Phenomenex Clinical Aplication Notebook

THE
CLINICAL
Application
Notebook
Edition 2
www.phenomenex.com/ClinicalResources
Table of Contents
Endocrinology and Biomarker Research........................................................................ 3
Unconjugated Bile Acids from Serum........................................................................................................4
Metanephrines from Plasma .....................................................................................................................8
Catacholamines from Urine using Dual Selectivities..................................................................................9
Catecholamines from Urine......................................................................................................................10
Testosterone from Serum.........................................................................................................................14
Steroid Panel............................................................................................................................................15
Cortisol, Cortisone, Prednisolone, and Prednisone from Urine...............................................................16
Total Cortisol from Plasma.......................................................................................................................21
Underivatized Methylmalonic Acid from Plasma .....................................................................................23
Underivatized Vitamin B1 and B6 from Whole Blood................................................................................26
Vitamin A and E from Serum....................................................................................................................34
Homocysteine..........................................................................................................................................37
Thiamine Monophosphate and Thiamine from Plasma and Breast Milk..................................................38
Therapeutic Drug Monitoring......................................................................................... 41

Digoxin and Digitoxin from Plasma..........................................................................................................42
Antidepressants from Urine......................................................................................................................45
Cortisone and Prednisolone from Plasma................................................................................................51
Immunosuppressants from Whole Blood.................................................................................................53
Comprehensive Drug Research Panel from Urine....................................................................................56
Comprehensive Drug Research Panel from Oral Fluid.............................................................................64
Automation of Comprehensive Drug Research Panel from Oral Fluid.....................................................71
Toxicology........................................................................................................................ 78
Ethyl Glucuronide and Ethyl Sulfate from Urine.......................................................................................79
Ethyl Glucuronide and Ethyl Sulfate from Urine by UHPLC.....................................................................82
Synthetic Cathinones from Urine.............................................................................................................85
Synthetic Cannabinoids from Urine.........................................................................................................87
Chiral Analysis of Synthetic Cannabinoids...............................................................................................89
THC-COOH and Barbiturates from Urine.................................................................................................92
Nicotine and Metabolites from Urine........................................................................................................95
Nicotine and Metabolites from Oral Fluid...............................................................................................101
Chiral Analysis of Amphetamines and Substituted Amphetamines.......................................................105
Sample Preparation....................................................................................................... 109

Whole Blood Pretreatment Procedures..................................................................................................110
Whole Blood Pretreatments for Basic Drugs.........................................................................................111
Maximizing Recoveries on Novum™ SLE...............................................................................................118
Simple, Fast Sample Clean Up with Phree™ Phospholipid Removal Products......................................120
Sensitivity Gains Using Strata®-X Microelution Solid Phase Extraction (SPE).......................................123
Improved Sensitivity of Hydrolyzed Urine Samples Using β-Gone™ β-Glucuronidase
Removal Products..................................................................................................................................125
Extend UHPLC Column Lifetime and Performance with Column Protection........................................127
Ordering Information..................................................................................................... 129
If Phenomenex products mentioned in this guide do not provide at least

an equivalent separation as compared to other products of the same
phase, size and dimensions, return the product with comparative data
2
within 45 days for a FULL REFUND. Phenomenex l WEB: www.phenomenex.com
Endocrinology
and Biomarker
Research
Phenomenex l WEB: www.phenomenex.com 3

Unconjugated Bile Acids from Serum
Shahana W. Huq and Simon Lomas
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA.
Sensitive and effective methods for the extraction and analysis of Sample Preparation
bile acids from human serum were developed using an Impact™ Product Name: Impact Protein Precipitation 2 mL Plate
protein precipitation plate and Kinetex® 2.6 µm Polar C18 HPLC/
UHPLC column. Part No.: CE0-7565
Size: 96-Well Plate
Overview
Add: 400 µL methanol into the wells of Impact plate
Bile acids are 24 carbon steroids formed in the liver from cho-
lesterol and are essential to solubilize and promote absorption 100 μL of Serum sample (spiked with internal
of dietary lipids and vitamins. The most abundant primary bile Load: standards and analytes at 200 ng/mL) directly into
the organic solvent in each well of the plate.
acids are cholic acid (CA) and chenodeoxycholic acid (CDCA),
while abundant secondary bile acids include deoxycholic acid 2 minutes at maximum speed (use a sealing mat
Vortex:
to prevent cross well contamination in the plate)
(DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA)
which are formed by deconjugation and dehydroxylation in the Wait:
Allow 5 minutes for completion
colon. Numerous analytical methods have emerged to determine of protein precipitation
bile acid concentration from plasma and serum in recent years.1-3 Centrifuge: Place the Impact plate on top
However, the major challenge of baseline separation between of a collection plate and centrifuge at 500 g
for 5 minutes or until filtrate is collected.
isobaric species remains, as LC-MS/MS alone doesn’t allow for
identification between them. In this application, we developed an Vacuum: Place the Impact plate onto a suitable
LC-MS/MS method for the quantitation of 8 unconjugated bile ac- 96-well sample manifold or robot. Ensure that a
Filter: 96-well collection plate is positioned inside the
ids utilizing a high efficiency Kinetex 2.6 µm Polar C18 core-shell manifold or under the Impact plate. Vacuum at 5 inch
column to ensure baseline resolution of each key isobaric group Hg for up to 5 minutes or until filtrate is collected.
of bile acids. For sample clean-up and extraction, a quick and
Positive Pressure: Place the Impact plate
easy method was developed using an Impact protein precipitation on top of a collection plate and apply 2-5
96-well plate. psi using a positive pressure manifold.
Materials and Methods Dispense 300 µL of mobile phase A (or water)

Dilute & inject: into the collection plate, vortex for 30 secs at
Reagents and Chemicals a high speed and inject on LC-MS/MS.
Double charcoal stripped human serum (DC Mass Spect Gold,
MSG 4000) was purchased from Golden West Biological (Te-
mecula, CA). HPLC-grade acetonitrile and methanol were pur-
chased from Honeywell (Morris Plains, NJ). All analytes and in-
ternal standards along with other chemicals were obtained from LC-MS/MS Conditions
the Sigma-Aldrich Company (St. Louis, MO). Purified water was Column: Kinetex 2.6 µm Polar C18
obtained using a Sartorius arium ® comfort II, courtesy of Sarto- Dimension: 100 x 2.1 mm
rious Corporation (Bohemia, NY). Part No.: 00D-4759-AN
Recommended Guard: AJ0-9530
Experimental Conditions Mobile Phase: A: 2 mM Ammonium Acetate (pH 6.9)
The LC-MS/MS method uses a Kinetex 2.6 µm Polar C18 core- B: Methanol/Acetonitrile (50:50)
shell 100 x 2.1 mm column with a 2 mM ammonium acetate (pH Gradient Time (min) B (%)
0 45
6.9) and methanol/acetonitrile (50:50) mobile phase. The LC 9 70
gradient resulted in a total run time of 9.5 minutes. Detection 9.5 70
was carried out on a SCIEX 4500 QTRAP ®, equipped with ESI 9.51 45
12 45
source operating under negative polarity. An Impact protein
Flow Rate: 0.4 mL/min
precipitation 96-well plate was used to extract the analytes Temperature: 50 ºC
from serum sample. To achieve reproducible and accurate re- Injection Volume: 5 uL
sults, a set of 8 stable isotope labelled bile acids were used as System: Agilent® 1260
internal standards (Table 1). A double charcoal stripped serum Detection: MS/MS (SCIEX 4500 QTRAP®), ESI-
sample was employed for extraction purposes to minimize po- Analytes: See Table 1
tential bias due to the presence of any endogenous bile acids.
For additional technical notes, visit www.phenomenex.com
4 Phenomenex l WEB: www.phenomenex.com

Results Figure 4. Representative XIC chromatogram displaying baseline separation
Figure 1. Chemical structure of the 8 bile acids from the analyte panel two
24194isomeric bile acids, GCDCA and GDCA
4.4e5
4.2e5
4.0e5
3.8e5
3.6e5
3.4e5
3.2e5
3.0e5
2.8e5
Intensity, cps
2.6e5
2.4e5
2.2e5
2.0e5
Cholic Acid (CA) Chenodeoxycholic Acid (CDCA) Deoxycholic Acid (DCA) 1.8e5
App ID 24194
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min
Table 1. Retention and Recovery of the Bile Acids
Analyte & IS name RT(min) % Recovery % CV (N=5)

Lithocholic Acid (LCA) Glycodeoxycholic Acid (GDCA) Glycochenodeoxycholic Acid
(GCDCA) UDCA 2.97 91 % 1.1
UDCA-D4 2.96
GCDCA 3.35 90 % 3.7
GCDCA-D4 3.34
CA 3.43 88 % 4.8
Ursodeoxycholic Acid (UDCA) Taurodeoxycholic Acid (TDCA) CA-D4 3.42
GDCA 3.72 90 % 4.4
GDCA-D4 3.71
Figure 2. Representative TIC chromatogram of unconjugated bile acids
extracted
24195 from human serum sample, utilizing an Impact PPT Plate TDCA 3.90 94 % 3.5
6.0e5 TDCA-D4 3.88
5.5e5
5.0e5 CDCA 5.75 90 % 4.5
4.5e5
4.0e5 CDCA-D4 5.74
Intensity, cps
3.5e5
3.0e5 DCA 6.03 88 % 4.6
2.5e5
App ID 24195
2.0e5 DCA-D4 6.02

1.5e5
1.0e5 LCA 8.90 90 % 6.9
5.0e4
0.0 LCA-D4 8.80
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min
Discussion
Figure 3. Representative XIC chromatogram displaying baseline separation The bile acid panel studied in this application contains several iso-
of24193
the three isomeric bile acids, UDCA, CDCA, and DCA meric analytes such as UDCA, CDCA, DCA, GCDCA and GDCA
2.6e5 that behave identically due to their structural similarity and can’t
2.5e5
2.4e5
2.3e5
be distinguished by mass spectrometry alone. Therefore, it is
imperative that optimized chromatographic conditions be estab-
2.2e5
2.1e5
1.9e5
1.8e5
1.7e5
1.6e5
lished to identify and confirm these isomers. As a result, a shallow
Intensity, cps
1.5e5
1.4e5
1.3e5
gradient from 45 % to 70 % was employed (for mobile phase B)
1.2e5
1.1e5 over 9 minutes to aid in the separation and sharpening of the iso-
1.0e5
meric peaks of interest (figure 3 and 4). Within the LC-MS method
App ID 24193
9.0e4
8.0e4
7.0e4
6.0e4
5.0e4
development a 2 mM ammonium acetate (pH 6.9) was found to
4.0e4
3.0e4
be more effective over 0.1 % formic acid as a modifier, resulting in
2.0e4
1.0e4
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min

a higher MS response for all analytes of interest. Additionally, the Conclusion
use of the Kinetex® 2.6 µm Polar C18 LC column provided the We conclude herewith, a combined simple and efficient sample
necessary resolution between the bile acids of interest by means extraction and LC-MS/MS method for quantitation of 8 unconju-
of a dual non-polar and polar selectivity and high column effi- gated bile acids. The Impact™ sample preparation solution pro-
ciency. duces fast turnaround that demands virtually no method develop-
ment and can easily be transcribed to automation. For analysis,
The serum concentration range expected for the analytes of inter- the Kinetex Polar C18 column provided the necessary increased
est in this assay are quite high (≥ 1500 ng/mL) and a 1:1 dilution retention and resolution between the bile acids of interest through
of the post extracted samples provided more than enough signal a combination of core-shell particle performance advantages and
for MS analysis. The dilution step prevents the peak distortion or a highly useful dual non-polar/polar selectivity.
early elution in the solvent front, circumventing the need for time
intensive evaporation and reconstitution of the samples. Table
1 shows high recovery (88 % to 94 %) for all BA analytes tested. References
The Coefficient of variation (CV) value for 5 replicate extraction 1. A. Zhu, W. Lu, E. Epure; Rapid Quantitation of 15 Major Bile Acids in Human
came down less than 7 %, demonstrating good reproducibility of Serum by UPLC-ESI-MS/MS; MEDPACE Bioanalytical Laboratories
the assay. The oleophobic membrane (organic solvent leak resis- 2. M. Schere, C. Gnewuch, G. Schmitz, G. Liebisch, Rapid quantitation of bile
tant for at least 30 minutes) loaded Impact plate, enables gradual acids and their conjugates in serum by liquid chromatography-tandem mass
spectrometry; Journal of Chromatography B, 877 (2009) 3920-3925.
protein precipitation and optimum recovery in one single step, by-
3. L. Luo, S. Schomaker, C. Houle, J. Aubrecht; Evaluation of Serum Bile Acid
passing the need for centrifugation.
Profiles as Biomarkers of Liver Injury in Rodents; Toxicological Sciences.

Choose Your
Sample Preparation Solution
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Phospholipid • • Impact™
Removal / • • •
Protein
• • • •
Product Recommendation
Precipitation β-Gone™
• • • • Phree™
QuEChERS • • •
• • • • • • roQ™
Simplified
Liquid Extraction • • • • • • • •
Novum™
Solid Phase Strata®/

Extraction Strata-X
Sample Preparation Selection and Users Guide

Over 50 pages to assist you in selecting and using the
appropriate technique
Get Your Copy - www.phenomenex.com/SPguide

Metanephrines from Plasma
Overview
This application describes a fast and effective method for extracting and analyzing
plasma catecholamines and metanephrines (PMETs). Using Strata®-X-CW solid
phase extraction (SPE), PMETS are extracted and analyzed on a thermally modified
fully porous Luna® Omega 3 µm Polar C18 which provides enhanced retention of
polar analytes.
SPE Protocol LC-MS/MS Conditions

96-Well Microelution Plate: Strata-X-CW, 2 mg/well Column: Luna Omega 3 µm Polar C18
Part No.: 8M-S035-4GA Dimensions: 50 x 4.6 mm
Condition: 200 μL Methanol Part No.: 00B-4760-E0
Equilibrate: 200 μL 50 mM Ammonium acetate, pH 7
Mobile Phase: A: 10 mM Ammonium Acetate
Load: 1 mL of pretreated sample (250 µL plasma and B: 0.1% Formic Acid in Acetonitrile
750 µL 50 mM Ammonium acetate, pH 7) Gradient: Time (min) % B
Wash 1: 200 μL of 50 mM Ammonium acetate, pH 7 0 0
Wash 2: 200 μL Acetonitrile/IPA (1:1) 3 100
Dry: 3 min under high vacuum Injection Volume: 1 µL
Elute: 2x 25 μL of Water/Acetonitrile/Formic Flow Rate: 0.4 mL/min
acid (85:10:5) Temperature: 22 °C
Inject: Dilute eluent with 100 μL of
Detection: MS/MS (SCIEX API 4000™)
0.1 % Formic acid in water
Analytes: 1. Norepinephrine
2. Epinephrine
3. Normetanephrine
4. Dopamine
5. Metanephrine
6. Serotonin
5 6
1.5e5
1.4e5
1.3e5
1.2e5
1.1e5
1.0e5
Intensity, cps
9.0e4
8.0e4
7.0e4
2
App ID 24343
6.0e4
5.0e4
3 4
4.0e4
3.0e4
2.0e4 1
1.0e4
0.0
1.0 1.5 2.0 2.5 3.0 3.5 min

Catacholamines from Urine using Dual Selectivities
Overview
This application describes a fast and effective method for extracting and analyzing urinary
catecholamines. Strata®-X-CW weak cation exchange solid phase extraction (SPE) was
utilized for the extraction of catecholamines from urine which was then ran on both a
thermally modified fully porous Luna® Omega 3 µm Polar C18 and a core-shell Kinetex
2.6 µm Polar C18 to display the differences in rentention profiles between these two
useful solid supports.
SPE Protocol LC-MS/MS Conditions
96-Well Microelution Plate: Strata-X-CW, 2 mg/well Column: 1) Luna Omega 3 µm Polar C18
Part No.: 8M-S035-4GA 2) Kinetex 2.6 µm Polar C18
Dimensions: 50 x 4.6 mm
Condition: 200 μL Methanol
Part No.: 1) 00B-4760-E0
Equilibrate: 200 μL 50 mM Ammonium acetate, pH 7
2) 00B-4759-E0
Load: 1 mL of pretreated sample (500 µL urine and Mobile Phase: A: 0.1 % Formic Acid in Water
500 µL 50 mM Ammonium acetate, pH 7) B: 0.1 % Formic Acid in Methanol
Wash 1: 200 μL of 50 mM Ammonium acetate, pH 7 Gradient: Time (min) %B
Wash 2: 200 μL Acetonitrile/IPA (1:1) 0 0
Dry: 3 min under high vacuum 3 100
Elute: 2x 25 μL of Water/Acetonitrile/Formic Injection Volume: 5 µL
acid (85:10:5) Flow Rate: 0.7 mL/min
Inject: Dilute eluent with 100 μL of Temperature: 22 °C
0.1 % Formic acid in water
Analytes: 1. Metanephrine
2. Normetanephrine
Figure 1. 3. 3-Methoxytyramine
1) Luna Omega 3 µm Polar C18
24171
3
6.0e5
5.5e5
5.0e5
4.5e5
4.0e5
Intensity, cps
3.5e5
3.0e5
2.5e5 2
2.0e5
App ID 24171
1.5e5
1.0e5 1
5.0e4
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
Figure 2.
241722.6 µm Polar C18
2) Kinetex
6.8e5 3
6.5e5
6.0e5
5.5e5
5.0e5
4.5e5
Intensity, cps
4.0e5
3.5e5
3.0e5
2
2.5e5
App ID 24172
2.0e5
1.5e5 1
1.0e5
5.0e4
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

Catecholamines from Urine
Overview Table 1. Recovery Values from 10 ng/mL to 63 pg/mL

Metanephrine and normetanephrine are both metabolites of epi-
nephrine and norepinephrine. In this technical note we will ex- Analyte Concentration Average % %CV (n=6)
plore how to use Strata®-X-CW Microelution SPE 96-Well Plates (ng/mL) Recovery
in conjunction with a Kinetex® Biphenyl HPLC column in order to Metanephrine
resolve an interference that coelutes with 3-Methoxytyramine on
10 102 5
a standard C18 HPLC column, while reaching low limits of quan-
tification for specific urinary catecholamines, metanephrine and 1 102 3
normetanephrine. 0.5 99 2
Materials and Methods 0.25 99 3

Sample Preparation 0.125 97 3
Urine Pretreatment: 500 µL of urine was diluted with 500 µL of 0.063 94 6
50 mM Ammonium acetate buffer, (pH 7). Urine was pre-spiked
from 10 ng/mL to 63 pg/mL with metanephrine, normetaneph- Normetanephrine
rine, and 3-methoxytyramine (standards provided by Cerril- 10 100 10
iant ®). 1 87 12
0.5 110 10
Solid Phase Extraction Method
0.25 89 9
96-Well Microelution: Strata-X-CW, 2 mg/well
Part No.: 8M-S035-4GA 0.125 110 13
Condition: 200 µL Methanol
Equilibrate: 200 µL 50 mM Ammonium acetate buffer, pH 7
0.063 108 15
Load: 1 mL of pretreated sample 3-Methoxytyramine
Wash 1: 200 µL of 50 mM Ammonium acetate buffer, pH 7
Wash 2: 200 µL Acetonitrile/IPA (1:1)
10 91 3
Elute: 2 x 25 µL of Water/Acetonitrile/Formic acid (85:10:5)* 1 89 6
Injection: Dilute eluent with 100 µL of 0.1 % Formic acid in water**
0.5 95 4
0.25 86 5
LC-MS/MS Conditions 0.125 87 6
Column: Kinetex 5 µm Biphenyl
Dimensions: 50 x 4.6 mm 0.063 92 7
Part No.: 00B-4627-E0
Mobile Phase: A: 0.1 % Formic acid in Water Figure 1. Chromatogram of unresolved interference for 3-methoxytyramine
B: 0.1 % Formic acid in Methanol
using C18 1ng/mL
Gradient: Time (min) B (%)
2.04
0 5 1.14e5
3 90 1.10e5
3.1 5 1.05e5
5 5 1.00e5
9.50e4
9.00e4
Injection Volume: 30 µL 8.50e4
Temperature: Ambient 8.00e4
Detection: MS/MS (SCIEX API 4000™) 7.50e4
7.00e4
Intensity, cps
6.50e4
6.00e4
5.50e4
5.00e4
4.50e4
4.00e4
3.50e4
3.00e4
2.50e4
App ID 23845
2.00e4
1.50e4
1.00e4
*Elution solvent optimized to minimize amount of organic to allow for minimal post elution dilution
5000.00
**Internal standard Metanephrine-D3 was included at 1 ng/mL in this portion of 100 µL diluent
0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

Figure 2. Chromatogram of resolved interference for 3-methoxytyramine
using Kinetex® Biphenyl at 1 ng/mL
1.90
2.0e5
1.9e5
1.8e5
1.7e5
1.6e5
1.5e5
1.4e5
1.3e5
1.2e5
Intensity, cps
1.1e5
1.0e5
9.0e4
8.0e4
7.0e4
6.0e4
5.0e4
4.0e4
App ID 23846
3.0e4
2.0e4
2.09
1.0e4
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Figure 3. 3-Methoxytyramine separated interference at 63 pg/mL on Kinetex Biphenyl

2.7e4 1.91
2.6e4
2.4e4
2.2e4
2.0e4
1.8e4
1.6e4
Intensity, cps
1.4e4
1.2e4
1.0e4 2.09
8000.0
6000.0
App ID 23847
4000.0
2000.0
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Figure 4. Representative Chromatogram of Urinary Catecholamines

4.4e5
4.2e5
4.0e5
3.8e5
3.6e5
3.4e5
3.2e5
3.0e5
2.8e5
2.6e5
Intensity, cps
2.4e5
2.2e5
2.0e5
1.8e5
1.6e5
1.4e5
1.2e5
1.0e5
App ID 23848
8.0e4
6.0e4
4.0e4
2.0e4
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min

Figure 5. Representative Calibration Curve for Metanephrine Figure 7. Representative Calibration Curve for 3-Methoxytyramine
1.25e6
1.25e6
1.20e6
1.20e6
1.15e6
1.15e6
1.10e6
1.10e6
1.05e6
1.05e6
1.00e6
1.00e6
9.50e5
9.50e5
9.00e5
9.00e5
8.50e5
8.50e5
8.00e5
8.00e5
7.50e5
7.50e5
7.00e5
Area, counts
7.00e5
Area, counts
6.50e5
6.50e5
6.00e5
6.00e5
5.50e5
5.50e5
5.00e5
5.00e5
4.50e5
4.50e5
4.00e5
4.00e5
3.50e5
3.50e5
3.00e5
3.00e5
2.50e5
2.50e5
2.00e5
2.00e5
1.50e5
1.50e5
1.00e5
1.00e5
5.00e4
5.00e4
0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 0.00
Concentration, ng/mL 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
Concentration, ng/mL
Figure 6. Representative Calibration Curve for Normetanephrine
1.25e6
1.20e6
1.15e6
1.10e6
1.05e6
1.00e6
9.50e5
9.00e5
8.50e5
8.00e5
7.50e5
7.00e5
Area, counts
6.50e5
6.00e5
5.50e5
5.00e5
4.50e5
4.00e5
3.50e5
3.00e5
2.50e5
2.00e5
1.50e5
1.00e5
5.00e4
0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
Concentration, ng/mL
Results and Discussion

HPLC: Figure 1 shows the Extracted-Ion Chromatography (XIC) 86 % and the highest % CV is for normetanephrine at 63 pg/mL
for 3-Methoxytyramine using a standard C18 50 x 4.6 HPLC col- (15 %).
umn. This figure shows only one, albeit tailing, peak. By contrast,
The 10 % acetonitrile elution is chosen purposefully. By minimizing
Figure 2 is the XIC for 3-Methoxytyramine using a Kinetex® Bi-
the amount of organic in the elution step, the need for post elution
phenyl HPLC column which clearly shows two distinct peaks,
dilution (to minimize peak distortion/splitting) is also decreased
one at 1.9 minutes as well as one at 2.09 minutes. This indicates
and therefore only a 2:1 dilution is required. By starting at 500 µL
that the biphenyl selectivity is able to resolve an interference in
(urine sample) and ending in 150 µL (elution volume plus diluent),
the sample that is not resolved with the standard C18 column.
the solid phase extraction process concentrated the samples 3x
Figure 3 shows the same separation as Figure 1, however it is at without the need for dry down and reconstitution.
a much lower level (63 pg/mL), making it even more important that
the interference is completely resolved. Figure 4 exhibits a chro- Conclusion
matogram for all analytes in the suite including Metanephrine-D3 In conclusion, we have shown that by implementing an SPE step
at 1 ng/mL. using Strata®-X-CW microelution in conjunction with a Kinetex
Biphenyl HPLC column we were able to maximize sensitivity and
SPE: Table 1 provides absolute recovery values for all three of the accuracy and display the utility of a method for detecting cate-
compounds analyzed, and Figures 5, 6 and 7 show representa- cholamines that can reach at least 63 pg/mL on an API 4000™
tive calibration curves for each analyte from 10 ng down to 63 pg LC-MS/MS (SCIEX).
where R > 0.9995. All absolute recovery values are greater than

TN-0076
Core-Shell Technology
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with Kinetex Core-Shell Technology
Shockingly better performance than your current
HPLC/UHPLC/PREP LC column. Guaranteed!*
1.3 1.7
µm
2.6
µm
3.5
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5
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™ ™ ™ ™
™
Find Your Kinetex Column:

www.phenomenex.com/Kinetex

Testosterone from Serum
Table 1.
LC Gradient Program
Testosterone was extracted from human serum by strong anion Step Total Time (min) Flow Rate (µL/min) B (%)
exchange polymeric SPE (solid phase extraction) and analyzed
0 0 400 10
using a Kinetex® 1.7 µm C18 column, 30 x 2.1 mm, in ESI+
1 2.5 400 90
mode. Kinetex sub-2 µm core-shell technology offers higher
efficiencies than traditional sub-2 µm columns, producing greater 2 3.5 400 90
chromatographic resolution, sensitivity, and higher peak capacities. 3 3.6 400 10
4 5 400 10
Overview
Testosterone is an androgenic steroid responsible for the develop- An API 5000™ (SCIEX) triple-quadrupole tandem mass spectrom-
ment of male reproductive organs, maintaining (or increasing) mus- eter is used for analysis equipped with an ESI probe operating in
cle mass, and bone density. As anabolic steroids, testosterone has positive polarity mode. Under an MRM mode, two channels were
been used (or abused) to increase muscle mass and enhance the monitored for Testosterone and Testosterone-D3 (Table 2).
athletic performance. The concentration of testosterone is lower
in the female population than men and in general diminishes with Table 2.
advancing age. MRM Transitions Used for Data Analysis
Materials and Methods Peak Name MRM Channel

Testo (1) 304.3 124.0
Sample Preparation
Testo (2) 304.3 112.0
The sample preparation is based on a simple solid phase extraction IS (Testo-D3 1) 307.3 124.0
method using strong anion exchange SPE (Strata®-X-A 30 mg/ IS (Testo-D3 2) 307.3 112.0
3 mL tubes) to produce a clean extract from human serum.
As demonstrated in Figure 1, the Kinetex 1.7 µm 30 x 2.1 mm
SPE Protocol UHPLC column efficiently separates testosterone from its isomeric
Cartridge: Strata-X-A, 30 mg/3 mL Tubes form epitestosterone. This column provides a very high degree
Part No.: 8B-S123-TBJ of selectivity, even in a short dimension, resulting in superior
Condition: 2 mL Methanol and 2 mL Water chromatographic separation in a short run time.
Load: In separate test tube, combine 0.25 mL serum
sample, 1 mL DI water and 0.1 mL working
internal standard and load on sorbent Figure 1.
Wash: 0.6 mL Methanol/Water (50:50)
Separation
19761 of Testosterone and Epitestosterone by LC-MS/MS
Dry: 2x 0.3 mL Methanol
Elute: 2x 0.3 mL Methanol 4.0e4 1
3.8e4
Final Prep and Analysis 3.6e4
Following evaporation of elution solvent @ 50-55 °C under gentle 3.4e4
nitrogen stream; Add 50 µL 25% hydroxylamine solution and heat 3.2e4
at 60-65 °C for 5-10 min, then add 200 µL 5% aqueous formic acid 3.0e4
2.8e4
and vortex the tubes. Transfer the solution to autosampler vials and 2.6e4
inject 25 µL on column. Inject 20 µL on HPLC / Mass Spectrometer 2.4e4
(MS) @ amu (ambient) 2.2e4
2.0e4
HPLC Conditions 1.8e4

2
Following the solid phase extraction, testosterone is derivatized to 1.6e4
1.4e4
form an oxime which is then analyzed, using a short-length 30mm 1.2e4 3
x 2.1 mm ID, 1.7 µm Kinetex C18 UHPLC column, in positive mode 1.0e4
App ID 19761
ESI LC-MS/MS under multiple-reactions-monitoring function1. 8000.0

6000.0
The mobile phase consisted of 0.1% formic acid with 1 mM am- 4000.0
2000.0
monium formate with no pH adjustment, in water (mobile phase A) 0.0
and acetonitrile (mobile phase B). A typical LC gradient (Table 1) is 0.5
49
1.0
98
1.5
146
2.0
194
2.5
243
3.0
291
3.5
340
4.0
388
4.5
436
min
used for the separation.

