supelclean™ 2 2. 2-Chlorophenol 104.7 ± 1.7
ENVI-Chrom P 8 4. 2-Bromophenol (Int. Std.) —
6 5. 3-Methylphenol 104.9 ± 1.6
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 10 6. 2-Nitrophenol 96.3 ± 2.8
sample/matrix . . . . . . . . . . . . . . . . . . . water spiked with phenol compounds 4
11
12 8. 2,4-Dichlorophenol 106.3 ± 2.2
SPE tube/cartridge . . . . . . . . Supelclean ENVI-Chrom P, 250 mg/6 mL (57225-U) 9. 4-Chloro-3-methylphenol 104.6 ± 2.0
10. 2,4,6-Trichlorophenol 103.9 ± 2.1
condition . . . . . . . . . . 6 mL methyl t-butyl or ethyl acetate; 6 mL acetonitrile. 13 11. 4-Nitrophenol 99.9 ± 4.1
Equilibrate with 6 mL DI water 12. 2,3,4,6-T etrachlorophenol 104.6 ± 1.5
13. Pentachlorophenol 97.7 ± 4.8
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 mL water sample. 0 2 4 6 8 10 12 14
Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 300 °C
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712-0073

GC Analysis of Polynuclear Aromatic liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D. FocusLiner™ with taper

sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . grilled hamburger extract
Hydrocarbons (PAHs) in Grilled Hamburger on Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005880
SPB®-608 (20 m x 0.18 mm I.D., 0.18 μm) after
QuEChERS Cleanup using Supel™ QuE Z-Sep, Fast 1. Naphthalene 11. Phenanthrene 21. Benzo[k]fluoranthene
GC Analysis 2. 2-Methylnaphthalene 12. Anthracene 22. Benzo[j]fluoranthene
3. 1-Methylnaphthalene 13. 1-Methylphenanthrene 23. Benzo[e[pyrene
Sample pretreatment: Place 5 g ground hamburger sample into 50 mL 4. Biphenyl 14. Fluoranthene-d10 (I.S.) 24. Benzo[a]pyrene
5. 2,6-Dimethylnaphthalene 15. Fluoranthene 25. Perylene-d12 (I.S.)
centrifuge tube (55248-U). Spike with 100 ng/g target PAHs and internal 6. Acenaphthylene 16. Pyrene 26. Perylene
standards. Let sit 10 min. Add 5 mL water and homogenize. 7. Acenaphthene 17. Retene 27. Indeno[1,2,3-cd]pyrene
This application demonstrates the use of dispersive SPE or QuEChERS for the 8. 2,3,5-Trimethylnaphthalene 18. Benzo[a]anthracene 28. Dibenz[a,h]anthracene
9. Fluorene 19. Chrysene & Triphenylene 29. Benzo[g,h,i]perylene
extraction and cleanup of grilled hamburger for GC-MS analysis of 10. Dibenzothiophene 20. Benzo[b]fluoranthene
polynuclear aromatic hydrocarbons (PAHs). Z-Sep sorbent was used in the
cleanup step and found to reduce background and yield recoveries >80% 23
for a majority of the PAHs tested.
sample/matrix . . . . . . . . . . . . grilled hamburger spiked with PAHs at 100 ng/g
extraction tube . . . . . . . . . . . . . Sodium chloride/magnesium sulfate (55294-U)
extraction process . . . . . . . . add 25 mL acetonitrile, contents of unbuffered salt 6
7 8 10
11 15 19
16
tube (55294-U); shake for 5 minutes; 1
4 5
centrifuge for 5 minutes at 3200 rpm 2 12
3 9 14
clean-up tube . . . . . . . . . . . . . . . . . . . . . . Supel QuE Z-Sep tube (55403-U) 13 17 18
21 24
clean-up process . . . . . transfer 3 mL of acetonitrile layer into a Supel QuE Z-Sep 20 25
cleanup tube (55403-U); shake 1 minute; centrifuge for 22 26
3 minutes at 3400 rpm; remove 1 mL supernatant for GC-MS analysis 27 28 29

column . . . . . . . . . . . . . . . . . . . . SPB-608, 20 m x 0.18 mm I.D. x 0.18 μm
oven . . . . . 60 °C (1 min), 25 °C/min to 275 °C, 10 °C/min to 300 °C (13 min)
inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 °C 6 8 10 12 14 16
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS-SIM Min
MSD interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL splitless (1 min.)

GC Analysis of Polynuclear Aromatic column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SPB-608, 20 m x 0.18 mm I.D. x 0.18 μm

oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 °C (1 min), 25 °C/min to 275 °C, 10 °C/min to 300 °C (13 min)
Hydrocarbons (PAHs) in Salmon on SPB®-608 inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 °C
(20 m x 0.18 mm I.D., 0.18 μm) after QuEChERS detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS, SIM
Cleanup using Supel™ QuE Z-Sep, Fast GC Analysis MSD interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 °C
Extraction solvent: acetonitrile, 25 mL carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 1.5 mL/min, constant
Clean-up volume: 3 mL injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL, splitless (1 min)
Clearn-up tube: Supel™ QuE Z-Sep (500 mg, part no. 55299-U, magnesium liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D. FocusLiner™ with taper
sulfate (450 mg, part no. 63135) Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005923
Stenerson, K.; Ye, M.; Halpenny, M., New Analytical Tools for the
Determination of Persistent Organic Pollutants (POPs) in Fatty Food and
Beverage Matrices Using QuEChERS Extraction/Cleanup and Gas Chroma- 1. Napthalene-d8 (I.S.) 11. Dibenzothiophene 21. Benzo[b]fluoranthene
2. Naphthalene 12. Phenanthrene 22. Benzo[k]fluoranthene
tography (GC) Analysis. Pittcon 2013. Sigma-Aldrich/Supelco publication 3. 2-Methylnaphthalene 13. Anthracene 23. Benzo[j]fluoranthene
T413034 4. 1-Methylnaphthalene 14. 1-Methylphenanthrene 24. Benzo[e]pyrene
Sample pretreatment: Place 5 g ground sample into 50 mL centrifuge tube. 5. Biphenyl 15. Fluoranthene-d10 (I.S.) 25. Benzo[a]pyrene
6. 2,6-Dimethylnaphthalene 16. Fluoranthene 26. Perylene-d12 (I.S.)
Spike with 100 ng/g target PAHs and internal standards. Let sit 30 min. Add 7. Acenaphthylene 17. Pyrene 27. Perylene
5 mL water and homogenize. 8. Acenaphthene 18. Retene 28. Indeno[1,2,3-cd]pyrene
9. 2,3,5-Trimethylnaphthalene 19. Benzo[a]anthracene 29. Dibenz[a,h]anthracene
This application demonstrates the use of dispersive SPE or QuEChERS for the 10. Fluorene 20. Chrysene & Triphenylene 30. Benzo[g,h,i]perylene
extraction and cleanup of raw salmon for GC-MS analysis of polynuclear
aromatic hydrocarbons (PAHs). Supel™ QuE Z-Sep sorbent was used in the
cleanup step and found to reduce background and yield recoveries >80%
for a majority of the PAHs tested. 20
16
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . Dispersive (QuEChERS) 7 8 11 12 17
9
5 6 15
sample/matrix . . . . . . . . . . . . . . . raw salmon spiked with PAHs at 100 ng/g 13
2 22
4 14 18 24
extraction process . . . . . . . . . . . add 25 mL acetonitrile and the contents of the 3 10 19 26
1
Supel™ QuE Non-buffered tube (55294-U); 25
shake for 5 minutes; centrifuge for 5 minutes at 3200 rpm 21 23
27
extraction tube . . . . . . . . . . . . . . . . . . . . . . Supel™ QuE Non-buffered tube, 29
28 30
magnesium sulfate/sodium chloride (55294-U)
clean-up tube . . Supel™ QuE Z-Sep (500 mg, part no. 55299-U), magnesium sulfate
4 6 8 10 12 14 16
(450 mg, part no. 63135) (55299-U, 63135)
Min
clean-up process . . transfer 3 mL of acetonitrile layer, Supel QuE Z-Sep (500 mg) and
MgSO4 (450 mg) into an extraction tube; (shake 1 minute; centrifuge for
3 minutes at 3400 rpm; remove 1 mL supernatant for GC-MS analysis)

GC Analysis of Polynuclear Aromatic Analyte % Recovery (± CV)

1. Acenaphthylene 66 ± 28
Hydrocarbons (PAHs) in Water on a 5% Phenyl 2. Fluorene 95 ± 7.5
3. Phenanthrene 95 ± 8
Column after SPE using Supelclean™ ENVI-18 4. Anthracene 95 ± 11
5. Pyrene 97 ± 7.1
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 6. Benzo(a)anthracene 94 ± 8.3
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Chrysene 101 ± 8.8
SPE tube/cartridge . . . . . . . . . . . . . Supelclean ENVI-18, 500 mg/6 mL (57064) 8. Benzo(b)fluoranthene 103 ± 10
9. Benzo(k)fluoranthene 103 ± 9.4
condition . . . . 2 x 6 mL toluene:methanol (10:1); 6 mL methanol; 6 mL DI water 11. Indeno(1,2,3-cd)pyrene 102 ± 10
sample addition . . . . . . . . . . . . . . . . . . 250 mL water sample at 10 mL/min 10. Benzo(a)pyrene 94 ± 11
drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 min under vacuum 1 2 12. Dibenzo(a,h)anthracene 104 ± 9.7
13. Benzo(ghi)perylene 101 ± 11
elution . . . . . . . . . . . . . . . . . . . . . . . . . 2 x 1 mL toluene:methanol (10:1) 3,4
column . . . . . . . . . . . . . . . . . 5% phenyl polysiloxane, 30m × 0.25 mm I.D., 5
0.25 μm film (Supelco equivalent, SLB-5ms, 28471-U)
oven . . . . . . . . . . . . . . . . . . . . . . . . 70 °C (2 min) to 280 °C at 8 °C/min
6,7
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FID, 310 °C
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL
8,9
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80-207
10
12
11 13
8 12 16 20 24 28
Min

GC Analysis of Semivolatiles in Drinking Water on 1. Acenapthene -d 10

mg/L*
5.0 24. Heptachlor epoxide
g/L*
2.0
a 5% Phenyl Column after SPE using ENVI™-18 DSK 2.
3.
Phenanthrene -d10
Chrysene -d 12
5.0
5.0
25.
26.
2,2',3',4,6-Pentachlorobiphenyl
g -Chlordane
2.0
2.0
4. Hexachlorocyclopentadiene 2.0 27. Pyrene 2.0
Sample Pre-treatment: 5. Dimethylphthalate 2.0 28. a -Chlordane 2.0
Adjust 1 L drinking water to pH <2 with 6 N HCl. Add 5 mL methanol and 6. Acenaphthylene 2.0 29. trans-Nonachlor 2.0
7. 2-Chlorobiphenyl 2.0 30. 2,2',4,4',5,6'-Hexachlorobiphenyl 2.0
mix thoroughly 8. Diethyphthalate 2.0 31. Endrin 2.0
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 9. Fluorene 2.0 32. Butylbenzylphthalate 2.0
10. 2,3-Dichlorobiphenyl 2.0 33. di(2-ethylhexyl)Adipate 2.0
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . acidified drinking water 11. Hexachlorobenzene 2.0 34. 2,2'3,3',4,4',6-Heptachlorobiphenyl 2.0
12. Simazine 2.0 35. Methoxychlor 2.0
SPE tube/cartridge . . . . . . . . . . . . . . . ENVI-18 DSK SPE Disk, 47 mm (57171) 13. Atrazine 2.0 36. 2,2'3,3',4,5',6,6'-Octachlorobiphenyl 2.0
condition . . . . . . . . . . . 5 mL dichloromethane; 5 mL methanol; 5 mL DI water 14. Pentachlorophenol 8.0 37. Benz(a)anthracene 2.0
15. g-BHC 2.0 38. Chrysene 2.0
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mL water sample 16. Phenanthrene 2.0 39. Di(2-ethylhexyl)phthalate 2.0
17. Anthracene 2.0 40. Benzo(b)fluoranthene 2.0
elution . . . . . . . . . . . . . . . . . . 2 x 5 mL acetonitrile rinsing internal chamber 18. 2,4,5-Trichlorobiphenyl 2.0 41. Benzo(k)fluoranthene 2.0
of sample reservoir thoroughly 19. Alachlor 2.0 42. Benzo(a)pyrene 2.0
20. Heptachlor 2.0 43. Perylene -d 12 5.0
column . . . . . . . . . . . . . . . . 5% phenyl polysiloxane, 30 m × 0.25 mm I.D., 21. di-n-Butylphthalate 2.0 44. Indeno(1,2,3 -cd)Pyrene 2.0
0.25 μm (Supelco equivalent, SLB-5ms, 28471-U) 22. 2,2',4,4'-Tetrachlorobiphenyl 2.0 45. Dibenz(a,h)anthracene 2.0
23. Aldrin 2.0 46. Benzo(ghi)perylene 2.0
oven . . . . . . . . . 40 °C to 160 °C (3 min), then to 300 °C (3 min) at 6 °C/min 1
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, scan range m/z = 45-450
carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 33 cm/sec
injection . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL split/splitless, 1 min delay 2
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749-0798 16

7
8
5
6 10 13, 14
9 15 18 21
17 19 22 27,28,29
4
11 20 3
23 25 37
12 1 26 36
24
30 31 34
33 38 39 43
32 35 41 42 44
45 46
40
600 1200 1800 2400

8:00 16:00 Min 24:00 32:00

GC Analysis of Sterols (Silylated Derivatives) in elution . . . . . . . . . . . . . . . . . . . . . . . esters fraction: 2.5 mL of hexane:ethyl acetate (90:10); free sterols fraction;
5 mL of hexane:ethyl acetate (90:10) and 3 mL x 5 of ethanol:diethyl ether:hexane (50:25:25)
Olive Oil (Free Sterol Fraction) on SLB®-5ms after eluate post-treatment . . . . . . . . . . . . Evaporate to dryness and perform silylation as described in the Analysis Note.
SPE using Discovery DSC-Si column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SLB-5ms, 20 m x 0.20 mm I.D., 0.20 μm (28564-U)
Sample pre-treatment oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 °C (1 min), 10 °C/min to 325 °C (10 min)
Weigh 0.25 gm oil into test tube. For spiked sample add 12.5 μL of inj. temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 °C
65-100 μg/mL mixed sterol std. Add 1 ml of hexane:ethyl acetate (90:10). detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MSD, rn/z 500 - 600
After SPE fractionation, evaporate solvent from each fraction, add 1 ml of MSD interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 °C
30% methanolic sodium methoxide:MeOH:MTBE solution (13:27:60), mix and carrier gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . helium, 0.6 mL/min, constant
let sit at room temp for 30 min. Add 1 ml of water and 2 mL of n-heptane.
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 μL, splitless
Withdraw aqueous phase (bottom) and replace w/ 1 mL of 1% citric acid
solution. Withdraw organic phase and dry over anhydrous sodium sulfate. liner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mm I.D. single taper
Silylation: Evaporate organic phase to dryness. Add 125 μL of Sylon BFT and sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Olive oil sample; fractionated, transesterified, and silylated
125 μL of pyridine. Heat at 70°C for 20 min. The sample is then ready for Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005816
GC-MS analysis.
Sterol content is one in a battery of tests used to analyze the composition 1. Cholesterol 5
and determine the grade and authenticity of olive oil. Test results are 2. Brassicasterol
compared against standards such as those established by the International 3. Campesterol
Olive Oil Council (IOC) and US Dept. of Agriculture. In vegetable oils, sterols 4. Stigmasterol
exist in the free form, or esterified to fatty acids. In this application, sterols 5. β-Sitosterol
were analyzed in olive oil separately as free and esterified. This was
accomplished by taking advantage of the polarity difference between these
two groups to fractionate them using silica gel SPE. In place of
saponification, the fractions were transesterified to liberate the esterified
sterols. The fractions were then silylated, and analyzed by GC-MS. The 3
silylated sterols all contained a significant molecular ion in their mass
spectum, which made it possible to perform peak identification free of 4
matrix interference. The sterols isolated in the free fraction are shown here. 1 2
sample/matrix . . . . . . . . olive oil spiked with a mixed sterol standard at 5 μg/g 18 20 22
SPE tube/cartridge . . . . . . . . . . . . . . . . Discovery DSC-Si, 1 g/6 mL (52656-U)
Min
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 x 5 mL hexane

HPLC Analysis of Acrylamide in Fried Potato Chips Efficiency of Recovery

on Discovery® HS F5 after SPE using Discovery® Concentration (µg/mL) %Recovery ± RSD (n=3)
DSC-MCAX and DSC-18 10 78 ± 7%
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 55 93 ± 5%

sample/matrix . . . . . Finely grind 2.0 g of potato chips, place in 20 mL vial and
add 10 mL water. Mix using vortex until mixture forms a thick paste.
(Place sample in centrifuge tubes and centrifuge at 16.1 rcf for 5 min.
Extract aqueous portion of sample leaving oil layer and solids.)
SPE tube/cartridge . . . . . . . . . . Discovery DSC-MCAX, 300 mg/3 mL (52784-U) Spiked Sample (38µg/kg) H2C
SPE tube/cartridge . . . . . . . . . . . . . . . . Discovery DSC-18, 1g/6mL (52606-U)
condition . . . . . . . . . . connect a Discovery DSC-MCAX SPE tube to the top of a
Discovery DSC-18 SPE tube using an SPE tube adapter,
pass 1 mL methanol, then 1 mL DI water through
O NH2
both SPE tubes until the tubes are dry
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL of aqueous extract
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL DI water Acrylamide
elution . . . . . . . . . . . . . removing upper MCAX SPE tube, pass 2 mL methanol
through lower DSC-18 SPE tube
mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100% ultra-pure water
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C Control Standard (55µg/kg)
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESI+
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003759
0 2 4 6 8 10 12 14
Min

HPLC Analysis of Aflatoxins B1, B2, G1 and G2 in 1. Aflatoxin G1

Corn on Ascentis® Express C18 after SPE using 2. Aflatoxin B1
2 3. Aflatoxin G2
Supel™ Tox AflaZea and Fluorescence Detection 4. Aflatoxin B2
Aflatoxins are closely related compounds produced as secondary metablo-
lites of fungi. They are found in grains and nuts like corn, wheat or peanuts.
Shown here is a rapid, sensitive method to extract these compounds from
corn using SPE cartridges specifically designed for aflatoxins, Supel Tox
1 4
AflaZea (aflatoxin zearalenone), which removes interferences from grains,
feeds, TMR samples, peanuts, peanut products and aqueous solutions. 3
Analysis was via HPLC on Ascentis Express Fused-Core C18 colums.
sample/matrix . . . . . . . . . 25 g corn meal in 100 mL acetonitrile:water (84:16) 0 2 4 6 8 10 12
SPE tube/cartridge . . . . . . . . . . . . . . . . Supel™ Tox AflaZea, 6 mL (55314-U) Min
elution . . . . . . . . . . . . . . . . . . . . collect sample in 5 mL silanized glass vial
eluate post-treatment . . . . transfer 200 μL sample, add 300 μL TFA derivatization
solution (70:20:10 water:TFA:acetic acid), heat in
microreactor 20 min at 65 °C, cool, add 580 μL deionized water, vortex
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.350 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 psi (22.4 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . FL, Ex 360 nm, Em 440 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005922

HPLC Analysis of Aflatoxins in Wheat on Ascentis® 1. Aflatoxin G2

Express C18 after SPE using Supel™ Tox AflaZea 2. Aflatoxin G1
3. Aflatoxin B2 4
Sample/matrix: 4. Aflatoxin B1
Weigh 25 grams of wheat meal into a 500 mL Erlenmeyer flask. Add 100 mL
of 84:16 acetonitrile:water, seal, and place on an orbital shaker at med-high 2
speed for 90 minutes. Filter the sample through a #2 qualitative filter. Spike
sample at 2 mg/mL using analytical standards. 3
sample/matrix . . . . . . . . . . . . 25 g wheat meal spiked with pesticide standards
SPE tube/cartridge . . . . . . . . . . . . . . . . . Supel™Tox AflaZea, 6 mL (55314-U)
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL 0 2 4 6 8 10 12 14
elution . . . . . . . . . . . . . . . . . . collect eluate in 5 mL silanized culture tubes
Min
eluate post-treatment . . . . . . . . . . . . . . combine 200 μL of sample eluate and
880 μL of DI water in a 2 mL autosampler vial
mobile phase . . . . . . . . . . . . . . . . . (A) water; (B) methanol; (C) acetonitrile;
(720:120:120, A:B:C) containing 0.780 g/L potassium bromide,
230 μL/L nitric acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.400 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4568 psi (315 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FLD 360 ex; 440 em
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
derivatization . . . . . . . . . . . . . . . . . . . via KOBRA electrochemical cell, 50 μA
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005907

HPLC Analysis of Aflatoxins on Ascentis® 1. Aflatoxin G2 4

Express C18 2. Aflatoxin G1
3. Aflatoxin B2
Aflatoxins are a complex of closely related toxins produced as secondary 4. Aflatoxin B1
metablolites of fungi. They are found in grains and nuts such as corn, wheat, 2
or peanuts. They are produced by aspergillus flavus and a. Parasiticus.
Members of the group include aflatoxin b1, aflatoxin b2, aflatoxin g1, 3
aflatoxin g2, aflatoxin m1, and aflatoxin m2.
mobile phase . . . . (A) water; (B) methanol; (C) acetonitrile; (720:120:120, A:B:C)
containing 0.780 g/L potassium bromide, 230 μL/L nitric acid
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.400 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4568 psi (315 bar) 0 2 4 6 8 10 12 14
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C Min
detector . . . . . . . . . . . . . . . . . FLD excitation = 360 nm, emission = 440 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 μL
sample . . . . . 200 μL spiked 84:16 acetonitrile:di water in 880 μL di water, Spike
concentrations: B1 = 16 ppb; B2 = 4 ppb; G1 = 16 ppb; B2 = 4 ppb
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005908

100 3.82
HPLC Analysis of Chloramphenicol in Milk on 95
RT: 0.00 - 5.99
90 SupelMIP SPE Extract of

Ascentis® C18 after SPE using SupelMIP® 85 Chloramphenicol Spiked Milk
3.78
SPE-Chloramphenicol 80
• Average recovery of 84%
Relative Abundance
75
• Increased sample cleanliness
70
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 65 • Reduced background and
improved signal-to-noise
sample addition . . . . . . . . . . . . . . . . . . . whole pasteurized milk spiked with 60
55
chloramphenicol at 15 ng/mL and 38 ng/mL 50
SPE tube/cartridge . . SupelMIP SPE - Chloramphenicol, 25 mg/10mL (LRC) (53210-U) 45

40
condition . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL methanol;1 mL DI water 1.03
35
30
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 25
washing . . . 2 x 1 mL MS-grade water, 1 mL 5% acetonitrile in 0.5% acetic acid, 20
15 3.67
2 x 1 mL MS-grade water, 1 mL 20% acetonitrile in 10 1.68
1% ammonium hydroxide. (dry SPE tubes for 15 min. under gentle 5 0.22 0.65 0.79 1.44 1.58 1.84
2.05
2.29 2.51 2.90 3.14 3.35 4.47 4.53 4.79 5.04 5.38 5.50 5.57
vacuum; 3 x 1 mL dichloromethane; dry SPE tubes 0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
for 1 min. under gentle vacuum)
100
RT: 0.00 - 5.99 1.99 3.81
elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . elute chloramphenicol with 95
Conventional SPE Extract of
2 x 1 mL methanol:acetic acid:MS-grade water (89:1:10, v/v/v) 90
Chloramphenicol Spiked Milk
85
eluate post-treatment . . . . evaporate combined eluate to dryness at 50 °C under 80 • Average recovery of 79%
nitrogen. reconstitute in 150 mL LC mobile phase prior to LC-MS analysis 75 1.97 • Higher background and
3.75
Relative Abundance
reduced signal-to-noise
column . . . . . . . Ascentis C18, 2.1 mm x 10 cm I.D., 3 μm particles (581301-U)
70
65 1.94 • Complete sample prep
mobile phase . . . . . . . . . (A) 100 mM ammonium acetate: (B) MS-grade water: 60 procedure involved protein
0.99
55 precipitation followed by SPE
(C) acetonitrile (10:60:30, A:B:C) 50 cleanup and three LLE Steps
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 mL/min 45

40
1.09 3.71
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 35
30 2.07
detector . . . . . . . . . . . . . . . . . . . . . . . . . . MS, ESI(-), 320-323 m/z range 25 1.91
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL 20 1.81
2.19 2.77
15 1.32 1.77 4.64 4.68
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G004433 10 1.38 2.43 2.67
3.31 3.59 4.23 4.36 5.00 5.12
5.28
0.16 0.79 2.91 5.81
5 0.44 3.47
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
Min

LC-MS/MS Analysis of Carbendazim in Orange 1 1. Carbendazim

Juice (Spiked) on Ascentis® Express C18 after
Supel™ QuE PSA/C18 QuEChERS Cleanup
Simple and fast extraction and analytical method were developed for
analysis of fungicide carbendazim in orange juice samples. QuEChERS
extraction method was used with PSA/C18 cleanup. Ascentis Express C18
column chromatography was coupled to MS/MS detection with limits of
detection at 0,1 ppb.
sample/matrix . . . . . . . . . . . . . orange juice spiked with carbendazim at 1 ppb
extraction tube . . . . . . . . . . . . . . . . Supel™ QuE Citrate (EN) Tube (55227-U)
extraction process . . . . . add 10 mL of orange juice with pulp to a 50 mL empty
extraction tube (55248-U); add carbendazim at 1 ppb; 0 1 2 3
add 10 mL acetonitrile; (shake for 1 minute; add contents of citrate extraction tube Min
(55227-U); shake for 1 min; centrifuge at 3200 rpm for 5 minutes)
clean-up process . . transfer 0.7 mL of the acetonitrile layer to the PSA/C18 cleanup
tube (55288-U); shake 1 minute; (centrifuge for 3 minutes at 3400 rpm;
remove 0.25 mL of the supernatant;
mix with 0.25 mL water for LC-MS analysis)
mobile phase . . . . . . . . . . . . . . . . . . A) 10 mM ammonium acetate in water;
(B) 10 mM ammonium acetate in methanol
gradient . . . . . . . . . 0-1 min: 30% B; 1.5-3.5 min: 100% B; 3.5-7 min: 30% B
flow rate . . . . . . . . . . . . . . . . . 0-1 min: 0.3 mL/min; 1.5-7 min: 0.5 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . MS, ESI(+), MRM, m/z 192/160, 192/132
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005619

LC-MS/MS of Pesticides in Oranges on Ascentis® tR(min) tR(min)

1. Methomyl 0.60 20. Quinalphos 7.23
Express C18 after Sample Prep using Supel™ Que 2. Trichlorfon 1.22 21. Edifenphos 7.28
Z-Sep/C18 QuEChERS sorbent 3. Carbendazim
4. Aldicarb
1.94
2.70
22. Etrimfos
23. Fenthion
7.33
7.34
Sample pretreatment: 10 g of pureed oranges homogenized with rind; 5. Parathion-methyl 5.87 24. Fenitrothion 7.36
6. Methabenzthiazuron 6.16 25. Diazinon 7.37
spike at 50 ppb by adding 16.75 μL of a pesticide mix containing each 7. Naled 6.37 26. Tolclofos-methyl 7.52
analyte at 30 μg/mL 8. Methidathion 6.38 27. Phorate 7.53
This demonstrates the application of the Z-Sep/C18 sorbent to the cleanup 9. Clethodim 6.42 28. Chlorpyrifos-methyl 7.67
10. Phosmet 6.50 29. EPN 7.68
of fruits in the LC-MS-MS analysis of polar pesticides including organo-
11. Ametryn 6.58 30. Terbufos 8.14
phosphates, carbamates and benzoylureas 12. Sethoxydim 6.61 31. Ethion 8.36
sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . Dispersive (QuEChERS) 13. Anilazine 6.78 32. Lufenuron 8.40
14. Fenxehamid 6.94 33. Spiromesifen 8.74
sample/matrix . . 10 g of pureed oranges (homogenized with rind); spike at 50 ppb 15. Mecarbam 7.00 34. Octhilinone 8.74
(add 16.75 μL of a custom made pesticide mix, each analyte at 30 μg/mL) 16. Oryzalin 7.06 35. Pyraclostrobin 8.75
17. Diflubenzuron 7.18 36. Carbophenothion 8.83
extraction tube . . . . . . . . . . . . . . . . Supel™ QuE Citrate (EN) Tube (55227-U) 18. Fenoxycarb 7.19 37. Flufenoxuron 9.02
extraction process . . add 10 mL acetonitrile; shake for 1 minute; add contents of a p
19. Iprobenfos 7.21 py
38. Fenpyroximate 9.10
Supel QuE citrate extraction tube (55227-U);
shake immediately for 1 minute; centrifuge at 3200 rpm for 5 minutes
clean-up process . . . . . . . . . . . transfer 0.7 mL of the acetonitrile layer into a
Supel QuE Z-Sep/C18 cleanup tube (55284-U); shake for 1 minute;
centrifuge at 5000 rpm for 5 minutes; (transfer 0.2 mL of the supernatant
into an empty 1.5 mL centrifuge tube; add 0.2 mL of water;
centrifuge at 5000 rpm for 2 minutes.)
mobile phase . . . . . . . . . . . . . . . . . (A) 10 mM ammonium acetate in water;
gradient . . . Hold at 30% B for 1 min; 30% to 80% B in 2 min; hold at 80% B
for 4 min; hold at 100% B for 3 min; hold at 30% B for 3 min
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2730 psi (188 bar) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C Min
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MS/MS, ESI positive
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005645

SPE Forensics and Toxicology
1. Amphetamine
Forensics and Toxicology
10
2. Methylamphetamine
DSC-MCAX SPE
8
HPLC Analysis of Amphetamines in Urine on
Discovery® HS F5 after SPE using Discovery®
6
mAU
DSC-MCAX and Standard C18
4
Sample Pre-Treatment: 1
1 mL human urine was spiked with 2 mg/mL amphetamine and 2
2
methylamphetamine. The spiked sample was diluted 1:1 with 1:1 with
0
50 mM ammonium acetate, pH 6.0.
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 0 2 4 6 8
sample/matrix . . . . . . . . . . . . . . . . . . human urine spiked amphetamine and Min
200
methylamphetamine at with 2 mg/mL
SPE tube/cartridge . . . . . . . . . . . Discovery DSC-MCAX, 100 mg/3mL (52783-U)
condition . . DSC-MCAX SPE tube: 1 mL methanol; 1 mL 50 mM ammonium acetate,
Standard C18
pH 6.0; C-18 SPE tube: 1 mL methanol; 1 mL DI water
mAU
100
washing . . . . . . DSC-MCAX SPE tube: 1 mL 50 mM ammonium acetate, pH 6.0;
1 mL 1M acetic acid; 1mL methanol;
C-18 SPE tube: 1 mL DI water; 1 mL 20% methanol
elution . . . . . . . . . . . . . DSC-MCAX SPE tube: 1 mL 5% ammonium hydroxide
0
in methanol; C-18 SPE tube: 1 mL methanol
eluate post-treatment . . evaporate to dryness with nitrogen at room temperature, 0 2 4 6 8
Min
reconstitute in mobile phase
Discussion:
column . . . . . Discovery HS F5, 15 cm x 4.6 mm I.D., 5 μm particles (567516-U) Note the Y-axis scale difference between DSC-MCAX and C18 SPE.
mobile phase . . (A) 10 mM ammonium acetate, pH 4.5: (B) methanol; (35:65, A:B) DSC-MCAX SPE offered a maximum background height of ~9 mAU.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL/min In contrast, standard C18 background levels were 20 times greater
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 °C than DSC-MCAX.
Also, on DSC-MCAX absolute recovery averaged at 100.3 and
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 210 nm 101.7%, for amphetamine and methylamphetamine, respectively.
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL On standard C18, absolute recovery averaged at 48 and 79% for
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003761 the two compounds.


Discovery DSC-8
External
Standard
3
um
Extract on
Excellent
E Peak Shape
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL
Application No.Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . G003766
Blank Serumm Low

L background/
Extract on minimal
m interferences
DSC-8 for
and
a resolution at
reduced
r run times
0 2 4 6
Min

HPLC Analysis of Furosemide (Lasix) in Horse Efficiency of Recovery

Serum on Discovery® C18 after SPE using Concentration Avg. Response %Recovery
Sample (μg/mL) Factor ± RSD (n=3)
Discovery® DSC-18 A 10.00 2.307 93.1 ± 3.1
Sample Pre-treatment: B 5.00 1.168 100.8 ± 1.4
C 1.00 0.107 97.4 ± 2.8
Neat horse serum was acidified with 10 mL 6 M HCl/mL serum. Internal
D 0.50 0.065 120.7 ± 1.3
standard (I.S.) blank was prepared by spiking acidified horse serum with E 0.10 0.009 132.8 ± 8.3
indapamide at 10 mg/mL. Sample A = 10 mg/mL furosemide in I.S. blank.
Sample B = 5 mg/mL furosemide in I.S. blank. Sample C = 0.5 mg/mL
furosemide in I.S. blank. Sample D = 0.1 mg/mL furosemide in I.S. blank.
Sample E = 0.05 mg/mL furosemide in I.S. blank.
sample/matrix . . . . . acidified horse serum spiked with indapamide at 10 mg/mL
SPE tube/cartridge . . . . . . . . . . . . . Discovery DSC-18, 50 mg/1 mL (52601-U) 1. Furosemide (3.683)
condition . . . . . . . . . 1 mL methanol; 1 mL 10 mM potassium phosphate, pH 3 2. Indapamide (4.586)
washing . . . . . . . . . . . . . . . . . . . . . . . . 10 mM potassium phosphate, pH 3
1
elution . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 60% methanol in DI water. serum blank
column . . . . . . . . Discovery C18, 15 cm x 4.6 mm I.D., 5 μm particles (504955)
mobile phase . . . . . . . . . . . . (A) 10 mM KH2PO 4, pH 3 (adjusted with H3 PO 4): 2 0.05 µg/mL spike
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
0.5 µg/mL spike
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 234 nm
5 µg/mL spike
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G003764
5 µg/mL ext. std.
0 2 4
Min

HPLC Analysis of 3-Methylpyrazole and Efficiency of Recovery

4-Methylpyrazole in Urine on Discovery® C18 after Discovery Leading Competitor
Compound (mg/mL) DSC-SCX (n=3) SCX (n=2)
SPE using Discovery® DSC-SCX % Recovery %RSD %Recovery %RSD
1. 3-methylpyrazole (1.0) 89.4 ±10.2% 67.1 ±20%
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 2. 4-methylpyrazole (1.0) 79.4 ±6.8% 50.5 ±30%
sample/matrix . . . . . . . . urine spiked with 3- and 4-methylpyrazole at 1 mg/mL
SPE tube/cartridge . . . . . . . . . . . . Discovery DSC-SCX, 500 mg/3 mL (52686-U)
SPE Procedure
condition . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL methanol; 2 mL DI water
1. Condition and equilibrate with
sample addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL 2 mL MeOH & 2 mL DI water.
washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mL DI water 2 2. Load 1 mL urine sample spiked with
elution . . . . 2 mL 5% methanol in 250 mM potassium phosphate buffer, pH 7.4 1 mg/mL of each analyte.
column . . . . . . . . . . . . Discovery C18, 15 cm × 4.6 mm, 5 μm preceded by a 1 3. Wash with 2 mL DI water.
2 cm guard column and 0.5 μm frit filter (504955) 4. Elute with 2 mL 5% MeOH in
mobile phase . . . . . . . . . . . . . . . MeOH:5 mM phosphate buffer, pH 6 (20:80) 250 mM phosphate buffer, pH 7.4.
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min 5. Quantify against external standards
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C via HPLC analyses.
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 220 nm
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 μL, dilute urine extract
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G001635
0 2 4 6 8 10 12 14
Min

LC-MS Analysis of 2C Amines Drugs of Abuse in Peak Compound Rentention m/z

Urine on Ascentis® Express HILIC following Sample 1 Bromo Dragonfly 2.03 294
2 DOI 2.18 322
Prep using Supel™-Select SCX SPE 3 2C-T-7 2.27 256.1
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 4 DOB 2.29 274.1
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . spiked urine, 1 mL 5 2C-B 2.54 260
SPE tube/cartridge . . Supel-Select SCX SPE 96-well Plate, 30 mg/well (575664-U) 6 2C-B-Fly 2.74 284
condition . . . . . . . . . . . 1 mL methanol, then 1 mL 0.1% formic acid in water 3, 4
washing . . 2 x 1 mL 10% methanol in water, 2 x 1 mL 1% formic acid in methanol 2
mobile phase . . . . . . 5 mM ammonium formate in acetonitrile:water (90:10, v/v) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min 5
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1570 psi (108 bar) 6
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL 0 2



400 Clenbuterol
CPS
100
0 1 2 3 4 5
Max 775 CPS
6000
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL 4000
CPS
3000
Clenbuterol
2000
1000
0
0 1 2 3 4 5
Min

LC-MS Analysis of Fluoroamphetamine Drugs of Peak Compound Rentention m/z

Abuse on Ascentis® Express HILIC after SPE using 1 2-Fluoroamphetamine 2.42 154.1
2 4-Fluoroamphetamine 2.58 154.1
Supel™-Select SCX 96-Well Plate 3 2-Fluoromethamphetamine 2.72 168.1
sample preparation . . . . . . . . . . . . . . . . . . . . . . SPE (Solid Phase Extraction) 4 4-Fluoromethamphetamine 2.97 168.1
sample/matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . urine, 1 mL 3
SPE tube/cartridge . . Supel-Select SCX SPE 96-well Plate, 30 mg/well (575664-U)
condition . . . . . . . . . . . 1 mL methanol, then 1 mL 0.1% formic acid in water 4
washing . . 2 x 1 mL 10% methanol in water, 2 x 1 mL 1% formic acid in methanol
1
mobile phase . . . . . . 5 mM ammonium formate in acetonitrile:water (90:10, v/v)
2
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 mL/min
pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1570 psi (108 bar)
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C 0 2
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TOF/MS Min
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 μL
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G006194

SPE Pharma and Biopharma
Pharma and Biopharma Concentration
Compound (μg/mL) % Recovery % RSD (n=6)
HPLC Analysis of Bronchodilators Theophylline 1. Theobromine 0.1 97.4 ±6.8
0.5 96.4 ±8.5
and Other Caffeine Metabolites from Serum on 1.0 96.1 ±5.0
Discovery® RP-AmideC16 after SPE using 2. Paraxanthine 0.1
0.5
96.2
95.2
±8.4
±8.7
Discovery® DSC-18 1.0 95.0 ±8.7
3. Theophylline 0.1 97.8 ±8.5
4. Caffeine 0.1 98.8 ±3.9
condition . . . . . . . . . . . . . . . . . . . . . . . . 2 mL methanol, then 2 mL water 0.5 95.6 ±6.7
0.50 μg/mL, or 1.0 μg/mL each analyte
sample preparation . . 1 mL methanol; evaporate to dryness with nitrogen stream at 1. Theobromine
room temperature; reconstitute in 200 μL mobile phase 2. Paraxanthine
containing 0.2 μg/mL or 7-methylxanthine (IS) 3. Theophylline
sample preparation . . . . . . . . . . . . . . . . . . . . . 2 mL 5% methanol in water; 4. Caffeine
dry tube 10 min with nitrogen stream IS 7-Methylxanthine
column . . Discovery RP-AmideC16, 15 cm × 4.6 mm, 5 μm particles, preceded by
a 2 cm RP-AmideC16 guard column and 0.5 μm frit filter (505013) 1
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min Serum Blank
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 °C
detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . UV, 272 nm 3
IS 2
injection . . . . . . . . . . . . . . . . . . . . 20 μL reconstituted porcine serum extract 4
Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G000594
Spiked Serum
(0.50 mg/mL)
0 2 4 6 8 10 12
Min

SPE Pharma and Biopharma

Actual
1 1.0258 0.0103 0.0092 89.32 0.9003
Dilute 2 mL of ethyl acetate-cream sample with 8 mL hexane:ethyl 4 1.1369 0.0114 0.0119 104.39 1.0442
acetate (2:1, v/v). 5 1.1390 0.0114 0.0113 99.12 1.0785
Average: 99.86 1.02
RSD: 6.99 0.06
flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min
column temp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 °C
Hydrocortisone
injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 μL
Hydrocortisone
1.0 2.0 3.0
Min

Indexes Analyte
Analyte Index
Page number groupings in this index indicate the appearance of applications within multiple techniques/market focus groups. Additionally, you can activate the Text Select tool (bottom
menu bar) and highlight the desired analyte. Right-click to copy the analyte name and paste it into the Search bar. Results for the active volume will appear on the left side of the window,
allowing you to browse the results. Because this index spans all three volumes, use the footer links to open a different volume to search for additional results.
Acenaphthylene ..................................................................................................................................................... 2375,310,1388,1411,1482,2028,

—1— 1408,2010,1409,2011,1451,1936,1480,2013,1481,2014,1483,2029,2365,2420,1506,2075,1507,2076,1517,1518,1524,1540,1541,1552,
1553,1554,1555,1556,1557,1558,1560,1561,1562,1563,1564,1567,1587
cis-11,14-Eicosadienoic acid methyl ester (C20:2) at 2 wt % ....................................................................................................... 1822,1922,2254
Acenaphthylene-d10 ....................................................................................................................................................................... 1484,2030,2366,2421
cis-8,11,14-Eicosatrienoic acid methyl ester (C20:3n6) at 2 wt % ............................................................................................... 1822,1922,2254
Acephate ................................................................................................................................................................................. 1347,1394,1798,1894,
cis-5,8,11,14,17-Eicosapentaenoic acid methyl ester (C20:5n3) at 2 wt % ................................................................................. 1822,1922,2254
1350,1401,1860,1995,1430,1641,1796,1892,1797,1893
cis-11,14,17-Eicosatrienoic acid methyl ester (C20:3n3) at 2 wt % ............................................................................................. 1822,1922,2254
Acesulfame .............................................................................................................................................................................................. 339,340,346,347
—A— Acetal ........................................................................................................................................................................................................................... 1969
Acetaldehyde .............................................................................................................................. 21,2326,1895,1969,2026,2027,2292,2464,2293,2294
A10 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 Acetaldehyde-2,4-dinitrophenylhydrazone ........................................................................................................................................... 22,2334,23,2335
A11 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 Acetaldehyde-2,4-DNPH ............................................................................................................................................................................................. 2337
A12 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 Acetaldehyde (2,4-DNPH derivative) .................................................................................................................................................................. 2336,24
A13 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 Acetaminophen ....................................................................................................................................................... 1105,183,850,869,870,871,912,971
A14 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 Acetamiprid ............................................................................................................................................................................................................ 1358,19
A15 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 Acetanilide ..................................................................................................................................................................................................................... 870
A16 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 Acetic acid ............................................................................... 1491,1581,2319,2331,1492,1583,2320,2333,1808,1908,2244,1809,1909,2245,
A17 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 1873,2031,2278,1969,1970,2404,1971,2405,2026,2027,2045,2350,2060,2443,2210,2211,2213
A18 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 Acetic acid, 8% .......................................................................................................................................................................................................... 1993
A20 (Polynucleotide) .......................................................................................................................................................................................... 1096,691 Acetochlor .......................................................................................................................................................................................................... 1888,2067
F(ab′)2 ........................................................................................................................................................................................................................... 673 Acetoin ............................................................................................................................................................................................................... 2056,2439
Acebutolol ................................................................................................................................................................................. 131,139,943,895,942,954 Acetol acetate .................................................................................................................................................................................................... 2056,2439
Acebutolol hydrochloride ................................................................................................................................................................... 2862,847,2863,848 Acetone ........................................... 21,2326,316,1431,1572,1490,1580,2318,2330,1492,1583,2320,2333,1493,2057,2370,2440,1504,2073,
Ace inhibitors ......................................................................................................................................................................................................... 123,849 1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1676,1725,1726,1727,1780,1969,2051,2435,2210,2211,2212,2452,2213,2214,2453,
Acenaphthalene ............................................................................................................................................................................................................. 311 2226,2456,2292,2464,2293,2294
Acenaphthene ............................................................................................................................................................................... 2375,310,311,685, Acetone-2,4-dinitrophenylhydrazone .................................................................................................................................................... 22,2334,23,2335
1408,2010,1409,2011,1451,1936,1480,2013,1481,2014,1483,2029,2365,2420,1517,1518,1524,1540,1541,1552,1553,1554,1555,1556,1557, Acetone-2,4-DNPH ...................................................................................................................................................................................................... 2337
1558,1560,1561,1562,1563,1564,1567,1587 Acetone (2,4-DNPH derivative) ........................................................................................................................................................................... 2336,24
Acenaphthene-d10 ........................................................................................................................... 1388,1411,1482,2028,1483,2029,2365,2420, Acetonitrile .............................................................................................................................................................................. 1490,1580,2318,2330,
1506,2075,1507,2076,1524,1550,1551,1552,1553,1554,1555,1556,1557,1564 2205,2206,2207,2450,2208,2209,2451,2224,2225,2455
Acenaphthene-d10 ........................................................................................................................................................................................... 1567,1587 Acetophenone ................................................................................................................................ 36,841,554,1117,1118,1550,1551,1564,1574,2328
Acenaphthene-d10 (I.S.) ........................................................................................................................................................................................... 1558 2-Acetylaminofluorene ...................................................................................................................................................................................... 1550,1551
3-O-Acetyl-α-boswellic acid ...................................................................................................................................... 1325,1326,1334,1335,1336,1337
3-O-Acetyl-β-boswellic acid ...................................................................................................................................... 1325,1326,1334,1335,1336,1337
i-1 Pub Date: August 2014 Vol 1: 1 thru 1428 Vol 2: 1429 thru 2468 Vol 3: 2469 thru 3042
Indexes Analyte
6-Acetylcodeine solution ...................................................................................................................................................................................... 264,484 Active amyl alcohol .......................................................................................................................................................................................... 2026,2027

15
N-Acetyl-DL-cysteine ......................................................................................................................................................................................... 2729,498 Adenine .................................................................................................................................................................................................................. 684,765
3-Acetyldeoxynivalenol ............................................................................................................................................................................... 12,358,17,394 Adenosine ................................................................................................................................................................................. 685,687,688,689,690,769
15-Acetyldeoxynivalenol ......................................................................................................................................................................................... 17,394 Adenosine diphosphate ................................................................................................................................................................................................ 690
15-Acetyldeoxynivalenol solution .......................................................................................................................................................................... 12,358 Adenosine diphosphoribose ......................................................................................................................................................................................... 690
Acetylene ................................................................................................................................................... 1639,1665,1723,1724,1734,1749,1750,1751 Adenosine monophosphate ......................................................................................................................................................................................... 690
Acetylene (430 ng on column) ............................................................................................................................................................................... 1752 Adenosine triphosphate ............................................................................................................................................................................................... 690
2-Acetylfuran ..................................................................................................................................................................................................... 2056,2439 Adrenomedullin rat ...................................................................................................................................................................................................... 738
3-O-Acetyl-11-keto-β-boswellic acid ....................................................................................................................... 1325,1326,1334,1335,1336,1337 Aflatoxin B1 .......................................................................................................................................................................... 1415,328,330,1416,329,331
N-Acetyl-DL-methionine ...................................................................................................................................................................................... 2730,499 Aflatoxin B1 solution .......................................................................................................................................................................................... 1414,327
2-Acetyl-1-methylpyrrole ................................................................................................................................................................................. 2056,2439 Aflatoxin B2 .......................................................................................................................................................................... 1415,328,330,1416,329,331
6-Acetylmorphine, 1.0 mg/mL solution ............................................................................................................................................................ 264,484 Aflatoxin B2 solution .......................................................................................................................................................................................... 1414,327
N-Acetylphenylalanine ........................................................................................................................................................................................ 2731,500 Aflatoxin G1 .......................................................................................................................................................................... 1415,328,330,1416,329,331
N-Acetylprocainamide ................................................................................................................................................................................................ 1121 Aflatoxin G1 solution .......................................................................................................................................................................................... 1414,327
N-Acetyl-4H-pyridine ........................................................................................................................................................................................ 2056,2439 Aflatoxin G2 .......................................................................................................................................................................... 1415,328,330,1416,329,331
2-Acetylpyrrole .................................................................................................................................................................................................. 2056,2439 Aflatoxin G2 solution .......................................................................................................................................................................................... 1414,327
Acetylsalicylic acid ................................................................................................................................................................................................... 37,279 Aflatoxin M1 .................................................................................................................................................................................................................. 331
Acetylsalicylic acid .............................................................................................................................................................................. 910,922,1141,1236 Aflatoxin M1 solution ................................................................................................................................................................................................... 396
N-Acetyl-DL-tryptophan ....................................................................................................................................................................................... 2732,501 AICAR ............................................................................................................................................................................................................................. 467
ac-GGALKALKGLK-amide ...................................................................................................................................................................................... 532,707 Air ................................................................................................................................................................................................................................. 1699
ac-GGGLGGAGGLK-amide ..................................................................................................................................................................................... 532,707 Ajmalicine .................................................................................................................................................................................................................... 1316
Acid phosphatase ......................................................................................................................................................................................................... 801 Alachlor .................................................................................................................................. 1388,1411,1482,2028,1506,2075,1507,2076,1886,2065
Acid red ......................................................................................................................................................................................................................... 388 Alanine ........................................................................................................................................................................................................................... 810
Acinathrin ........................................................................................................................................................................................................... 1886,2065 D-Alanine .............................................................................................................................................................................................................. 2733,503
ac-IVYIHPR ............................................................................................................................................................................................................. 531,932 L-Alanine ............................................................................................................................................................................................ 2733,503,1897,2304
ac-KVYIHPI ............................................................................................................................................................................................................. 531,932 DL-Alanine ............................................................................................................................................................................................ 2734,504,2735,505
ac-KYALKALKGLK-amide ....................................................................................................................................................................................... 532,707 β-Alanine ....................................................................................................................................................................................................................... 810
ac-KYGLGGAGGLK-amide ...................................................................................................................................................................................... 532,707 DL-Alanine (AQC derivatives) ............................................................................................................................................................................. 2738,517
ac-RGAGGLGLGK-amide ............................................................................................................................................................................................... 771 DL-Alanine (t-BOC derivatives) .......................................................................................................................................................................... 2751,533
ac-RGGGGLGLGK-amide ................................................................................................................................................................................................ 771 DL-Alanine, FMOC derivatives ............................................................................................................................................................................ 2773,588
ac-RGVGGLGLGK-amide ................................................................................................................................................................................................ 771 Alanylvaline .......................................................................................................................................................................................................... 2736,506
ac-RGVVGLGLGK-amide ................................................................................................................................................................................................ 771 Albumin ........................................................................................................................................................................................................... 748,750,752
Acrolein ................................................................................................................................................................................................................... 21,2326 Albumin from chicken egg white ....................................................................................................................................................... 207,738,761,762
Acrolein-2,4-dinitrophenylhydrazone .................................................................................................................................................... 22,2334,23,2335 Bovine Serum Albumin .......................................................................................................................................................... 734,735,736,737,744,825
Acrolein (2,4-DNPH derivative) ........................................................................................................................................................................... 2336,24 Albumin (bovine serum) ............................................................................................................................................................................................. 826
ac-RVYIHPI ............................................................................................................................................................................................................. 531,932 Albumin chicken egg (44.3 kDa) .............................................................................................................................................................................. 493
Acrylamide ....................................................................................................................................................................................................................... 28 Albumin dimer (bovine serum) ................................................................................................................................................................................. 826
Acrylic acid .......................................................................................................................................................................... 28,1277,368,85,1278,369,86 Albuterol ............................................................................................................................................................................................................... 2864,852
Acrylonitrile ........................................................................................................................................................................................................ 1504,2073 Alcohol dehydrogenase ........................................................................................................................................................................................ 748,750
Indexes Analyte
Aldicarb ........................................................................................................................................................................................ 1391,305,316,1419,406 7-Aminoclonazepam solution ..................................................................................................................................................................................... 833

Alditol acetates ........................................................................................................................................................................................................... 2036 7-Aminoclonazepam-d4 solution ................................................................................................................................................................................ 833
Aldosterone solution ........................................................................................................................................................................................... 1374,257 2-Amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline .................................................................................................................................................... 63
Aldrin ............................... 1381,1395,1438,1924,1382,1396,1439,1925,1388,1411,1482,2028,1466,1740,1858,1989,2356,2384,2392,2406, 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline ........................................................................................................................................................ 63
1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1524,1532,1571,1591,2325,2347,1604 2-Amino-4,6-dinitrotoluene ......................................................................................................................................................................................... 296
Alidochlor ........................................................................................................................................................................................................... 1888,2067 2-Amino-4,6-dinitrotoluene ........................................................................................................... 1455,1695,2297,2354,2383,2466,1456,1696,2298
Aliphatic hydrocarbons .............................................................................................................................................................................................. 1640 4-Amino-2,6-dinitrotoluene ......................................................................................................................................................................................... 297
Allene .................................................................................................................................................................................................................. 1665,1723 4-Amino-2,6-dinitrotoluene ........................................................................................................... 1455,1695,2297,2354,2383,2466,1456,1696,2298
Allethrin-1 .......................................................................................................................................................................................................... 1887,2066 7-Aminoflunitrazepam solution .................................................................................................................................................................................. 833
Allethrin-2 .......................................................................................................................................................................................................... 1887,2066 7-Aminoflunitrazepam-d7 solution ............................................................................................................................................................................. 833
Allocimene ................................................................................................................................................................................................ 2095,2131,2161 Aminoglutethimide .............................................................................................................................................................................................. 2868,861
Allura Red AC ............................................................................................................................................................................................................... 388 DL-Aminoglutethimide ...................................................................................................................................................................... 2868,861,1340,3023
Allyl alcohol ................................................................................................................................................................................................................ 1895 2-Aminoheptane (N-TFA derivatives) ............................................................................................................................................................ 1645,2488
Allyl chloride ..................................................................................................................................................................................................... 1504,2073 3-Aminoheptane (N-TFA derivatives) ............................................................................................................................................................ 1645,2488
2-Allyl-5-methylpyrazine ............................................................................................................................................................... 1871,2022,2395,2417 3-Aminoheptane (N-Trifluoroacetyl derivative) ............................................................................................................................................ 1651,2492
alpha-Hydroxyalprazolam solution ............................................................................................................................................................................. 833 1-Aminoindan ..................................................................................................................................................................................... 2717,2869,408,862
alpha-Terpinyl acetate + Citronellyl acetate ......................................................................................................................................................... 2096 5-Amino-2-methylheptane (N-TFA derivatives) ........................................................................................................................................... 1645,2488
Alprazolam ................................................................................................................................................................. 136,936,137,937,138,938,875,876 2-Amino-3-methyl-3H-imidazo[4,5-f]quinoline .......................................................................................................................................................... 63
Alprazolam solution .............................................................................................................................................................................................. 490,833 3-Amino-2-methylpentane (N-TFA derivatives) ........................................................................................................................................... 1645,2488
Alprazolam-d5 solution ................................................................................................................................................................................................ 833 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine .................................................................................................................................................. 63
Alprenolol ............................................................................................................................................................................................................. 131,1020 2-Aminopentane (N-TFA derivatives) ............................................................................................................................................................ 1645,2488
Alprenolol hydrochloride .................................................................................................................................................. 2865,855,2866,856,2867,857 4-Aminophenol ............................................................................................................................................................................................................. 870
AM2201 ....................................................................................................................................................................................................................... 1226 4-Aminophenyl sulfone ............................................................................................................................................................................................. 1199
AM2201 4-Hydroxypentyl metabolite solution .............................................................................................................................................. 1225,838 Aminopterin ........................................................................................................................................................................................................... 863,864
AM2233 (459.11 m/z) ...................................................................................................................................................................................... 1227,839 2-Aminopyridine ............................................................................................................................................................................................ 866,897,902
Amaranth ....................................................................................................................................................................................................................... 388 2-Amino-9H-pyrido[2,3-b]indole .................................................................................................................................................................................. 63
Ametryn ........................................................................................................................... 1419,406,1466,1740,1858,1989,2356,2384,2392,2406, Aminorex solution ........................................................................................................................................................................................................ 454
1489,1788,1877,2039,2369,2391,2397,2425,1506,2075,1507,2076,1889,2068 3-aminotoluene ........................................................................................................................................................................................................... 1673
Amiloride ................................................................................................................................................................................................................ 130,893 5-Amino-1,3,3-trimethylcyclohexanemethylamine enantiomers
Amino acids .................................................................................................................................................................................................................. 509 (N-TFA derivatives) .............................................................................................................................................. 1652,2493,1653,2494,1654,2495
2-Aminoanthraquinone .............................................................................................................................................................................................. 1673 Amiodarone hydrochloride .......................................................................................................................................................................................... 256
Aminoazobenzene ....................................................................................................................................................................................................... 1673 Amiodarone hydrochloride solution ................................................................................................................. 1370,229,230,1378,266,267,1379,268
4-Aminobenzoic acid ............................................................................................................................................................ 1129,187,748,750,751,752 Amitraz ............................................................................................................................................................................................................... 1890,2069
p-Aminobenzoic acid (0.14 kDa) ............................................................................................................................................................................... 493 Amitriptyline ............................................................................................. 1164,210,1167,212,239,811,1168,1169,1170,2279,2303,2459,2468,2289
3-Aminobenzotrifluoride ............................................................................................................................................................................................ 1655 Amitriptyline hydrochloride ............................................................................................................................................................ 1368,209,1165,2288
4-Aminobiphenyl ..................................................................................................................................................................................... 1550,1551,1673 Amlodipine .................................................................................................................................................................................................................... 931
2-Aminobutane (N-TFA derivatives) .............................................................................................................................................................. 1645,2488 Amlodipine besylate ........................................................................................................................................................................................... 2870,865
γ-Aminobutyric acid ..................................................................................................................................................................................................... 467 Ammelide .................................................................................................................................................................. 117,318,397,2078,2079,2080,2081
Aminocarb ........................................................................................................................................................................................................... 1,295,294 Amobarbital .......................................................................................................................................................................... 1200,269,485,1201,270,486
Indexes Analyte
Amoxicillin .......................................................................................................................................................................................................... 1009,1041 Anthracene ......................................................................... 2375,310,311,1388,1411,1482,2028,1408,2010,1409,2011,1451,1936,1480,2013,