Column: Kinetex 1.7 μm C18 100 Å
Part No.: 00A-4475-AN
Mobile Phase: A: 0.1% Formic Acid +1 mM Ammonium Formate in Water
B: 0.1% Formic Acid +1 mM Ammonium Formate in Acetonitrile
Gradient: Time (min) B (%) Flow Rate: 0.4 mL/min
0 10 Temperature: 55 °C
Detection: LC-MS/MS (SCIEX API 5000), ESI+
2.5 90 Analyte: 1. Testosterone
3.5 90 2. Testosterone – D3 (IS)
3. Epitestosterone
3.6 10

Steroid Panel
Overview LC-MS/MS Conditions

This application note describes the separation and LC-MS/MS Column: Kinetex® 5 µm EVO C18
analysis of a 12 steroid panel. The steroid panel was analyzed Dimensions: 100 x 2.1 mm
in a single LC-MS/MS application however they can also be Part No.: 00D-4633-AN
analyzed in three separate panels as shown in the representative Recommended Guard: AJ0-9298
Mobile Phase: A: 5 mM Ammonium Formate in Water
chromatograms. B: 100 % Methanol
Gradient: Time (min) %B
0 30
8 95
8 95
8.51 30
10 30
Temperature: 22 °C
Detection: MRM (SCIEX API 5000™), APCI+
Representative Chromatograms of Steroids
Three separate chromatograms are depicted to better show the separation between the 12 steroids however they were run together in a single
LC-MS/MS run
2.3e6
1.7e6 2.2e6
2.1e6
1.6e6
2.0e6
1.5e6 1.9e6
1.4e6 1.8e6
1.7e6
1.3e6
1.6e6
1.2e6 1.5e6
Analytes Analytes
1.1e6 1.4e6
1. Cortisol (Hydrocortisone) 1. DHEA-SO4
Intensity, cps
1.3e6
1.0e6 2. Cortisone 2. Androstenedione
1.2e6
Intensity, cps
9.0e5 3. 17-Deoxycortisol (Corticosterone) 1.1e6

3. Testosterone
8.0e5 4. 11-Deoxycortisol 1.0e6
4. DHEA
9.0e5 5. Epi-Testosterone
7.0e5
8.0e5
6.0e5
7.0e5
5.0e5 6.0e5
App ID 23273
4.0e5 5.0e5
App ID 23272
4.0e5
3.0e5
3.0e5
2.0e5 2.0e5
1.0e5 1.0e5
0.0
0.0 1. 0 2. 0 3. 0 4. 0 5. 0 6. 0 7. 0 8. 0 9. 0 min
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
1.7e6
1.6e6
1.5e6
1.4e6
1.3e6
1.2e6
1.1e6 Analytes
1.0e6
1. 17-OH-Progesterone
2. 17-OH-Pregnenolone
Intensity, cps
9.0e5 3. Progesterone
8.0e5
7.0e5
6.0e5
5.0e5
4.0e5
App ID 23274
3.0e5
2.0e5
1.0e5
0.0
1. 0 2. 0 3. 0 4. 0 5. 0 6. 0 7. 0 8. 0 9. 0 min

Cortisol, Cortisone, Prednisolone, and Prednisone from Urine
The existing methods for the quantification of cortisol, cortisone, two isomers of cortisone and prednisolone, were successful-
prednisolone, and prednisone are very diverse. While liquid- ly resolved by using the Kinetex® core-shell Biphenyl HPLC/
liquid extraction, protein precipitation, and “dilute-and-shoot” UHPLC column.
procedures offer quick and dirty methodologies, they risk in-
creases in instrument downtime and analytical column costs.
We evaluated a variety of silica-based and polymer-based SPE
sorbents, each of which provides a different retention mecha-
nism. The evaluation showed that the Strata®-X polymer-based
SPE sorbent, with a unique elution solvent has been found to be
a robust, reproducible, and cost effective sample preparation
solution for the laboratory, while providing a LLOQ of 10.0 ng/
mL in human urine for all four corticosteroids. Experimental Conditions
Overview SPE Conditions

Cortisol is a corticosteroid hormone that stimulates anti-in-
flammatory and anti-stress pathways as a response to stress. 96-Well Plate: Strata-X 60 mg/well
Part No.: 8E-S100-UGB
Cortisone, another corticosteroid, is the inactive metabolite of Condition: 1 mL Methanol
cortisol. Prednisolone is also an isomer of cortisone, making Equilibrate: 1 mL Water
chromatographic and spectrometric analysis difficult. Load sample: 300 μL human urine diluted in 300 μL Water with 1 μg/mL IS
(Cortisol D4)
Wash 1: 1 mL Water
As such, a highly specific method is needed to accurately quan- Wash 2: 1 mL 10 % Methanol in Water
titate urinary cortisol and cortisone. In this study, we evaluate Elute: 2x 500 μL of 2 % Formic Acid in Ethyl acetate/Isopropanol (85:15)
selected SPE sorbents, and optimize the best performing Stra- Dry Down : To dryness under a gentle Nitrogen stream at 50 °C
Reconstitute : 100 μL of 10 mM Ammonium acetate/10 mM Ammonium acetate
ta-X SPE extraction methods to reach acceptable recoveries of in Methanol (50:50)
four corticosteroids. The separation of all analytes, especially
Cortisol (m.w. 362.4) Cortisone (m.w. 360.4)
Prednisone (m.w. 358.4) Prednisolone (m.w. 360.4)


Experimental Conditions cont’d Figure 2.
Wash Solvent Optimization
LC-MS/MS Conditions 140 QCL (30 ng/mL)
LC-MS/MS was performed using a Kinetex 2.6 μm core-shell ®
Biphenyl HPLC/UHPLC column, 50 x 3.0 mm (P/N 00B-4622-Y0) 120
on an Agilent® 1200SL LC system (Agilent Technologies, Palo

Alto, CA, USA) with an upper pressure limit of 400 bar, equipped 100
with a binary pump, autosampler and interfaced with an API

4000™ triple quadrupole mass spectrometer (AB SCIEX, 80
Recovery %
Framingham, MA, USA). The ionization source was electrospray
ionization (ESI) analyzed in positive ion mode (Table 1). 60
40
Column: Kinetex 2.6 µm Biphenyl
Dimensions: 50 x 3.0 mm selected
Part No.: 00B-4622-Y0 20
Mobile Phase: A: 10 mM Ammonium acetate in Water
B: 10 mM Ammonium acetate in Methanol 0
Gradient: Time (min) %B 0% 10% 20% 30% 40% 50% 80% 100%
0.01 40
0.5 40
2 90 140 QCH (1,600 ng/mL)
3 90
3.01 40
5 40 120
Column Temperature: 40 °C
Injection Volume: 10 µL 100
Detection: MS/MS (AB SCIEX API 4000), ESI+
Instrument: Agilent 1200SL with binary pumps
80
Recovery %
Table 1.
Mass Transitions and Analyte Retention Times 60
Analyte Q1 Mass (Da) Q3 Mass (Da) Analyte Retention

Time (min) 40
Cortisone 1 361 163 3.36 selected

Cortisone 2 361 121 3.36 20
Cortisol 1 363 121 3.31

Cortisol 2 363 309 3.31 0
Prednisolone 1 361 147 3.19 0% 10% 20% 30% 40% 50% 80% 100%
Prednisolone 2 361 173 3.19

% Methanol
Prednisone 1 359 147 3.23
Cortisone Cortisol Prednisone Prednisolone
Prednisone 2 359 237 3.23
Cortisol-D4 1 367 121 3.22
Cortisol-D4 2 367 331 3.22
Figure 1.
Recovery of Cortisone and Cortisol using Various SPE Sorbents
100
90
80
70
Recovery (%)
60
50
40
30
20 Visit www.phenomenex.com/ClinicalResources
10 for more technical information and guides
0
Strata C18-E Strata-X Strata-X-C Strata-X-A
Cortisone Cortisol

Experimental Conditions cont’d
Figure 3. Table 2.
Recovery using Strata®-X Across Low (QCL, 30 ng/mL) and High (QCH, Accuracy and Precision
1600 ng/mL) QC Concentrations
LL0Q QCL QCM QCH

100
Nominal Conc. (ng/mL) 10 30 500 1600
80
Cortisone
Recovery (%)
60
Mean Conc. Fund (ng/mL) 10.1 30.4 527 1678
40 STDV 0.649 2.32 8.18 50.8
CV% 6.40 7.64 1.55 3.02
20
Accuracy (%) 101 101 106 105
0
Cortisone Cortisol Prednisone Prednisolone
Cortisol
QCL QCH
STDV 0.399 2.41 6.65 17.9
CV% 3.82 7.71 1.28 1.08
Figure 4. Accuracy (%) 105 104 104 104
22164
Linear Regression of Cortisol
2.4
Prednisolone
2.2 Cortisol (10.0 -2000 ng/mL)
2.0 r = 0.9998 Mean Conc. Fund (ng/mL) 10.8 33.1 537 1587
1.8 STDV 0.973 3.49 14.9 66.5
1.6 CV% 8.99 10.5 2.78 4.19
Analyte Area / IS Area
1.4
Accuracy (%) 108 110 107 99.3
1.2
1.0 Prednisone
0.8
App ID 22164
0.6
0.4
STDV 0.816 1.97 14.1 45.0
0.2 CV% 7.97 6.31 2.63 2.77
0.0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
Accuracy (%) 102 104 108 101
Analyte Conc. / IS Conc.

Figure 5.
Cortisol, Cortisone, Prednisolone, and Prednisone at LLOQ (10 ng/mL)
22166
22165
3.23 3.35
1.8e4 8.5e4
1.7e4 8.0e4
1.6e4 7.5e4
1.5e4
7.0e4
1.4e4
Cortisone Cortisone
Intensity, cps
6.5e4
1.3e4
6.0e4
1.2e4
5.5e4
Intensity, cps
1.1e4
5.0e4
1.0e4
4.5e4
9000.0
App ID 22166
App ID 22165
4.0e4
8000.0
3.5e4
7000.0
3.0e4
6000.0
2.5e4
5000.0
4000.0 2.0e4
0.60 2.93
3000.0 1.5e4
2000.0 1.0e4
0.53 3.05 3.36
1000.0 5000.0
0.0 0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min
22167
22168
3.31 3.19
7480
6000
7000
5500
6500
5000 6000
4500 Prednisone 5500

Prednisolone
5000
Intensity, cps
4000
Intensity, cps
4500
3500 1.08
4000
3000 2.46 3500
3.36
App ID 22168
App ID 22167
2500 3000
2500
2000
2000
1500
1500
1000 0.72 2.65 3.02 3.13
4.00
1.00 1.40 1000
0.60 1.69 1.76 2.09 2.22 3.573.61 3.79 2.65 3.42 3.67
500 4.08 500 0.69 3.94
0 0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min
Figure
22169
6.
Separation of Corticosteroids (1600 ng/mL)
1
6.9e5
6.5e5
1. Prednisolone
6.0e5
2. Prednisone 2
5.5e5
5.0e5
Intensity, cps
4.5e5
4.0e5
App ID 22169
3.5e5
3.0e5
2.5e5
2.0e5
1.5e5
1.0e5
5.0e4
0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min
22170
4
1.10e6
1.00e6 3. Cortisol
9.00e5 4. Cortisone
8.00e5
Intensity, cps
7.00e5 3
6.00e5
App ID 22170
5.00e5
4.00e5
3.00e5
2.00e5
1.00e5
0.00
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min

Figure 7.
Resolution of Isobaric Compounds, Prednisolone and Cortisone
2
4.4e5
4.2e5 1. Prednisolone
4.0e5
3.8e5
2. Cortisone
3.6e5
3.4e5
3.2e5
3.0e5
2.8e5 1
Intensity, cps
2.6e5
2.4e5
2.2e5
2.0e5
1.8e5
App ID 22678
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 min
Results and Discussion Conclusions

The selected SPE media was compared based on manufacturer A fast, robust LC-MS/MS method was developed for the
recommended procedures for each sorbent. Strata®-X provided extraction and quantitation of cortisone, cortisol, prednisone,
the best recovery (Figure 1) while low recoveries of analytes and prednisolone from urine using Phenomenex Strata-X 96-
were observed using manufacturer recommended procedures well plates. Using a unique elution solvent, analyte recoveries
for the other sorbents. Optimization of the second strong wash were maximized. After extraction, a Kinetex core-shell Biphenyl
solvent was performed to maximize recovery using Strata-X. HPLC/UHPLC column offered optimal separation and sensitiv-
10% Methanol in water was found to have better recovery ity along with baseline resolution between the isobaric com-
across all the concentrations and analytes (Figure 2). Recover- pounds cortisone and prednisolone. The assay achieved an
ies of four corticosteroids were acceptable at both high and low LLOQ for all four corticosteroids from urine of 10 ng/mL, prov-
concentration QC’s (Figure 3). ing that the method was sensitive enough to detect low levels
of urinary corticosteroids.
Assay linearity of all analytes was acceptable from 10.0 – 2,000
ng/mL. A representative calibration curve for cortisol is shown
in Figure 4, resulting in a correlation coefficient of 0.9998.
Accuracy and precision of 4 level QCs for all 4 compounds was References
between 99.3-110 % with CV% at 1.08-10.55 %, respectively 1. Hoehn K, Marieb EN (2010). Human Anatomy & Physiology. San Francisco:
(Table 2). Benjamin Cummings. ISBN 0-321-60261-7.
2. Mune T et al. (1995) Human hypertension caused by mutations in the
The Kinetex® core-shell Biphenyl HPLC/UHPLC column offered kidney isozyme of 11 beta-hydroxysteroid dehydrogenase. Nat Genet 10:
excellent sensitivity of all 4 compounds at LLOQ (10 ng/mL) 394-399
(Figure 5) as well as separation of the two pairs of cortisone/ 3. Turner, S. T. Monogenic forms of low-renin hypertension. Nature Clinical
cortisol and prednisone/prednisolone (Figure 6). In addition Practice Nephrology, 624-630.
to excellent sensitivity and separation, the Kinetex core-shell
Biphenyl HPLC/UHPLC column achieved separation of cortisone
and prednisolone which are isobaric compounds (Figure 7).

Total Cortisol from Plasma
Figure 1. Structure of Cortisol

Existing methods for cortisol analysis are very diverse. This study
focuses on developing an optimized sample preparation proce-
dure for cortisol from plasma. The evaluation resulted in a rapid,
clean, and reproducible method using Novum™ Simplified Liquid
OH
Extraction (SLE) 96-well plates.
Overview
Cortisol or hydrocortisone is a corticosteroid secreted by the ad-
O
renal cortex. Cortisol is synthesized from cholesterol and may be
found in the blood bound to globulin or in free-form. Cortisol has OH3
an anti-inflammatory effect and aids in carbohydrate metabolism, OH
renal function, and the promotion of gluconeogenesis.
The aim of this work is to introduce a fast and effective plasma

cortisol extraction procedure using Novum SLE which results in
a clean extract with high recovery. To determine the most effec-
tive procedure, two different extraction solvents were examined;
dichloromethane (methylene chloride or DCM) and a more polar
elution solvent, methyl tert-butyl ether (MTBE)/ethyl acetate (EtO-
Ac) (50:50).
Analytes
1. Cortisol
2. Cortisol-D4 as Internal Standard

Experimental Conditions
Extraction Procedure
Sample Pre-treatment Figure 2. Representative chromatogram of plasma cortisol (25 ng/
• Dilute 200 µL of human plasma (spiked with cortisone at mL) extracted by a more polar solvent combination (MTBE/EtOAc
25 ng/mL) with 200 µL of 50 mM sodium phosphate dibasic (50:50)) (notice the region enclosed in red circle).
209
heptahydrate, pH unadjusted. Vortex briefly (3-5 sec). 1.20e5
1.10e5
Cortisol
SLE Protocol
1.00e5
96-Well Plate: Novum™ SLE MAX
9.00e4
Part No.: 8E-S138-5GA
8.00e4
Pretreated sample and pulse vacuum
7.00e4
Intensity, cps
Load: (~5” Hg) for 5-10 seconds or until sample has
complete entered the sorbent. Wait 5 minutes. 6.00e4
255
900 μL of elution solvent onto the Novum media 5.00e4
and allow the solvent to elute by gravity
4.00e4
App ID 22790
(~5 min elution time) and collect the eluant.
3.00e4
Repeat with another 900 µL of elution solvent
Elute: and collect the eluant. Apply vacuum at 5” of Hg 2.00e4
for 20-30 secs to complete the extraction.
1.00e4 233 271
Note: Prolonged application of vacuum will
0.0
result in elution of plasma from the Novum SLE 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
media and into the final extracted solvent.
Evaporate the final extract to complete dryness Figure 3. Representative chromatogram of plasma cortisol (25
Dry down:
under a slow stream of N2 at 40°C. ng/mL) extraction using DCM as an extraction solvent (notice the
The dry residue in 200 µL of initial mobile region enclosed in red circle).
Reconstitute: 2.13
phase fortified with cortisol-D4 1.7e5
1.6e5 Cortisol
1.5e5
1.4e5
HPLC Conditions MS/MS Conditions 1.3e5
Column: Kinetex® 2.6 µm Biphenyl CAD: 7.00 1.2e5
Dimensions: 50 x 2.1 mm CUR: 25.00 1.1e5
Part No.: 00B-4622-AN GS1: 50.00
Recommended Guard: AJ0-9209 GS2: 50.00 1.0e5
Intensity, cps
Mobile Phase: A: 10 mM Ammonium acetate in Water IS: 5000.00 9.0e4

B: 10 mM Ammonium acetate in Methanol TEM: 600.00 8.0e4
Gradient: Time (min) % B
7.0e4
0 50
6.0e4
2 95
App ID 22787
3.1 95 5.0e4
3.11 50 4.0e4
5 50 3.0e4
2.0e4
Temperature: Ambient
2.57
Injection: 10 µL 1.0e4
2.81 3.05
Detection: MS/MS (SCIEX API 5000™) 0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Table 1. Absolute recovery of cortisol using the optimized Novum

Analyte RT, min Q1, Da Q3, Da CE SLE procedure (DCM elution solvent)
Cortisol 120.0 27 Analyte % Absolute Recovery %CV (N=8)
2.13 363.1
309.1 22
Cortisol 79% 6.8
Cortisol-D4 2.13 367.1 121.1 28

Cortisol was extracted from plasma using Novum SLE 96-well
plates. In order to determine the most effective extraction meth-
od in terms of cleanup and recovery, two different elution solvents
were examined. The first elution solvent, a more polar combina-
tion of MTBE/EtOAc (50:50), resulted in acceptable Cortisol re-
coveries however the resulting chromatogram (Figure 2) revealed
that matrix contaminants were also eluted from the the Novum
SLE sorbent. The second elution solvent, DCM, resulted in a
much cleaner extract (Figure 3) and an absolute recovery of 79 %
(Table 1).
Conclusion
The Novum SLE extraction with DCM shows superior cleanliness
as it reduces the late eluting peaks in the chromatogram. The
prescribed procedure provides reproducible recoveries of cortisol
from human plasma.

Underivatized Methylmalonic Acid from Plasma
Overview
Methylmalonic acid (MMA) is a small dicarboxylic acid. This hy-
drophilic molecule can present chromatographic challenges both
in achieving adequate retention under reversed phase conditions
as well as resolution from the isomeric/isobaric species such as
succinic acid, especially at low analyte concentrations. To combat Sample Pretreatment
these challenges, many published LC-MS/MS methods require a Combine 0.5 mL of 1% aqueous acetic acid and 50 µL of internal
sample derivatization step. In this technical note, we present a standard with 100 µL blank, standard, or sample.
fast, reproducible LC-MS/MS method to analyze underivatized
MMA by utilizing a unique Luna® Omega 1.6 µm PS C18. The
method runtime is 5 minutes including column re-equilibration. SPE Method
For sample preparation procedure we used Strata®-X-AW solid Cartridge: Strata-X-AW 30 mg/1 mL
Part No.: 8B-S038-TAK
phase extraction to produce a clean sample from plasma. Analyte Condition: 1 mL of methanol
detection was performed using negative mode electrospray ion- Equilibrate: 1 mL of 1 % acetic acid in water
ization of a triple quadrupole MS. Load: Pretreated sample (see above)
Wash: 0.5 mL of methanol/water (50:50)
Materials Dry: 5 to 10 minutes at max vacuum (or positive pressure)
Methylmalonic acid and methyl-D3-malonic acid standards Elute: 2 x 0.6 mL 2 % ammonium hydroxide in methanol
were purchased from Cerilliant (Round Rock, TX). Succinic Dry Down Extract: Evaporate solvent to dryness @ 45-50 °C under a gentle stream
of nitrogen
acid, formic acid, acetic acid, ammonium hydroxide, and am-
Reconstitute: 200 µL of mobile phase A (0.1 % formic acid in water)
monium acetate were purchased from Sigma-Aldrich (St. Louis,
MO). HPLC-grade acetonitrile and methanol were purchased
from Honeywell (Morris Plains, NJ). Purified water was obtained
using a Sartorius® arium ® comfort II filtration system (Göttin-
gen, Germany). Egg albumin was sourced from eggs purchased
from a local grocery store (Torrance, CA). Quality controls in LC Conditions
serum were purchased from UTAK (Valencia, CA). Pooled hu- Column: Luna Omega 1.6 µm PS C18
man K 2EDTA plasma was purchased from BioreclamationIVT Dimensions: 50 x 2.1 mm
(Westbury, NY). Part No.: 00B-4752-AN
Experimental Conditions Mobile Phase: A: 0.1% Formic acid in Water
B: 0.1% Formic acid in Acetonitrile
The calibration curve was prepared using a matrix of 2% egg al-
bumin in 50 mM ammonium acetate, pH 7, since the plasma ob- 0.01 2
tained for this project was not MMA-free. Seven points across a 2 90
linear range of 50 nmol/L to 5000 nmol/L were tested. The internal 3 90
3.01 2
standard was prepared in 50:50 water/acetonitrile with MMA-D3 5 2
at 50 µmol/L. Flow Rate: 0.4 mL/min
Injection Volume: 5 µL
Analyte recovery was tested at two concentrations using pooled Temperature: 40°C
plasma spiked with MMA at 250 nmol/L and 750 nmol/L above
the endogenous MMA level. Percent recovery for each concentra-
tion level was calculated as the mean area ratio of pre-extraction MS/MS Conditions
spiked samples divided by the mean area ratio of post-extraction Detector: SCIEX 4000 QTRAP®
spiked samples. Mode: Negative Ionization Mode
Scan Type: MRM
A separate set of plasma samples was spiked with succinic acid Curtain Gas (CUR): 10.0 psi
at 1.5 µg/mL above the endogenous level to test chromatographic Collision Gas (CAD): Medium
resolution of a higher concentration of succinic acid from MMA. IonSpray Voltage (IS): -4500 V
Temperature (TEM): 600 °C
All studies were performed with N=3 replicates.
Ion Source Gas 1 (Gas1): 50 psi
Ion Source Gas 2 (Gas2): 50 psi
Interface Heater (ihe): On

MRM Transitions
24051
ID Q1 Q3 Time DP EP CE CXP 2.5e4

2.4e4
Mass Mass (msec) (volts) (volts) (volts) (volts) 2.3e4
2.2e4
(Da) (Da) 2.1e4
2.0e4
1.9e4
MMA 1 117.0 72.9 100 -32 -10 -13 -15 1.8e4
1.7e4
Intensity, cps
1.6e4
1.5e4
MMA 2 117.0 54.9 100 -32 -10 -34 -15 1.4e4
App ID 24051
1.3e4
1.2e4
MMA-D3 120.1 75.9 100 -36 -10 -13 -15 1.1e4
1.0e4
9000.0
8000.0
7000.0
6000.0
Results and Discussion 5000.0

4000.0
3000.0
In this method, succinic acid was well-resolved chromatographi- 2000.0

1000.0
0.0
cally from MMA, including the samples spiked with succinic acid 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min
at roughly 10-fold (1.5 µg/mL) above the endogenous level of the Figure 2. Representative chromatogram of the lowest calibrator (Cal. 1), 50
MMA in pooled plasma (Figure 1). Calibration curves for extract- nmol/L MMA, extracted. Peaks in order of elution: interference (0.34 min),
ed samples covered a range from 50 nmol/L to 5000 nmol/L with succinic acid (1.00 min), and MMA (1.25 min).
r2 = 0.999 (Figure 3). Signal-to-noise for the lowest calibrator was
>25 with accuracy ≥90% (Figure 2 and Table 1). Precision and Table 1. A seven point calibration curve from 50 nmol/L to 5000 nmol/L
accuracy met our criteria, with %CV ≤15% and accuracy within was evaluated.
±15% for all replicates (Tables 1, 2). Analyte recovery was demon-
Sample Concentration Average Accuracy % Analyte
strated at two concentrations with percent recovery of 114% for Name (nmol/L) Calculated (%) CV Signal-to-
~700 nmol/L and 102% for ~1160 nmol/L, respectively (Table 3). Concentration Noise
Precision for all plasma samples, including the ~385 nmol/L un- N=3 (nmol/L)
spiked plasma sample, were within the acceptable range at ±10% Cal. 1 50 47 93.9 7.59 32
(Table 3). Cal. 2 100 101 101 13.8 61
We were able to analyze MMA and avoid a derivatization step by Cal. 3 250 262 105 7.45 124
using a mixed-mode UHPLC column that contains both a posi- Cal. 4 500 495 99.1 3.97 221
tively charged ligand for retention of acidic compounds and a C18 Cal. 5 1000 1025 103 4.17 443
ligand for added selectivity. MMA was more than adequately re- Cal. 6 2500 2467 98.7 2.41 1035
tained and well-resolved from succinic acid on a Luna Omega 1.6
Cal. 7 5000 5003 100 2.82 1907
µm PS C18, 50 x 2.1 mm column. Through the efficiency gained
by using a 1.6 µm particle, a fast 5-minute runtime was achieved,
including column re-equilibration. Table 2. Two levels of quality controls (200 nmol/L and 1000 nmol/L) were
evaluated.
A SPE procedure was developed to ensure a good degree of sam-
ple cleanliness to accommodate the small 1.6 µm particle size. By Sample Name Target Value Calculated %CV Published
(nmol/L) Concentration Range
selecting SPE as the sample preparation technique, we were able (nmol/L) (nmol/L)*
to use a small sample volume of only 100 µL of plasma. The weak
Low QC - 1 200 205
anion exchange mechanism of the sorbent selectively retained
methylmalonic acid and produced good analyte recovery. Low QC - 1 200 205 5.44 170-230
Low QC - 3 200 212