Amphetamine ........................................................................................................................................................................................................ 189,448 1481,2014,1483,2029,2365,2420,1506,2075,1507,2076,1517,1518,1524,1540,1541,1552,1553,1554,1555,1556,1557,1558,1560,1561,1562,
(-)-Amphetamine ................................................................................................................................................................................................ 2718,409 1563,1564,1567,1587
Amphetamine ........................................................................................................................................................... 411,412,480,2234,2285,2457,2460 Antibiotics ...................................................................................................................................................................................................................... 882
D-Amphetamine hemisulfate salt solution ................................................................................................................................................................ 432 Antibody, monoclonal .................................................................................................................................................................................................. 677
(+)-Amphetamine ....................................................................................................................................................................................... 2718,409,458 Antihistamines ............................................................................................................................................................................................................... 891
d-Amphetamine .......................................................................................................................................................... 2195,2231,2282,2621,2711,2847 Antihypertensives .......................................................................................................................................................................................................... 131
l-Amphetamine ........................................................................................................................................................... 2195,2231,2282,2621,2711,2847 Antioxidants ....................................................................................................................................................................................................... 1898,2182
dl-Amphetamine (AQC derivatives) ................................................................................................................................................................. 2719,413 Antiretrovirals ................................................................................................................................................................................................................ 911
d-Amphetamine (N-TFA derivative) ............................................................... 2196,2232,2283,2622,2712,2848,2197,2233,2284,2623,2713,2849 Apigen 7-glucoside .................................................................................................................................................................................................... 1276
d,l-Amphetamine (N-TFA derivatives) ........................................................................................................................................ 2235,2286,2458,2461 Apigenin 6,8-di-C-glucoside ....................................................................................................................................................................................... 353
Ampicillin ........................................................................................................................................................................................................... 1009,1041 Apigenin 7-glucoside ........................................................................................................................................................................................ 1266,1288
Ampicillin Sodium ...................................................................................................................................................................................................... 1230 Apimaysin .................................................................................................................................................................................................................... 1246
n-Amyl acetate ................................................................................................................................................................................................. 1675,1725 Apohemoglobin, carboxymethylated, tryptic digest ................................................................................................................................................ 542
n-Amyl acetate isomers ................................................................................................................................................................................... 1726,1727 Aprobarbital ........................................................................................................................................................ 414,925,416,927,417,929,418,930,928
n-Amyl alcohol ........................................................................................................................................................................................................... 2210 Aprotinin ................................................................................................................................................................................... 742,748,749,750,751,752
n-Amyl alcohol ........................................................................................................................................................................................................... 2211 Aprotinin from bovine lung ....................................................................................................................................................................................... 762
n-Amyl alcohol ..................................................................................................................................................................... 2212,2452,2213,2214,2453 aqpZ ............................................................................................................................................................................................................................. 663
β-Amylase from sweet potato ................................................................................................................................................................................... 744 Aquaporin Z .................................................................................................................................................................................................................. 663
Amylbenzene ................................................................................................................................................................................................................... 33 Arabinose ................................................................................................................................................................................................................ 383,386
tert-Amyl methyl ether .......................................................................................................................................................................... 1594,1721,1722 D-(−)-Arabinose ........................................................................................................................................................................................................... 379
Amyloid β-protein fragment 10-35 .......................................................................................................................................................................... 511 Arabitol ....................................................................................................................................................................................................... 386,2036,2037
(+)-Anabasine hydrochloride solution ...................................................................................................................................................................... 245 Arachidic acid .......................................................................................................................................................................................... 1808,1908,2244
Ancymidol .................................................................................................................................................................................................. 2477,3,18,2486 Arachidic acid methyl ester (C20:0) ............................................. 1637,1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251,
5-α-Androstane (I.S.) ................................................................................................................................................................................................. 1526 1836,1945,1837,1954,1839,1959,1840,1960,1842,1962,1844,1964
(E)-Anethole ................................................................................................................................................................................................................ 2161 Arachidic acid methyl ester (C20:0) at 4 wt % .............................................................................................................................. 1822,1922,2254
Angelicin ...................................................................................................................................................................................................................... 1262 Arachidonic acid methyl ester (C20:4n6) .......................................................................................................................................... 1815,1915,2247
Angiotensin ............................................................................................................................................................................................................ 513,873 Arachidonic acid methyl ester (C20:4n6) ................................................................................... 1817,1917,2249,1818,1918,2250,1819,1919,2251
Angiotensin I .......................................................................................................................................................................... 126,512,513,873,874,1188 Arachidonic acid methyl ester (C20:4n6) at 2 wt % ...................................................................................................................... 1822,1922,2254
Angiotensin (1-7) amide ............................................................................................................................................................................................ 788 Aramite #1 .................................................................................................................................................................................................................. 1552
Angiotensin II ................................................................................................................................................... 126,512,513,873,708,719,788,874,1188 Aramite #2 .................................................................................................................................................................................................................. 1552
Angiotensin III trifluoroacetate salt hydrate ...................................................................................................................... 126,512,513,873,874,1188 D-Arginine ............................................................................................................................................................................................................. 2745,524
Anilazine ......................................................................................................................................................................... 1419,406,1445,1679,1826,1931 L-Arginine ............................................................................................................................................................................................................. 2745,524
Aniline ........................................................................................................................................................................ 32,492,1484,2030,2366,2421, DL-Arginine hydrochloride .................................................................................................................................................................................. 2744,523
1550,1551,1552,1553,1554,1555,1556,1557,1558,1655,1672,1673 DL-Arginine, FMOC derivatives ........................................................................................................................................................................... 2774,589
Anilines ........................................................................................................................................................................................................................ 1655 Argon ........................................................................................................................................................................................................................... 1745
Anilofos ............................................................................................................................................................................................................... 1885,2064 Arg8-vasopressin ..................................................................................................................................................................................... 709,720,710,721
o-Anisidine ................................................................................................................................................................................................................... 1673 Aroclor 1016 ................................................................................................................................................................................... 1533,1534,1535,1537
Anisole ................................................................................................................................................................ 2210,2211,2212,2452,2213,2214,2453 Aroclor 1248 ............................................................................................................................................................................................................... 1538
Indexes Analyte
Aroclor 1254 ............................................................................................................................................................................................................... 1539 Azinphos-methyl ............................................................................. 1350,1401,1860,1995,1449,1683,1830,1935,1542,1543,1885,2064,2004,2413

Aroclor 1260 ................................................................................................................................................................................... 1533,1534,1535,1537 Azobenzene ............................................................................................................................ 1524,1552,1553,1554,1555,1556,1557,1558,1564,1673
Aromadendrene .......................................................................................................................................................................................................... 2131 Azoxystrobin ........................................................................................................................... 1430,1641,1796,1892,1446,1680,1827,1932,1886,2065
Arotinolol .............................................................................................................................................................................................................. 2871,920
L-Ascorbic acid ..................................................................................................................................................... 122,1234,1301,1302,1303,1304,1309 —B—
Asparagine ..................................................................................................................................................................................................................... 819 Baccatin III ...................................................................................................................................................................................... 1104,1153,1154,1220
D-Asparagine ........................................................................................................................................................................................................ 2747,527
Bacitracin A .......................................................................................................................................................................................................... 395,1109
L-Asparagine ............................................................................................................................................................... 508,509,2747,527,727,1897,2304
Baicalein ......................................................................................................................................................................................................................... 352
DL-Asparagine monohydrate .............................................................................................................................................................................. 2746,526
Balan ................................................................................................................................................................................................ 1467,1990,2357,2407
DL-Asparagine, FMOC derivatives ...................................................................................................................................................................... 2775,590
Barban ......................................................................................................................................................................................... 1,295,294,302,2374,303
Aspartame ............................................................................................................................................................................................... 346,347,348,349 Barbital ............................................................................................................... 1200,269,485,1201,270,486,414,925,416,927,417,929,418,930,928
Aspartic acid .......................................................................................................................................................................................................... 509,819 Beclomethasone ............................................................................................................................................................................................................ 898
D-Aspartic acid ..................................................................................................................................................................................................... 2748,528
Behenic acid ............................................................................................................................................................................................. 1808,1908,2244
L-Aspartic acid ........................................................................................................................................................................... 2748,528,727,1897,2304
Behenic acid methyl ester ........................................................................................................................................ 1818,1918,2250,1819,1919,2251
DL-Aspartic acid ................................................................................................................................................................................................... 2749,529
Behenic acid methyl ester (C22:0) .................................................................................................................................................... 1815,1915,2247
DL-Aspartic acid potassium salt ........................................................................................................................................................................ 2872,921
Behenic acid methyl ester (C22:0) ................................................................................................................ 1817,1917,2249,1840,1960,1842,1962
DL-Aspartic acid, FMOC derivatives .................................................................................................................................................. 2776,591,2777,592
Behenic acid methyl ester (C22:0) at 4 wt % ................................................................................................................................. 1822,1922,2254
Aspirin ................................................................................................................................................................................................................... 1105,183 Benalaxyl ...................................................................................................................................................................... 1449,1683,1830,1935,1888,2067
Aspon ............................................................................................................................................................................................... 1542,1543,1544,1545 Bendroflumethiazide ............................................................................................................................................................................................ 2875,933
Asp-Phe .......................................................................................................................................................................................................................... 788 Benfluralin .................................................................................................................................................................... 1430,1641,1796,1892,1886,2065
Asp-Phe methyl ester .......................................................................................................................................................................................... 339,340 Benfuracarb ..................................................................................................................................................................................... 1446,1680,1827,1932
Astaxanthin .................................................................................................................................................................... 1239,344,1241,1242,1243,1317 Benfuresate ......................................................................................................................................................................................................... 1890,2069
Atenolol .................................................................................................................................. 130,893,139,943,140,2633,2720,419,2873,923,941,942 Benoxaprofen ....................................................................................................................................................................................................... 2876,934
Atorvastatin ................................................................................................................................................................................................... 1148,194,964 Benzaldehyde .............................................................................................................................. 21,2326,1491,1581,2319,2331,1564,1574,2328,
Atraton ............................................................................................................................................................................................ 1506,2075,1507,2076 1878,2044,2398,2430,1879,2046,2399,2431,1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436,1883,2054,2402,2437,
Atrazine ..................................................................................................................................... 5,110,15,111,16,20,1391,305,1357,1393,315,316, 1896,2403
1388,1411,1482,2028,1466,1740,1858,1989,2356,2384,2392,2406,1467,1990,2357,2407,1489,1788,1877,2039,2369,2391,2397,2425, Benzaldehyde-2,4-dinitrophenylhydrazone .......................................................................................................................................... 22,2334,23,2335
1506,2075,1507,2076,1544,1545,1564,1886,2065 Benzaldehyde-2,4-DNPH ............................................................................................................................................................................................ 2337
Atrazine-desethyl ..................................................................................................................................................................................... 5,110,15,111,16 Benzaldehyde (2,4-DNPH derivative) ................................................................................................................................................................. 2336,24
Atrazine-desethyl-desisopropyl-2-hydroxy ........................................................................................................................... 397,2078,2079,2080,2081 Benzalkonium chloride ...................................................................................................................................................................... 277,994,1114,1195
Atrazine-desisopropyl ................................................................................................................................................................................. 110,15,111,16 Benzamide ..................................................................................................................................................................................................................... 492
DL-Atrolactic acid hemihydrate ............................................................................................................................................................. 2568,34,2569,35
Benz[a]anthracene ........... 2375,310,1387,1410,1479,2012,1388,1411,1482,2028,1483,2029,2365,2420,1484,2030,2366,2421,1506,2075,
Atropine ................................................................................................................................................................................................................ 2874,924 1507,2076,1524,1552,1553,1554,1555,1556,1557,1564,1795,1891
Aviation gasoline pattern .......................................................................................................................................................................................... 1599 Benz[a]anthracene solution ...................................................................................................................................................................... 311,1451,1936
Azaconazole ....................................................................................................................................................................................................... 1886,2065 Benzene ............................................................................................................................................ 36,841,554,1117,1118,1493,2057,2370,2440,
Azamethiphos .................................................................................................................................................................................................... 1890,2069 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1500,1586,2321,2343,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,
DL-7-Azatryptophan hydrate .............................................................................................................................................................................. 2750,530
1522,1527,1528,1529,1530,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1592,1593,1594,1595,1596,1597,
3′-Azido-3′-deoxythymidine ........................................................................................................................................................................................ 911 1600,1658,1601,1667,1602,1668,1610,1611,1612,1621,1640,1676,1725,1726,1727,1794,2200,2201,2202,2448,2203,2204,2449,2224,
Azinphos-ethyl .......................................................................................................................................... 1350,1401,1860,1995,1542,1543,1890,2069 2226,2456
Indexes Analyte
Benzene-d6 ............................................................................................................................................................................................................... 36,841 Benzo[a]pyrene ........................................................................................................................... 2375,310,311,1388,1411,1482,2028,1408,2010,

Benzidine ......................................................................................................................................... 1524,1552,1553,1554,1555,1556,1557,1558,1673 1409,2011,1434,1435,1451,1936,1480,2013,1481,2014,1483,2029,2365,2420,1506,2075,1507,2076,1517,1518,1524,1540,1541,1552,1553,
Benzo[a]anthracene ..................................................................................................... 1408,2010,1409,2011,1480,2013,1481,2014,1517,1518,1540 1554,1555,1556,1557,1558,1560,1561,1562,1563,1564,1567,1587,1795,1891
Benzo[a]anthracene .................................................................................................................................................................................................... 1541 Benzo[e]pyrene ............................................................................................................................................................ 1408,2010,1409,2011,1567,1587
Benzo(a)anthracene .................................................................................................................................................................................................... 1558 Benzo[a]pyrene-d12 ........................................................................................................................................................................................... 1795,1891
Benzo[a]anthracene .................................................................................................................................................... 1560,1561,1562,1563,1567,1587 Benzoylecgonine .................................................................................................................................................................................................... 427,464
Benzoate ........................................................................................................................................................................................................................ 338 Benzoylecgonine solution ............................................................................................................................................................................................ 481
Benzocaine .............................................................................................................................................................................................................. 125,872 Benzyl acetate ................................................................................................................................................................................ 2111,2184,2183,2446
Benzo[b]fluoranthene .................................................................................................................................... 2375,310,311,1388,1411,1482,2028, Benzyl alcohol ........................................................................................................................................................................ 1484,2030,2366,2421,
1408,2010,1409,2011,1434,1435,1451,1936,1480,2013,1481,2014,1483,2029,2365,2420,1506,2075,1507,2076,1517,1518,1524,1540, 1550,1551,1552,1553,1554,1555,1556,1557,1558,1564
1541,1552,1553,1554,1555,1556,1557,1558,1560,1561,1562,1563,1564,1567,1587,1795,1891 Benzyl benzoate ............................................................................................................................................................................. 1478,1769,2009,2192
Benzo[j]fluoranthene ............................................................................................................. 1408,2010,1409,2011,1451,1936,1540,1541,1795,1891 Benzyl butyl phthalate ............................................................................................................................ 1483,2029,2365,2420,1507,2076,1556,2191
Benzo[k]fluoranthene ................................................................................................................. 2375,310,311,1388,1411,1482,2028,1408,2010, Benzyl chloride ............................................................................................................................................................................... 1568,1588,2322,2344
1409,2011,1434,1435,1451,1936,1480,2013,1481,2014,1483,2029,2365,2420,1506,2075,1507,2076,1517,1518,1524,1540,1541,1552,1553, Benzyl 4-hydroxybenzoate .......................................................................................................................................................................................... 284
1554,1555,1556,1557,1558,1560,1561,1562,1563,1564,1567,1587,1795,1891 Benzylisoeugenol ............................................................................................................................................................................................... 2111,2184
Benzoic acid ..................................................................................................................................................................................... 37,1105,183,279,280 4-Benzyl-2-oxazolidinone ..................................................................................................................................................................................... 2570,39
Benzoic acid ............................................................................................................................................... 334,332,335,339,340,346,347,348,349, Benzylparaben ................................................................................................................................................................................................ 282,283,286
910,1236,1552,1553,1554,1555,1556,1557,1558,1564 Benzyl salicylate ................................................................................................................................................................................................ 2183,2446
Benzoic acid, 0.7% .................................................................................................................................................................................................... 1993 α-cis-Bergamotene ......................................................................................................................................................................... 2050,2434,2096,2131
Benzoin ........................................................................................................................................................................................... 2877,38,939,2878,940 α-trans-Bergamotene ........................................................................................................................................................................................ 2050,2434
Benzoin methyl ether ...................................................................................................................................................................................... 1237,3029 trans-α-Bergamotene ................................................................................................................................................................................................. 2096
Benzo[ghi]perylene ............................................................................................................................................................................................. 2375,310 α-trans-Bergamotene ................................................................................................................................................................................................. 2096
Benzo[ghi]perylene ....................................................................................................................................................................................................... 311 trans-α-Bergamotene ........................................................................................................................................................................................ 2099,2105
Benzo[ghi]perylene ........................................................................................................................................................................ 1388,1411,1482,2028 α-trans-Bergamotene ........................................................................................................................................................................................ 2106,2120
Benzo[ghi]perylene ......................................................................................................................... 1408,2010,1409,2011,1429,1434,1435,1451,1936 trans-α-Bergamotene ................................................................................................................................................................................................. 2121
Benzo[g,h,i]perylene ....................................................................................................................................................................... 1480,2013,1481,2014 β-trans-Bergamotene ........................................................................................................................................................................................ 2096,2138
Benzo[ghi]perylene ......................................................................................................................................................................... 1483,2029,2365,2420 Bergamot oil ............................................................................................................................................................................................................... 2113
Benzo[ghi]perylene ........................................................................................................................................................................................... 1506,2075 Bergapten .................................................................................................................................................................................................................... 1262
Benzo[ghi]perylene ......................................................................................................................................................................... 1507,2076,1517,1518 Beta blockers ......................................................................................................................................................................................................... 139,943
Benzo[ghi]perylene ..................................................................................................................................................................................................... 1524 beta-Lactoglobulin A .................................................................................................................................................................................................... 744
Benzo[ghi]perylene ............................................................................................................................................................................................ 1540,1541 Betamethasone .............................................................................................................................................................................................................. 169
Benzo[ghi]perylene ........................................................................................................................................................................ 1552,1553,1554,1555 beta-Pinene ..................................................................................................................................................................................... 2047,2175,2351,2353
Benzo[ghi]perylene ..................................................................................................................................................................................................... 1556 Betaxolol ............................................................................................................................................................................................................. 1114,1195
Benzo[ghi]perylene ..................................................................................................................................................................................................... 1557 BHA ..................................................................................................................................................................................................................... 1898,2182
Benzo[ghi]perylene ............................................................................................................................................................... 1558,1560,1561,1562,1563 α-BHC .......... 1382,1396,1439,1925,1469,1994,2359,2409,1506,2075,1513,1524,1532,1571,1591,2325,2347,1604,1694,1835,1941,2296,2693
Benzo[ghi]perylene ..................................................................................................................................................................................................... 1564 β-BHC .................................................................... 1382,1396,1439,1925,1466,1740,1858,1989,2356,2384,2392,2406,1469,1994,2359,2409,
Benzo[ghi]perylene ............................................................................................................................................................................................ 1567,1587 1506,2075,1507,2076,1512,1513,1514,1516,1524,1532,1571,1591,2325,2347,1604,1889,2068
Benzo[ghi]perylene ........................................................................................................................................................................................... 1795,1891 γ-BHC .............................. 1381,1395,1438,1924,1382,1396,1439,1925,1388,1411,1482,2028,1466,1740,1858,1989,2356,2384,2392,2406,
Benzophenone ............................................................................................................................................................................................................... 840 1469,1994,2359,2409,1513,1516,1524,1532,1571,1591,2325,2347,1604
Indexes Analyte
δ-BHC .......................................................................................................... 1382,1396,1439,1925,1466,1740,1858,1989,2356,2384,2392,2406, Bis(4-Methyl-2-pentyl) phthalate ................................................................................................................................................ 1478,1769,2009,2192

1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1532,1604,1889,2068 Bis(4-methyl-2-pentyl) phthalate ...................................................................................................................................... 1865,2008,2394,2416,2191
BHT ............................................................................................................................................................................................................ 1898,2182,1942 (1R)-2,2′-Bis[(S)-(4-methylphenyl)sulfinyl]-1,1′-binaphthalene .............................................................................................................................. 42
Bicycloelemene ............................................................................................................................................................................... 2050,2434,2124,2131 (1S)-2,2′-Bis[(S)-(4-methylphenyl)sulfinyl]-1,1′-binaphthalene ............................................................................................................................... 42
Bicyclogermacrene ............................................................................................................................................. 2050,2434,2131,2137,2138,2142,2170 Bisphenol A .................................................................................................................................................................................................... 299,341,300
Bifenazate ........................................................................................................................................................................................................... 1889,2068 Bitertanol ...................................................................................................................................................................... 1448,1682,1829,1934,1889,2068
Bifenox ................................................................................................................................................................................................................ 1886,2065 Bivalirudin ...................................................................................................................................................................................................................... 820
Bifenthrin ......................................................................................................................................................................................... 1404,2001,1886,2065 Blood alcohols ............................................................................................................................................................................................................. 2293
Bilirubin .......................................................................................................................................................................................................................... 142 Bolstar ................................................................................................................................................................................................................. 1544,1545
Biliverdin ........................................................................................................................................................................................................................ 142 Bolstar (Sulprofos) ............................................................................................................................................................................................ 1542,1543
1,1'-Binaphthyl-2,2'-diyl hydrogenphosphate ................................................................................................................................................... 2571,41 Bombesin ................................................................................................................................................................................................. 709,710,720,721
Bioallethrin-1 ..................................................................................................................................................................................................... 1887,2066 Borneol ...................................................................................................................................................................................................... 2105,2106,2120
Bioallethrin-2 ..................................................................................................................................................................................................... 1887,2066 (−)-Bornyl acetate ........................................................................................................................................................................................... 2111,2184
Biogenic amines ............................................................................................................................................................................................................ 145 Bornyl acetate ............................................................................................................................................................................................................. 2131
Biotin ................................................................................................................................................................... 1238,3030,1300,1306,1307,1308,1333 α-Boswellic acid .......................................................................................................................................................... 1325,1326,1334,1335,1336,1337
2,2′-Biphenol ................................................................................................................................................................................................................. 492 β-Boswellic acid .......................................................................................................................................................... 1325,1326,1334,1335,1336,1337
Biphenyl ...................................................................................................................................................................................... 842,845,1408,2010, β-Bourbonene ........................................................................................................................................................................ 2095,2136,2137,2138,2167
1409,2011,1441,1927,1442,1928,1443,1929,1564,1889,2068 (B) Bovine serum albmin (BSA) ...................................................................................................................................................................... 1185,808
4-Biphenylacetic acid ................................................................................................................................................................................................. 1174 Bovine serum albumin (BSA) ..................................................................................................................................................................................... 829
(E)-α-Bisabolene ...................................................................................................................................................................................... 2050,2434,2096 Bovine serum albumin (BSA) dimer, 132 kD ......................................................................................................................................................... 735
(Z)-α-Bisabolene ................................................................................................................................................................................................ 2096,2121 Bradykinin ......................................................................................................................................................................................... 709,710,720,721,788
β-Bisabolene ............................................................................................................................................. 2050,2434,2096,2099,2105,2106,2120,2121 Bradykinin, fragment 1-5 .................................................................................................................................................................................... 709,720
(E)-γ-Bisabolene ............................................................................................................................................................................. 2050,2434,2096,2120 Brassicasterol ................................................................................................................................................................................... 1354,1412,1875,2034
(Z)-γ-Bisabolene ................................................................................................................................................................................................ 2096,2120 Bromacil .................................................................................................... 2478,6,2879,945,1467,1990,2357,2407,1506,2075,1507,2076,1889,2068
α-Bisabolol ...................................................................................................................................................................................... 2050,2434,2096,2120 4-Bromoacetanilide ....................................................................................................................................................................................................... 492
epi-β-Bisabolol ............................................................................................................................................................................................................ 2096 Bromobenzene ........................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549
Bis(2-butoxyethyl) phthalate ........................................................................................................................................................ 1478,1769,2009,2192 2-Bromobutane ............................................................................................................................................................................... 1706,2217,2514,2854
Bis(2-n-butoxyethyl) phthalate ........................................................................................................................................... 1865,2008,2394,2416,2191 Bromobutide ...................................................................................................................................................................................................... 1886,2065
Bis(2-chloroethoxy)methane .......................................................................................................... 1524,1552,1553,1554,1555,1556,1557,1558,1564 α-Bromo-γ-butyrolactone ................................................................................................................................................................................. 1656,2496
Bis(2-chloroethyl) ether ................................................................................................................. 1524,1552,1553,1554,1555,1556,1557,1558,1564 Bromochloromethane ............. 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549,1565,1566
Bis-(2-chloroisopropyl) ether ........................................................................................................ 1524,1552,1553,1554,1555,1556,1557,1558,1564 Bromodichloromethane ...................................................................................................................................... 1494,2058,2371,2441,1497,2061,
3,6-Bis(2-chlorophenyl)-1,2,4,5-tetrazine ....................................................................................................................................................... 1889,2068 1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344
Bisdemethoxycurcumin ............................................................................................................................................................................................... 1331 4-Bromo-2,5-dimethoxyphenethylamine hydrochloride solution .......................................................................................................... 1424,457,483
Bis(2-ethoxyethyl) phthalate ......................................................................................................... 1478,1769,2009,2192,1865,2008,2394,2416,2191 4-Bromodiphenyl ether .................................................................................................................................................................................... 1556,1558
Bis(2-ethylhexyl) adipate .............................................................................................................................................................. 1506,2075,1507,2076 (R)-(-)-Bromo-DragonFLY hydrochloride solution ........................................................................................................................... 228,1424,457,483
Bis(2-ethylhexyl) phthalate .................................................................................................................................................. 1478,1769,2009,2192, 4-Bromofluorobenzene .................................................................................................................................................................. 1504,2073,1519,1520
1483,2029,2365,2420,1506,2075,1507,2076,1524,1552,1553,1554,1555,1556,1557,1558,1564,1865,2008,2394,2416,2191 4-Bromofluorobenzene ............................................................................................................................................................................................. 1546
7,12-Bis(hydroxymethyl)benz[a]anthracene ............................................................................................................................................... 175,304,438 4-Bromofluorobenzene ......................................................................................................................................................... 1547,1548,1549,1565,1566
Bis(2-methoxyethyl) phthalate ..................................................................................................................................................... 1865,2008,2394,2416
Indexes Analyte
Bromoform ............................................................................... 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072, n-Butane ............................................................................................................................................................................................................. 1592,1665

1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344 n-Butane ...................................................................................................................................................................................................................... 1723
2-Bromohexadecanoic acid ................................................................................................................................................................................ 2755,538 n-Butane ...................................................................................................................................................................................................................... 1751
Bromomethane .............................................................. 1494,2058,2371,2441,1496,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072, n-Butane (215 ng on column) ................................................................................................................................................................................ 1752
1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1569,1589,2323,2345 1,4-Butanediol ................................................................................................................................. 1703,1975,2185,1704,1976,2186,1705,1977,2187
2-Bromo-3-methylbutyric acid ............................................................................................................................................................................ 2572,43 2,3-Butanediol .............................................................................................................. 1703,1975,2185,1704,1976,2186,1705,1977,2187,2060,2443
1-Bromo-2-nitrobenzene (I.S.) ........................................................................................................................................................................ 1544,1545 2,3-Butanedione ................................................................................................................................................................................................ 2056,2439
2-Bromooctanoic acid methyl ester ............................................................................................................................................................... 1657,2497 Butanetriol ................................................................................................................................................................................................................... 1638
2-Bromopentane ............................................................................................................................................................................. 1706,2217,2514,2854 (S)-(−)-1,2,4-Butanetriol .......................................................................................................................................................................................... 1632
2-Bromophenol .................................................................................................................................................. 1359,1386,1407,1475,1755,2005,1531 Butanetriol (I.S.) ...................................................................................................................................................................................... 1631,1633,1634
4-Bromophenyl phenyl ether .......................................................................................................................... 1524,1552,1553,1554,1555,1557,1564 Butanoic acid ..................................................................................................................................................................................................... 1971,2405
Bromophos ................................................................................................................................................................... 1440,1677,1824,1926,1886,2065 Butanol ................................................................................................................................................................................... 1490,1580,2318,2330,1969
Bromophos-ethyl ...................................................................................................................................... 1350,1401,1860,1995,1447,1681,1828,1933 n-Butanol ..................................................................................................................................................................................................................... 2211
Bromopropylate ................................................................................................................................................................................................. 1886,2065 n-Butanol ........................................................................................................................................................................................................... 2212,2452
Brompheniramine .................................................................................................................................................................................................. 890,891 iso-Butanol .......................................................................................................................................................... 2210,2211,2212,2452,2213,2214,2453
Bromuconazole-1 ............................................................................................................................................................................................... 1889,2068 sec-Butanol .................................................................................................................................................................................................................. 1644
Bromuconazole-2 ............................................................................................................................................................................................... 1889,2068 sec-Butanol .................................................................................................................................................................................................................. 1676
BSA (bovine serum albumin) ..................................................................................................................................................................................... 758 sec-Butanol ............................................................................................................................................................................ 1725,1726,1727,1969,2211
BSA carboxyamidomethylated tryptic digest ............................................................................................................................................................ 541 sec-Butanol ............................................................................................................................................................................ 2212,2452,2213,2214,2453
BSA dimer ..................................................................................................................................................................................................................... 734 tert-Butanol .............................................................................................................................................................................................. 1644,1721,1722
Buphedrone ................................................................................................................................................................................................ 434,2049,2433 1-Butanol ............................................................................................................................... 1431,1572,1644,1666,1676,1725,1726,1727,1895,2210
Buphedrone hydrochloride solution .............................................................................................................................................. 243,468,434,472,476 1-Butanol solution ................................................................................................................................................................................... 2213,2214,2453
Buphedrone ephedrine metabolite hydrochloride solution ............................................................................................................................. 470,474 2-Butanol ............................................................................................................................................................ 1507,2076,1593,1895,2292,2464,2294
Bupirimate .......................................................................................................................................................................................................... 1887,2066 R-(-)-2-Butanol ................................................................................................................................................................................................. 1661,2500
Bupivacaine hydrochloride monohydrate .................................................................................................... 2880,947,2881,948,2882,949,2198,2850 S-(+)-2-Butanol ................................................................................................................................................................................................ 1661,2500
Buprenorphine solution ................................................................................................................................................................................................ 692 R-(-)-2-Butanol (O-Acetyl derivative) ............................................................................................................................................................ 1659,2498
Buprenorphine glucuronide solution .......................................................................................................................................................................... 692 S-(+)-2-Butanol (O-Acetyl derivative) .......................................................................................................................................................... 1659,2498
Buprofezin .......................................................................................................................................................................................................... 1886,2065 R-(-)-2-Butanol (O-TFA derivative) ................................................................................................................................................................ 1660,2499
Bupropion hydrochloride ..................................................................................................................................................................... 132,907,1189,231 S-(+)-2-Butanol (O-TFA derivative) .............................................................................................................................................................. 1660,2499
Buspirone ................................................................................................................................................................................................................ 914,918 sec-Butanone ............................................................................................................................................................................................................... 2210
Buspirone hydrochloride ............................................................................................................................................................................................ 1202 2-Butanone ................................................................................................................................................ 1504,2073,1525,1546,1547,1548,1549,
Butabarbital ........................................................................................................................................................ 414,925,415,926,417,929,418,930,928 1565,1566,1568,1588,2322,2344,1676,1726,1727,2056,2439,2210,2211,2212,2452,2213,2214,2453
Butachlor ...................................................................................................................................................................... 1506,2075,1507,2076,1888,2067 2-Butanone-2,4-dinitrophenylhydrazone .............................................................................................................................................. 22,2334,23,2335
1,3-Butadiene .............................................................................................................................................................. 1568,1588,2322,2344,1665,1723 2-Butanone (2,4-DNPH derivative) ..................................................................................................................................................................... 2336,24
Butafencil ............................................................................................................................................................................................................ 1888,2067 1-Butene ............................................................................................................................................................................................................. 1665,1723
Butalbital ................................................................................................................................................................ 1200,269,485,1201,270,486,415,926 cis-2-Butene ............................................................................................................................................................................................. 1612,1665,1723
Butamifos ..................................................................................................................................................................... 1430,1641,1796,1892,1885,2064 trans-2-Butene ................................................................................................................................................................................................... 1665,1723
Butanal ......................................................................................................................................................................................................................... 1969 3-Butenone ........................................................................................................................................................................................................ 2056,2439
Butane ....................................................................................................................................................................................................... 1610,1611,1612 l-Buthionine sulfoximine .............................................................................................................................................................................................. 121
Indexes Analyte
Butorphanol ......................................................................................................................................................................................... 2883,950,2884,951 Butylparaben .................................................................................................................................................................................................................. 284

2-Butoxyethanol ....................................................................................................................................................................................... 1703,1975,2185 Butyl paraben ................................................................................................................................................................................................. 282,283,286
2-Butoxyethanol (butyl Cellosolve®) ..................................................................................................................................................... 1704,1976,2186 Butylparaben .................................................................................................................................................................................................................. 289
2-(2-Butoxyethoxy)ethanol ..................................................................................................................................................................... 1703,1975,2185 Butyl paraben ............................................................................................................................................................................................... 290,291,1106
2-(2-Butoxyethoxy)ethanol (butyl CARBITOL™) .................................................................................................................................. 1704,1976,2186 Butyolne ......................................................................................................................................................................................................................... 435
Butyl acetate ............................................................................................. 1676,1876,2035,2396,2423,1969,2210,2211,2212,2452,2213,2214,2453 Butyraldehyde ........................................................................................................................................................................................................ 21,2326
n-Butyl acetate .................................................................................................................................................. 1431,1572,1675,1725,1726,1727,1780 Butyraldehyde-2,4-dinitrophenylhydrazone ......................................................................................................................................... 22,2334,23,2335
sec-Butyl acetate ........................................................................................................................................................................................................ 1780 Butyraldehyde-2,4-DNPH ........................................................................................................................................................................................... 2337
n-Butyl alcohol ........................................................................................................................................................................................................... 1780 Butyraldehyde (2,4-DNPH derivative) ................................................................................................................................................................ 2336,24
sec-Butyl alcohol ......................................................................................................................................................................................................... 1780 Butyric acid ......................................................................................................... 1808,1908,2244,1809,1909,2245,1873,2031,2278,1969,1970,2404
Butylamine ................................................................................................................................................................................................................... 1776 Butyric acid, 0.7% ..................................................................................................................................................................................................... 1993
sec-Butylamine ................................................................................................................................................................................................... 1650,2491 Butyric acid methyl ester (C4:0) ..................................................................... 1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251
N-Butylaniline .................................................................................................................................................................................................................. 32 Butyric acid methyl ester (C4:0) at 4 wt % ..................................................................................................................................... 1822,1922,2254
Butylate ........................................................................................................................................................................................... 1506,2075,1890,2069 β-Butyrolactone .................................................................................................................................................... 322,1662,2501,1663,2502,1664,2503
Butylbenzene ............................................................................................................................................................................. 33,1546,1547,1548,1549 γ-Butyrolactone .................................................................................................................................................................................................. 2056,2439
n-Butylbenzene ................................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1612,2300,2467
iso-Butylbenzene ......................................................................................................................................................................................................... 1610 —C—
sec-Butylbenzene ....................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549 C1 ................................................................................................................................................................................................................................... 718
sec-Butylbenzene ........................................................................................................................................................................................................ 1610 C10:0 ................................................................................................................................................ 1823,1923,2255,1951,1955,1956,2258,1958,2261
sec-Butylbenzene ........................................................................................................................................................................................................ 1612 C10:1 ............................................................................................................................................................................. 1951,1955,1956,2258,1958,2261
tert-Butylbenzene ...................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549 C11:0 ................................................................................................................................................ 1823,1923,2255,1951,1955,1956,2258,1958,2261
N-Butylbenzene ........................................................................................................................................................................................................... 1610 C12:0 ................................................................................................................................................ 1823,1923,2255,1951,1955,1956,2258,1958,2261
Butylbenzyl phthalate ...................... 1388,1411,1482,2028,1478,1769,2009,2192,1506,2075,1524,1552,1553,1554,1555,1557,1558,1564, C13:0 ................................................................................................................................................ 1823,1923,2255,1951,1955,1956,2258,1958,2259
1865,2008,2394,2416
C13:0 iso ................................................................................................................................................................................................... 1951,1955,1958
Butyl cellosolve .............................................................................................................................................................................. 1725,1726,1727,1780
C13:0 anteiso ........................................................................................................................................................................................... 1951,1955,1958
Butylcyclohexane ............................................................................................................................................................................................... 1528,1530
C14:0 ................................................. 1823,1923,2255,1870,2021,1872,2023,2277,1942,1943,1951,1952,1953,1955,1956,2258,1958,2259,2261
2-sec-Butyl-4,6-dinitrophenol ........................................................................................................................... 1477,1757,2007,2364,2389,2415,1531
C14:0 iso ................................................................................................................................................................................ 1951,1955,1956,2258,1958
tert-Butyl ethyl ether ................................................................................................................................................................................................. 1721
C14:1 .......................................................................................................................................................... 1823,1923,2255,1951,1955,1956,2258,2261
Butyl hexanoate .......................................................................................................................................................... 1876,2035,2396,2423,1971,2405
C14:1n9c ...................................................................................................................................................................................................................... 1958
tert-Butylhydroquinone ..................................................................................................................................................... 276,337,1282,292,1898,2182
C14:0 methyl ester .................................................................................................................................................................................................... 1947
tert-Butylhydroxyanisole ........................................................................................................................................................................................ 276,337
C15:0 ................................................................................................................................................ 1823,1923,2255,1951,1955,1956,2258,1958,2261
3-tert-Butyl-4-hydroxyanisole ............................................................................................................................................................................ 1282,292
C15:0 iso ...................................................................................................................................................................... 1951,1955,1956,2258,1958,2261
Butyl 4-hydroxybenzoate ........................................................................................................................ 1748,1998,2190,2223,2387,2412,2447,2454
C15:1 ......................................................................................................................................................................................................... 1823,1923,2255
tert-Butylhydroxytoluene ....................................................................................................................................................................................... 276,337
C15:0 anteiso ............................................................................................................................................................... 1951,1955,1956,2258,1958,2261
(±)-Butyl-γ-lactone ....................................................................................................................................................................... 1729,2118,2529,2676
C15:0 methyl ester .................................................................................................................................................................................................... 1947
Butyl 2-methylbutanoate .............................................................................................................................................................. 1876,2035,2396,2423
C16:0 ................................................. 1823,1923,2255,1870,2021,1872,2023,2277,1942,1943,1951,1952,1953,1955,1956,2258,1958,2259,2261
tert-Butyl methyl ether ............................................... 1504,2073,1527,1565,1566,1568,1588,2322,2344,2210,2211,2212,2452,2213,2214,2453
C16:0 iso ................................................................................................................................................................................ 1951,1955,1956,2258,1958
Butylone hydrochloride .................................................................................................................................................................................... 2049,2433
C16:1 ......................................................................................................................................................................................................... 1823,1923,2255
Butylone hydrochloride solution ................................................................................................................................................... 243,468,434,470,475
C16:1ω7 ....................................................................................................................................................................................................................... 2259
Indexes Analyte
C16:1n10t .................................................................................................................................................................................................................... 1958 C18:2 Δ 9t,12c ........................................................................................................................................................................................ 1799,1899,2241

C16:1n7 ........................................................................................................................................................................ 1870,2021,1942,1943,1952,1953 C18:2 D 9t, 12t ....................................................................................................................................................................................... 1801,1901,2243
C16:1n7c ...................................................................................................................................................................................................................... 1958 C18:2 Δ 9t,12t ......................................................................................................................................................................................... 1799,1899,2241
C16:1n7 methyl ester ................................................................................................................................................................................................ 1947 C18:3 ............................................................................................................................................................................................................................ 2259
C16:1n9c ....................................................................................................................................................................... 1951,1955,1956,2258,1958,2261 C18:3ω6 ....................................................................................................................................................................................................................... 2259
C16:1n9t ............................................................................................................................................................................................................. 1951,1955 C18:3 D 9c, 12c, 15c ............................................................................................................................................................................. 1801,1901,2243
C16:2n4 ........................................................................................................................................................................ 1870,2021,1942,1943,1952,1953 C18:3 Δ 9c,12c,15c ................................................................................................................................................................................. 1799,1899,2241
C16:3n4 ........................................................................................................................................................................ 1870,2021,1942,1943,1952,1953 C18:3 Δ 9c,12c,15t ................................................................................................................................................................................. 1799,1899,2241
C16:0 methyl ester .................................................................................................................................................................................................... 1947 C18:3 D 9c, 12t, 15c ............................................................................................................................................................................. 1801,1901,2243
C17:0 ................................................................................................................................................ 1823,1923,2255,1951,1955,1956,2258,1958,2261 C18:3 Δ 9c,12t,15c ................................................................................................................................................................................. 1799,1899,2241
C17:0 iso ...................................................................................................................................................................... 1951,1955,1956,2258,1958,2261 C18:3 Δ 9c,12t,15t .................................................................................................................................................................................. 1799,1899,2241
C17:1 .......................................................................................................................................................... 1823,1923,2255,1951,1955,1956,2258,1958 C18:3 D 9t, 12c, 15c ............................................................................................................................................................................. 1801,1901,2243
C17:1n9c ...................................................................................................................................................................................................................... 2261 C18:3 Δ 9t,12c,15c ................................................................................................................................................................................. 1799,1899,2241
C17:0 anteiso ............................................................................................................................................................... 1951,1955,1956,2258,1958,2261 C18:3 D 9t, 12c, 15t .............................................................................................................................................................................. 1801,1901,2243
C18:0 ................................................. 1823,1923,2255,1870,2021,1872,2023,2277,1942,1943,1951,1952,1953,1955,1956,2258,1958,2259,2261 C18:3 Δ 9t,12c,15t .................................................................................................................................................................................. 1799,1899,2241
C18:0 iso ...................................................................................................................................................................... 1951,1955,1956,2258,1958,2261 C18:3 D 9t, 12t, 15c .............................................................................................................................................................................. 1801,1901,2243
C18:1 ............................................................................................................................................................................................................................ 2259 C18:3 Δ 9t,12t,15c .................................................................................................................................................................................. 1799,1899,2241
C18:1 D 11c ............................................................................................................................................................................................. 1801,1901,2243 C18:3 D 9t, 12t, 15t .............................................................................................................................................................................. 1801,1901,2243
C18:1 Δ 11c ............................................................................................................................................................................................. 1799,1899,2241 C18:3 Δ 9t,12t,15t .................................................................................................................................................................................. 1799,1899,2241
C18:1 Δ 11t ............................................................................................................................................................................................. 1799,1899,2241 C18:4 ............................................................................................................................................................................................................................ 2259
C18:1 D 12c ............................................................................................................................................................................................. 1801,1901,2243 C18:1n10c .................................................................................................................................................................................................................... 1958
C18:1 Δ 12c ............................................................................................................................................................................................. 1799,1899,2241 C18:1n10t .............................................................................................................................................................................. 1951,1955,1956,2258,1958
C18:1 Δ 12t ............................................................................................................................................................................................. 1799,1899,2241 C18:1n11c .................................................................................................................................................................... 1951,1955,1956,2258,1958,2261
C18:1 D 13c ............................................................................................................................................................................................. 1801,1901,2243 C18:1n11t ..................................................................................................................................................................... 1951,1955,1956,2258,1958,2261
C18:1 Δ 13c ............................................................................................................................................................................................. 1799,1899,2241 C18:1n12c .................................................................................................................................................................... 1951,1955,1956,2258,1958,2261
C18:1 Δ 13t ............................................................................................................................................................................................. 1799,1899,2241 C18:1n12t ................................................................................................................................................................................................. 1955,1956,2258
C18:1 Δ 6c ............................................................................................................................................................................................... 1799,1899,2241 C18:1n13c .................................................................................................................................................................... 1951,1955,1956,2258,1958,2261
C18:1 Δ 6t ............................................................................................................................................................................................... 1799,1899,2241 C18:1n13t .................................................................................................................................................................................................................... 1955
C18:1 Δ 7c ............................................................................................................................................................................................... 1799,1899,2241 C18:1n14c ....................................................................................................................................................................................... 1951,1955,1958,2261
C18:1 Δ 7t ............................................................................................................................................................................................... 1799,1899,2241 C18:1n14t .................................................................................................................................................................................................................... 1955
C18:1 D 9c ............................................................................................................................................................................................... 1801,1901,2243 C18:1n15c .......................................................................................................................................................................................................... 1951,1955
C18:1 Δ 9c ............................................................................................................................................................................................... 1799,1899,2241 C18:1n15t .................................................................................................................................................................................................................... 1955
C18:1 Δ 9t ............................................................................................................................................................................................... 1799,1899,2241 C18:1n16t .............................................................................................................................................................................. 1951,1955,1956,2258,1958
C18:2 ............................................................................................................................................................................................................................ 2259 C18:1n5c ............................................................................................................................................................................................................. 1955,1958
C18:2 D 9c, 12c ...................................................................................................................................................................................... 1801,1901,2243 C18:1n5t ...................................................................................................................................................................................................................... 1951
C18:2 Δ 9c,12c ........................................................................................................................................................................................ 1799,1899,2241 C18:1n6c .......................................................................................................................................................................................... 1955,1956,2258,1958
C18:2 D 9c, 12t ...................................................................................................................................................................................... 1801,1901,2243 C18:1n6t .......................................................................................................................................................................................... 1951,1955,1958,2261
C18:2 Δ 9c,12t ........................................................................................................................................................................................ 1799,1899,2241 C18:1n7 ........................................................................................................................................................................ 1870,2021,1942,1943,1952,1953
C18:2 D 9t, 12c ...................................................................................................................................................................................... 1801,1901,2243 C18:1n7c ................................................................................................................................................................................................... 1955,1956,2258
Indexes Analyte
C18:1n7 methyl ester ................................................................................................................................................................................................ 1947 C20:4n3 ........................................................................................................................................................................ 1870,2021,1942,1943,1952,1953

C18:1n7t ....................................................................................................................................................................... 1951,1955,1956,2258,1958,2261 C20:4n6 .................................................................................................................................. 1823,1923,2255,1870,2021,1942,1956,2258,1958,2261
C18:1n8c ...................................................................................................................................................................................................................... 1955 C20:5n3 ...................................................................................................... 1823,1923,2255,1872,2023,2277,1942,1951,1952,1956,2258,1958,2261
C18:1n8t ................................................................................................................................................................................ 1951,1955,1956,2258,2261 C20:5n3 (EPA) ................................................................................................................................................................................ 1870,2021,1943,1953
C18:1n9 ............................................................................................................................................ 1870,2021,1872,2023,2277,1942,1943,1952,1953 C20:5n3 methyl ester ................................................................................................................................................................................................ 1947
C18:1n9c .......................................................................................................................................... 1823,1923,2255,1951,1955,1956,2258,1958,2261 C21:0 ............................................................................................................................................................................. 1823,1923,2255,1956,2258,1958
C18:1n9 methyl ester ................................................................................................................................................................................................ 1947 C21:5n3 ..................................................................................................................................................................................................... 1872,2023,2277
C18:1n9t .......................................................................................................................................... 1823,1923,2255,1951,1955,1956,2258,1958,2261 C22:0 ................................................................................................................................................................... 1823,1923,2255,1872,2023,2277,1958
C18:2n6 ............................................................................................................................................ 1870,2021,1872,2023,2277,1942,1943,1952,1953 C22:2 ......................................................................................................................................................................................................... 1823,1923,2255
C18:2n6c ................................................................................................................................................................................................... 1823,1923,2255 C22:5ω3 ....................................................................................................................................................................................................................... 2259
C18:2n6 methyl ester ................................................................................................................................................................................................ 1947 C22:6ω3 ....................................................................................................................................................................................................................... 2259
C18:2n6t ................................................................................................................................................................................................... 1823,1923,2255 C22:1LyLn ...................................................................................................................................................................................................................... 324
C18:2n9c,11t ................................................................................................................................................................ 1951,1955,1956,2258,1958,2261 C22:1n9 ..................................................................................................................................................................................................... 1823,1923,2255
C18:2n9c,12c ................................................................................................................................................................ 1951,1955,1956,2258,1958,2261 C22:1n9 methyl ester ................................................................................................................................................................................................ 1947
C18:2n9c,12t .......................................................................................................................................................................... 1951,1955,1956,2258,2261 C22:4n6 .............................................................................................................................................................................................................. 1951,2261
C18:2n9t,12c ................................................................................................................................................................................... 1951,1956,2258,2261 C22:5n3 ......................................................................... 1823,1923,2255,1870,2021,1872,2023,2277,1942,1943,1952,1953,1956,2258,1958,2261
C18:3n3 ......................................................................................................................... 1823,1923,2255,1942,1951,1952,1955,1956,2258,1958,2261 C22:5n3 methyl ester ................................................................................................................................................................................................ 1947
C18:3n3 (ALA) ............................................................................................................................................................................... 1870,2021,1943,1953 C22:6n3 ............................................................................................................... 1823,1923,2255,1872,2023,2277,1942,1951,1952,1956,2258,2261
C18:3n3 methyl ester ................................................................................................................................................................................................ 1947 C22:6n3 (DHA) ............................................................................................................................................................................... 1870,2021,1943,1953
C18:3n4 ........................................................................................................................................................................ 1870,2021,1942,1943,1952,1953 C22:6n3 methyl ester ................................................................................................................................................................................................ 1947
C18:3n6 ............................................................................................................................................ 1823,1923,2255,1942,1951,1955,1956,2258,2261 C23:0 ................................................................................................................................................................... 1823,1923,2255,1872,2023,2277,1958
C18:4n3 ............................................................................................................................................ 1870,2021,1872,2023,2277,1942,1943,1952,1953 C23:1 ............................................................................................................................................................................................................................ 1958
C18:4n3 methyl ester ................................................................................................................................................................................................ 1947 C24:0 ................................................................................................................................................................... 1823,1923,2255,1872,2023,2277,1958
C18:3 D 9c, 12t, 15t and C18:3 D 9c, 12c, 15t .............................................................................................................................. 1801,1901,2243 C24:1LyLn ...................................................................................................................................................................................................................... 324
C18:1 D 12t, C18:1 D 6c, C18:1 D 7c and C18:1 D13t .................................................................................................................. 1801,1901,2243 C24:1n9 ..................................................................................................................................................... 1823,1923,2255,1872,2023,2277,1958,2261
cis-C18:1 FAME isomers ............................................................................................................................................................... 1802,1902,1805,1905 C25:0 ............................................................................................................................................................................................................................ 1958
trans-C18:1 FAME isomers ........................................................................................................................................................... 1802,1902,1805,1905 C26:0 ......................................................................................................................................................................................................... 1956,2258,2261
C18:0 methyl ester .................................................................................................................................................................................................... 1947 C3 ................................................................................................................................................................................................................................... 718
C19:1 ............................................................................................................................................................................................................................ 1958 C4 ................................................................................................................................................................................................................................... 718
C2 ................................................................................................................................................................................................................................... 718 C4:0 ..................................................................................................................................................................... 1823,1923,2255,1951,1955,1958,2261
C20:0 ............................................................................................................................. 1823,1923,2255,1872,2023,2277,1951,1955,1958,2259,2261 C40-C46 esters .................................................................................................................................................................................................. 1884,2063
C20:2 ......................................................................................................................................................................................................... 1823,1923,2255 C5-C11 alkanes ........................................................................................................................................................................................................... 1596
C20:4ω6 ....................................................................................................................................................................................................................... 2259 C5-C11 hydrocarbons ................................................................................................................................................................................................. 1593
C20:1n9 ............................................................................................ 1823,1923,2255,1870,2021,1872,2023,2277,1942,1943,1952,1953,1958,2261 C5-C9 alkanes ............................................................................................................................................................................................................. 1597
C20:1n9c ............................................................................................................................................................................................................. 1956,2258 C6+ .............................................................................................................................................................................................................................. 1628
C20:1n9 methyl ester ................................................................................................................................................................................................ 1947 C6:0 .................................................................................................................................................. 1823,1923,2255,1951,1955,1956,2258,1958,2261
C20:2n6 ..................................................................................................................................................................................................... 1956,2258,2261 C7:0 .............................................................................................................................................................................................................................. 1958
C20:3n3 .................................................................................................................................. 1823,1923,2255,1870,2021,1942,1943,1952,1953,2261 C8:0 .................................................................................................................................................. 1823,1923,2255,1951,1955,1956,2258,1958,2261
C20:3n6 ............................................................................................................................................................... 1823,1923,2255,1956,2258,1958,2261 C9:0 ........................................................................................................................................................................................................... 1951,1955,1958
Indexes Analyte
Cadaverine ..................................................................................................................................................................................................................... 143 Caprolactam ................................................................................................................................................................................................................. 1564