3.6e5
3.4e5 High QC - 1 1000 1260
3.2e5
3.0e5
2.8e5 High QC - 2 1000 1180 4.82 870-1170
Intensity, cps
2.6e5
2.4e5
2.2e5 High QC - 3 1000 1140
App ID 23962
2.0e5
1.8e5
1.6e5 *Manufacturer’s recommended ranges
1.4e5
1.2e5 23963
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4 0.83
0.80
0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min 0.75
0.70
0.65
Figure 1. Representative chromatogram of an extracted sample. Pooled 0.60
human plasma was spiked with standards to 1.5 µg/mL of succinic acid 0.55
0.50
and 750 nmol/L of methylmalonic acid above the endogenous concentra-
App ID 23963
0.45
0.40
tions and processed by solid phase extraction. Peaks in order of elution: 0.35
plasma interference (0.81 min), succinic acid (1.00 min), methyl-D3-malonic 0.30
0.25
acid (1.20 min), and methylmalonic acid (1.23 min). 0.20
0.15
0.10
0.05
0.00
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
Figure 3. Calibration curve plot using a linear regression with 1/x weighting
factor. r2 = 0.999

Table 3. Analyte recovery was tested at two concentrations of spiked
plasma.
Sample Name Spike Average Calculated % %

(nmol/L) Concentration CV Recovery
N=3 (nmol/L)
Prespiked
250 nmol/L 250 696 9.44 114
in plasma
Prespiked
750 nmol/L 750 1157 2.00 102
in plasma
Extracted
unspiked 0 385 3.01 N/A
plasma
Conclusion
This work here demonstrates a selective SPE procedure and LC-
MS/MS method for quantitation of MMA in plasma. The mixed-
mode stationary phase of Luna® Omega PS C18 was successfully
able to chromatographically resolve underivatized MMA from suc-
cinic acid and a plasma interference peak with better than base-
line resolution. The UHPLC method has a 5-minute runtime (in-
cluding column re-equilibration). A method utilizing Strata® X-AW
weak anion exchange SPE sorbent was developed to produce a
clean extract from plasma. The reproducible method demonstrat-
ed good analyte recovery for MMA.
References
1. Schmedes, Anne, Brandslund, Ivan, Analysis of
Methylmalonic Acid in Plasma by Liquid Chromatography–
Tandem Mass Spectrometry, Clinical Chemistry, April 2006
2. Mineva, Ekaterina M., Zhang, Mindy, Rabinowitz, Daniel J.,

Phinney, Karen W., and Pfeiffer, Christine M., An LC-MS/MS
method for serum methylmalonic acid suitable for monitoring
vitamin B12 status in population surveys, Anal Bioanal Chem,
April 2015
3. Meyer, E., Lambert, W.E., and De Leenheer, A., Succinic

Acid Is Not a Suitable Indicator of Suxamethonium Exposure
in Forensic Blood Samples, Journal of Analytical Toxicology,
Vol. 21, March/April 1997
4. Kishnir, Mark M., Komaromy-Hiller, Gabor, Shushan, Bori,

Urry, Francis M., Roberts, William L., Analysis of Dicarboxylic
Acids by Tandem Mass Spectrometry. High-Throughput
Visit www.phenomenex.com/ClinicalResources
Quantitative Measurement of Methylmalonic Acid in Serum, for more technical information and guides
Plasma, and Urine, Clinical Chemistry, Nov 2001

Underivatized Vitamin B1 and B6 from Whole Blood
Overview LC-MS/MS Method Conditions

Vitamins B1 and B6 are two water-soluble vitamins with clinical Column: Gemini® 5 µm C18
research interest. Thiamine Diphosphate (TDP) is the main biolog-
Part No.: 00B-4435-E0
ically active form of Vitamin B1 and is required for various meta-
bolic functions. Pyridoxal-5-phosphate (PLP) is the main biolog- Mobile Phase: A: 10 mM Ammonium bicarbinate in water, pH 8.8
ically active form of Vitamin B6 and is a coenzyme for a number B: Methanol
of transamination reactions. Analyzing these micronutrients is im- Gradient: Time (min) B (%)
portant for diagnosing potential deficiencies, which often occurs 0.01 0
1.5 0
in the elderly community due to malnutrition, bypass surgery, and 5 60
disease. Chromatographically, these are very challenging analytes 6.5 60
for a reversed phase separation as they have negative values for 6.51 0
the octanol/water partition coefficients. The structure of TDP and 9 0
PLP are shown in Figure 1. The most common method for TDP
Injection Volume 10 µL
and PLP analysis is a HPLC based assay with a pre-column deri-
Instrument: Agilent® 1260 LC
vatization with alkaline potassium ferricyanide and semicabazide,
Detection: MS/MS (SCIEX API 4500™), ESI+
followed by fluorescence detection. This procedure is time and Sample: 1. Pyridoxal 5-phosphate (PLP)
labor intensive and uses toxic reagent. Another approach uses ion 2. Thiamine Diphosphate (TDP)
pairing reagents, however these are not alternatives that are com- 3. Pyridoxine D2
patible with mass spectrometry. Presented here is a simple meth- 4. Thiamine-13C4
od that does not involve derivatization or ion pairing reagents.
The method involves whole blood extraction at lower pH, cost ef-
fective internal standards, no derivatization and a reversed phase Figure 1. Structure of TDP and PLP
LC-MS/MS compatible method for the analysis of polar TDP and
PLP. The assay is evaluated for precision, accuracy, linear range, NH2 H O
and the results meet acceptance criteria. + O- C
-O H2
N N C
P OH
Experimental Conditions – S O OH O
O
Optimized Sample Extraction Method N P O OH
+
Human whole blood samples were frozen immediately at -20 °C O P OH N CH3
TDP PLP H
after collection. It is important to freeze the sample for at least logP: - 5.80 O
logP: - 2.09
24 hours prior to analysis in order to prevent the analyte from
decomposition, especially TDP.
1. Pipette 100 µL of thawed hemolyzed blood into a 1.8 mL Table 1. MRM Transitions
centrifuge tube
ID Q1 Mass Q3 Mass Dwell DP CE
(Da) (Da) (msec)
2. Add 300 µL of working internal standard (20 ng/mL of Pyridox
ine-D2 and 50 ng/mL of Thiamine-13C4 in DI water) and mix for PLP 1 248.1 149.8 125 75 15
30 seconds PLP 2 248.1 94.1 125 75 25
3. Add 30 µL of 70 % HClO4 and mix for 1 minute to precipitate TDP 1 425.1 122.1 25 60 15
proteins TDP 2 425.1 81.0 25 60 52
TDP 3 425.1 303.9 25 70 20
4. Centrifuge sample at 14,000 rpms for 10 minutes to pellet the
protein Thiamine-13C4 1 270.1 122.9 75 40 15
Thiamine-13C4 2 270.1 148.1 75 40 15
5. Transfer 200 µL of supernatant into an autosampler vial for LC
MS/MS analysis Pyridoxine D2 1 172.1 155.0 75 40 15
Pyridoxine D2 2 172.1 136.0 75 40 15
Note: Since the analytes are light sensitive, the extraction steps
were performed in amber color centrifuge tube and were pro-
tected from light.

Table 2. Accuracy and Precision
Run 1 Run 2
TDP PLP TDP PLP
Nominal Conc. 100 ng/mL
Sample ID Conc. Found (ng/mL) Conc. Found (ng/mL)
QC1_1 103 88.5 118 95.8
QC1_2 80.5 103 89.1 74.5
QC1_3 94.8 98.5 105 88.3
QC1_4 87.2 110 102 106
QC1_5 85.5 102 86.7 88
QC1_6 93.6) 73.6 110 85.8
Nominal Conc. 200 ng/mL
QC2_1 222 170 215 176
QC2_2 226 201 217 190
QC2_3 218 196 259 260
QC2_4 211 215 192 209
QC2_5 216 202 228 205
QC2_6 255 228 227 160
Mean Conc. Found (ng/mL) 96.3 92.8 224 201
STDV 11.3 11.7 18.2 26.8
CV% 11.8 12.6 8.13 13.3
Accuracy (%) 96.3 92.8 112 101
Figure 2. Representative Chromatogram in Whole Blood at LLOQ (20 ng/mL)
4500
TDP 3.0e5 Thiamine 13C4 (IS)
4000 2.8e5
3.4e5
3.2e5
2.6e5
3500 3.0e5
2.4e5
2.8e5
2.2e5
2.6e5
3000
2.4e5
2.0e5
2.2e5
2500 1.8e5
Intensity, cps
2.0e5
cps
Intensity, cps
1.6e5
1.8e5
Intensity,
2000
1.4e5
1.6e5
1.4e5
1.2e5
1500 1.2e5
1.0e5
1.0e5
1000 8.0e4
8.0e4
6.0e4
6.0e4
500 4.0e4
4.0e4
2.0e4
2.0e4
0.0
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 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
0.0
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
2000 PLP
1.5e4 2.0e5
1800
1.4e4
1.3e4
1.8e5
2.2e5 Pyridoxine D2 (IS)
1600
1.2e4 1.6e5
2.0e5
1.1e4
1400 1.4e5
1.8e5
1.0e4
9000.0
1200 1.6e5
1.2e5
cpscps
cps
8000.0 1.4e5
Intensity,
Intensity,
1.0e5
1000
7000.0
Intensity,
cps
1.2e5
6000.0 8.0e4
Intensity,
800 1.0e5
5000.0
6.0e4
4000.0 8.0e4
600
App ID 23549
3000.0 4.0e4
6.0e4
2000.0
400
2.0e4
4.0e4
1000.0
0.0
200 2.0e4
0.0
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 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
0.0
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
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

Figure 3. Representative Chromatogram in Whole Blood at ULOQ (250 ng/mL)
1.3e4
4500
1.2e4
TDP Thiamine13C4 (IS)
4000 3.4e5
1.1e4
3.2e5
1.0e4
3500 3.0e5
2.8e5
9000.0
3000 2.6e5
8000.0 2.4e5
2.2e5
2500
7000.0
cps
Intensity, cps
2.0e5
Intensity, cps
Intensity,
6000.0 1.8e5
2000
1.6e5
5000.0
1.4e5
1500 1.2e5
4000.0
1.0e5
3000.0
1000 8.0e4
6.0e4
2000.0
500 4.0e4
1000.0 2.0e4
0.0
0
0.0
0.0
0.0 0.5
0.5 1.0
1.0 1.5
1.5 2.0
2.0 2.52.53.03.03.53.54.04.04.5 4.55.0 5.0
5.5 5.5
minmin 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
Pyridoxine D2 (IS)
PLP
1.5e4 2.0e5
1.4e4
1.8e5
1.3e4
1.2e4 1.6e5
1.1e4
1.4e5
1.0e4
9000.0 1.2e5
Intensity, cps
Intensity, cps
8000.0
1.0e5
7000.0
6000.0 8.0e4
5000.0
App ID 23550
6.0e4
4000.0
3000.0 4.0e4
2000.0
2.0e4
1000.0
0.0
0.0
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 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
Figure 4. Interference Peak Analysis during TDP Detection Figure 5. Plot Demonstrating Suitability of Internal Standard
5760 9.5e5
9.0e5
5500 8.5e5
8.0e5
7.5e5
7.0e5
5000 6.5e5
6.0e5
IS Peak Area, counts
5.5e5
5.0e5
4500 4.5e5
4.0e5 Pyridoxine D2 HCL
3.5e5
4000 3.0e5
2.5e5
2.0e5 25 % of mean response
1.5e5
3500 1.0e5
5.0e4
0.0
Intensity, cps
5 10 15 20 25 30 35 40 45 50 55 60
3000
Interference 425.1 > 303.9 mz 1.5e6
Index
1.4e6
2500 1.3e6
1.2e6
1.1e6
2000
1.0e6
9.0e5
8.0e5
App ID 23556
1500 7.0e5
6.0e5
5.0e5
Thiamine13C4 HCL
1000 4.0e5
3.0e5
2.0e5
500 1.0e5
0.0
5 10 15 20 25 30 35 40 45 50 55 60
0 Index
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

Figure 6. Representative Calibration Curve of TDP in Whole Blood Figure 7. Representative Calibration Curve of PLP in Whole Blood
0.064
0.044
0.060 0.042
0.040
0.055 0.038
0.036
0.050
0.034
0.045 0.032
0.030
0.040 0.028

0.026
0.035
0.024
0.030 0.022
0.020
0.025 0.018
0.016
0.020
0.014
0.015 Dynamic range: 20-250 ng/mL 0.012 Dynamic range: 20-250 ng/mL
0.010
0.010 r = 0.9939 8.000e-3
r = 0.9953
6.000e-3
5.000e-3
4.000e-3
0.000 2.000e-3
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 0.000
Analyte Conc. / IS Conc. 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250
Figure 8. Matrix Effects on PLP Analysis: Water vs. Whole Blood
3.6e4
3.4e4
3.2e4
Water Step
Total Time Flow Rate
A (%) B (%)
3.0e4 (min) (mL/min)
2.8e4 0 0.01 0.5 100 0
2.6e4 1 1.5 0.5 100 0
2.4e4 2 1.8 0.5 40 60
Intensity, cps
2.2e4 3 5 0.5 40 60
2.0e4 4 5.5 0.5 100 0
1.8e4 Whole Blood 5 8 0.5 100 0
1.6e4
1.4e4
1.2e4
1.0e4
8000.0
App ID 23557
6000.0
4000.0
2000.0
0.0
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
Figure 9. Flow Rate Alteration to Separate Interference Peaks During PLP Detection
3.8e4
3.6e4
3.4e4
3.2e4
3.0e4
2.8e4
2.6e4 Interference 248.1 > 149.8 mz
2.4e4
Intensity, cps
2.2e4
2.0e4 Flow Rate: 0.6 mL/min
1.8e4
1.6e4
1.4e4
1.2e4
1.0e4
8000.0
6000.0
4000.0
2000.0
0.0
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
1.4e4
1.3e4
1.2e4
1.1e4
1.0e4
9000.0
Interference 248.1 > 149.8 mz
Intensity, cps
8000.0
7000.0
App ID 23555
6000.0
5000.0
4000.0
3000.0
2000.0
1000.0
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min 5.0 5.5 min

Figure 10. Matrix Effect on PLP Detection
23551
1.5e6
1.4e6
1.3e6
1.2e6 Blood (blank)

1.1e6
1.0e6
9.0e5
Intensity, cps
8.0e5
7.0e5 Solvent (blank) PLP (200 ng/mL)

6.0e5
5.0e5
4.0e5
3.0e5
App ID 23551
2.0e5
1.0e5
0.0
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
Figure
23552 11. Matrix Effect on TDP Detection
7.3e4
7.0e4
6.5e4
Blood (blank)
6.0e4
5.5e4
5.0e4
4.5e4
Intensity, cps
TDP(200 ng/mL)
4.0e4
Solvent (blank)
3.5e4
3.0e4
2.5e4
2.0e4
1.5e4
App ID 23552
1.0e4
5000.00
0.0
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

Figure 12. Endogenous Levels of PLP in Six Individual Lots of Blank Whole Blood
520
500
450
Average Endogenous Level: ~400 cps (Height)

400
350
300
Intensity, cps
250
200
150
100
App ID 23553
50
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.0 5.0 5.5 min
Figure 13. Endogenous Levels of TDP in Six Individual Lots of Blank Whole Blood
23554
1.19e4
1.10e4
1.00e4
9000.00
Average Endogenous Level: ~ 6000 cps (Height)
8000.00
Intensity, cps
7000.00
6000.00
5000.00
4000.00
3000.00
2000.00
App ID 23554
1000.00
0.00
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

Presented here is a simplified extraction procedure for whole using the whole blood calibration curve as it is the best practice
blood samples followed by a LC-MS/MS compatible reversed for TDP and PLP analysis.
phase method for both TDP and PLP. Both analytes are highly
polar (refer to structures in Figure 1.) and retaining both com- Challenging matrices, such as whole blood, usually require a
pounds under reversed phase conditions is challenging. Gemini® rigorous sample cleanup due to the presence of matrix interfer-
5 µm C18 is a reversed phase HPLC column with an extreme pH ences. Especially due to the fact that whole blood as a matrix
resistance that offers flexibility to explore pH beyond conventional offers variability. In order to account for this variability and to
silica columns capabilities. For the present work, Gemini 5 µm demonstrate method flexibility, experiments were performed with
C18, 50 x 4.6 mm was employed under high pH mobile phase change in mobile phase flow rate. The respective chromatograms
conditions. The gradient started with 100 % aqueous mobile are shown in Figure 9, which shows the separation of interfer-
phase to promote retention of TDP and PLP and representative ence peaks with changes in the mobile phase flow rate. Further
chromatogram at Lower Limit Of Quantitation (LLOQ ) and Upper to demonstrate the success of the sample cleanup, matrix ef-
Limit Of Quantitation (ULOQ) are presented in Figures 2 and 3. fects were monitored during the method development process
These chromatograms provide evidence of successful retention by performing post column infusion tests as shown in Figures
of both the analytes on Gemini C18 column despite the use of 10 and 11. Certainly, endogenous levels of TDP and PLP in the
any ion pairing reagent. In addition to improved reversed phase blank whole blood can affect the LLOQ of the assay and for the
retention, interference peaks were also resolved from the analyte same reason, it is important to evaluate the level of endogenous
peaks as shown in Figure 4. interference. Six individual lots of commercially purchased human
whole blood blanks were evaluated to determine the endogenous
The extraction was performed under acidic conditions prior to levels of TDP and PLP. The average endogenous level of six lots
LC-MS/MS which not only helped with the stability of TDP and was ~6000 cps (peak height) for TDP and ~400 cps (peak height)
PLP, but also allowed the extraction to take place at room tem- for PLP when quantified by MS/MS (SCIEX API 4500™) as shown
perature. Two sets of evaluation batches were run individually on in Figures 12 and 13.
2 different days, on each day 2 concentration of QCs 100 ng/mL
and 200 ng/ml were run 6 times to ensure precision and accuracy. Overall, the method development involved changing the extraction
Accuracy and precision data are presented in Table 2. The as- pH, selecting the correct reversed phase column, and optimiza-
say is accurate with a recovery of 92.8-112.0 % and precise with tion of gradient profile. In addition, selection of cost effective
% CV of 8.13-12.6 % for human whole blood matrix. The method internal standards produced a time and cost efficient alternative
utilized cost effective alternative for internal standard which are for the separation of TDP and PLP. Aside from being efficient, the
commercially available. The response of the isotope internal stan- method proved to be reproducible and gave consistent results for
dards is proven consistent within ±25 % of the mean response of more than 500 injections on the LC-MS/MS.
the standards and QCs for all samples as shown in Figure 5.
Conclusion
The dynamic ranges of both TDP and PLP in whole blood are A simple and rapid assay method is presented for the quantitation
presented in Figures 6 and 7 and the developed method proved of Vitamin B1 and B6 in human whole blood by LC-MS/MS. The
to be linear from 20 to 250 ng/mL. Water as a matrix was also method is accurate with recoveries from 92.8-112.0 % and with
analyzed and was compared with chromatograms of whole blood %CV of 8.13-12.6 % it proves to be precise and the method is
extract as shown in Figure 8. It is clearly evident that, DI water linear from 20-250 ng/mL of the analytes. Matrix effects and res-
matrix will have clean chromatography but when calibration curve olution of interferences from analyte peaks were also monitored
is performed on DI water to quantitate TDP and PLP from whole for TDP and PLP, resulting in an acceptable detection of TDP and
blood, there will be matrix interference that will not be accounted PLP. This assay is simple, time saving, cost effective and automa-
as shown in Figure 8. For the same reason, we highly recommend tion friendly.

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Vitamin A and E from Serum

We have developed a simple and reliable method to extract vita- Column: Kinetex 5 µm EVO C18
min A and E from human serum, using Novum™ SLE in conjunction
Part No.: 00D-4633-AN
with Kinetex® 5 µm EVO C18, 100 x 2.1 mm HPLC column. A fast
and effective LC-MS/MS method was developed to obtain the best
Mobile Phase: A: Water
selectivity of the vitamin A and E along with its two vitamers alpha
B: Isopropanol/acetonitrile (1:1)
and gamma Tocopherol. A cleaner background was observed for
both alpha and gamma under negative ionization mode. By tak-
0 65
ing advantage of the polarity switching technique (from positive to 3 95
negative ionization) after vitamin A elutes from the column, a much 4 95
cleaner
H C background,
3 CH 3
CH resolved
CH 3 in a higher signal for both vitamers of
3 4.1 65
vitamin E under APCI mode. 5.5 65
OH Flow Rate: 0.6 mL/min
CH3
Structures andCHLogP Values
CH
of Vitamin A and E Instrument: Agilent® 1260
H 3C CH3 3 3
H 3C CH3 CH3 CH3 Detection: MS/MS (SCIEX API 5000™), APCI

OH
OH
1. CH3
CH3
HO The organo silica chemistry of Kinetex EVO C18 enhanced the
interaction between the stationary phase and liposoluble vitamins,
2. O resulting in greater selectivity for the two vitamers a and g
tocopherol (Fig. 1).
HO
HO
The recovery (analyte recovery more than 92 % with CV
3. between 2-9 %) of the three vitamins were best optimized under
O
O a neutral load condition (Fig. 2) followed by elution with a mixture
HOA (retinol) 2. g-Tocopherol (vitamin E) 3. a-Tocopherol (vitamin E)
1. Vitamin of ethyl acetate and acetone in the ratio of 90 to 10 (Fig. 3).
O
Extraction of the human serum samples in the method
development, required use of a larger bed mass of SLE Novum
media. The extent of cleanliness was much improved switching
HO from Novum MINI to MAX (Fig. 4)1.
HO
O The extraction efficiency of Novum was improved by increasing

O the surface area (larger bed mass of Novum MAX). The
partitioning of target analytes from aqueous into the organic
extraction solvent is highly dependant on mass transfer rate.
More the surface area, better the aqueous can interact with the
extraction solvent and higher the analyte transfer rate.

Larger elution volume applicability of Novum ™ MAX media,
ensured effective partitioning of target analytes from aqueous into
the organic.
The linearity curve generated for vitamin A extraction from
human serum, extended 1,000 fold dynamic range, covering the
expected range (Fig 5). The linear regression value of 0.999 or
greater reflects the robustness of the method.
Presence of endogenous level vitamin E (a and g tocopherol)
observed in serum samples obtained from three different suppliers
(Fig. 6). A food grade, albumin enriched, egg white was utilized
to generate the linearity curve, that did not show any endogenous
level (Fig. 6). The extracted curve for a and g tocopherol shows
good linearity and correlation over 1,000 fold conc. range
(Fig. 7 & 8).
Figure 1. Figure 3.
LC-MS/MS Analysis of Vitamin A and E Using Dual Polarity Technique in MS Optimize (Novum MINI) Elution Condition Under Neutral Load)
23286 23393
3.6e6
3 3.4e6
3. 9e6
App ID 23393
3.2e6
3. 8e6 3.0e6
APCI+ APCI- 2.8e6
Intensity, cps
3. 6e6 2.6e6
3. 4e6
2.4e6
2.2e6
2.0e6
Hexane/IPA (90:10)
3. 2e6 1.8e6
1.6e6
3. 0e6 1.4e6
1.2e6
1.0e6
2. 8e6 2 8.0e5
6.0e5
2. 6e6 4.0e5
2.0e5
2. 4e6 0.0
Intensity, cps
2. 2e6 1 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
98 194 291 388 485 581 657 735 813 891 969 min
2. 0e6
1. 8e6
1. 6e6
App ID 23286
23395
1. 4e6 3.6e6
3.4e6
1. 2e6
App ID 23395
3.2e6
3.0e6
1. 0e6 2.8e6
2.6e6
8. 0e5
Ethyl acetate/acetone (90:10)
In te ns ity , cp s
2.4e6
2.2e6
6. 0e5 2.0e6
1.8e6
4. 0e5 1.6e6
1.4e6
2. 0e5 1.2e6
1.0e6
0.0 8.0e5
6.0e5
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min 4.0e5
2.0e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
98 194 291 388 485 581 657 735 813 891 969 min
Figure 2. 23396
Optimize Load Condition (Novum MINI, P/N:8E-S138-FGA) 6.0e6
23390 5.5e6
5.0e6
1.14e5
1.10e5
4.5e6
1.00e5 4.0e6
In te n s ity , c p s
Acidic
App ID 23396
9.00e4 3.5e6
8.00e4 3.0e6 Methylene chloride/IPA (95:5) 3
App ID 23390
7.00e4 2.5e6
6.00e4 2.0e6 2
5.00e4 1.5e6
4.00e4 1.0e6
1
3.00e4 5.0e5
2.00e4 0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
98 194 291 388 485 581 657 735 813 891 969 min
1.00e4
0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
98 194 291 388 485 581 657 735 813 891 969 min
3.2e5
3.0e5
2.8e5
Figure 4.
2.6e5
Basic
2.4e5
2.2e5
Recovery and Extent of Cleanliness Using the Optimized Large Load and Large
2.0e5
Elution on Novum MAX
App ID 23391
1.8e5
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
Improved recovery and cleanliness (92-110 %; CV=2-9 %) from Novum MAX
4.0e4
2.0e4
0.0
.
120 10
23394 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
8.5e5
98 194 291 388 485 581 657 735 813 891 969 min
9
8.0e5
7.5e5
100 8
App ID 23401
7.0e5
Neutral
6.5e5
6.0e5
80 7
5.5e5
6
App ID 23394
5.0e5
4.5e5
4.0e5
3.5e5 60 5
3.0e5
2.5e5 4
2.0e5
40 % Recovery
1.5e5
1.0e5 3
5.0e4 % CV
0.0 2
0.5
98
1.0
194
1.5
291
2.0
388
2.5
485
3.0
581
3.5
657
4.0
735
4.5
813
5.0
891
5.5
969 min
20
1
0 0
Vitamin A α-Tocopherol (Vit E) γ-Tocophero l(Vit E)

Figure 5. Figure 7.
Linearity Curve of Vitamin A Extracted Samples on Novum™ MAX Linearity Curve of a-Tocopherol Extracted Samples on Novum™ MAX
(Matrix: doubly-charcoal stripped serum) (Matrix: Egg White Albumin)
1.6e7 460
440
1.5e7 420
1.4e7 400
1.3e7
380
360 a-Tocopherol (Vitamin E)
1.2e7 340
Vitamin A 320

1.1e7 300
1.0e7 280
Area, counts
260
9.0e6
App ID 23403
240
8.0e6 220
App ID 23402
200
7.0e6 180
160
6.0e6 140
5.0e6 120
100
4.0e6 80
3.0e6 60
40
2.0e6 20
1.0e6 0
0.0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Concentration, µg/mL
Figure 8.
Figure 6. Linearity Curve of g-Tocopherol Extracted Samples on Novum MAX
Presence of Endogenous Level of Vitamin E (a and g) from Serum (Three (Matrix: Egg White Albumin)
Different Sources)
279
260
1. Vitamin A (retinol); 2. g-Tocopherol (vitamin E); 3. a-Tocopherol 240
(vitamin E) 220 g-Tocopherol (Vitamin E)