β-Cadinene .................................................................................................................................................................................................................. 2103 Caprylic acid ............................................................................................................................................................................................. 1808,1908,2244
γ-Cadinene ................................................................................................................................................................................................................... 2131 Caprylic acid methyl ester ........................................................................................................................................ 1818,1918,2250,1819,1919,2251
δ-Cadinene ................................................................................................................................................ 2050,2434,2096,2124,2131,2142,2161,2166 Caprylic acid methyl ester (C8:0) ............................................................................................................................ 1815,1915,2247,1817,1917,2249
(+)-δ-Cadinene .......................................................................................................................................................................................................... 2170 Caprylic acid methyl ester (C8:0) at 4 wt % ................................................................................................................................... 1822,1922,2254
Cadin-4-en-10-ol .............................................................................................................................................................................................. 2095,2131 Captafol ........................................................................................................................................................................................... 1448,1682,1829,1934
Cadusafos ........................................................................................................................................................................................ 1350,1401,1860,1995 Captan ........................................................................... 1347,1394,1798,1894,1430,1641,1796,1892,1571,1591,2325,2347,1797,1893,1889,2068
Cadusafos-1 ........................................................................................................................................................................................................ 1885,2064 Captofol .............................................................................................................................................................................................................. 1889,2068
Cadusafos-2 ........................................................................................................................................................................................................ 1885,2064 Captopril ........................................................................................................................................................................................... 123,849,129,892,896
Cafenstrole .......................................................................................................................................................................................................... 1886,2065 Carbamazepine .............................................................................................................................................................................................................. 851
Caffeic acid ........................................................................................................................................................................................................... 376,1259 Carbamazepine solution ............................................................................................................................................................................................. 1228
Caffeine .................................. 130,893,1362,1427,148,946,149,1105,183,334,335,339,340,342,343,346,347,348,349,850,1119,1275,2288,2289 Carbaryl .................................................................................................................................. 1347,1394,1798,1894,1445,1679,1826,1931,1797,1893
Caffeoylferuloylquinic acid (3C,4F-QA) .................................................................................................................................................................... 1275 Carbazole ...................................................................................................................... 1524,1540,1541,1552,1553,1554,1555,1556,1557,1558,1564
Caffeoylferuloylquinic acid (3F,4C-QA) .................................................................................................................................................................... 1275 Carbendazim .............................................................................................................................................................. 110,15,111,16,1418,404,1419,406
Caffeoylferuloylquinic acid (3F,5C-QA) .................................................................................................................................................................... 1275 Carbetamide ....................................................................................................................................................................................................... 1890,2069
Caffeoylferuloylquinic acid (4C,5F-QA) .................................................................................................................................................................... 1275 S-(−)-Carbidopa ................................................................................................................................................................................. 162,2634,163,2635
Caffeoylquinic acid (1-CQA) ...................................................................................................................................................................................... 1275 Carbinoxamine ............................................................................................................................................................................................................... 890
Caffeoylsinapylquinic acid (CSQA) ............................................................................................................................................................................ 1275 Carbofuran ............................................................................................................................................. 1,295,294,302,1391,305,1445,1679,1826,1931
Caftaric acid ........................................................................................................................................................................................................... 376,377 Carbon dioxide ......................................................................................................................................... 1622,1628,1639,1747,1749,1750,1751,1753
α-Calacorene ................................................................................................................................................................................................................ 2124 Carbon dioxide (215 ng on column) ..................................................................................................................................................................... 1752
Calcium pantothenate ....................................................................................................................................... 1299,1300,1306,1307,1308,1332,1333 Carbon disulfide ............................... 1504,2073,1565,1566,1568,1588,2322,2344,1582,2332,1685,1686,2381,1687,2382,1871,2022,2395,2417
Calphostin C ........................................................................................................................................................................................................... 151,539 Carbon disulfide (1 ppm) ......................................................................................................................................................................................... 1783
Campesterol .................................................................................................................................................................................... 1354,1412,1875,2034 Carbon disulfide (solvent) ............................................................................................................................................................................... 1486,1487
Campesterol solution ........................................................................................................................................................................................ 1874,2033 Carbonfenthion .................................................................................................................................................................................................. 1887,2066
Camphene .................................................. 2050,2434,2095,2096,2099,2103,2105,2106,2111,2184,2120,2121,2131,2137,2138,2142,2166,2170 Carbonic anhydrase ................................................................................................................................................................. 742,748,749,750,751,752
Campherenol ................................................................................................................................................................................... 2050,2434,2096,2120 Carbonic Anhydrase from bovine erythrocytes ................................................................................................................................................. 738,744
Camphor .............................................................................................................. 1670,1910,2085,2199,2504,2665,2691,2851,2096,2103,2120,2166 Carbon monoxide ........................................................................................................................................................................... 1639,1749,1750,1753
Cannabidiol ............................................................................................................................................................................................................. 421,459 Carbon Monoxide ....................................................................................................................................................................................................... 1754
Cannabidiol solution ..................................................................................................................................................................................................... 487 Carbon monoxide (215 ng on column) ................................................................................................................................................................. 1752
Cannabinol .............................................................................................................................................................................................................. 421,459 Carbon tetrachloride .................................................................................. 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,
Cannabinol solution ...................................................................................................................................................................................................... 487 1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1674,1676,1725,
Capric acid methyl ester ........................................................................................................................................... 1818,1918,2250,1819,1919,2251 1726,1727,2200,2201,2202,2448,2203,2204,2449,2224
Capric acid methyl ester (C10:0) ............................................................................................................................. 1815,1915,2247,1817,1917,2249 Carbonyl sulfide .............................................................................................................................................................................. 1686,2381,1687,2382
Capric acid methyl ester (C10:0) at 4 wt % .................................................................................................................................... 1822,1922,2254 Carbonyl sulfide (1 ppm) ......................................................................................................................................................................................... 1783
Caproic acid ................................................................................................................................................................. 1808,1908,2244,1873,2031,2278 Carbophenothion ........................................................................................................................................................... 1419,406,1542,1543,1544,1545
Caproic acid methyl ester ......................................................................................................................................... 1818,1918,2250,1819,1919,2251 Carboxin ...................................................... 1466,1740,1858,1989,2356,2384,2392,2406,1471,1746,1861,1997,2361,2386,2393,2411,1506,2075
Caproic acid methyl ester (C6:0) ....................................................................................................................................................... 1815,1915,2247 Carboxine ............................................................................................................................................................................................................ 1507,2076
Caproic acid methyl ester (C6:0) ......................................................................................................................................................... 1817,1917,2249 Cardiac drugs ................................................................................................................................................................................................................ 954
Caproic acid methyl ester (C6:0) at 4 wt % .................................................................................................................................... 1822,1922,2254 2-Caren-10-al ................................................................................................................................................................................. 2047,2175,2351,2353
Indexes Analyte
3-Carene ............................................................................................................................................................................................................. 2099,2170 Cephapirin .............................................................................................................................................................................................................. 882,956

γ-3-Carene ......................................................................................................................................................................................................... 2111,2184 Cetuximab ERBITUX® .................................................................................................................................................................................................... 549
δ-3-Carene ............................................................................................................................................................................. 2050,2434,2120,2131,2142 Chenodeoxychloic acid ................................................................................................................................................................................................. 234
Carfentrazone-ethyl ........................................................................................................................................................................................... 1888,2067 Chenodeoxycholic acid .......................................................................................................................................................................................... 141,234
(±)-Carnitine hydrochloride .............................................................................................................................................................................. 2756,547 Chinomethionate ............................................................................................................................................................................................... 1889,2068
Carnosic acid ............................................................................................................................................................................................................... 1284 Chlomethoxyfen ................................................................................................................................................................................................. 1890,2069
Carnosol ....................................................................................................................................................................................................................... 1284 Chloramphenicol .............................................................................................................................................................................. 127,877,320,403,879
α-Carotene ......................................................................................................................................................................................... 1239,344,1240,1317 DL-threo-Chloramphenicol-d5 ................................................................................................................................................................................ 320,403
β-Carotene ......................................................................................................................................................................................... 1239,344,1240,1317 Chlorbenzilate .................................................................................................................................................................................................... 1888,2067
Carotenoids .................................................................................................................................................................................................................. 1243 cis-Chlordane .................................................................................................................................................................................. 1347,1394,1798,1894
cis-Carveol ................................................................................................................................................................................................. 2103,2131,2166 cis-Chlordane ................................................................ 1432,1671,1811,1911,2469,2505,2657,2692,1458,1707,1851,1979,2470,2515,2658,2694
trans-Carveol ...................................................................................................................................................................................................... 2095,2167 cis-Chlordane ..................................................................................................................................................................................................... 1797,1893
trans-Carveol acetate ................................................................................................................................................................................................. 2167 α-Chlordane ......... 1382,1396,1439,1925,1388,1411,1482,2028,1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1532,1604
Carvone ................................................................................................................................................................. 322,2096,2103,2120,2138,2166,2167 γ-Chlordane .......... 1382,1396,1439,1925,1388,1411,1482,2028,1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1532,1604
D-Carvone ......................................................................................... 2086,2666,2087,2667,2090,2670,2091,2671,2092,2672,2093,2673,2094,2674 Chlordiazepoxide ..................................................................................................................................................................................... 915,916,917,919
L-Carvone ......................................................................................... 2088,2668,2089,2669,2090,2670,2091,2671,2092,2672,2093,2673,2094,2674 Chloreneb ........................................................................................................................................................................................ 1448,1682,1829,1934
(E)-Caryophyllene .............................................................................................................................................................................................. 2050,2434 Chlorfenapyr ....................................................................................................................................................................................................... 1886,2065
(−)-trans-Caryophyllene ............................................................................................................................................................................................ 2105 Chlorfenson ........................................................................................................................................................................................................ 1886,2065
(E)-Caryophyllene ....................................................................................................................................................................................................... 2120 Chlorfenvinphos ........................................................................................................................................................................................ 1542,1543,1545
(−)-trans-Caryophyllene ............................................................................................................................................................................................ 2170 Chlorfenvinphos-Z ............................................................................................................................................................................................. 1885,2064
(E)-Caryophyllene .................................................................................................................. 2095,2096,2121,2124,2131,2137,2138,2161,2166,2167 Chlorfenviphos-E ................................................................................................................................................................................................ 1885,2064
β-Caryophyllene ................................................................................................................................................. 2099,2103,2106,2111,2184,2136,2142 Chlorhexidine .......................................................................................................................................................................................................... 281,957
9-epi-(E)-Caryophyllene ............................................................................................................................................................................................. 2131 Chloridazon ........................................................................................................................................................................................................ 1890,2069
Caryophyllene oxide .................................................................................................................................................... 2124,2131,2137,2138,2142,2161 Chlorimipramine ............................................................................................................................................................................. 2279,2303,2459,2468
Catalase from bovine liver .......................................................................................................................................................................................... 744 Chlormefos ......................................................................................................................................................................................................... 1890,2069
Catechin ....................................................................................................................................................................................................................... 1244 Chlorneb ............................................................................................................................................................................................................. 1506,2075
(+)-Catechin ................................................................................................................................................................................................................. 376 Chloroacetonitrile ............................................................................................................................................................................................... 1504,2073
(+)-Catechin hydrate ................................................................................................................................................................................................ 1259 p-Chloroamphetamine ........................................................................................................................................................................................ 1172,213
Catechin gallate .......................................................................................................................................................................................................... 1244 2-Chloroaniline .................................................................................................................................................................................................. 1655,1672
Catecholamine Mix 1 (Epinephrines) solution ......................................................................................................................................................... 271 3-Chloroaniline ............................................................................................................................................................................................................ 1564
Catecholamine Mix 2 (Metanephrines) solution ..................................................................................................................................................... 271 4-Chloroaniline ................................................................................................... 1484,2030,2366,2421,1552,1553,1554,1555,1556,1557,1558,1673
Cedrol ................................................................................................................................................................................................................. 2111,2184 Chlorobenzene ...................................................... 1493,2057,2370,2440,1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,
Cedryl acetate .................................................................................................................................................................................................... 2111,2184 1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1676,1725,
Cefadroxil ................................................................................................................................................................................................................ 882,956 1726,1727,1794,2205,2206,2207,2450,2208,2209,2451
Cefsulodin ............................................................................................................................................................................................................... 882,956 Chlorobenzene-d5 .............................................................................................................................................. 1500,1586,2321,2343,1519,1520,1523
Celecoxib ........................................................................................................................................................................................................................ 898 Chlorobenzene-d5 ..................................................................................................................................................................................................... 1546
Cellobiose ....................................................................................................................................................................................................................... 383 Chlorobenzene-d5 ..................................................................................................................................................................................................... 1547
Cellosolve acetate ....................................................................................................................................................................................................... 1780 Chlorobenzene-d5 ........................................................................................................................................................................... 1548,1549,1565,1566
Cephalomannine ............................................................................................................................................................................. 1104,1153,1154,1220 Chlorobenzilate ................................................................................................................................ 1506,2075,1507,2076,1509,1510,1511,1550,1551
Indexes Analyte
2-Chlorobiphenyl ...................................................................................................................................... 1388,1411,1482,2028,1506,2075,1507,2076 Chloropyrifos-methyl ...................................................................................................................................................................... 1885,2064,2004,2413

1-Chlorobutane .................................................................................................................................................................................................. 1504,2073 Chloropyriphos-methyl ................................................................................................................................................................... 1447,1681,1828,1933
2-Chlorobutane ............................................................................................................................................................................... 1706,2217,2514,2854 Chloroquine diphosphate salt ............................................................................................................................................................................ 2885,958
Chlorodibromomethane ............................................................................................... 1498,2062,2373,2444,1502,2071,1503,2072,1505,2074,1523 Chlorothalonil ................................................................................................................................... 1347,1394,1798,1894,1430,1641,1796,1892,
4-Chlorodiphenyl ether .................................................................................................................................................................................... 1556,1558 1506,2075,1507,2076,1509,1510,1511,1571,1591,2325,2347,1797,1893
Chloroethane .............................................................................................................................................. 1494,2058,2371,2441,1496,1497,2061, o-Chlorotoluene ........................................................................................................................................................................................ 1504,2073,1547
1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344 p-Chlorotoluene ........................................................................................................................................................................................ 1504,2073,1547
2-Chloroethyl vinyl ether .............................................................................................................. 1494,2058,2371,2441,1519,1520,1521,1522,1523 2-Chlorotoluene ..................................................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1505,2074,1546,1548,1549
3-Chloro-4-fluoroaniline ................................................................................................................................................................................... 1655,1672 4-Chlorotoluene ..................................................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1505,2074,1546,1548,1549
Chloroform ............................................................................... 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072, Chlorotoluron ..................................................................................................................................................................................................................... 5
1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1674,1676,1725,1726, Chlorpheniramine .................................................................................................................................................... 850,858,859,871,890,912,971,1145
1727,2205,2206,2207,2450,2208,2209,2451,2226,2456 (±)-Chlorpheniramine .................................................................................................................................................................................................. 891
Chlorofos .......................................................................................................................................................................................... 1430,1641,1796,1892 (±)-Chlorpheniramine maleate salt .............................................................................................................................................. 2886,959,1341,3024
Chlorogenic acid (5-CQA) ......................................................................................................................................................................................... 1275 Chlorpiriphos-methyl ...................................................................................................................................................................... 1446,1680,1827,1932
Chloromethane .............................................................. 1494,2058,2371,2441,1496,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072, Chlorpromazine ...................................................................................................................................................................................................... 165,578
1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1569,1589,2323,2345 Chlorpropham .................................................................................................................... 1,295,294,302,2374,303,1506,2075,1507,2076,1886,2065
3-Chloro-4-methylaniline ........................................................................................................................................................................................... 1672 Chlorpropylate .................................................................................................................................................................................................... 1889,2068
4-Chloro-2-methylaniline ........................................................................................................................................................................................... 1673 Chlorpyrifos ............................................................ 308,1347,1394,1798,1894,1430,1641,1796,1892,1507,2076,1542,1543,1585,2342,1797,1893
4-Chloro-4-methylaniline ........................................................................................................................................................................................... 1655 Chlorpyrifos-methyl .......................................................................................................................................................................... 1419,406,1542,1543
4-Chloro-3-methylphenol .................................................................... 90,1359,1386,1407,1475,1755,2005,1476,1756,2006,2363,2388,2414, Chlorthal-dimethyl ......................................................................................................................................................................... 1507,2076,1887,2066
1477,1757,2007,2364,2389,2415,1524,1531,1552,1553,1554,1555,1556,1557,1558,1564 Chlorthalidone ............................................................................................................................................................. 851,2887,960,2888,961,2889,962
3-(Chloromethyl)pyridine ........................................................................................................................................................................................... 1673 Chlorthalonil ....................................................................................................................................................................................................... 1889,2068
2-Chloronaphthalene ...................................................................................................................... 1524,1552,1553,1554,1555,1556,1557,1558,1564 Chlorthiophos ..................................................................................................................................................................................................... 1890,2069
Chloroneb ................................................................................................... 1430,1641,1796,1892,1449,1683,1830,1935,1507,2076,1509,1510,1511 Chlortoluron ................................................................................................................................................................................................. 110,15,111,16
4-Chloro-2-nitroaniline ..................................................................................................................................................................................... 1655,1672 Chlorzoxazone ................................................................................................................................................................................................................ 963
2-Chlorophenol .............................................................................................................................................. 90,1359,1386,1407,1475,1755,2005, Cholecalciferol ............................................................................................................................................................................................... 1293,217,420
1476,1756,2006,2363,2388,2414,1477,1757,2007,2364,2389,2415,1524,1531,1552,1553,1554,1555,1556,1557,1558,1564 Cholecalciferol (D3) .................................................................................................. 1292,216,1294,218,1297,219,1251,1252,1255,1256,1253,1298
2-Chlorophenol-3,4,5,6-d4 ......................................................................................................................................................................................... 1556 5α-Cholestane .................................................................................................................................................................................................... 1874,2033
2-Chlorophenol-d4 .......................................................................................................................... 1524,1550,1551,1552,1553,1554,1555,1557,1564 Cholesterol ....................................................................................................................................................................................... 1354,1412,1875,2034
2-chlorophenol-d4 ...................................................................................................................................................................................................... 1558 Cholic acid .............................................................................................................................................................................................................. 141,234
2-(2-Chlorophenoxy)propionic acid .......................................................................................................................................................... 2479,7,2481,8 Chrysene .......................................................... 2375,310,311,1388,1411,1482,2028,1408,2010,1409,2011,1451,1936,1480,2013,1481,2014,
2-(4-Chlorophenoxy)propionic acid ............................................................................................................................................................. 2482,9,2480 1483,2029,2365,2420,1506,2075,1507,2076,1517,1518,1524,1540,1541,1552,1553,1554,1555,1556,1557,1558,1560,1561,1562,1563,1564,
p-Chlorophenylalanine ........................................................................................................................................................................................ 1172,213 1567,1587,1795,1891
4-Chloro-DL-phenylalanine ............................................................................................................................................................... 2758,552,2290,2462 Chrysene-d12 ................................................................................................................. 1388,1411,1482,2028,1483,2029,2365,2420,1506,2075,
2-Chloro-DL-phenylanine ..................................................................................................................................................................................... 2757,551 1507,2076,1524,1550,1551,1552,1553,1554,1555,1556,1557,1564
4-Chlorophenyl phenyl ether ........................................................................................................................... 1524,1552,1553,1554,1555,1557,1564 Chrysene-d12 .................................................................................................................................................................................................... 1567,1587
3-Chloropropane ................................................................................................................................................................................................ 1497,2061 Chrysene-d12 (I.S.) .................................................................................................................................................................................................... 1558
Chloropropylate ........................................................................................................................................................................................ 1509,1510,1511 Chrysin ............................................................................................................................................................................................................................ 352
Chloropyrifos .................................................................................... 1350,1401,1860,1995,1351,1403,1862,2000,1506,2075,1544,1545,1885,2064 α-Chymotrypsin .............................................................................................................................................................................................. 754,755,756
Indexes Analyte
Chymotrypsinogen A .................................................................................................................................................................................................... 759 Cocaethylene .......................................................................................................................................................................................................... 462,464

α-Chymotrypsinogen A ................................................................................................................................................................................................ 739 Cocaethylene solution ......................................................................................................................................................................................... 1203,488
β-Chymotrypsinogen ...................................................................................................................................................................................... 754,755,756 Cocaine ................................................................................................................................................. 462,464,2289,2290,2462,2291,2463,2295,2465
Cihalothrin ....................................................................................................................................................................................... 1447,1681,1828,1933 Cocaine solution ............................................................................................................................................................................................................ 454
Cimetidine .................................................................................................................................................................................................................... 1173 Cocaine hydrochloride ........................................................................................................................................................................ 432,1203,488,2288
1,4-Cineole ......................................................................................................................................................................................................... 2105,2106 Cocapropylene .................................................................................................................................................................................................... 2295,2465
1,8-Cineole ...................................................................................................................................................................................... 2111,2184,2136,2142 Codeine ......................................... 30,124,868,153,423,154,424,407,853,427,428,429,1233,854,871,912,1119,2288,2289,2290,2462,2291,2463
Cinmethylin ........................................................................................................................................................................................................ 1890,2069 Codeine solution .............................................................................................................................................................................. 264,484,481,490,692
Cinnamic alcohol ............................................................................................................................................................................................... 2111,2184 Codeine β-D-glucuronide ....................................................................................................................................................................... 153,423,154,424
Cinnamyl alcohol ....................................................................................................................................................................................... 492,2180,2181 Codeine-6-β-D-glucuronide solution ........................................................................................................................................................................... 692
Cinnamyl cinnamate, mixture of isomers ..................................................................................................................................................... 2111,2184 Cod liver oil ................................................................................................................................................................................................................ 1946
Cinoxacin ..................................................................................................................................................................................................... 1005,356,1199 Conalbumin .................................................................................................................................................................................................................... 740
CIPC .................................................................................................................................................................................................. 1350,1401,1860,1995 Conalbumin from chicken egg white ....................................................................................................................................................................... 761
Ciprofibrate ........................................................................................................................................................................................................... 2890,965 α-Copaene ....................................................................................................................................................................................... 2103,2124,2161,2166
Ciprofloxacin ........................................................................................................................................................ 1004,355,1005,356,878,880,881,1013 (−)-α-Copaene ........................................................................................................................................................................................................... 2170
Citalopram hydrobromide .................................................................................................................................................................................. 2891,966 β-Copaene ................................................................................................................................................................................................................... 2170
Citric acid ............................................................................................................................................................................. 68,1277,368,85,1278,369,86 Coronene ............................................................................................................................................................................................................ 1567,1587
Citronella ...................................................................................................................................................................................................................... 2103 Corticosterone ............................................................................................................................................................................................... 169,1204,272
CitronellaI ..................................................................................................................................................................... 2050,2434,2096,2120,2142,2166 Corticosterone solution ....................................................................................................................................................................................... 1374,257
Citronellal ..................................................................................................................................................................................................................... 2099 Cortisone .............................................................................................................................................................................................. 1290,204,1204,272
(R)-(+)-Citronellal ...................................................................................................................................................................................................... 2170 Cortisone 21-acetate ............................................................................................................................................................................ 203,1204,272,453
(±)-Citronellal ................................................................................................................................................................................................... 2183,2446 Cotinine .......................................................................................................................................................................................................................... 246
Citronellol ......................................................................................................................................... 2096,2103,2112,2120,2166,2180,2181,2183,2446 Coumachlor ............................................................................................................................................................................................ 2574,46,2895,972
Citronellyl acetate .............................................................................................................................................. 2050,2434,2096,2099,2120,2131,2166 Coumafuryl ............................................................................................................................................................................................................. 2575,47
Citronellyl formate ...................................................................................................................................................................................................... 2112 Coumaphos ................................................ 1347,1394,1798,1894,1350,1401,1860,1995,1351,1403,1862,2000,1542,1543,1544,1545,1797,1893
Citronyllyl acetate ....................................................................................................................................................................................................... 2103 p-Coumaric acid ......................................................................................................................................................................................... 376,1205,1259
DL-Citrulline ........................................................................................................................................................................................................... 2759,553 Coumarin .................................................................................................................................................................................................. 2111,2184,2181
DL-Citrulline, FMOC derivatives .......................................................................................................................................................................... 2778,593 Coumaroylquinic acid (CQA) ..................................................................................................................................................................................... 1275
Clenbuterol hydrochloride ............................................................ 140,2633,2720,419,1192,235,2647,2725,460,2894,422,969,2892,967,2893,968 Courmarin .................................................................................................................................................................................................................... 2180
Clethodim ............................................................................................................................................................................................................. 1419,406 CP-47,497 C8 homolog (1,1-Dimethyloctyl-2-[(1R,3S)-3-hydroxycyclohexyl]-5-(2-methyloctan-2-yl)phenol) ...................................... 449,452
Clobazam .............................................................................................................................................................................................................. 1190,232 CP-47,497 (2-[(1R,3S)-3-Hydroxycyclohexyl]- 5-(2-methyloctan-2-yl)phenol) .................................................................................................. 449
Clobetasol propionate .................................................................................................................................................................................................. 167 CP-47,497 (2-[(1R,3S)-3-Hydroxycyclohexyl]-5-(2-methyloctan-2-yl)phenol) .................................................................................................... 452
Clomeprop .......................................................................................................................................................................................................... 1886,2065 Creatine Kinase ............................................................................................................................................................................................................. 556
Clomipramine ....................................................................................................................................................................................................... 1164,210 o-Cresol .......................................................................................................................................................... 48,1283,1556,1558,1676,1726,1727,1794
Clonazepam ............................................................................... 128,887,135,935,136,936,137,937,138,938,1191,233,875,876,888,889,2291,2463 m-Cresol ..................................................................................................................................................................................... 48,1676,1726,1727,1794
Clorazepate .............................................................................................................................................................................................. 128,887,888,889 p-Cresol .......................................................................................................................................................... 48,1283,1556,1558,1676,1726,1727,1794
Clothianidin ......................................................................................................................................................................................... 1358,19,1890,2069 Crimidine ............................................................................................................................................................................................................ 1890,2069
Cloxacillin ........................................................................................................................................................................................................... 1009,1041 Crotonaldehyde ...................................................................................................................................................................................................... 21,2326
Clozapine ................................................................................................................................................................................................................ 908,909 Crotonaldehyde-2,4-dinitrophenylhydrazone ....................................................................................................................................... 22,2334,23,2335
Indexes Analyte
Crotonaldehyde (2,4-DNPH derivative) .............................................................................................................................................................. 2336,24 Cyfluthrin-1 ........................................................................................................................................................................................................ 1886,2065

Crotoxyphos ..................................................................................................................................................................................... 1542,1543,1544,1545 Cyfluthrin-2 ........................................................................................................................................................................................................ 1886,2065
Crufomate (Ruelene) ............................................................................................................................................................................................. 2576,49 Cyfluthrin-3 ........................................................................................................................................................................................................ 1886,2065
Crypto-chlorogenic acid (4-CQA) ............................................................................................................................................................................. 1275 Cyfluthrin-4 ........................................................................................................................................................................................................ 1886,2065
α-Cubebene ........................................................................................................................................................................................................ 2124,2161 Cyfluthrin isomers ....................................................................................................................................................... 1351,1403,1862,2000,1404,2001
β-Cubebene .............................................................................................................................................................................................. 2124,2161,2170 Cyhalofop-butyl ................................................................................................................................................................................................. 1886,2065
Cubenol ........................................................................................................................................................................................................................ 2124 Cyhalothrin ......................................................................................................................................................................................................... 1404,2001
Cumene .......................................................................................................................... 1492,1583,2320,2333,1493,2057,2370,2440,1546,1547, Cyhalothrin-1 ..................................................................................................................................................................................................... 1887,2066
1548,1549,1565,1566,1612,1621,1640,2210,2212,2452,2213,2214,2453 Cyhalothrin-2 ..................................................................................................................................................................................................... 1887,2066
Cuminic aldehyde .......................................................................................................................................................................... 2047,2175,2351,2353 Cylert .............................................................................................................................................................................................................................. 974
α-Curcumene ............................................................................................................................................................................................................... 2096 Cymene ............................................................................................................................................................................................ 2047,2175,2351,2353
Curcumin ...................................................................................................................................................................................................................... 1331 o-Cymene .................................................................................................................................................................................................................... 2131
Cyanazine ................................................................................................................................................................. 5,110,15,111,16,1391,305,316, p-Cymene ................................................................................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1505,2074,
1444,1678,1825,1930,1466,1740,1858,1989,2356,2384,2392,2406,1489,1788,1877,2039,2369,2391,2397,2425,1506,2075,1507,2076, 1546,1547,1548,1549,2050,2434,2056,2439,2095,2096,2099,2103,2105,2106,2120,2121,2124,2131,2136,2137,2138,2142,2166,2167,2170
1886,2065 p-Cymen-8-ol .................................................................................................................................................................................................... 2131,2142
Cyanidin 3-(6-acetylglucoside) ................................................................................................................................................................................... 336 Cypermethrin-1 .................................................................................................................................................................................................. 1887,2066
Cyanidin chloride .............................................................................................................................................................................................. 1320,1321 Cypermethrin-2 .................................................................................................................................................................................................. 1887,2066
Cyanidin 3-glucoside .................................................................................................................................................................................................... 336 Cypermethrin-3 .................................................................................................................................................................................................. 1887,2066
Cyanocobalamin ............................................................................................................................................................................................. 742,748,751 Cypermethrin-4 .................................................................................................................................................................................................. 1887,2066
Cyanocobalamin (B12) ...................................................................................................................................... 1299,1300,1306,1307,1308,1332,1333 Cypermethrin isomer ............................................................................................................................... 1351,1403,1862,2000,1449,1683,1830,1935
Cyanofenphos ..................................................................................................................................................................................................... 1887,2066 Cyproconazole-1 ................................................................................................................................................................................................ 1888,2067
Cyanophos .......................................................................................................................................................................................................... 1885,2064 Cyproconazole-2 ................................................................................................................................................................................................ 1888,2067
Cyanuric acid ................................................................................................................................................................... 117,318,2078,2079,2080,2081 Cyprodinil .................................................................................................................................................. 1347,1394,1798,1894,1797,1893,1888,2067
Cycloate ..................................................................................................................................................... 1448,1682,1829,1934,1506,2075,1507,2076 D-Cysteine ............................................................................................................................................................................................................. 2760,557
Cyclobarbital ......................................................................................................................................................................... 1200,269,485,1201,270,486 L-Cysteine .......................................................................................................................................................................................... 2760,557,1897,2304
Cyclodecane .................................................................................................................................................................................... 1491,1581,2319,2331 DL-Cysteine ............................................................................................................................................................................................................ 2761,558
Cyclodextrin .............................................................................................................................................................................................................. 360,66 L-Cystine ............................................................................................................................................................................................................. 1897,2304
β-Cyclodextrin ............................................................................................................................................................................................................... 367 Cytidine ............................................................................................................................................................................................. 686,687,688,689,769
Cyclohexane ............................................................................ 1565,1566,1568,1588,2322,2344,1592,1612,2205,2206,2207,2450,2208,2209,2451 Cytochrome c ................................................................................................................................................................................................................ 739
trans-1,2-Cyclohexanediol ................................................................................................................................................................................ 1688,2506 Cytochrome c (bovine) ................................................................................................................................................................................................ 560
Cyclohexanol ................................................................................................................................................................................... 1676,1725,1726,1727 Cytochrome c from chicken heart ............................................................................................................................................... 704,732,733,738,762
Cyclohexanone ....................................................................................................................... 1613,1614,1615,1616,1617,1618,1624,1625,1626,1780 Cytochrome c from equine heart ............................................................................................................................................ 1130,746,747,1184,805
3-Cyclohexene-1-carbonitrile ........................................................................................................................................................................... 1582,2332 Cytochrome c, species variants ........................................................................................................................................................................... 540,759
Cyclopentane ............................................................................................................................................................................................................... 1592 Cytochrome c (equine) ................................................................................................................................................................................................ 560
Cyclopenta[cd]pyrene ....................................................................................................................................................................................... 1795,1891 Cytochrome c (rabbit) ................................................................................................................................................................................................. 560
Cyclopentene ............................................................................................................................................................................................................... 1612 Cytochrome c, tryptic digest ...................................................................................................................................................................................... 562
Cyclophosphamide monohydrate ...................................................................................................................................................................... 2896,973 Cytosine ........................................................................................................................................................................................................... 684,685,765
Cyclopropane ...................................................................................................................................................................................................... 1665,1723
Cyflufenamid ...................................................................................................................................................................................................... 1886,2065
Indexes Analyte
10-Deacetyl-7-(β-xylosyl)paclitaxel .......................................................................................................................................................................... 1154

—D— 10-Deacetyl-7-(β-xylosyl)paclitaxel C ...................................................................................................................................................................... 1154
2,4-D ......................................................................................................................................................................................... 1355,1389,2,298,306,307 Decachlorobiphenyl ................................................................. 1382,1396,1439,1925,1441,1927,1442,1928,1443,1929,1469,1994,2359,2409,
1512,1513,1514,1516,1532,1533,1534,1535,1538,1539,1604
(R,R)-DACH-ACR (N-(2-acryloylamino-(1R,2R)-cyclohexyl)-acrylamide) ........................................................................................................ 2577,50
2,2',3,3',4,4',5'5',6,6'-Decachlorobiphenyl (No. 209) ................................................................................................................................... 1402,1999
(S,S)-DACH-ACR (N-(2-acryloylamino-(1R,2R)-cyclohexyl)-acrylamide) ........................................................................................................ 2577,50
Decachlorobiphenyl (surr.) ........................................................................................................................................................................................ 1537
Dacthal .................................................................................................................................................................................. 1355,1389,2,298,1506,2075
(E,E)-2,4-Decadienal ......................................................................................................................................................................................... 2103,2166
Dacthal (dimethyl) .................................................................................................................................................................................. 1509,1510,1511
Decanal ........................................................................................................................... 1431,1572,1490,1580,2318,2330,1491,1581,2319,2331,
DAG32 1:2 ......................................................................................................................................................................................................... 1849,1974
1492,1583,2320,2333,1878,2044,2398,2430,1879,2046,2399,2431,1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436,
DAG32 1:3 ......................................................................................................................................................................................................... 1849,1974
1883,2054,2402,2437,1971,2405,2045,2350,2050,2434,2096,2099,2103,2105,2106,2111,2184,2120,2166,2170
DAG34 1:2 ......................................................................................................................................................................................................... 1849,1974
Decane ....................................................................................................... 1431,1572,1486,1487,1526,1528,1530,1595,1597,1601,1667,1602,1668
DAG34 1:3 ......................................................................................................................................................................................................... 1849,1974
n-Decane ................................................................................................................................................................................................... 1612,2311,2312
DAG36 1:2 ......................................................................................................................................................................................................... 1849,1974
n-decane ...................................................................................................................................................................................................................... 2313
DAG36 1:3 ......................................................................................................................................................................................................... 1849,1974
n-decane ...................................................................................................................................................................................................................... 2314
Daidzein ...................................................................................................................................................................................... 1270,361,1271,362,1272
Decane (C10) .............................................................................................................................................................................................................. 1636
Daidzin ........................................................................................................................................................................................ 1270,361,1271,362,1272
Decanoic acid ........................................................................................................................................................................ 1873,2031,2278,1970,2404
Dalapon ................................................................................................................................................................................................................... 306,307
δ-Decanolactone enantiomers ...................................................................................................................................................... 1690,2104,2508,2675
DAMME ................................................................................................................................................................................................................... 166,583
trans-2-Decenal ........................................................................................................................................................................................................... 2170
Danofloxacin ............................................................................................................................................................................................... 1004,355,1013
Decylamine .................................................................................................................................................................................................................. 1776
Dansyl-DL-glutamic acid bis(cyclohexylammonium) salt ............................................................................................................................... 2762,563
DEF (from oxidation of Merphos in the injection port) ............................................................................................................................ 1544,1545
Dansyl-DL-leucine cyclohexylammonium salt ................................................................................................................................................... 2763,564
(L)-Dehydroascorbic acid ........................................................................................................................................................................................... 1234
Dansyl-DL-methionine cyclohexylammonium salt .......................................................................................................................... 2764,565,2765,566
Dehydrocholic acid ....................................................................................................................................................................................................... 141
Dansyl-DL-norleucine cyclohexylammonium salt ............................................................................................................................................. 2766,567
cis-Dehydrolinalool oxide ........................................................................................................................................................................................... 2131
Dansyl-DL-phenylalanine cyclohexylammonium salt ....................................................................................................................................... 2767,568
3,4-Dehydro-DL-proline ....................................................................................................................................................................................... 2769,570
Dansyl-DL-valine cyclohexylammonium salt .................................................................................................................................................... 2768,569
Delphinidin 3-(6-acetylglucoside) ............................................................................................................................................................................... 336
Dazomet isomer ............................................................................................................................................................................. 1446,1680,1827,1932
Delphinidin chloride .......................................................................................................................................................................................... 1320,1321
2,4-DB ..................................................................................................................................................................................................................... 306,307
Delphinidin 3-glucoside ............................................................................................................................................................................................... 336
2,4-DB methyl ester ............................................................................................................................................................................................. 306,307
Deltamethrin ............................................................................................................................................. 1351,1403,1862,2000,1449,1683,1830,1935
3,4′-DDD .................................................................................................................................................... 1466,1740,1858,1989,2356,2384,2392,2406
Deltamethrin-1 .................................................................................................................................................................................................. 1886,2065
4,4′-DDD ................................................................................. 1382,1396,1439,1925,1469,1994,2359,2409,1506,2075,1513,1516,1524,1532,1604
Deltamethrin-2 .................................................................................................................................................................................................. 1886,2065
4,4′-DDE .............................................................................................................. 1347,1394,1798,1894,1404,2001,1507,2076,1512,1514,1797,1893
Demeclocycline ............................................................................................................................................................................................................ 1157
2,4′-DDT ............................................................................................................................................................................................................. 1795,1891
Demethoxycurcumin ................................................................................................................................................................................................... 1331
4,4′-DDT ....... 1351,1403,1862,2000,1382,1396,1439,1925,1404,2001,1466,1740,1858,1989,2356,2384,2392,2406,1469,1994,2359,2409,
Demeton-O ...................................................................................................................................................................................... 1542,1543,1544,1545
1506,2075,1507,2076,1512,1513,1514,1516,1524,1532,1571,1591,2325,2347,1604,1795,1891
Demeton S ......................................................................................................................................................................................................... 1542,1543
10-DeacetyIpaclitaxel .................................................................................................................................................................................................. 1153
Demeton-ethyl ................................................................................................................................................................................ 1350,1401,1860,1995
10-Deacetylbaccatin III ............................................................................................................................................................................................... 1154
Demeton-s-methyl ......................................................................................................................................................................... 1444,1678,1825,1930
10-Deacetyl-7-epipaclitaxel .............................................................................................................................................................................. 1153,1154
Demeton-S-methyl ............................................................................................................................................................................................ 1887,2066
10-deacetyl-7-epipaclitaxel ....................................................................................................................................................................................... 1220
Demeton-s-methylsulfon ............................................................................................................................................................... 1444,1678,1825,1930
10-Deacetylpaclitaxel ........................................................................................................................................................................................ 1104,1154
Demoxepam .................................................................................................................................................................................................. 1190,232,916
10-deacetyl paclitaxel ................................................................................................................................................................................................ 1220
Indexes Analyte
2′-Deoxyadenosine monohydrate ........................................................................................................................................................................ 572,688 Diacetal ........................................................................................................................................................................................................................ 1969

5′-Deoxyadenosine ........................................................................................................................................................................................................ 689 Diacetone alcohol ....................................................................................................................................................................................................... 1725
Deoxycholic acid .................................................................................................................................................................................................... 141,234 Dialifor ............................................................................................................................................................................................. 1444,1678,1825,1930
Deoxycholic acid-2,2,4,4-d4 ......................................................................................................................................................................................... 234 Dialifos ................................................................................................................................................................................................................ 1890,2069
11-Deoxycorticosterone solution ........................................................................................................................................................................ 1374,257 Diallate-1 ..................................................................................................................................................................................................................... 1550
2′-Deoxycytidine ........................................................................................................................................................................................................... 572 Diallate-2 ..................................................................................................................................................................................................................... 1550
2′-Deoxycytidine hydrate .............................................................................................................................................................................. 687,688,689 Diallate isomer ............................................................................................................................................................................................................ 1551
Deoxydenosine ............................................................................................................................................................................................................... 687 1,4-Diaminobutane ....................................................................................................................................................................................................... 143
(±)-Deoxyephedrine hydrochloride .................................................................................................................................................................... 427,458 2,6-Diamino-4-chloropyrimidine (I.S.) .................................................................................................................................................. 2078,2079,2080
2′-Deoxyguanosine monohydrate ................................................................................................................................................................ 572,687,688 2,6-Diamino-4-chloropyrimidine (I.S) ...................................................................................................................................................................... 2081
3′-Deoxyguanosine ....................................................................................................................................................................................................... 689 4,4′-Diaminodiphenylmethane .................................................................................................................................................................................. 1673
Deoxynivalenol ..................................................................................................................................................................................... 12,358,17,394,331 2,4-Diaminotoluene .................................................................................................................................................................................................... 1655
Deoxyribonucleosides .................................................................................................................................................................................................... 572 Diamyl phthalate ........................................................................................................................................................................... 1478,1769,2009,2192
12-Deoxywithastramonolide ....................................................................................................................................... 1235,1310,1311,1312,1313,1314 o-Dianisidine ................................................................................................................................................................................................................ 1673
S-(+)-Deprenyl .............................................................................................................................................................................. 2215,2256,2624,2852 Diastereomeric compounds, proprietary ................................................................................................................................................................ 1338
R-(−)-Deprenyl hydrochloride .................................................................................................................................................................................... 905 Diazepam ............................................................................................................................ 128,887,135,935,136,936,137,937,138,938,1190,232,
R-(-)-Deprenyl (Selegiline) ........................................................................................................................................................... 2215,2256,2624,2852 1207,273,489,842,875,876,888,889,916,2288,2289,2290,2462,2291,2463
Dermorphin .................................................................................................................................................................................................................... 425 Diazepam solution ....................................................................................................................................................................................... 490,833,1228
N-Desethylamiodarone hydrochloride solution ............................................................................................... 1370,229,230,1378,266,267,1379,268 Diazepam-d5 solution ................................................................................................................................................................................................... 833
Desethyl atrazine .......................................................... 1466,1740,1858,1989,2356,2384,2392,2406,1489,1788,1877,2039,2369,2391,2397,2425 Diazinon ............................................... 1419,406,1347,1394,1798,1894,1350,1401,1860,1995,1430,1641,1796,1892,1447,1681,1828,1933,
Desipramine .................................................................................................................................. 1164,210,1166,211,1167,212,1165,1169,1170,2289 1466,1740,1858,1989,2356,2384,2392,2406,1471,1746,1861,1997,2361,2386,2393,2411,1506,2075,1507,2076,1542,1543,1544,1545,
Desipramine hydrochloride .............................................................................................................................................................................. 1206,2288 1585,2342,1797,1893,1885,2064,2004,2413
Desisopropyl atrazine ................................................... 1466,1740,1858,1989,2356,2384,2392,2406,1489,1788,1877,2039,2369,2391,2397,2425 Dibenz[a,h]acridine .................................................................................................................................................................................. 1540,1541,1550
Desmediphan ..................................................................................................................................................................................................... 1888,2067 Dibenz[a,j]acridine ................................................................................................................................................................................... 1540,1541,1551
32-Desmethoxyrapamycin ............................................................................................................................................................................................ 188 Dibenz[a,h]anthracene ........................................................................................................................................................................................ 2375,310
N-Desmethylchlordiazepoxide ...................................................................................................................................................................................... 916 Dibenz[a,h]anthracene .................................................................................................................................................................................................. 311
Desmethyldiazepam .......................................................................................................................... 136,936,137,937,138,938,1207,273,489,875,876 Dibenz[a,h]anthracene ............................................................................................................................. 1387,1410,1479,2012,1388,1411,1482,2028
N-Desmethyldiazepam ........................................................................................................................................................................... 135,935,915,916 Dibenz[a,h]anthracene ...................................................................................................................................................................................... 1408,2010
Desmethyldiltiazem ....................................................................................................................................................................................................... 896 Dibenz[a,h]anthracene .......................................................................................................... 1409,2011,1483,2029,2365,2420,1506,2075,1507,2076
Desmethyldoxepin ............................................................................................................................................................................ 1167,212,1169,1170 Dibenz[a,h]anthracene ............................................................................................................................................................................................... 1517
N-Desmethylnefopam (nefopam metabolite) .................................................................................................................................................. 2898,976 Dibenz[a,h]anthracene .......................................................................................................... 1524,1552,1553,1554,1555,1556,1557,1564,1795,1891
N-Desmethyltamoxifen ............................................................................................................................................................................................... 1152 Dibenzo(a,h)anthracene .................................................................................................................................................................................... 1434,1435
Desmethyl venlafaxine ......................................................................................................................................................................... 132,907,1189,231 Dibenzo[a,h]anthracene .................................................................................................................................................................................... 1451,1936
Devrinol ................................................................................................................................................................................................................... 10,2483 Dibenzo[a,h]anthracene .............................................................................................................................................. 1480,2013,1481,2014,1518,1540
Dexamethasone ............................................................................................................................................................................................................. 169 Dibenzo[a,h]anthracene ............................................................................................................................................................................................. 1541
Dexamisole ...................................................................................................................................................................................................... 455,456,454 Dibenzo(a,h)anthracene ............................................................................................................................................................................................. 1558
Dextromethorphan ........................................................................................................................................................................................................ 971 Dibenzo[a,h]anthracene .............................................................................................................................................. 1560,1561,1562,1563,1567,1587
Dextromethorphan hydrobromide ..................................................................................................................................................................... 2899,977 7H-Dibenzo[c,g]carbazole .......................................................................................................................................................................................... 1540
Dextrose ......................................................................................................................................................................................................................... 387 7H-Dibenzo[c,g]carbazole .......................................................................................................................................................................................... 1541
Indexes Analyte
Dibenzofuran ....................................................................................................... 1484,2030,2366,2421,1552,1553,1554,1555,1556,1557,1558,1564 1,2-Dichlorobenzene ............................................................... 1494,2058,2371,2441,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,

Dibenzo(a,e)pyrene ..................................................................................................................................................................................................... 1540 1505,2074,1519,1520,1522,1523,1524,1546,1547,1548,1549,1552,1553,1554,1555,1556,1557,1558,1564,1565,1566,1568,1588,2322,2344,
Dibenzo[a,e]pyrene .................................................................................................................................................................................. 1541,1795,1891 1569,1589,2323,2345,1725,1726,1727
Dibenzo[a,h]pyrene ........................................................................................................................................................................ 1540,1541,1795,1891 1,2-Dichlorobenzene-d4 ..................................................................................................................................... 1498,2062,2373,2444,1504,2073,
Dibenzo[a,i]pyrene ......................................................................................................................................................................... 1540,1541,1795,1891 1505,2074,1524,1550,1551,1552,1553,1554,1555,1556,1557
Dibenzo[a,l]pyrene ............................................................................................................................................................................................ 1795,1891 1,3-Dichlorobenzene ............................................................... 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,
Dibenzothiophene ........................................................................................................................................................................... 1408,2010,1409,2011 1504,2073,1505,2074,1519,1520,1522,1524,1546,1547,1548,1549,1552,1553,1554,1555,1556,1557,1558,1564,1565,1566,
N,N-Dibenzyl-DL-tartaric diamide ......................................................................................................................................................................... 2578,51 1568,1588,2322,2344,1569,1589,2323,2345
1,5-di-epi-β-Bourbonene .................................................................................................................................................................................. 2138,2167 1,4-Dichlorobenzene ............................................................... 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,
Dibrom® .......................................................................................................................................................................... 1419,406,1544,1545,1887,2066 1504,2073,1505,2074,1519,1520,1522,1524,1546,1547,1548,1549,1552,1553,1554,1555,1556,1557,1558,1564,1565,1566,
Dibromfos ........................................................................................................................................................................................ 1350,1401,1860,1995 1568,1588,2322,2344,1569,1589,2323,2345,1676,1794
Dibromochloromethane ............................. 1494,2058,2371,2441,1497,2061,1504,2073,1519,1520,1522,1546,1547,1548,1549,1565,1566, 1,4-Dichlorobenzene-d4 .................................................................................................................................... 1493,2057,2370,2440,1519,1520,1524
1568,1588,2322,2344 1,4-Dichlorobenzene-d4 ........................................................................................................................................................................................... 1546
Dibromochloropropane ............................................................................................................................................................................ 1509,1510,1511 1,4-Dichlorobenzene-d4 ........................................................................................................................................................................................... 1547
1,2-Dibromo-3-chloropropane ........................................................................................................ 1497,2061,1498,2062,2373,2444,1502,2071, 1,4-Dichlorobenzene-d4 ........................................................................... 1548,1549,1550,1551,1552,1553,1554,1555,1556,1557,1564,1567,1587
1503,2072,1504,2073,1505,2074,1546,1547,1548,1549,1565,1566 1,4-dichlorobenzene-d4 (I.S.) ................................................................................................................................................................................... 1558
1,2-Dibromoethane ....................................................................................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072, 1,2-dichlorobenzene-d4 (surr.) ................................................................................................................................................................................. 1558
1505,2074,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345 3,3′-Dichlorobenzidine ....................................................................................... 1524,1552,1553,1554,1555,1556,1557,1558,1564,1655,1672,1673
Dibromofluoromethane ............................................................................................................................................... 1519,1520,1546,1547,1548,1549 2,6-Dichlorobenzonitrile .................................................................................................................................................................................... 1887,2066
Dibromomethane ....................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549 p-Dichlorobenzophenone ............................................................................................................................................ 1347,1394,1798,1894,1797,1893
1,2-Dibromomethane ..................................................................................................................................................................... 1504,2073,1546,1547 2,3-Dichlorobiphenyl ................................................................................................................................ 1388,1411,1482,2028,1506,2075,1507,2076
3,5-Dibromo-p-tyrosine ...................................................................................................................................................................................... 2770,573 2,6-Dichlorobiphenyl ...................................................................................................................................................................... 1441,1927,1442,1928
2,6-Di-tert-butyl-4-hydroxymethylphenol ........................................................................................................................................................ 1282,292 2,6-dichlorobiphenyl ......................................................................................................................................................................................... 1443,1929
3,5-Di-tert-butyl-4-hydroxytoluene ................................................................................................................................................................... 1282,292 3,3′-Dichlorobiphenyl ................................................................................................................................................................................................. 1501
n-Dibutylnitrosoamine .................................................................................................................................................................... 1484,2030,2366,2421 3,4′-Dichlorobiphenyl ................................................................................................................................................................................................. 1501
Dibutyl phthalate ............................................................................................... 1478,1769,2009,2192,1507,2076,1556,1865,2008,2394,2416,2191 3,4-Dichlorobiphenyl .................................................................................................................................................................................................. 1501
Di-n-butyl phthalate ....................................................................................................................... 1388,1411,1482,2028,1483,2029,2365,2420, 3,5-Dichlorobiphenyl .................................................................................................................................................................................................. 1501
1506,2075,1524,1552,1553,1554,1555,1557,1558,1564 4,4'-Dichlorobiphenyl ..................................................................................................................................................................... 1441,1927,1442,1928
Dicaffeic acid ............................................................................................................................................................................................................... 1275 4,4’-dichlorobiphenyl ........................................................................................................................................................................................ 1443,1929
Dicaffeoylquinic acid (1,5-di-CQA) ........................................................................................................................................................................... 1275 4,4′-Dichlorobiphenyl ................................................................................................................................................................................................. 1501
Dicaffeoylquinic acid (3,4-di-CQA) ........................................................................................................................................................................... 1275 1,1-Dichloro-2,2-bis(4-chlorophenyl)ethane ............................................................................................................ 1404,2001,1507,2076,1512,1514
Dicaffeoylquinic acid (3,5-di-CQA) ........................................................................................................................................................................... 1275 1,1-Dichloro-2,2-bis(4-chlorophenyl)ethene ........................................... 1382,1396,1439,1925,1466,1740,1858,1989,2356,2384,2392,2406,
Dicaffeoylquinic acid (4,5-di-CQA) ........................................................................................................................................................................... 1275 1469,1994,2359,2409,1506,2075,1513,1516,1524,1532,1571,1591,2325,2347,1604
Dicamba ................................................................................................................................................................................................... 1355,1389,2,298 1,4-Dichlorobutane ......................................................................................................................................................................... 1494,2058,2371,2441
Dichlofenthion .............................................................................................................................................................. 1350,1401,1860,1995,1885,2064 cis-1,4-Dichloro-2-butene .............................................................................................................................................................. 1546,1547,1548,1549
Dichlofluanid .................................................................................... 1347,1394,1798,1894,1449,1683,1830,1935,1797,1893,1889,2068,2004,2413 trans-1,4-Dichloro-2-butene ...................................................................................................................................... 1504,2073,1546,1547,1548,1549
Dichlofluanid metabolite .................................................................................................................................................................................. 1889,2068 Dichlorodifluoromethane .......................................................................... 1496,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1505,2074
3,4-Dichloroaniline ............................................................................................................................................................................................ 1655,1672 1,1-Dichloroethane .................................................................. 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,
2,6-Dichlorobenzamide ..................................................................................................................................................................................... 1890,2069 1504,2073,1505,2074,1519,1520,1521,1522,1523,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1674,1676,
1725,1726,1727
Indexes Analyte
1,2-Dichloroethane ................................................................................................................................................................. 1494,2058,2371,2441, trans-1,3-Dichloropropene ................................................................................................................................. 1494,2058,2371,2441,1497,2061,

1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566, 1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,
1568,1588,2322,2344,1569,1589,2323,2345,1674,1676,1725,1726,1727,2200,2201,2202,2448,2203,2204,2449,2224 1568,1588,2322,2344,1569,1589,2323,2345
1,2-Dichloroethane-d4 ............................................................................................................................................................................. 1519,1520,1546 1,2-Dichlorotetrafluoroethane ....................................................................................................................................................... 1568,1588,2322,2344
1,2-Dichloroethane-d4 .............................................................................................................................................................................................. 1547 Dichlorvos .................................................................................................................................. 308,1347,1394,1798,1894,1350,1401,1860,1995,
1,2-Dichloroethane-d4 ................................................................................................................................................................... 1548,1549,1565,1566 1506,2075,1507,2076,1542,1543,1544,1545,1585,2342,1797,1893,1885,2064
1,1-Dichloroethene ................................................................................................................................... 1504,2073,1546,1547,2202,2448,2204,2449 2,4-Dichorophenol ...................................................................................................................................................................................................... 1524
cis-1,2-Dichloroethene .......................................................................................................... 1497,2061,1504,2073,1546,1547,1569,1589,2323,2345 Diclobutrazole .................................................................................................................................................................................................... 1886,2065
trans-1,2-Dichloroethene ................................................................................................................................... 1494,2058,2371,2441,1497,2061,1522 Diclocymet-1 ...................................................................................................................................................................................................... 1889,2068
trans-1,3-Dichloroethene .................................................................................................................................................................................. 1504,2073 Diclocymet-2 ...................................................................................................................................................................................................... 1889,2068
1,1-Dichloroethylene ............................................ 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1505,2074, Diclofenac ................................................................................................................................................................................................. 851,899,978,979
1519,1520,1521,1522,1523,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1674,1676,1725,1726,1727,2200,2201,2203, Dicloran ............................................................................................................................................................... 1509,1510,1511,1886,2065,2004,2413
2224 Dicofol .................................................................................................................. 1347,1394,1798,1894,1448,1682,1829,1934,1797,1893,2004,2413
cis-1,2-Dichloroethylene ........................... 1493,2057,2370,2440,1498,2062,2373,2444,1502,2071,1503,2072,1505,2074,1548,1549,1565,1566 Dicrotophos ............................................................................................................................ 1350,1401,1860,1995,1542,1543,1544,1545,1887,2066
cis-1,2-dichloroethylene ................................................................................................................................................................. 1568,1588,2322,2344 Dicyclohexyl phthalate ................................................................................................................... 1478,1769,2009,2192,1865,2008,2394,2416,2191
cis-1,2-Dichloroethylene ............................................................................................................................................................................................. 2205 Didymin .......................................................................................................................................................................................................................... 354
cis-1,2-Dichloroethylene ............................................................................................................................................. 2206,2207,2450,2208,2209,2451 Dieldrin .................................................................. 1347,1394,1798,1894,1382,1396,1439,1925,1466,1740,1858,1989,2356,2384,2392,2406,
trans-1,2-Dichloroethylene ................................................................................................................................. 1498,2062,2373,2444,1502,2071, 1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1524,1532,1571,1591,2325,2347,1604
1503,2072,1505,2074,1519,1520,1523,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1674,1676,1725,1726,1727 Diethofencarb ..................................................................................................................................................................................................... 1888,2067
Dichlorofenthion ............................................................................................................................................................................. 1542,1543,1544,1545 Diethylamine ................................................................................................................................................................ 1648,2379,1649,2380,2225,2455
Dichlorofluoromethane .......................................................................................................... 1504,2073,1546,1547,1548,1549,1565,1566,1697,1698 N,N-Diethylaniline ........................................................................................................................................................................................................... 32
2,6-Dichloroindophenol sodium salt hydrate ................................................................................................................................................ 1888,2067 2,6-Diethylaniline .............................................................................................................................................................................................. 1655,1672
Dichloromethane ..................................................................................................................... 1491,1581,2319,2331,1519,1520,1546,1547,1548, 1,3-Diethylbenzene ........................................................................................................................................................................................... 1610,1612
1549,1565,1566,1568,1588,2322,2344,1674,1676,1726,1727,2205,2207,2450,2209,2451 1,4-Diethylbenzene ..................................................................................................................................................................................................... 1612
2,6-Dichloro-4-nitroaniline ............................................................................................................................................................ 1449,1683,1830,1935 Diethylcyanamide .............................................................................................................................................................................................. 1582,2332
2,4-Dichlorophenol ... 90,1359,1386,1407,1475,1755,2005,1476,1756,2006,2363,2388,2414,1477,1757,2007,2364,2389,2415,1531,1552, Diethyl disulfide .......................................................................................................................................................................................................... 1685
1553,1554,1555,1556,1557,1558,1564 Diethylene glycol .................................................................................................................................................................. 1383,1457,1705,1977,2187
2,6-Dichlorophenol ................................................................ 1476,1756,2006,2363,2388,2414,1477,1757,2007,2364,2389,2415,1531,1550,1551 Diethyl ether ................................................................................................................ 1504,2073,1525,2210,2211,2212,2452,2213,2214,2453,2224
[3-(3,5-Dichlorophenyl)-2,4-dioxoimidazolidinyl]-N-(methylethyl)carboxamide ....................................................................................... 1889,2068 N,N-Diethylformamide ...................................................................................................................................................................................... 1582,2332
1,1-Dichloropropane ....................................................................................................................................................................... 1502,2071,1503,2072 Di(2-ethylhexyl) adipate ............................................................................................................................................................... 1388,1411,1482,2028
1,2-Dichloropropane ............................................................... 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072, Di(2-ethylhexyl) phthalate ............................................................................................................................................................ 1388,1411,1482,2028
1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345 3,5-Diethyl-2-methylpyrazine ....................................................................................................................................................... 1871,2022,2395,2417
1,3-Dichloropropane ........................................................................................................................................... 1497,2061,1498,2062,2373,2444, N,N-Diethyl-2-(1-naphthyloxy)propanamide ................................................................................................................................................. 1507,2076
1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549 O,O-Diethyl O-(4-nitrophenyl) phosphate .................................................................................................................................. 1350,1401,1860,1995
2,2-Dichloropropane .................................. 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549 Diethyl phthalate ............................................................................................................................. 1388,1411,1482,2028,1478,1769,2009,2192,
1,1-Dichloropropene ........................................................................................... 1497,2061,1498,2062,2373,2444,1504,2073,1505,2074,1546,1547 1483,2029,2365,2420,1506,2075,1507,2076,1524,1552,1553,1554,1555,1556,1557,1558,1564,1865,2008,2394,2416,2191
1,1-Dichloro-1-propene .................................................................................................................................................................................... 1548,1549 2,3-Diethylpyrazine ........................................................................................................................................................................ 1871,2022,2395,2417
cis-1,3-Dichloropropene .......................................................... 1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072, 2,6-Diethylpyrazine ........................................................................................................................................................................ 1871,2022,2395,2417
1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345 Diethyl succinate ......................................................................................................................................................................................................... 1895
Indexes Analyte
Diethyl sulfide ............................................................................................................................................................................................................. 1685 Dimethoate ..................... 1350,1401,1860,1995,1351,1403,1862,2000,1466,1740,1858,1989,2356,2384,2392,2406,1470,1996,2360,2410,

2,5-Diethyltetrahydrofuran ...................................................................................................................................................................... 2137,2138,2167 1471,1746,1861,1997,2361,2386,2393,2411,1542,1543,1544,1545,1550,1551,1885,2064
Difenoconazole-1 ............................................................................................................................................................................................... 1886,2065 Dimethomorph-1 ............................................................................................................................................................................................... 1890,2069
Difenoconazole-2 ............................................................................................................................................................................................... 1886,2065 Dimethomorph-2 ............................................................................................................................................................................................... 1890,2069
Diflubenzuron ....................................................................................................................................................................................................... 1419,406 Di-6,6'-Methoxy-bi(2-naphthol) .......................................................................................................................................................................... 2580,53
Diflunisal ........................................................................................................................................................................................................................ 899 (S)-(6,6′-Dimethoxybiphenyl-2,2′-diyl)bis[bis(3,5-di-tert-butylphenyl)phosphine] .................................................................................... 106,2612
Difluorobenzene .......................................................................................................................................................................................................... 1523 (±)-2,5-Dimethoxy-4-bromoamphetamine hydrochloride solution ..................................................................................................... 228,1424,457
1,4-Difluorobenzene .......................................................................................................................................................................................... 1565,1566 1,2-Dimethoxyethane ........................................................................................................................................ 2205,2206,2207,2450,2208,2209,2451
Digitonenin .................................................................................................................................................................................................................. 1289 (±)-2,5-Dimethoxy-4-Iodoamphetamine hydrochloride solution ......................................................................................................... 228,1424,457
Digitoxin ......................................................................................................................................................................................................................... 263 2,5-Dimethoxy-4-(n)-propylthiophenethylamine hydrochloride solution ............................................................................................. 1424,457,483
Digoxin .......................................................................................................................................................................................................... 256,263,1289 2,5-Dimethoxytetrahydrofuran ...................................................................................................................................................... 1692,2509,1693,2510
Dihexyl phthalate ................................................................................................................................................................. 1865,2008,2394,2416,2191 2,5-Dimethoxytetrahydrofuran, meso-isomer ................................................................................................................................................ 1693,2510
Di-n-Hexyl phthalate ..................................................................................................................................................................... 1478,1769,2009,2192 Dimethylacetamide ............................................................................................................................................ 2205,2206,2207,2450,2208,2209,2451
1,6-Dihydrocarveol ...................................................................................................................................................................................................... 2167 Dimethyl adipate ........................................................................................................................................................................................................ 1691
Dihydrocarveyl acetate ............................................................................................................................................................................................... 2167 Dimethylamine ............................................................................................................................................................. 1648,2379,1649,2380,2225,2455
(Z)-Dihydrocarvone ..................................................................................................................................................................................................... 2167 Dimethylaminoazobenzene ........................................................................................................................................................................................ 1673
(E)-Dihydrocarvone ..................................................................................................................................................................................................... 2167 4-Dimethylaminoazobenzene ........................................................................................................................................................................... 1550,1551
2,3-Dihydrofarnesol ..................................................................................................................................................................................................... 2131 N,N-Dimethylaniline .............................................................................................................................................................................. 32,842,1655,1672
2,3-Dihydro-5-methylfuran .............................................................................................................................................................................. 2056,2439 2,4-Dimethylaniline .................................................................................................................................................................................................... 1673
5α-Dihydrotestosterone (DHT) solution ..................................................................................................................................................................... 258 2,6-Dimethylaniline ........................................................................................................................ 1655,1672,1673,2311,2312,2313,2314,2316,2317
5a-Dihydrotestosterone glucuronide ........................................................................................................................................................................... 258 Dimethyl azelate ......................................................................................................................................................................................................... 1691
3,4-Dihydroxybenzylamine ........................................................................................................................................................................................... 955 2,5-Dimethylbenzaldehyde ................................................................................................................................................................................... 21,2326
4,4′-Dihydroxybiphenyl ................................................................................................................................................................................................ 492 7,12-Dimethylbenz[a]anthracen-trans-8,9-diol .......................................................................................................................................... 175,304,438
3,4-Dihydroxy-DL-phenylalanine ........................................................................................................................................................ 2900,980,2901,981 7,12-Dimethylbenz[a]anthracene .............................................................................................................................................. 175,304,438,1550,1551
3,4-Dihydroxy-phenylacetic acid ............................................................................................................................................................................... 1275 7,12-Dimethylbenz[a]anthracene-trans-10,11-diol .................................................................................................................................... 175,304,438
3,4-Dihydroxy-D-phenylalanine ......................................................................................................................................................... 162,2634,163,2635 7,12-Dimethylbenz[a]anthracen-2-ol .......................................................................................................................................................... 175,304,438
3,4-Dihydroxy-L-phenylalanine .................................................................................................................................................. 145,162,2634,163,2635 7,12-Dimethylbenz[a]anthracen-3-ol .......................................................................................................................................................... 175,304,438
3,4-Dihydroxyphenylglycol ........................................................................................................................................................................................... 180 7,12-Dimethylbenz[a]anthracen-4-ol .......................................................................................................................................................... 175,304,438
2,3-Dihydroxy-3-phenylpropionic acid ............................................................................................................................................... 2579,2902,52,982 3,3′-Dimethylbenzidine ........................................................................................................................................................ 1552,1553,1554,1555,1557
1α,25-Dihydroxyvitamin D2 solution ............................................................................................................................................... 1323,260,1324,261 7,8-Dimethylbenzo[g]pteridine-2,4(1H,3H)-dione, Compound A. ................................................................................................................ 1219,274
1α,25-Dihydroxyvitamin D3 solution ............................................................................................................................................... 1323,260,1324,261 1,1-Dimethylbiguanide hydrochloride ........................................................................................................................................................................ 398
Diisobutyl phthalate ................................................................................................................................. 1478,1769,2009,2192,1865,2008,2394,2416 Dimethyl-bisphenol A ........................................................................................................................................................................................... 299,341
Diisopropyl phthalate ..................................................................................................................................................................... 1478,1769,2009,2192 2,2-Dimethylbutane ................................................................................................................................................................................. 1592,1610,1612
Diltiazem ........................................................................................................................................................................................................................ 896 2,3-Dimethylbutane ....................................................................................................................................................................... 1592,1610,1611,1612
Dimepiperate ...................................................................................................................................................................................................... 1888,2067 Dimethylcyclohexanol ........................................................................................................................................................................................ 2045,2350
Dimethametryn .................................................................................................................................................................................................. 1888,2067 1,1-Dimethylcyclopentane .......................................................................................................................................................................................... 1592
Dimethenamid ................................................................................................................................................................................................... 1888,2067 trans-1,2-Dimethylcyclopentane ................................................................................................................................................................................ 1592
Dimethipin .......................................................................................................................................................................................................... 1886,2065 cis-1,3-Dimethylcyclopentane .................................................................................................................................................................................... 1592
Dimethoat ........................................................................................................................................................................................ 1448,1682,1829,1934 trans-1,3-Dimethylcyclopentane ................................................................................................................................................................................ 1592
Indexes Analyte
2,2-Dimethyl-3-decene .................................................................................................................................................................. 1491,1581,2319,2331 3,7-Dimethyl-2,6-octadienyl acetate ............................................................................................................... 2050,2434,2105,2106,2113,2120,2170

Dimethyl disulfide ....................................................................................................... 1685,1686,2381,1687,2382,1871,2022,2395,2417,2051,2435 2,2-Dimethylpentane .................................................................................................................................................................................................. 1592
1,2-Dimethyl-4-ethylbenzene .......................................................................................................................................................................... 1610,1612 2,3-Dimethylpentane ............................................................................................................................................................................... 1592,1610,1612
1,3-Dimethyl-4-ethylbenzene .......................................................................................................................................................................... 1610,1612 2,4-Dimethylpentane .................................................................................................................................................. 1431,1572,1592,1610,1611,1612
1,3-Dimethyl-5-ethylbenzene .................................................................................................................................................................................... 1612 3,3-Dimethylpentane .................................................................................................................................................................................................. 1592
1,4-Dimethyl-2-ethylbenzene .......................................................................................................................................................................... 1610,1612 α,α-Dimethylphenethylamine ........................................................................................................................................................................... 1550,1551
Dimethylformaldehyde ...................................................................................................................................................................................... 2056,2439 2,4-Dimethylphenol .............................................................................. 90,1359,1386,1407,1475,1755,2005,1476,1756,2006,2363,2388,2414,
Dimethylformamide ............................................................................................ 1676,1725,1726,1727,2205,2206,2207,2450,2208,2209,2451,2224 1477,1757,2007,2364,2389,2415,1524,1531,1552,1553,1554,1555,1556,1557,1558,1564
2,5-Dimethylfuran ............................................................................................................................................................................................. 2056,2439 2,6-Dimethylphenol .................................................................................................................................................... 2311,2312,2313,2314,2316,2317
Dimethyl glutarate ..................................................................................................................................................................................................... 1691 5,5-Dimethyl-4-phenyl-2-oxazolidinone ............................................................................................................................................................. 2581,54
2,2-Dimethylheptane ........................................................................................................................................................................................ 1592,1612 Dimethyl phthalate ............................................................................ 492,1388,1411,1482,2028,1478,1769,2009,2192,1483,2029,2365,2420,
2,3-Dimethylheptane .................................................................................................................................................................................................. 1592 1506,2075,1507,2076,1524,1552,1553,1554,1555,1556,1557,1558,1564,1691,1865,2008,2394,2416,2191
2,4-Dimethylheptane .................................................................................................................................................................................................. 1592 Dimethyl pimelate ...................................................................................................................................................................................................... 1691
2,5-Dimethylheptane .................................................................................................................................................................................................. 1592 2,3-Dimethylpyrazine .................................................................................................................................................. 1871,2022,2395,2417,2056,2439
3,4-Dimethylheptane .................................................................................................................................................................................................. 1592 2,5-Dimethylpyrrole .......................................................................................................................................................................................... 2056,2439
3,5-Dimethylheptane .................................................................................................................................................................................................. 1592 2,5-Dimethylresorcinol ............................................................................................................................................................................................ 152,44
2,4-Dimethylheptanen ................................................................................................................................................................................................ 1612 Dimethyl sebacate ...................................................................................................................................................................................................... 1691
2,6-Dimethylheptanen ................................................................................................................................................................................................ 1612 Dimethyl styrene ............................................................................................................................................................................ 1490,1580,2318,2330
2,2-Dimethylhexane .................................................................................................................................................................................................... 1592 Dimethyl suberate ...................................................................................................................................................................................................... 1691
2,3-Dimethylhexane ....................................................................................................................................................................... 1592,1610,1611,1612 Dimethyl succinate ..................................................................................................................................................................................................... 1691
2,4-Dimethylhexane ....................................................................................................................................................................... 1592,1610,1611,1612 Dimethyl sulfide ....................................................................................................................................................................................... 1685,1686,2381
2,5-Dimethylhexane ....................................................................................................................................................................... 1592,1610,1611,1612 Dimethyl sulfide (1 ppm) ......................................................................................................................................................................................... 1783
3,3-Dimethylhexane .................................................................................................................................................................................................... 1592 Dimethyl sulfoxide .......................................................................................................................... 2210,2211,2212,2452,2213,2214,2453,2225,2455
3,4-Dimethylhexane .......................................................................................................................................................................................... 1592,1612 Dimethyl terepthalate ................................................................................................................................................................................................ 1691
3,4-Dimethyl-1-hexene .............................................................................................................................................................................................. 1612 6,7-Dimethyl-8-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]pteridine-2,4(3H,8H)-dione, Compound C ........................................................ 1219,274
3,4-Dimethyl-trans-2-hexene .................................................................................................................................................................................... 1612 2,3-Dimethyltyrosine ........................................................................................................................................................................................... 2771,574
3,4-Dimethyl-1-hexenen ............................................................................................................................................................................................ 1612 1,3-Dimethyluric acid ................................................................................................................................................................................................... 150
Dimethyl isophthalate ................................................................................................................................................................................................ 1691 1,7-Dimethyluric acid ................................................................................................................................................................................................... 149
Dimethyl malonate ..................................................................................................................................................................................................... 1691 Dimethylvinphos-E ............................................................................................................................................................................................ 1885,2064
3,4-Dimethylmethcathinone hydrochloride solution ................................................................................................................................................ 471 Dimethylvinphos-Z ............................................................................................................................................................................................ 1885,2064
2,6-Dimethylnaphthalene .............................................................................................................................................................. 1408,2010,1409,2011 1,7-Dimethylxanthine ......................................................................................................................................... 1362,1427,148,946,149,150,1315,802
Dimethylnaphthalenes ...................................................................................................................................................................................... 1610,1611 Dimetridazole ................................................................................................................................................................................................................. 906
4,4-Dimethyl-2-neopentyl-1-pentene ...................................................................................................................................................................... 1721 Diniconazole ....................................................................................................................................................................................................... 1890,2069
1,3-Dimethyl-2-nitrobenzene ........................................................................................................................................................ 1506,2075,1507,2076 1,3-Dinitrobenzene ............................................................................................................................... 296,465,1455,1695,2297,2354,2383,2466,
4,8-Dimethyl-1,3-(E),7-nonatriene ........................................................................................................................................................................... 2096 1456,1696,2298,1550,1551
4,8-Dimethyl-(3E)-1,3,7-nonatriene ......................................................................................................................................................................... 2131 1,4-Dinitrobenzene ............................................................................................................................................................................................ 1550,1551
cis-3,7-Dimethyl-2,6-octadien-1-ol ........................................................................................................................... 2096,2099,2103,2111,2184,2120 2,4-Dinitrophenol .............. 90,1476,1756,2006,2363,2388,2414,1477,1757,2007,2364,2389,2415,1524,1531,1552,1553,1554,1555,1556,1557
Indexes Analyte
2,4-dinitrophenol ........................................................................................................................................................................................................ 1558 Disulfoton ...................................................... 308,1350,1401,1860,1995,1466,1740,1858,1989,2356,2384,2392,2406,1470,1996,2360,2410,

2,4-Dinitrophenol ........................................................................................................................................................................................................ 1564 1471,1746,1861,1997,2361,2386,2393,2411,1506,2075,1507,2076,1542,1543,1544,1545,1550,1551,1887,2066
2,4-Dinitrotoluene .................................................................................................... 296,465,1455,1695,2297,2354,2383,2466,1456,1696,2298, Ditalimfos ........................................................................................................................................................................................................... 1890,2069
1506,2075,1507,2076,1524,1552,1553,1554,1555,1556,1557,1558,1564 Ditalimphos ..................................................................................................................................................................................... 1445,1679,1826,1931
2,6-Dinitrotoluene ........................................................................................................... 297,1455,1695,2297,2354,2383,2466,1456,1696,2298, Diterpenes .......................................................................................................................................................................................................... 1884,2063
1506,2075,1507,2076,1524,1552,1553,1554,1555,1556,1557,1558,1564 Dithiopyr ....................................................................................................................................................................... 1430,1641,1796,1892,1886,2065
Dinonyl phthalate ................................................................................................................................................................. 1865,2008,2394,2416,2191 Diuretics ......................................................................................................................................................................................................................... 985
Di-n-Nonyl phthalate ..................................................................................................................................................................... 1478,1769,2009,2192 Diuron ....................................................................................................................................... 1,295,5,110,15,111,16,294,302,1391,305,314,375,317
Dinoseb ........................................................................................................................ 1355,1389,2,298,1476,1756,2006,2363,2388,2414,1550,1551 DMB-NANA ............................................................................................................................................................................................................ 575,987
Dinotefuran ............................................................................................................................................................................................................. 1358,19 DMB-NGNA ............................................................................................................................................................................................................. 575,987
Dioctyl phthalate ............................................................................................................................................................................ 1865,2008,2394,2416 DMT-2′O-TC-rG(ib) Phosphoramidite ......................................................................................................................................................................... 686
Di-n-octyl phthalate ................................................................................................................................................................................................... 1434 DMT-2′O-TC-rU Phosphoramidite ............................................................................................................................................................................... 686
Di-n-octyl phthalate ................................................................................................................................................................................................... 1435 cis-13,16-Docosadienoic acid methyl ester (C22:2) ................................................................................................................... 1815,1915,2247,
Di-n-Octyl phthalate ...................................................................................................................................................................... 1478,1769,2009,2192 1817,1917,2249,1818,1918,2250,1819,1919,2251
Di-n-octyl phthalate ....................................................................................................................... 1483,2029,2365,2420,1524,1552,1553,1554,1555 cis-13,16-Docosadienoic acid methyl ester (C22:2) at 2 wt % ..................................................................................................... 1822,1922,2254
Di-n-octyl phthalate ................................................................................................................................................................................................... 1556 cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester ...................... 1814,1914,2246,1816,1916,2248,1867,2018,2274,1868,2019,2275,
Di-n-octyl phthalate ................................................................................................................................................................................................... 1557 1869,2020,2276,2262
Di-n-octyl phthalate ................................................................................................................................................................................................... 1558 cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester (C22:6n3) ............ 1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251
Di-n-octyl phthalate ......................................................................................................................................................................................... 1564,2191 cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester (C22:6n3) at 2 wt % ........................................................................... 1822,1922,2254
Diolein .......................................................................................................................................................................................................................... 1633 Docosane ............................................................................................................................................................................................................ 1526,1637
Dioleoylglycerol ........................................................................................................................................................................................................... 1632 Docosane (C22) .......................................................................................................................................................................................................... 1636
Diols ........................................................................................................................................................................................................... 1703,1975,2185 (E,Z)-2,6-Dodecadienal ..................................................................................................................................................................................... 2103,2166
Diosmetin 6,8-di-C-glucoside ...................................................................................................................................................................................... 353 Dodecanal .............................................................................................................................................................................. 1971,2405,2103,2106,2166
Dioxabenzofos .................................................................................................................................................................................................... 1885,2064 Dodecane ....................................................................... 1431,1572,1486,1487,1491,1581,2319,2331,1526,1596,1597,1601,1667,1602,1668,1637
1,4-Dioxane ............................. 1550,1568,1588,2322,2344,1640,1675,1676,1725,1726,1727,2205,2206,2207,2450,2208,2209,2451,2226,2456 n-Dodecane ........................................................................................................................................................................................................ 2311,2312
Dioxathion ....................................................................................................................................................................................... 1544,1545,1890,2069 n-dodecane ........................................................................................................................................................................................................ 2313,2314
Dioxibrassinin ..................................................................................................................................................................................................... 1250,3031 Dodecane (C12) .......................................................................................................................................................................................................... 1636
Dioxin ............................................................................................................................................................................................... 1436,1575,1812,1912 Dodecane (I.S.) ........................................................................................................................................................................................................... 2308
Dipentyl phthalate ................................................................................................................................................................ 1865,2008,2394,2416,2191 Dodecanoic acid .................................................................................................................................................................... 1873,2031,2278,1970,2404
Diperodon hydrochloride ................................................................................................................................................................... 2903,983,2904,984 Dodecanone ....................................................................................................................................................................................................... 1971,2405
Diphenamid .................................................................................................................................................................. 1506,2075,1507,2076,1888,2067 (E)-2-Dodecenal .......................................................................................................................................................................................................... 2103
Diphenhydramine ........................................................................................................................................................................................... 67,846,2289 2-(E)-Dodecenal .......................................................................................................................................................................................................... 2166
Diphenhydramine hydrochloride ............................................................................................................................................................................... 2288 5-Dodecene ........................................................................................................................................................................................................ 1582,2332
Diphenylamine ................................................................................................................................................... 1404,2001,1550,1551,1673,1888,2067 Dodecyl gallate ...................................................................................................................................................................................................... 276,337
Diphenyl phthalate ............................................................................................................................................................... 1865,2008,2394,2416,2191 Dopamine ............................................................................................................................................................................................................... 144,955
Dipropylene glycol .......................................................................................................................... 1703,1975,2185,1704,1976,2186,1705,1977,2187 Dopamine hydrochloride .............................................................................................................................................................................................. 145
Dipyridamole ................................................................................................................................................................................................................. 931 Dopamine hydrochloride solution .............................................................................................................................................................................. 271
Diquat ..................................................................................................................................................................................................... 13,1356,1392,312 Dorzolamide ......................................................................................................................................................................................................... 2905,988
Dothiepin .............................................................................................................................................................................................................. 2906,989
Indexes Analyte
Dotriacontane ..................................................................................................................................................................................................... 1486,1487 Enalapril ................................................................................................................................................................................................... 123,849,129,892

Doxepin .................................................................................................................................. 1368,209,1164,210,1166,211,1167,212,1165,1169,1170 Enantiomers ........................................................................................................................................................................... 1694,1835,1941,2296,2693
Doxylamine ............................................................................................................................................................................... 858,859,871,890,912,971 β- Endorphin ................................................................................................................................................................................................................. 788
Drugs of abuse ........................................................................................................................................................................................................... 2289 Endosulfan I ......................................................... 1351,1403,1862,2000,1382,1396,1439,1925,1466,1740,1858,1989,2356,2384,2392,2406,
Dual .................................................................................................................................................................................................. 1467,1990,2357,2407 1469,1994,2359,2409,1506,2075,1513,1516,1524,1532,1604,1886,2065
Durene ........................................................................... 1431,1572,1595,1596,1597,1610,1612,1613,1614,1615,1616,1617,1618,1624,1625,1626 α-Endosulfan ................................................................................................................................................................................... 1507,2076,1512,1514
Dyfonate .......................................................................................................................................................................................... 1350,1401,1860,1995 β-Endosulfan ................................................................................................................................................................................... 1507,2076,1512,1514
Endosulfan II ........................................................................... 1382,1396,1439,1925,1404,2001,1466,1740,1858,1989,2356,2384,2392,2406,
—E— 1469,1994,2359,2409,1506,2075,1513,1516,1524,1532,1604,1886,2065
Endosulfan sulfate ............................ 1347,1394,1798,1894,1382,1396,1439,1925,1404,2001,1466,1740,1858,1989,2356,2384,2392,2406,
Eberconazole ........................................................................................................................................................................................................ 112,2614
1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1524,1532,1604,1797,1893
EDDP (2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) ............................................................................................................ 2723,439,464,479
Endotoxifen (I) ............................................................................................................................................................................................................ 1152
Edifenphos ......................................................................................................................................................................................... 1419,406,1885,2064
Endotoxifen (II) ........................................................................................................................................................................................................... 1152
Edinphos .......................................................................................................................................................................................... 1350,1401,1860,1995
Endrin .............................. 1381,1395,1438,1924,1382,1396,1439,1925,1388,1411,1482,2028,1466,1740,1858,1989,2356,2384,2392,2406,
cis-11,14-Eicosadienoic acid methyl ester (C20:2) ....................................... 1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251
1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1524,1532,1604
Eicosane ................................................................................................................................................................................. 1486,1487,1526,2316,2317
Endrin aldehyde ..................................................................................................................................................................... 1382,1396,1439,1925,
Eicosane (C20) .................................................................................................................................................................................................. 1636,1637
1466,1740,1858,1989,2356,2384,2392,2406,1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1532,1604
Eicosanoate (C20:0) .................................................................................................................................................................................................... 1636
Endrin ketone ............................................................................................. 1382,1396,1439,1925,1466,1740,1858,1989,2356,2384,2392,2406,
Eicosanoic acid ......................................................................................................................................................................................... 1873,2031,2278
1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1532,1604
cis-5,8,11,14,17-Eicosapentaenoic acid methyl ester (C20:5n3) ................. 1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251
Enilconazol (Imazalil) ................................................................................................................................................................. 11,2484,2582,112,2614
cis-8,11,14-Eicosatrienoic acid methyl ester ..................................................................... 1814,1914,2246,1816,1916,2248,1867,2018,2274,2262
Enrofloxacin ................................................................................................................................................................................ 1004,355,1005,356,1013
cis-11,14,17-Eicosatrienoic acid methyl ester ......................................................................................................... 1814,1914,2246,1816,1916,2248
Ephedrine ................................................................................................................................................................................. 992,2234,2285,2457,2460
cis-11,14,17-Eicosatrienoic acid methyl ester (C20:3n3) ............................. 1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251
(±)-Ephedrine ............................................................................................................................................................................................................... 441
cis-8,11,14-Eicosatrienoic acid methyl ester (C20:3n6) ............................... 1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251
(±)-Ephedrine hydrochloride ............................................................................................................................................................................ 2908,993
cis-11-Eicosenoic acid methyl ester ................................................................................... 1818,1918,2250,1819,1919,2251,1837,1954,1840,1960
Epicatechin ................................................................................................................................................................................................................... 1244
cis-13-Eicosenoic acid methyl ester ..................................................................................................................................................... 1868,2019,2275
(−)-Epicatechin gallate ............................................................................................................................................................................................. 1244
cis-11-Eicosenoic acid methyl ester (C20:1) ....................................................................................... 1836,1945,1839,1959,1842,1962,1844,1964
Epigallocatechin .......................................................................................................................................................................................................... 1244
cis-11-Eicosenoic acid methyl ester (C20:1n9) ...................................................................................................... 1815,1915,2247,1817,1917,2249
(−)-Epigallocatechin gallate ..................................................................................................................................................................................... 1244
cis-11-Eicosenoic acid methyl ester (C20:1n9) at 2 wt % ............................................................................................................. 1822,1922,2254
Epimedin A ................................................................................................................................................................................................................. 1330
Elaidic acid .................................................................................................................................................................................................................... 357
Epimedin B .................................................................................................................................................................................................................. 1330
Elaidic acid methyl ester (C18:1n9t) ................................................................................................................................................. 1815,1915,2247
Epimedin C .................................................................................................................................................................................................................. 1330
Elaidic acid methyl ester (C18:1n9t) .......................................................................................... 1817,1917,2249,1818,1918,2250,1819,1919,2251
Epimedin A (PHY80586) ........................................................................................................................................................................................... 1267
Elaidic acid methyl ester (C18:1n9t) at 2 wt % .............................................................................................................................. 1822,1922,2254
Epimedin B (PHY80587) ........................................................................................................................................................................................... 1267
Elastase ........................................................................................................................................................................................................................... 582
Epimedin C (PHY80588) ........................................................................................................................................................................................... 1267
β-Elemene .......................................................................................................................................................................................................... 2096,2142
Epinastine ....................................................................................................................................................................................................................... 994
+ β-Elemene .............................................................................................................................................................................................................. 2170
(−)-Epinephrine .................................................................................................................................................................................................... 144,145
cis-β-Elemene .............................................................................................................................................................................................................. 2131
Epinephrine ..................................................................................................................................................................................................... 254,955,994
δ-Elemene ....................................................................................................................................................................................... 2096,2131,2142,2161
7-Epi-paclitaxel ........................................................................................................................................................................................ 1104,1153,1154
α-Elemol ...................................................................................................................................................................................................................... 2161
7-epipaclitaxel ............................................................................................................................................................................................................. 1220
EMDP (2-Ethyl-5-methyl-3,3-diphenylpyrroline) ..................................................................................................................................................... 479
Indexes Analyte
Epitestosterone ........................................................................................................................................................................................ 197,198,200,201 Ethafluralin ......................................................................................................................................................................................................... 1887,2066

EPN ......................................................................................................................... 1419,406,1350,1401,1860,1995,1542,1543,1544,1545,1885,2064 Ethane ........................................................................................................................................................ 1628,1639,1665,1723,1724,1749,1750,1751
Eptam® ............................................................................................................................................................................................ 1467,1990,2357,2407 Ethane (430 ng on column) .................................................................................................................................................................................... 1752
EPTC ................................................................................................................................................................................................. 1506,2075,1890,2069 Ethanethiol ................................................................................................................................................................................................ 1685,1687,2382
Eptifibatide ..................................................................................................................................................................................................................... 820 Ethanol .................................................................. 1431,1572,1568,1588,2322,2344,1593,1596,1629,1630,1676,1725,1726,1727,1780,1969,
(±)-Equol .................................................................................................................................................................................. 1270,361,1271,362,1272 2026,2027,2060,2443,2111,2184,2210,2211,2212,2452,2213,2214,2453,2226,2456,2292,2464,2293,2294
Erbitux (Cetuximab) ..................................................................................................................................................................................................... 782 Ethanol denatured ................................................................................................................................................................................... 1525,1595,1597
Erectile Dysfunction Drugs ........................................................................................................................................................................................... 996 2-Ethenyl-5-methylpyrazine .......................................................................................................................................................... 1871,2022,2395,2417
Ergocalciferol ........................................................................................................................................................................................................ 1293,217 2-Ethenyl-6-methylpyrazine .......................................................................................................................................................... 1871,2022,2395,2417
Ergocalciferol (D2) ................................................................................................................................ 1292,216,1294,218,1297,219,1255,1256,1253 Ethion ............................................................................... 1419,406,1350,1401,1860,1995,1440,1677,1824,1926,1542,1543,1544,1545,1885,2064
Eriocitrin .................................................................................................................................................................................................................. 353,359 Ethofenprox ..................................................................................................................................................................................... 1430,1641,1796,1892
Erioglaucine disodium salt .......................................................................................................................................................................................... 388 Ethoprop .......................................................................................................................................................................................... 1506,2075,1542,1543
Erucic acid methyl ester (C22:1) ................................................................................................................................................................... 1842,1962 Ethoprophos .............................................................................. 308,1350,1401,1860,1995,1449,1683,1830,1935,1507,2076,1544,1545,1885,2064
Erucic acid methyl ester (C22:1n9) .................................................................................................................................................... 1815,1915,2247 Ethoprophos (I.S.) ....................................................................................................................................................... 1347,1394,1798,1894,1797,1893
Erucic acid methyl ester (C22:1n9) ............................................................................................. 1817,1917,2249,1818,1918,2250,1819,1919,2251 2-Ethoxyethanol ........................................................................................ 1383,1457,1703,1975,2185,1725,2205,2206,2207,2450,2208,2209,2451
Erucic acid methyl ester (C22:1n9) at 2 wt % ................................................................................................................................ 1822,1922,2254 2-Ethoxyethanol (Cellosolve®) ............................................................................................................................................ 1726,1704,1976,2186,1727
Erythromycin ............................................................................................................................................................................................................... 1194 2-(2-Ethoxyethoxy)ethanol ..................................................................................................................................................................... 1703,1975,2185
Erythromycin C ........................................................................................................................................................................................................... 1194 2-(2-Ethoxyethoxy)ethanol (CARBITOL™) ............................................................................................................................................. 1704,1976,2186
Erythromycin estolate ................................................................................................................................................................................................ 1194 2-Ethoxyethyl acetate ............................................................................................................................................................................. 1725,1726,1727
Erythromycin ethyl succinate .................................................................................................................................................................................... 1194 Ethoxyquin ......................................................................................................................................................................................... 1282,292,1898,2182
Erythropoietin, main peak ........................................................................................................................................................................................... 584 Ethychlozate ....................................................................................................................................................................................................... 1890,2069
Erythrosin B sodium salt ............................................................................................................................................................................................. 388 Ethyl acetate ........................................................ 1431,1572,1485,1778,2032,2367,2390,2422,1568,1588,2322,2344,1675,1676,1725,1726,
Esfenvalerate ...................................................................................................................................................................................................... 1890,2069 1727,1780,1895,1969,2026,2027,2056,2439,2060,2443,2111,2184,2210,2211,2212,2452,2213,2214,2453,2224
Esprocarb ............................................................................................................................................................................................................ 1888,2067 Ethyl alcohol ...................................................................................................................................................................................................... 2056,2439
α-Estradiol ...................................................................................................................................................................................... 155,555,196,202,2257 N-Ethylaniline .................................................................................................................................................................................................................. 32
β-Estradiol ...................................................................................................................................................................................... 155,555,196,202,1186 Ethyl azinphos ................................................................................................................................................................................................... 1544,1545
17β-Estradiol ............................................................................................................................................................................................................... 2257 Ethyl behenate .................................................................................................................................................................................................. 2111,2184
β-Estradiol 3,17-disulfate ..................................................................................................................................................................................... 155,555 Ethylbenzene ........... 102,1493,2057,2370,2440,1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,
β-Estradiol 3-(β-D-glucuronide) .......................................................................................................................................................................... 155,555 1505,2074,1519,1520,1521,1522,1527,1528,1529,1530,1546,1547,1548,1549,1565,1566,1569,1589,2323,2345,1592,1593,1594,1595,1596,
β-Estradiol 17-β-D-glucuronide ........................................................................................................................................................................... 155,555 1597,1600,1658,1601,1667,1602,1668,1610,1611,1612,1613,1614,1615,1616,1617,1618,1621,1624,1625,1626,1640,1676,1725,1727,1780,
β-Estradiol 3-(β-D-glucuronide) 17-sulfate ....................................................................................................................................................... 155,555 1794,2300,2467
β-Estradiol 3-sulfate ............................................................................................................................................................................................. 155,555 3-Ethylbenzene .................................................................................................................................................................................................. 1431,1572
Estriol .............................................................................................................................................................................................. 155,555,196,202,2257 4-Ethylbenzene .................................................................................................................................................................................................. 1431,1572
Estriol 3-(β-D-glucuronide) .................................................................................................................................................................................. 155,555 2-Ethyl-1-butanol ....................................................................................................................................................................................................... 1666
Estriol 3-sulfate ..................................................................................................................................................................................................... 155,555 Ethyl tert-butyl ether ................................................................................................................................................................................................. 1722
Estrogenic compounds ............................................................................................................................................................................................... 2257 Ethyl butyrate .......................................................................................................................................................................................... 1969,2060,2443
Estrone .................................................................................................................................................................................. 155,555,196,202,1186,2257 N-Ethylcathinone hydrochloride solution ........................................................................................................................................................... 472,476
Estrone 3-(β-D-glucuronide) ................................................................................................................................................................................ 155,555 Ethyl cellosolve ........................................................................................................................................................................................................... 1780
Estrone 3-sulfate ................................................................................................................................................................................................... 155,555 Ethyl chloride ................................................................................................................................................................................. 1569,1589,2323,2345
Indexes Analyte
Ethylcyclohexane ......................................................................................................................................................................................................... 1612 2-Ethyl-3-methylpyrazine .............................................................................................................................................................. 1871,2022,2395,2417

Ethylcyclopentane ....................................................................................................................................................................................................... 1592 2-Ethyl-5-methylpyrazine ........................................................................................................................................... 1871,2022,2395,2417,2056,2439
3-Ethylcyclopentane ................................................................................................................................................................................................... 1612 2-Ethyl-6-methylpyrazine ........................................................................................................................................... 1871,2022,2395,2417,2056,2439
2-Ethyl-3,5-dimethylpyrazine ....................................................................................................................................................... 1871,2022,2395,2417 Ethyl methyl sulfide ................................................................................................................................................................................................... 1685
3-Ethyl-2,5-dimethylpyrazine ....................................................................................................................................................... 1871,2022,2395,2417 Ethyl octanoate ................................................................................................................................................................................................. 2060,2443
S-Ethyl-N,N-dipropylthiocarbamate ................................................................................................................................................................. 1507,2076 Ethylone ...................................................................................................................................................................................................... 435,2049,2433
Ethylene ..................................................................................................................................................... 1639,1665,1723,1724,1734,1749,1750,1751 Ethylone hydrochloride ................................................................................................................................................................... 243,468,434,470,475
Ethylene (430 ng on column) ................................................................................................................................................................................. 1752 Ethylparaben .......................................................................................................................................................................................................... 282,283
Ethylene glycol ............................................................. 1383,1457,1703,1975,2185,1704,1976,2186,1705,1977,2187,2205,2206,2208,2225,2455 Ethyl paraben ................................................................................................................................................................................................................ 286
Ethylene glycol butyl ether ................................................................................................................................................................... 1383,1457,1676 Ethylparaben ......................................................................................................................................................................................... 289,290,291,1106
Ethylene glycol monophenyl ether ............................................................................................................................................................................ 284 Ethyl parathion ......................................................................................................................................... 1440,1677,1824,1926,1470,1996,2360,2410
Ethylene sulfide .............................................................................................................................................................................. 1492,1583,2320,2333 3-Ethylpentane ............................................................................................................................................................................................................ 1592
Ethyl formate ............................................................................................ 1675,1676,1725,1726,1727,1780,2210,2211,2212,2452,2213,2214,2453 Ethyl propionate ......................................................................................................................................................................................................... 1969
Ethyl gallate .................................................................................................................................................................................................................. 376 2-Ethylpyrazine ............................................................................................................................................................ 1871,2022,2395,2417,2056,2439
Ethyl geranoate .......................................................................................................................................................................................................... 2131 3-Ethylpyridine .................................................................................................................................................................................................. 2056,2439
Ethyl-β-D-glucuronide ................................................................................................................................................................................................... 467 Ethyl stearate ..................................................................................................................................................................................................... 2111,2184
3-Ethylheptane .................................................................................................................................................................................................. 1592,1612 Ethyltoluene ................................................................................................................................................................................................................. 1621
4-Ethylheptane ............................................................................................................................................................................................................ 1592 2-Ethyltoluene ................................................................................................................................................................................................... 1431,1572
3-Ethylhexane ............................................................................................................................................................................................................. 1592 Ethylxylene ................................................................................................................................................................................................................... 1621
Ethyl hexanoate ................................................................................................................................................................................................ 2060,2443 Etobenzanid ........................................................................................................................................................................................................ 1888,2067
2-Ethylhexanoic acid ......................................................................................................................................... 1694,1835,1941,2296,2693,1970,2404 Etobenzanid metabolite ................................................................................................................................................................................... 1888,2067
2-Ethyl-1-hexanol .......................................................................................................................................................................... 1492,1583,2320,2333 Etodolac ......................................................................................................................................................................................................... 898,2909,997
2-Ethylhexyl alcohol ................................................................................................................................................................................................... 1728 Etofenprox .......................................................................................................................................................................................................... 1888,2067
N-(2-Ethylhexyl)-5-norbornene-2,3-dicarboximide ....................................................................................................................................... 1507,2076 Etoxazole ............................................................................................................................................................................................................ 1888,2067
Ethyl 4-hydroxybenzoate ........................................................................................... 37,279,284,1236,1748,1998,2190,2223,2387,2412,2447,2454 Etoxazole metabolite ........................................................................................................................................................................................ 1888,2067
Ethyl isobutyrate ......................................................................................................................................................................................................... 1969 Etridiazole .................................................................................................. 1430,1641,1796,1892,1506,2075,1507,2076,1509,1510,1511,1887,2066
Ethyl isocyanate .......................................................................................................................................................................................................... 2338 Etrimfos ........................................................................................................................................................ 1419,406,1440,1677,1824,1926,1885,2064
Ethyl lactate ................................................................................................................................................................................................................ 1895 Eucalyptol .............................................................................................................................................................................. 2121,2124,2137,2138,2167
(±)-Ethyl-γ-lactone ....................................................................................................................................................................... 1729,2118,2529,2676 Eugenol ............................................................................................................................................................................................ 2180,2181,2183,2446
Ethyl mercaptan ................................................................................................................................................................................................ 1686,2381
Ethyl mercaptan (1 ppm) ........................................................................................................................................................................................ 1783 —F—
Ethyl methacrylate ............................................................................................................................................................................................ 1504,2073 F(ab')2 ............................................................................................................................................................................................................. 675,676,678
Ethyl methanesulfonate .................................................................................................................................................................................... 1550,1551 Famophos ...................................................................... 1466,1740,1858,1989,2356,2384,2392,2406,1471,1746,1861,1997,2361,2386,2393,2411
1-ethyl-4-methylbenzene .............................................................................................................................................................. 1568,1588,2322,2344 Famotidine ................................................................................................................................................................................................................... 1173
cis-1,2-Ethylmethylcyclopentane ............................................................................................................................................................................... 1592 Famoxadone ....................................................................................................................................................................................................... 1889,2068
trans-1,2-Ethylmethylcyclopentane ........................................................................................................................................................................... 1592 Famphos .......................................................................................................................................................................................... 1470,1996,2360,2410
cis-1,3-Ethylmethylcyclopentane ............................................................................................................................................................................... 1592 Famphur ........................................................................................... 1350,1401,1860,1995,1470,1996,2360,2410,1542,1543,1544,1545,1550,1551
trans-1,3-Ethylmethylcyclopentane ........................................................................................................................................................................... 1592 (E,E)-Farnesal .............................................................................................................................................................................................................. 2131
3-Ethyl-3-methylpentane ........................................................................................................................................................................................... 1612 (2E,6Z)-Farnesal .......................................................................................................................................................................................................... 2131
Indexes Analyte
α-Farnesene ........................................................................................................................................................................... 1876,2035,2396,2423,2105 Fensulfothion .......................................................................................................................... 1350,1401,1860,1995,1542,1543,1544,1545,1885,2064

trans-α-Farnesene ....................................................................................................................................................................................................... 2106 Fentanyl ...................................................................................................................................................................................................... 427,1003,2289
(E,E)-α-Farnesene ........................................................................................................................................................ 2095,2096,2103,2131,2142,2166 Fentanyl solution .......................................................................................................................................................................................................... 481
trans-β-Farnesene ....................................................................................................................................................................................................... 2170 Fentanyl citrate salt ................................................................................................................................................................................................... 2288
(E)-β-Farnesene .................................................................................................................................................................... 2096,2121,2131,2142,2167 Fenthion ........................................................................... 1419,406,1350,1401,1860,1995,1445,1679,1826,1931,1542,1543,1544,1545,1885,2064
(Z)-β-Farnesene .......................................................................................................................................................................................................... 2096 Fenuron .................................................................................................................................................................................. 1,295,294,302,314,375,317
(Z,E)-Farnesol ..................................................................................................................................................................................................... 2166,2103 τ-Fenvalerate ................................................................................................................................................................................... 1447,1681,1828,1933
(Z,Z)-Farnesol .............................................................................................................................................................................................................. 2131 Fenvalerate-1 ..................................................................................................................................................................................................... 1886,2065
Farnesyl acetate .......................................................................................................................................................................................................... 2131 Fenvalerate-2 ..................................................................................................................................................................................................... 1886,2065
Fast Green FCF ............................................................................................................................................................................................................. 388 Fenvalerate isomer ......................................................................................................................................................................... 1448,1682,1829,1934
Fatty acids ................................................................................................................................................................................................ 1808,1908,2244 Fermizone ........................................................................................................................................................................................................... 1890,2069
Fd′ .................................................................................................................................................................................................................................. 768 Ferredoxin-NADP+ Reductase from Spinacia oleracea (spinach) ........................................................................................................................... 586
Fenamiphos ................................................................................................ 1447,1681,1828,1933,1466,1740,1858,1989,2356,2384,2392,2406, Ferritin ............................................................................................................................................................................................................. 742,750,752
1471,1746,1861,1997,2361,2386,2393,2411,1506,2075,1507,2076,1885,2064 Feruloylquinic acid (3-FQA) ...................................................................................................................................................................................... 1275
Fenarimol ..................................................................................................................................................................... 1506,2075,1507,2076,1886,2065 Feruloylquinic acid (4-FQA) ...................................................................................................................................................................................... 1275
Fenazaquin ...................................................................................................................................................................................... 1446,1680,1827,1932 Feruloylquinic acid (5-FQA) ...................................................................................................................................................................................... 1275
Fenbuconazole ................................................................................................................................................................................................... 1889,2068 Fexofenadine ............................................................................................................................................................................................................... 1160
Fenchlorphos ................................................................................................................................................................ 1449,1683,1830,1935,1886,2065 Fipronil ........................................................................................................................................................... 2583,57,1446,1680,1827,1932,1886,2065
Fenchlorvos .................................................................................................................................................................................................................... 308 Flavanone ..................................................................................................................................................................... 1257,3032,1258,3033,1346,3042
Fenchyl alcohol ........................................................................................................................................................................................................... 2131 Flavone ........................................................................................................................................................................................................................... 352
α-Fenchyl alcohol ....................................................................................................................................................................................................... 2106 Flavonoids .................................................................................................................................................................................................................... 1259
(1R)-endo-(+)-Fenchyl alcohol ................................................................................................................................................................................ 2105 (±)-Flecainide solution ...................................................................................................................................... 1370,229,230,1378,266,267,1379,268
Fenfluramine ........................................................................................................................................................................................................ 1172,213 Fluacrypyrim ....................................................................................................................................................................................................... 1887,2066
Fenhexamid .......................................................................................................................................................................................................... 1419,406 Fluazifop-p-butyl ............................................................................................................................................................................ 1446,1680,1827,1932
Fenitrothion ............................... 1419,406,1350,1401,1860,1995,1430,1641,1796,1892,1447,1681,1828,1933,1542,1543,1544,1545,1885,2064 Fluconazole solution .............................................................................................................................................................................................. 831,832
Fenofibric Acid ..................................................................................................................................................................................................... 2910,999 Flucythrinate-1 ................................................................................................................................................................................................... 1886,2065
Fenoldopam ......................................................................................................................................................................................................... 1191,233 Flucythrinate-2 ................................................................................................................................................................................................... 1886,2065
Fenoprofen ......................................................................................................................................................................................................... 1000,2911 Fludioxonil .......................................................................................................................................................................................................... 1889,2068
Fenoprofen methyl ester ................................................................................................................................................................................. 1001,2912 Flufenoxuron ........................................................................................................................................................................................................ 1419,406
Fenoprop (2-(2,4,5-Trichlorophenoxy)propionic acid) ........................................................................................................................................... 1119 Flumethasone ................................................................................................................................................................................................................ 167
Fenoterol hydrobromide ................................................................................................................................................................................... 1002,2913 Flumioxazin ........................................................................................................................................................................................................ 1888,2067
Fenothiocarb ...................................................................................................................................................................................................... 1888,2067 Flunitrazepam solution ......................................................................................................................................................................................... 490,833
Fenoxanil-1 ........................................................................................................................................................................................................ 1888,2067 Flunitrazepam-d7 .......................................................................................................................................................................................................... 833
Fenoxanil-2 ........................................................................................................................................................................................................ 1888,2067 Fluometuron .................................................................................................................................................................................. 1,295,294,302,314,375
Fenoxaprop-ethyl ............................................................................................................................................................................................... 1889,2068 Fluoranthene ............................................................................................................................... 2375,310,311,1408,2010,1409,2011,1451,1936,
Fenoxycarb ........................................................................................................................................................................................ 1419,406,1888,2067 1480,2013,1481,2014,1517,1518,1524,1540,1541,1552,1553,1554,1555,1556,1557,1558,1560,1561,1562,1563,1564,1567,1587
Fenpropathrin .................................................................................................................................................................................................... 1886,2065 Fluoranthene-d10 ............................................................................................................................................................................................... 1409,2011
Fenpropimorph ............................................................................................................................................................ 1447,1681,1828,1933,1890,2069 Fluoranthene-d10M (I.S.) .................................................................................................................................................................................. 1408,2010
Fenpyroximate ..................................................................................................................................................................................................... 1419,406 Fluoranthrene .................................................................................................................................................................................. 1483,2029,2365,2420
Indexes Analyte
Fluorene ........................................................... 2375,310,311,1388,1411,1482,2028,1408,2010,1409,2011,1451,1936,1480,2013,1481,2014, Fluticasone propionate ................................................................................................................................................................................................. 167

1483,2029,2365,2420,1506,2075,1507,2076,1517,1518,1524,1540,1541,1552,1553,1554,1555,1556,1557,1558,1560,1561,1562,1563,1564, Flutolanil ............................................................................................................................................................................................................. 1886,2065
1567,1587,1795,1891 Flutriafol .......................................................................................................................................................................................... 1444,1678,1825,1930
Fluorene-d10 ..................................................................................................................................................................................................... 1567,1587 Fluvalinate-1 ...................................................................................................................................................................................................... 1886,2065
2-Fluoroadenine-9-β-D-arabinofuranoside .......................................................................................................................................................... 168,587 Fluvalinate-2 ...................................................................................................................................................................................................... 1886,2065
(±)-2-Fluoroamphetamine hydrochloride solution ........................................................................................................................................ 466,1426 Fluvastatin ..................................................................................................................................................................................................... 1148,194,964
4-Fluoroamphetamine .................................................................................................................................................................................................. 458 Fluvoxamine ................................................................................................................................................................................................................... 157
(±)-4-Fluoroamphetamine hydrochloride solution ........................................................................................................................................ 466,1426 2C-B-FLY hydrochloride solution ....................................................................................................................................................... 228,1424,457,483
Fluorobenzene .............................................................................................................. 1498,2062,2373,2444,1504,2073,1505,2074,1519,1520,1546 Folic acid .................................................................................................................................... 863,864,1300,1302,1303,1304,1306,1307,1308,1333
Fluorobenzene ........................................................................................................................................................................................................... 1547 Folinic acid calcium salt hydrate ................................................................................................................................................ 1260,3034,1261,3035
Fluorobenzene .................................................................................................................................................................................................... 1548,1549 Folpet ................................................................................................................... 1347,1394,1798,1894,1449,1683,1830,1935,1797,1893,1889,2068
2-Fluorobiphenyl ............................................................................................................................................................................ 1517,1518,1524,1540 Fonofos ......................................................................................................................................................................... 1448,1682,1829,1934,1885,2064
2-Fluorobiphenyl ....................................................................................................................................................................................................... 1541 Fonophos ......................................................................................................................................................................................... 1542,1543,1544,1545
2-Fluorobiphenyl ................................................................................................................... 1550,1551,1552,1553,1554,1555,1556,1557,1558,1564 Formaldehyde ........................................................................................................................................................................................................ 21,2326
5-Fluorocytosine ..................................................................................................................................................................................................... 168,587 Formaldehyde solution ..................................................................................................................................................................................... 1584,2341
5-Fluoro-2′-deoxyuridine ...................................................................................................................................................................................... 168,587 Formaldehyde-2,4-dinitrophenylhydrazone ......................................................................................................................................... 22,2334,23,2335
(±)-2-Fluoromethamphetamine hydrochloride solution ............................................................................................................................... 466,1426 Formaldehyde-2,4-DNPH ........................................................................................................................................................................................... 2337
(±)-4-Fluoromethamphetamine hydrochloride solution ............................................................................................................................... 466,1426 Formaldehyde (2,4-DNPH derivative) ................................................................................................................................................................. 2336,24
3-Fluoromethcathinone ..................................................................................................................................................................................... 2049,2433 Formaldehyde (~38 %) ........................................................................................................................................................................................... 1699
3-Fluoromethcathinone hydrochloride solution .......................................................................................................................... 243,468,434,471,473 Formamide ............................................................................................................................................................................................... 2205,2206,2208
4-Fluoromethcathinone ..................................................................................................................................................................................... 2049,2433 Formoterol fumarate dihydrate ................................................................................................................................................................................ 1208
4-Fluoromethcathinone hydrochloride solution .......................................................................................................................... 243,468,434,471,473 Formothion ................................................................................................................................................................... 1350,1401,1860,1995,1887,2066
1-Fluoronaphthalene ...................................................................................................................................................................... 1517,1518,1540,1541 Fosthiazate ...................................................................................................................................................................................... 1350,1401,1860,1995
2-Fluorophenol ............................................................................................................. 1524,1550,1551,1552,1553,1554,1555,1556,1557,1558,1564 Fosthiazate-1 ...................................................................................................................................................................................................... 1885,2064
p-Fluorophenyl methyl sulfoxide ........................................................................................................................................................................ 2584,58 Fosthiazate-2 ...................................................................................................................................................................................................... 1885,2064
6-Fluorotryptophan ............................................................................................................................................................................................. 1172,213 Free fatty acids ........................................................................................................................................................................................ 1808,1908,2244
5-Fluorouracil ......................................................................................................................................................................................................... 168,587 Freon® 11 .................................................................................................................................................................................................................... 1698
Fluoxetine .......................................................................................................................................... 67,157,239,811,2216,2264,2299,2625,2715,2853 Freon 12 ...................................................................................................................................................................................................................... 1697
Fluoxetine hydrochloride ..................................................................................................................................... 837,1006,2914,1007,2915,1008,1010 Freon® 12 .................................................................................................................................................................................................................... 1698
Fluoxetine hydrochloride solution ............................................................................................................................................................................ 1223 Freon® 21 .................................................................................................................................................................................................................... 1698
Fluoxetine (Prozac®) ............................................................................................................................................................................................. 846,931 Freon 22 ...................................................................................................................................................................................................................... 1697
Fluphenazine dihydrochloride ............................................................................................................................................................. 132,907,1189,231 Freon® 22 .................................................................................................................................................................................................................... 1698
Fluquinconazole ................................................................................................................................................................................................. 1886,2065 Freon® 113 solution .................................................................................................................................................................................................. 2224
Flurazepam solution ..................................................................................................................................................................................................... 833 Freon 114 .................................................................................................................................................................................................................... 1697
Flurbiprofen ..................................................................................................................................................................................... 1011,2916,1012,2917 Freon® 114 .................................................................................................................................................................................................................. 1698
Fluridon .............................................................................................................................................................................................................. 1507,2076 Freon 114a .................................................................................................................................................................................................................. 1697
Flusilazole ........................................................................................................................................................................................................... 1888,2067 Freon® 114a (impurity) ............................................................................................................................................................................................. 1698
Flusilazole metabolite ....................................................................................................................................................................................... 1888,2067 Fructose ........................................................................................................................................................................................................... 333,381,387
Fluthiacet-methyl .............................................................................................................................................................................................. 1890,2069 D-(−)-Fructose .............................................................................................................................................................................................................. 380
Indexes Analyte
Fucitol ................................................................................................................................................................................................................. 2036,2037 Gensenoside Rb2 ......................................................................................................................................................................................................... 1265