23397 200

180
3
160
App ID 23404
6.0e6
140
5.5e6
120
5.0e6
100
4.5e6
80
4.0e6
60
App ID 23397
3.5e6
40
3.0e6
2 20
2.5e6 0
1
2.0e6 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
1.5e6 Analyte Conc. / IS Conc.
1.0e6
5.0e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
98 194 291 388 485 581 657 735 813 891 969 min
Unadulterated Serum (Interference: Vit. A, a and g)

References
23398
3
1. S Huq, S Sadjadi, and S Orlowicz, “A Fast and Effective Quantitation
5.4e6 Method for Vitamin A and E from Human Serum Using Novum™ SLE in
5.0e6
4.5e6
Conjunction with a Kinetex® EVO C18 Column.” Mass Spec Application
4.0e6 for Clinical Laboratory Conference, US, 2016
3.5e6
App ID 23398
3.0e6
2.5e6
2
2.0e6
1.5e6
1.0e6
1
5.0e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
98 194 291 388 485 581 657 735 813 891 969 min
Charcoal-Stripped Serum (Interference: Vit. A, a and g)
23399
3
3.2e6
3.0e6
2.8e6
2.6e6
2.4e6
2.2e6
2.0e6
App ID 23399
1.8e6
1.6e6
1.4e6
2
1.2e6
1.0e6
8.0e5
6.0e5
4.0e5
2.0e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
98 194 291 388 485 581 657 735 813 891 969 min
Doubly Charcoal-Stripped Serum (Interference: a and g)
23400
1.10e6
1.05e6
1.00e6
9.50e5
App ID 23400
9.00e5
8.50e5
8.00e5
7.50e5
7.00e5
6.50e5
Intensity, cps
6.00e5
5.50e5
5.00e5
4.50e5
4.00e5
3.50e5
3.00e5
2.50e5
2.00e5
1.50e5
1.00e5
5.00e4
0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
98 194 291 388 485 581 657 735 813 891 969 min
Food grade Egg White Albumin (Interference: None)

Homocysteine
Conditions
Homocysteine is a sulphur containing amino acid (C4H9NO2S)
found in blood plasma and serum.1 This application illustrates
H
a rapid retention and resolution of Homocysteine using a Luna®
Omega 1.6 µm PS C18 column. Luna Omega PS C18 is a unique +
H3N C COO -
mixed-mode stationary phase that provides incredibly useful
polar and non-polar retention. The surface of the PS C18 contains
a positive charge which aids in the retention of acidic compounds CH2 CH2 SH
through ionic interactions, while the C18 ligand promotes general
reversed phase retention. This mixed-mode selectivity allows for Homocysteine
greater separation between compounds with varying functional
groups. With its positive surface (PS) the Luna Omega PS C18
provides valuable increase in retention of acids like Homocysteine
through ionic/polar interactions.
Figure 1.
Representative Chromatogram of Homocysteine
1.8e6 1
1.7e6
1.6e6
1.5e6
LC-MS/MS Methodology
Column: Luna Omega 1.6 μm PS C18
1.4e6
1.3e6
1.2e6
1.1e6
Mobile Phase: A: 0.1 % Formic Acid in Water
Intensity, cps
1.0e6 B: 0.1 % Formic Acid in Methanol

9.0e5 Gradient: Time (min) %B
8.0e5 0 2
7.0e5 2 30
6.0e5 2.01 90
App ID 23844
5.0e5 2.5 90
4.0e5 2.51 2
3.0e5 3 2
2.0e5 Flow Rate: 0.4 mL/min
1.0e5 Temperature: 22 °C
0.0 Detection: MS/MS (SCIEX Triple Quad™ 4500)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 min Sample: 1. Homocysteine

Thiamine Monophosphate and Thiamine from Plasma and Breast Milk
Column: Gemini® NX-C18 3 μm

Overview Dimensions: 100 x 3.0 mm
Thiamine monophosphate (TMP) and thiamine were extracted Part No.:00D-4453-Y0
from human plasma and human breast milk by performing a Mobile Phase: A: 25 mM Disodium Phosphate, 10 % Methanol (pH 7.0)
B: 25 mM Disodium Phosphate, 70 % Methanol (pH 7.0)
rapid protein precipitation using Impact Protein Precipitation
Plates followed by HPLC analysis using a Gemini® 3 μm NX-C18 0 97
100 x 3.0 mm HPLC column with fluorescence detection. Impact 0.25 75
technology offers easy, fast protein removal while providing max- 0.75 75
3 65
imized recovery of the target analytes. The Gemini 3 μm NX-C18 4 0
HPLC column produced excellent chromatographic resolution, 5 0
sensitivity, and high peak capacities. 5.1 97
8 97
Materials and Methods Flow Rate: 0.75 mL/min
Injection: 20 µL
Protein Precipitation Temperature: 25 °C
Detection: Fluorescence (Excitation: 375 nm, Emission: 435 nm)
1. Place the Impact™ plate onto a suitable 96-well sample
manifold
2. Dispense 100 µL of human plasma or breast milk into Figure 1.
Protein Precipitation Using Impact Protein Precipitation Plates
each well of the Impact plate
3. Add 300 µL of methanol to each well of the Impact plate
4. Mix 3 times by aspirating with a pipette tip
5. Apply vacuum to filter the sample and collect the purified
filtrate in a collection plate
Transfer 50 µL to an autosampler vial (or allow it to remain in the

collection plate). Add 50 µL of water and 50 µL of derivatizing
reagent (15 % sodium hydroxide solution plus 200 µL of 30 mM
K3Fe(CN)6). Cover the vial or collection plate with a lid or sealing
mat, respectively. Vortex for 15 seconds. Put the vial or collection Proteins
plate into an autosampler. The sample is now ready to be injected Target Analyte
onto the HPLC-FLD.
LC Conditions
An Agilent® 1100 HPLC system (Agilent Technologies, Inc., Santa
Clara, CA, USA) was used with a Shimadzu® RF-20A Promi- Figure 2.
nence® Fluorescence Detector (Shimadzu, Japan) for LC/FLD 200
21874 nmol/L of TMP and thiamine in water filtered by an Impact Protein
Precipitation Plate
analysis.
TMP
mAU
68
66
Thiamine
64
62
60
58
56
App ID 21874
54
52
0 2 4 6 8 min

Figure 3a. Figure 5.
2.5 nmol/L of TMP and thiamine in water filtered by an Impact™ Protein Lower level TMP (37.2 nmol/L) and thiamine (66.4 nmol/L) in human breast
Precipitation
21875 Plate milk
21879
mAU
mAU
54.0 58
58
56
54
TMP
53.0
52
Thiamine
50
Thiamine
TMP
App ID 21875
48
App ID 21879
52.0
46
0 2 4 6 8 min
0 2 4 6 8 min
Figure 3b. Figure 6.

Comparison of TMP and thiamine at 25 nmol/L vs. blank control (overlay Standard curve of TMP filtered by an Impact Protein Precipitation Plate at
and expended chromatograms for human plasma filtered by an Impact a concentration
21876
range of 0 to 200 nmol/L
Protein Precipitation Plate) 80
21880
mAU
R2 = 0.9997 •
70
54
60
53.5 50
Peak area (mAU)
TMP
53 40
Thiamine
App ID 21880
30
52.5
Blue = blank control

Red = standards @ 25 nmol/L
20
•
52
App ID 21876
10
1.5 2 2.5 3 3.5 4 4.5 5 min
•
0 •••
50 100 150 200 250
Figure 4.
-10
Higher level TMP (93.6 nmol/L) and thiamine (240.8 nmol/L) in human TMP Concentraton (nmoL/L)
breast
21878
milk
Table 1.
Thiamine
Standard curve (6-point) of TMP
mAU
TMP Standard Curve (6-point)
58
Concentration (nmol/L) Peak Area (mAU)

56
0 0
TMP 2.5 0.8
54 5 1.5
10 3.1
52 50 19.4
200 73.6
50
Table 2.
Recovery of TMP and thiamine from human plasma after cleanup with an
48
Impact Protein Precipitation Plate
App ID 21878
Recovery for Human Plasma TMP Recovery for Human Plasma Thiamine
46
Added TMP Observed Recovery Added Thiamine Observed Recovery
(nmol/L (nmol/L) (%) (nmol/L) (nmol/L) (%)
0 2 4 6 8 min
0 3.0 0 5.0
5 7.0 87.5 5 10.8 108.0
10 12.8 98.5 10 18.4 122.7
Mean 93.0 Mean 115.4

Table 3. This ensured that the precipitated protein was left within the wells
Accuracy studies for TMP and thiamine from human plasma after cleanup of the Impact plate while protein free sample was allowed to pass
with an Impact™ Protein Precipitation Plate through the filter and into a collection plate (Figure 1).
TMP Spiked Plasma Controls Thiamine Spiked Plasma Controls
After the protein precipitation step, the plasma and breast milk
Control No. Expected Results Accuracy Expected Results Accuracy samples were derivatized and analyzed by HPLC-FLD using a
(nmol/L) (nmol/L) (%) (nmol/L) (nmol/L) (%) Gemini® 3 μm NX-C18 HPLC column. The Gemini 3 μm NX-C18
HPLC column contains a unique silica-organic layer that is graft-
Control 1 40 38.7 96.8 40 34.2 85.5
ed onto the base silica which mechanically strengthens the parti-
Control 2 20 20.0 99.8 20 19.9 99.4 cle while providing excellent efficiencies. Efficiency and resolution
were necessary in this analysis because the separation of TMP and
Control 3 10 10.7 107.0 10 8.2 81.8 thiamine (Figures 2, 3a, and 3b) was crucial in order to accurately
quantify each compound in plasma (Figure 7) and breast milk (Fig-
Mean 101.2 Mean 88.9 ures 4 and 5).
Figure 7. The reproducibility of our analysis was determined by producing

Overlay of endogenous and spiked TMP and thiamine in plasma filtered by a standard curve of TMP at a concentration range of 0 to 200
Impact
21877 nmol/L, resulting in a correlation coefficient of R2 = 0.9997
mAU (Figure 6). Thiamine was also subjected to a linearity curve at a
concentration range of 0 to 200 nmol/L, resulting in a correlation
47
coefficient of R2 = 0.9993 (not shown). Even at low levels of de-
48.75
tection, our method proved to be reproducible for both TMP and
48.5 thiamine.
48.25 TMP
Thiamine
Compared with a water blank, the lower level TMP and thiamine
46
standards (2.5 nmol/L) can be clearly distinguished (Figure 3b),
App ID 21877
45.75
showing that our extraction and HPLC method are extremely sen-
45.5
sitive. Endogenous levels of TMP and thiamine were also studied
45.25 in plasma (Figure 7), which showed that 3 nmol/L and 5 nmol/L
were present, respectively.
2 2.5 3 3.5 4 4.5 5 min
Blue = endogenous TMP (3 nmol/L) and thiamine (5 nmol/L)

Resulting recoveries of both target compounds averaged 93 %
Red = spiked TMP (5 nmol/L) and thiamine (5 nmol/L) for human plasma TMP and 115 % for thiamine (Table 2). Accu-
Green = spiked TMP (10 nmol/L) and thiamine (10 nmol/L) racy studies resulted in an average accuracy of 101.2 for TMP
and 88.9 for thiamine (Table 3), suggesting that our method not
Table 4. only provided acceptable recoveries but was also accurate and
Retention time reproducibility studies
reproducible.
Retention Time (RT)
Retention times (RT) were stable by comparing several retention
Inj. No. TMP (min) Thiamine (min) times between injection number 100 and 490, resulting in % CV’s
100 2.487 4.032 of 1.58 % for TMP and 1.18 % for thiamine (Table 4).
150 2.534 4.133
Conclusion
180 2.528 4.143 Protein precipitation has always been a popular sample prepa-
240 2.491 4.122 ration method however the process can be improved upon. Us-
ing Impact Protein Precipitation Plates, TMP and thiamine were
400 2.411 4.045 cleaned up from plasma and breast milk providing benefits such
420 2.452 4.061 as minimal method development and processing time as well as
490 2.44 4.04
ease of use. The resulting cleanup method can also be automat-
ed, allowing laboratories to save time by increasing productivity
Mean 2.4818 4.0822 while improving reproducibility and reducing the risk of human
STEDV 0.0393 0.0483 error. Preparation by automated protein precipitation is also a
versatile sample preparation technique as the resulting extract
%CV 1.58 1.18
can be analyzed by several different methods including HPLC-UV,
Results and Discussion LC-MS/MS, and HPLC-FLD. Using HPLC-FLD, our separation
When developing a method for the analysis of TMP and thia- method on the Gemini 3 μm NX-C18 HPLC column provided ex-
mine, it was important that the method be rapid, sensitive, and cellent resolution of TMP and thiamine and was sensitive enough
accurate. Traditionally, a protein precipitation step is used for fast to detect down to low levels.
cleanup of plasma or breast milk samples. Protein precipitation is References
normally performed using a centrifuge tube or a 96-well collection
1. Wolfgan Stuetz, Verena Ilona Carrara, Rose McGready, Sue Jean Lee, Hans
plate; however this process requires that supernatant be collect- Konrad Biesalski and Francois Henry Noste. PLOS ONE, 2012.
ed while being careful not to disrupt pelleted protein in the bottom
of the tube or collection plate. This step was greatly simplified by
using Impact Protein Precipitation Plates. The Impact plate allows
for the analysis of 96 samples at once, eliminates the transfer
steps that are commonly associated with protein precipitation,
and can also be automated. Protein precipitation was performed
within the wells of the Impact plate and sample was not allowed
to pass through the filter of the plate until vacuum was applied.

Therapeutic
TN-0083
Drug
Monitoring

Digoxin and Digitoxin from Plasma
A LC-MS/MS method has been developed for the rapid analysis MS/MS procedure outlined below.
of digoxin and digitoxin in plasma effective over a concentration
range of 0.25 to 10 ng/mL, which covers the commonly accepted Two high (7.5 ng/mL) and two low (0.75 ng/mL) QC sample solu-
therapeutic levels for samples. The method described here uses tions were prepared in plasma. Each QC sample was then pre-
Strata®-X for sample clean up and concentration via solid phase pared for LC-MS/MS analysis using the same sample preparation
extraction and a Kinetex® C8 core-shell column for fast and sensitive and SPE procedure used for the calibration curve. Each of the two
LC-MS/MS analysis. replicates was then analyzed in duplicate resulting in four data
points for each QC sample concentration.
Equipment and Materials

Overview Agilent® 1200 Series HPLC (Agilent Technologies Inc., Santa
Digoxin and digitoxin are cardiac glycosides that were originally
Clara, CA, USA) was interfaced with API 4000™ MS/MS with ESI
extracted from the foxglove plant, Digitalis lanata. These two gly-
TurboIonSpray® (SCIEX, Foster City, CA, USA) operated in posi-
cosides have profound effects on heart activity and are thought
tive ionization mode (ESI+).
to function by altering the mechanisms that regulate the cardiac
action potential. Functionally, the net effect of digoxin or digitoxin Sample Preparation
administration is a decrease in heart rate. Digitoxin is eliminated The plasma samples were prepared as follows:
from the body via the liver, whereas digoxin is eliminated via renal
activity. 1. 1.0 mL of plasma sample was spiked with 10 µL of 40 ng/mL
oleandrin internal standard and transferred to a small test tube.
We describe here a simple and accurate method for the analy-
sis of digoxin and digitoxin in plasma which utilizes solid phase 2. The sample was diluted with 2 mL of D.I. water; sample ready
extraction using Strata-X SPE tubes followed by rapid LC-MS/ for SPE.
MS analysis using a Kinetex core-shell C8 column. In comparison
to methods that use liquid:liquid extraction, the proposed SPE
method has the additional benefit of reduced waste and the ability Solid Phase Extraction
to analyze many samples concurrently. The prepared plasma sample is cleaned up and concentrated
using SPE.
Materials and Methods Cartridge: Strata-X 30 mg/3 mL
Reagents and Chemicals Part No.: 8B-S100-TBJ
Condition: 2 mL Methanol (1-2 mL/min)
All reagents and solvents were HPLC or analytical grade. HPLC
Equilibrate: 2 mL of 10 mM Ammonium acetate in water
Grade acetonitrile was purchased from Honeywell, Burdick & Note: Do not let sorbent run dry
Jackson (Muskegon, MI); Milli-Q water was used to prepare the Load: 3 mL of previously prepared plasma sample (1-2 drops/
buffer solutions used for SPE. Digoxin, digitoxin and oleandrin sec)
(internal standard) were purchased from Sigma-Aldrich (Part Wash: 1 mL of Methanol/10 mM ammonium acetate (50:50)
Numbers D6770, 851736 and 09640, respectively). in water
Dry: >10” Hg for 5-10 minutes to remove residual water
The 10 mM ammonium acetate solutions were prepared by weigh- Elute: 2 mL methanol (ca. 1 drop/sec)
ing out 0.7708 g of ammonium acetate and dissolving it in 1.0 L of Drydown: Nitrogen gas at 50 ºC
water and 1.0 L of methanol. Reconstitute: 100 µL of 10 mM ammonium acetate in water/10 mM ammo-
nium acetate (50:50) in methanol, sample is ready for analysis.
The internal standard (IS) working solution (40 ng/mL) was pre- Note: After reconstituting transfer sample to an autosampler
pared by dissolving 2.5 mg of Oleandrin in 2.5 mL of methanol vial with an insert (e.g. Phenomenex, Part Number AH0-
to make a 1 mg/mL solution. The 1 mg/mL IS solution was then 4604) to ensure the sample will be injected accurately.
diluted to 40 ng/mL in 50:50 methanol/water. A 1 mg/mL digoxin
and digitoxin stock solution was prepared by dissolving 2.5 mg
of digoxin and digitoxin in 2.5 mL of methanol. From the 1 mg/
mL stock solution a 100 ng/mL solution was made by diluting
in 50:50 methanol/water. From the 100 ng/mL digoxin and digi-
toxin solution the calibration standards were prepared by serial
dilution, yielding calibration standards at 100, 50, 25, 10, 5 and
2.5 ng/mL.
The calibration curve was generated by spiking 100 µL of each

calibration standard into 900 µL of blank plasma, yielding a 1.0
mL initial sample volume. Each spiked plasma standard was pre-
pared for analysis following the sample preparation, SPE and LC-

LC-MS/MS Conditions helps to concentrate the analytes and remove potential matrix in-
Column: Kinetex® 2.6 μm C8 100 Å terfering compounds present in plasma. Strata®-X is a n-vinylpyr-
Dimensions: 50 x 2.1 mm rolidone functionalized polystyrene divinyl benzene polymer sor-
Part No.: 00B-4497-AN
Recommended Guard: AJ0-8784 bent that possesses both hydrophobic and hydrophilic character,
Mobile Phase: A: 10 mM Ammonium acetate in water allowing for the retention of digoxin and digitoxin and removal
B: 10 mM Ammonium acetate in methanol of unretained matrix compounds. Due to the strong retention of
Gradient: Time (sec) B %
0 50 the analytes on the Strata-X sorbent, an aggressive wash step
2.5 100 (50:50 methanol/ammonium acetate in water) was used to remove
2.51 50 weakly retained matrix interference. This results in a much cleaner
5 50
Flow Rate: 0.4 mL/min sample extract for analysis and highlights one of the major bene-
Inj. Volume: 40 µL fits of the Strata-X sorbent.
Temperature: 30 ºC (column)
MS/MS Detection: MS/MS (SCIEX API 4000™), ESI+ Figure 1.
Digoxin/digitoxin (0.25 ng/mL) in plasma extracted using Strata-X
MS/MS Conditions (1 = Digoxin, 2 = Oleandrin (IS), 3 = Digitoxin)
Ionization ESI
2300 1 2 3
2200
Polarity Positive 2100
2000
1900
Scan Type MRM 1800
1700
1600
Neb 60 1500
Intensity, cps
1400
1300
Drying gas 40 1200
1100
1000
Collision Gas (CAD) 3
App ID 19691
900
800
700
Temperature (TEM) 350 600
500
Curtain Gas (CUR) 12 400
300
200
IS 5500 100
0
Entrance Potential (EP) 10 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Peak Analyte Retention Q1 Q3 Time DP CE CXP A standard calibration curve was generated over the concentra-
No. Time (min) (msec) tion range of 0.25 ng/mL to 10 ng/mL by plotting the relative re-
1 Digoxin 1.90 798.4 651.4 100 61 19 16 sponse (peak area of the analyte/peak area of the oleandrin IS)
versus concentration. The standard calibration curve was linear
2 Oleandrin (IS) 2.35 577.2 373.2 100 40 19 12 over the calibration range with an r2 value of 0.9999 for digoxin
3 Digitoxin 2.75 782.4 635.4 100 61 17 17 and an r2 value of 0.9998 for digitoxin (Figures 2 and 3).
Figure 2.
Results and Discussion Digoxin calibration curve – relative response (peak area of the analyte
The use of the Kinetex® C8 core-shell technology column al- peak/peak area of the oleandrin IS) versus concentration (0.25 – 10 ng/mL)
lowed for very fast elution of digoxin and digitoxin at 1.9 and 2.75 –extracted from plasma using Strata-X; r2 value = 0.9999.
minutes, respectively (Figure 1). In ESI positive mode, digoxin
and digitoxin were detected by monitoring the 798.4/651.4 and 46
782.4/635.4 mass transitions, respectively. Peak areas for both 44

42
analytes were normalized using oleandrin as an internal standard, 40
which was detected by monitoring the 577.2/373.2 mass transi- 38

36
tion. 34
32
Because of the very low analyte concentrations in plasma sam- 30
ples that need to be monitored, the only feasible detection tech-

Analyte Area/IS Area
28
26
nique is mass spectrometry. Therefore, the column technology 24
which is utilized is of utmost importance since the analyst can 22
use the column to improve sample throughput and sensitivity. As 20

18
demonstrated, the Kinetex C8 column delivers a large amount of 16
sensitivity while keeping analysis time to a minimum. As shown in 14
Figure 1, digoxin and digitoxin are well resolved from each other 12
10
and the internal standard (oleandrin); the signal-to-noise is also 8
excellent at the lower end of the calibration range (0.25 ng/mL), 6

4
allowing the typical therapeutic range to be monitored. 2
Solid phase extraction is often used for sample preparation of 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
Analyte Conc./IS Conc.
7.0 7.5 8.0 8.5 9.0 9.5 10.0
plasma samples prior to chromatographic analysis because it

Figure 3. Figure 4.
Digitoxin calibration curve – relative response (peak area of the analyte Suppression study for Digoxin/Digitoxin (2 µg/mL) and internal standard
peak/peak area of the oleandrin IS) versus concentration (0.25 – 10 ng/mL) infused through T-splitter with a blank plasma SPE extract injection.
–extracted from plasma using Strata®-X; r2 value = 0.9998
a. Blank Plasma
34 2.4e5
2.3e5
32 2.2e5
2.1e5
30 2.0e5
1.9e5
28 1.8e5
1.7e5
1.6e5
26 1.5e5
1.4e5
24 1.3e5
Intensity, cps
1.2e5
22 1.1e5
App ID 19695
1.0e5
Analyte Area/IS Area
9.0e4
20
8.0e4
7.0e4
18 6.0e4
5.0e4
16 4.0e4
3.0e4
14 2.0e4
1.0e4
0.0
12 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
50 99 147 196 245 294 342 391 440
10
6
b. Digoxin
4 0.02
2.0e5
1.9e5
2 1.8e5
1.7e5
1.6e5
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 1.5e5 0.09
Analyte Conc./IS Conc. 1.4e5 1.23 1.65 3.82 4.34 4.46 4.61
1.3e5 0.75 0.80 4.18
1.83 2.70 3.77
1.2e5 2.55 3.06 3.14
1.1e5 2.40
Intensity, cps
The extracted QC samples were made at two concentrations, one 1.0e5

9.0e4
App ID 19696
low (0.75 ng/mL) and one high (7.5 ng/mL). The QC samples were 8.0e4
7.0e4
treated in the same way as the calibration curve standards, i.e. 6.0e4
5.0e4
spiked with standard, diluted and then extracted with the Strata-X 4.0e4
3.0e4
solid phase extraction cartridges before LC-MS/MS analysis. Ex- 2.0e4
1.0e4
cellent reproducibility was obtained for both the low and high end 0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
50 99 147 196 245 294 342 391 440
QC samples with RSD values < 6 % (Table 1).
Table 1. c. Digitoxin
QC samples at low (0.75 ng/mL) and high (7.5 ng/mL) concentrations 1.5e5
0.03
1.4e5
Analyte QC concentration (ng/mL) RSD (%) (n=4) 1.3e5

0.05
1.2e5
0.75 1.3 1.1e5 0.18 0.23

Digoxin 1.0e5 3.81
7.5 5.8 9.0e4
0.80
0.88
1.29
1.53 1.78
1.82 3.88 4.25 4.33 4.47
1.11 2.66
2.55 3.15
8.0e4
Intensity, cps
0.75 2.9 2.40 3.73
Digitoxin 7.0e4
7.5 4.4
App ID 19696
6.0e4
5.0e4
4.0e4
The suppression study (Figure 4) shows that the digitoxin 3.0e4

2.0e4
peak elutes in an optimal portion of the chromatographic run 1.0e4
(Figure 4c) where there is no signal suppression or enhance- 0.0

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
50 99 147 196 245 294 342 391 440 488
ment. On the other hand, digoxin elutes in a portion of the run
(Figure 4b) where some suppression is present. However, the
amount of suppression is minimal and still allows for adequate Conclusion
signal and excellent reproducibility. This overall method pro- The fast run times and and high sensitivity provided by Kinetex®
vides a very linear correlation between concentration and peak core-shell columns along with thorough clean-up provided by
response over the calibration range (0.25 – 10 ng/mL) and thera- Strata-X SPE makes the proposed method very suitable for ana-
peutic dosage range for digoxin (0.8 – 2.0 ng/mL) in serum. lysis of digoxin and digitoxin in a high-throughput environment.