Fumaric acid ............................................................................................................................................................................. 1277,368,85,1278,369,86 Gensenoside Rc ........................................................................................................................................................................................................... 1265
Fumonisin B1 solution ................................................................................................................................................................................................. 331 Gensenoside Rd .......................................................................................................................................................................................................... 1265
Fumonisin B2 solution ................................................................................................................................................................................................. 331 Gensenoside Re ........................................................................................................................................................................................................... 1265
2-Furaldehyde .................................................................................................................................................................................................... 1896,2403 Gensenoside Rf ........................................................................................................................................................................................................... 1265
Furaltadone .............................................................................................................................................................................................. 127,877,879,906 Gensenoside Rg1 ......................................................................................................................................................................................................... 1265
Furametpyr ......................................................................................................................................................................................................... 1889,2068 Gentisic acid ........................................................................................................................................................................................................ 869,1275
Furametpyr metabolite ..................................................................................................................................................................................... 1889,2068 (±)-Geosmin ............................................................................................................................................ 1488,2038,2368,2424,1499,2070,2376,2445
Furan ................................................................................................................................................................................................ 1436,1575,1812,1912 Geranial ............................................................................................ 2050,2434,2099,2103,2105,2106,2120,2121,2131,2142,2166,2170,2180,2181
Furazolidone .................................................................................................................................................................................................................. 906 Geranic acid methyl ester ......................................................................................................................................................................................... 2142
Furfural ................. 59,60,1878,2044,2398,2430,1879,2046,2399,2431,1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436, Geraniol ............................................................................................................................................ 2050,2434,2099,2103,2112,2120,2131,2142,2166
1883,2054,2402,2437,1969,2056,2439 Geranium oil ............................................................................................................................................................................................................... 2112
Furfuryl acetate ................................................................................................................................................................................................. 2056,2439 Geranyl acetate .................................................................................................................................................. 2096,2099,2103,2121,2131,2142,2166
Furfuryl alcohol ............................................................................................................................................................................. 59,60,1895,2056,2439 Geranyl acetone .......................................................................................................................................................................................................... 2131
Furfuryl ether ..................................................................................................................................................................................................... 2056,2439 Geranyl butyrate ......................................................................................................................................................................................................... 2112
Furfuryl formate ................................................................................................................................................................................................ 2056,2439 Geranyl formate ................................................................................................................................................................................................ 2112,2131
2-Furfurylfuran ................................................................................................................................................................................................... 2056,2439 (E,E)-Geranyl linalool ................................................................................................................................................................................................. 2131
2-Furfuryl-5-methyl sulfide ............................................................................................................................................................................. 2056,2439 Geranyl propanoate .................................................................................................................................................................................................... 2131
Furfurylpyrrole .................................................................................................................................................................................................... 2056,2439 Geranyl propionate ........................................................................................................................................................................................... 2050,2434
Furoin ...................................................................................................................................................................................................................... 2585,61 Geranyl tiglate ............................................................................................................................................................................................................ 2112
Furosemide ................................................................................................................................................................................................... 985,986,1014 Germacrene D ........................................................................................................................ 2095,2096,2103,2131,2137,2138,2161,2166,2167,2170
1-(2-Furyl)-3-butanone .................................................................................................................................................................................... 2056,2439 Germacrene D .......................................................................................................................................................................................... 2050,2434,2136
2-Furyl methyl ketone .............................................................................................................................................................................................. 59,60 Ginsenoside Rb1 ................................................................................................................................................................................................ 1245,1263
Ginsenoside-Rb1 from Panax ginseng (Korean ginseng) root ............................................................................................................................. 1264
—G— Ginsenoside Rb2 ................................................................................................................................................................................................ 1245,1263
Gabapentin solution ..................................................................................................................................................................................................... 836 Ginsenoside Rb2 ......................................................................................................................................................................................................... 1264
Galactitol ............................................................................................................................................................................................................ 2036,2037 Ginsenoside Rc .................................................................................................................................................................................................. 1245,1263
Galactose ........................................................................................................................................................................................................................ 383 Ginsenoside-Rc from Panax ginseng (Korean ginseng) root ............................................................................................................................... 1264
D-(+)-Galactose ............................................................................................................................................................................................................ 379
Ginsenoside Rd ........................................................................................................................................................................................ 1245,1263,1264
β-Galactosidase from Escherichia coli ........................................................................................................................................................................ 761 Ginsenoside Re .................................................................................................................................................................................................. 1245,1263
Galangin ....................................................................................................................................................................................................................... 1329 Ginsenoside-Re from Panax ginseng (Korean ginseng) root ............................................................................................................................... 1264
Gallic acid ............................................................................................................................................................................................................ 376,1259 Ginsenoside Rf ......................................................................................................................................................................................... 1245,1263,1264
Gallocatechin ............................................................................................................................................................................................................... 1244 Ginsenoside Rg1 ................................................................................................................................................................................................ 1245,1263
(−)-Gallocatechin gallate .......................................................................................................................................................................................... 1244 Ginsenoside Rg1 ......................................................................................................................................................................................................... 1264
(G) gamma-Globulin .......................................................................................................................................................................................... 1185,808 γ-Globulin ........................................................................................................................................................................................................ 207,757,826
Gasoline ........................................................................................................................................................................................................................ 1627 γ-Globulin bovine blood (150 kDa) .......................................................................................................................................................................... 493
Genistein .................................................................................................................................................................................... 1270,361,1271,362,1272 γ-Globulins from human blood ................................................................................................................................................................. 1129,187,825
Genistin ...................................................................................................................................................................................... 1270,361,1271,362,1272 Globulol ........................................................................................................................................................................................................................ 2131
Gensenoside Rb1 ......................................................................................................................................................................................................... 1265 Glucitol ................................................................................................................................................................................................................ 2036,2037
Indexes Analyte
Glucose ...................................................................................................................................................................................... 333,367,381,383,384,385 Glycyrrhetinic acid ...................................................................................................................................................................................................... 1289

D-(+)-Glucose ........................................................................................................................................................................................................ 379,382 Gly-Phe .......................................................................................................................................................................................................................... 719
D-(+)-Glucose monohydrate ....................................................................................................................................................................................... 380 Gly-Tyr ............................................................................................................................................................................................................................ 708
Glucose-6-phosphate Dehydrogenase from baker's yeast (S. cerevisiae) ............................................................................................................ 737 Goal .................................................................................................................................................................................................. 1467,1990,2357,2407
Glucose-6-phosphate Dehydrogenase from Leuconostoc mesenteroides ............................................................................................................... 744 Grepafloxacin .......................................................................................................................................................................................................... 880,881
Glutamic acid ................................................................................................................................................................................................................ 819 Guaiacol glyceryl ether carbamate ................................................................................................................................................................. 1059,2946
D-Glutamic acid ................................................................................................................................................................................................... 2794,612 Guaia-6,9-diene .......................................................................................................................................................................................................... 2112
L-Glutamic acid .......................................................................................................................................................... 508,509,2794,612,727,1897,2304 Guanine ................................................................................................................................................................................................................... 684,765
DL-Glutamic acid monohydrate ......................................................................................................................................................................... 2793,611 Guanosine ................................................................................................................................................................................................ 687,688,689,769
DL-Glutamic acid (FMOC derivatives) ............................................................................................................................................................... 2779,594 Guanosine diphosphate ................................................................................................................................................................................................ 690
L-Glutamic Dehydrogenase solution from bovine liver ........................................................................................................................................... 761 Guanosine triphosphate ............................................................................................................................................................................................... 690
Glutamine ........................................................................................................................................................................................................ 508,509,819 Guthion® ........................................................................................................................................................................................................................ 308
D-Glutamine .......................................................................................................................................................................................................... 2796,614
L-Glutamine ................................................................................................................................................................................ 2796,614,727,1897,2304 —H—
DL-Glutamine ........................................................................................................................................................................................................ 2795,613
Halfenprox .......................................................................................................................................................................................................... 1886,2065
Glutamine derivative ......................................................................................................................................................................................... 1897,2304 Halocarbon 11 ................................................................................................................................................................................ 1569,1589,2323,2345
DL-Glutamine (FMOC derivatives) ..................................................................................................................................................................... 2780,595
Halocarbon 12 ................................................................................................................................................................................ 1569,1589,2323,2345
Glutathione S-Transferase from equine liver ............................................................................................................................................................ 615 Halocarbon 113 .............................................................................................................................................................................. 1569,1589,2323,2345
DL-Glutethimide .......................................................................................................................................................................................... 1015,2721,431
Halocarbon 114 .............................................................................................................................................................................. 1569,1589,2323,2345
Glycerin ............................................................................................................................................................................................................... 1633,1638 Haloperidol ............................................................................................................................................................................................ 834,837,931,1212
Glycerol ............................................................................................................................................................................................ 1632,1705,1977,2187 Haloperidol chloro analog ........................................................................................................................................................................................... 837
Glyceryl tridecanoate .................................................................................................................................................................................................. 1632 Haloperidol metabolite II ..................................................................................................................................................................................... 834,837
Glyceryl trilinoleate ................................................................................................................................................................................. 390,391,392,393 Haptoglobin Human, Phenotype 1-1 ........................................................................................................................................................................ 761
Glyceryl trioleate ........................................................................................................................................................................... 390,391,392,393,1632 α-HCH (α-hexachlorocyclohexane) ......................................................................................................... 1462,1711,1855,1983,2474,2519,2662,2698
(R)-(+)-Glycidol ................................................................................................................................................................................................ 1700,2511 α-HCH (α-hexachlorocyclohexane) enantiomers ......................................................................... 1458,1707,1851,1979,2470,2515,2658,2694,
(S)-(−)-Glycidol ................................................................................................................................................................................................ 1700,2511 1459,1708,1852,1980,2471,2516,2659,2695,1460,1709,1853,1981,2472,2517,2660,2696,1461,1710,1854,1982,2473,2518,2661,2697,
R-(+)-Glycidol ................................................................................................................................................................................ 1701,2512,1702,2513 1463,1712,1856,1984,2475,2520,2663,2699,1464,1713,1857,1985,2476,2521,2664,2700
S-(-)-Glycidol .................................................................................................................................................................................. 1701,2512,1702,2513 Heliotropine ..................................................................................................................................................................................... 2111,2184,2183,2446
Glycine ................................................................................................................................................................................................................ 1897,2304 Hemocyanin ................................................................................................................................................................................................................... 749
Glycine-leucine-alanine ....................................................................................................................................................................................... 2831,783 Hemoglobin human ..................................................................................................................................................................................................... 617
Glycitein ...................................................................................................................................................................................... 1270,361,1271,362,1272 Hemoglobin S ............................................................................................................................................................................................................... 619
Glycitin ....................................................................................................................................................................................... 1270,361,1271,362,1272 Hemoglobin A0 ............................................................................................................................................................................................................. 619
Glycocholic acid ..................................................................................................................................................................................................... 237,238 Hemoglobin A2 ............................................................................................................................................................................................................. 619
Glycocholic acid hydrate .............................................................................................................................................................................................. 234 Hemoglobin S (human) .............................................................................................................................................................................................. 618
Glycodeoxycholic acid monohydrate .......................................................................................................................................................................... 234 Heneicosane (C21) ..................................................................................................................................................................................................... 1636
Glycodeoxycholic-2,2,4,4-d4 acid ................................................................................................................................................................................ 234 Heneicosanoic acid methyl ester (C21:0) ...................................................... 1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251
Glycolithocholic acid ..................................................................................................................................................................................................... 234 Heneicosanoic acid methyl ester (C21:0) at 2 wt % ...................................................................................................................... 1822,1922,2254
Glycols ....................................................................................................................................................................................................... 1703,1975,2185 2,2′,3,4,4′,5′,6-Heptabromodiphenyl ether solution .................................................................................................................................... 1450,1684
Glycoursodeoxycholic acid ........................................................................................................................................................................................... 234
Indexes Analyte
Heptachlor ............................................................ 1351,1403,1862,2000,1381,1395,1438,1924,1382,1396,1439,1925,1388,1411,1482,2028, 3,3',4,4',5,5'-Hexachlorobiphenyl .................................................................................................................................................. 1441,1927,1442,1928

1466,1740,1858,1989,2356,2384,2392,2406,1469,1994,2359,2409,1506,2075,1507,2076,1512,1513,1514,1516,1524,1532, 3,3’,4,4’,5,5’-hexachlorobiphenyl ..................................................................................................................................................................... 1443,1929
1571,1591,2325,2347,1604 3,3′,4,4′,5,5′-Hexachlorobiphenyl ............................................................................................................................................................................. 1501
Heptachlor epoxide ....... 1347,1394,1798,1894,1381,1395,1438,1924,1382,1396,1439,1925,1388,1411,1482,2028,1469,1994,2359,2409, 2,2',3,4,4',5'-Hexachlorobiphenyl (No. 138) .................................................................................................................................................. 1402,1999
1506,2075,1513,1516,1524,1532,1571,1591,2325,2347,1604,1797,1893 2,2',4,4',5,5'-Hexachlorobiphenyl (No. 153) .................................................................................................................................................. 1402,1999
Heptachlor exo-epoxide ................................................................................................................................................................ 1507,2076,1512,1514 2,3,3',4,4',5-Hexachlorobiphenyl (No. 156) ................................................................................................................................................... 1402,1999
Heptachlor expoxide ................................................................................................................................ 1466,1740,1858,1989,2356,2384,2392,2406 2,3,3',4,4',5'-Hexachlorobiphenyl (No. 157) .................................................................................................................................................. 1402,1999
2,2′,3,3′,4,4′,6-Heptachlorobiphenyl ...................................................................................................... 1388,1411,1482,2028,1506,2075,1507,2076 3,3',4,4',5,5'-Hexachlorobiphenyl (No. 169) .................................................................................................................................................. 1402,1999
2,2',3,4',5,6,6'-Heptachlorobiphenyl ............................................................................................................................................. 1441,1927,1442,1928 Hexachlorobutadiene ................................................................................. 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,
2,2’,3,4’,5,6,6’-heptachlorobiphenyl ................................................................................................................................................................ 1443,1929 1505,2074,1524,1546,1547,1552,1553,1554,1555,1557,1558,1564,1569,1589,2323,2345
2,3,3',4,4',5,5'-Heptachlorobiphenyl ............................................................................................................................................. 1441,1927,1442,1928 Hexachloro-1,3-butadiene ................................................................................................................................. 1548,1549,1556,1568,1588,2322,2344
2,3,3',4,4',5,5'-Heptachlorobiphenyl (No. 189) ............................................................................................................................................. 1402,1999 α-1,2,3,4,5,6-Hexachlorocyclohexane ...................................................... 1351,1403,1862,2000,1466,1740,1858,1989,2356,2384,2392,2406,
2,2',3,4,4',5,5'-Heptachlorobipheny (No. 180) .............................................................................................................................................. 1402,1999 1507,2076,1512,1514,1516,1889,2068
Heptadecane ............................................................................................................................................................................................. 1637,2316,2317 Hexachlorocyclopentadiene ................................................................................................................................ 1388,1411,1482,2028,1506,2075,
Heptadecane (C17) .................................................................................................................................................................................................... 1636 1507,2076,1524,1552,1553,1554,1555,1556,1557,1558,1564
Heptadecanoic acid methyl ester (C17:0) ..................................................... 1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251 Hexachloroethane .................................................................................................................. 1504,2073,1552,1553,1554,1555,1556,1557,1558,1564
Heptadecanoic acid methyl ester (C17:0) at 2 wt % ..................................................................................................................... 1822,1922,2254 Hexachloropropene ............................................................................................................................................................................................ 1550,1551
cis-10-Heptadecenoic acid methyl ester (C17:1) .......................................... 1815,1915,2247,1817,1917,2249,1818,1918,2250,1819,1919,2251 Hexachlorothane ......................................................................................................................................................................................................... 1524
cis-10-Heptadecenoic acid methyl ester (C17:1) at 2 wt % .......................................................................................................... 1822,1922,2254 Hexaconazole ..................................................................................................................................................................................................... 1889,2068
Heptanal .................................................................................................................................................................................................... 1971,2405,2099 Hexacosane .................................................................................................................................................................................................................. 1526
Heptane ................................................................ 1431,1572,1486,1487,1595,1601,1667,1602,1668,1610,2210,2211,2212,2452,2213,2214,2453 Hexadecane ............................................................................................................................................... 1431,1572,1486,1487,1526,1637,2316,2317
n-Heptane .............................................................................................................................. 1529,1568,1588,2322,2344,1592,1611,1612,1629,1630 Hexadecane (C16) ...................................................................................................................................................................................................... 1636
Heptanoic acid .......................................................................................... 1808,1908,2244,1809,1909,2245,1873,2031,2278,1970,2404,1971,2405 Hexadecanoate (C16:0) .............................................................................................................................................................................................. 1636
Heptanol .......................................................................................................................................................................................... 1490,1580,2318,2330 Hexadecanoic acid ................................................................................................................................................................................... 1873,2031,2278
1-Heptanol ................................................................................................................................................................................................................... 1895 Hexadecanolide .................................................................................................................................................................................................. 2111,2184
Heptanone .......................................................................................................................................................................................................... 1971,2405 cis-11-Hexadecenal ........................................................................................................................................................................................... 1896,2403
4-Heptanone ...................................................................................................................................................................................................... 2056,2439 Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) ...................................................................................................................................... 1456,1696,2298
Heptenophos ................................................................................................................................................................................... 1449,1683,1830,1935 Hexaldehyde-2,4-dinitrophenylhydrazone ............................................................................................................................................ 22,2334,23,2335
n-Heptyl acetate ......................................................................................................................................................................................................... 2096 Hexaldehyde-2,4-DNPH .............................................................................................................................................................................................. 2337
Heptylamine ................................................................................................................................................................................................................ 1776 Hexaldehyde (2,4-DNPH derivative) ................................................................................................................................................................... 2336,24
Heroin ............................................................................................................................................................ 428,432,2288,2289,2290,2462,2291,2463 Hexamethylene diisocyanate ...................................................................................................................................................................... 2340,27,2338
Heroin solution ............................................................................................................................................................................................................. 490 Hexamethyl phosphoramide (HMPA) ............................................................................................................................................................ 1544,1545
Hesperidin ....................................................................................................................................................................................................... 353,354,359 Hexanal ..................... 21,2326,1490,1580,2318,2330,1491,1581,2319,2331,1492,1583,2320,2333,1971,2405,2045,2350,2051,2435,2056,2439
2,3',4,4',5,5'-Hexachlorbiphenyl (No. 167) .................................................................................................................................................... 1402,1999 Hexane ........................................................................................................................................................................... 1431,1572,1486,1487,1595,
Hexachlorobenzene .............................................. 1347,1394,1798,1894,1351,1403,1862,2000,1381,1395,1438,1924,1388,1411,1482,2028, 1601,1667,1602,1668,1610,1611,1612,1676,1725,1726,1727,2111,2184,2205,2206,2207,2450,2208,2209,2451,2224
1404,2001,1506,2075,1507,2076,1524,1552,1553,1554,1555,1556,1557,1558,1564,1571,1591,2325,2347,1797,1893 n-Hexane ............................................................................................................................................................................... 1568,1588,2322,2344,1592
2,2′,4,4′,5,6′-Hexachlorobiphenyl ........................................................................................................... 1388,1411,1482,2028,1506,2075,1507,2076 1,2-Hexanediol ................................................................................................................................................................................................... 1646,2489
2,2',4,4',6,6'-Hexachlorobiphenyl .................................................................................................................................................. 1441,1927,1442,1928 1,2-Hexanediol (O-TFA derivatives) ............................................................................................................................................................... 1647,2490
2,2’,4,4’,6,6’-hexachlorobiphenyl ..................................................................................................................................................................... 1443,1929 2,3-Hexanedione ............................................................................................................................................................................................... 2056,2439
Indexes Analyte
3,4-Hexanedione ............................................................................................................................................................................................... 2056,2439 1,2,3,4,6,7,8-HpCDD ................................................................................................................................. 1436,1575,1812,1912,1437,1576,1813,1913

N,N'-1,6-Hexanediylbis(4-(2-pyridinyl)-1 piperazinecarboxamide) ........................................................................................................................... 25 1,2,3,4,6,7,8-HpCDF ................................................................................................................................. 1436,1575,1812,1912,1437,1576,1813,1913
Hexanoic acid .............................................................................................................................................................. 1970,2404,1971,2405,2045,2350 1,2,3,4,7,8,9-HpCDF ................................................................................................................................. 1436,1575,1812,1912,1437,1576,1813,1913
Hexanol ...................................................................................................................................................... 1876,2035,2396,2423,1969,2060,2443,2142 HU-210, HU-211 ((6a,10a)- 9-(Hydroxymethyl)-6,6-dimethyl-
n-Hexanol .................................................................................................................................................................................................................... 2131 3-(2-methyloctan-2-yl)- 6a,7,10,10a-tetrahydrobenzo [c]chromen-1-ol) ..................................................................................................... 449
1-Hexanol .................................................................................................................................................................................................................... 1895 HU-210, HU-211 ((6a,10a)-9-(Hydroxymethyl)-6,6-dimethyl-3-(2-methyloctan-
2-Hexanone ......................................................................................................... 1504,2073,1546,1547,1548,1549,1565,1566,1676,1725,1726,1727 20-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromen-1-ol) ....................................................................................................................................... 452
3-Hexanone ........................................................................................................................................................................................................ 2056,2439 Human Growth Hormone human .............................................................................................................................................................................. 628
Hexatriacontane ................................................................................................................................................................................................. 1486,1487 α-Humulene ................................................................................................................. 2050,2434,2096,2099,2103,2124,2131,2142,2161,2166,2170
Hexazinone ............................................................................................................................................. 5,1467,1990,2357,2407,1506,2075,1507,2076 1,2,3,4,7,8-HxCDD .................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913
(2E)-Hexenal ............................................................................................................................................................................................................... 2131 1,2,3,6,7,8-HxCDD .................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913
(E)-2-Hexenal .............................................................................................................................................................................................................. 2142 1,2,3,7,8,9-HxCDD .................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913
cis-2-Hexene ................................................................................................................................................................................................................ 1612 1,2,3,4,7,8-HxCDF ..................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913
(Z)-2-Hexenol .............................................................................................................................................................................................................. 2142 1,2,3,6,7,8-HxCDF ..................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913
(Z)-3-Hexenol .............................................................................................................................................................................................................. 2142 1,2,3,7,8,9-HxCDF ..................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913
(2E)-Hexenyl acetate ................................................................................................................................................................................................. 2131 2,3,4,6,7,8-HxCDF ..................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913
(E)-2-Hexenyl acetate ................................................................................................................................................................................................ 2142 Hyaluronidase ................................................................................................................................................................................................................ 801
(Z)-3-Hexenyl acetate ................................................................................................................................................................................................ 2142 Hydrobenzoin .......................................................................................................................................................................................... 2586,64,2587,65
(3Z)-Hexenyl butanoate ............................................................................................................................................................................................ 2131 Hydrocarbons ............................................................................................................................................................................................................... 1592
Hexobarbital ................................................................................................................................................................................. 415,926,2287,2714,843 Hydrochlorothiazide ........................................................................................................................................................................................ 130,893,851
Hexyl acetate ..................................................................................................................................................... 1876,2035,2396,2423,2060,2443,2096 Hydrocodone .......................................................................................................................................................................................................... 427,429
Hexylamine .................................................................................................................................................................................................................. 1776 Hydrocodone solution ............................................................................................................................................................................ 264,484,481,692
Hexylcinnamaldehyde ....................................................................................................................................................................................... 2180,2181 Hydrocortisone ...................................................................................................................................................................................... 199,205,1204,272
α-Hexylcinnamaldehyde ................................................................................................................................................................................... 2183,2446 Hydrocortisone 21-acetate ................................................................................................................................................................................... 203,453
Hexyl 2-ethylhexyl phthalate ....................................................................................................................................................... 1478,1769,2009,2192 Hydrogen ..................................................................................................................................................................................................................... 1639
Hexyl hexanoate ............................................................................................................................................................................. 1876,2035,2396,2423 Hydrogen sulfide ............................................................................................................................................................................................... 1687,2382
Hexyl 2-methylbutanoate ............................................................................................................................................................. 1876,2035,2396,2423 Hydrogen sulfide (1 ppm) ....................................................................................................................................................................................... 1783
Hippuric acid .......................................................................................................................................................................................................... 170,433 Hydromorphone ............................................................................................................................................................................................................ 429
Histamine ....................................................................................................................................................................................................................... 143 Hydromorphone solution .............................................................................................................................................................................. 264,484,692
D-Histidine ............................................................................................................................................................................................................ 2797,620 Hydromorphone-3-β-D-glucuronide ............................................................................................................................................................................ 692
L-Histidine .......................................................................................................................................................................................... 2797,620,1897,2304 Hydroquinidine ....................................................................................................................................................................................................... 67,1209
DL-Histidine ........................................................................................................................................................................................................... 2798,621 Hydroquinidine hydrochloride ................................................................................................................................................................................... 1218
HMX ........................................................................................................................................................................................................................ 296,465 Hydroquinone .............................................................................................................................................................................................................. 1283
DL-Homatropine .............................................................................................................................................................................. 1016,2918,1017,2919 4-Hydroxybenzoic acid .................................................................................................................................................................................. 37,279,1236
DL-Homophenylalanine ........................................................................................................................................................................................ 2799,622 (2S,3S)-Hydroxybupropion hydrochloride .......................................................................................................................................... 132,907,1189,231
Homovanillic acid .................................................................................................................................................................... 1279,370,87,1281,372,89 3-Hydroxybutyric acid methyl ester ............................................................................................................................................ 1714,2265,2522,2626
Homoveratric acid .......................................................................................................................................................................................... 1280,371,88 2-Hydroxycaproic acid methyl ester ........................................................................................................................................... 1715,2266,2523,2627
Hotrienol ......................... 1878,2044,2398,2430,1879,2046,2399,2431,1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436, HYDROXYCHLOROQUINE ............................................................................................................................................................................................... 1019,2920
1883,2054,2402,2437 6-Hydroxychlorzoxazone ............................................................................................................................................................................................... 963
Indexes Analyte
25-Hydroxycholecalciferol ................................................................................................................................................................................... 1318,241 2-Hydroxy-2-trifluoromethyl propionic acid ethyl ester ............................................................................................................................. 1719,2527
Hydroxycitronellal .............................................................................................................................................................................................. 2180,2181 2-Hydroxy-2-trifluoromethyl propionic acid methyl ester .......................................................................................................................... 1720,2528
trans-3'-Hydroxycotinine .............................................................................................................................................................................................. 246 5-Hydroxytryptophan ..................................................................................................................................................................................... 190,191,192
2-Hydroxy-7,12-dimethylbenz[a]anthracene (t-diol) ................................................................................................................................ 175,304,438 5-Hydroxy-L-tryptophan ............................................................................................................................................................................................... 467
β-(Hydroxyethyl)theophylline ...................................................................................................................................................................................... 149 1α-Hydroxyvitamin D3 ....................................................................................................................................................................... 1323,260,1324,261
5-Hydroxyflavone .......................................................................................................................................................................................................... 352 3-epi-25-Hydroxyvitamin D3 ......................................................................................... 1247,159,1248,160,1249,161,1269,172,1323,260,1324,261
7-Hydroxyflavone .......................................................................................................................................................................................................... 352 25-Hydroxyvitamin D2 ........................................................................................................................................................................................ 1318,241
L-α-Hydroxyglutaric acid .............................................................................................................................................................................................. 240 25-Hydroxyvitamin D2 solution .................................................................................... 1247,159,1248,160,1249,161,1268,171,1319,242,1323,260
D-α-Hydroxyglutaric acid disodium salt ................................................................................................................................ 255,2649,2843,3041,821 25-Hydroxyvitamin D3 solution .................................................. 1247,159,1248,160,1249,161,1268,171,1269,172,1319,242,1323,260,1324,261
DL-α-Hydroxyglutaric acid disodium salt ................................................................................................................................................................... 240 3-epi-25-Hydroxyvitamin D2 solution .............................................................................................................................................. 1323,260,1324,261
L-α-Hydroxyglutaric acid disodium salt ................................................................................................................................. 255,2649,2843,3041,821 Hydroxyzine dihydrochloride ............................................................................................................................................................................ 1021,2921
5-Hydroxyindoleacetic acid ........................................................................................................................................................................... 190,191,192 Hyperoside .......................................................................................................................................................................................................... 1267,1330
17β-Hydroxy-17-methylandrosta-1,4-dien-3-one .................................................................................................................................................... 195 Hypoxanthine ................................................................................................................................................................................................................. 690
2-Hydroxy-2-methyl butyric acid ethyl ester ............................................................................................................................ 1716,2267,2524,2628
2-Hydroxy-2-methyl butyric acid methyl ester ........................................................................................................................ 1717,2268,2525,2629 —I—
5-Hydroxymethyl-2-furaldehyde .............................................................................................................................................................................. 59,60 Ibuprofen ................................................................................................................................................................... 850,851,899,1022,2922,1093,1094
3-Hydroxy-5-methylisoxazole .......................................................................................................................................................................... 1890,2069 (R)-(−)-Ibuprofen .......................................................................................................................................................................... 2218,2270,2631,2855
7-Hydroxymethyl-12-methylbenz[a]anthracene ......................................................................................................................................... 175,304,438 (S)-(+)-Ibuprofen .......................................................................................................................................................................... 2218,2270,2631,2855
7-Hydroxymethyl-12-methylbenz[a]anthracene-trans-3,4-diol ................................................................................................................ 175,304,438 Icariin (PHY89714) ..................................................................................................................................................................................................... 1267
7-Hydroxymethyl-12-methylbenz[a]anthracene-trans-8,9-diol ................................................................................................................ 175,304,438 Icaritin .......................................................................................................................................................................................................................... 1330
7-Hydroxymethyl-12-methylbenz[a]anthracene-trans-10,11-diol ............................................................................................................ 175,304,438 Icaritin (PHY80478) .................................................................................................................................................................................................... 1267
7-Hydroxymethyl-12-methylbenz[a]anthracen-2-ol .................................................................................................................................. 175,304,438 Icarrin ........................................................................................................................................................................................................................... 1330
7-Hydroxymethyl-12-methylbenz[a]anthracen-3-ol .................................................................................................................................. 175,304,438 Ifosfamide ........................................................................................................................................................................................................... 1023,2923
7-Hydroxymethyl-12-methylbenz[a]anthracen-4-ol .................................................................................................................................. 175,304,438 Imazalil ......................................................................................................................................................................... 1347,1394,1798,1894,1797,1893
8-(Hydroxymethyl)-7-methyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4(3H,10H)- dione, Compound D ....... 1219,274 Imidacloprid .................................................................................................................................................................................................... 1358,19,319
4-Hydroxy-4-methyl-2-pentanone .................................................................................................................. 1492,1583,2320,2333,1676,1726,1727 Imidan ................................................................................................................................................................................................................ 2004,2413
2-(Hydroxymethyl)piperidine ........................................................................................................................................................................... 1584,2341 Imipramine .............................................................. 1368,209,1164,210,1166,211,1167,212,239,811,1165,1168,1169,1170,2279,2303,2459,2468
Hydroxymetoprolol .............................................................................................................................................................................................. 894,1020 Imipramine hydrochloride ......................................................................................................................................................................................... 1206
2-Hydroxyoctanoic acid methyl ester ......................................................................................................................................... 1718,2269,2526,2630 Imiquimod ..................................................................................................................................................................................................... 133,913,1214
5-Hydroxyomeprazole ................................................................................................................................................................... 1372,247,248,249,250 Immunoglobulin G ......................................................................................................................................................................................... 675,676,678
4-Hydroxyphencyclidine ............................................................................................................................................................................................... 867 Immunoglobulin G (human) ...................................................................................................................................................................................... 494
4-Hydroxyphenolarsonic acid .......................................................................................................................................................................................... 4 Impurity ............................................................................................................... 1446,1680,1827,1932,1447,1681,1828,1933,1448,1682,1829,1934
L-4-Hydroxyproline ............................................................................................................................................................................................ 1897,2304
Indan ................................................................................................................................................................................................................... 1612,1621
Hydroxyrisperidon ................................................................................................................................................................................. 132,907,1189,231 Indane .......................................................................................................................................................................................................................... 1610
N-Hydroxysuccinimide .................................................................................................................................................................................................. 494 Indanofan ........................................................................................................................................................................................................... 1890,2069
4-Hydroxytamoxifen (I) ............................................................................................................................................................................................. 1152 Indapamide ............................................................................................................................................................. 985,986,1014,1024,2924,1025,2925
4-Hydroxytamoxifen (II) ............................................................................................................................................................................................ 1152 Indeno[1,2,3-cd]pyrene ................................................................................................... 2375,310,311,1387,1410,1479,2012,1388,1411,1482,2028
6β-Hydroxytestosterone solution ................................................................................................................................................................................ 258 Indeno[1,2,3-cd]pyrene ........................................................................................................................... 1408,2010,1409,2011,1480,2013,1481,2014
β-Hydroxytheophylline ........................................................................................................................................................................................ 1315,802 Indeno[1,2,3-cd]pyrene ............................................................................................... 1483,2029,2365,2420,1506,2075,1507,2076,1517,1518,1524
Indexes Analyte
Indeno[1,2,3-cd]pyrene .............................................................................................................................................................................................. 1540 Isobergapten ................................................................................................................................................................................................................ 1262

Indeno[1,2,3-cd]pyrene ..................................................................................................................................... 1541,1552,1553,1554,1555,1556,1557 Isobornyl acetate ..................................................................................................................................................................................... 2050,2434,2096
Indeno(1,2,3-cd)pyrene .............................................................................................................................................................................................. 1558 Isobutanal .................................................................................................................................................................................................................... 1969
Indeno[1,2,3-cd]pyrene .................................................................................................................................................................................... 1560,1561 Isobutane ..................................................................................................................................................................................................................... 1592
Indeno[1,2,3-cd]pyrene .............................................................................................................................................................................................. 1562 Isobutanol ..................................................................................................................................................................... 1725,1969,2026,2027,2060,2443
Indeno[1,2,3-cd]pyrene .............................................................................................................................................................................................. 1563 Isobutyl acetate ............................................................................... 1675,1676,1725,1726,1727,1780,1969,2210,2211,2212,2452,2213,2214,2453
Indeno[1,2,3-cd]pyrene .............................................................................................................................................................................................. 1564 Isobutyl alcohol ........................................................................................................................................................................................................... 1780
Indeno[1,2,3-cd]pyrene .................................................................................................................................................................................... 1567,1587 Isobutylbenzene .......................................................................................................................................................................................................... 1612
Indeno[1,2,3-cd]pyrene .................................................................................................................................................................................... 1795,1891 2-Isobutyl-3-methoxypyrazine ................................................................................................................ 1488,2038,2368,2424,1499,2070,2376,2445
Indeno[1,2,3-c,d]pyrene solution ................................................................................................................................................. 1434,1435,1451,1936 Isobutyl 2-methylbutenoate ......................................................................................................................................................... 1876,2035,2396,2423
Indigo carmine .............................................................................................................................................................................................................. 388 Isobutyric acid .................................................................................................... 1808,1908,2244,1809,1909,2245,1873,2031,2278,1969,1970,2404
Indomethacin .................................................................................................................................................................................................. 899,900,901 Isocaproic acid ................................................................................................................................ 1808,1908,2244,1809,1909,2245,1873,2031,2278
Indoprofen .......................................................................................................................................................................................................... 1026,2926 Isocarbophos ................................................................................................................................................................ 1350,1401,1860,1995,1887,2066
Indoxacarb .................................................................................................................................................................... 1445,1679,1826,1931,1888,2067 Isocyanic acid .............................................................................................................................................................................................................. 2338
Inhibin α-Subunit Fragment 1-32 human ............................................................................................................................................................... 632 Isodrin ................................................................................................................................................................................................................. 1550,1551
Inhibin α-Subunit Fragment 1-32 porcine ............................................................................................................................................................... 633 Isofenphos .............................................................................................................................. 1430,1641,1796,1892,1444,1678,1825,1930,1885,2064
Inosine .................................................................................................................................................................................................................... 686,690 Isoleptospemone ......................................................................................................................................................................................................... 2124
Inosine monophosphate ............................................................................................................................................................................................... 690 D-Isoleucine .......................................................................................................................................................................................................... 2801,641
Inositol ................................................................................................................................................................................................................ 2036,2037 L-Isoleucine ........................................................................................................................................................................................ 2801,641,1897,2304
Insulin from bovine pancreas .......................................................................................................................................................... 1028,636,1031,639 DL-Isoleucine ......................................................................................................................................................................................................... 2800,640
Insulin human ............................................................................................................................................................................. 635,1028,636,1031,639 DL-allo-Isoleucine ................................................................................................................................................................................................. 2737,507
Insulin from porcine pancreas ......................................................................................................................................................... 1028,636,1031,639 DL-Isoleucine (FMOC derivatives) ...................................................................................................................................................................... 2781,596
Insulin, bovine .............................................................................................................................................................................................. 559,1030,638 (−)-Isomenthol .............................................................................................................................................................................. 2126,2189,2655,2678
Insulin, human ................................................................................................................................................................................... 1029,637,1030,638 (+)-Isomenthol .............................................................................................................................................................................. 2126,2189,2655,2678
Insulin, porcine ................................................................................................................................................................................... 1029,637,1030,638 Isomenthone ................................................................................................................................................................................................................ 2112
2-Iodobutane .................................................................................................................................................................................. 1706,2217,2514,2854 d-Isomenthone ............................................................................................................................................................................................................ 2136
Iodomethane ................................................................................................................................................................................... 1546,1547,1548,1549 α-Isomethylionone ...................................................................................................................................................................................................... 2181
Iodophenfos ..................................................................................................................................................................................... 1350,1401,1860,1995 Isooctane (solvent) ......................................................................................................................... 1613,1614,1615,1616,1617,1618,1624,1625,1626
α-Ionone ..................................................................................................................................................................................................... 322,2111,2184 Isopentanal .................................................................................................................................................................................................................. 1969
Ionox 100 ............................................................................................................................................................................................ 276,337,1898,2182 Isopentane .......................................................................................................................................................................................................... 1592,1721
Ipratropium .................................................................................................................................................................................................................... 119 Isopentanol ......................................................................................................................................................................................................... 2060,2443
Ipratropium bromide monohydrate ......................................................................................................................................................................... 1134 Isopentyl acetate .................................................................................................................................................................. 1675,1676,1726,1727,1895
Iprobenfos .......................................................................................................................................................................................... 1419,406,1888,2067 Isophenfos ....................................................................................................................................................................................... 1350,1401,1860,1995
Iprodione ................................................................................................................................................... 1430,1641,1796,1892,1444,1678,1825,1930 Isophenfos-methyl .......................................................................................................................................................................... 1350,1401,1860,1995
Iprodione metabolite ........................................................................................................................................................................................ 1889,2068 Isophorone ................................................................................................. 1506,2075,1507,2076,1524,1552,1553,1554,1555,1556,1557,1558,1564
Isazofos ............................................................................................................................................................................................ 1350,1401,1860,1995 Isophorone diisocyanate (isomer 1) ........................................................................................................................................................................ 2338
Isazophos ............................................................................................................................................................................................................ 1885,2064 Isophorone diisocyanate (isomer 2) ........................................................................................................................................................................ 2338
Isoamyl acetate .................................................................................................................................................. 1725,1780,1876,2035,2396,2423,1969 Isopinocampheol ................................................................................................................................................................................................ 1642,2487
Isoamyl alcohol ..................................................................................................................................................................... 1725,2026,2027,2060,2443 Isopromethazine ................................................................................................................................................................................................ 1032,2927
Indexes Analyte
Isopropamide iodide ................................................................................................................................................................................................... 1134 JWH-018 5-Hydroxypentyl metabolite solution .................................................................................................................................... 1225,838,1226
Isopropanol ............................................................................................................................. 1431,1572,1648,2379,1725,1969,2224,2226,2456,2293 JWH-018 (Naphthalen-1-yl-(1-pentylindol-3-3yl)methanone) .............................................................................................................................. 452
Isopropyl acetate ................................................................................................ 1675,1676,1725,1726,1727,2210,2211,2212,2452,2213,2214,2453 JWH-018 5-Pentanoic acid metabolite solution .................................................................................................................... 450,451,1225,838,1226
Isopropyl alcohol ........................................................................................................................................................................................................ 1780 JWH-019 ...................................................................................................................................................................................................................... 1226
4-Isopropylaniline .............................................................................................................................................................................................. 1655,1672 JWH-073 ...................................................................................................................................................................................................................... 1226
Isopropylbenzene ....................................... 1493,2057,2370,2440,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074 JWH-073 solution .................................................................................................................................................................................. 450,451,449,482
Isopropylbenzene (Cumene) .......................................................................................................... 1613,1614,1615,1616,1617,1618,1624,1625,1626 JWH-073 (328.18 m/z) ..................................................................................................................................................................................... 1227,839
2-Isopropyl-3-methoxypyrazine .............................................................................................................. 1488,2038,2368,2424,1499,2070,2376,2445 JWH-073 4-Butanoic acid metabolite solution ...................................................................................................................... 450,451,1225,838,1226
p-Isopropyltoluene ............................................................................................................................................................................................. 1504,2073 JWH-073 3-hydroxybutyl metabolite ................................................................................................................................................................. 450,451
(±)-Isoproterenol hemisulfate salt ................................................................................................................................................................. 1033,2928 JWH-073 3-Hydroxybutyl metabolite ........................................................................................................................................................................ 452
Isoprothiolane .............................................................................................................................................................. 1430,1641,1796,1892,1886,2065 JWH-073 3-Hydroxybutyl metabolite ............................................................................................................................................................ 1225,838
Isoproturon ................................................................................................................................................................................. 5,110,15,111,16,314,375 JWH-073 3-Hydroxybutyl metabolite ...................................................................................................................................................................... 1226
Isoquercitrin ............................................................................................................................................................................................................ 376,377 JWH-073 3-Hydroxybutyl metabolite solution ................................................................................................................................................. 449,482
Isosafrole ............................................................................................................................................................................................................. 1550,1551 JWH-073 4-Hydroxybutyl metabolite solution ...................................................................................................................................... 1225,838,1226
Isosafrole-2 .................................................................................................................................................................................................................. 1550 JWH-073 (Naphthalen-1-yl-(1-butylindol-3-yl)methanone) .................................................................................................................................. 452
Isosilybin A ............................................................................................................................................................................................... 1266,1276,1288 JWH-122 4-Hydroxypentyl metabolite solution ............................................................................................................................................. 1225,838
Isosilybin B ............................................................................................................................................................................................... 1266,1276,1288 JWH-122 5-Hydroxypentyl metabolite solution ............................................................................................................................................. 1225,838
Isovaleraldehyde .................................................................................................................................................................................................... 21,2326 JWH-200 (1-(2-Morpholin-4-ylethyl)indol-3-yl)-naphthalen-1-ylmethanone) .................................................................................................... 449
Isovaleric acid ............................................................................................................... 1808,1908,2244,1809,1909,2245,1873,2031,2278,1970,2404 JWH-200 ((1-(2-Morpholin-4-ylethyl)indol-3-yl)-naphthalen-1-ylmethanone) ........................................................................................... 452,482
isovitexin (PHY89233) ............................................................................................................................................................................................... 1305 JWH-203 (340.164 m/z) ................................................................................................................................................................................... 1227,839
Isoxathion ........................................................................................................................................................................................................... 1887,2066 JWH-210 (370.23 m/z) ..................................................................................................................................................................................... 1227,839
Itraconazole solution ............................................................................................................................................................................................. 831,832 JWH-210 4-Hydroxypentyl metabolite solution ............................................................................................................................................. 1225,838
IVYIHPR ................................................................................................................................................................................................................... 531,932 JWH-210 5-Hydroxypentyl metabolite solution ............................................................................................................................................. 1225,838
JWH-250 4-Hydroxypentyl metabolite ............................................................................................................................................................ 1225,838
—J— JWH-250 5-Hydroxypentyl metabolite ........................................................................................................................................................ 1225,838
Jacobsen Catalyst (N,N'-Bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminomanganese(III) chloride) ............................ 2588,69,2589,70 JWH-250 (2-(2-Methoxyphenyl)-1-(1-pentylindol-3-yl)ethanone) ....................................................................................................................... 449
(Z)-Jasmone ................................................................................................................................................................................................................ 2167 JWH-250 (2-(2-Methoxyphenyl)-1-(1-penylindol-3-yl)ethanone) ................................................................................................................. 452,482
cis-Jasmone ........................................................................................................................................................................................................ 2111,2184
(E)-Jasmone ................................................................................................................................................................................................................ 2131
—K—
(Z)-Jasmone ....................................................................................................................................................................................................... 2137,2138 Kaempferol-3-O-glycoside ......................................................................................................................................................................................... 1275
Jet fuel ......................................................................................................................................................................................................................... 1622 40 kDa PEG molecule attached to protein identified as peak 1 ........................................................................................................................ 777
JMH-081 ...................................................................................................................................................................................................................... 1226 Kepone ......................................................................................................................................................................................................................... 1550
JMH-122 ...................................................................................................................................................................................................................... 1226 Kerosene ...................................................................................................................................................................................................................... 1620
JWH-015 (328.18 m/z) ..................................................................................................................................................................................... 1227,839 (±)-Ketamine hydrochloride .................................................................................................................................................................... 1034,2929,436
JWH-018 ...................................................................................................................................................................................................................... 1226 11-Keto-β-boswellic acid ........................................................................................................................................... 1325,1326,1334,1335,1336,1337
JWH-018 solution .................................................................................................................................................................................. 450,451,449,482 Ketoconazole .............................................................................................................................................................................. 112,2614,157,1036,2931
JWH-018 4-hydroxypentyl metabolite ...................................................................................................................................................................... 450 Ketoconazole solution ........................................................................................................................................................................................... 831,832
JWH-018 4-Hydroxypentyl metabolite ................................................................................................................................................... 1225,838,1226 Ketoprofen ........................................................................................................................... 173,2636,2722,437,899,1037,2932,1038,2933,1093,1094
JWH-018 4-Hydroxypentyl metabolite solution ....................................................................................................................................................... 451 Ketorolac tris salt .............................................................................................................................................................................................. 1039,2934
Indexes Analyte
Kitazine ............................................................................................................................................................................................ 1350,1401,1860,1995 Leu-enkephalin ........................................................................................................................................................................................ 709,710,720,721

Krypton ........................................................................................................................................................................................................................ 1745 [Leu5]-Enkephalin ......................................................................................................................................................................................................... 719
KVYIHPI ................................................................................................................................................................................................................... 531,932 Levamisol hydrochloride ....................................................................................................................................................................................... 455,456
Levamisole ....................................................................................................................................................................................... 2290,2462,2291,2463
—L— (−)-Levamisole hydrochloride solution ..................................................................................................................................................................... 454
Labetalol ........................................................................................................................................................................................................................ 131 Levofloxacin ............................................................................................................................................................................................................ 880,881
Labetalol hydrochloride .................................................................................................................................................................................... 1040,2935 Levothyroxine sodium hydrate ................................................................................................................................................................................... 174
α-Lactalbumin from bovine milk ............................................................................................................................................................................... 744 Lidocaine .................................................................................................................................................................................... 125,872,1187,2288,2289
Lactic acid ............................................................................................................................................... 1273,2590,3036,71,1277,368,85,1278,369,86 Lidocaine solution ............................................................................................................................................... 1370,229,230,1378,266,267,1379,268
D-(−)-Lactic acid ................................................................................................................................................................................................ 244,2648
Lignoceric acid methyl ester ..................................................................................................................................... 1818,1918,2250,1819,1919,2251
L-(+)-Lactic acid ................................................................................................................................................................................................. 244,2648
Lignoceric acid methyl ester (C24:0) ................................................................................................... 1815,1915,2247,1817,1917,2249,1842,1962
DL-Lactic acid ................................................................................................................................................................ 68,1274,2591,2706,3037,363,72
Lignoceric acid methyl ester (C24:0) at 4 wt % ............................................................................................................................. 1822,1922,2254
L-Lactic Dehydrogenase from rabbit muscle ............................................................................................................................................................ 761
Lime oil ....................................................................................................................................................................................................................... 2106
β-Lactoglobulin (bovine), carboxymethylated, tryptic digest ................................................................................................................................ 642 Limonene ............................................................. 1574,2328,2047,2175,2351,2353,2095,2096,2103,2121,2131,2137,2138,2142,2166,2167,2180
β-Lactoglobulin tryptic digest ..................................................................................................................................................................................... 812 (R)-(+)-Limonene ....................................................... 1431,1572,1491,1581,2319,2331,2050,2434,2056,2439,2112,2120,2170,2181,2183,2446
Lactones ........................................................................................................................................................................................... 1729,2118,2529,2676 d-Limonene ................................................................................................................................................................................................................. 2106
Lactose .................................................................................................................................................................................................................... 380,387 l-Limonene .................................................................................................................................................................................................................. 2136
α-Lactose ................................................................................................................................................................................................................ 384,385 δ-Limonene ........................................................................................................................................................................................................ 2099,2105
Lamivudine .................................................................................................................................................................................................................... 911 cis-Limonene epoxide ................................................................................................................................................................................................ 2170
Lansoprazole ................................................................................................................................................................................... 1042,1043,2936,1173 trans-Limonene epoxide ............................................................................................................................................................................................ 2170
Lasalocid A ........................................................................................................................................................................................................... 215,1174 Limonene oxide ........................................................................................................................................ 1730,1986,2125,2188,2530,2654,2677,2701
Lauric acid methyl ester ............................................................................................................................................ 1818,1918,2250,1819,1919,2251 cis-Limonene oxide ..................................................................................................................................................... 2050,2434,2096,2103,2120,2166
Lauric acid methyl ester (C12:0) .................................................................... 1815,1915,2247,1817,1917,2249,1836,1945,1837,1954,1839,1959 trans-Limonene oxide .......................................................................................................................................................... 2096,2103,2120,2131,2166
Lauric acid methyl ester (C12:0 ..................................................................................................................................................................... 1844,1964 l-cis Limonene oxide ............................................................................................................................... 1730,1986,2125,2188,2530,2654,2677,2701
Lauric acid methyl ester (C12:0) at 4 wt % .................................................................................................................................... 1822,1922,2254 l-trans Limonene oxide ........................................................................................................................... 1730,1986,2125,2188,2530,2654,2677,2701
Lauric aldehyde ....................................................................................................................................................................................... 2099,2105,2170 Linalol ........................................................................................................................................................................................................................... 2103
Lauryl gallate ....................................................................................................................................................................................................... 1282,292 Linalool ....................................................................................................................................................... 2050,2434,2056,2439,2095,2096,2099,
2105,2106,2112,2120,2121,2124,2131,2136,2137,2138,2142,2161,2166,2167,2170,2180,2181,2183,2446
Lavandulol .......................................................................................................................................................................................................... 2111,2184
α-Linalool ........................ 1878,2044,2398,2430,1879,2046,2399,2431,1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436,
LC .................................................................................................................................................................................................................................... 768
1883,2054,2402,2437
Lecithinase-A ................................................................................................................................................................................................................. 704
cis-Linalool oxide .............................................................................................................................................................................................. 2131,2142
Lemon oil .................................................................................................................................................................................................................... 2120
Linalyl acetate ................................................................................................................................. 2056,2439,2096,2111,2184,2131,2142,2183,2446
Lenacil ................................................................................................................................................................................................................. 1890,2069
Linalyl isobutanoate ................................................................................................................................................................................................... 2120
Leptophos ..................................................................................................................................................................... 1542,1543,1544,1545,1890,2069
Linalyl propionate .................................................................................................................................................................................... 2096,2131,2142
Leptospermone ............................................................................................................................................................................................................ 2124
Lindane ................. 1347,1394,1798,1894,1351,1403,1862,2000,1381,1395,1438,1924,1382,1396,1439,1925,1404,2001,1797,1893,1889,2068
D-Leucine .............................................................................................................................................................................................................. 2803,644
Lindane solution ............................................................................................................................................................................. 1507,2076,1512,1514
L-Leucine ............................................................................................................................................................................................ 2803,644,1897,2304
Linoleate (C18:2) ........................................................................................................................................................................................................ 1636
DL-Leucine ............................................................................................................................................................................................................. 2802,643
Linoleic acid .................................................................................................................................................................................................................. 357
DL-Leucine (FMOC derivatives) .......................................................................................................................................................................... 2782,597
Linoleic acid methyl ester ......................................................................................................................................... 1818,1918,2250,1819,1919,2251
Leu-Enk .......................................................................................................................................................................................................................... 788
Indexes Analyte
Linoleic acid methyl ester (C18:2) ................................................................................................................................................................ 1842,1962 Lycopene ..................................................................................................................................................................................................... 1239,344,1317
Linoleic acid methyl ester (C18:2n6c) ............................................................ 1637,1815,1915,2247,1817,1917,2249,1836,1945,1837,1954, Lypressin ........................................................................................................................................................................................................................ 820
1839,1959,1840,1960,1844,1964,1845,1965,1846,1966,1847,1967 D-Lysine ................................................................................................................................................................................................................ 2805,648
Linoleic acid methyl ester (C18:2n6c) at 2 wt % ........................................................................................................................... 1822,1922,2254 L-Lysine .............................................................................................................................................................................................. 2805,648,1897,2304
Linolelaidic acid ............................................................................................................................................................................................................ 357 DL-Lysine ............................................................................................................................................................................................................... 2806,649
Linolelaidic acid methyl ester ................................................................................................................................... 1818,1918,2250,1819,1919,2251 DL-Lysine (FMOC derivatives) ............................................................................................................................................................................ 2783,598
Linolelaidic acid methyl ester (C18:2n6t) ............................................................................................................... 1815,1915,2247,1817,1917,2249 Lysozyme ................................................................................................................................................................................................. 754,755,756,759
Linolelaidic acid methyl ester (C18:2n6t) at 2 wt % ...................................................................................................................... 1822,1922,2254 Lysozyme from chicken egg white .............................................................................. 651,705,732,733,738,743,1130,746,747,761,762,1184,805
Linolenate (C18:3) ...................................................................................................................................................................................................... 1636
α-Linolenic acid methyl ester ................................................................ 1637,1818,1918,2250,1819,1919,2251,1840,1960,1846,1966,1847,1967 —M—
γ-Linolenic acid methyl ester ................................................................................................................................... 1818,1918,2250,1819,1919,2251
Malathion .............................................................. 1350,1401,1860,1995,1351,1403,1862,2000,1466,1740,1858,1989,2356,2384,2392,2406,
Linolenic acid methyl ester (C18:3) .............................................................................................................................................................. 1842,1962 1471,1746,1861,1997,2361,2386,2393,2411,1542,1543,1544,1545,1585,2342,1885,2064
Linolenic acid methyl ester (C18:3n3) ................................................................................................. 1836,1945,1837,1954,1839,1959,1844,1964 Maleic acid .............................................................................................................................................................................................. 850,890,891,954
α-Linolenic acid methyl ester (C18:3n3) ................................................................................................................ 1815,1915,2247,1817,1917,2249 Malic acid ........................................................................................................................................................................................................................ 68
α-Linolenic acid methyl ester (C18:3n3) at 2 wt % ....................................................................................................................... 1822,1922,2254 Maltitol ........................................................................................................................................................................................................................... 382
γ-Linolenic acid methyl ester (C18:3n6) ................................................................................................................ 1815,1915,2247,1817,1917,2249 Maltoheptaose ............................................................................................................................................................................................................... 364
α-Linolenic acid methyl ester (C18:3n6) at 2 wt % ....................................................................................................................... 1822,1922,2254 Maltohexaose ................................................................................................................................................................................................................. 364
Linuron ..................................................................................................................... 1,295,5,110,15,111,16,294,302,2374,303,1391,305,314,375,317 Maltopentaose ............................................................................................................................................................................................................... 364
Lipoxidase from Glycine max (soybean) ................................................................................................................................................................... 646 Maltose .................................................................................................................................................................................................... 364,384,385,386
Lisinopril ................................................................................................................................................................................................................. 129,892 Maltose solution ........................................................................................................................................................................................................... 387
Lithocholic acid ...................................................................................................................................................................................................... 141,234 D-(+)-Maltose monohydrate ...................................................................................................................................................................................... 380
LLLn ......................................................................................................................................................................................................................... 392,393 Maltotetraose ................................................................................................................................................................................................................. 364
LLnLn ...................................................................................................................................................................................................................... 392,393 Maltotriose ............................................................................................................................................................................................... 364,384,385,386
LnPP ............................................................................................................................................................................................................................... 391 Malvidin 3-(6-acetylglucoside) ................................................................................................................................................................................... 336
Lomefloxacin ................................................................................................................................................................. 1004,355,1005,356,878,880,881 Malvidin 3-(6-caffeoylglucoside) ................................................................................................................................................................................ 336
Lopinavir ........................................................................................................................................................................................................................ 911 Malvidin chloride .............................................................................................................................................................................................. 1320,1321
Lorazepam solution ............................................................................................................................................................................................. 833,1224 Malvidin 3-(6-cumarylglucoside) ................................................................................................................................................................................ 336
(±)-Lorazepam ........................................................................................................................................... 1191,233,1044,2937,1045,2938,1046,2939 Malvidin 3-glucoside .................................................................................................................................................................................................... 336
Lorazepam glucuronide solution ....................................................................................................................................................................... 833,1224 DL-Mandelic acid .............................................................................................................................................................................. 1053,2656,288,2940
Losartan potassium .................................................................................................................................................................................................... 1211 Mannitol ............................................................................................................................................................................................................. 2036,2037
Lovastatin ...................................................................................................................................................................................................... 1148,194,964 Mannose ................................................................................................................................................................................................................. 364,383
LSD ............................................................................................................................................................................................................ 2291,2463,2301 D-(+)-Mannose ............................................................................................................................................................................................................. 379
LSD TMS ...................................................................................................................................................................................................................... 2301 Maysin .......................................................................................................................................................................................................................... 1246
Luciferin ................................................................................................................................................................................... 2592,2804,647,73,2593,74 MCHM isomer #1 ............................................................................................................................................................................................. 1465,2355
Lufenuron ............................................................................................................................................................................................................. 1419,406 MCHM isomer #2 ............................................................................................................................................................................................. 1465,2355
Lumiflavine (7,8,10-trimethylbenzo[g]pteridine-2,4(3H,10H)-dione), Compound B. ................................................................................ 1219,274 MCPA-thioethyl ................................................................................................................................................................................................. 1889,2068
Luteinizing hormone releasing hormone ........................................................................................................................................................... 710,721 MCPB .................................................................................................................................................................................................................. 1890,2069
Luteinizing hormone releasing hormone (LHRH) ............................................................................................................................................ 709,720 MCPB-ethyl ........................................................................................................................................................................................................ 1890,2069
Luteolin .......................................................................................................................................................................................................................... 352 MDA ................................................................................................................................................................................................. 2234,2285,2457,2460
Indexes Analyte
MDAI hydrochloride solution ............................................................................................................................................................................... 472,476 Mephedrone hydrochloride .............................................................................................................................................................................. 2049,2433

MDA (3,4-methylenedioxyamphetamine) .......................................................................................................................................................... 411,412 Mephedrone hydrochloride solution ............................................................................................................................................. 243,468,434,472,476
MDEA ............................................................................................................................................................................................... 2234,2285,2457,2460 (±)-Mephenytoin .............................................................................................................................................................................................. 1054,2941
MDMA ............................................................................................................................................................................................. 2234,2285,2457,2460 Mephobarbital ................................................................................................................................................ 414,925,417,929,418,930,928,1055,2942
(±)-MDMA solution .................................................................................................................................................................................................... 481 (±)-Mephobarbital ..................................................................................................................................................................... 415,926,2287,2714,843
MDMA (Ecstacy, methylenedioxymethamphetamine) .............................................................................................................................. 411,412,427 Mephobarbital (methylphenobarbital) ........................................................................................................................................................... 1064,2951
MDPBP hydrochloride solution ................................................................................................................................................................................... 469 Mepivacaine hydrochloride ..................................................................................................................................................................... 1056,2943,1187
Mecarbam ............................................................................................................................................................................................................ 1419,406 Mepronil ............................................................................................................................................................................................................. 1888,2067
Meclizine ........................................................................................................................................................................................................................ 860 Meptazinol ......................................................................................................................................................................................................... 1057,2944
Mecoprop methyl ester ................................................................................................................................................................ 1430,1641,1796,1892 Mercarbam ......................................................................................................................................................................................................... 1887,2066
Mefanamic acid ............................................................................................................................................................................................................ 899 Merphos .................................. 1466,1740,1858,1989,2356,2384,2392,2406,1471,1746,1861,1997,2361,2386,2393,2411,1507,2076,1542,1543
Mefenacet ........................................................................................................................................................................................................... 1889,2068 Mesitylene ................................................................................................. 1527,1546,1547,1548,1549,1568,1588,2322,2344,1610,1611,1612,1621
Melamine .................................................................................................................................................................. 397,398,405,2078,2079,2080,2081 Mesityl oxide ......................................................................................................................................................................... 1676,1725,1726,1727,1780
Mellitin ........................................................................................................................................................................................................................... 788 Mesoridazine .......................................................................................................................................................................................................... 165,578
Membrane protein ....................................................................................................................................................................................................... 663 Mesoridazine benzenesulfonate ................................................................................................................................................................................ 1212
Menadione (K3) ................................................................................................................................................................................................ 1252,1298 Metalaxyl .............................................................................................................................................................. 319,1430,1641,1796,1892,1888,2067
trans-p-Mentha-2,8-dien-1-ol .................................................................................................................................................................................. 2131 Metanephrine ................................................................................................................................................................................................................ 254
cis-p-Menth-2-en-1-ol ..................................................................................................................................................................................... 2131,2142 Metaproterenol hemisulfate salt ..................................................................................................................................................................... 1058,2945
trans-p-Menth-2-en-1-ol ........................................................................................................................................................................................... 2131 Metaxaxil ......................................................................................................................................................................................... 1444,1678,1825,1930
Menthofuran ...................................................................................................................................................................................................... 2136,2137 Metazachlor ................................................................................................................................................................................. 5,1447,1681,1828,1933
Menthol ....................................................................................................................................................................................................................... 2095 Met-enkephalin ............................................................................................................................................................................... 709,710,719,720,721
(−)-Menthol ......................................................................................................................................................................... 2126,2189,2655,2678,2136 Methabenzthiazuron ......................................................................................................................................................................................... 5,1419,406
Menthol ................................................................................................................................................................................. 2137,2138,2167,2183,2446 Methacrifos ................................................................................................................................................................... 1440,1677,1824,1926,1885,2064
iso-Menthol ........................................................................................................................................................................................................ 2137,2138 Methacrolein-2,4-dinitrophenylhydrazone ........................................................................................................................................... 22,2334,23,2335
neo-Menthol ...................................................................................................................................................................................................... 2137,2138 Methacrolein (2,4-DNPH derivative) ................................................................................................................................................................... 2336,24
(+)-Menthol ................................................................................................................................................................................... 2126,2189,2655,2678 Methacrylic acid ................................................................................................................................................................................................ 1582,2332
(−)-Menthone ................................................................................................................................................................................................... 2111,2184 Methacrylonitrile ................................................................................................................................................................................................ 1504,2073
Menthone .................................................................................................................................................................................................................... 2112 Methadone ........................................................................................................................................................................... 2723,439,445,464,479,2289
(−)-Menthone ............................................................................................................................................................................................................ 2136 (±)-Methadone hydrochloride ............................................................................................................................................... 176,2637,2724,440,2288
Menthone ........................................................................................................................................................................................................... 2137,2138 Methamidiphos ............................................................................................................................................................ 1347,1394,1798,1894,1797,1893
iso-Menthone ..................................................................................................................................................................................................... 2137,2138 Methamidophos .............................................................................................................................................................................. 1350,1401,1860,1995
Menthyl acetate ........................................................................................................................................................................................................ 2137 Methamphetamine ................................................................................................................................................................................................ 189,448
Menthyl acetate ................................................................................................................................................................................................ 2138,2167 (-)-Methamphetamine ........................................................................................................................................................................................ 2718,409
iso-Menthyl acetate ........................................................................................................................................................................................ 2137,2138 Methamphetamine ......................................................................................................................................... 411,412,480,2234,2285,2457,2460,2289
neo-Menthyl acetate ...................................................................................................................................................................................... 2137,2138 (+)-Methamphetamine ...................................................................................................................................................................................... 2718,409
Mepenzolate bromide ......................................................................................................................................................................................... 119,1134 d-Methamphetamine .................................................................................................................................................. 2195,2231,2282,2621,2711,2847
Meperidine ..................................................................................................................................................................................................................... 427 l-Methamphetamine ................................................................................................................................................... 2195,2231,2282,2621,2711,2847
Meperidine solution ..................................................................................................................................................................................................... 481 (±)-Methamphetamine solution ................................................................................................................................................................................ 481
mephedrone .................................................................................................................................................................................................................. 435 Methamphetamine (TFA derivative) ............................................................................................ 1694,1835,1941,2296,2693,2235,2286,2458,2461
Indexes Analyte
Methandienone ....................................................................................................................................................................................... 197,198,200,201 2-Methoxy-5-methylaniline ....................................................................................................................................................................................... 1673

Methane .............................................................................................................. 1622,1628,1639,1665,1723,1724,1734,1749,1750,1751,1753,1754 2-Methoxy-5-nitroaniline ........................................................................................................................................................................................... 1673
Methane (430 ng on column) ................................................................................................................................................................................ 1752 2-Methoxyphenol .............................................................................................................................................................................................. 2056,2439
Methanethiol ...................................................................................................................................................................................................... 1687,2382 Methoxyverapamil ................................................................................................................................................................................................. 952,953
Methanol ........................................................................ 1160,1485,1778,2032,2367,2390,2422,1493,2057,2370,2440,1500,1586,2321,2343, (±)-Methoxyverapamil hydrochloride ..................................................................................................................................................................... 1222
1629,1630,1676,1721,1722,1725,1726,1727,1728,1780,1895,1969,2026,2027,2205,2206,2207,2450,2208,2209,2451,2226,2456,2292,2464, Methscopolamine .......................................................................................................................................................................................................... 119
2293,2294 Methyl acetate ................................................................................................................................. 1565,1566,1675,1676,1725,1726,1727,1780,
Methanol, 0.01% .............................................................................................................................................................................................. 1635,2378 1969,2056,2439,2136,2210,2211,2212,2452,2213,2214,2453
Methanol (solvent) ........................................................................................................................................................................ 1525,1528,1529,1530 Methylacrylate ................................................................................................................................................................................................... 1504,2073
Methanol (~15 %) ................................................................................................................................................................................................... 1699 1-Methyladenosine ....................................................................................................................................................................................................... 686
Methapyrilene ..................................................................................................................................................................................... 858,859,1550,1551 Methylamine ................................................................................................................................................................ 1648,2379,1649,2380,2225,2455
Methapyrilene HCl ...................................................................................................................................................................................................... 1213 Methyl anthranilate .................................................................................................................................................................................................... 2131
Methcathinone .............................................................................................................................................................................................................. 441 Methyl arachidate .................. 1814,1914,2246,1816,1916,2248,1820,1920,2252,1843,1963,1850,1978,1866,2015,2273,1867,2018,2274,2237
Methedrone ................................................................................................................................................................................................................... 435 Methyl arachidonate .......................................................................................................................................... 1814,1914,2246,1816,1916,2248,
Methedrone hydrochloride ............................................................................................................................................................................... 2049,2433 1820,1920,2252,1848,1968,2263,1859,1992,2272,1867,2018,2274,1868,2019,2275,1869,2020,2276,2262
Methedrone hydrochloride solution .............................................................................................................................................. 243,468,434,469,477 DL-threo-β-Methylaspartic acid .......................................................................................................................................................................... 2810,667
Methidathion ............................................................................................................................................... 1419,406,1350,1401,1860,1995,1885,2064 Methyl azinphos ................................................................................................................................................................................................ 1544,1545
Methidation ..................................................................................................................................................................................... 1448,1682,1829,1934 Methyl behenate ...................................................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252,1843,1963,1850,1978
Methiocarb ....................................................................................................................................................................................... 1,295,294,2004,2413 Methyl benzoate .......................................................................................................................................................................................... 1207,273,489
D-Methionine ........................................................................................................................................................................................................ 2808,665 α-Methylbenzylamine ........................................................................................................................................................................................... 2594,75
L-Methionine ............................................................................................................................................................................ 2808,665,1231,1897,2304 α-Methylbenzylamine (N-3,5-Dinitrobenzoyl derivative) ................................................................................................................................. 2595,76
DL-Methionine ...................................................................................................................................................................................... 2807,664,2809,666 (±)-α-Methylbenzyl butyrate ...................................................................................................................................................... 1759,2149,2541,2680
DL-Methionine (t-BOC derivatives) .................................................................................................................................................................... 2752,534 2-Methylbutanal ............................................................................................................................................................................. 1896,2403,2056,2439
DL-Methionine (FMOC derivatives) .................................................................................................................................................................... 2784,599 3-Methylbutanal ....................................................................................................................................... 1871,2022,2395,2417,1896,2403,2056,2439
Methionine methyl ester (N-trifluoroacetyl derivatives) .......................................................................................................... 1987,2305,2702,2726 2-Methylbutane .................................................................................................................................................................... 1610,1611,1612,1665,1723
L-Methionine sulfone ............................................................................................................................................................................................ 118,813 2-Methyl-1-butanol ........................................................................................................................................... 1644,1876,2035,2396,2423,2060,2443
L-Methionine sulfoxide ................................................................................................................................................................................................. 813 2-Methyl-2-butanol .................................................................................................................................................................................................... 1644
L-Methionine sulfoximine .............................................................................................................................................................................. 118,121,813 3-Methylbutanol .......................................................................................................... 1676,1726,1727,1895,2210,2211,2212,2452,2213,2214,2453
Methomyl ................................................................................................................................................................... 1,295,294,302,1391,305,1419,406 3-Methyl-1-butanol .................................................................................................................................................................................................... 1644
Methoprene ........................................................................................................................................................................................................ 1889,2068 3-Methyl-2-butanol .................................................................................................................................................................................................... 1969
Methorphan ....................................................................................................................................................................................................... 1060,2947 3-Methyl-2-butanone ................................................................................................................................................. 1485,1778,2032,2367,2390,2422
Methotrexate ........................................................................................................................................................................................... 863,864,903,904 2-Methyl-1-butene ..................................................................................................................................................................................................... 1612
Methoxychlor .......................................................................................................................................................................... 1351,1403,1862,2000, 2-Methyl-2-butene .................................................................................................................................................................................. 1612,1721,1722
1381,1395,1438,1924,1382,1396,1439,1925,1388,1411,1482,2028,1466,1740,1858,1989,2356,2384,2392,2406,1469,1994,2359,2409, Methyl tert-butyl ether .................................................................................................................................... 1493,2057,2370,2440,1594,1722,2224
1506,2075,1507,2076,1509,1510,1511,1512,1513,1514,1516,1532,1571,1591,2325,2347,1604 Methyl t-butyl ether-(α,α′-14C) ............................................................................................................................................................................... 1528
2-Methoxyethanol ............................................... 1383,1457,1703,1975,2185,1725,1726,1727,2205,2206,2207,2450,2208,2209,2451,2225,2455 Methyl tert-butyl ether (MTBE) ............................................................................................................................................................................... 1530
2-Methoxyethanol (methyl Cellosolve®) .............................................................................................................................................. 1704,1976,2186 3-Methylbutyl hexanoate .............................................................................................................................................................. 1876,2035,2396,2423
Methoxymaysin ........................................................................................................................................................................................................... 1246 Methyl butyl ketone ......................................................................................................................................... 2205,2206,2207,2450,2208,2209,2451
3-Methoxymethcathinone hydrochloride ............................................................................................................................................................ 469,477 methyl n-butyl ketone .................................................................................................................................................................. 1568,1588,2322,2344
Indexes Analyte
Methyl butyrate ........................................................................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252,1971,2405 Methyl cis,cis-11,14-eicosadienoate .......................................................................................................................... 1814,1914,2246,1816,1916,2248

3-Methyl butyrate ...................................................................................................................................................................................................... 2124 Methyl eicosanoate ........................................................................................................................................................................ 2236,2238,2239,2240
Methyl caprate ...................................................................................................................................................................... 1820,1920,2252,1850,1978 Methyl eicosapentaenoate ......................................................................................................................................... 1848,1968,2263,1859,1992,2272
Methyl caproate .......................................................................................................................................................... 1809,1909,2245,1820,1920,2252 Methyl all-cis-5,8,11,14,17-eicosapentaenoate .................................................................. 1814,1914,2246,1816,1916,2248,1820,1920,2252,
Methyl caprylate ............................................................................................. 350,56,1814,1914,2246,1816,1916,2248,1820,1920,2252,1850,1978 1867,2018,2274,1868,2019,2275,1869,2020,2276,2262
3-Methylcatechol ..................................................................................................................................................................................................... 152,44 Methyl cis-8,11,14,17-eicosatetraenoate .................................................................................................................. 1868,2019,2275,1869,2020,2276
4-Methylcatechol ..................................................................................................................................................................................................... 152,44 Methyl eicosatrienoate ............................................................................................................................................... 1848,1968,2263,1859,1992,2272
Methyl cellosolve ........................................................................................................................................................................................................ 1780 Methyl cis-8,11,14-eicosatrienoate ........................................................................................................................................................ 1820,1920,2252
Methyl chavicol .......................................................................................................................................................................................................... 2161 Methyl cis-11,14,17-eicosatrienoate ...................................................................................................................................................... 1820,1920,2252
Methyl chloro isothiazolinone .................................................................................................................................................................... 1210,293,321 Methyl eicosenoate .................................................................................................................................. 1843,1963,1848,1968,2263,1866,2015,2273
Methyl chloropyrifos ......................................................................................................................................................................................... 1544,1545 Methyl cis-9-eicosenoate ........................................................................................................................................................................ 1868,2019,2275
Methyl chlorpyrifos ..................................................................................................................................................... 1347,1394,1798,1894,1797,1893 Methyl cis-11-eicosenoate .... 1814,1914,2246,1816,1916,2248,1820,1920,2252,1850,1978,1867,2018,2274,1868,2019,2275,1869,2020,2276
3-Methylcholanthrene .................................................................................................................................................................... 1540,1541,1550,1551 Methyl elaidate .................................................................................................. 1814,1914,2246,1816,1916,2248,1820,1920,2252,1850,1978,2237
5-Methylchrysene .............................................................................................................................................................................................. 1795,1891 4,4′-Methylenebis(N,N-dimethylaniline) .................................................................................................................................................................. 1673
Methylcyclohexane .......................................................................................................................... 1565,1566,2205,2206,2207,2450,2208,2209,2451 Methylenebis(phenyl isocyanate) ........................................................................................................................................................................ 2340,27
2-Methylcyclohexanol ................................................................................................................................................................................................. 1725 Methylene chloride ................................................................. 1493,2057,2370,2440,1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,
2-Methylcyclohexanol isomers ........................................................................................................................................................................ 1726,1727 1500,1586,2321,2343,1502,2071,1503,2072,1504,2073,1505,2074,1522,1569,1589,2323,2345,1725,1780,2206,2208,2224,2226,2456
Methylcyclopentane .......................................................................................................................................................................................... 1592,1612 Methylene chloride (impurity) ................................................................................................................................................................................. 1529
3-Methylcyclopentene ................................................................................................................................................................................................ 1612 Methylene chloride (solvent) ................................................................................................................................................................. 1526,2308,2311
2-Methyl-2-cyclopentenone ............................................................................................................................................................................. 2056,2439 (±)-3,4-Methylenedioxymethamphetamine hydrochloride solution ..................................................................................................................... 432
2′-O-Methylcytidine ...................................................................................................................................................................................................... 686 3,4-Methylenedioxypyrovalerone ..................................................................................................................................................................... 2049,2433
3-Methylcytidine ........................................................................................................................................................................................................... 686 3,4-Methylenedioxypyrovalerone HCl (MDPV) solution ............................................................................................................. 243,468,434,470,478
5-Methylcytidine ........................................................................................................................................................................................................... 686 4,4’-Methylenediphenyl diisocyanate ....................................................................................................................................................................... 2338
Methyl decanoate .............................................................................................................. 350,56,1808,1908,2244,1814,1914,2246,1816,1916,2248 N,N'-(Methylenediphenylene)bis(4-(2-pyridi nyl)-1-piperazinecarboxamide) ......................................................................................................... 25
2-Methyl-4,6-dinitrophenol ...................... 1476,1756,2006,2363,2388,2414,1477,1757,2007,2364,2389,2415,1524,1531,1552,1553,1554, (±)-4-Methylephedrine hydrochloride solution ................................................................................................................................................ 470,474
1555,1556,1557,1558,1564 Methyl erucate ............................................................. 1814,1914,2246,1816,1916,2248,1820,1920,2252,1843,1963,1850,1978,1868,2019,2275
Methyl cis-13,16-docosadienoate ................................................................................................. 1814,1914,2246,1816,1916,2248,1820,1920,2252 2,4-D methyl ester ......................................................................................................................................................... 306,307,1571,1591,2325,2347
Methyl docosahexaenoate .......................................................................................................................................... 1848,1968,2263,1859,1992,2272 2,4,5-T methyl ester ............................................................................................................................................................................................. 306,307
Methyl cis-4,7,10,13,16,19-docosahexaenoate .................................................................................................................................... 1820,1920,2252 4-Methylethcathinone hydrochloride solution ................................................................................................................................................... 471,478
Methyl docosanoate ................................................................................................................................................................................ 1866,2015,2273 2-Methyl-6-ethylaniline .................................................................................................................................................................................... 1655,1672
Methyl docosapentaenoate ........................................................................................................................................ 1848,1968,2263,1859,1992,2272 1-Methyl-2-ethylbenzene ................................................................................................................................................................................. 1610,1612
Methyl cis-4,7,10,13,16-docosapentaenoate ........................................................................................................................................ 1867,2018,2274 1-Methyl-3-ethylbenzene ....................................................................................................................................................................... 1610,1611,1612
Methyl all-cis-7,10,13,16,19-docosapentaenoate .............................................................. 1867,2018,2274,1868,2019,2275,1869,2020,2276,2262 1-Methyl-4-ethylbenzene ................................................................................................................................................................................. 1610,1612
Methyl docosatetraenoate .......................................................................................................................................... 1848,1968,2263,1859,1992,2272 2-Methyl-4-ethylhexane ............................................................................................................................................................................................ 1592
Methyl cis-11-docosenoate .................................................................................................................................................................... 1868,2019,2275 Methyl ethyl ketone ................................................................................................................................ 1431,1572,1593,1725,1780,2051,2435,2224
Methyl dodecanoate ................................................................................................................... 350,56,1808,1908,2244,2236,2237,2238,2239,2240 2-Methyl-3-ethylpentane ........................................................................................................................................................................................... 1592
Methyl eicosadienoate ............................................................................................................................................................................ 1859,1992,2272 3-Methyl-3-ethylpentane ........................................................................................................................................................................................... 1592
Methyl cis-8,11-eicosadienoate ............................................................................................................................................................. 1867,2018,2274 Methyl eugenol .......................................................................................................................................................................................................... 2161
Methyl cis-11,14-eicosadienoate ........................................................................................................................................................... 1820,1920,2252 Methyl formate ........................................................................................................................................ 1675,1676,1725,1726,1727,1780,2060,2443
Indexes Analyte
N-Methyl-2-formylpyrrole ................................................................................................................................................................................ 2056,2439 Methyl 3-hydroxydodecanoate ..................................................................................................................................................... 2236,2238,2239,2240

5-Methyl-2-furaldehyde ............................................................................................................................................................................................ 59,60 Methyl (±)-3-hydroxydodecanoate ......................................................................................................................................................................... 2237
2-Methylfuran .................................................................................................................................................................................................... 2056,2439 Methyl 2-hydroxyhexadecanoate ........................................................................................................................................ 2236,2237,2238,2239,2240
5-Methylfurfural ................................................................................................................................................................................................ 2056,2439 7-Methyl-12-hydroxymethylbenz[a]anthracene ......................................................................................................................................... 175,304,438
1-(5-Methyl-2-furyl)-2-propanone ................................................................................................................................................................. 2056,2439 7-Methyl-12-hydroxymethylbenz[a]anthracene-trans-8,9-diol ................................................................................................................ 175,304,438
Methyl geranate ......................................................................................................................................................................................................... 2096 7-Methyl-12-hydroxymethylbenz[a]anthracen-2-ol .................................................................................................................................. 175,304,438
Methyl geranoate .................................................................................................................................................................................... 2050,2434,2120 7-Methyl-12-hydroxymethylbenz[a]anthracen-3-ol .................................................................................................................................. 175,304,438
7-Methylguanosine ....................................................................................................................................................................................................... 686 7-Methyl-12-hydroxymethylbenz[a]anthracen-4-ol .................................................................................................................................. 175,304,438
Methyl heneicosanoate .................................................................................................................. 1814,1914,2246,1816,1916,2248,1820,1920,2252 Methyl 2-hydroxytetradecanoate ........................................................................................................................................ 2236,2237,2238,2239,2240
Methyl heptadecanoate ..................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252,1850,1978,2236,2237,2238,2239,2240,2262 Methyl 3-hydroxytetradecanoate ........................................................................................................................................ 2236,2237,2238,2239,2240
Methyl cis-10-heptadecenoate ..................................................................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252 2-Methylimidazole ........................................................................................................................................................................................................ 365
2-Methylheptane ..................................................................................................................................................................................... 1592,1610,1612 4(5)-Methylimidazole ................................................................................................................................................................................................... 365
3-Methylheptane ..................................................................................................................................................................................... 1592,1610,1612 2-Methylindane ........................................................................................................................................................................................................... 1612
4-Methylheptane ............................................................................................................................................................................................... 1592,1612 Methylindan isomer ................................................................................................................................................................................................... 1621
Methylheptanol .................................................................................................................................................................................................. 1971,2405 Methylionone ..................................................................................................................................................................................................... 2183,2446
2-Methyl-1-heptene ................................................................................................................................................................................................... 1612 2-Methylisoborneol .................................................................................................................................. 1488,2038,2368,2424,1499,2070,2376,2445
6-Methyl-5-hepten-2-ol ......................................................................................................................................................................... 1642,2487,2131 Methyl isocyanate ...................................................................................................................................................................................................... 2338
6-Methyl-5-hepten-2-one ....................................................................... 1491,1581,2319,2331,1582,2332,2045,2350,2096,2111,2184,2131,2142 2-Methyl-5-isopropylpyrazine ....................................................................................................................................................... 1871,2022,2395,2417
Methyl cis-9,12-hexadecadienoate ........................................................................................................................................................ 1869,2020,2276 2-Methyl-4-isothiazolin-3-one ................................................................................................................................................................... 1210,293,321
Methyl hexadecanoate ..................................................................................................................................................................................... 2239,2240 Methyl jasmonate ............................................................................................................................................................................................. 2111,2184
Methyl cis-6,9,12,15-hexadecatetraenoate ........................................................................................................................................... 1869,2020,2276 Methyl DL-lactate .......................................................................................................................................................................................................... 322
Methyl cis-6,9,12-hexadecatrienoate ........................................................................................................................................... 1869,2020,2276,2262 (±)-Methyl-γ-lactone .................................................................................................................................................................... 1729,2118,2529,2676
Methyl cis-9-hexadecenoate .................................................................................................................................................................. 2236,2237,2238 Methyl laurate ............................................................................................................. 1814,1914,2246,1816,1916,2248,1820,1920,2252,1850,1978
Methyl cis-9-hexadecenoate ............................................................................................................................................................................ 2239,2240 Methyl lignocerate ................................................................................................................................... 1820,1920,2252,1843,1963,1859,1992,2272
Methylhexanal ................................................................................................................................................................................................... 1971,2405 Methyl linoleate ............................................................................ 350,56,1808,1908,2244,1814,1914,2246,1816,1916,2248,1820,1920,2252,
2-Methylhexane ....................................................................................................................................................................................... 1592,1610,1611 1850,1978,1859,1992,2272,1866,2015,2273,1867,2018,2274,1868,2019,2275,1869,2020,2276,2262
3-Methylhexane .............................................................................................................................................................................. 1592,1610,1611,1612 Methyl linolelaidate ....................................................................................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252
Methyl hexanoate ....................................................................................................................................................... 1814,1914,2246,1816,1916,2248 Methyl linolenate ...................................................................................................... 350,56,1808,1908,2244,1814,1914,2246,1816,1916,2248,
5-Methyl-2-hexanone ............................................................................................................................................................................. 1676,1725,1727 1820,1920,2252,1843,1963,1866,2015,2273,1868,2019,2275,1869,2020,2276,2262
2-Methyl-1-hexene ..................................................................................................................................................................................................... 1612 Methyl α-linolenate ...................................................................................................................................................................................................... 357
5-Methyl-1-hexene ..................................................................................................................................................................................................... 1612 Methyl γ-linolenate ............................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252,1859,1992,2272,1867,2018,2274
2-Methylhexenes ........................................................................................................................................................................................................ 1612 Methyl (R)-(−)-mandelate .................................................................................................................... 1733,2221,2533,2858,1785,2172,2558,2688
o-Methylhippuric acid ........................................................................................................................................................................................... 170,433 Methyl (S)-(+)-mandelate ..................................................................................................................... 1733,2221,2533,2858,1785,2172,2558,2688
m-Methylhippuric acid ......................................................................................................................................................................................... 170,433 Methyl DL-mandelate ................................................................................................................................................................................................... 322
p-Methylhippuric acid ........................................................................................................................................................................................... 170,433 R-(-)-Methyl mandelate ......................................................................................................................... 1731,2219,2531,2856,1732,2220,2532,2857
Methyl 4-hydroxybenzoate .............................................................................................................. 284,1748,1998,2190,2223,2387,2412,2447,2454 S-(+)-Methyl mandelate ........................................................................................................................ 1731,2219,2531,2856,1732,2220,2532,2857
Methyl 2-hydroxydecanoate ......................................................................................................................................................... 2236,2238,2239,2240 Methyl mercaptan ............................................................................................................................................................................................ 1686,2381
Methyl (±)-2-hydroxydecanoate ............................................................................................................................................................................. 2237 Methyl mercaptan (1 ppm) ..................................................................................................................................................................................... 1783
Methyl 2-hydroxydodecanoate ........................................................................................................................................... 2236,2237,2238,2239,2240 Methyl methacrylate ........................................................................................................................................................................................ 1504,2073
Indexes Analyte
Methyl methanesulfonate ................................................................................................................................................................................ 1550,1551 Methyl palmitoleate ........................................................................... 357,1808,1908,2244,1814,1914,2246,1816,1916,2248,1820,1920,2252,

α-Methyl-DL-methionine ............................................................................................................................................................................................... 810 1850,1978,1868,2019,2275,1869,2020,2276,2262
Methyl (E)-α-(methoxyimino)-2-(2-methylphenoxymethyl)phenylacetate ............................................................................................... 1886,2065 Methyl paraben .............................................................................................................................................................................................. 282,283,286
Methyl N-methyl anthranilate ..................................................................................................................................................... 2103,2131,2142,2166 Methyl Paraben .............................................................................................................................................................................................. 289,290,291
Methyl cis-9,10-methylenehexadecanoate ........................................................................................................................ 2236,2237,2238,2239,2240 Methyl Paraben .......................................................................................................................................................................................................... 1106
Methyl cis-9,10-methyleneoctadecanoate ......................................................................................................................... 2236,2237,2238,2239,2240 Methyl paraoxon ............................................................................................................................................................................................... 1506,2075
Methyl 15-methylhexadecanoate ....................................................................................................................................... 2236,2237,2238,2239,2240 Methyl parathion .................................. 308,309,1440,1677,1824,1926,1466,1740,1858,1989,2356,2384,2392,2406,1470,1996,2360,2410,
Methyl 14-methylpentadecanoate ..................................................................................................................................... 2236,2237,2238,2239,2240 1471,1746,1861,1997,2361,2386,2393,2411,1542,1543,1550,1551
Methyl 12-methyltetradecanoate ....................................................................................................................................... 2236,2237,2238,2239,2240 Methyl pentadecanoate ......................................................................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252,
Methyl 13-methyltetradecanoate ....................................................................................................................................... 2236,2237,2238,2239,2240 1850,1978,2236,2237,2238,2239,2240,2262
Methyl myristate ........................................................ 350,56,1814,1914,2246,1816,1916,2248,1820,1920,2252,1843,1963,1848,1968,2263, Methyl cis-10-pentadecenoate ..................................................................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252
1850,1978,1859,1992,2272,1868,2019,2275,1869,2020,2276,2236,2237,2238,2262 2-Methylpentane ............................................................................................................................................... 1528,1529,1530,1592,1610,1611,1612
Methyl myristoleate .................................................................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252,1850,1978 3-Methylpentane ......................................................................................................................................................... 1592,1610,1611,1612,2051,2435
1-Methylnaphthalene ............................................................ 1408,2010,1409,2011,1550,1551,1593,1594,1595,1596,1610,1611,1612,2300,2467 1-Methylpentan-1-ol .................................................................................................................................................................................................. 1969
1-Methylnaphthalene solution .................................................................................................................................................................................. 1597 2-Methyl-1-pentanol .................................................................................................................................................................................................. 1969
2-Methylnaphthalene ............................................................................................................. 1408,2010,1484,2030,2366,2421,1552,1553,1554, 4-Methyl-2-pentanol .................................................................................................................................................................................................. 1666
1555,1556,1557,1564,1593,1594,1596,1597,2300,2467 4-Methyl-2-pentanone ................................................. 1431,1572,1504,2073,1525,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,
2-Methylnaphthalene solution .................................................................................................................................................................................. 1595 1676,1725,1726,1727,2051,2435,2210,2211,2212,2452,2213,2214,2453,2224
2-Methylnaphthalene (β) .......................................................................................................................................... 1409,2011,1558,1610,1611,1612 3-Methyl-2-pentene ................................................................................................................................................................................................... 1612
Methyl nervonate ........................................................................................................................................................ 1820,1920,2252,1859,1992,2272 trans-3-Methyl-2-pentene ......................................................................................................................................................................................... 1612
2-Methyl-5-nitroaniline .............................................................................................................................................................................................. 1673 4-Methylpenten-2-one .................................................................................................................................................................. 1871,2022,2395,2417
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) ......................................................................................................................................... 245 1-Methylphenanthrene .................................................................................................................................................................. 1408,2010,1409,2011
4-(Methylnitrosoamino)-1-(3-pyridinyl)-1-butanone ............................................................................................................................................... 246 Methylphenidate ................................................................................................................ 1138,2997,446,2222,2271,2302,2632,2716,2859,844,974
Methyl nonadecanoate ........................................................................................................................................................ 2236,2237,2238,2239,2240 Methylphenidate hydrochloride .............................................................................. 1062,177,2638,2949,1061,2948,442,1063,2950,443,1072,2959
3-Methylnonane .......................................................................................................................................................................................................... 1612 Methylphenidate (Ritalin) ............................................................................................................................................................. 2234,2285,2457,2460
Methyl cis-9,12-octadecadienoate ............................................................................................................................................... 2236,2238,2239,2240 2-Methylphenol .......................................................................................... 1359,1386,1407,1475,1755,2005,1476,1756,2006,2363,2388,2414,
Methyl octadecanoate ................................................................................................................................................................... 2236,2238,2239,2240 1477,1757,2007,2364,2389,2415,1531,1552,1553,1554,1555,1557,1564,1725
Methyl cis-9-octadecanoate .......................................................................................................................................................... 2236,2238,2239,2240 3-Methylphenol .......................................................................................... 1359,1386,1407,1475,1755,2005,1476,1756,2006,2363,2388,2414,
Methyl trans-9-octadecanoate ..................................................................................................................................................... 2236,2238,2239,2240 1477,1757,2007,2364,2389,2415,1531,1550,1551,1725
2-Methyloctane ........................................................................................................................................................................................................... 1592 4-Methylphenol ................................................................................................................................................... 1476,1756,2006,2363,2388,2414,
3-Methyloctane ................................................................................................................................................................................................. 1592,1612 1477,1757,2007,2364,2389,2415,1531,1552,1553,1554,1555,1557,1564,1725
4-Methyloctane ................................................................................................................................................................................................. 1592,1612 N,N'-(2-Methyl-1,3-phenylene)bis(4-(2-pyridinyl)-1-piperazinecarboxamide) ........................................................................................................ 25
Methyl oleate .............. 350,56,1808,1908,2244,1814,1914,2246,1816,1916,2248,1820,1920,2252,1843,1963,1850,1978,1866,2015,2273, N,N'-(4-Methyl-1,3-phenylene)bis(4-(2-pyridinyl)-1-piperazinecarboxamide) ........................................................................................................ 25
1867,2018,2274,1868,2019,2275,1869,2020,2276,2237,2262 4-Methyl-m-phenylenediamine ................................................................................................................................................................................ 1672
Methylone ...................................................................................................................................................................................................................... 435 5-Methyl-5-phenylhydantoin ............................................................................................................................ 2596,77,2597,78,2811,668,1066,2953
Methylone hydrochloride .................................................................................................................................................... 243,468,434,472,2049,2433 4-Methyl-5-phenyl-2-oxazolidinone ................................................................................................................................................................... 2598,79
Methyl palmitate .......................................................................... 350,56,1808,1908,2244,1814,1914,2246,1816,1916,2248,1820,1920,2252, 1-Methyl-3-phenylpropylamine (4-Phenyl-2-butylamine) (N-TFA derivatives) ....................................................................................... 1738,2537
1843,1963,1850,1978,1859,1992,2272,1867,2018,2274,1868,2019,2275,1869,2020,2276,2236,2237,2238,2262 α-Methyl-α-phenylsuccinimide ........................................................................................................................................................... 2599,80,2812,669
Methyl phenyl sulfoxide ................................................................................................................................................................... 1067,2600,2954,81
Indexes Analyte
2-Methylpropanal .............................................................................................................................................................................................. 1896,2403 (±)-Metoprolol (+)-tartrate salt ........................................................................................... 140,2633,2720,419,1069,2956,1070,2957,1071,2958

2-Methylpropane .................................................................................................................................................................. 1610,1611,1612,1665,1723 Metoxuron .......................................................................................................................................................................................................................... 5
2-Methyl-1-propanol .................................................................................................................................................. 1644,1666,1676,1726,1727,1895 Metribuzin ................................................................................................................................................... 1391,305,1506,2075,1507,2076,1889,2068
2-Methylpropene ..................................................................................................................................................................................... 1612,1665,1723 Metronidazole solution ......................................................................................................................................................................................... 831,832
2-Methyl-6-propenylpyrazine ....................................................................................................................................................... 1871,2022,2395,2417 Mevinolin from Aspergillus sp. ................................................................................................................................................................................... 157
Methylpropyl butanoate ................................................................................................................................................................ 1876,2035,2396,2423 Mevinphos .................................................................................................................................................................... 1506,2075,1507,2076,1542,1543
2-Methyl-5-propylpyrazine ........................................................................................................................................................... 1871,2022,2395,2417 Mevinphos-1 ...................................................................................................................................................................................................... 1885,2064
2-Methylpyrazine ........................................................................................................................................................ 1871,2022,2395,2417,2056,2439 Mevinphos-2 ...................................................................................................................................................................................................... 1885,2064
3-Methylpyrazole ................................................................................................................................................................................................. 1423,444 Mevinphos isomers ........................................................................................................................................................................................... 1544,1545
4-Methylpyrazole ................................................................................................................................................................................................. 1423,444 Mexacarbate ........................................................................................................................................................................................................ 1,295,294
1-Methylpyrrole ........................................................................................................................................................... 1871,2022,2395,2417,2056,2439 MGK 264 ............................................................................................................................................................................................................ 1506,2075
n-Methylpyrrolidone .......................................................................................................................................... 2205,2206,2207,2450,2208,2209,2451 Mianserin hydrochloride .................................................................................... 1073,2960,1074,2961,1075,2962,1076,2963,1077,2964,1078,2965
2-Methylresorcinol ................................................................................................................................................................................................... 152,44 Miconazole ........................................................................................................................................................................................ 112,2614,1079,2966
Methyl salicylate ............................................................................................................................................................................ 2045,2350,2183,2446 (±)-Miconazole nitrate salt ............................................................................................................................................................................ 1080,2967
Methylscopolamine ................................................................................................................................................................................................ 885,886 Midazolam solution ...................................................................................................................................................................................................... 833
Methyl stearate ...................................................................................................................................... 350,56,1808,1908,2244,1814,1914,2246, Milk proteins .......................................................................................................................................................................................................... 366,671
1816,1916,2248,1820,1920,2252,1843,1963,1866,2015,2273,1867,2018,2274,1869,2020,2276,2237,2262 Minocycline .................................................................................................................................................................................................................. 1157
Methyl stearidonate ....................................................................................................................... 1866,2015,2273,1868,2019,2275,1869,2020,2276 Mirex ................................................................................................................................................................................................ 1571,1591,2325,2347
Methylstyrene ................................................................................................................................................................................. 1492,1583,2320,2333 Molinate ................................................................................................................................. 1448,1682,1829,1934,1506,2075,1507,2076,1886,2065
17α-Methyltestosterone solution ............................................................................................................................................................... 1374,257,258 Monensin .............................................................................................................................................................................................................. 215,1174
Methyl tetracosanoate ................................................................................................................................................ 1814,1914,2246,1816,1916,2248 3-Monoacetylmorphine (m/z 328) NMID766 .......................................................................................................................................................... 490
Methyl cis-15-tetracosenoate ........................................................................................................................... 1814,1914,2246,1816,1916,2248,2237 2-Monochlorobiphenyl ................................................................................................................................................................... 1441,1927,1442,1928
Methyl tetradecanoate ..................................................................................................................................................................................... 2239,2240 2-monochlorobiphenyl ...................................................................................................................................................................................... 1443,1929
2-Methylthiophene ..................................................................................................................................................................................................... 1685 4-Monochlorobiphenyl ................................................................................................................................................................... 1441,1927,1442,1928
Methyl tricosanoate ........................................................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252,1866,2015,2273 4-monochlorobiphenyl ...................................................................................................................................................................................... 1443,1929
Methyl tridecanoate ..................................................... 1814,1914,2246,1816,1916,2248,1820,1920,2252,1850,1978,2236,2237,2238,2239,2240 Monocrotophos ................................................................................ 1350,1401,1860,1995,1444,1678,1825,1930,1542,1543,1544,1545,1887,2066
3-Methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one ......................................................................................................................... 2180 Monolinolein (C18:2) ................................................................................................................................................................................................. 1638
α-Methyl-m-tyrosine ........................................................................................................................................................................................... 2813,670 Monolinolenin (C18:3) ............................................................................................................................................................................................... 1638
Methyl undecanoate ................................. 1814,1914,2246,1816,1916,2248,1820,1920,2252,1850,1978,1970,2404,2236,2237,2238,2239,2240 Monolinuron ...................................................................................................................................................................................................................... 5
1-Methyluric acid .................................................................................................................................................................................................. 149,150 Monoolein .......................................................................................................................................................................................................... 1632,1633
5-Methyluridine ............................................................................................................................................................................................................. 686 Monoolein (C18:1) ............................................................................................................................................................................................ 1634,1638
1-Methylxanthine ................................................................................................................................................................................. 149,150,1315,802 Monopalmitin (C16:0) ...................................................................................................................................................................................... 1634,1638
3-Methylxanthine .......................................................................................................................................................................................................... 150 Monostearin (C18:0) ......................................................................................................................................................................................... 1634,1638
7-Methylxanthine ................................................................................................................................................ 1362,1427,148,946,149,150,1315,802 Monuron ............................................................................................................................................................... 1,295,294,302,1391,305,314,375,317
Methyprylon ........................................................................................................................................................................................................... 415,926 Morphine ....................................................................................................................................................................... 124,868,427,428,429,2288,2289
Metobromuron ................................................................................................................................................................................................................... 5 Morphine solution .................................................................................................................................................. 1082,178,264,484,481,490,491,692
Metolachlor ......................................................................................................................... 5,1351,1403,1862,2000,1506,2075,1507,2076,1888,2067 Morphine glucuronide .................................................................................................................................................................................................. 692
Metoprolol ............................................................................................................................................................................................. 894,941,954,1020 Morphine-3-β-D-glucuronide solution ....................................................................................................................................................... 1082,178,491
Metoprolol tartrate solution ...................................................................................................................................................................................... 1228 Morphine-6-β-D-glucuronide solution .............................................................................................................................................................. 1082,178
Indexes Analyte
Mosapride citrate salt dihydrate .................................................................................................................................................................... 1083,2968 Naphthalene-d8 (I.S) ................................................................................................................................................................................................. 1558
Musk T ............................................................................................................................................................................................................... 2111,2184 1,4-Naphthoquinone ......................................................................................................................................................................................... 1550,1551
Musk ambrette .................................................................................................................................................................................................. 2111,2184 1-Naphthylamine ............................................................................................................................................................................................... 1550,1551
Musk ketone ...................................................................................................................................................................................................... 2111,2184 2-Naphthylamine ............................................................................................................................................... 1484,2030,2366,2421,1550,1551,1673
Musk xylol ......................................................................................................................................................................................................... 2111,2184 1-(1-Naphthyl)ethylamine ............................................................................................................................................................................... 1339,2618
α-Muurolene ................................................................................................................................................................................................................ 2131 (±)-1-(1-Naphthyl)ethylamine ........................................................................................................................................................ 1086,2601,2969,82
τ-Muurolol ................................................................................................................................................................................................................... 2161 1-Naphthyl-N-methylcarbamate ............................................................................................................................ 1,295,294,302,1391,305,2004,2413
Myclobutanil ............................................................................................................................................. 1447,1681,1828,1933,1886,2065,2004,2413 N-(1-Naphthyl)thiourea ...................................................................................................................................................................................... 1391,305
Myoglobin .......................................................................................................................................................................... 207,740,754,755,756,757,826 Naphyrone hydrochloride ............................................................................................................................................................................................. 469
Myoglobin from equine heart .................................................................................................................................................................................... 825 Napropamide ..................................................................................................................................... 313,1430,1641,1796,1892,1506,2075,1888,2067
Myrcene ...................................................................................................... 2047,2175,2351,2353,2050,2434,2095,2096,2099,2103,2105,2106, Naproxen .......................................................................................................................................................................... 850,899,1087,2970,1093,1094
2111,2184,2120,2121,2124,2131,2136,2137,2138,2142,2161,2166,2167,2170,2183,2446 Narasin ........................................................................................................................................................................................................................... 215
β-Myrcene .......................................................................................................................................................................................................... 2056,2439 Naringin ......................................................................................................................................................................................................................... 359
Myricetin ............................................................................................................................................................................................................... 352,1259 Narirutin .......................................................................................................................................................................................................... 353,354,359
Myristic acid ............................................................................................................................................................................................. 1808,1908,2244 Natriuretic Peptide, C-Type ............................................................................................................................................................................. 1889,2068
Myristic acid methyl ester ..................................................................................................................... 1818,1918,2250,1819,1919,2251,1837,1954 Nebivolol ............................................................................................................................................................................................................ 1088,2971
Myristic acid methyl ester (C14:0) .............................................. 1815,1915,2247,1817,1917,2249,1836,1945,1839,1959,1842,1962,1844,1964 Neburon ........................................................................................................................................................................ 1,295,294,302,2374,303,314,375
Myristic acid methyl ester (C14:0) at 4 wt % ................................................................................................................................. 1822,1922,2254 Nefazodone .................................................................................................................................................................................................................... 157
Myristoleic acid methyl ester ................................................................................................................................... 1818,1918,2250,1819,1919,2251 Neo-chlorogenic acid ................................................................................................................................................................................................. 1275
Myristoleic acid methyl ester (C14:1) ..................................................................................................................... 1815,1915,2247,1817,1917,2249 Neodihydrocarveol ...................................................................................................................................................................................................... 2167
Myristoleic acid methyl ester (C14:1) at 2 wt % ............................................................................................................................ 1822,1922,2254 (−)-Neomenthol ............................................................................................................................................................................ 2126,2189,2655,2678
(E)-Myroxide ............................................................................................................................................................................................................... 2120 Neomenthol ................................................................................................................................................................................................................. 2136
trans-Myrtanol ............................................................................................................................................................................................................ 2131 (+)-Neomenthol ............................................................................................................................................................................ 2126,2189,2655,2678
Neon ............................................................................................................................................................................................................................. 1745
—N— Neotame .................................................................................................................................................................................................. 346,347,348,349
NAD+ .............................................................................................................................................................................................................................. 690 Neral ........................................................................................ 2050,2434,2096,2099,2103,2105,2106,2120,2121,2131,2142,2166,2170,2180,2181
NADH .............................................................................................................................................................................................................................. 690 Nereistoxin oxalate ............................................................................................................................................................................................ 1890,2069
NADP+ ........................................................................................................................................................................................................................... 690 Nerol ................................................................................................................................................................................................................... 2131,2142
NADPH ............................................................................................................................................................................................................................ 690 Nerolidol ............................................................................................................................................................................................................. 2111,2184
Naled .................................................................................................................................................................................................................. 1542,1543 (E)-Nerolidol ............................................................................................................................................................................................. 2096,2131,2142
Nandrolone ....................................................................................................................................................................................... 195,197,198,200,201 Nervonic acid methyl ester ....................................................................................................................................... 1818,1918,2250,1819,1919,2251
Naphthalene ........................................................................................................................... 2375,310,311,842,845,1117,1408,2010,1409,2011, Nervonic acid methyl ester (C24:1n9) .................................................................................................................... 1815,1915,2247,1817,1917,2249
1451,1936,1480,2013,1481,2014,1483,2029,2365,2420,1493,2057,2370,2440,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072, Nervonic acid methyl ester (C24:1n9) at 2 wt % ........................................................................................................................... 1822,1922,2254
1504,2073,1505,2074,1517,1518,1524,1527,1528,1530,1540,1541,1546,1547,1548,1549,1552,1553,1554,1555,1556,1557,1558,1560,1561, Neryl acetate ...................................................................................................... 2050,2434,2099,2103,2105,2106,2120,2121,2131,2142,2166,2170
1562,1563,1564,1567,1587,1582,2332,1593,1594,1595,1596,1597,1611,1612,1613,1614,1615,1616,1617,1618,2300,2467 Neurotensin .................................................................................................................................................................................................................... 788
Naphthalene-d8 ........................................................................................................................ 842,1409,2011,1477,1757,2007,2364,2389,2415, Nevirapine ............................................................................................................................................................................................................ 911,1214
1524,1550,1551,1552,1553,1554,1555,1556,1557,1564 New Coccine ................................................................................................................................................................................................................. 388
Naphthalene-d8 ............................................................................................................................................................................................... 1567,1587 Niacin ........................................................................................................................................................................................................................... 1301
Naphthalene-d8 ................................................................................................................................................................................................. 1795,1891 Niacinamide ............................................................................................................................................................................. 122,1299,1300,1332,1333
Indexes Analyte
Niacinamide (Vitamin B3) ............................................................................................................................................................................ 334,335,332 N-Nitrosodi-n-propylamine ................................................... 1468,1744,1991,2358,2385,2408,1524,1552,1553,1554,1555,1556,1557,1558,1564

Nicardipine hydrochloride ............................................................................................................................................................. 1089,2972,1090,2973 N-Nitrosomethylethylamine ..................................................................................................................... 1468,1744,1991,2358,2385,2408,1550,1551
Nicotinamide ................................................................................................................................................................ 1302,1303,1304,1306,1307,1308 N-Nitrosomorpholine ......................................................................................................................................................................................... 1550,1551
Nicotine ........................................................................................................................................................................................................................ 2289 N-Nitrosopiperidine .................................................................................................................................. 1468,1744,1991,2358,2385,2408,1550,1551
(±)-Nicotine ................................................................................................................................................................................................................ 2288 N-Nitrosopyrrolidine .......................................................................................................................................................................................... 1550,1551
S(−)-Nicotine solution ................................................................................................................................................................................................. 245 Nitrothal-isopropyl ............................................................................................................................................................................................. 1887,2066
Nicotinic acid ................................................................................................................................... 1300,1302,1303,1304,1306,1307,1308,1309,1333 2-Nitrotoluene .......................................................................................................................... 297,1455,1695,2297,2354,2383,2466,1456,1696,2298
Nifedipine ............................................................................................................................................................................................................. 896,1215 3-Nitrotoluene .......................................................................................................................... 297,1455,1695,2297,2354,2383,2466,1456,1696,2298
Nitenpyram ............................................................................................................................................................................................................. 1358,19 4-Nitrotoluene .......................................................................................................................... 297,1455,1695,2297,2354,2383,2466,1456,1696,2298
Nitralin ................................................................................................................................................................................................................ 1889,2068 5-Nitro-o-toluidine ............................................................................................................................................................................................ 1550,1551
Nitrazepam ........................................................................................................................................................................... 135,935,1191,233,916,1091 Nitrous oxide ............................................................................................................................................................................................................... 1743
Nitrazepam solution ..................................................................................................................................................................................................... 833 Nivalenol ................................................................................................................................................................................................................... 17,394
2-Nitroaniline ..................................................................................... 492,1484,2030,2366,2421,1552,1553,1554,1555,1556,1557,1558,1564,1673 Nivalenol hydrate .................................................................................................................................................................................................... 12,358
3-Nitroaniline ............................................................................................ 1484,2030,2366,2421,1552,1553,1554,1555,1556,1557,1558,1564,1673 Nizatidine ..................................................................................................................................................................................................................... 1173
4-Nitroaniline ............................................................................................ 1484,2030,2366,2421,1552,1553,1554,1555,1556,1557,1558,1564,1673 trans-Nonachlor ..................................................................................................................... 1388,1411,1482,2028,1507,2076,1571,1591,2325,2347
Nitrobenzene ................................................................................................................................................ 296,1455,1695,2297,2354,2383,2466, 2,2',3,3',4,4',5,5',6-Nonachlorobiphenyl ....................................................................................................................................... 1441,1927,1442,1928
1456,1696,2298,1504,2073,1524,1552,1553,1554,1555,1556,1557,1558,1564 2,2’,3,3’,4,4’,5,5’,6-nonachlorobiphenyl .......................................................................................................................................................... 1443,1929
Nitrobenzene-d5 ........................................................................................................... 1524,1550,1551,1552,1553,1554,1555,1556,1557,1558,1564 2,2',3,3',4,5,5',6,6'-Nonachlorobiphenyl ....................................................................................................................................... 1441,1927,1442,1928
4-Nitrocatechol ......................................................................................................................................................................................................... 152,44 2,2’,3,3’,4,5,5’,6,6’-nonachlorobiphenyl .......................................................................................................................................................... 1443,1929
Nitrofen ............................................................................................................................................................................................................... 1889,2068 Nonadecane ................................................................................................................................................................................................................. 1637
Nitrofurantoin .................................................................................................................................................................................................. 127,877,879 Nonadecane (C19) ...................................................................................................................................................................................................... 1636
Nitrogen .................................................................................................................................. 1622,1627,1628,1639,1743,1747,1749,1750,1751,1753 Nonanal .......................................................................................................................... 1431,1572,1490,1580,2318,2330,1491,1581,2319,2331,
Nitrogen (bulk) ................................................................................................................................................................................................. 1752,1754 1878,2044,2398,2430,1879,2046,2399,2431,1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436,1883,2054,2402,2437,
Nitromethane ..................................................................................................................................................... 2205,2206,2207,2450,2208,2209,2451 1971,2405,2045,2350,2050,2434,2096,2099,2111,2184,2120,2166,2170
2-Nitrophenol ................................................................................................................................................. 90,1359,1386,1407,1475,1755,2005, Nonane ................................................................................................................ 1431,1572,1486,1487,1528,1530,1595,1601,1667,1602,1668,1610
1476,1756,2006,2363,2388,2414,1477,1757,2007,2364,2389,2415,1524,1531,1552,1553,1554,1555,1556,1557,1558,1564 n-Nonane ........................................................................................................................................................................................................... 1592,1612
4-Nitrophenol ................................................................................................................................................. 90,1359,1386,1407,1475,1755,2005, Nonane (C9) ............................................................................................................................................................................................................... 1636
1476,1756,2006,2363,2388,2414,1477,1757,2007,2364,2389,2415,1524,1531,1552,1553,1554,1555,1556,1557 Nonanoic acid .................................................................................................................................. 1492,1583,2320,2333,1878,2044,2398,2430,
4-nitrophenol .............................................................................................................................................................................................................. 1558 1879,2046,2399,2431,1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436,1883,2054,2402,2437,1970,2404
4-Nitrophenol .............................................................................................................................................................................................................. 1564 Nonanol ........................... 1878,2044,2398,2430,1879,2046,2399,2431,1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436,
4-Nitrophenyl β-D-maltoside ....................................................................................................................................................................................... 119 1883,2054,2402,2437,2103
Nitropropane ................................................................................................................................................................................................................ 1676 Nonan-3-ol .................................................................................................................................................................................................................. 2138
2-Nitropropane ............................................................................................................................................................ 1504,2073,1725,1726,1727,1780 Nonanone ........................................................................................................................................................................................................... 1971,2405
4-Nitroquinoline N-oxide ................................................................................................................................................................................. 1550,1551 trans-2-Nonenal ................................................................................................................................................................................................ 1896,2403
(S)-N-Nitrosoanabasine (NAB) solution ..................................................................................................................................................................... 245 1-Nonene ..................................................................................................................................................................................................................... 1612
N-Nitrosodi-n-butylamine ........................................................................................................................ 1468,1744,1991,2358,2385,2408,1550,1551 2-Nonen-1-ol .................................................................................................................................................................................. 1492,1583,2320,2333
N-Nitrosodiethylamine ............................................................................................................................. 1468,1744,1991,2358,2385,2408,1550,1551 Nonyl acetate ................................................................................................................................................................................. 2050,2434,2096,2099
N-Nitrosodimethylamine .......................................................................... 1468,1744,1991,2358,2385,2408,1552,1553,1554,1555,1556,1557,1558 Nonylamine ................................................................................................................................................................................................................. 1776
N-Nitrosodiphenylamine ........................................................ 1468,1744,1991,2358,2385,2408,1524,1552,1553,1554,1555,1556,1557,1558,1564 Nonylphenol ...................................................................................................................................................................................................... 83,299,341
Indexes Analyte
Nootkatone .................................................................................................................................................................................................................. 2096