Antidepressants from Urine
Table 1.
According to the pharmacological mechanism of action (MOA), LC-MS/MS was performed using a Kinetex® 2.6 µm core-shell
antidepressants are divided into seven classes. These include tri- C18 50 x 3.0 mm HPLC/UHPLC column and a Shimadzu® Nexe-
cyclic antidepressants (TCAs), selective serotonin reuptake inhib- ra® UHPLC system (Kyoto, Japan) with an upper pressure limit of
itors (SSRIs) and serotonin and norepinephrine reuptake inhibitors 1300 bar. A triple quadrupole API 3200 system (AB SCIEX, Fram-
(SNRIs), among others. ingham, MA), equipped with an electrospray source, was used
for mass spectrometric detection. The MS spectra were recorded
LC-MS/MS methodology over immunoassay is preferred because in multiple-reactions monitoring and scheduled MRM™ algorithm.
of its selectivity and robustness. In this study, a simple sample MRM Transitions and Ionization Source Parameters are listed in
preparation procedure and a rapid, sensitive, specific Ultra-High
Performance Liquid Chromatography – Tandem Mass Spectrom-
etry (UHPLC-MS/MS) method has been developed for quantify- Table 1.
ing several antidepressants, including amitriptyline, bupropion,
Column: Kinetex 2.6 µm C18
citalopram, clomipramine, doxepin, duloxetine, fluoxetine, imip- Dimensions: 50 x 3.0 mm
ramine, paroxetine, and venlafaxine as well as five major metab- Part No.: 00B-4462-Y0
olites, nortriptypline, norfluoxetine, desipramine, hydroxybupropi- Recommended Guard: AJ0-8775
Mobile Phase: A: 2 mM Ammonium acetate with 0.075 % (v/v) acetic acid (pH 4.5)
on, and o-desmethylvenlafaxine, in human urine samples. B: 2 mM Ammonium acetate with 0.075 % (v/v) acetic acid and acetonitrile
Instrument: Shimadzu Nexera UHPLC
Reagents and Chemicals Detector: MS/MS (SCIEX API 3200)
Primary reference standards and deuterated internal standards Gradient: Time (min) % B
were purchased (Cerilliant Corporation, Round Rock, TX). A 0 18
0.4 18
mixture of standard solution was prepared in acetonitrile and 1.1 45
then added to drug-free urine to make multi-concentration levels 1.7 60
of calibrators. The internal standards mixture includes amitripty- 2.1 95
2.5 95
line-d3, clomipramine-d3, desipramine-d3, doxepin-d3, imipra- 2.54 18
mine-d3, nortriptyline-d3, bupropion-d9, fluoxetine-d6, paroxe- 3.5 18
tine-d6, and venlafaxine-d6, and it was prepared in acetonitrile Ionization Source Parameters:
and added in urine samples, standards, and controls. Gas 1 & Gas 2 50
CAD 6
Sample Preparation Cur 35
IS 3000
A simple “dilute and shoot” urine sample extraction was carried
Temp 550
out by a Tomtec™ Quadra 4™ liquid hander (Hamden, CT) in
96-well collection plates to increase throughput. Samples were
treated with beta-glucuronidase to hydrolyze glucuronide conju-
gates, followed by dilution and centrifugation.
LC-MS/MS Conditions
LC-MS/MS was performed using a Kinetex® 2.6 µm core-shell
C18 50 x 3.0 mm HPLC/UHPLC column and a Shimadzu® Nexe-
ra® UHPLC system (Kyoto, Japan) with an upper pressure limit of
1300 bar. A triple quadrupole API 3200™ system (AB SCIEX, Fram-
ingham, MA), equipped with an electrospray source, was used
for mass spectrometric detection. The MS spectra were recorded
in multiple-reactions monitoring and scheduled MRM™ algorithm.
MRM Transitions and Ionization Source Parameters are listed in

Table 1. Results
MRM Transitions & Retention Times Linearity was evaluated by analyzing samples at 10 concentration
levels over the reportable range of assay for three days. Linear
Analyte Peak Name Q1 Q3
Analyte Retention Time Regression using the Analyst® software (“1 / x*x” weighting) was
(min) used to determine slope and correlation coefficient. Results are
Amitriptyline 278.0 91.0 2.29 shown in Table 2. Intra-day and inter-day precision and accuracy
were determined by analyzing quintuplicate samples at three lev-
Bupropion 240.1 184.1 1.88 els of concentrations. Results are shown in Table 3 and Table 4.
Citalopram 325.1 109.1 2.03 The carry-over was also estimated by injecting blank samples im-
mediately following the highest concentration, and was less than
Clomipramine 315.1 86.2 2.42
15 % of lower limit of quantitation (LLOQ), which can be accept-
Desipramine 267.1 208.2 2.17 able. Bench-top stability was assessed by reinjection of same
samples within 24-48 hours, and was within compliance range. In
o-Desmethylvenlafaxine 264.1 107.2 1.27 addition, cross reactivity was investigated against all our current
test panels of drugs.
Doxepin 280.0 107.1 2.08
Duloxetine 298.0 154.2 2.21

Table 2.
Fluoxetine 310.1 148.0 2.30 Summary of Reportable Range and Linearity Correlation
Hydroxybupropion 256.1 238.1 1.57 Reportable Linearity

Cut-off Correlation Data
Drug Range
Imipramine 281.0 86.2 2.23 (ng/mL)
(ng/mL) Slope (r) y-intercept
Norfluoxetine 296.0 134.1 2.24
Amtriptyline 50 12.5-7500 0.97 0.997 27
Nortriptyline 264.1 233.2 2.22
Bupropin 10 2.5-1500 1.02 0.998 2.45
Paroxetine 330.0 192.2 2.12 Citalopram 10 2.5-1500 1.04 0.993 3.89
Venlafaxine 278.1 260.2 1.81 Clomipramine 50 12.5-7500 1.11 0.995 2.21
Desipramine 50 12.5-7500 1.11 0.998 41.28
Figure 1. o-Desmethylvenlafaxine 10 2.5-1500 0.9 0.992 9.04

Representative chromatogram for ten antidepressants and five of their
major metabolites Doxepin 50 12.5-7500 0.89 0.998 69.57
1.20e6 Duloxetine 5 12.5-7500 0.94 0.994 32.93

1.10e6
Fluoxetine 20 12.5-7500 1.05 0.995 0.95
1.00e6
Hydroxybupropion 10 2.5-1500 0.99 0.993 1.05
9.00e5
8.00e5 Imipramine 50 12.5-7500 0.94 0.997 31.65

Intensity, cps
7.00e5
Norfluoxetine 10 12.5-7500 1.16 0.998 86.59
6.00e5
Nortriptyline 50 12.5-7500 0.88 0.993 85.48
5.00e5
4.00e5
Paroxetine 10 12.5-7500 1.03 0.997 14.56
3.00e5 Venlafaxine 20 2.5-1500 1.03 0.999 4.54

2.00e5
1.00e5
0.00
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 min

Table 3.
Intra-day Precision and Accuracy
Intra-day
Day 1 Day 2 Day 3
Drug Concentration (ng/mL) CV (%) Accuracy (%) CV (%) Accuracy (%) CV (%) Accuracy (%)
100 5.9 109.0 2.7 102.4 5.4 97.0
Amitriptyline 250 3.3 109.7 3.4 111.0 4.3 108.9
750 4.0 102.3 3.0 103.1 7.2 97.8
20 8.7 105.2 7.3 108.9 11.7 94.5
Bupropion 50 5.5 108.5 5.4 111.4 5.1 109.4
150 5.8 96.6 6.5 102.2 2.3 101.5
20 6.0 111.4 3.7 104.3 6.8 100.4
Citalopram 50 5.1 109.7 5.7 110.2 3.9 97.9
150 4.7 103.6 5.0 105.7 3.9 91.8
100 2.1 102.2 2.3 103.2 7.4 94.3
Clomipramine 250 3.5 101.5 2.9 105.5 7.4 100.8
750 4.1 99.7 2.6 106.8 5.0 98.5
100 7.5 101.6 5.0 104.2 5.9 97.4
Desipramine 250 3.5 107.1 4.5 112.3 6.1 106.1
750 8.7 98.2 3.4 104.4 4.6 95.9
20 7.3 104.2 1.9 111.4 2.8 100.3
o-Desmethylvenlafaxine 50 4.0 115.2 1.5 119.1 4.1 113.1
150 1.0 100.1 2.0 109.0 9.2 103.2
100 0.7 110.8 3.5 108.2 5.6 103.3
Doxepin 250 3.1 115.4 1.4 118.0 5.0 114.6
750 0.7 100.4 2.3 106.8 1.9 98.4

Table 4.
Inter-day Precision and Accuracy
Inter-day
Drug Concentration (ng/mL) CV (%) Accuracy (%)
100 6.7 102.8
Amitriptyline 250 3.5 109.9
750 5.3 101.0
20 10.5 102.9
Bupropion 50 5.1 109.8
150 5.3 100.4
20 6.9 105.4
Citalopram 50 7.3 106.0
150 7.8 100.1
100 5.8 99.9
Clomipramine 250 5.1 102.6
750 5.3 101.8
100 6.4 101.1
Desipramine 250 5.1 108.5
750 6.5 99.6
20 6.2 105.3
o-Desmethylvenlafaxine 50 3.8 115.8
150 6.3 104.4
100 4.6 107.4
Doxepin 250 3.5 116.0
750 4.1 102.0
100 6.7 108.5
Duloxetine 250 7.2 110.9
750 6.9 101.5

Table 4. (continued) Discussion and Conclusion
Inter-day Precision and Accuracy Accuracy and precision for all antidepressants and metabolites in
reportable range were obtained. The intra-day and inter-day vari-
ability was less than 20 %. Negative cut-off values and reportable
Inter-day
ranges for urinary analysis can be acceptable.
Drug Concentration (ng/mL) CV (%) Accuracy (%)
100 6.9 100.3
Fluoxetine 250 7.1 102.5
750 6.3 102.9
20 5.0 103.7
Hydroxybupropion 50 4.3 112.3
150 6.2 103.2
100 8.1 107.3
Imipramine 250 5.3 115.2
750 8.1 102.4
100 7.5 96.1
Norfluoxetine 250 9.4 100.1
750 7.5 100.2
100 7.5 107.2
Nortriptyline 250 7.2 112.1
750 6.8 104.3
100 6.2 105.4
Paroxetine 250 5.8 106.0
750 5.6 100.3
20 8.9 100.2
Venlafaxine 50 5.6 110.7
150 5.1 100.3

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Cortisone and Prednisolone from Plasma

Corticosteroids exhibit anti-inflammatory properties and are Column: Kinetex 2.6 µm Biphenyl
widely abused within the sports industry. Hence there is a need Dimensions: 50 x 3.0 mm
Part No.: 00B-4622-Y0
for more sensitive analytical tools to detect and confirm these
classes of drugs. Chromatographic separation is essential for
Mobile Phase: A: 10 mM Ammonium acetate in Water
detection and confirmation of corticosteroids because cortisone B: 10 mM Ammonium acetate in Methanol
and prednisolone are isomeric compounds. A simple automated Gradient: Time (min) %B
extraction method using a Tecan Freedom EVO® 100 liquid han- 0 50
dler and Novum™ Simplified Liquid Extraction (SLE) 96-well plate 2 95
is employed to extract corticosteroids from plasma samples. A 3.1 95
Kinetex® 2.6 µm, 50 x 3 mm core-shell Biphenyl HPLC/UHPLC 3.11 50
5 50
column is used to successfully separate these two compounds
which will be beneficial for detection and confirmation of the two
Inj. Volume: 10 µL
isomers. Temperature: Ambient
Detection: MS/MS (SCIEX, API 5000™)
Figure 1.
Structure of cortisone (a) and prednisolone (b)
Table 1.
MRM transitions & retention times for cortisone and prednisolone
Peak # Analyte RT, min Q1, Da Q3, Da CE
1 Cortisone 2.66 361.1 121.2 28

361.1 163.2
2 Cortisol-D4 (IS) 2.69 367.1 121.1 28
a. Cortisone b. Prednisolone
3 Prednisolone 2.92 361.1 121.2 28
361.1 163.2
Table 2.
Extraction Procedure Recovery of cortisone and prednisolone in human plasma after extraction
with Novum SLE
Sample pre-treatment Analyte % Absolute %CV
• Dilute 150 µL of human plasma (spiked with 25 ng/mL Recovery (N=16)
and 125 ng/mL of cortisone and prednisolone
Cortisone 99 3.6
respectively) with 150 µL of 50 mM sodium phosphate
dibasic heptahydrate, pH unadjusted. Mix briefly Prednisolone 95 5.4
(3-5 sec).
SLE Protocol
96-Well Plate: Novum™ SLE MINI
Part No.: 8E-S138-FGA
Pretreated sample and pulse vacuum (~5” Hg)
Load: for 20 seconds or until sample has complete
entered the sorbent. Wait 5 minutes.
Dispense 1000 µL of ethyl acetate onto the Novum
SLE media and allow the solvent to elute by gravity (~5
min elution time) and collect the eluant. Apply vacuum
Elute: at 5” of Hg for 45 secs to complete the extraction.
result in elution of plasma from the Novum SLE
Evaporate the final extract to complete dryness
Dry down:
under a slow stream of N2 at 40°C.
The dry residue in 150 µL of initial mobile
Reconstitute:
phase fortified with cortisol-D4.

1.5e5 2
1.4e5
1.3e5
1.2e5
1.1e5
1.0e5
Intensity, cps
9.0e4
3
8.0e4
7.0e4
6.0e4
5.0e4 1
4.0e4
App ID 22785
3.0e4
2.0e4
1.0e4
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Results and Discussion Conclusions

Prednisolone and Cortisone were extracted from plasma samples The automated extraction of cortisone and prednisolone from
using Novum™ SLE 96-well plates. In an effort to streamline the human plasma using Novum SLE 96-well plates along with Tecan’s
process, the extraction was automated using a Tecan Freedom Freedom EVO 100 liquid handler yields quantitative recovery of both
EVO® 100 liquid handler. The Novum SLE protocol followed a cortisone and prednisolone. The analytical method using a Kinetex
simple load, wait, elute procedure which allowed us to process 96 core-shell Biphenyl HPLC/UHPLC column with MS/MS detection
samples in less than 15 minutes. By automating the procedure, shows good selectivity for the two isomeric compounds.
we ensured that the method was consistent and reproducible
because there is no chance for human error.
The extracted sample was then analyzed by LC-MS/MS using

a Kinetex® core-shell Biphenyl column (Figure 2). Separation
of the two isomeric compounds was achieved, allowing us to
accurately detect and confirm the presence of both cortisone and
prednisolone. Recoveries for cortisone and prednisolone were
99 % and 95 % with CV values of 3.6 and 5.4 (N=16), respectively
(Table 2). This indicates that our extraction method was both
acceptable and accurate.
to view webinars

TN-1169
APPLICATIONS
LC-MS/MS Analysis of Immunosuppressants from Whole Blood
using Aeris™ WIDEPORE XB-C18
Immunosuppressants Core-Shell
from Whole BloodHPLC/UHPLC Columns
Cyclosporine A, tacrolimus, sirolimus, and everolimus are four of that not all labs are equipped to operate.8, 9, 10 Herein, we present a
the most commonly administered immunosuppressant drugs and simple and rapid method for the analysis of immunosuppressants
play a central role in the success of tissue and organ transplants. from whole blood that utilizes a simple protein precipitation step
These drugs are most typically analyzed from whole blood using followed directly by LC-MS/MS analysis using a wide-pore core-
LC-MS/MS. However, because of the analytical challenges posed shell HPLC column. This fast, simple method shows excellent pre-
when working with whole blood, many of the published methods cision and accuracy down to the µg/L concentration range.
rely upon complex and/or expensive extraction steps utilizing off-
line solid phase extraction, on-line solid phase extraction, or the Materials and Methods
use of pre-columns prior to the actual analytical column. In this cur-
rent work, we present a rapid and effective method for the analysis Reagents
of these four immunosuppressants from whole blood that use a
simple protein precipitation step followed by direct injection onto The whole blood used in this study was obtained from Bioreclama-
a wide-pore core-shell HPLC column (Aeris™ WIDEPORE 3.6 µm tion LLC (Westbury, NY). Methanol (LC/MS grade) was purchased
XB-C18). The method displays excellent accuracy and is sensitive from J. T. Baker (Center Valley, PA). Deionized water was used for
down to the low μg/L (ng/mL) range. buffers and sample dilutions. Tacrolimus, everolimus, sirolimus,
and cyclosporine A were obtained from Sigma-Aldrich Chemical
Overview Co. (St. Louis, MO, USA). The internal standard used for CsA was
cyclosporine D (Cerilliant), and the internal standard for the other
Immunosuppressants are a class of drugs that inhibit the body’s immunosuppressants was ascomycin (Cerilliant, Round Rock, TX).
immune response and are typically administered to prevent the Unless stated otherwise, all other reagents used in this study were
rejection of transplanted organs (e.g. kidney) or tissue (e.g. bone purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
marrow), and may also be used to treat various autoimmune dis-
orders such as Crohn’s Disease or rheumatoid arthritis. The first Whole Blood Protein Precipitation
effective immunosuppressant drug was cyclosporine A (or CsA), an To perform the protein precipitation, 0.2 mL whole blood (spiked
undecapeptide, initially discovered by researchers at the pharma- with analytes and internal standards) was placed into a 1.5 mL
ceutical company Sandoz.1 Since the development of CsA, many polypropylene microcentrifuge tube. 400 µL of MeOH/2 % zinc
other immunoppressant drugs have been developed, including the sulfate (80:20) dissolved in water was added to the whole blood
macrolides tacrolimus (FK506), serolimus (also known as rapamy- sample. This mixture was then vortexed vigorously for 10-20 sec-
cin), and everolimus. onds and then centrifuged at 14,000 rpm for 10 minutes at room
While all of these drugs ultimately act to suppress the immune re- temperature. The supernatant (~0.5 mL) was transferred to a new
sponse, they each exert their effects through different mechanisms. autosampler vial, and then directly injected into the LC-MS/MS with
Cyclosporine A binds to the protein cyclophillin, and the resulting a 20 µL injection volume.
CsA-cyclophillin complex blocks the calcineurin-mediated tran- Optional: Solid Phase Extraction (SPE)
scription of the interleukin 2 (IL-2) gene in antigen activated T cells,
thus preventing the growth, differentiation, and proliferation of T In this publication, we present a simple method that uses protein
cells that mediate the immune response.2,3 Tacrolimus binds to the precipitation and LC-MS/MS to analyze these immunosuppres-
protein FKBP12 (FK506 binding protein), and the resulting complex sants. For users with LC-MS/MS systems that are not as sensitive
prevents the cascade of reactions that ultimately lead to a reduc- as the API 5000™ (AB SCIEX, Framingham, MA) used in the current
tion in IL-2 transcription.3 Unlike CsA and tacrolimus, which block study, or for researches or analysts seeking much lower levels of
synthesis of IL-2, sirolimus and everolimus exert their activity by detection and quantitation, we also include an off-line solid phase
blocking the response of T-cells to IL-2.4 extraction method of cyclosporine A from whole blood. Using a
vacuum manifold, a 30 mg/3 mL Strata® -X-CW (weak cation-ex-
Because of of their
their potent
potent immunosuppressant
immunosuppressant effects effectsandandrelatively
relatively change) solid phase extraction cartridge (Phenomenex, Torrance,
therapeutic index,
narrow therapeutic index, therapeutic
therapeutic drugdrug monitoring
monitoring ofofpatients
patients CA.) was conditioned with 1 mL of 100 % methanol, followed by
is required inin order
order to
to insure
insure the
the efficacy
efficacyof ofthe
thetreatment,
treatment,andandalsoalso 1 mL of 25 mM ammonium bicarbonate (pH 8.3). The protein pre-
to minimize
minimizetoxic sideside
toxic effects.
effects.
5, 6
Liquid
5, 6 chromatography
Liquid coupled
chromatography cipitated whole blood sample was loaded onto the SPE bed and
to tandem
coupled to mass
tandem spectrometry (LC-MS/MS)
mass spectrometry has become
(LC-MS/MS) the ana-
has become drawn through the SPE cartridge at a slow flow rate (~1 mL/min).
lyticalana-lytical
the method of choice
methodfor of the analysis
choice of forimmunosuppressants.
the analysis of The cartridge was then washed with 0.4 mL of the 25 mM ammoni-
These drugs must be monitored
immunosuppressants. These drugs from whole
must blood, which poses
be monitored froma um bicarbonate, followed by a second wash using 0.4 mL of meth-
sampleblood,
whole preparation
whichchallenge
poses aassample matrix effects can confound
preparation challengeanal-as anol/water (50:50). Under high vacuum, the SPE bed was dried for
yses through
matrix effects ion
cansuppression and/or enhancement,
confound anal-yses through ion and can also
suppression 4-5 minutes, and then the analytes were eluted from the cartridge
affect the
and/or reproducibility
enhancement, andandcan accuracy
also affectof the
analytical methods.
reproducibility andTo using 200 µL of 100 % methanol. This elution step was repeated,
overcome of
accuracy theanalytical
challengesmethods.
posed when working with
To overcome thewhole blood,
challenges and the resulting extracts were combined (400 µL) and evaporat-
many methods
posed when workingthat have
withbeen
wholedeveloped
blood, manyfor immunosuppressant
methods that have ed to dryness under a gentle stream of nitrogen at 40-45 °C. The
analysis
been involve off-line
developed solid-phase extraction7,
for immunosuppressant analysiswhich can be
involve time-
off-line extract residue was re-suspended with 400 µL of methanol/5 mM
consuming and
solid-phase expensive,
extraction 7 or complex
, which can on-line extraction methods
be time-consuming and ammonium formate (pH 3.2) (35:65) and transferred to a glass au-
expensive, or complex on-line extraction methods tosampler vial for LC-MS/MS analysis.
For additional technical notes, visit www.phenomenex.com Page 1 of 4
TN-1169
LC-MS/MSAnalysis
LC/MS/MS Conditions 23:1 (2.5 µg/L), sirolimus 34:1 (2.5 µg/L), everolimus 13:1 (2.5 µg/L).
Given the relatively high signal-to-noise ratios, it is clear that, if
Analysis was performed using an API 5000™ mass spectrometer necessary, it would most likely be possible to accurately identify
(AB SCIEX, Framingham, MA.) coupled to an Agilent® 1260 UHPLC and quantify the target immunosuppressants at significantly lower
system (Agilent Technologies; Santa Clara, CA.). The analytical col- levels than were used in the present study.
umn was an Aeris™ WIDEPORE 3.6 µm XB-C18 column (50 mm x
2.1mm), with a SecurityGuard™ ULTRA guard cartridge (both from
Phenomenex, Torrance, CA.). Mobile phase A consisted of 5 mM Quantitation
ammonium formate (no pH adjustment) dissolved in deionized wa-
ter, and mobile phase B consisted of 5 mM ammonium formate dis- Absolute recovery values (compared to a pure neat standard)
solved in methanol. The analysis was performed using a simple, ranged from 73 % for serolimus to 103 % for tacrolimus, with RSD
rapid gradient going from 35 % B to 95 % B over 1 minute, holding % values for four replicates ranging between 1.3 % and 8.8 % (Ta-
at 95 % B for 1 minute, and then re-equilibrating at the initial 35 % ble 2). Precision and accuracy values are given in Table 3 for high
B for 2 minutes between injections. The flow rate was 700 µL per and low concentration QC samples. Accuracy values ranged from
minute, and the column was maintained at 75 °C. 85.4 % to 114 %, with precision (or impresicion) values of 6.00 %
or lower.
Multiple reaction monitoring (MRM) of the immunosuppressants
was performed using electrospray in positive ion mode. The source
was operated at 400 °C with an electrospray voltage of 4000. Ion Table 2.
source parameters were as follows: curtain gas 25, GS1 60, GS2 Absolute percent recovery of the immunosuppressants from precipitated
45, CAD gas. MRM transitions for the analytes are shown in Table 1. whole blood
Analyte
AnalyteName
Name Conc.
Conc.(μg/L)
(µg/L) %
%Recovery %RSD
Recovery % RSD(N=4)
(N=4)
CyclosporineAA
Cyclosporine 500
500 91.0
91.0 6.40
6.40
Table 1. Everolimus
Everolimus 50
50 77.0
77.0 8.80
8.80
MRM transitions for the immunosuppressants and the internal standards
Serolimus
Serolimus 50
50 73.0
73.0 1.30
1.30
Analyte Name Q1, Da Q3, Da Tacrolimus
Tacrolimus 50
103.0 103.0
926.6 3.20
3.20
Analyte Name Q1, Da Q3, Da
Ascomycin 1
Ascomycin 1
809.6
809.6
756.7
756.7
Ascomycin
Ascomycin 2 2 809.6
809.6 564.5
564.5
Table 3.
Everolimus
Everolimus 1 1 975.8
975.8 908.6
908.6 Precision and accuracy data for QC samples
Everolimus
Everolimus 2 2 975.8
975.8 926.6926.6
Analyte
AnalyteName
Name Conc.
Conc.(μg/L)
(µg/L) %
% CV
CV %Accuracy
% Accuracy)
Sirolimus 1
Sirolimus 1 931.6
931.6 864.6864.6
150
150 5.40
5.40 113.8
113.8
Sirolimus 2
Sirolimus 2 931.6
931.6 882.8882.8 Cyclosporine A
Cyclosporine A
750
750 4.30
4.30 114.4
114.4
Tacrolimus 1
Tacrolimus 1 821.7
821.7 786.4786.4
Tacrolimus 2 821.7 768.5 15
15 4.40
4.40 95.8
95.8
Tacrolimus 2 821.7 768.5 Tacrolimus
Tacrolimus
Cyclosporin A 1 1220.1 1202.9 75
75 4.50
4.50 98.3
98.3
Cyclosporin A 1 1220.1 1202.9 15 2.90 100.1
Cyclosporin A 2 1220.1 425.1 Sirolimus
15 2.90 100.1
Cyclosporin A 2 1220.1 425.1 Sirolimus 75 2.70 85.5
Cyclosporin D 1 1233.9 1216.9 75 2.70 85.5
Cyclosporin D 1 1233.9 1216.9 15 0.90 108.5
Cyclosporin D 2 1233.9 1198.7 Everolimus 15 0.90 108.5
Cyclosporin D 2 1233.9 1198.7 Everolimus 75 3.80 95.7
75 3.80 95.7
Chromatography
Figure 1 contains representative extracted ion chromatograms

(XIC) for the MRMs of the selected immunosuppressants and the
two internal standards obtained from a spiked, protein precipitated
whole blood sample (50 ng/mL for everolimus, sirolimus, tacroli-
mus; 500 ng/mL for cyclosporine A). Flow before 0.8 minutes and
after 2.5 minutes was diverted to waste. All of the immunosup-
pressants display excellent chromatography, and are eluted in a
cycle time of 4 minutes. The total elution window for the immu-
nosuppressants is less than 1 minute, allowing for extremely high
sample-throughput for analysts that utilize multiplexing technology.
Comparison with the protein precipitated matrix blank (Figure 2)
shows little or no matrix interference for each of the MRM transi-
tions monitored. Signal-to-noise ratio for each of the analytes at
the lowest levels monitored were: CsA 140:1 (25 µg/L), tacrolimus
Page 2 of 4
TN-1169
App ID 22098
1.29e5 Figure 1. Representative extracted ion

1.48 Cyclosporine D 2.0e5 1.45 Cyclosporine A
chromatograms (XIC) of spiked whole
Intensity, cps
Intensity, cps
1.00e5
1.5e5
blood extract (50 µg/L for Everolimus,
1.0e5
5.00e4 Sirolimus, Tacrolimus; 500 µg/L for
5.0e4
Cyclosporin A)
0.00 0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min 0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
1.5e4 2.4e4
1.37 Everolimus 2.0e4
1.35 Sirolimus
Intensity, cps
Intensity, cps
1.0e4 1.5e4
1.0e4
5000.0
5000.0
0.0 0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min 0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
3.0e4
1.32 Tacrolimus 6.0e4 1.32 Ascomycin
App ID 22098
Intensity, cps
Intensity, cps
2.0e4 4.0e4
1.0e4 2.0e4
0.0 0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min 0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
App ID 22099
1300 1260
0.80 0.80
Figure 2. Representative extracted ion
Intensity, cps
Intensity, cps
1000 1000
Cyclosporine D Cyclosporine A
chromatograms (XIC) for the protein
500 500
precipitated whole blood matrix blank
0 0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min 0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
360 280
0.81 250 0.81 2.19
300
Intensity, cps
Intensity, cps
200
Everolimus Sirolimus
200 150
100
100
50
0 0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min 0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
1340 6960
0.81 6000
Intensity, cps
Intensity, cps
App ID 22099
1000
Tacrolimus 4000
Ascomycin
500
2000
0 0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min 0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
Conclusions References
In this work, we present a simple and effective method for the 1. Svarstad, HC Bugge, and SS Dhillion. 2000. Biodiversity and Conservation 9
analysis of four commonly used immunosuppressants obtained (11): 1521–1541.
from whole blood samples. Using a simple protein precipitation step, 2. S. Matsuda and S. Koyasu. 2000. Immunopharmacology 47: 119-125.
we were able to achieve a quantitation of 25 µg/L for CsA, 2.5 µg/L 3. Liu J, Farmer J, Lane W, Friedman J, Weissman I, Schreiber S (1991).
for tacrolimus, 2.5 µg/L for sirolimus, and 2.5 µg/L for everolimus. Cell 66 (4): 807–15.
Signal-to-noise ratios at the lowest level analyzed using this method 4. SN Seghal. 2003. Transplant Proc. 35(3): 7S-14S.
were greater than 13, indicating that the method is most likely 5. Christians U, Klawitter J, Clavijo CF. Kidney Int Suppl. 2010 Mar;(115):S1-7.
applicable to even lower levels of detection and quantitation. The
6. Barry Kahan, Paul Keown, Gary Levy, and Atholl Johnson. 2002. Clinical
use of a unique wide-pore core-shell column (Aeris™ WIDEPORE Therapeutics 24(3) 330-350.
3.6 µm XB-C18) provided excellent chromatography for these
7. Karapirli M, Kizilgun M, Yesilyurt O, Gul H, Kunak ZI, Akgul EO, Macit E,
relatively high molecular weight molecules, and also possesses a Cayci T, Gulcan Kurt Y, Aydin I, Yaren H, Seyrek M, Cakir E, Yaman H.
surface chemistry that is stable at the elevated temperature used ScientificWorldJournal. 2012;2012:571201.
in this assay (75 °C). 8. Christians U, Jacobsen W, Serkova N, Benet LZ, Vidal C, Sewing KF, Manns
MP, Kirchner GI. J Chromatogr B Biomed Sci Appl. 2000 Oct 1;748(1):41-53.
9. Deters M, Kirchner G, Resch K, Kaever V. Clin Chem Lab Med. 2002
Mar;40(3):285-92.
10. Buchwald A, Winkler K, Epting T. BMC Clin Pharmacol. 2012 Jan 11;12:2.
doi: 10.1186/1472-6904-12-2.
For additional technical notes, visit www.phenomenex.com Page 3 of 4
Comprehensive Drug Research Panel from Urine
Using a 30 x 2.1 mm Kinetex® 2.6 µm Biphenyl LC Column, our Materials and Methods
team successfully analyzed 48 drug analytes in less than 4 minutes.
Reagents and Chemicals
A full ESI+/ESI- 52 drug panel was performed in 7 minutes and
Analytical reference standards and Surine negative synthetic urine
30 seconds—including re-equilibration. This rapid analysis showed
were purchased from Cerilliant Corporation (Round Rock, TX,
great resolution and no loss in sensitivity compared to slower
USA). IMCSzyme purified β-glucuronidase was purchased from
methods.
IMCS (Columbia, SC, USA).
Overview
Sample Preparation
Abuse of pain management drugs has reached an epidemic level
Calibrators were prepared in synthetic urine.
across the United States. According to the US Center for Disease
Control, use of and non-intentional deaths from opioids have
Urine samples underwent a 60 minute incubated enzymatic
quadrupled since 19991. In the first 3 months of 2016, deaths from
hydrolysis at 55 °C and were diluted 40-fold with mobile phase prior
heroin and fentanyl-laced heroin have surpassed 2015 death tolls
to injection. Samples were centrifuged for 20 minutes at 18,000 rcf
in several cities in the United States2,3. These increases have led to
before and after incubation.
increased pressure on researchers.
Sample prep methods, such as sorbent-based β-glucuronidase
Labs demand methods that can help them analyze these samples
removal or protein precipitation, were outside of the scope of this
as fast as technology will allow without compromising confidence
method development. To avoid system downtime and premature
in their results. Large research panels, from urine, covering
column death, we recommend more extensive sample prep
several classes of drugs have become standard in the industry.
procedures.
In order to satisfy their customers’ needs, labs commonly test for
amphetamines, benzodiazepines, opioids, drugs of abuse, tricyclic
antidepressants, barbiturates, nicotine, and THC metabolites. A Experimental Conditions
list of analytes is outlined in Tables 1 and 2. UHPLC analysis was performed using a Shimadzu® Nexera® X2 LC-
30 (Kyoto, Kyoto Prefecture, Japan) with an upper pressure limit of
Sample preparation, LC-MS/MS analysis, and data review are all 1300 bar, equipped with a binary pump and autosampler. Detection
steps which contribute to a sample’s turnaround time. In our work, by tandem mass spectrometry was performed using a SCIEX
we focused on shortening the LC runtime to under 8 minutes per QTRAP® 6500 (Framingham, MA, USA) with ESI configuration. Two
sample while maintaining resolution of all critical pairs including separate methods were developed for analysis of compounds by
isobaric/isomeric compounds like morphine and hydromorphone, positive and negative electrospray ionization. Method conditions
codeine and hydrocodone, and 6-MAM and naloxone. are listed on page 2. The Kinetex 2.6 µm Biphenyl Core-Shell
HPLC/UHPLC column was used to perform the separation. The
Biphenyl chemistry was chosen for its robustness, excellent peak
shape, and strong selectivity for this group of analytes. By using a
30 x 2.1 mm column, instead of the 50 mm column typically used
for these types of panels, we decreased retention times and system
backpressure thereby allowing the flow rate to be increased.

Positive ESI Panel Table 1. (continued)
LC Conditions List of ESI+ Analytes and Transitions.
Column: Kinetex® 2.6 µm Biphenyl
Dimensions: 30 x 2.1 mm Analyte Q1 (m/z) Q3 (m/z)
SRecommended Guard: AJ0-9209 Carisoprodol 2 261.1 97.0
Mobile Phase: A: 10 mM Ammonium Formate in Water (pH unadjusted)
B: 1.0 % Formic Acid in Methanol Citalopram 1 325.1 109.1
0 5 Citalopram 2 325.1 262.0
1.2 30
1.3 55 Codeine 1 300.1 115.1
2.3 70
Codeine 2 300.1 152.1
2.4 95
2.7 95 Cotinine 1 177.1 80.0
2.72 5
4 5 Cotinine 2 177.1 98.0
Temperature: 30 °C Diazepam 1 285.1 193.1
Diazepam 2 285.1 154.1
MS/MS Conditions EDDP 1 278.1 234.1
Conditions: SCIEX 6500 QTRAP®
Mode: Positive electrospray ionization EDDP 2 278.1 186.1
Scan Type: MRM
Curtain Gas (CUR): 30 Fentanyl 1 337.2 188.1
Gas 1 (GS1): 70
Gas 2 (GS1): 80
Fentanyl 2 337.2 105.1
IS: 2000 V Fluoxetine 1 310.1 44.1
Interface Heater: ON Fluoxetine 2 310.1 148.2
Collision Gas (CAD): Medium
Entrance Potential (EP): 10 V Gabapentin 1 172.1 137.2
Gabapentin 2 172.1 95.1
Hydrocodone 1 300.0 199.1
Table 1. Hydrocodone 2 300.0 128.1
List of ESI+ Analytes and Transitions.
Hydromorphone 1 286.1 185.1
Analyte Q1 (m/z) Q3 (m/z) Hydromorphone 2 286.1 128.1
6-MAM 1 328.1 165.1 Imipramine 1 281.1 86.0
6-MAM 2 328.1 211.1 Imipramine 2 281.1 58.1
7-Aminoclonazepam 1 286.0 222.1 Lorazepam 1 321.1 275.1
7-Aminoclonazepam 2 286.0 121.0 Lorazepam 2 321.1 229.1
alpha-Hydroxyalprazolam 1 325.2 297.1 MDMA 1 194.1 163.1
alpha-Hydroxyalprazolam 2 325.2 216.1 MDMA 2 194.1 135.1
Alprazolam 1 309.1 205.1 Meperidine 1 248.1 174.1
Alprazolam 2 309.1 281.0 Meperidine 2 248.1 220.1
Amitriptyline 1 278.1 191.0 Meprobamate 1 219.1 158.1
Amitriptyline 2 278.1 202.0 Meprobamate 2 219.1 97.0
Amphetamine 1 136.1 119.1 Methadone 1 310.2 105.1
Amphetamine 2 136.1 91.0 Methadone 2 310.2 265.1
Benzoylecgonine 1 290.1 168.1 Methamphetamine 1 150.1 91.0
Benzoylecgonine 2 290.1 105.1 Methamphetamine 2 150.1 119.1
Buprenorphine 1 468.1 396.2 Methylphenidate 1 234.1 84.1
Buprenorphine 2 468.1 55.2 Methylphenidate 2 234.1 56.0
Carisoprodol 1 261.1 176.1 Morphine 1 286.1 152.1
Comparative separations may not be representative of all applications.

Table 1. (continued) Table 1. (continued)
List of ESI+ Analytes and Transitions List of ESI+ Analytes and Transitions
Analyte Q1 (m/z) Q3 (m/z) Analyte Q1 (m/z) Q3 (m/z)

Morphine 2 286.1 165.2 Imipramine-D3 284.1 89.0
Naloxone 1 328.2 212.0 Lorazepam-D4 325.0 105.9
PCP 1 244.1 91.0 MDMA-D5 199.1 165.0
PCP 2 244.1 159.1 Meperidine-D4 252.2 224.1
Pregabalin 1 160.1 55.1 Meprobamate-D7 226.1 165.1
Pregabalin 2 160.1 97.1 Methadone-D3 313.2 105.0
Sertraline 1 306.1 159.0 Methamphetamine-D5 155.1 92.0
Sertraline 2 306.1 275.1 Methylphenidate-D9 243.2 93.0
Tapentadol 1 222.1 107.0 Morphine-D3 289.2 152.0
Tapentadol 2 222.1 121.0 Naloxone-D5 333 258.0
Temazepam 1 301.1 255.1 Norbuprenorphine-D3 417.2 83.2
Temazepam 2 301.1 177.1 Nordiazepam-d5 276.1 140.1
Tramadol 1 264.1 58.1 Norfentanyl-D5 238.2 84.0
Tramadol 2 264.1 42.1 Norhydrocodone-D3 289.1 202.0
Zolpidem 1 308.1 235.1 Noroxycodone-D3 305.1 190.0
Zolpidem 2 308.1 219.0 Nortriptyline-D3 267.1 233.0
Zolpidem-4- O-Desmethyl-cis- 256.3 64.1
carboxylic Acid 1 338.1 265.0 tramadol-D6
Zolpidem-4- Oxycodone-D3 319.1 244.1
carboxylic Acid 2 338.1 292.9
Oxymorphone-D3 305.1 230.0
6-MAM-D3 331.1 165
Phencyclidine-D5 249.1 96.0
7-Aminoclonazepam-D4 290.1 226.2
Pregabalin-D6 166.1 148.3
Alpha-hydroxyalprazolam-D5 330.1 302.0
Tapentadol-D3 225.1 107.0
Alprazolam-D5 314.1 286.0
Temazepam-D5 306.1 260.1
Amitriptyline-D3 281.1 202.0
Tramadol-13C, D3 268.3 58.1
Amphetamine-D5 141.1 96.0
Zolpidem-D7 315.1 242.1
Benzoylecgonine-D3 293.1 171.1
Buprenorphine-D4 472.3 400.1
Carisoprodol-D7 268.1 183.1
Citalopram-D6 331.1 109.0
Codeine-D3 303.2 152.1
Cotinine-D3 180.1 80.0
Diazepam-D5 290.1 198.1
EDDP-D3 281.0 234.1
Fentanyl-D5 342.2 105.1
Gabapentin-D10 182.2 164.2
Hydrocodone-D3 303.0 199.1
Hydromorphone-D3 289.0 185.0

Negative ESI Panel Table 2.
List of ESI- Analytes and Transitions
LC Conditions
Dimensions: 30 x 2.1 mm Analyte Q1 (m/z) Q3 (m/z)
Recommended Guard: AJ0-9209 Butalbital 1 223.0 42.0
Mobile Phase: A: 10 mM Ammonium Formate in Water (pH unadjusted)
B: 0.1 % Formic Acid in Methanol Butalbital 2 223.0 180.0
0 10
Phenobarbital 1 231.1 42.1
0.2 10 Phenobarbital 2 231.1 188.0
2.5 90
2.9 90 Secobarbital 1 237.0 42.0
3 10
3.5 10 Secobarbital 2 237.0 194.0
THC-COOH 1 343.1 245.0
Temperature: 40 °C
Injection Volume: 25 µL THC-COOH 2 343.1 191.2
MS/MS Conditions Butalbital-D5 228.0 42.0
Detector: SCIEX 6500 QTRAP®
Mode: Negative Electrospray Ionization Secobarbital-D5 242.0 42.0
Scan Type: MRM
THC-COOH-D9 352.1 254.0
Curtain Gas (CUR): 30
Gas 1 (GS1): 60
Gas 2 (GS1): 65
IS: -4500 V
Interface Heater: ON
Collision Gas (CAD): Medium The chromatographic conditions produced good peak shape
Entrance Potential (EP): -10 V and separation of analytes. Total run time for both panels was
7 minutes 30 seconds, with 48 of the 52 drugs analyzed in
positive ESI mode in just under 4 minutes (Figure 1), and the
remaining analytes analyzed in negative ESI mode in 3 minutes
and 30 seconds (Figure 2). Figures 3-5 demonstrate baseline
resolution for isobaric compounds morphine/hydromorphone/
norhydrocodone, codeine/hydrocodone, and oxymorphone/
noroxycodone, and near baseline resolution for naloxone/6-MAM
and EDDP/amitriptyline (Figures 6 and 7).
A six-point calibration curve was prepared in synthetic urine

with a linear range of 40% to 10x the cutoffs listed in Tables 3
and 4. An additional two points at 25x and 50x the cutoff were
run to extend the range for selected opiates. Data analysis was
performed using using SCIEX MultiQuant™ Software.
The methods were fully tested with the parameters tested

including precision and accuracy, LOD, and carryover studies.

Table 3.
ESI+ Retention Times and Cutoffs
Retention Cutoff Retention Cutoff

List of Analytes (ESI+) List of Analytes (ESI+)
Time (min) (ng/mL) Time (min) (ng/mL)
1 6-MAM 1.7 10 41 PCP 2.2 50
2 7-Aminoclonazepam 2.0 100 42 Pregabalin 0.4 100
3 alpha-hydroxyalprazolam 2.5 100 43 Sertraline 2.5 50
4 Alprazolam 2.7 100 44 Tapentadol 1.8 100
5 Amitriptyline 2.4 50 45 Temazepam 2.6 100
6 Amphetamine 1.1 100 46 Tramadol 1.9 100
7 Benzoylecgonine 1.8 50 47 Zolpidem 2.1 20
8 Buprenorphine 2.2 20 48 Zolpidem-4-carboxylic Acid 1.9 20
9 Carisoprodol 2.1 200
10 Citalopram 2.1 50 Table 4.
11 Codeine 1.6 100 ESI- Retention Times and Cutoffs.
12 Cotinine 1.7 100 Retention Cutoff

List of Analytes (ESI-) Time (min) (ng/mL)
13 Diazepam 2.8 100
1 Butalbital 1.7 100
14 EDDP 2.3 100
2 Phenobarbital 1.6 100
15 Fentanyl 2.1 10
3 Secobarbital 2 100
16 Fluoxetine 2.2 50
4 THC-COOH 2.7 50
17 Gabapentin 0.7 100
18 Hydrocodone 1.7 100
19 Hydromorphone 1.2 100
20 Imipramine 2.3 50
21 Lorazepam 2.3 100
22 MDMA 1.6 50
23 Meperidine 1.9 100
24 Meprobamate 1.9 100
25 Methadone 2.5 100
26 Methamphetamine 1.4 100
27 Methylphenidate 1.9 10
28 Morphine 1.0 100
29 Naloxone 1.6 50
30 Norbuprenorphine 2.0 20
31 Nordiazepam 2.5 100
32 Norfentanyl 1.8 10
33 Norhydrocodone 1.6 100
34 Noroxycodone 1.5 100
35 Normorphine 0.4 100
36 Nortriptyline 2.3 50
37 O-Desmethyltramadol 1.6 100
38 Oxycodone 1.7 100
39 Oxymorphone 1.1 100
40 Paroxetine 2.3 50

Figure 1.
TIC of ESI+ panel
1.03e7
1.00e7
9.50e6
9.00e6
8.50e6
8.00e6
7.50e6
7.00e6
6.50e6
Intensity, cps
6.00e6
5.50e6
5.00e6
4.50e6
4.00e6
3.50e6
3.00e6
2.50e6
2.00e6
App ID 23560
1.50e6
1.00e6
5.00e5
0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
Figure 2.
TIC of ESI- panel
3.0e4
2.8e4
2.6e4
2.4e4
2.2e4
2.0e4
Intensity, cps
1.8e4
1.6e4
App ID 23566
1.4e4
1.2e4
1.0e4
8000.0
6000.0
4000.0
2000.0
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 min
Figure 3.
XIC of morphine, hydromorphone, norhydrocodone (m/z = 286.1)
2.4e6
2.2e6
2.0e6
1.8e6
Hydromorphone
Intensity, cps
1.6e6
1.4e6 Norhydrocodone
App ID 23561
1.2e6
1.0e6
8.0e5
6.0e5
4.0e5 Morphine
2.0e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min

Figure 4.
XIC of codeine & hydrocodone (m/z = 300.1)
2.1e6
2.0e6
1.9e6
1.8e6
1.7e6
1.6e6 Hydrocodone
1.5e6
1.4e6
Intensity, cps
1.3e6
1.2e6
App ID 23562
1.1e6
1.0e6
9.0e5
8.0e5
7.0e5
6.0e5
5.0e5
4.0e5
Codeine
3.0e5
2.0e5
1.0e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
Figure 5.
XIC of oxymorphone & noroxycodone (m/z = 302.1)
1.29e6
1.20e6
1.10e6
1.00e6 Oxymorphone
9.00e5 Noroxycodone
Intensity, cps
8.00e5
App ID 23563
7.00e5
6.00e5
5.00e5
4.00e5
3.00e5
2.00e5
1.00e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
Figure 6.
XIC of naloxone & 6-MAM (m/z = 328.2)
Conclusion
4.9e5
4.5e5 Naloxone In this work we demonstrated how
3.5e5
research facilities can improve runtimes
3.0e5 without sacrificing results. The increases
Intensity, cps
in throughput may allow labs to better

App ID 23564
2.5e5
2.0e5 respond to the increases in demand

1.5e5
without costly investment in capital
6-MAM
1.0e5
equipment.
5.0e4
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min
References
1. Centers for Disease Control and Preven-
tion. Morbidity and Mortality Weekly Report.
Available from URL:http://www.cdc.gov/mmwr/
Figure 7. preview/mmwrhtml/mm6043a4.htm?s_cid=m-
XIC of EDDP & amitriptyline (m/z = 278.1) m6043a4_w#fig2. Accessed March 29, 2016.
2. Truong, T. (2016, March 28). Fentanyl deaths
4.4e6
4.2e6 surpass 2015 totals. WWLTV. Available from URL
4.0e6
3.8e6 http://www.wwltv.com/news/local/orleans/fen-
3.6e6
3.4e6 tanyl-deaths-surpass-2015-totals-1/107048722.
3.2e6 EDDP
3.0e6
2.8e6
Accessed April 7, 2016.
2.6e6
3. Stephenson, Crocker. (2016, April 7). Fentanyl-re-
App ID 23565
2.4e6
2.2e6
2.0e6
1.8e6
lated deaths spike to 30 in Milwaukee County in
1.6e6
1.4e6
‘16. Milwaukee Wisconsin Journal Sentinel. Avail-
1.2e6
1.0e6 able from URL http://www.jsonline.com/news/
8.0e5
6.0e5
Amitriptyline health/fentanyl-related-deaths-spike-to-30-in-mil-
4.0e5
2.0e5 waukee-county-in-16-b99701948z1-374873441.
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 min html.
Accessed April 7, 2016.

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Traditional Protein Precipitation vs.
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20011
4.5e6
4.0e6
3.5e6
Intensity, cps
3.0e6
2.5e6
2.0e6
1.5e6
ID 20011
App ID: 20011
1.0e6
App
5.0e5
0
0
1 2 3 4 5 6 7 8 10
9 11 min
~ 50x Zoom
9.0e4
8.5e4
8.0e4
7.5e4
7.0e4
6.5e4
6.0e4
Intensity, cps
5.5e4
5.0e4
4.5e4
4.0e4
3.5e4
3.0e4
2.5e4
Traditional Protein
Precipitation
2.0e4 Phospholipids
1.5e4 Removed with Phree Phree Phospholipid
Removal Plates
1.0e4
5000.0
0.0
0
1 2 3 4 5 6 7 8 10
9 11 min
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Comprehensive Drug Research Panel from Oral Fluid
Overview Materials and Methods

Drug testing in oral fluid has steadily gained popularity over other
sample types such as urine and whole blood. One reason for this Reagents and Chemicals
popularity is the easy non-intrusive sample collection procedure. Analytical reference standards and human saliva were purchased
Collection of oral fluid is especially suited for the road-side or from Cerilliant (Round Rock, TX, USA) and BioreclamationIVT
work place drug screening where access to a proper medical (Chastertown, MD, USA) respectively. The Intercept i2® and
facility or personnel may be limited. To address this increasing Quantisal® oral fluid collection devices were obtained from Ora-
need, many companies are manufacturing oral fluid collection Sure Technologies, Inc. (Bethlehem, PA) and Immunanalysis Cor-
devices with a collection applicator and a preservative (or ex- poration (Pomona, CA). All other chemicals were obtained from
traction) solution. The device buffer solution contains a number the Sigma-Aldrich Company (St. Louis, MO). Water purification
of excipients such as antibacterial agents and surfactants to via Sartorius Arium® Comfort II (Goettinger, Germany).
prevent bacterial growth and increase the analytes stability during
the sample transit to testing laboratories. The buffer solution
poses challenges, such as ion suppression for LC-MS analysis.
Here, we present a sample preparation procedure to significantly
reduce the effects of the device buffer solution while maintaining
a reproducible and consistent recovery of analytes.
Analysis Workflow
Collection Sample
Device Vial LC-MS Analysis
-A
-A
-C
-C

Sample Preparation
Sample Collection
Oral fluid specimens (from both Quantisal® and Intercept i2®
devices) were collected by placing the cellulose pad (on a plastic
stick) orally until the indicator window turns blue. The saturated
pad on the stick is then placed into the transport tube containing
the buffer solution.
Sample Pretreatment
For Intercept i2 device Remove plastic nipple at end of transport tube, place in centrifuge tube and centrifuge at 600 g for 15 min to collect
the supernatant. Transfer 0.5 mL of it into a vial to perform SPE extraction as below.
For Quantisal collection device Gently vortex the transport tube for 5-10 seconds before transferring 0.5 mL to a vial for SPE extraction. If solution is
left to settle for 1-2 minutes, centrifugation is not necessary.
SPE Method
Step Basic analyte extraction Acidic analyte extraction
Product Name: Strata®-X-C, 30 mg in 3 mL cartridge Strata-X-A, 30 mg in 3 mL cartridge
Part No.: 8B-S029-TBJ 8B-S123-TBJ
Condition: 1 mL 100 % Methanol 1 mL 100 % Methanol
Equilibrate: 1 mL DI Water 1 mL DI Water
Weak Wash: 1 mL DI Water 1 mL DI Water
Strong Wash: 1 mL 50:50 Acetone: Water 1 mL 50:50 Acetone : Water
Dry down: 3–4 minutes at maximum vacuum (15” Hg or higher) 3–4 minutes at maximum vacuum (15” Hg or higher)
Elute: 2 x 500 µL Methanol:Acetonitrile:Ammonium hydroxide (5:5:2) 2 x 500 µL Methanol:Acetonitrile:Formic acid (50:50:5)
Dry down: Evaporate to dryness under gentle Nitrogen @ 45-50 °C Evaporate to dryness under gentle Nitrogen @ 45-50 °C
Reconstitute: With 125 µL initial Mobile Phase With 125 µL initial Mobile Phase
Combine into a single sample vial
LC-MS/MS Conditions
Positive ESI Panel Negative ESI Panel
Column: Kinetex® 2.6 μm Biphenyl Column: Kinetex 2.6 μm Biphenyl

Dimensions: 50 x 3.0 mm Dimensions: 50 x 3.0 mm
Part No.: 00B-4622-Y0 Part No.: 00B-4622-Y0
Recommended Guard: AJ0-9208 Recommended Guard: AJ0-9208
Mobile Phase: A: 0.1 % Formic acid in Water Mobile Phase: A: 10 mM Ammonium formate in Water
B: 0.1 % Formic acid in Methanol B: 100 % Methanol
Gradient: Time (min) %B Gradient: Time (min) %B
0 10 0 10
4 95 4 95
5.5 95 5 95
5.51 10 5.01 10
7.5 10 7 10
Flow Rate: 0.5 mL/min Flow Rate: 0.5 mL/min
Temperature: Ambient Temperature: Ambient
Injection Volume: 10 µL Injection Volume: 10 µL
Detector: MS/MS (SCIEX 5000™ Triple Quad) Detector: MS/MS (SCIEX 5000 Triple Quad)
Detection Mode: ESI+ Detection Mode: ESI-

Results
Figure 1.
Representative TIC of ESI+ for Comprehensive Drug Panel Analytes
5.0e6
4.0e6
3.0e6
Intensity, cps
2.0e6
App ID 23719
1.0e6
0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 min
Figure 2.
Representative TIC of ESI- for Comprehensive Drug Panel Analytes
1.8e5
1.6e5
1.4e5
1.2e5
Intensity, cps
1.0e5
8.0e4
6.0e4
App ID 23720
4.0e4
2.0e4
0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 min

Table 1.
Absolute recovery with extraction condition and ionization for comprehensive drug panel analytes
from both devices (Acids on Strata ®-X-A, Bases and Neutrals on Strata-X-C).
Quantisal® Intercept i2®