Norbornanol ................................................................................................................................................................................................................. 2120 —O—
Norbuprenorphine solution .......................................................................................................................................................................................... 692 OCDD .......................................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913
Norbuprenorphine glucuronide solution .................................................................................................................................................................... 692 OCDF .......................................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913
Norclomipramine ................................................................................................................................................................................ 1164,210,1166,211 Ochratoxin A ................................................................................................................................................................................................................. 331
Norcotinine ..................................................................................................................................................................................................................... 245 (Z)-β-Ocimene ................................................................................................................................................................................................... 2050,2434
Nordiazepam solution .................................................................................................................................................................................................. 833 (E)-β-Ocimene .................................................................................................... 2050,2434,2096,2103,2120,2121,2131,2137,2138,2142,2166,2167
Nordiazepam-d5 solution ............................................................................................................................................................................................. 833 trans-β-Ocimene ......................................................................................................................................................................................................... 2170
Nordihydroguaiaretic acid .................................................................................................................................................................... 276,337,1282,292 (Z)-β-Ocimene .......................................................................................................................................... 2167,2096,2121,2131,2137,2138,2142,2166
Nordoxepin ................................................................................................................................................................ 1164,210,1166,211,915,1165,1168 2,2',3,3',4,4',5,5'-Octachlorobiphenyl ........................................................................................................................................... 1441,1927,1442,1928
Norephedrine ................................................................................................................................................................................................................. 992 2,2’,3,3’,4,4’,5,5’-octachlorobiphenyl ............................................................................................................................................................... 1443,1929
Norepinephrine ........................................................................................................................................................................................ 144,180,254,955 2,2′,3,3′,4,5′,6,6′-Octachlorobiphenyl .................................................................................................... 1388,1411,1482,2028,1506,2075,1507,2076
Norethandrolone .............................................................................................................................................................................. 195,197,198,200,201 2,2',3,3',5,5',6,6'-Octachlorobiphenyl ........................................................................................................................................... 1441,1927,1442,1928
Norfentanyl .................................................................................................................................................................................................................... 464 2,2’,3,3’,5,5’,6,6’-octachlorobiphenyl ............................................................................................................................................................... 1443,1929
Norfloxacin .................................................................................................................................................................................................... 1005,356,878 Octachloronaphthalene .................................................................................................................................................................. 1571,1591,2325,2347
Norfluoxetine hydrochloride ............................................................................................................................................................................. 1008,1010 Octacosane ................................................................................................................................................................................................ 1486,1487,1526
Norflurazon ...................................................................................................................................................................................... 1506,2075,1507,2076 Octadecadienoic acid .................................................................................................................................................................................................. 1784
Norgestrel ............................................................................................................................................................................................................. 181,2639 Octadecane ................................................................................................................................................................... 1486,1487,1526,1637,2316,2317
DL-Norleucine ........................................................................................................................................................................................................ 2814,681
Octadecane (C18) ....................................................................................................................................................................................................... 1636
DL-Norleucine, FMOC derivatives ....................................................................................................................................................................... 2785,600
Octadecanoate (C18:0) .............................................................................................................................................................................................. 1636
Normeperidine ............................................................................................................................................................................................................... 427 Octadecanoic acid .................................................................................................................................................................................... 1873,2031,2278
Normeperidine solution ................................................................................................................................................................................................ 481 cis-11-Octadecenoic methyl ester ................................................................................................ 1867,2018,2274,1868,2019,2275,1869,2020,2276
Normetanephrine .......................................................................................................................................................................................................... 254 3,5-octadien-2-one ........................................................................................................................................................................................... 2045,2350
Normorphine .......................................................................................................................................................................................................... 124,868 Octanal ..................................... 1490,1580,2318,2330,1491,1581,2319,2331,1971,2405,2050,2434,2096,2099,2103,2111,2184,2120,2166,2170
Normorphine solution .................................................................................................................................................................................................. 491 Octane ........................................................................................................ 1431,1572,1486,1487,1592,1595,1601,1667,1602,1668,1610,1971,2405
(±)-Nornicotine solution ............................................................................................................................................................................................. 246 n-Octane ...................................................................................................................................................................................................................... 1612
Noroxycodone ................................................................................................................................................................................................................ 462 1,2-Octanediol ................................................................................................................................................................................................... 1646,2489
Norphenylephrine hydrochloride ........................................................................................................................................... 2815,682,1092,2602,2974 1,2-Octanediol (O-TFA derivatives) ................................................................................................................................................................ 1647,2490
Norsertraline ......................................................................................................................................................................................................... 1196,252 Octanoic acid ................................................................................................................................... 1873,2031,2278,1970,2404,1971,2405,2060,2443
Nortriptyline ................................................................................................................. 1368,209,1164,210,1166,211,1167,212,1165,1168,1169,1170 Octanol ................................................................................................................................................................................... 2099,2103,2120,2136,2166
DL-Norvaline .......................................................................................................................................................................................................... 2816,683
1-Octanol ............................................................................................................................................................ 2170,2311,2312,2313,2314,2316,2317
DL-Norvaline (FMOC derivatives) ....................................................................................................................................................................... 2786,601
Octan-3-ol ................................................................................................................................................................................................ 2137,2138,2167
(±)-Norverapamil hydrochloride .............................................................................................................................................................................. 1222 δ-Octanolactone ............................................................................................................................................................................................................ 322
Noscapine .......................................................................................................................................................................................................... 30,407,853 2-Octanone .................................................................................................................................................................. 2311,2312,2313,2314,2316,2317
Noscapine hydrochloride hydrate ............................................................................................................................................................................... 490 Octan-3-one ................................................................................................................................................................................................................ 2138
NSAIDs .......................................................................................................................................................................................................................... 1094 1-Octen-3-ol ................................................................................................................................................................................................................ 2136
Nuarimol .......................................................................................................................................................................................... 1447,1681,1828,1933 Octyl acetate ............................................................................................................................................................................................ 2050,2434,2096
Nucleic acids ................................................................................................................................................................................................................. 684 3-Octyl acetate ........................................................................................................................................................................................................... 2167
Octylamine ................................................................................................................................................................................................................... 1776
Indexes Analyte
Octyl gallate .......................................................................................................................................................................................... 276,337,1282,292 Oxo-isooctyl alcohols .................................................................................................................................................................................................. 1728

2-Octyl-4-isothiazolin-3-one .............................................................................................................................................................................. 1419,406 Oxpoconazole ..................................................................................................................................................................................................... 1890,2069
Octylphenol ...................................................................................................................................................................................................................... 83 Oxpoconazole fumarate; furmate portion ...................................................................................................................................................... 1890,2069
Oenothein B ......................................................................................................................................................................................................... 1095,182 Oxprenolol ................................................................................................................................................................................................... 954,1101,2979
Oenothein B isomer ............................................................................................................................................................................................ 1095,182 Oxprenolol hydrochloride .............................................................................................................................................................. 1102,2980,1103,2981
Ofloxacin ............................................................................................................................................................................................................... 1004,355 Oxycodone ....................................................................................................................................................................................................... 427,428,462
Oleate (C18:1) ............................................................................................................................................................................................................ 1636 Oxycodone solution ................................................................................................................................................................................ 264,484,481,692
Oleic acid ................................................................................................................................................................................................................ 351,357 4,4′-Oxydianiline ......................................................................................................................................................................................................... 1673
Oleic acid methyl ester ..................................... 1637,1818,1918,2250,1819,1919,2251,1837,1954,1840,1960,1845,1965,1846,1966,1847,1967 Oxyfluorfen ......................................................................................................................................................................................................... 1887,2066
Oleic acid methyl ester (C18:1n9c) ............................................. 1815,1915,2247,1817,1917,2249,1836,1945,1839,1959,1842,1962,1844,1964 Oxygen ................................................................................................................................................................................... 1639,1747,1749,1753,1754
Oleic acid methyl ester (C18:1n9c) at 4 wt % ................................................................................................................................ 1822,1922,2254 Oxymetholone ............................................................................................................................................................................................................... 195
OLL ........................................................................................................................................................................................................... 390,391,392,393 Oxymorphone-D3 solution ........................................................................................................................................................................................... 692
OLLn ......................................................................................................................................................................................................... 390,391,392,393 Oxymorphone-3-β-D-glucuronide solution ................................................................................................................................................................ 692
OLO ................................................................................................................................................................................................................................. 324 Oxyphenonium bromide ............................................................................................................................................................................................ 1134
Omeprazole ........................................................................................................................................................ 1372,247,248,249,250,1097,2975,1173 Oxytetracycline ............................................................................................................................................................................................................ 1157
Omeprazole sulfone ...................................................................................................................................................................... 1372,247,248,249,250 Oxytocin ............................................................................................................................................................................................ 709,710,720,721,820
Omethoate ............................................................................................................................. 1350,1401,1860,1995,1447,1681,1828,1933,1887,2066
OOL ........................................................................................................................................................................................................... 390,391,392,393 —P—
OOLn ....................................................................................................................................................................................................................... 390,391 Paarlan ............................................................................................................................................................................................. 1467,1990,2357,2407
OOP ......................................................................................................................................................................................................................... 390,391 Paclitaxel ......................................................................................................................................................................................... 1104,1153,1154,1220
OOPo ............................................................................................................................................................................................................................... 390 Paclobutrazole .................................................................................................................................................................................................... 1888,2067
OPP ..................................................................................................................................................................................................................... 1890,2069 Palmitelaidic acid .......................................................................................................................................................................................................... 357
Ordram ............................................................................................................................................................................................ 1467,1990,2357,2407 Palmitic acid .......................................................................................................................................................................................................... 351,357
DL-Ornithine monohydrochloride ....................................................................................................................................................................... 2817,693
Palmitic acid methyl ester ................................................... 1637,1818,1918,2250,1819,1919,2251,1837,1954,1840,1960,1846,1966,1847,1967
Oryzalin ................................................................................................................................................................................................................. 1419,406 Palmitic acid methyl ester (C16:0) .............................................. 1815,1915,2247,1817,1917,2249,1836,1945,1839,1959,1842,1962,1844,1964
Ovalbumin ....................................................................................................................................................................................................... 757,760,826 Palmitic acid methyl ester (C16:0) at 6 wt % ................................................................................................................................ 1822,1922,2254
(O) Ovalbumin ..................................................................................................................................................................................................... 1185,808 Palmitic acid methyl ester .............................................................................................................................................................................. 1845,1965
Ovalbumin (257-264) chicken ................................................................................................................................................................... 1129,187,825 Palmitoleic acid methyl ester ............................................................................................. 1818,1918,2250,1819,1919,2251,1837,1954,1840,1960
Oxabetrinil .......................................................................................................................................................................................................... 1889,2068 Palmitoleic acid methyl ester (C16:1) ......................................................................................................................................... 1815,1915,2247,
Oxadiazon ............................................................................................................................... 1448,1682,1829,1934,1467,1990,2357,2407,1887,2066 1817,1917,2249,1836,1945,1839,1959,1844,1964
Oxadixyl .............................................................................................................................................................................................................. 1889,2068 Palmitoleic acid methyl ester (C16:1) at 2 wt % ........................................................................................................................... 1822,1922,2254
Oxalide ................................................................................................................................................................................................................ 2111,2184 Papaverine ........................................................................................................................................................................................ 30,407,853,1233,854
Oxamniquine ...................................................................................................................................................................................................... 1098,2976 Papaverine hydrochloride ............................................................................................................................................................................................ 490
Oxamyl ......................................................................................................................................................................................................... 1,295,294,302 Parabens ......................................................................................................................................................................................................................... 285
Oxazepam ..................................................................... 136,936,137,937,138,938,1190,232,1207,273,489,875,876,915,916,1099,2977,1100,2978 Paracetamol .......................................................................................................................................................................................................... 910,1145
Oxazepam solution .............................................................................................................................................................................................. 833,1224 Paraoxon-methyl ............................................................................................................................................................................................... 1507,2076
Oxazepam-d5 solution .................................................................................................................................................................................................. 833 Paraquat ................................................................................................................................................................................................. 13,1356,1392,312
Oxazepam glucuronide solution ........................................................................................................................................................................ 833,1224 Parathion ..................................................................................................... 1350,1401,1860,1995,1466,1740,1858,1989,2356,2384,2392,2406,
Oxazepam-d5 glucuronide ........................................................................................................................................................................................... 833 1471,1746,1861,1997,2361,2386,2393,2411,1544,1545,1550,1551,1585,2342,2004,2413
Indexes Analyte
Parathion-methyl ............................................................................... 1419,406,1350,1401,1860,1995,1449,1683,1830,1935,1544,1545,1885,2064 2,2’,4,6,6’-pentachlorobiphenyl ....................................................................................................................................................................... 1443,1929

Paraxanthine ........................................................................................................................................................................................ 1362,1427,148,946 3,3',4,4',5-Pentachlorobiphenyl .................................................................................................................................................... 1441,1927,1442,1928
Paroxetine ............................................................................................................................................................................................................... 239,811 3,3’,4,4’,5-pentachlorobiphenyl ....................................................................................................................................................................... 1443,1929
Paroxetine hydrochloride hemihydrate .................................................................................................................................................................... 1216 3,3′,4,4′,5-Pentachlorobiphenyl ................................................................................................................................................................................ 1501
PBDE 8 ............................................................................................................................................................................................................... 1450,1684 3,3′,4,5,5′-Pentachlorobiphenyl ................................................................................................................................................................................ 1501
PBDE 17 ............................................................................................................................................................................................................. 1450,1684 2,2',4,5,5'-Pentachlorobiphenyl (No. 101) .................................................................................................................................................... 1402,1999
PBDE 28 ............................................................................................................................................................................................................. 1450,1684 2,3,3',4,4'-Pentachlorobiphenyl (No. 105) .................................................................................................................................................... 1402,1999
PBDE 66 ............................................................................................................................................................................................................. 1450,1684 2,3,4,4',5-Pentachlorobiphenyl (No. 114) ..................................................................................................................................................... 1402,1999
PBDE 100 ........................................................................................................................................................................................................... 1450,1684 2,3',4,4',5-Pentachlorobiphenyl (No. 118) .................................................................................................................................................... 1402,1999
PBDE 138 ........................................................................................................................................................................................................... 1450,1684 2',3,4,4',5-Pentachlorobiphenyl (No. 123) .................................................................................................................................................... 1402,1999
PBDE 153 ........................................................................................................................................................................................................... 1450,1684 3,3',4,4',5-Pentachlorobiphenyl (No. 126) .................................................................................................................................................... 1402,1999
PBDE 154 ........................................................................................................................................................................................................... 1450,1684 Pentachloroethane .................................................................................................................................... 1504,2073,1546,1547,1548,1549,1550,1551
PBDE 190 ........................................................................................................................................................................................................... 1450,1684 Pentachloronitrobenzene ............................................................................................. 1351,1403,1862,2000,1509,1510,1511,1550,1551,1887,2066
PBDE 209 ........................................................................................................................................................................................................... 1450,1684 Pentachlorophenol ............. 1355,1389,2,298,90,1359,1386,1407,1475,1755,2005,1388,1411,1482,2028,1476,1756,2006,2363,2388,2414,
pBR322 Hae III Digest ................................................................................................................................................................................................. 695 1477,1757,2007,2364,2389,2415,1506,2075,1507,2076,1524,1531,1552,1553,1554,1555,1556,1557,1558,1564,1571,1591,2325,2347
PCB congeners ................................................................................................................................................................................ 1385,1474,1472,2362 Pentachlorophenol (acylated) ....................................................................................................................................................... 2040,2426,2042,2428
1,2,3,7,8-PCDD .......................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913 Pentacosane ................................................................................................................................................................................................................. 1637
1,2,3,7,8-PCDF .......................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913 Pentacosane (C25) ..................................................................................................................................................................................................... 1636
2,3,4,7,8-PCDF .......................................................................................................................................... 1436,1575,1812,1912,1437,1576,1813,1913 Pentadecane ....................................................................................................................................................... 1431,1572,1602,1668,1637,2316,2317
PCP ...................................................................................................................................................................................................................... 2291,2463 Pentadecane (C15) ..................................................................................................................................................................................................... 1636
Pebulat ............................................................................................................................................................................................................... 1507,2076 Pentadecanoic acid methyl ester ............................................................................................................................. 1818,1918,2250,1819,1919,2251
Pebulate .............................................................................................................................................................................................................. 1506,2075 Pentadecanoic acid methyl ester (C15:0) ............................................................................................................... 1815,1915,2247,1817,1917,2249
PEG 40, 000Da ............................................................................................................................................................................................................. 696 Pentadecanoic acid methyl ester (C15:0) at 2 wt % ...................................................................................................................... 1822,1922,2254
PEG 26, 100Da ............................................................................................................................................................................................................. 696 cis-10-Pentadecenoic acid methyl ester .................................................................................................................. 1818,1918,2250,1819,1919,2251
PEG 81, 400Da ............................................................................................................................................................................................................. 696 cis-10-Pentadecenoic acid methyl ester (C15:1) ................................................................................................... 1815,1915,2247,1817,1917,2249
PEG 50, 630Da ............................................................................................................................................................................................................. 696 cis-10-Pentadecenoic acid methyl ester (C15:1) at 2 wt % .......................................................................................................... 1822,1922,2254
PEG 8, 730Da ............................................................................................................................................................................................................... 696 Pentafluorophenol ....................................................................................................................................................................................................... 1531
70 kDa protein (undisclosed drug candidate) ......................................................................................................................................................... 777 Pentamethylbenzene .................................................................................................................................................................................................. 1621
Pelargonidin chloride ........................................................................................................................................................................................ 1320,1321 2,2,4,6,6-Pentamethyl-3-heptene ............................................................................................................................................................................. 1721
Pencanazole ................................................................................................................................................................. 1347,1394,1798,1894,1797,1893 Pentanal ..................................................................................................... 1490,1580,2318,2330,1491,1581,2319,2331,1969,1971,2405,2051,2435
Penconazole ....................................................................................................................................................................................................... 1887,2066 Pentane ............................................. 1528,1530,1592,1595,1601,1667,1602,1668,1610,1611,1612,1721,2210,2211,2212,2452,2213,2214,2453
Pendimethalin ........................................................................................................................ 1430,1641,1796,1892,1449,1683,1830,1935,1887,2066 n-Pentane .................................................................................................................................................................................................................... 1665
DL-Penicillamine ................................................................................................................................................................................................. 1108,2982 n-Pentane .................................................................................................................................................................................................................... 1723
Penicillin G ................................................................................................................................................................................................. 184,1009,1041 1,2-Pentanediol ................................................................................................................................................................................................. 1646,2489
Penicillin V ......................................................................................................................................................................................................... 1009,1041 1,2-Pentanediol (O-TFA derivatives) .............................................................................................................................................................. 1647,2490
2,2′,4,4′,5-Pentabromodiphenyl ether solution ............................................................................................................................................ 1450,1684 2,3-Pentanedione .............................................................................................................................................................................................. 2056,2439
Pentachlorobenzene .................................................................................................................................................... 1550,1551,1571,1591,2325,2347 Pentanol ....................................................................................................................................................................................................................... 1969
2,2′,3′,4,6-Pentachlorobiphenyl .............................................................................................................. 1388,1411,1482,2028,1506,2075,1507,2076 1-Pentanol .......................................................................................................................................................................................................... 1644,1666
2,2',4,6,6'-Pentachlorobiphenyl .................................................................................................................................................... 1441,1927,1442,1928 2-Pentanol .......................................................................................................................................................................................................... 1644,1969
Indexes Analyte
3-Pentanol .......................................................................................................................................................................................................... 1644,1969 Perylene-d12 (I.S.) ..................................................................................................................................................................................................... 1558

Pentedrone hydrochloride .................................................................................................................................................................................... 471,478 Pesticides ..................................................................................................................................................................................................................... 1515
1-Pentene .................................................................................................................................................................................................................... 1612 Petunidin 3-(6-acetylglucoside) .................................................................................................................................................................................. 336
cis-2-Pentene ..................................................................................................................................................................................................... 1612,1721 Petunidin 3-(6-cumarylglucoside) .............................................................................................................................................................................. 336
trans-2-Pentene ................................................................................................................................................................................................. 1612,1721 Petunidin 3-glucoside .................................................................................................................................................................................................. 336
3-Penten-2-one ................................................................................................................................................................................................. 2056,2439 α-Phellandrene .................................................... 2047,2175,2351,2353,2050,2434,2096,2099,2103,2105,2106,2120,2121,2131,2142,2166,2170
2-Pentenyl hexanoate ................................................................................................................................................................... 1876,2035,2396,2423 β-Phellandrene ......................................................................................................................................................................................... 2121,2138,2167
Pentobarbital ...................................................................................................................................................... 414,925,416,927,417,929,418,930,928 Phenacetin .................................................................................................................................................. 1372,247,248,249,250,870,1198,1550,1551
Pentoxazone ....................................................................................................................................................................................................... 1889,2068 Phenanthrene ........................................................................................................... 2375,310,311,1388,1411,1482,2028,1408,2010,1409,2011,
Pentyl acetate ............................................................................................................................................................................................................. 1676 1451,1936,1480,2013,1481,2014,1483,2029,2365,2420,1506,2075,1507,2076,1517,1518,1524,1540,1541,1552,1553,1554,1555,1556,1557,
Pentylamine ................................................................................................................................................................................................................. 1776 1558,1560,1561,1562,1563,1564,1567,1587,1795,1891
Pentyl butanoate ............................................................................................................................................................................ 1876,2035,2396,2423 Phenanthrene-d10 ......................................................................................................... 1388,1411,1482,2028,1483,2029,2365,2420,1506,2075,
Pentyl hexanoate ........................................................................................................................................................ 1876,2035,2396,2423,1971,2405 1507,2076,1524,1550,1551,1552,1553,1554,1555,1556,1557,1564
Pentylone hydrochloride .............................................................................................................................................................................................. 469 Phenanthrene-d10 ............................................................................................................................................................................................ 1567,1587
Peonidin 3-(6-acetylglucoside) ................................................................................................................................................................................... 336 Phenanthrene-d10 .............................................................................................................................................................................................. 1795,1891
Peonidin chloride .............................................................................................................................................................................................. 1320,1321 Phenanthrene-d10 (I.S.) ............................................................................................................................................................................................ 1558
Peonidin 3-(6-cumarylglucoside) ............................................................................................................................................................................... 336 Phencyclidine ................................................................................................................................................................................................................. 867
Peonidin 3-O-glucoside chloride ................................................................................................................................................................................ 336 Phenethyl alcohol ....................................................................................................................................................................................................... 2131
Peppermint oil ............................................................................................................................................................................................................ 2136 Phenmedipham ................................................................................................................................................................................................. 1890,2069
Perilla alcohol .......................................................................................................................................................................................... 2050,2434,2170 Phenobarbital .................................................................................. 1200,269,485,1201,270,486,414,925,416,927,417,929,418,930,928,2288,2289
Perilla aldehyde .................................................................................................................................................................... 2050,2434,2103,2120,2166 Phenol .. 48,90,1117,1283,1359,1386,1407,1475,1755,2005,1476,1756,2006,2363,2388,2414,1477,1757,2007,2364,2389,2415,1531,1552,
(−)-Perillaaldehyde .................................................................................................................................................................................................... 2170 1553,1554,1555,1556,1557,1558,1564
(+)-Perillaaldehyde .................................................................................................................................................................................................... 2096 Phenol-d6 ...................................................................................................................... 1524,1550,1551,1552,1553,1554,1555,1556,1557,1558,1564
Perillyl acetate ............................................................................................................................................................................................................ 2131 Phenols ......................................................................................................................................................................................................................... 1531
Peritone ........................................................................................................................................................................................................................ 2136 Phenothrin-1 ...................................................................................................................................................................................................... 1890,2069
Permanent gases ........................................................................................................................................................................................................ 1749 Phenothrin-2 ...................................................................................................................................................................................................... 1890,2069
cis-Permethrin ........................................................................................................................................... 1347,1394,1798,1894,1507,2076,1797,1893 2-Phenoxyethanol ................................................................................................................................................................................... 282,283,285,286
cis/trans-Permethrin .......................................................................................................................................................................................... 1506,2075 Phenoxymethylpenicillinic acid ................................................................................................................................................................................... 184
trans-Permethrin ...................................................................................................................................... 1347,1394,1798,1894,1507,2076,1797,1893 1-Phenoxy-2-propanol ................................................................................................................................................. 284,282,283,285,286,1465,2355
Permethrin-1 ...................................................................................................................................................................................................... 1887,2066 Phensuximide .................................................................................................................................................................................. 1111,2983,1112,2984
Permethrin-2 ...................................................................................................................................................................................................... 1887,2066 Phentermine .................................................................................................................................................................................... 2234,2285,2457,2460
Permethrin isomer ................................................................................................................................................................................... 1509,1510,1511 Phenthoate ................................................................................................................................................................... 1350,1401,1860,1995,1885,2064
Permethrin (isomers) solution ........................................................................................................................................................................ 1404,2001 Phentoat .......................................................................................................................................................................................... 1448,1682,1829,1934
Permetrin ......................................................................................................................................................................................... 1445,1679,1826,1931 Phenylacetaldehyde ....... 1878,2044,2398,2430,1879,2046,2399,2431,1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436,
Peroxidase ...................................................................................................................................................................................................................... 722 1883,2054,2402,2437,1896,2403
Perylene ........................................................................................................................................................................ 1408,2010,1409,2011,1567,1587 Phenylacetic acid ............................................................................................................................................................................................................ 91
Perylene-d12 ............................................................................ 1388,1411,1482,2028,1408,2010,1409,2011,1434,1435,1483,2029,2365,2420, D-Phenylalanine ................................................................................................................................................................................................... 2818,723
1506,2075,1507,2076,1524,1550,1551,1552,1553,1554,1555,1556,1557,1564 L-Phenylalanine ................................................................................................................................................................................. 2818,723,1897,2304
Perylene-d12 ..................................................................................................................................................................................................... 1567,1587 DL-Phenylalanine .................................................................................................................................................................................................. 2819,724
Indexes Analyte
DL-Phenylalanine (AQC derivatives) .................................................................................................................................................................. 2739,518 Phosfolan ......................................................................................................................................................................................... 1350,1401,1860,1995

DL-Phenylalanine (t-BOC derivatives) ............................................................................................................................................................... 2753,535 Phosmet ........................................................................... 1419,406,1350,1401,1860,1995,1445,1679,1826,1931,1542,1543,1544,1545,1887,2066
DL-Phenylalanine (FMOC derivatives) ............................................................................................................................................................... 2787,602 Phosphamidon ................................................................................................................................................................................ 1350,1401,1860,1995
Phenylbutazone ........................................................................................................................................................................................................... 1217 Phosphamidon .............................................................................................................................................................................. 1350,1401,1860,1995
4-Phenylcyclohexene ...................................................................................................................................................................... 1491,1581,2319,2331 Phosphamidon ............................................................................................................................................................. 1542,1543,1544,1545,1885,2064
Phenylenediamine ....................................................................................................................................................................................................... 1550 L-α-Phosphatidylcholine ............................................................................................................................................................................................... 399
p-Phenylenediamine ................................................................................................................................................................................................... 1551 L-α-Phosphatidylethanolamine from egg yolk ......................................................................................................................................................... 399
Phenylephrine ............................................................................................................................................................................................. 850,1113,2821 Phosphatidylinositol ammonium salt solution ......................................................................................................................................................... 399
Phenylethanol ................................................................................................................................................................................. 1759,2149,2541,2680 L-α-Phosphatidyl-L-serine from Glycine max (soybean) ......................................................................................................................................... 399
1-Phenylethanol ............................................................................................................................................................................................................ 322 Phospholipase-A ............................................................................................................................................................................................................ 801
(R)-(+)-1-Phenylethanol .............................................................................................................................................................. 1760,2150,2542,2681 Phospholipase B ............................................................................................................................................................................................................ 801
(±)-(1-Phenyl)ethanol .................................................................................................................................................................. 1759,2149,2541,2680 Phosphorylase b from rabbit muscle ........................................................................................................................................................................ 744
Phenylethyl Alcohol ................................................................................... 1878,2044,2398,2430,1879,2046,2399,2431,1880,2048,2400,2432, Phthalate esters .............................................................................................................................................................................. 1478,1769,2009,2192
1881,2052,2349,2352,1882,2053,2401,2436,1883,2054,2402,2437 Phthalide ............................................................................................................................................................................................................ 1886,2065
Phenylethyl alcohol ........................................................................................................................................................................ 2056,2439,2111,2184 Phylloquinone (K1) ................................................................................................................................................................................. 1255,1256,1253
1-Phenylethylamine .......................................................................................................................................................................................... 1761,2543 Phytol .................................................................................................................................................................................................................. 2111,2184
R-(+)-1-Phenylethylamine (N-Chloroacetyl derivative) ........................................................................................................... 1763,2545,1764,2546 Picloram ................................................................................................................................................................................................... 1355,1389,2,298
S-(-)-1-Phenylethylamine (N-Chloroacetyl derivative) ............................................................................................................. 1763,2545,1764,2546 2-Picoline ........................................................................................................................................................................................................... 1550,1551
R-(+)-1-Phenylethylamine (N-TFA derivative) ................................................................ 1762,2544,1765,2547,1766,2548,1767,2549,1768,2550 2-Picoline (2-Methyl pyridine) ................................................................................................................................................................................. 1673
S-(-)-1-Phenylethylamine (N-TFA derivative) .................................................................. 1762,2544,1765,2547,1766,2548,1767,2549,1768,2550 Pilocarpine .......................................................................................................................................................................................................... 1114,1195
Phenylethyl benzoate ....................................................................................................................................................................................... 2111,2184 Pindolol ........................................................................................................................................ 131,139,943,896,895,941,942,1115,2985,1116,2986
N-(1-Phenylethyl)maleimide ................................................................................................................................................................................ 2604,92 α-Pinene ...................................................................................................................................................................................................................... 2137
Phenylethyl phenylacetate ............................................................................................................................................................................... 2111,2184 (−)-α-Pinene ..................................................................................................................................................................................................... 1431,1572
3-Phenyl furan ................................................................................................................................. 1878,2044,2398,2430,1879,2046,2399,2431, α-Pinene ................................................................................................................................. 1431,1572,2047,2175,2351,2353,2050,2434,2095,2096
1880,2048,2400,2432,1881,2052,2349,2352,1882,2053,2401,2436,1883,2054,2402,2437 α-Pinene ........................................................................ 2096,2099,2103,2105,2106,2112,2120,2121,2124,2131,2136,2138,2142,2166,2167,2170
Phenyl isocyanate ....................................................................................................................................................................................................... 2338 β-Pinene .................................................................................................................................................... 2050,2434,2056,2439,2096,2097,2099,2103
DL-3-Phenyllactic acid ....................................................................................................................................................... 1285,2605,2707,3038,373,93 (−)-β-Pinene .............................................................................................................................................................................................................. 2105
Phenyl methyl ketone ................................................................................................................................................................... 1492,1583,2320,2333 β-Pinene .................................................................................................... 2111,2184,2120,2121,2124,2131,2136,2137,2138,2142,2166,2167,2169
4-Phenyl-2-oxazolidinone ...................................................................................................................................................................... 2606,94,2607,95 (−)-β-Pinene .............................................................................................................................................................................................................. 2170
(±)-1-Phenylpentanol ................................................................................................................................................................... 1759,2149,2541,2680 d-β-Pinene .......................................................................................................... 1771,2152,2552,2683,1773,2154,2554,2685,1775,2156,2556,2687
1-Phenyl-2-propanol ......................................................................................................................................................................................... 1642,2487 l-β-Pinene ........................................................................................................... 1771,2152,2552,2683,1773,2154,2554,2685,1775,2156,2556,2687
2-Phenyl-2-propanol ...................................................................................................................................................................... 1492,1583,2320,2333 d-α-Pinene (R-(+)-α-Pinene) ................................................................................................................ 1770,2151,2551,2682,1772,2153,2553,2684
2-Phenylpropionic acid ...................................................................................................................................................................... 1286,3039,323,374 l-α-Pinene (S-(-)-α-Pinene) ................................................................................................................... 1770,2151,2551,2682,1772,2153,2553,2684
Phenylsuccinic anhydride ..................................................................................................................................................................................... 2608,96 (−)-trans-Pinocarveol ................................................................................................................................................................................................ 2095
Phloxine B ..................................................................................................................................................................................................................... 388 Pioglitazone hydrochloride .......................................................................................................................................................................................... 840
Phorate ........... 1419,406,1350,1401,1860,1995,1447,1681,1828,1933,1466,1740,1858,1989,2356,2384,2392,2406,1470,1996,2360,2410, Pipecolinic acid ................................................................................................................................................................................... 185,2640,2822,726
1471,1746,1861,1997,2361,2386,2393,2411,1542,1543,1544,1545,1550,1551,1885,2064 Pipenzolate bromide .................................................................................................................................................................................................. 1134
Phosalone .................................................................................................................................................. 1350,1401,1860,1995,1885,2064,2004,2413 Piperacillin .......................................................................................................................................................................................................... 1009,1041
Phosdrin ........................................................................................................................................................................................... 1350,1401,1860,1995 trans-Piperitol .............................................................................................................................................................................................................. 2131
Indexes Analyte
Piperitone .................................................................................................................................................................................................. 2137,2138,2167 Procyanidin B3 ............................................................................................................................................................................................................ 1287

Piperonylbutoxide ........................................................................................................................................................................... 1404,2001,1889,2068 Procymidone ................................................................................................................................................................................... 1887,2066,2004,2413
Piperophos .......................................................................................................................................................................................................... 1885,2064 Profenfos .......................................................................................................................................................................................... 1350,1401,1860,1995
Pirenzepine .................................................................................................................................................................................................................. 1173 Profenofos ..................................................................................................................................................................... 1449,1683,1830,1935,1885,2064
Pirimicarb ........................................................................................................................................................................................ 1446,1680,1827,1932 Progesterone .................................................................................................................................................................................................................. 851
Pirimiphos-methyl ....................................................................................................................................................... 1448,1682,1829,1934,1885,2064 Progesterone solution ......................................................................................................................................................................................... 1374,257
Pirmiphos-ethyl .............................................................................................................................................................................. 1350,1401,1860,1995 Proglumide sodium salt ................................................................................................................................................................ 1122,2987,1123,2988
Pirmiphos-methyl ........................................................................................................................................................................... 1350,1401,1860,1995 Prohydrojasmon-1 ............................................................................................................................................................................................. 1890,2069
Piroxicam ................................................................................................................................................................................... 866,897,899,900,901,902 Prohydrojasmon-2 ............................................................................................................................................................................................. 1890,2069
PLL ............................................................................................................................................................................................................ 390,391,392,393 D-Proline ............................................................................................................................................................................................................... 2823,729
PLLn ........................................................................................................................................................................................................................ 392,393 L-Proline ............................................................................................................................................................................................. 2823,729,1897,2304
PLO .......................................................................................................................................................................................................................... 390,391 DL-Proline ............................................................................................................................................................................ 2824,730,2825,731,2844,830
PLP .......................................................................................................................................................................................................................... 390,391 DL-Proline (FMOC derivatives) ........................................................................................................................................................................... 2788,603
POL ................................................................................................................................................................................................................................. 393 D-Proline methyl ester (N-Acetyl derivative) ............................................................................................................................. 2016,2306,2704,2727
POLn ........................................................................................................................................................................................................................ 390,391 L-Proline methyl ester (N-Acetyl derivative) ............................................................................................................................. 2016,2306,2704,2727
POL+SLL ........................................................................................................................................................................................................................ 392 Promazine ............................................................................................................................................................................................................... 165,578
Poly-DL-alanine .............................................................................................................................................................................................................. 207 Promecarb ....................................................................................................................................................................................... 1449,1683,1830,1935
Poly-DL-alanine, 1,000-5,000 Da ......................................................................................................................................................................... 757,758 Promethazine ............................................................................................................................................................................................................... 1124
Polybrominated diphenyl ether (PBDE) ......................................................................................................................................................... 1450,1684 Promethazine hydrochloride ............................................................................................................................................................................ 1125,2989
Poly(ethylene glycol) ...................................................................................................................................................................................................... 97 Promethazine sulfoxide .................................................................................................................................................................................... 1126,2990
POO ................................................................................................................................................................................................................................. 393 Prometon ................................. 1466,1740,1858,1989,2356,2384,2392,2406,1489,1788,1877,2039,2369,2391,2397,2425,1506,2075,1507,2076
PoOLn ...................................................................................................................................................................................................................... 390,391 Prometryn ............. 1466,1740,1858,1989,2356,2384,2392,2406,1489,1788,1877,2039,2369,2391,2397,2425,1506,2075,1507,2076,1888,2067
PoOO ............................................................................................................................................................................................................................... 391 Pronamide ....................................................................................................................................................................................... 1506,2075,1550,1551
POP .................................................................................................................................................................................................................. 390,391,392 Propachlor ......................................................................... 1391,305,316,1467,1990,2357,2407,1506,2075,1507,2076,1509,1510,1511,1888,2067
PoPO ............................................................................................................................................................................................................................... 390 Propanal ....................................................................................................................................................................................................................... 1969
Posaconazole solution ........................................................................................................................................................................................... 831,832 Propane ............................................................................................................................................................... 1592,1612,1628,1665,1723,1724,1751
PPL .......................................................................................................................................................................................................................... 392,393 Propane (215 ng on column) ................................................................................................................................................................................. 1752
Pravastatin ............................................................................................................................................................................................. 1148,194,851,964 1,2-Propanediol ............................................................................................................................... 1383,1457,1646,2489,1705,1977,2187,2045,2350
Prazepam ..................................................................................................................................................................................................................... 2289 1,3-Propanediol ........................................................................................................................................................... 1703,1975,2185,1704,1976,2186
Prednisolone ......................................................................................................................................................................... 169,199,1290,204,851,1120 1,2-Propanediol (O-TFA derivatives) .............................................................................................................................................................. 1647,2490
Prednisone .............................................................................................................................................................................. 199,203,1290,204,205,453 1-Propanethiol ............................................................................................................................................................................................................. 1685
Pregabalin solution ....................................................................................................................................................................................................... 836 2-Propanethiol ............................................................................................................................................................................................................. 1685
Pretilachlor ......................................................................................................................................................................................................... 1888,2067 Propanil ....................................................................................................................................................................................................... 313,1889,2068
Prifenox-2 ........................................................................................................................................................................................................... 1887,2066 Propanoic acid, 2-methyl-, 3-hydroxy-2,4,4-trimethylpentyl ester ........................................................................................ 1491,1581,2319,2331
Prilocaine ................................................................................................................................................................................................................ 125,872 Propanol .......................................................................................................................................................................................... 1490,1580,2318,2330
Procainamide hydrochloride ............................................................................................................................................................................ 1121,1187 n-Propanol ................................................................................................................................................................................................ 1649,2380,1676
Procaine hydrochloride ................................................................................................................................................................................................. 490 1-Propanol .............................................................................................................................. 1431,1572,1644,1666,1725,1726,1727,1895,1969,
Procyanidin B1 ............................................................................................................................................................................................................ 1287 2026,2027,2210,2211,2212,2452,2213,2214,2453,2225,2455,2292,2464,2293,2294
Procyanidin B2 .................................................................................................................................................................................................... 376,1287 2-Propanol .................................................................... 1568,1588,2322,2344,1676,1726,1727,1895,2210,2211,2212,2452,2213,2214,2453,2294
Indexes Analyte
2-Propanol, 0.0785% (I.S.) ............................................................................................................................................................................. 1635,2378 R-(+)-Propylene oxide .................................................................................................................................................................................... 1777,2557

Propantheline ................................................................................................................................................................................................................ 119 S-(-)-Propylene oxide ...................................................................................................................................................................................... 1777,2557
Propantheline bromide .............................................................................................................................................................................................. 1134 Propylene (~95 %) .................................................................................................................................................................................................. 1723
Propaphos .......................................................................................................................................................................................................... 1885,2064 Propyl gallate ..................................................................................................................................................................... 276,337,1282,292,1898,2182
Propargite-1 ....................................................................................................................................................................................................... 1889,2068 Propyl 4-hydroxybenzoate ...................................................................................................................... 1748,1998,2190,2223,2387,2412,2447,2454
Propargite-2 ....................................................................................................................................................................................................... 1889,2068 Propyl isocyanate ........................................................................................................................................................................................................ 2338
Propazine ................................................................................................................................................................... 110,15,111,16,1357,1393,315, (±)-Propyl-γ-lactone ..................................................................................................................................................................... 1729,2118,2529,2676
1466,1740,1858,1989,2356,2384,2392,2406,1467,1990,2357,2407,1489,1788,1877,2039,2369,2391,2397,2425,1506,2075,1507,2076 Propyl paraben ............................................................................................................................................................................................... 282,283,286
Propetamphos ........................................................................................................................................... 1350,1401,1860,1995,1449,1683,1830,1935 Propylparaben ............................................................................................................................................................................................................... 289
Propham ............................................................................................................................... 1,295,294,302,2374,303,1391,305,1444,1678,1825,1930 Propyl paraben ............................................................................................................................................................................................. 290,291,1106
Propiconazole-1 ................................................................................................................................................................................................. 1888,2067 Propyltoluene .............................................................................................................................................................................................................. 1621
Propiconazole-2 ................................................................................................................................................................................................. 1888,2067 Propyne ........................................................................................................................................................................................... 1665,1723,1724,1751
Propiconazole isomers ................................................................................................................................................................... 1430,1641,1796,1892 Propyne (215 ng on column ................................................................................................................................................................................... 1752
Propionaldehyde .................................................................................................................................................................................................... 21,2326 Propyzamide ....................................................................................................... 1430,1641,1796,1892,1446,1680,1827,1932,1507,2076,1887,2066
Propionaldehyde-2,4-dinitrophenylhydrazone ..................................................................................................................................... 22,2334,23,2335 Prothiofos ........................................................................................................................................................................................................... 1885,2064
Propionaldehyde-2,4-DNPH ....................................................................................................................................................................................... 2337 Prothiophos .................................................................................................................................................................................................................... 308
Propionaldehyde (2,4-DNPH derivative) ............................................................................................................................................................ 2336,24 Prothoate ......................................................................................................................................................................................... 1448,1682,1829,1934
Propionic acid ..................................................................................................... 1808,1908,2244,1809,1909,2245,1873,2031,2278,1969,1970,2404 Protriptyline ....................................................................................................................................................................................... 1167,212,1169,1170
Propionic acid, 0.7% ................................................................................................................................................................................................. 1993 Protriptyline hydrochloride ........................................................................................................................................................................................ 1165
Propionitrile ........................................................................................................................................................................................................ 1504,2073 Prowl® ............................................................................................................................................................................................. 1467,1990,2357,2407
Propoxur .............................................................................................................................................. 1,295,294,302,1347,1394,1798,1894,1797,1893 Pseudoephedrine ......................................................................................................... 850,871,912,992,1132,2994,1145,1160,2234,2285,2457,2460
Propoxyphene ........................................................................................................................................................................................................ 427,445 Pseudolimonene .......................................................................................................................................................................................................... 2138
(+)-Propoxyphene solution ........................................................................................................................................................................................ 481 Pseudouridine ................................................................................................................................................................................................................ 686
Propranolol ....................................................................................................................................................................................... 139,943,931,942,954 Psoralen ....................................................................................................................................................................................................................... 1262
Propranolol hydrochloride solution .......................................................................................................................................................................... 1228 Pulegone ............................................................................................................................................................. 2047,2175,2351,2353,2136,2137,2138
DL-Propranolol hydrochloride ........................................................................................................................................................................... 1128,2992 Pullulan MW 10,000 ............................................................................................................................................................................................ 378,764
(±)-Propranolol hydrochloride ............................................................................................................................................. 837,1127,2991,1342,3025 Pullulan MW 113,000 .......................................................................................................................................................................................... 378,764
Propyl acetate .......................................................................................................................................... 1676,2210,2211,2212,2452,2213,2214,2453 Pullulan MW 210,000 .......................................................................................................................................................................................... 378,764
n-Propyl acetate ....................................................................................................................................... 1675,1725,1726,1727,1780,1969,1971,2405 Pullulan MW 1,320 .............................................................................................................................................................................................. 378,764
n-Propyl alcohol ......................................................................................................................................................................................................... 1780 Pullulan MW 342 ................................................................................................................................................................................................. 378,764
Propylamine ................................................................................................................................................................................................................. 1776 Pullulan MW 366,000 .......................................................................................................................................................................................... 378,764
N-Propylaniline ................................................................................................................................................................................................................ 32 Pullulan MW 6,000 ............................................................................................................................................................................................ 378,764
Propylbenzene .............................................................. 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1505,2074,1548,1549,1610,1621 Pullulan MW 21,700 ............................................................................................................................................................................................ 378,764
n-Propylbenzene ................................................................................................................................................................... 1504,2073,1546,1547,1612 Pullulan MW 48,800 ............................................................................................................................................................................................ 378,764
iso-Propylbenzene ....................................................................................................................................................................................................... 1610 Pullulan MW 805,000 .......................................................................................................................................................................................... 378,764
α-Propyldopacetamide ........................................................................................................................................................................................ 1172,213 Pyraclofos ........................................................................................................................................................................................................... 1885,2064
Propylene .................................................................................................................................................................................................. 1665,1724,1751 Pyraclostrobin .................................................................................................................................................................................... 1419,406,1890,2069
Propylene (215 ng on column ................................................................................................................................................................................ 1752 Pyraflufen-ethyl ................................................................................................................................................................................................. 1890,2069
Propylene glycol .......................................................................................................................................................... 1703,1975,2185,1704,1976,2186 4-Pyran-4-one ................................................................................................................................................................................................... 2056,2439
Indexes Analyte
Pyrazine .............................................................................................................................................................................................................. 2056,2439 Quinapril hydrochloride .............................................................................................................................................................................................. 1211

Pyrazophos ......................................................................................................................................................................................................... 1886,2065 Quinidine ............................................................................................................................................ 30,67,256,407,853,846,1233,854,931,1135,1218
Pyrazoxyfen ........................................................................................................................................................................................................ 1889,2068 Quinine ............................................................................................................................................................................................. 30,407,853,1233,854
Pyrene ..... 2375,310,311,1388,1411,1482,2028,1408,2010,1409,2011,1451,1936,1480,2013,1481,2014,1483,2029,2365,2420,1506,2075, Quinoclamine ..................................................................................................................................................................................................... 1886,2065
1507,2076,1517,1518,1524,1540,1541,1552,1553,1554,1555,1556,1557,1558,1560,1561,1562,1563,1564,1567,1587 Quinoxyfen ......................................................................................................................................................................................................... 1887,2066
Pyrene-d10 ....................................................................................................................................................................................... 1484,2030,2366,2421 Quizalofop-ethyl ................................................................................................................................................................................................ 1890,2069
Pyrene-d10 ........................................................................................................................................................................................................ 1567,1587
Pyributicarb ........................................................................................................................................................................................................ 1887,2066 —R—
Pyridaben ..................................................................................................................................................................... 1447,1681,1828,1933,1887,2066
Rabeprazole ........................................................................................................................................................................................................ 1136,2996
Pyridafenthion .................................................................................................................................................................................................... 1885,2064
Ranitidine ..................................................................................................................................................................................................................... 1173
Pyridaphenthion ........................................................................................................................................ 1350,1401,1860,1995,1430,1641,1796,1892
Rapamycin ..................................................................................................................................................................................................................... 188
Pyridine ....................................................... 1550,1551,1552,1553,1554,1555,1556,1557,1558,2056,2439,2205,2206,2207,2450,2208,2209,2451
Rapeseed oil ...................................................................................................................................................................................................... 1843,1963
Pyridoglutethimide ............................................................................................................................................................................................ 1133,2995
RCS-4 (322.19 m/z) ........................................................................................................................................................................................... 1227,839
Pyridoxine ....................................................................................................................................................................... 122,1301,1302,1303,1304,1309
RCS-8 (376.246 m/z) ......................................................................................................................................................................................... 1227,839
Pyridoxine hydrochloride .................................................................................................................................. 1299,1300,1306,1307,1308,1332,1333
RDX ......................................................................................................................................................................................................................... 296,465
Pyridoxine hydrochloride (Vitamin B6) ....................................................................................................................................................... 334,335,332
Residual solvents ........................................................................................................................................................................................................ 2224
1-(2-Pyridyl)piperazine ......................................................................................................................................................................................... 2340,27
Resmethrin ......................................................................................................................................................................................................... 1888,2067
Pyrifenox-1 ......................................................................................................................................................................................................... 1887,2066
Resorcinol ........................................................................................................................................................................................................ 152,44,1283
Pyrimethamine ............................................................................................................................................................................................................ 1199
cis-Resveratrol ................................................................................................................................................................................. 2024,2418,2025,2419
Pyrimethanil ....................................................................................................................................................................................................... 1890,2069
trans-Resveratrol ............................................................................................................................................................................. 2024,2418,2025,2419
Pyrimidifen ......................................................................................................................................................................................................... 1887,2066
Retene .............................................................................................................................................................................................. 1408,2010,1409,2011
Pyriminobac-E-methyl ...................................................................................................................................................................................... 1887,2066
Retinol .......................................................................................................................................................................................................................... 1296
Pyriminobac-Z-methyl ...................................................................................................................................................................................... 1887,2066
Retinol palmitate .............................................................................................................................................................................................. 1255,1256
Pyrimiphos-ethyl ............................................................................................................................................................................ 1446,1680,1827,1932
Retinyl acetate ............................................................................................................................................................. 1255,1256,1295,1296,1327,1328
Pyriproxyfen ....................................................................................................................................................................................................... 1889,2068
Retinyl palmitate ........................................................................................................................................................................................................ 1328
Pyrocatechol .................................................................................................................................................................................................... 152,44,1283
RGAGGLGLGK-amide ..................................................................................................................................................................................................... 771
DL-Pyroglutamic acid ........................................................................................................................................................................................... 2826,766
Rhamnitol ........................................................................................................................................................................................................... 2036,2037
Pyroquilon .......................................................................................................................................................................................................... 1889,2068
Ribitol .......................................................................................................................................................................................................... 386,2036,2037
Pyrovalerone hydrochloride solution .......................................................................................................................................................................... 472
(−)-Riboflavin ...................................................................................................................................................... 1219,274,1300,1302,1303,1304,1333
Pyrrole ................................................................................................................................................................................................................ 2056,2439
Riboflavin (B2) ......................................................................................................................................................................................... 1306,1307,1308
Pyrrole-2-carboxaldehyde ................................................................................................................................................................................. 2056,2439
Riboflavin (Vitamin B2) ................................................................................................................................................................................. 334,335,332
α-Pyrrolidinovalerophenone hydrochloride solution ................................................................................................................................................. 470
Ribonuclease ................................................................................................................................................................................................... 754,755,756
Ribonuclease A ....................................................................................................................................................................................... 739,758,759,826
—Q— Ribonuclease A ........................................................................................................................................................................................................... 829
Quercetin .............................................................................................................................................................................................................. 352,1259 Ribonuclease A from bovine pancreas .................................. 1129,187,704,732,733,734,735,736,737,738,743,1130,746,747,762,1184,805,825
Quercetin-3-O-glycoside ............................................................................................................................................................................................. 1275 (R) Ribonuclease A ............................................................................................................................................................................................. 1185,808
Quercitrin .................................................................................................................................................................................................. 1259,1266,1276 Ribonuclease A bovine pancreas (13.7 kDa) ........................................................................................................................................................... 493
Quinalphos ................................................................................................................................................... 1419,406,1350,1401,1860,1995,1885,2064 Ribonucleosides ............................................................................................................................................................................................................. 769
Quinapril ................................................................................................................................................................................................................. 123,849 Ritalinic acid ............................................................................................................................................................................................... 1138,2997,446
Indexes Analyte
Ritonavir ......................................................................................................................................................................................................................... 911 α-Selinene .......................................................................................................................................................................................................... 2103,2166

Robenidine .............................................................................................................................................................................................................. 281,957 β-Selinene .................................................................................................................................................................................................................... 2124
Rolitetracycline ............................................................................................................................................................................................................ 1157 Se-Methyl-seleno-DL-cysteine ........................................................................................................................................................................... 1068,2955
Ro-Neet ........................................................................................................................................................................................... 1467,1990,2357,2407 Semivolatiles ...................................................................................................................................................................................................... 1506,2075
Ronnel .................................................................................................................. 1350,1401,1860,1995,1542,1543,1544,1545,1571,1591,2325,2347 Sencor .............................................................................................................................................................................................. 1467,1990,2357,2407
Rose bengal ................................................................................................................................................................................................................... 388 Serine ............................................................................................................................................................................................................................. 819
cis-Rose oxide ............................................................................................................................................................................................................. 2112 D-Serine ................................................................................................................................................................................................................. 2827,773
trans-Rose oxide ......................................................................................................................................................................................................... 2112 L-Serine ........................................................................................................................................................................ 508,509,727,2827,773,1897,2304
Rosmarinic acid ........................................................................................................................................................................................................... 1284 DL-Serine .............................................................................................................................................................................................. 2828,774,2829,775
Roxarsone ........................................................................................................................................................................................................................... 4 DL-Serine (AQC derivatives) .............................................................................................................................................................. 2740,519,2741,520
Ruelene (cruformate) .......................................................................................................................................................................................... 100,2609 DL-Serine (FMOC derivatives) ............................................................................................................................................................................ 2789,604
Rutin ................................................................................................................................................................................................................... 1259,1275 Serotonin ................................................................................................................................................................................................................ 191,192
Rutin hydrate ......................................................................................................................................................................................................... 376,377 Sertraline ....................................................................................................................................................................................................... 157,1196,252
RVYIHPI ................................................................................................................................................................................................................... 531,932 Sertraline hydrochloride ................................................................................................................................................................................... 1142,3000
β-Sesquiphellandrene .............................................................................................................................................................................. 2096,2131,2142
—S— cis-Sesquisabinene hydrate ........................................................................................................................................................................................ 2096
Sabinene .............................................................. 2050,2434,2056,2439,2096,2099,2103,2120,2121,2131,2136,2137,2138,2142,2161,2166,2167 Sethoxydim .......................................................................................................................................................................................................... 1419,406
(+)-Sabinene .............................................................................................................................................................................................................. 2170 Sibutramine hydrochloride monohydrate ................................................................................................................................... 1143,3001,1144,3002
cis-Sabinene hydrate ...................................................................................................................................................................................... 2050,2434 Siduron .................................................................................................................................................. 1,295,294,302,2374,303,1430,1641,1796,1892
cis-Sabinene hydrate ............................................................................................................................................................ 2096,2120,2137,2138,2167 Siduron A ............................................................................................................................................................................................................... 314,375
trans-Sabinene hydrate ................................................................................................................................................................. 2103,2136,2138,2166 Siduron B ............................................................................................................................................................................................................... 314,375
Safrole .......................................................................................................................................................................................................................... 1550 Silafluofen ........................................................................................................................................................................................................... 1889,2068
Salbutamol ......................................................................................................................................................................................................... 1140,2999 Sildenafil ................................................................................................................................................................................................................. 995,996
Salicylic acid ................................................................................................................ 37,1279,370,87,1281,372,89,279,910,922,978,979,1141,1236 Silibinin ........................................................................................................................................................................................................................ 1266
Salinomycin ................................................................................................................................................................................................................. 1174 Siloxane .............................................................................................................................................................................................................. 1574,2328
Salinomycin SV ............................................................................................................................................................................................................. 215 Silybin A ............................................................................................................................................................................................................ 1276,1288
β-Santalene ................................................................................................................................................................................................................. 2096 Silybin B ................................................................................................................................................................................................... 1266,1276,1288
(Z)-β-Santelene ................................................................................................................................................................................................. 2050,2434 Silychristin ................................................................................................................................................................................................. 1266,1276,1288
Saquinavir ...................................................................................................................................................................................................................... 911 Silydianin .................................................................................................................................................................................................. 1266,1276,1288
Sarafloxacin ................................................................................................................................................................................ 1004,355,1005,356,1013 Simazine ....................... 5,110,15,111,16,1391,305,1357,1393,315,316,1351,1403,1862,2000,1388,1411,1482,2028,1430,1641,1796,1892,
1466,1740,1858,1989,2356,2384,2392,2406,1467,1990,2357,2407,1489,1788,1877,2039,2369,2391,2397,2425,1507,2076,1544,1545,
Sauvagine ....................................................................................................................................................................................................................... 788
1888,2067
scFC ................................................................................................................................................................................................................................ 673
Simeconazole ..................................................................................................................................................................................................... 1886,2065
scFc .................................................................................................................................................................................................................. 675,676,678
Simetryn ............... 1466,1740,1858,1989,2356,2384,2392,2406,1489,1788,1877,2039,2369,2391,2397,2425,1506,2075,1507,2076,1889,2068
scFC ................................................................................................................................................................................................................................ 768
Simstatin ............................................................................................................................................................................................................... 1148,194
Scopolamine .................................................................................................................................................................................................... 119,885,886
Simvastatin .................................................................................................................................................................................................................... 964
Sebuthylazin ....................................................................................................................................................................................................................... 5
α-Sinensal ................................................................................................................................................................................................. 2103,2131,2166
Secobarbital ........................................................................................................................................................ 414,925,416,927,417,929,418,930,928
β-Sinensal .................................................................................................................................................................................................................... 2131
Secodehydroabietic acid ............................................................................................................................................................................................ 1784
Sirolimus solution ....................................................................................................................................................................................................... 1229
Selegiline (Deprenyl) .................................................................................................................................................................... 189,448,480,2897,975
Indexes Analyte
β-Sitosterol ................................................................................................................................................................... 1354,1412,1875,2034,1874,2033 trans-Stilbene oxide ............................................................................................................................................................................................ 101,2610

SLL ........................................................................................................................................................................................................................... 391,393 Stirofos ................................................................................................................................................................................................................ 1506,2075
SLO ................................................................................................................................................................................................................................. 324 Stirophos ................................................................................................................................................................................................... 1542,1544,1545
Sodium glycochenodeoxycholate ................................................................................................................................................................................ 234 Storophos ..................................................................................................................................................................................................................... 1543
Sodium gycochenodeoxycholate .......................................................................................................................................................................... 237,238 Strigol ...................................................................................................................................................................................................................... 14,2485
Sodium taurochenodeoxycholate ......................................................................................................................................................................... 234,237 Strychnine ......................................................................................................................................................................................... 30,407,853,1233,854
Sodium taurodeoxycholate hydrate ........................................................................................................................................................................... 234 Styrene .............................................................................................................................................. 1497,2061,1498,2062,2373,2444,1502,2071,
Sodium taurodeoxycholate hydrate ........................................................................................................................................................................... 237 1503,2072,1504,2073,1505,2074,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1676,1725,1726,1727,1794,
Sodium taurolithocholate ............................................................................................................................................................................................. 234 1795,1891
Sodium tauroursodeoxycholate ................................................................................................................................................................................... 234 Substance P ........................................................................................................................................................................................................... 709,720
SOL ................................................................................................................................................................................................................... 390,391,393 Sucrose ....................................................................................................................................................................... 333,380,381,382,383,384,385,387
SOLn ............................................................................................................................................................................................................................... 391 Sudan I ................................................................................................................................................................................................................... 120,400
Solone ............................................................................................................................................................................................................................ 205 Sudan II .................................................................................................................................................................................................................. 120,400
SOL+POO ....................................................................................................................................................................................................................... 392 Sudan III ................................................................................................................................................................................................................. 120,400
Solvents ........................................................................................................................................................................................................................ 2224 Sudan IV ................................................................................................................................................................................................................. 120,400
SOO ......................................................................................................................................................................................................................... 390,391 Sudan Orange G ................................................................................................................................................................................................... 120,400
SOP .......................................................................................................................................................................................................................... 390,391 Sudan red G .......................................................................................................................................................................................................... 120,400
Sorbate ........................................................................................................................................................................................................................... 338 Sudan Red 7B ....................................................................................................................................................................................................... 120,400
Sorbic acid ............................................................................................................................ 1279,370,87,1281,372,89,280,334,332,335,346,347,910 Sufulfotep .................................................................................................................................................. 1466,1740,1858,1989,2356,2384,2392,2406
Sorbic acid, 0.7% ....................................................................................................................................................................................................... 1993 Sulconazole nitrate salt .................................................................................................................................................................................... 1149,3005
(±)-Sotalol hydrochloride ........................................................................................................................... 140,2633,2720,419,1146,3003,1147,3004 Sulfacetamide ........................................................................................................................................................................................ 1151,779,883,884
Soybean oil ........................................................................................................................................................................................................ 1874,2033 Sulfadiazine ........................................................................................................................................................................................... 1151,779,883,884
Sparfloxacin ............................................................................................................................................................................................................ 880,881 Sulfamerazine .............................................................................................................................................................................. 1151,779,883,884,1150
Spathulenol ..................................................................................................................................................................................... 2095,2096,2131,2142 Sulfamethazine ............................................................................................................................................................................................................ 1150
Spearmint oil .............................................................................................................................................................................................................. 2169 Sulfamethizole ....................................................................................................................................................................................... 1151,779,883,884
Spermidine ..................................................................................................................................................................................................................... 143 Sulfanilamide ............................................................................................................................................................................... 1151,779,883,884,1150
Spermine ........................................................................................................................................................................................................................ 143 Sulfanitran ...................................................................................................................................................................................................................... 906
Sphingomyelin ............................................................................................................................................................................................................... 399 Sulfathiazole ................................................................................................................................................................................. 1151,779,883,884,1150
Spirodiclofen ...................................................................................................................................................................................................... 1890,2069 Sulfolane ............................................................................................................................................................. 2205,2206,2207,2450,2208,2209,2451
Spiromesifen ......................................................................................................................................................................................................... 1419,406 Sulfonamide positional isomers .................................................................................................................................................................................. 103
Stavudine ....................................................................................................................................................................................................................... 911 Sulfopropyl acrylate ........................................................................................................................................................................................................ 29
Stearic acid ............................................................................................................................................................................................................ 351,357 Sulfopropyl methylacrylate ............................................................................................................................................................................................ 29
Stearic acid methyl ester .................................. 1637,1818,1918,2250,1819,1919,2251,1837,1954,1840,1960,1845,1965,1846,1966,1847,1967 Sulfotep .. 1350,1401,1860,1995,1448,1682,1829,1934,1470,1996,2360,2410,1471,1746,1861,1997,2361,2386,2393,2411,1542,1543,1544,
Stearic acid methyl ester (C18:0) ................................................ 1815,1915,2247,1817,1917,2249,1836,1945,1839,1959,1842,1962,1844,1964 1545,1550,1551,1890,2069
Stearic acid methyl ester (C18:0) at 4 wt % ................................................................................................................................... 1822,1922,2254 Sulfotepp ...................................................................................................................................................................................................................... 1551
Steroids .......................................................................................................................................................................................................................... 195 Sulfur dioxide ................................................................................................................................................................................. 1687,2382,2060,2443
Sterol ester triterpene alcohols ....................................................................................................................................................................... 1884,2063 Sulfur dioxide (5 ppm) ............................................................................................................................................................................................. 1783
Sterols ................................................................................................................................................................................................................. 1874,2033 Sulindac .......................................................................................................................................................................................................................... 899
Stigmasterol ................................................................................................................................................................. 1354,1412,1875,2034,1874,2033 Sulprofos ............................................................................................................................................................................................................. 1887,2066
Indexes Analyte
Sunset Yellow FCF ....................................................................................................................................................................................................... 388 Taxifolin ..................................................................................................................................................................................................... 1266,1276,1288

Superoxide dismutase .................................................................................................................................................................................................. 780 Taxols ........................................................................................................................................................................................................................... 1154
Superoxide Dismutase from bovine erythrocytes ............................................................................................................................................. 738,744 Taylor-Wharton Dewar accessories ..................................................................................................................................................................... 705,743
Surfactants ....................................................................................................................................................................................................................... 83 2,3,7,8-TCDD ................................................................................................................................................................................... 1437,1576,1813,1913
Sutan ................................................................................................................................................................................................ 1467,1990,2357,2407 2,3,7,8-TCDF .............................................................................................................................................. 1436,1575,1812,1912,1437,1576,1813,1913
Swep ................................................................................................................................................................................................. 1,295,294,1889,2068 Tebuconazole ..................................................................................................................................................................................................... 1888,2067
Synephrine ..................................................................................................................................................................................................................... 992 Tebufenpyrad ..................................................................................................................................................................................................... 1888,2067
(±)-Synephrine ................................................................................................................................................................................................... 2830,781 Tebupirimifos ..................................................................................................................................................................................................... 1890,2069
Syringic acid .......................................................................................................................................................................................... 1280,371,88,1259 Tebuthiuron ..................................................................................................................................................................................... 1506,2075,1507,2076
Tecnazene ........................................................................................................................................................................................................... 1887,2066
—T— Tefluthrin ............................................................................................................................................................................................................ 1887,2066
2,4,5-T ....................................................................................................................................................................................... 1355,1389,2,298,306,307 Temazepam .......................................................................................................................................................................... 1190,232,1207,273,489,916
T26 ............................................................................................................................................................................................................................... 1972 Temazepam solution ........................................................................................................................................................................................... 833,1224
T28 ............................................................................................................................................................................................................................... 1972 Temazepam glucuronide lithium salt solution ...................................................................................................................................................... 1224
T30 ............................................................................................................................................................................................................................... 1972 Temephos .................................................................................................................................................. 1350,1401,1860,1995,1448,1682,1829,1934
T32 ............................................................................................................................................................................................................................... 1972 Temsirolimus ...................................................................................................................................................................................................... 1221,1229
T34 ............................................................................................................................................................................................................................... 1972 Tenylchlor ........................................................................................................................................................................................................... 1888,2067
T36 ............................................................................................................................................................................................................................... 1972 TEPP .................................................................................................................................................................................................................... 1542,1543
T38 ............................................................................................................................................................................................................................... 1972 Terbacil ................................................................................................................................... 1467,1990,2357,2407,1506,2075,1507,2076,1888,2067
T40 ............................................................................................................................................................................................................................... 1972 Terbucarb ..................................................................................................................................................................... 1430,1641,1796,1892,1890,2069
T42 ............................................................................................................................................................................................................................... 1972 Terbufos .......... 1419,406,1350,1401,1860,1995,1466,1740,1858,1989,2356,2384,2392,2406,1471,1746,1861,1997,2361,2386,2393,2411,
1506,2075,1507,2076,1542,1543,1544,1545,1885,2064
T44 ...................................................................................................................................................................................................................... 1972,1973
Terbumeton ........................................................................................................................................................... 110,15,111,16,1447,1681,1828,1933
T46 ...................................................................................................................................................................................................................... 1972,1973
Terbumeton-desethyl .................................................................................................................................................................................. 110,15,111,16
T48 ................................................................................................................................................................................................... 1849,1974,1972,1973
Terbutaline hemisulfate salt ............................................................................................................................................................................ 1155,3006
T50 ................................................................................................................................................................................................... 1849,1974,1972,1973
Terbuthylazin-desethyl ................................................................................................................................................................................ 110,15,111,16
T52 ................................................................................................................................................................................................... 1849,1974,1972,1973
Terbuthylazine ............................................................................................................................................................................................. 110,15,111,16
T54 ................................................................................................................................................................................................... 1849,1974,1972,1973
Terbutryn ................................................................................................................. 110,15,111,16,1466,1740,1858,1989,2356,2384,2392,2406,
Tadalafil .................................................................................................................................................................................................................. 995,996
1489,1788,1877,2039,2369,2391,2397,2425,1506,2075,1507,2076,1888,2067
Tamoxifen ........................................................................................................................................................................................................... 1152,1223
Terbutylazine ...................................................................................................................................................................................................................... 5
Tartaric acid ..................................................................................................................................................................................................................... 68
Terfenadine ........................................................................................................................................................................................................ 1156,3007
L-Tartaric acid ........................................................................................................................................................................... 1277,368,85,1278,369,86
o-Terphenyl ...................................................................................................................................................................................................................... 33
DL-Tartaric acid ................................................................................................................................................................. 104,1291,2611,2708,3040,389
p-Terphenyl-d14 .............................................................................................................................................................................. 1550,1551,1552,1556
Tartrazine ....................................................................................................................................................................................................................... 388
Terphenyl-d14 .............................................................................................................................................................. 1524,1553,1554,1555,1557,1564
Taurine ..................................................................................................................................................................................................... 333,342,343,381
Terphenyl-d14 (surr.) ................................................................................................................................................................................................. 1558
Taurochenodeoxycholic acid ........................................................................................................................................................................................ 238
o-Terphenyl (surr.) ..................................................................................................................................................................................................... 1526
Taurocholic acid ............................................................................................................................................................................................. 234,237,238
α-Terpinene ................................................................................................................................................ 2050,2434,2056,2439,2096,2097,2099,
Taurocholic acid sodium salt hydrate ....................................................................................................................................................................... 234
2103,2105,2111,2184,2120,2121,2131,2136,2137,2138,2142,2166,2167,2169,2170
Taurodeoxycholic acid .................................................................................................................................................................................................. 238
β-Terpinene .............................................................................................................................................................................................. 2103,2106,2136
Taurodeoxycholic acid-2,2,4,4-d4 ................................................................................................................................................................................ 234
Indexes Analyte
γ-Terpinene .................................................................................................................... 2047,2175,2351,2353,2050,2434,2056,2439,2095,2096, 1,1,1,2-Tetrachloroethane ...... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549,1674,1676

2099,2105,2120,2121,2124,2131,2137,2138,2142,2161,2166,2167,2170 1,1,2,2-Tetrachloroethane ..................................................................................................................................................... 1494,2058,2371,2441,
Terpinen-1-ol ..................................................................................................................................................................................................... 2105,2106 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,
(-)-Terpinen-4-ol ............................................................................................................................................................................ 1785,2172,2558,2688 1568,1588,2322,2344,1569,1589,2323,2345,1674,1676,1725,1726,1727
Terpinen-4-ol ............................................................................................ 2050,2434,2099,2103,2106,2120,2121,2131,2136,2142,2161,2166,2167 Tetrachloroethene ........................................................................................................................................................ 1546,1547,1674,1676,1726,1727
(+)-Terpinen-4-ol .................................................................................................................................... 1785,2172,2558,2688,2095,2096,2105,2170 Tetrachloroethylene ........ 1493,2057,2370,2440,1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,
α-Terpineol ....................................... 2050,2434,2095,2099,2103,2105,2106,2112,2120,2121,2124,2131,2136,2142,2166,2167,2170,2183,2446 1505,2074,1519,1520,1521,1522,1523,1548,1549,1565,1566,1568,1588,2322,2344,1725
β-Terpineol .................................................................................................................................................................................................................. 2105 2,3,4,5-Tetrachlorophenol .......................................................................................................................................................................................... 1531
γ-Terpineol ................................................................................................................................................................................................................... 2105 2,3,4,6-Tetrachlorophenol ......................................................................... 1359,1386,1407,1475,1755,2005,1476,1756,2006,2363,2388,2414,
Terpinolene ................................................ 2050,2434,2096,2099,2103,2105,2106,2120,2121,2124,2131,2136,2137,2138,2142,2166,2167,2170 1477,1757,2007,2364,2389,2415,1531,1550,1551
α-Terpinyl acetate .......................................................................................................................................................................... 2103,2131,2142,2166 2,3,5,6-Tetrachlorophenol ....................................................................................................................................................................... 1531,1550,1551
(±)-α-Terpinyl acetate .............................................................................................................................................................................................. 2095 Tetrachloroterephthalic acid .................................................................................................................................................................. 1355,1389,2,298
Testosterone .............................................................................................................................................................................. 195,197,198,200,201,206 Tetrachloro-m-xylene ........................................................................................................................................ 1513,1516,1532,1533,1538,1539,1604
Testosterone solution .................................................................................................................................................................................. 1374,257,258 2,4,5,6-Tetrachloro-m-xylene ........................................................................................................................................................ 1512,1514,1534,1535
1-Testosterone ............................................................................................................................................................................................................... 206 Tetrachlorvinphos ........................................................................................................................................................................... 1507,2076,1885,2064
Testosterone glucuronide ............................................................................................................................................................................................. 258 2,3,4,6-Tetrachorophenol (acylated) ............................................................................................................................................ 2040,2426,2042,2428
2,2′,4,4′-Tetrabromodiphenyl ether solution ................................................................................................................................................ 1450,1684 Tetraconazole ..................................................................................................................................................................................................... 1888,2067
2,3′,4′,6-Tetrabromodiphenyl ether solution ................................................................................................................................................ 1450,1684 Tetracontane ...................................................................................................................................................................................................... 1486,1487
1,2,4,5-Tetrachlorobenzene .............................................................................................................................................................................. 1550,1551 Tetracosane ..................................................................................................................................................................................... 1486,1487,1526,1637
2,2′,4,4′-Tetrachlorobiphenyl .................................................................................................................. 1388,1411,1482,2028,1506,2075,1507,2076 Tetracosane (C24) ...................................................................................................................................................................................................... 1636
2,2',6,6'-Tetrachlorobiphenyl ......................................................................................................................................................... 1441,1927,1442,1928 Tetracyclines ................................................................................................................................................................................................................ 1157
2,2’,6,6’-tetrachlorobiphenyl ............................................................................................................................................................................ 1443,1929 Tetradecanal ....................................................................................................................................................................................................... 2103,2166
3,3',4,4'-Tetrachlorobiphenyl ......................................................................................................................................................... 1441,1927,1442,1928 Tetradecane .................................................................................................................. 1431,1572,1486,1487,1526,1596,1597,1602,1668,1637,2120
3,3’,4,4’-tetrachlorobiphenyl ............................................................................................................................................................................ 1443,1929 n-Tetradecane ............................................................................................................................................................................................................. 2131
3,3′,4,4′-Tetrachlorobiphenyl ..................................................................................................................................................................................... 1501 Tetradecane (C14) ...................................................................................................................................................................................................... 1636
3,3′,4,5-Tetrachlorobiphenyl ...................................................................................................................................................................................... 1501 Tetradecanoic acid ................................................................................................................................................................................... 1873,2031,2278
3,3′,4,5′-Tetrachlorobiphenyl ..................................................................................................................................................................................... 1501 n-Tetradecanol ............................................................................................................................................................................................................ 2131
3,3′,5,5′-Tetrachlorobiphenyl ..................................................................................................................................................................................... 1501 (E)-2-Tetradecenal ...................................................................................................................................................................................................... 2103
3,4,4′,5-Tetrachlorobiphenyl ...................................................................................................................................................................................... 1501 1-Tetradecene ............................................................................................................................................................................................................. 2120
2,2',5,5'-Tetrachlorobiphenyl (No. 52) ........................................................................................................................................................... 1402,1999 Tetradifon ..................................................................................................................................................................... 1449,1683,1830,1935,1887,2066
3,3',4,4'-Tetrachlorobiphenyl (No. 77) ........................................................................................................................................................... 1402,1999 Tetraethylene glycol ................................................................................................................................................................................ 1705,1977,2187
3,4,4',5-Tetrachlorobiphenyl (No.81) .............................................................................................................................................................. 1402,1999 Tetraethyl pyrophosphate (TEPP) ................................................................................................................................................................... 1544,1545
1,2,3,4-Tetrachlorodibenzo-p-dioxin ...................................................................................................... 1452,1577,1831,1937,1453,1578,1832,1938 Δ8-Tetrahydrocannabinol .............................................................................................................................................................................................. 459
1,2,3,7-Tetrachlorodibenzo-p-dioxin ...................................................................................................... 1452,1577,1831,1937,1453,1578,1832,1938 Δ9-Tetrahydrocannabinol ...................................................................................................................................................................................... 421,459
1,2,3,8-Tetrachlorodibenzo-p-dioxin ...................................................................................................... 1452,1577,1831,1937,1453,1578,1832,1938 4,5,6,7-Tetrahydro-3,6-dimethylbenzofuran ............................................................................................................................................................ 2138
1,2,7,8-Tetrachlorodibenzo-p-dioxin ...................................................................................................... 1452,1577,1831,1937,1453,1578,1832,1938 Tetrahydrofuran ................................ 1504,2073,1568,1588,2322,2344,1675,1676,1725,1726,1727,1780,2210,2211,2212,2452,2213,2214,2453
1,4,7,8-Tetrachlorodibenzo-p-dioxin ...................................................................................................... 1452,1577,1831,1937,1453,1578,1832,1938 cis-(+/-)-3,3a,8,8a-Tetrahydro-2H-indeno[1,2-d]oxazol-2-one ...................................................................................................................... 2573,45
2,3,7,8-Tetrachlorodibenzo-p-dioxin solution ................................................. 1436,1575,1812,1912,1452,1577,1831,1937,1453,1578,1832,1938 1,2,3,4-Tetrahydro-1-naphthylamine (N-acetyl derivatives) ....................................................................................................................... 1786,2559
Tetrachloroethane ........................................................................................................................................................................... 1569,1589,2323,2345 1,2,3,4-Tetrahydro-1-naphthylamine (N-TFA derivatives) ........................................................................................................................... 1787,2560
Indexes Analyte
3a,4,5,6-Tetrahydrosuccinimido[3,4-b]acenaphthen-10-one .......................................................................................................................... 107,2613 α-Thujene ............................................................ 2137,2050,2434,2095,2099,2103,2113,2120,2121,2124,2131,2138,2142,2161,2166,2167,2170

Tetrahydrothiophene ................................................................................................................................................................................................... 1685 Thymidine ................................................................................................................................................................................................ 572,687,688,689
Tetralin ................................................................................................................................................................ 2205,2206,2207,2450,2208,2209,2451 Thymine .................................................................................................................................................................................................................. 684,765
Tetramethylbenzene .......................................................................................................................................................................................... 1621,1624 Thymol ............................................................................................................................................................................................. 2103,2137,2138,2166
1,2,3,4-Tetramethylbenzene ...................................................................................................................................................................................... 1612 Thymol methyl ester ................................................................................................................................................................................................. 2103
1,2,3,5-Tetramethylbenzene ............................................................................................................................................................................. 1610,1612 Thymol methyl ether ................................................................................................................................................................................................. 2166
1,2,4,5-Tetramethylbenzene ............................................................................................................................................................................. 1593,1594 Thyroglobulin ................................................................................................................................ 207,742,748,749,750,751,752,757,758,786,826,829
Tetratetracontane ............................................................................................................................................................................................... 1486,1487 Thyroglobulin from bovine thyroid ............................................................................................................................. 1129,187,734,735,736,737,825
Tetryl ..................................................................................................................................................................... 297,1455,1695,2297,2354,2383,2466 (T) Thyroglobulin ................................................................................................................................................................................................ 1185,808
(±)-Thalidomide ............................................................................................................................................................................ 1158,3008,1159,3009 Thyroglobulin (640 kDa) ............................................................................................................................................................................................. 493
THBP ..................................................................................................................................................................................................... 276,337,1898,2182 Thyroglobulin aggregate .............................................................................................................................................................................................. 786
THC .................................................................................................................................................................................................. 2290,2462,2291,2463 Thyroglobulin dimer ..................................................................................................................................................................................................... 493
(−)-Δ8-THC solution .................................................................................................................................................................................................... 487 Thyroglobulin trimer .................................................................................................................................................................................................... 493
(−)-Δ9-THC solution .................................................................................................................................................................................................... 487 D-Thyroxine ................................................................................................................................................................................................... 208,2834,787
Theobromine ............................................................................................................................................................... 1362,1427,148,946,149,150,1275 L-Thyroxine .................................................................................................................................................................................................... 208,2834,787
Theophylline ......................................................................................................................................................................... 1362,1427,148,946,149,150 L-Thyroxine (T4) solution ............................................................................................................................................................................................ 822
Thiabendazole .............................................................................................................................................................. 1347,1394,1798,1894,1797,1893 Tillam ............................................................................................................................................................................................... 1467,1990,2357,2407
Thiacloprid .............................................................................................................................................................................................................. 1358,19 Timolol .................................................................................................................................................................................................................... 941,954
Thiamethoxam ............................................................................................................................................................................. 1358,19,319,1890,2069 Toclofos-methyl ........................................................................................................................................................... 1430,1641,1796,1892,1885,2064
Thiamine ....................................................................................................................................................................... 1300,1301,1302,1303,1304,1309 Tocopherol ................................................................................................................................................................................................................... 1295
Thiamine hydrochloride ................................................................................................................................................................... 122,1306,1307,1308 α-Tocopherol ...................................................................................................................................................................................................... 1295,1296
Thiobencarb ........................................................................................................................................................................................................ 1887,2066 (±)-α-Tocopherol .......................................................................................................... 1322,253,1251,1252,1255,1256,1253,1254,1298,1327,1328
Thiocyclam ......................................................................................................................................................................................................... 1889,2068 (+)-γ-Tocopherol .............................................................................................................................. 1322,253,1251,1252,1295,1296,1298,1327,1328
2-Thiocytidine dihydrate .............................................................................................................................................................................................. 686 δ-Tocopherol ................................................................................................................................................................ 1255,1256,1295,1296,1327,1328
Thiodicarb ........................................................................................................................................................................................ 1430,1641,1796,1892 DL-α-Tocopherol acetate ................................................................................................................................................................................... 1327,1328
Thiometon .................................................................................................................................................................... 1350,1401,1860,1995,1887,2066 Tocopheryl acetate ............................................................................................................................................................................................ 1295,1296
Thionazin ..................................................................................................... 1466,1740,1858,1989,2356,2384,2392,2406,1470,1996,2360,2410, α-Tocopheryl acetate ................................................................................................................................................................................................. 1295
1471,1746,1861,1997,2361,2386,2393,2411,1542,1543,1550,1551 Tokuthion ........................................................................................................................................................................................ 1542,1543,1544,1545
Thionazin (Zinophos) ........................................................................................................................................................................................ 1544,1545 Tolban .............................................................................................................................................................................................. 1467,1990,2357,2407
Thiophene .................................................................................................................................................................................................................... 1685 Tolclofos-methyl .................................................................................................................................................................................................. 1419,406
Thioredoxin from Escherichia coli ............................................................................................................................................................................... 744 Tolfenpyrad ........................................................................................................................................................................................................ 1890,2069
Thioridazine ............................................................................................................................................................................................. 165,578,908,909 o-Tolidine .................................................................................................................................................................................................. 1556,1558,1673
cis-Thiothixene ....................................................................................................................................................................................................... 165,578 Tolmetin ......................................................................................................................................................................................................................... 899
Threonine ................................................................................................................................................................................................................ 509,819 Tolperisone hydrochloride ................................................................................................................................................................................ 1161,3010
D-Threonine .......................................................................................................................................................................................................... 2832,784 o-Tolualdehyde ...................................................................................................................................................................................................... 21,2326
L-Threonine ......................................................................................................................................................................... 508,727,2832,784,1897,2304 m-Tolualdehyde ..................................................................................................................................................................................................... 21,2326
DL-Threonine ......................................................................................................................................................................................................... 2833,785 p-Tolualdehyde ...................................................................................................................................................................................................... 21,2326
DL-Threonine (AQC derivatives) ............................................................................................................................................................... 2567,2742,521 m-Tolualdehyde 2,4-dinitrophenylhydrazone ...................................................................................................................................... 22,2334,23,2335
DL-Threonine (FMOC derivatives) ...................................................................................................................................................................... 2790,605 m-Tolualdehyde (2,4-DNPH derivative) ............................................................................................................................................................. 2336,24
Indexes Analyte
Toluene ............................................................................................... 36,841,554,842,1117,1118,1493,2057,2370,2440,1494,2058,2371,2441, 2,4,6-Trichloroanisole ............................................................................................................ 1488,2038,2368,2424,2040,2426,2042,2428,2043,2429

1497,2061,1498,2062,2373,2444,1500,1586,2321,2343,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1527,1528,1529, 1,2,3-Trichlorobenzene ............................. 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549
1530,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1592,1593,1594,1595,1596,1597,1600,1658,1601,1667, 1,2,4-Trichlorobenzene ........................................................... 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,
1602,1668,1610,1611,1612,1613,1614,1615,1616,1617,1618,1621,1624,1625,1626,1640,1676,1725,1726,1727,1780,1794,2205,2206, 1524,1546,1547,1548,1549,1552,1553,1554,1555,1556,1557,1558,1564,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345
2207,2450,2208,2209,2451,2300,2467 2,4′,5-Trichlorobiphenyl ........................................................................................................................... 1388,1411,1482,2028,1506,2075,1507,2076
Toluene-d8 ......................................................................................................................................... 842,1519,1520,1546,1547,1548,1549,1565,1566 2,4,6-Trichlorobiphenyl .................................................................................................................................................................. 1441,1927,1442,1928
2,4-Toluene diisocyanate ........................................................................................................................................................................................... 2338 2,4,6-trichlorobiphenyl ...................................................................................................................................................................................... 1443,1929
2,6-Toluene diisocyanate ........................................................................................................................................................................................... 2338 3,3′,4-Trichlorobiphenyl ............................................................................................................................................................................................. 1501
p-Toluic acid ............................................................................................................................................................................. 1279,370,87,1281,372,89 3,3′,5-Trichlorobiphenyl ............................................................................................................................................................................................. 1501
o-Toluidine ........................................................................................................................ 108,109,1484,2030,2366,2421,1550,1551,1655,1672,1673 3,4,4'-Trichlorobiphenyl ................................................................................................................................................................. 1441,1927,1442,1928
m-Toluidine ............................................................................................................................................................................................................ 108,109 3,4,4’-trichlorobiphenyl ..................................................................................................................................................................................... 1443,1929
p-Toluidine .................................................................................................................................................................................................... 108,109,1673 3,4,4′-Trichlorobiphenyl ............................................................................................................................................................................................. 1501
Tolyfluanide ..................................................................................................................................................................................... 1445,1679,1826,1931 3,4,5-Trichlorobiphenyl ............................................................................................................................................................................................... 1501
Tolylene-2,4-diisocyanate ..................................................................................................................................................................................... 2340,27 3,4′,5-Trichlorobiphenyl ............................................................................................................................................................................................. 1501
Tolylene-2,6-diisocyanate ..................................................................................................................................................................................... 2340,27 2,3',6-Trichlorobiphenyl (No. 28) ................................................................................................................................................................... 1402,1999
Tolylfluanid ................................................................................................................................................ 1347,1394,1798,1894,1797,1893,1889,2068 1,1,1-Trichloroethane ........................................... 1431,1572,1493,2057,2370,2440,1494,2058,2371,2441,1497,2061,1498,2062,2373,2444,
Tolylfluanid metabolite ..................................................................................................................................................................................... 1889,2068 1500,1586,2321,2343,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,
Tonalid ................................................................................................................................................................................................................ 2111,2184 1568,1588,2322,2344,1569,1589,2323,2345,1674,1676,1725,1726,1727,2200,2201,2202,2448,2203,2204,2449,2224
2,4,5-TP ................................................................................................................................................................................................... 1355,1389,2,298 1,1,2-Trichloroethane ............................................................................................................................................................. 1494,2058,2371,2441,
Tramadol hydrochloride ................................................................................................................................................................................... 1162,3011 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566
Trazodone ............................................................................................................................................................................................................... 914,918 1,1,2-trichloroethane ...................................................................................................................................................................... 1568,1588,2322,2344
Triademefon ....................................................................................................................................................................................................... 1506,2075 1,1,2-Trichloroethane ..................................................................................................................................................................... 1569,1589,2323,2345
Triadimefon .................................................................................................................................................................. 1507,2076,1887,2066,2004,2413 Trichloroethene .................................................................................................................................................................................................. 1504,2073
Triadimenol ........................................................................................................................................................................................................ 1163,3012 Trichloroethylene ................................................................................................................................................. 1431,1572,1494,2058,2371,2441,
Triadimenol-1 .................................................................................................................................................................................................... 1887,2066 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1505,2074,1519,1520,1521,1522,1523,1546,1547,1548,1549,1565,1566,
Triadimenol-2 .................................................................................................................................................................................................... 1887,2066 1568,1588,2322,2344,1569,1589,2323,2345,1674,1676,1725,1726,1727,2205,2206,2207,2450,2208,2209,2451,2224,2226,2456
Triallate ............................................................................................................................................................................................................... 1886,2065 Trichlorofluoromethane ....................................................................................... 1494,2058,2371,2441,1496,1497,2061,1498,2062,2373,2444,
Triamcinolone acetonide .............................................................................................................................................................................................. 167 1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1697,1698
Triazophos .......................................................................................................................................................................................................... 1885,2064 Trichloronate ............................................................................................................................................. 1350,1401,1860,1995,1542,1543,1544,1545
2,4,6-Tribromophenol ......................................... 1476,1756,2006,2363,2388,2414,1524,1531,1550,1551,1552,1553,1554,1555,1556,1557,1564 2,3,4-Trichlorophenol ................................................................................................................................................................................................. 1531
2,4,6-tribromophenol (surr.) ..................................................................................................................................................................................... 1558 2,3,5-Trichlorophenol ................................................................................................................................................................................................. 1531
Tribufos ............................................................................................................................................................................................................... 1886,2065 2,4,5-Trichlorophenol ................................................................................. 1476,1756,2006,2363,2388,2414,1477,1757,2007,2364,2389,2415,
Tributyl phosphate .................................................................................................................................................................................. 1545,1585,2342 1531,1552,1553,1554,1555,1556,1557,1558,1564,1571,1591,2325,2347
Tributyl phosphate (surr.) ......................................................................................................................................................................................... 1544 2,4,6-Trichlorophenol ........................................................................... 90,1359,1386,1407,1475,1755,2005,1476,1756,2006,2363,2388,2414,
Tricaprin ....................................................................................................................................................................................................................... 1638 1477,1757,2007,2364,2389,2415,1524,1531,1552,1553,1554,1555,1556,1557,1558,1564
Tricaprin (I.S.) .......................................................................................................................................................................................... 1631,1633,1634 2,4,6-Trichlorophenol (acylated) .................................................................................................................................................. 2040,2426,2042,2428
Trichlamide ......................................................................................................................................................................................................... 1889,2068 1,1,1-Trichloropropane ................................................................................................................................................................... 1500,1586,2321,2343
Trichlorfon ...................................................................................................................................................................... 1419,406,1542,1543,1544,1545 1,2,3-Trichloropropane .............................. 1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1546,1547,1548,1549
2,4,5-Trichloroaniline ........................................................................................................................................................................................ 1655,1672 1,1,2-Trichloro-1,2,2-trifluoroethane ......................................................................................................................... 1565,1566,1568,1588,2322,2344
Trichlorphan .................................................................................................................................................................................... 1350,1401,1860,1995
Indexes Analyte
Tricosane ...................................................................................................................................................................................................................... 1637 1,2,3-Trimethylbenzene ...................................................................................................................................... 1431,1572,1497,2061,1593,1594,

Tricosanoate (C23) ..................................................................................................................................................................................................... 1636 1595,1596,1597,1610,1611,1612,1621,2300,2467
Tricosanoic acid methyl ester ................................................................................................................................... 1818,1918,2250,1819,1919,2251 1,2,4-Trimethylbenzene ................................................................................................................... 1497,2061,1498,2062,2373,2444,1502,2071,
Tricosanoic acid methyl ester (C23:0) ..................................................................................................................... 1815,1915,2247,1817,1917,2249 1503,2072,1504,2073,1505,2074,1527,1528,1529,1530,1546,1547,1548,1549,1568,1588,2322,2344,1569,1589,2323,2345,1593,1594,1595,
Tricosanoic acid methyl ester (C23:0) at 2 wt % ............................................................................................................................ 1822,1922,2254 1596,1597,1610,1611,1613,1614,1615,1616,1617,1618,1621,1624,1625,1626,2300,2467
Tri-o-cresyl phosphate (TOCP) ........................................................................................................................................................................ 1544,1545 1,3,5-Trimethylbenzene ............................................... 1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1569,1589,2323,2345
Tricyclazone ........................................................................................................................................................................................................ 1507,2076 2,2,3-Trimethylbutane ................................................................................................................................................................................................ 1592
Tricyclene ......................................................................................................................................................................................... 2050,2434,2120,2131 2,4,5-Trimethylheptane .............................................................................................................................................................................................. 1612
Tricyclic antidepressants ..................................................................................................................................................................................... 1164,210 3,3,4-Trimethylheptane .................................................................................................................................................................................... 1610,1612
Tridecane ................................................................................................................................ 1431,1572,1596,1597,1600,1658,1602,1668,1621,1637 3,3,5-Trimethylheptane .............................................................................................................................................................................................. 1612
n-Tridecane .................................................................................................................................................................................................................. 1613 2,2,5-Trimethylhexane ................................................................................................................................................................................................ 1592
n-Tridecane ......................................................................................................................................................... 1614,1615,1616,1617,1618,1625,1626 2,3,5-Trimethylhexane ................................................................................................................................................................................................ 1592
n-Tridecane ........................................................................................................................................................................................................ 2311,2312 2,3,5-Trimethylnaphthalene .......................................................................................................................................................... 1408,2010,1409,2011
n-tridecane ......................................................................................................................................................................................................... 2313,2314 2,2,3-Trimethylpentane .............................................................................................................................................................................................. 1592
Tridecane (C13) .......................................................................................................................................................................................................... 1636 2,2,4-Trimethylpentane ........................................................................................................................... 1431,1572,1528,1529,1530,1610,1611,1612
Tridecanoic acid methyl ester ................................................................................................................................... 1818,1918,2250,1819,1919,2251 2,3,4-Trimethylpentane ........................................................................................................................................................................... 1610,1611,1612
Tridecanoic acid methyl ester (C13:0) .................................................................................................................... 1815,1915,2247,1817,1917,2249 2,4,4-Trimethyl-1-pentene/triisobutylene ................................................................................................................................................................ 1721
Tridecanoic acid methyl ester (C13:0) at 2 wt % ........................................................................................................................... 1822,1922,2254 (±)-1-(2,4,6-Trimethylphenyl)ethanol ............................................................................................................................... 1694,1835,1941,2296,2693
2-(E)-Tridecenal .......................................................................................................................................................................................................... 2166 Trimethylpyrazine ........................................................................................................................................................................... 1871,2022,2395,2417
Tridemorph ......................................................................................................................................................................................................... 1888,2067 2,3,5-Trimethylpyrazine .................................................................................................................................................................................... 2056,2439
Triethylamine ......................................................................................................................................................................... 1648,2379,1649,2380,1673 Trimipramine ............................................................................................................................................... 1167,212,1169,1170,2279,2303,2459,2468
Triethylene glycol ................................................................................................................................................................. 1383,1457,1705,1977,2187 Trimipramine solution ........................................................................................................................................................................................ 1368,209
O,O,O-Triethyl phosphorothioate ............................................................................................................................... 1470,1996,2360,2410,1550,1551 Trimipramine maleate salt ..................................................................................................................................................................... 1165,1171,3013
Triethyl thiophosphate ................................................. 1466,1740,1858,1989,2356,2384,2392,2406,1471,1746,1861,1997,2361,2386,2393,2411 1,3,5-Trinitrobenzene .................................................................................... 296,465,1455,1695,2297,2354,2383,2466,1456,1696,2298,1550,1551
Trifloxystrobin .................................................................................................................................................................................................... 1887,2066 2,4,6-Trinitrophenylmethylnitramine (Tetryl) ....................................................................................................................................... 1456,1696,2298
Trifluoperazine ........................................................................................................................................................................................................ 165,578 Trinitrotoluene .............................................................................................................................................................. 1455,1695,2297,2354,2383,2466
3-(Trifluoromethyl)aniline .......................................................................................................................................................................................... 1672 2,4,6-Trinitrotoluene ................................................................................................................................................................. 296,465,1456,1696,2298
(±)-γ-(Trifluoromethyl)benzyl alcohol ........................................................................................................................................ 1759,2149,2541,2680 Triolein ......................................................................................................................................................................................................................... 1633
Trifluorothymidine ................................................................................................................................................................................................. 168,587 Tripelennamine .............................................................................................................................................................................................................. 860
α,α,α-Trifluorotoluene ....................................................................................................................................................................................... 1528,1530 Tripelennamine hydrochloride ................................................................................................................................................................................... 1213
Trifluralin .................................................... 1351,1403,1862,2000,1446,1680,1827,1932,1467,1990,2357,2407,1506,2075,1507,2076,1887,2066 Triphenylene .............................................................................................................................................................................. 33,1408,2010,1409,2011
2′,4′,5′-Trihydroxybutyrophenone ..................................................................................................................................................................... 1282,292 Triphenyl phosphate ................................................................................................................................................... 1506,2075,1507,2076,1544,1545
3,3′,5-Triiodo-L-thyronine sodium salt ...................................................................................................................................................................... 174 Triprolidine ............................................................................................................................................................................................... 858,859,860,891
3,3′,5-Triiodo-L-thyronine (T3) solution .................................................................................................................................................................... 822 tRNA hydrolysate .......................................................................................................................................................................................................... 789
3,3′,5′-Triiodo-L-thyronine (Reverse T3) solution .................................................................................................................................................... 822 Troger’s Base ...................................................................................................................................................................... 113,2615,114,2616,115,2617
Triisobutylene .............................................................................................................................................................................................................. 1721 Trovafloxacin .......................................................................................................................................................................................................... 880,881
Trimethylamine .................................................................................................................................................................................................. 2225,2455 α-Trypsin ........................................................................................................................................................................................................................ 790
2,4,5-Trimethylaniline ................................................................................................................................................................................................ 1673 β-Trypsin ........................................................................................................................................................................................................................ 790
Trypsin Inhibitor .................................................................................................................................................................................................... 740,760
Indexes Analyte
Trypsin Inhibitor from Glycine max (soybean) ................................................................................................................................................. 738,761 Uridine ...................................................................................................................................................................................................... 687,688,689,769
Trypsinogen ................................................................................................................................................................................................................... 759 Ursodeoxycholic acid .................................................................................................................................................................................................... 234
Tryptamine ..................................................................................................................................................................................................................... 810
Tryptophan .............................................................................................................................................................................................. 190,191,192,810 —V—
D-Tryptophan ........................................................................................................................................................................................................ 2835,793
Vacor .............................................................................................................................................................................................................................. 313
L-Tryptophan ..................................................................................................................................................................................... 2835,793,1897,2304
Valacyclovir ............................................................................................................................................................................................................. 133,913
DL-Tryptophan ...................................................................................................................................................................................................... 2836,794
Valencene ........................................................................................................................................................................................................... 2050,2434
Tryptophan derivative ....................................................................................................................................................................................... 1897,2304 (+)-Valencene ............................................................................................................................................................................................................ 2170
DL-Tryptophan (FMOC derivatives) .................................................................................................................................................................... 2791,606
Valeraldehyde ......................................................................................................................................................................................................... 21,2326
DL-Tryptophan methyl ester (3,5-DNP derivatives) ........................................................................................................................................ 2772,577
Valeraldehyde-2,4-dinitrophenylhydrazone ......................................................................................................................................... 22,2334,23,2335
Two PEG molecules attached to protein identified as peak 1 .......................................................................................................................... 777 Valeraldehyde-2,4-DNPH ........................................................................................................................................................................................... 2337
Tyramine ......................................................................................................................................................................................................... 143,145,810 Valeraldehyde (2,4-DNPH derivative) ................................................................................................................................................................. 2336,24
D-Tyrosine ............................................................................................................................................................................................................. 2837,795
Valeric acid ................................................................................................................... 1808,1908,2244,1809,1909,2245,1873,2031,2278,1970,2404
L-Tyrosine .......................................................................................................................................................................................... 2837,795,1897,2304
D-Valine ................................................................................................................................................................................................................ 2841,799
DL-Tyrosine ............................................................................................................................................................................................................ 2840,798
L-Valine .............................................................................................................................................................................................. 2841,799,1897,2304
DL-o-Tyrosine ........................................................................................................................................................................................................ 2838,796
DL-Valine .............................................................................................................................................................................................. 2792,607,2842,800
DL-m-Tyrosine ....................................................................................................................................................................................................... 2839,797
DL-Valine (AQC derivatives) ............................................................................................................................................................................... 2743,522
DL-Tyrosine (t-BOC derivatives) ......................................................................................................................................................................... 2754,536
Val-Tyr-Val ............................................................................................................................................................................................................. 708,719
Tyr-Tyr-Tyr ..................................................................................................................................................................................................................... 788 Vanadium oxides tetroxide ......................................................................................................................................................................................... 825
Vanillic acid ................................................................................................................................................................................................................. 1259
—U— Vanillin ................................................................................................................................................................................................................ 2111,2184
Uncosane ..................................................................................................................................................................................................................... 1637 Vardenafil ............................................................................................................................................................................................................... 995,996
Undecanal .............................................................................................................................. 1490,1580,2318,2330,2050,2434,2103,2120,2166,2170 [deamino-Cys1, D-Arg8]-Vasopressin acetate salt hydrate ..................................................................................................................................... 820
Undecane ............................................................. 1431,1572,1484,2030,2366,2421,1486,1487,1595,1597,1600,1658,1601,1667,1602,1668,1637 Verapamil ....................................................................................................................................................................................................................... 896
n-Undecane ........................................................................................................................................................................................................ 2311,2312 Verapamil hydrochloride .............................................................................................................................................................................................. 256
n-undecane ........................................................................................................................................................................................................ 2313,2314 Verapamil hydrochloride solution ............................................................................................................................................................................ 1222
Undecane (C11) .......................................................................................................................................................................................................... 1636 (±)-Verapamil ....................................................................................................................................................................................................... 952,953
Undecanoic acid methyl ester .................................................................................................................................. 1818,1918,2250,1819,1919,2251 (R)-(+)-Verbenone ............................................................................................ 1789,2173,2227,2561,2689,2860,1790,2174,2228,2562,2690,2861
Undecanoic acid methyl ester (C11:0) .................................................................................................................... 1815,1915,2247,1817,1917,2249 (S)-(-)-Verbenone .............................................................................................. 1789,2173,2227,2561,2689,2860,1790,2174,2228,2562,2690,2861
Undecanoic acid methyl ester (C11:0) at 2 wt % .......................................................................................................................... 1822,1922,2254 Vernam ............................................................................................................................................................................................ 1467,1990,2357,2407
Undecanone ....................................................................................................................................................................................................... 1971,2405 Vernolat .............................................................................................................................................................................................................. 1507,2076
1-Undecene ................................................................................................................................................................................................................. 1612 Vernolate ............................................................................................................................................................................................................ 1506,2075
Uniconoazole P .................................................................................................................................................................................................. 1887,2066 Vigabatrin solution ....................................................................................................................................................................................................... 836
UR-144 ......................................................................................................................................................................................................................... 1226 Vinclozolin ................................................................................................................................................. 1445,1679,1826,1931,1887,2066,2004,2413
Uracil .................................................................................. 36,841,67,207,1200,269,485,1207,273,489,492,684,734,735,736,737,749,757,758,765 Vinyl chloride ............................................................................................................................................ 1494,2058,2371,2441,1496,1497,2061,
Uracil ............................................................................................................................................................................................................................ 786 1498,2062,2373,2444,1502,2071,1503,2072,1504,2073,1505,2074,1519,1520,1522,1546,1547,1548,1549,1565,1566,1569,1589,2323,2345
Uracil ................................................................................................................................................................................................... 825,829,1117,1118, 2-Vinyl-5-methylfuran ...................................................................................................................................................................................... 2056,2439
(U) Uracil ............................................................................................................................................................................................................. 1185,808 Virginiamycin M1 ................................................................................................................................................................................................ 395,1109
Uric acid ........................................................................................................................................................................................................................ 149 Viridiflorol .............................................................................................................................................................................. 2095,2136,2137,2138,2167
Indexes Analyte
Vitamin E ..................................................................................................................................................................................................................... 1295

Vitamin D2 solution ............................................................................................................................................................................................ 1322,253 —X—
Vitamin D3 ..................................................................................................................................................................................................................... 420 Xanthine ......................................................................................................................................................................................................................... 149
Vitamin D3 solution ............................................................................................................................................................................................ 1322,253 Xanthophyll .................................................................................................................................................................... 1239,344,1241,1242,1243,1317
Vitamin A (acetate) ......................................................................................................................................................................................... 1295,1296 Xanthosine ..................................................................................................................................................................................................................... 690
Vitamin A alcohol ...................................................................................................................................................................................................... 1296 Xanthotoxin ................................................................................................................................................................................................................. 1262
Vitamin E acetate ............................................................................................................................................................................................. 1295,1296 Xenon ........................................................................................................................................................................................................................... 1745
Vitamin D3 solution ................................................................................................................................................................................................... 1254 XLR-11 ......................................................................................................................................................................................................................... 1226
Vitamin E acetate ................................................................................................................................................................................... 1255,1256,1253 o-Xylene ...................................................................... 98,116,1493,2057,2370,2440,1497,2061,1498,2062,2373,2444,1502,2071,1503,2072,
Vitamin K1 ............................................................................................................................................................................................................. 275,835 1504,2073,1505,2074,1527,1528,1529,1530,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1592,1593,1594,
Vitamin K1 2,3 epoxide ....................................................................................................................................................................................... 275,835 1595,1596,1597,1600,1658,1601,1667,1602,1668,1610,1611,1612,1621,1640,1676,1725,1726,1727,1780,1794,1795,1891,2205
Vitamins, water soluble ............................................................................................................................................................................................ 1301 o-Xylene ....................................................................................................................................................................................................................... 2206
vitexin 4"-O-glucoside (PHY82682) ......................................................................................................................................................................... 1305 o-Xylene ............................................................................................................................................................................................................. 2207,2450
vitexin (PHY89290) .................................................................................................................................................................................................... 1305 o-Xylene ....................................................................................................................................................................................................................... 2208
vitexin 2"-O-rhamnoside (PHY89291) .................................................................................................................................................................... 1305 o-Xylene .......................................................................................................................................................................................... 2209,2451,2300,2467
Volatile fatty acids .................................................................................................................................................................................. 1808,1908,2244 m-Xylene ........................................................................................ 98,116,1493,2057,2370,2440,1497,2061,1498,2062,2373,2444,1502,2071,
Volatiles .............................................................................................................................................................................................................. 1503,2072 1503,2072,1504,2073,1505,2074,1527,1528,1529,1530,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,1569,1589,2323,2345,1592,
Voriconazole solution ............................................................................................................................................................................................ 831,832 1593,1594,1595,1596,1597,1600,1658,1601,1667,1602,1668,1611,1612,1621,1640,1676,1725,1726,1727,1780,1794,1795,1891,2205
m-Xylene ..................................................................................................................................................................................................................... 2206
—W— m-Xylene ............................................................................................................................................................................................................ 2207,2450
Warfarin™ ........................................................................ 1175,220,1176,221,1177,222,2641,3014,1178,223,2642,3015,1179,224,2643,3016, m-Xylene ..................................................................................................................................................................................................................... 2208
1180,225,2644,3017,1183,227,2646,3020,1182,3019,1343,3026,1344,2652,3027,1345,2653,3028 m-Xylene ......................................................................................................................................................................................... 2209,2451,2300,2467
Warfarin solution ............................................................................................................................................................. 1375,259,1377,265,2651,3022 p-Xylene ...................................................................................... 98,102,116,842,845,1493,2057,2370,2440,1497,2061,1498,2062,2373,2444,
(R)-(+)-Warfarin .............................................................................................................................................................................. 1376,262,2650,3021 1502,2071,1503,2072,1504,2073,1505,2074,1527,1528,1530,1546,1547,1548,1549,1565,1566,1568,1588,2322,2344,
(S)-(−)-Warfarin .............................................................................................................................................................................. 1376,262,2650,3021 1569,1589,2323,2345,1592,1593,1594,1595,1596,1597,1600,1658,1601,1667,1602,1668,1611,1612,1613,1614,1615,1616,1617,1618,1621,
Water ..................................................................................................................................................................................... 1622,1627,1699,1750,1751 1624,1625,1626,1640,1676,1725,1726,1727,1780,1794,1795,1891,2205
R-(-)-Wieland-Miescher Ketone ................................................................................................................................ 1791,2563,1792,2564,1793,2565 p-Xylene ....................................................................................................................................................................................................................... 2206
S-(+)-Wieland-Miescher Ketone .............................................................................................................................. 1791,2563,1792,2564,1793,2565 p-Xylene ............................................................................................................................................................................................................. 2207,2450
Withaferin A ................................................................................................................................................................ 1235,1310,1311,1312,1313,1314 p-Xylene ....................................................................................................................................................................................................................... 2208
Withanolide A ............................................................................................................................................................. 1235,1310,1311,1312,1313,1314 p-Xylene .......................................................................................................................................................................................... 2209,2451,2300,2467
Withanolide B ............................................................................................................................................................. 1235,1310,1311,1312,1313,1314 p-Xylene-d10 .................................................................................................................................................................................................................. 842
Withanone ............................................................................................................................................................................. 1310,1311,1312,1313,1314 Xylitol ............................................................................................................................................................................................................................. 386
Withanoside V ............................................................................................................................................................................................................ 1310 Xylose ....................................................................................................................................................................................................... 383,384,385,386
Withanoside IV ..................................................................................................................................................................... 1235,1311,1312,1313,1314 D-(+)-Xylose ................................................................................................................................................................................................................. 379
Withanoside V ...................................................................................................................................................................... 1235,1311,1312,1313,1314 7-(β-Xylosyl)paclitaxel ................................................................................................................................................................................................ 1154

Withanoside IV ........................................................................................................................................................................................................... 1310 Xylylcarb ............................................................................................................................................................................................................. 1890,2069
Indexes Analyte
—Y— Trademarks
Yohimbine hydrochloride ................................................................................................................................................................................. 1232,1316 The following trademarks and registered trademarks are accurate to the best of our
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Zeaxanthin .................................................................................................................................................................................................. 1239,344,1317
Zuclopenthixol ....................................................................................................................................................................................... 132,907,1189,231 Advanced Materials Technology, Inc. — Fused-Core®
American Society for Testing and Materials — ASTM®
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Chattem — Allegra®
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Chromatography

Volume 1 Volume 2 Volume 3

EPA Method 632: HPLC Analysis of Pesticides on 3 4 10 15

column . . . . . . . Ascentis C18, 25 cm x 4.6 mm I.D., 5 μm particles (581325-U) 5 7

mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . (A) water; (B) acetonitrile 1

1 Pub Date: August 2014

HPLC Analysis of Acidic Herbicides in Water on a 1. Tetrachloroterephthalic acid

2 Pub Date: August 2014

HPLC Analysis of Ancymidol Enantiomers on

3 Pub Date: August 2014

HPLC Analysis of Animal Feed Additives on 1 1. 4-hydroxyphenolarsonic acid

4 Pub Date: August 2014

HPLC Analysis of Atrazine Herbicides on Ascentis® 1. Desethylatrazine 10. Diuron

5 Pub Date: August 2014

HPLC Analysis of Bromacil Enantiomers on Astec® H

6 Pub Date: August 2014

HPLC Analysis of 2(2-Chlorophenoxy) Propionic O

Acid Enantiomers on Astec® CHIROBIOTIC® T H3C *

flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL/min

7 Pub Date: August 2014

HPLC Analysis of 2(2-Chlorophenoxy) Propionic O

Acid Enantiomers on Astec® CHIROBIOTIC® TAG O *

8 Pub Date: August 2014

HPLC Analysis of 2(4-Chlorophenoxy) Propionic O

Acid Enantiomers on Astec® CHIROBIOTIC® TAG O *

9 Pub Date: August 2014

HPLC Analysis of Devrinol (Napropamide) CH3

Enantiomers on Astec® CHIROBIOTIC® V N

10 Pub Date: August 2014

HPLC Analysis of Enilconazol (Imazalil) Cl

11 Pub Date: August 2014

HPLC Analysis of Fusarium Mycotoxins on 1. Nivalenol

12 Pub Date: August 2014

HPLC Analysis of Paraquat and Diquat on 1. Diquat

13 Pub Date: August 2014

HPLC Analysis of Strigol Enantiomers on Astec® H3 C CH 3 H

column . . CYCLOBOND I 2000 RSP, 25 cm x 4.6 mm I.D., 5 μm particles (20324AST) H O

14 Pub Date: August 2014

15 Pub Date: August 2014

HPLC Analysis of 16 Triazine and Phenyl Urea 1. Atrazine desisopropyl 9. Chlortoluron

16 Pub Date: August 2014

HPLC Analysis of B-Trichothecenes on Ascentis® 1. Nivalenol

17 Pub Date: August 2014

LC-MS Analysis of Ancymidol Enantiomers on N OH CH3

column . . . . . . . . . . . . . . . CYCLOBOND I 2000 HP-RSP, 25 cm x 4.6 mm I.D., N

18 Pub Date: August 2014

LC-MS/MS Analysis of Neonicotinoid Pesticides in 1, 2 1. Dinotefuran

19 Pub Date: August 2014

UHPLC Analysis of Atrazine on Titan™ C18 1. Atrazine, 8.27 1

column . . . . . . . . Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U)

20 Pub Date: August 2014

21 Pub Date: August 2014

HPLC Analysis of 13 Aldehyde/Ketone DNPH 1. Formaldehyde-2,4-DNPH

22 Pub Date: August 2014

HPLC Analysis of 13 Aldehyde/Ketone DNPH 1. Formaldehyde-2,4-DNPH

23 Pub Date: August 2014

HPLC Analysis of Aldehydes and Ketones as DNPH R

Derivatives on Ascentis® Express C18 HN 1. Formaldehyde-2,4-dnph

Application No. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G005382