Analyte Extraction Cartridge Ionization %Rec (%CV) %Rec (% CV)
1 6-MAM Strata-X-C ESI+ 67 (2.6) 79 (5.0)
2 7-Aminoclonazepam Strata-X-C ESI+ 54 (6.6) 45 (2.8)
3 a-Hydroxyalprazolam Strata-X-C ESI+ 54 (1.5) 69( 8.9)
4 Alprazolam Strata-X-C ESI+ 58 (5.0) 76 (7.1)
5 Amitriptyline Strata-X-C ESI+ 66 (5.6) 59 (12.8)
6 Amphetamine Strata-X-C ESI+ 65 (4.2) 60 (5.7)
7 Benzoylecgonine Strata-X-C ESI+ 58 (0.9) 82 (4.9)
9 Citalopram Strata-X-C ESI+ 58 (3.1) 104 (5.1)
10 Codeine Strata-X-C ESI+ 66 (8.6) 86 (11.3)
11 Fentanyl Strata-X-C ESI+ 56 (3.8) 84 (8.7)
12 Fluoxetine Strata-X-C ESI+ 47 (2.7) 67 (12.0)
13 Gabapentin Strata-X-C ESI+ 67 (5.8) 86 (6.0)
14 Hydrocodone Strata-X-C ESI+ 57 (4.0) 68 (7.3)
15 Hydromorphone Strata-X-C ESI+ 49 (8.0) 63 (3.6)
16 Imipramine Strata-X-C ESI+ 57 (0.5) 76 (7.4)
17 Lorazepam Strata-X-A ESI+ 64 (5.5) 89 (7.4)
18 MDMA Strata-X-C ESI+ 54 (2.7) 72 (10.0)
19 Meperidine Strata-X-C ESI+ 52 (2.7) 57 (7.3)
21 Methadone Strata-X-C ESI+ 80 (1.8) 85 (1.2)
22 Methamphetamine Strata-X-C ESI+ 66 (2.1) 73 (6.5)
23 Cotinine Strata-X-C ESI+ 52 (7.7) 62 (9.9)
24 Diazepam Strata-X-C ESI+ 54 (4.0) 75 (5.4)
25 EDDP Strata-X-C ESI+ 46 (8.0) 67 (13.8)
26 Methylphenidate Strata-X-C ESI+ 70(4.1) 72 (7.5)
27 Morphine Strata-X-C ESI+ 64 (2.2) 82 (3.3)
28 Nordiazepam Strata-X-C ESI+ 53 (2.4) 81 (5.5)
29 Norfentanyl Strata-X-C ESI+ 58 (3.7) 86 (5.1)
30 Norhydrocodone Strata-X-C ESI+ 57 (6.9) 76 (8.0)
31 Normorphine Strata-X-C ESI+ 53 (18.3) 73 (15.0)
32 Noroxycodone Strata-X-C ESI+ 52 (3.7) 67 (6.6)
33 Nortriptyline Strata-X-C ESI+ 62 (13.4) 82 (5.4)
34 Methyltramadol Strata-X-C ESI+ 74 (2.9) 89 (7.9)
35 Oxycodone Strata-X-C ESI+ 55 (5.5) 65 (4.8)
36 Oxymorphone Strata-X-C ESI+ 57 (5.8) 73 (9.5)
37 PCP Strata-X-C ESI+ 65 (6.96) 87 (11.1)
38 Paroxetine Strata-X-C ESI+ 53 (5.4) 73 (7.2)
39 Pregabalin Strata-X-C ESI+ 73 (2.6) 86 (4.3)
40 Sertaline Strata-X-C ESI+ 54 (17.4) 59 (2.6)
41 Tapentadol Strata-X-C ESI+ 64 (5.3) 92 (2.7)
42 Temazepam Strata-X-C ESI+ 50 (5.08) 54 (8.9)
43 Tramadol Strata-X-C ESI+ 80 (4.4) 83 (3.3)
44 Zolpidem Strata-X-C ESI+ 64 (2.4) 82 (4.3)
45 Zolpidem 4Carboxy Strata-X-C ESI+ 60 (5.1) 82 (4.3)
46 Butalbital Strata-X-A ESI- 60 (1.2) 93 (1.5)
47 Secobarbital Strata-X-A ESI- 65 (8.1) 74 (9.5)
48 Phenobarbital Strata-X-A ESI- 68 (1.3) 77 (5.8)
49 THC-COOH Strata-X-A ESI- 80 (4.0) 63 (9.5)

Discussion
In this method, we sought to develop a clean extraction meth- The intensity of interference peaks far exceeded the signal
od with high recovery and minimal interference. One way to obtained from the injection of a 100 % Methanol, as shown in see
demonstrate the effectiveness of a sample preparation technique Figures 3 and 4.
is to compare the total ion current of the extracted sample to the
When comparing Q1 scan of neat solution to the extract from
device buffer solution. We tested the buffer solutions from both
the method, interferences were successfully removed. There is
collection devices well beyond the analyte window. A Q1 scan,
strong evidence that removing buffer excipients improves the
performed on a SCIEX QTRAP® system, from 100 to 2,000 m/z
robustness of the LC-MS analysis. For further detail regarding our
revealed that in its untreated state both buffer solutions showed
method development please find our MSACL 2016 poster at:
strong interference—specifically two clusters of peaks, one group
in the mid-section of the chromatogram and another late-eluting www.phenomenex.com/MSACLOralFluidPoster.
group. On a closer examination, both groups of peaks displayed
MS spectra consistent with homologues or polymeric species.
Figure 3.
Quantisal® representative LC-MS chromatogram of buffer solution and
MS spectra of circled peaks. Blue trace: neat buffer solution, Green
trace: Blank (Methanol), and Red trace: Extracted sample.
6.5e9
6.0e9
5.5e9
5.0e9 Device Buffer Solution
4.5e9
Extracted Sample
4.0e9
Intensity, cps
3.5e9 Blank (Methanol)
3.0e9
2.5e9
2.0e9
App ID 23721
1.5e9
1.0e9
5.0e8
0.0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 min
4.1e6
4.0e6 6.5e6
3.8e6
3.6e6 6.0e6
3.4e6 5.5e6
3.2e6
3.0e6 5.0e6
Intensity, cps
2.8e6 4.5e6
2.6e6
Intensity, cps
2.4e6 4.0e6
2.2e6 3.5e6
2.0e6
1.8e6 3.0e6
1.6e6 2.5e6
1.4e6
1.2e6 2.0e6
1.0e6 1.5e6
8.0e5
6.0e5 1.0e6
4.0e5 5.0e5
2.0e5
0 0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z, Da m/z, Da

Figure 4.
Intercept i2® representative LC-MS chromatogram of buffer solution
and MS spectra of circled peaks. Blue trace: neat buffer solution, Green
trace: Blank (Methanol), and Red trace: Extracted sample.
8.0e9
7.0e9
Device Buffer Solution
6.0e9 Extracted Sample
5.0e9 Blank (Methanol)

Intensity, cps
4.0e9
3.0e9
2.0e9
App ID 23722
1.0e9
0.0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 min
5.4e6 7.2e6 7.0e6 7.0e6
5.0e6 7.0e6
6.5e6 6.5e6
6.5e6
4.5e6 6.0e6 6.0e6
6.0e6
5.5e6 5.5e6
4.0e6 5.5e6
5.0e6 5.0e6
5.0e6
3.5e6
4.5e6
Intensity, cps
Intensity, cps
Intensity, cps
Intensity, cps
4.5e6 4.5e6
3.0e6 4.0e6 4.0e6
4.0e6
2.5e6 3.5e6 3.5e6 3.5e6
3.0e6 3.0e6 3.0e6
2.0e6
2.5e6 2.5e6 2.5e6
1.5e6 2.0e6 2.0e6 2.0e6
1.0e6 1.5e6 1.5e6 1.5e6

1.0e6 1.0e6 1.0e6
5.0e5
5.0e5 5.0e5 5.0e5
0.0 0.0 0.0 0.0
200 400 600 800 1000 1200 1400 1600 1800 2000 200 400 600 800 1000 1200 1400 1600 1800 2000
200 400 600 800 1000 1200 1400 1600 1800 2000 200 400 600 800 1000 1200 1400 1600 1800 2000
m/z, Da m/z, Da m/z, Da m/z, Da
This SPE procedure was applied to a comprehensive drug Conclusion

panel consisting of 49 analytes representing a wide range To remove the harmful effect of excipients in the buffer solution,
acidic, basic and neutral compounds. In order to achieve an aggressive organic wash was necessary. The result is a very
recovery of all analytes and sufficiently remove the excipi- clean extract where a majority of the excipients were removed.
ents from the buffer solution an aggressive organic wash was To maximize recovery of a comprehensive list of drugs we utilized
required. The recovery of acidic analytes improved greatly by the strength of 2 sample prep devices—Strata X-A and Strata
using Strata®-X-A (anion exchange) procedure (Table 2). Both X-C. This sample procedure can provide a consistent level of
extraction procedures together exhibited excellent recovery accuracy and precision.
and reproducibility (4 replicates) for all probe compounds. The
combination of cleanliness and recovery is the primary driver
of a dual extraction method.

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Automation of Comprehensive Drug Research Panel from Oral Fluid
Overview Sample Prep Automation

There is a growing interest in oral fluid testing over other test The method, found on page 2, can be amenable to many different
matrices such as urine and blood because it is non-intrusive, automation platforms. We selected single carrier configuration with
convenient, and observable – making adulteration or substitution inserts that process up to eight samples in parallel with industry
difficult. In our previous technical note (www.phenomenex.com/ standard 3 mL cartridges. This selection accommodates loading
TN100) we developed a unique sample preparation procedure that of 40 SPE cartridges simultaneously. Because all wash solvents
encompasses a wide range of illicit and pain management drugs used in the extraction steps for both acidic and basic compounds
that result in a very clean extract with high recovery.1,2 Here, we were the same, automation allowed for simultaneous extraction.
extend our scope in automating the procedure by utilizing a liquid For the elution solvent, the liquid handler loaded two elution solvent
handler. The speed of data analysis has improved significantly in separately for the cationic and anionic sorbent media respectively.
the past decade due to increased detection capabilities of mass The duration and amount of positive pressure application to the
spectrometry coupled with liquid chromatography. Despite these extracted cartridges are all software controlled. Calibrators for the
advances, the biggest time constraint in sample processing is the 7-point linearity curve were prepared by serial dilution of the spiked
manual process around sample preparation. One survey attributes oral fluid. The curve spanned a total of seven concentration levels.
61% of an analytical chemist’s time is spent on sample process- The QC samples for extraction were prepared at two concentration
ing3. Automated liquid handling can increase lab productivity and levels. Addition of acidic (methanolic) solution to the eluted samples
circumvents human error. were necessary to prevent loss of free bases in the dry down step.
Materials and Methods

Analytical reference standards and human saliva were purchased
from Cerilliant® Corporation (Round Rock, TX) and Bioreclama-
tionIVT® (Chestertown, MD). The Quantisal oral fluid collection
devices were obtained from Immunalysis Corporation (Pomona,
CA). All other chemicals were obtained from the Sigma-Aldrich
Company (St. Louis, MO). D.I. water via Sartorius® arium® Comfort
II, courtesy of Sartorius Corporation (Bohemia, NY). Liquid Handling
via Tecan Freedom EVO® 100. (San Jose, CA)
Methods
Sample collection
1.0 mL of saliva was pipetted onto the application tip of the oral
fluid collection device. The saturated pad was then placed into the
transport tube containing the buffer solution.
Sample pretreatment
The Quantisal applicator tip that absorbed about 1 mL of oral fluid
was transferred to the transport tube containing the preservative
buffer and left for 1 to 2 hours. The transport tube was placed
directly on the automation platform. Liquid handler pipetted 0.5 mL
from the top of the sample, to avoid transfer of debris onto the SPE
cartridge.

SPE Method LC-MS/MS Conditions
Step Basic analyte extraction Acidic analyte extraction Positive ESI Panel
Column: Kinetex® 2.6 μm Biphenyl
Strata®-X-C, 30 mg in 3 mL
Product Name: Strata-X-A, 30 mg in 3 mL cartridge Dimensions: 50 x 3.0 mm
cartridge
Part No.: 00B-4622-Y0
Part No.: 8B-S029-TBJ 8B-S123-TBJ Recommended Guard: AJ0-9208
Mobile Phase: ‌
A: 0.1 % Formic acid in Water
Condition: 1 mL 100 % Methanol 1 mL 100 % Methanol B: 0.1 % Formic acid in Methanol
Equilibrate: 1 mL DI Water 1 mL DI Water 0 10
4 95
Combine 0.5 mL of pretreated Combine 0.5 mL of pretreated sample 5 95
sample with 1 mL 1 % formic acid, with 1 mL 1 % ammonium hydroxide, 5.01 10
Load:
mix/vortex 5-10 sec and load on mix/vortex 5-10 sec and load on 7.5 10
Strata-X-C. Strata-X-A. Flow Rate: 0.5 mL/min
Wash 1: 1 mL DI Water 1 mL DI Water Injection Volume: 10 µL
Detection: MS/MS (SCIEX API 5000™), ESI+
Wash 2: 1 mL Acetone/Water (50:50) 1 mL Acetone/Water (50:50)
2-3 minutes under positive

Dry down: 2-3 minutes under positive pressure
pressure
2 x 500 µL Methanol/Acetonitrile/
2 x 500 µL Methanol/Acetonitrile/
Elute: 28-30 % Ammonium Hydroxide
Conc Formic acid (50:50:5)
(5:5:2)
Negative ESI Panel
Wash 3: 30 µL of 50 mM HCl/Methanol – Column: Kinetex 2.6 μm Biphenyl
Evaporate to dryness under gentle Evaporate to dryness under gentle
Dry down: Part No.: 00B-4622-Y0
stream N2 @ 45-50 °C stream of N2 @ 45-50 °C
Reconstitute: With 125 µL initial mobile phase With 125 µL initial mobile phase Mobile Phase: ‌ 0.1 mM Ammonium formate in Water
A:
B: 100 % Methanol
Combine both fractions into a single autosampler vial Gradient: Time (min) %B
0 10
4 95
5 95
LC-MS/MS Conditions 5.01 10
The LC-MS/MS method utilized a Kinetex® Biphenyl 2.6 µm, 50 7 10
x 3.0 mm column (Part No.: 00B-4622-Y0) with a simple mobile Flow Rate: 0.5 mL/min
phase consisting of 0.1 % formic acid in water and methanol. A fast
LC gradient resulted in total run time of 5 min. The detection was
Detection: MS/MS (SCIEX API 5000), ESI-
carried out on a SCIEX API 5000™ equipped with ESI source. For
basic compounds the MS was operated under positive polarity and
in a separate injection, all acidic compounds (except lorazepam)
were analyzed in negative polarity.

Table 1.
Precision, accuracy and linear regression data for analytes
Analyte R2 QC1 (ng/mL) % Accuracy (QC1) % CV (QC1) QC2 (ng/mL) % Accuracy (QC2) % CV (QC2)
6-MAM 0.9987 2.5 107.2 15.7 8 85.9 7.6
7-Aminoclonazepam 0.9981 25 96.74 8.77 80 90.45 7.8
α-Hydroxyalprazolam 0.9971 25 92.84 12.49 80 91.78 10.28
Alprazolam 0.9995 25 98.39 14.06 80 93.4 5.5
Amitriptyline 0.9976 12.5 100.76 9.38 40 103.53 8.75
Amphetamine 0.9996 25 93.74 15.18 80 85.25 11.15
Benzoylecgonine 0.9978 25 93.95 10.27 80 88.24 14.55
Buprenorphine 0.9952 5 90.33 11.31 16 92.66 6.92
Citalopram 0.9992 12.5 89.47 4.18 40 95.41 5.38
Codeine 0.9992 25 95.05 12.27 80 92.15 16.05
Diazepam 0.9952 25 90.87 9.4 80 86.49 7.8
Fentanyl 0.9965 2.5 95.93 6.48 8 98.47 3.95
Fluoxetine 0.9952 25 93.98 4.83 80 89.29 5.7
Gabapentin 0.9946 25 92.21 13.38 80 97.81 10.66
Hydrocodone 0.9952 25 97.84 8.4 80 92.83 10.26
Hydromorphone 0.9975 25 99.73 7.7 80 90.15 0.86
Imipramine 0.9994 12.5 95.36 6.07 40 94.11 7.06
Lorazepam 0.9973 25 111.67 9.5 80 85.03 3.57
MDMA 0.9983 25 90.14 4.15 80 88.12 7.46
Meperidine 0.9959 25 99.74 6.56 80 99.49 8.2
Methadone 0.9993 25 95.93 7 80 88.25 7.33
Methamphetamine 0.9959 25 101.3 7.8 80 85.42 11.56
Methylphenidate 0.9991 2.5 97.78 12.07 8 90.79 9.1
Morphine 0.9995 25 105.74 8.5 80 100.87 7.9
Norbuprenorphine 0.9961 5 99.81 3.76 16 92.06 6.48
Nordiazepam 0.999 25 94.38 5.14 80 89.54 6.07
Norfentanyl 0.9998 2.5 107.31 11.47 8 88.97 2.67
Norhydrocodone 0.998 25 95.71 8.55 80 87.82 13.4
Noroxycodone 0.9988 25 107.26 6.26 80 94.07 6.85
Normorphine 0.9969 25 102.69 14.4 80 93.35 9.23
Nortriptyline 0.997 12.5 109.43 6.9 40 90.08 7.17
O-Desmethyltramadol 0.9991 25 104.9 3.15 80 95.5 11.2
Oxycodone 0.9972 25 94.88 4.66 80 87.75 10.59
Oxymorphone 0.9977 12.5 97.02 5.09 40 96.31 7.19
Paroxetine 0.9977 12.5 97.02 5.09 40 96.31 7.19
PCP 0.9974 12.5 96.77 4.58 40 100.83 3.04
Pregabalin 0.9992 25 96.2 10.98 80 103.13 5.11
Sertraline 0.9972 12.5 93.91 6.09 40 93.83 4.75
Tramadol 0.9973 25 101.84 4.49 80 103.45 7.92
Tapentadol 0.9978 25 95.16 8.07 80 93.96 8.48
Zolpidem 0.9987 5 93.27 7.1 16 91.19 6.8
Zolpidem-p-carboxylic 0.999 5 94.35 4.04 16 95.71 13.1
Butalbital 0.9992 25 96.3 11.18 80 90.81 3.96
Phenobarbital 0.9978 25 101.16 7.89 80 102.15 3.01
Secobarbital 0.9995 25 99 5.58 80 96.79 2.64
THC-COOH 0.9992 12.5 104.28 6.17 40 112.24 4.97

Figure 1.
Calibration curve of extracted samples representing dynamic range of
morphine (1-300ng/mL); R=0.9996.
10.8
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
Figure 2.
6 MAM (0.1-30ng/mL); R=0.9987.
1.00
0.95
0.90
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Figure 3.
zolpidem (0.5-60 ng/mL); R=0.9987.
9.8
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5 10 15 20 25 30 35 40 45 50 55 60
Figure 4.
norfentanyl (0.25-30 ng/mL); R=0.9998.
4.0
3.8
3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Figure 5.
butalbital (1-300 ng/mL); R=0.9992.
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
Figure 6.
THC-COOH (0.5 ng/mL-150 ng/mL); R=0.9990.
6.2
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Results and Discussion Conclusion
Calibration curves for extracted samples in this study covered Oral fluid is a complex matrix and LC-MS/MS analysis requires
a range from 0.1 ng/mL to 300 ng/mL (Figures 1-6). At least good recovery and a clean extract. Automated sample prep on a
five points per analyte were used for calibration curve. Beyond liquid handling robot helps rapidly increase throughput and reduce
300 ng/mL we encountered saturation of the MS for most of the human error. This procedure provides good dynamic calibration
analytes. Several analytes, zolpidem, norfentanyl, butalbital and range with good precision and accuracy with little human interven-
THC-COOH (Figures 3-6), displayed non-linear curve. A quadrat- tion.
ic calibration curve with 1/x weighting was applied to all analytes
in this assay. Table 1 shows, the correlation coefficient value (R) References:
in all cases were greater than 0.995. The precision and accura- 1. S. Huq, S. Sadjadi, L. Snow; “A Superior Sample Preparation of Comprehen-
cy for low and high QCs spanned from 3-15 % and 85-112 % sive Drug Panel Analytes from Oral Fluid Collection Devices; Phenomenex-
TN-0100
respectively for all four replicates at each concentration level.
2. S. Sadjadi, S. Huq, L. Snow; “An Investigation into Removing the Excipients
from Select Oral Fluids Collection Devices by SPE and LC-MS Detection;”
Mass Spec Application for Clinical Laboratory Conference, 2016
3. LC|GC Editors. “Overview of Sample Preparation” LC|GC November 01,
2015 Volume 33, Issue 11 (pg 46-51). Accessed on Sept 12, 2016 from
http://www.chromatographyonline.com/overview-sample-preparation.
Want even more info?


TN-1152
Toxicology

Ethyl Glucuronide and Ethyl Sulfate from Urine
Overview Luna Omega 5 µm Polar C18 Method Development
There is rarely a “silver bullet” in method development. A method For the analyses performed using the Luna Omega 5 µm Polar
that works great for one lab, may not work for another lab for a va- C18 column, we identified two mobile phase options. The first
riety of reasons. Difference in systems, samples, and the nature method consisted of a classical LC-MS mobile phase of water
of mass spectrometry can negatively affect transferring a method. with 0.1 % formic acid and methanol with 0.1 % formic acid. Rep-
In this technical note, we offer three options for the analysis of resentative XIC for the spiked urine sample are shown in Figure 1.
ethyl glucuronide (EtG) and ethyl sulfate (EtS) from urine to help
alleviate typical concerns seen with method transfer. For the second mobile phase option, we used 5 mM ammonium
formate (adjusted to pH 3.3 using formic acid) as the aqueous
EtS and EtG are direct metabolites of alcohol (ethanol). Both me- component and 0.1 % formic acid in acetonitrile as the organ-
tabolites can be detected up to 3 days after alcohol consump- ic solvent. Representative XIC for the spiked urine sample are
tion.1 Additionally, these analytes are non-volatile and therefore shown in Figure 2.
more stable in stored specimens than alcohol.
While the long detection window and sample stability in urine of- LC-MS/MS Conditions #1
fer strong benefits over blood alcohol level testing, the two com- Column: Luna Omega 5 µm Polar C18
pounds themselves pose several challenges. First, both com- Part No.: 00B-4754-E0
pounds are very polar and therefore don’t retain well on common Recommended Guard: AJ0-7601
reversed phase LC stationary phases. Secondly, sensitivity is Mobile Phase: A: 0.1% Formic acid in Water
generally low under negative ionization mode (ESI-) due to urinary B: 0.1% Formic acid in Methanol
matrix interferences (isobaric or not) that suppress analyte signal. Gradient: Time (min) B (%)
0 0
3 90
Ideally, a forensic toxicologist would want a method that retains 3.1 0
these polar compounds, gives excellent efficiency, sensitivity and Flow Rate: 0.7 mL/min
peak shape, and separates common urine interferences. Here Injection Volume: 5 µL
we offer three novel methods on two stationary phases—Luna® Temperature: 25°C
Omega 5 µm Polar C18 and Luna Omega 5 µm PS C18—that Detector: MS/MS (SCIEX API 4000™)
give increased retention, efficiency, and peak shape. While both
of these novel stationary phases can provide stability in 100 % Analyte Retention Time (min)
aqueous mobile phases and enhanced retention for highly polar
EtS 1.36
molecules such as EtG and EtS that do not typically retain well
on conventional C18 phases, the primary distinction between the EtG 2.05
two phases is that the Luna Omega 5 µm PS C18 phase contains LC-MS/MS Conditions #2
a positively-charged surface group that can interact via polar and Column: Luna Omega 5 µm Polar C18
ionic mechanisms with polar acids like EtS and result in dramatic Dimensions: 50 x 4.6 mm
shifts in selectivity and retention. Part No.: 00B-4754-E0
Materials and Methods Mobile Phase: A: 5 mM Ammonium formate (pH 3.3)
Analytical reference standards and human urine were pur- B: 0.1% Formic acid in Acetonitrile
chased from Cerilliant Corporation (Round Rock, TX, USA) and Gradient: Time (min) B (%)
0 5
BioreclamationIVT (Chestertown, MD, USA). All other chemi- 3 90
cals were obtained from the Sigma-Aldrich Company (St. Louis, 3.1 5
MO). Flow Rate: 0.7 mL/min
Experimental Conditions Temperature: 25°C
Detector: MS/MS (SCIEX API 4000)
All LC-MS/MS analyses were performed using Luna Omega 5 µm
Polar C18 and Luna Omega 5 µm PS C18 columns on an Agilent®
1200 LC system (Agilent Technologies, Palo Alto, CA, USA) and Analyte Retention Time (min)
SCIEX API 4000 (SCIEX, Framingham, MA, USA). EtS 1.21
EtG 1.86
Sample Preparation
Urine was spiked with EtG and EtS to a final concentration of
500 ng/mL. The spiked urine was then diluted 10-fold using the
same aqueous buffer that was used for the LC method on which
the samples were run (either water with 0.1 % formic acid or 5
mM ammonium formate pH 3.3). It is critical to match the diluent
with the buffer that is used for the LC method in order to optimize
chromatographic behavior.
1
The Role of Biomarkers in the Treatment of Alcohol Use Disorders (2012). SAMHSA Advisory. Volume 11 Issue 2.
Available at: http://store.samhsa.gov/shin/content/SMA12-4686/SMA12-4686.pdf

Figure 1. XIC for EtS and EtG Using LC-MS/MS Conditions #1
min
App ID 23898
min
Figure 2. XIC for EtS and EtG Using LC-MS/MS Conditions #2
min
App ID 23899
min

Using the Luna® Omega 5 µm Polar C18, both mobile phase op- PS C18 Results and Discussion
tions yield similar retention behavior for the EtG and EtS peaks. While EtG elutes at approximately the same time as the previously
In addition, in both cases, the EtS peak is well-resolved from the described methods, EtS retention is dramatically increased us-
large isobaric urinary interference that elutes early in the run. The ing the Omega PS C18 column—and actually eluted at about the
principle difference between the two methods is that, in our hands, same time or slightly later than the EtG peak. This dramatic shift
the method using 5 mM ammonium formate pH 3.3 appears to re- in retention moves the EtS analyte further from the large urinary
sult in fewer potential interferences in the EtG transitions (Figure isobaric interference. It should be noted that, as a method de-
2). Customer should evaluate both options and determine which velopment tip, adjusting the pH of the mobile phase can be used
works best in the context of their sample preparation procedures, to modify the retention of that EtS peak. Increasing the mobile
samples, and instrumentation. phase pH will result in further increasing the retention of the EtS,
while decreasing the mobile phase pH will reduce the retention
Luna Omega 5 µm PS C18 Method Development of the EtS peak. This can be a valuable tool in fine-tuning your
Using the second LC column option, Luna Omega 5 µm PS method.
C18, which contains the positively charged functional groups on
the surface of the silica, we found that the ideal selectivity was This enhanced retention of EtS is due to polar and ionic interac-
achieved when using the 5 mM ammonium formate buffer system. tions with the unique positive surface charge of the Luna Ome-
Representative XIC are shown in Figure 3. ga PS C18 phase chemistry. This unique mixed-mode retention
mechanism can be sensitive to pH fluctuations caused by the
LC-MS/MS Conditions plug of urine injected onto the column, so it was necessary to use
Column: Luna Omega 5 µm PS C18 the ammonium formate buffer as the aqueous component of the
Dimensions: 50 x 4.6 mm mobile phase because of its improved buffering capacity (as com-
Part No.: 00B-4753-E0 pared to simple water/formic acid). If we ran the same separation
Recommended Guard: AJ0-7606 using just water with 0.1 % formic acid as the aqueous buffer, we
Mobile Phase: A: 5 mM Ammonium formate (pH 3.3)
B: 0.1 % Formic acid in Acetonitrile
found that the retention of EtS was greatly increased, but it was
Gradient: Time (min) B (%) subject to peak distortion and not suitable for accurate quantita-
0 0 tion at injection volumes greater than ~5 µL.
3 90
3.1 0
Flow Rate: 0.7 mL/min Conclusion
Injection Volume: 5 µL In this technical note, we offer three possible solutions for the
Temperature: 25°C separation of EtS and EtG in urine. Evaluating different methods
Detector: MS/MS (SCIEX API 4000™) on different columns is the best way to identify the solution that
works best for your lab. In our method development, on our sys-
Analyte Retention time (min) tems, we have determined that using Luna Omega PS C18 with
EtS 1.95 an ammonium formate buffer provided the best retention of EtS
and the least amount of interference, but your results may vary
EtG 1.88
depending upon the specifics of your laboratory. However, we
are confident that one of these solutions, if not all, will work well
for you.
Figure 3. XIC of EtS and EtG from Urine using Luna Omega 5 µm PS C18
min
App ID 23900
App ID 23900
min

Ethyl Glucuronide and Ethyl Sulfate from Urine by UHPLC
Overview Experimental Conditions

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are metabolites LC-MS/MS method was performed using a Luna Omega 1.6 µm
of ethanol and are slowly eliminated in the urine. EtG has been Polar C18 column on a Shimadzu® Nexera® X2 with LC30A
measured up to 80 hours after heavy ethanol consumption. EtS can solvent module (Shimadzu Scientific Instruments, Columbia, MD,
also be used as an indicator of recent alcohol consumption. USA) and an upper pressure limit of 1000 bar. MS analysis was
performed using a SCIEX 4000 QTRAP® LC-MS/MS system (Sciex,
Toxicology laboratories have adopted LC-MS methodologies for
Framingham, MA, USA).
the quantitative analysis of both EtG and EtS. However, many labs
struggle in developing methods that are accurate and quantitative.
This is due in part because both metabolites are very polar in nature
(log P of EtG= 1.51, EtS= 0.62). With acceptable limits of quan- LC-MS/MS Conditions
titation being 500 ng/mL for EtG and 250 ng/mL for EtS1, an LC LC Column: Luna Omega 1.6 µm Polar C18
column with good polar selectivity is critical since EtG and EtS can Dimensions: 100 x 2.1 mm
elute close to matrix interferences. Part No.: 00D-4748-AN
In this study, we present a method for fast, accurate and reproduc- Mobile Phase: A: 10 mM Ammonium Formate in 0.1% Formic Acid,
ible quantitation and determination of both metabolites using Luna® pH unadjusted (~3.2)
Omega Polar C18, a novel sub-2 µm UHPLC column with polar B: Methanol/Acetonitrile 50:50
selectivity and 100% aqueous stability. The efficiency and
0.01 0
selectivity of this novel sub-2 µm phase allows for an accurate and
1 0
sensitive method while meeting throughput needs common for 2 25
many toxicology labs. 2.01 95
3 95
Temperature: 40°C
LC System: Shimadzu Nexera X2
Detection: MS/MS (SCIEX 4000 QTRAP®)
Ethyl-β-D-glucuronide, ethyl sulfate, and deuterated standards
Table 1.
(EtS-D5, EtG-D5) were purchased from Cerilliant (Cerilliant Corpora-
Primary and Secondary EtG/EtS MRM Transitions
tion, Round Rock, TX, USA).
Table 1. Primary and Secondary EtG/EtS MRM Transitions
Sample Preparation
ID Q1 Q3
Negative human urine was collected and spiked with ethyl-β-D-glu-
curonide and ethyl sulfate to prepare a stock standard at 20,000 EtG 1 (Primary) 221.2 75.0
ng/mL. This standard was serially diluted in urine to make a 5 point EtS 1 (Primary) 124.9 80.1
calibration curve. Two positive samples were also run. EtG_D5 226.1 85.1
Each standard and sample was diluted 10-fold with mobile phase EtS_D5 130.0 80.1
A (10 mM ammonium formate in 0.1% formic acid, pH unadjusted)
EtG 2 (Secondary) 221.2 85.1
and spiked with internal standard as follows: 100µL urine + 885µL
mobile phase A + 10µL EtS-D5 (10µg/mL) + 5µL EtG-D5 (100µg/ EtS 2 (Secondary) 124.9 97.0
mL).

Result and Discussion Table 3. EtG Quantitation
The use of the Luna® Omega 1.6 µm Polar C18 column allowed for Analyte Calculated
the fast elution of EtG and EtS in less than 2 min (Fig. 1), with total Accuracy
Sample Concentration Concentration
(%)
run times including column cleaning less than 5 minutes. This fast (ng/mL) (ng/mL)
separation allows for multiplexing techniques to increase through- Primary Standard 1 50 54 107.0
put. Note that the retention of EtS is ~0.9 minutes, and EtG is ~3 Primary Standard 2 100 94 94.0
minutes. This ensures that low responding EtG elutes away from
Primary Standard 3 500 482 96.4
major a matrix component that responds in the EtS channel, which
is a known cause for signal suppression. Primary Standard 4 1000 1030 103.0
Calibration curves were generated over a concentration of 50 ng/
Secondary Standard 1 50 54 108.0
mL to 5000 ng/mL. A quadratic regression was used to determine
relative response versus concentration using peak area of EtG and Secondary Standard 2 100 91 91.4
EtS/peak area of IS, with 1/X weighting factor), Correlation coef- Secondary Standard 3 500 502 100.0
ficient for EtS calibration curves are 0.997 and 0.9994 for primary Secondary Standard 4 1000 1000 100.0
and secondary MRM’s, respectively. Correlation coefficient for EtG Secondary Standard 5 5000 5000 100.0
calibration curves are 0.9998 and 0.9998 for primary and secondary
MRM’s, respectively.
Quantitative data for EtS and EtG standards are summarized in
Table 2 and Table 3. Sample concentrations were calculated using
the primary MRM channels, and results are summarized in Table 4. Table 4. Sample Results
Calculated
Sample Analyte Analyte Peak IS Peak Area
Concentration
Name Peak Name Area (counts) (counts)
(ng/mL)
Sample 1 ETS-1 763 6.75E+04 6.76E+04
Table 2. EtS Quantitation Sample 2 ETS-1 405 4.14E+04 7.50E+04

Sample 1 ETG-1 5000 4.69E+04 6.35E+04
Analyte Calculated
Accuracy Sample 2 ETG-1 2230 2.66E+04 5.93E+04
Sample ID Concentration Concentration
(%)
(ng/mL) (ng/mL)

Primary Standard 4 1000 987 98.7 Figure
Figure 1.
1. Extracted Ion Chromatograms for EtG/EtS and their Deuterated Internal Standards (EtG-D5 and EtS-D5),
Extracted Ion Chromatograms for EtG/EtS and their Deuterated Internal
500ng/mL
Primary Standard 5 5000 5000 100.0 (App ID 23368)
Standards (EtG-D5 and EtS-D5), 500ng/mL
5.8e4
4.0e4
2.0e4
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
2930
2000
1000
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
App ID 23268
5.8e4
4.0e4
2.0e4
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
Figure 2. Extracted Ion Chromatograms for Positive Sample 1

(App ID 23369)¬
3.4e4
3.0e4
Intensity, cps
2.0e4
1.0e4
Phenomenex l WEB: www.phenomenex.com 0.0

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min 83
2930
2000
1000
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
5.8e4
4.0e4
2.0e4
0.0
Figure 2. 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min Figure 3.
Extracted Ion Chromatograms for Positive Sample 1 Extracted Ion Chromatograms for EtG and EtS, 100 ng/mL
Figure 2. Extracted Ion Chromatograms for Positive Sample 1
(App ID 23369)¬
3.6e4 1
3.4e4
3.2e4 2
3.0e4
3.4e4
3.0e4
2.8e4
2.6e4
Intensity, cps
2.4e4
Intensity, cps
2.0e4 2.2e4
2.0e4
1.0e4 1.8e4
1.6e4
App ID 23597
0.0
1.2e4
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min 1.0e4
8000
6000
4000
2000
0
1 2 3 min
1.00e4
Intensity, cps
5000.00
Conclusions
A method using a Luna Omega 1.6 µm Polar C18, a new UHPLC
0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
column with excellent polar selectivity, can be used for accurate
and quantitative analysis for ethanol metabolites ethyl glucuronide
and ethyl sulfate. The method shows good linearity and accuracy
App ID 23269
from the concentration ranges of 50 to 5000 ng/mL, which is within

3.4e4
3.0e4
the specifications for most analytical laboratory needs.
Intensity, cps
2.0e4 Another method was also presented here as proof of concept for
1.0e4 mobile phase optimization. This method also showed good sepa-
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
ration for both EtG and EtS, with run times of less than 5 minutes,
and could be used for further studies for increasing sensitivity.
Further Method Development References:

Another method was implemented using different mobile phase 1. Jatlow, Peter I. et al. “Ethylglucuronide and Ethyl Sulfate Assays in Clinical
conditions, to compensate for differences in instrumentation and Trials, Interpretation and Limitations: Results of a Dose Ranging Alcohol
Challenge Study and Two Clinical Trials.” Alcoholism, clinical and experimental
method requirements.
140
research 38.7 (2014): 2056–2065. PMC. Web. 4 May 2016.
120
LC-MS/MS method was performed on an Agilent 1260 (Agilent
mAU
100
Technologies, Santa Clara, CA, USA) with an upper pressure limit
80
of 600 bar. MS analysis was performed using a SCIEX
mAU API 4000™
60
MS/MS.
40
20
0 LC-MS/MS Conditions 0
0 2 4Column:
6 Luna
8 Omega
min. 1.6 µm Polar C18 0 2 4 6 8 min.
Mobile Phase: A: 10 mM Ammonium Formate pH 3
0.1% Formic Acid, pH unadjusted (~3.2)
B: Acetonitrile with 0.1% Formic Acid
0 0
1 50
1.1 0
5 0
Temperature: 30°C
Instrument: : Agilent® 1260
Detection: MS/MS (SCIEX API 4000)

Synthetic Cathinones from Urine

This application features an expanded bath salt panel that includes Column: Luna Omega 3 µm Polar C18
recently DEA-scheduled bath salts 4-MEC, alpha-PVP, butylone, Dimension: 50 x 2.1 mm
3-FMC, and 4-FMC1 from urine. This method is a simple dilute and Part No.: 00B-4760-AN
shoot sample preparation followed by HPLC using Luna® Omega Recommended Guard: AJ0-7600
Mobile Phase: A: 0.1 % Formic acid in Water
3 µm Polar C18. Polar C18 phase chemistry achieved great peak B: 0.1 % Formic acid in Methanol
shape and resolution of isomeric pairs 3-MEC/4-MEC and ethy- Gradient: Time (min) B (%)
lone/butylone. 0.01 15
2 75
Sample Preparation 3 75
3.1 15
“Dilute-and-Shoot” method without enzymatic hydrolysis. Donor 4.5 controller stop
urine sample was spiked with standards at 25 ng/mL and diluted Flow Rate: 0.5 mL/min
1:4 with mobile phase A prior to injection. Temperature: 40 °C
Instrument: Agilent® 1260
Detection: MS/MS (SCIEX 4500 Triple Quad), ESI+
Sample: 1. Methcathinone 13. Ethylone
2. Buphedrone 14. alpha-PVP
3. Mephedrone 15. Pentylone
4. 3-FMC/4-FMC 16. 4-chloro-alpha-PPP
Extracted Ion Chromatogram of Bath Salts in Urine 5. 3-MEC 17. Pyrovalerone
6. 4-MEC 18. N-Ethylpentylone
4.7e6 7. Methedrone 19. PV8
4.6e6 8. 4-CMC 20. 4-chloro-alpha-PVP
4.4e6 9. alpha-PPP 21. 3,4-MDPV
4.2e6 10. Methylone 22. 3,4-tetramethylene-alpha-PVP
4.0e6 11. 4-CEC
3.8e6 12. Butylone
3.6e6
3.4e6
3.2e6
3.0e6
Intensity, cps
2.8e6
2.6e6
2.4e6
2.2e6
2.0e6
1.8e6
1.6e6
1.4e6
1.2e6
1.0e6
8.0e5
App ID 24205
6.0e5
4.0e5
2.0e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
1
Office of the Federal Register. Schedules of Controlled Substances: Placement of 10 Synthetic Cathinones into Schedule I. March 01, 2017. Available at
https://www.gpo.gov/fdsys/pkg/FR-2017-03-01/pdf/2017-03974.pdf.

Resolution of Isobaric Pairs
3-MEC / 4-MEC
4.7e6
4.6e6
4.4e6
4.2e6 3-MEC
4.0e6
3.8e6
3.6e6
3.4e6
3.2e6
3.0e6
2.8e6
Intensity, cps
2.6e6
2.4e6 4-MEC
2.2e6
2.0e6
1.8e6
1.6e6
1.4e6
1.2e6
1.0e6
8.0e5
App ID 24206
6.0e5
4.0e5
2.0e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Ethylone & Butylone

4.8e5
4.6e5
4.4e5
4.2e5 Ethylone
4.0e5
3.8e5
3.6e5
3.4e5
3.2e5
3.0e5
Intensity, cps
2.8e5
2.6e5
2.4e5
2.2e5
2.0e5
1.8e5
1.6e5
1.4e5
Butylone
1.2e5
1.0e5
8.0e4
App ID 24207
6.0e4
4.0e4
2.0e4
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

Synthetic Cannabinoids from Urine
Using Kinetex® Core-Shell Technology, a fast HPLC/UHPLC LC-MS/MS Conditions

screening method for synthetic cannabinoid urinary metabolites Table 1. Cannabinoid Peak Identification
was developed. Additional comprehensive confirmatory methods
for HPLC and UHPLC instrumentation were generated using Luna Peak No. Analyte Q1 Q3 Mode
and Kinetex LC columns. 1 JWH 073 N-butanoic acid metabolite-d5 363.1 159.1/155.2 +ve
Overview 2 JWH 073 N-butanoic acid metabolite 358.2 155.1/230.1 +ve

Synthetic cannabinoids are widely abused compounds that are 3 JWH 018 N-pentanoic acid metabolite-d4 376.2 155.1 +ve
DEA listed. Following use, the metabolized synthetic cannabi-
4 JWH 018 N-pentanoic acid metabolite 372.2 268.1/155.1 +ve
noids are excreted in the user’s urine. Hence, the predominant
approach for testing synthetic cannabinoid use is urine analysis. 5 JWH 073 N-(4-hydroxybutyl) metabolite 344.2 155.2/241.2 +ve
6 JWH 073 N-(3-hydroxybutyl) metabolite 344.2 155.2/241.2 +ve
Due to the related structures of the drug metabolites, there are
7 JWH 018 N-(5-hydroxypentyl) metabolite 358.3 155.2/230.2 +ve
a limited number of unique mass-transitions for the triple-quad-
rupole mass-spectrometer to monitor (Table 1). Therefore, it is 8 JWH 018 N-(4-hydroxypentyl) metabolite 358.3 155.2/230.2 +ve
critical that analytes requiring quantification are resolved chro- 9 AM2201 N-(4-hydroxypentyl) metabolite 376.1 155 +ve
matographically. This limitation can affect a laboratory’s ability to
10 AM694 436.1 231/309.2 +ve
rapidly process samples.
11 AM2201-d5 365.2 156.2/203.1 +ve
Depending on lab goals and instrumentation, there are different 12 AM2201 360.2 155.1/232.2 +ve
ways to perform the chromatographic analysis. Presented here is
a screening method, a low pressure confirmatory method, and a 13 JWH-073 328.2 155.2/200.2 +ve
UHPLC compatible method for the analysis of synthetic cannabi- 14 JWH-018 342.2 155.1/214.2 +ve
noid urinary metabolites.
Materials and Methods HPLC/UHPLC Screening Method

Standards were purchased from Cayman Chemicals, Ann Arbor, Column: Kinetex 2.6 µm C18
MI. Dimensions: 50 x 2.1 mm
Samples were analyzed using an Agilent® 1200 series HPLC Recommended Guard: AJ0-8782
(Agilent Technologies, Palo Alto, CA, USA.) coupled with an API Mobile Phase: A: 10 mM Ammonium Formate
B: Acetonitrile/Methanol (50:50)
5000™ triple-quadrupole mass-spectrometer (AB SCIEX, Fram-
ingham, MA, USA.). 0 40
5 90
8 90
Backpressure: 170 bar
Inj. Volume: 0.5 µL
Detection: MS/MS (SCIEX API 5000), ESI+
Sample: Synthetic cannabinoid urinary metabolites at 50 ng/mL (Table 1)
Figure 1. Synthetic cannabinoids on Kinetex 2.6 µm C18

22880
1.4e6 7,8
1.3e6
1.2e6
1.1e6
1.0e6 5
9.0e5
Intensity, cps
6
8.0e5
7.0e5
App ID 22880
6.0e5 11, 12
10
5.0e5
4.0e5 1,2 3,4
Go to www.phenomenex.com/ClinicalResources 3.0e5
2.0e5 9 13
to view technical resources 1.0e5 14

0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 min

HPLC Confirmatory Method Sample Pretreatment
Column: Luna® 3 µm C18(2) Combine 1 mL Human Urine sample (spiked with analytes at 50
Dimensions: 150 x 3.0 mm ng/mL), 2 mL of 100 mM sodium acetate buffer, pH 5.0, 25 μL
Part No.: 00F-4251-Y0 β-D-glucuronidase (Patella Vulgata from Sigma, 100KU). Vortex
10-15 secs, followed by incubation for 2 hours in a shaker at 55
Mobile Phase: A: 10 mM Ammonium Formate
B: Acetonitrile
°C to complete hydrolysis of the glucuronides.
0 45
10 52
11 95 SPE Protocol
14 95
Flow Rate: 0.7 mL/min Cartridge: Strata®-X-Drug B, 60 mg/3 mL
Part No.: 8B-S128-UBJ
Backpressure: 267 bar Condition: Not Required
Inj. Volume: 1.0 µL Load: Hydrolyzed sample (approx. 3 mL)
Temperature: Ambient Wash 1: 2 mL 100 mM Sodium acetate buffer, pH 5.0
Detection: AMS/MS (SCIEX API 5000™), ESI+ Wash 2: 2 mL Acetonitrile/ 100 mM Sodium acetate buffer,
pH 5.0 (30:70)
Sample: Synthetic cannabinoid urinary metabolites at 50 ng/mL (Table 1) Dry: >10” Hg for 5-10 minutes to remove residual water
Elute: 2 mL Ethyl acetate/Isopropanol (85:15)
Figure 2. Synthetic cannabinoids on Luna 3 µm C18(2)
22881 Dry down: Nitrogen gas at 45 °C
10
Reconstitute: 0.5 mL of initial mobile phase
1.04e6
1.00e6
9.50e5
9.00e5
8.50e5
8.00e5
7.50e5
7.00e5
The fast screening method utilizing a Kinetex 2.6 µm C18 column
11,12
Intensity, cps
6.50e5 1,2
6.00e5
5.50e5
3.4
allows for elution of all target compounds in less than 6 minutes.
App ID 22881
5.00e5
4.50e5
4.00e5
5
6
However, with a coelution of the 4- and 5-hydroxypentyl metabo-
3.50e5
3.00e5 7 8 13 lites, a positive result for either of these isomers would require the
2.50e5
2.00e5 use of one of the two confirmatory methods mentioned previously.
1.50e5 14
1.00e5
5.00e4
0.00
9
Both of these methods obtain resolution of all compounds, in-
cluding the difficult to resolve 4- and 5-hydroxypentyl metabo-
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 min
lites. The method involving the Luna 3 µm C18(2) is suitable for
HPLC instrumentation with lower pressure limitations, since it
has an initial backpressure of 267 bar. Meanwhile, the second
UHPLC Confirmatory Method confirmatory method utilizes a Kinetex 2.6 µm C18 that’s suitable
Column: Kinetex® 2.6 µm C18 for instrumentation with higher pressure limits such as UHPLC
Dimensions: 150 x 3.0 mm systems. By utilizing higher pressure rated instrumentation and
Part No.: 00F-4462-Y0 a high efficiency Kinetex 2.6 µm core-shell HPLC column, this
Recommended Guard: AJ0-8775 second confirmatory method obtains a much faster analysis time,
Mobile Phase: A: 10 mM Ammonium Formate
less than 10 minutes, than the lower pressure method. Addition-
B: Acetonitrile
ally, the Kinetex 5 µm C18 offers scientists another HPLC solu-
0 45 tion as selectivity would match with that of the Kinetex 2.6 µm
7 50 C18, but with efficiency levels on par to the Luna 3 µm C18(2) and
8 95
10 95
backpressures similar to a 5 µm fully porous media.
Backpressure: 375 bar
Inj. Volume: 1.0 µL
Temperature: Ambient Conclusion
Detection: MS/MS (SCIEX API 5000), ESI+ Synthetic cannabinoid urine analysis by LC-MS/MS focuses on
Sample: Synthetic cannabinoid urinary metabolites at 25 ng/mL (Table 1) the difficult to resolve cannabinoid metabolites. These drug-use
markers can be resolved using both fully porous Luna 3 µm HPLC
Figure 3. Synthetic cannabinoids on a Kinetex 2.6 µm C18
22882
media and the even higher efficiency Kinetex 2.6 µm HPLC/UH-
PLC media. In such cases, the high efficiency Kinetex 2.6 µm
1.7e6 10 11,12
1.6e6 HPLC/UHPLC column is recommended, since it obtains excellent
1.5e6
1.4e6
resolving power with limited backpressure.
1.3e6
1.2e6
Intensity, cps
1.1e6
1.0e6
1,2
9.0e5
3,4
App ID 22882
8.0e5
5
7.0e5 13
6
6.0e5
5.0e5 7
8
4.0e5 14
3.0e5
2.0e5
1.0e5 9
0.0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 min

Chiral Analysis of Synthetic Cannabinoids
In this technote, we describe a new targeted metabolomic approach Reagents and chemicals were obtained from Sigma-Aldrich
for assessing human synthetic cannabinoid exposure and pharma- (St. Louis, MO), Fisher Scientific (Pittsburgh, PA) and Hemostat
cology in blood and urine samples. The method utilizes a solid phase Laboratories (Dixon, CA). All sample and analytical standards in-
extraction (SPE) step followed by chiral LC-MS/MS analysis using cluding chiral isomers of JWH-018-(ω-1)-OH and AM2201-(ω-1)-
a Lux® polysaccharide-based chiral stationary column providing a OH were synthesized and provided by Cayman Chemical (Ann
reliable and reproducible method that can be transferred to clinical Arbor, MI). Strata®-X-Drug B polymeric strong cation-exchange
research, forensic, and toxicology labs for analytical testing. solid phase extraction cartridges, Lux Cellulose-3 analytical
column and SecurityGuard™ were obtained from Phenomenex
Overview (Torrance, CA). Samples were prepared using a Gilson Nebula
Herbal mixtures labeled as “K2” or “Spice” are often marketed as 215 solid phase extraction system (Middleton, WI) and analyzed
legal marijuana substitutes to circumvent existing regulations and using an Agilent® 1200 Series quaternary liquid chromatography
to avoid detection in standard drug screens. These products com- system (Santa Clara, CA) interfaced with an API 4000™ QTRAP®
monly contain the synthetic cannabinoid parent drugs JWH-018 tandem mass spectrometer (AB SCIEX, Framingham, MA). The
(Figure 1, Parent Drug 1) and AM2201 (Figure 1, Parent Drug 2), operation of the HPLC system and mass spectrometer was con-
both aminoalkylindoles and potent cannabinoid receptor agonists. trolled by Analyst® software (version 1.5.1, AB SCIEX, Framing-
ham, MA).
Unfortunately, little is known about the metabolism and toxicolo-
gy of these new drugs, but several studies have identified the (ω
Sample Pretreatment
-1)-hydroxyl metabolites enantiomers (Figure 1, Metabolites 1a/1b
or 2a/2b), (ω)-hydroxyl (Metabolite 3) and (ω)-carboxyl (Metabolite Urine sample
4) as primary biomarkers. These metabolites are also known to re- See Reference 1 for internal standard preparation and complete
tain significant in vitro and in vivo pharmacological activity, which experimental details.
may offer a mechanistic explanation of the adverse effects associ-
ated with synthetic cannabinoid use. Since the (ω-1)-hydroxyl me- Blood sample
tabolites of JWH-018 and AM2201 are chiral molecules, analytical Pipette 50 µL of blood into 950 µL 0.1M sodium acetate buffer
procedures capable of low level quantification of specific enantio- (pH 5.0) and spike with 10 µL of internal standard (IS) solution.
meric metabolites are required to further understand the metabolic The sample was then subjected to the SPE method described
and toxicological consequences of synthetic cannabinoid use. below.
SPE Protocol
This technote describes a novel LC-MS/MS method and SPE pro-
cedure capable of simultaneously resolving enantiomers as well as Cartridge: Strata-X-Drug B, 30 mg/3 mL
parent compounds and other related metabolites. Part No.: 8B-S128-TBJ
Condition: NOT REQUIRED
Equilibrate: NOT REQUIRED
Load: 1 mL pretreated sample
Wash: 1 mL Sodium acetate buffer
Materials and Methods Wash: 1 mL Sodium acetate buffer/Acetonitrile (70:30)
Figure 1. Elute: 5 mL Ethyl acetate/Isopropanol (85:15)
Dry: Dry down completely under a stream of nitrogen @ 60 °C
Parent drugs and metabolic oxidation compound structures. The circled Reconstitute: 100 µL Ethanol
compounds are chiral metabolites.

LC-MS/MS Conditions
Column: Lux® 3 μm Cellulose-3 Figures 2 and 3 represent LC-MS/MS chromatograms produced
Part No.: 00F-4492-B0
from 10 ng/mL and 5 ng/mL (respectively) synthetic cannabinoid
Recommended Guard: AJ0-8621 quality control samples in human urine and blood (all synthetic
Mobile Phase: A: 20 mM Ammonium bicarbonate cannabinoids standard were provided by Cayman Chemical). The
B: Acetonitrile chromatography was similar in all standards. The different color
Gradient: Time (min) B (%) tracings are representative of the Specific Reaction Monitoring
0 40 (SRM) experiments for each specific metabolite (see Table 1 for
10 95
SRM transitions).
12 95
15 40 Figure 2.
16 40 LC-MS/MS chromatograms produced from a representative 10 ng/mL
Flow Rate: 0.5 mL/min quality control sample prepared in pooled human urine
Temperature: 40 °C
( ω)-hydroxyl 3
JWH-018 1
Table 1.
AM2201 2
Mass Spectrometry Parameters for Selective Reaction Monitoring (SRM)
AM2201 ( ω − 1 ) 2a/2b
Analyte Q1 (m/z) Q3 (m/z)
( ω)-carboxyl 4
155*
AM2201 360 JWH-018 ( ω − 1 ) 1a/1b
127†
155*
(R)-(-)-AM2201-(ω-1)-OH 376
127†
155* 2e+4
(S)-(+)-AM2201-(ω-1)-OH 376
127† R R
7500 S
155* S
JWH-018 342
127† 5000
155* 2e+4
JWH-018-(ω)-OH 358
127† 2500
Signal (cps)
155*
JWH-018-(ω)-COOH 372
127†
155* 1e+4
(R)-(-)-JWH-018-(ω-1)-OH 358
127†
155*
App ID 22141
(S)-(+)-JWH-018-(ω-1)-OH
HPLC Analytical Method 358
127†
5e+3
*Quantification Ion †
Confirmation Ion

JWH-018 is metabolized in humans to form the (ω)-monohy-
2 4 6 8 min
droxylated, (ω)-carboxylated, and (ω-1)-monohydroxylated me-
Figure 3.
tabolites. AM2201 exposure leads to the formation of common LC-MS/MS chromatograms produced from a representative 5 ng/mL
(ω)-JWH-018 metabolites but also the distinct (ω-1)-monohy- quality control sample prepared in blood
droxylated AM2201 metabolites (Figure 1). A targeted metabo-
lomic approach that simultaneously measures each primary me-
ω)--hydroxyl 3
(ω
tabolite including the enantiomeric (ω-1)-metabolites is required
JWH- 018 1
to facilitate future clinical studies designed to understand the
AM2201 2
relationship between drug metabolism and clinical symptoms
documented after JWH-018 and AM2201 use. This new chiral AM2201 ( ω − 1 ) 2a/2b
LC-MS/MS approach achieves this requirement by resolving all ( ω)-carboxyl 4
metabolites of interest, including the R and S enantiomers of the JWH-018
- ( ω − 1 ) 1a/1b
(ω-1)-monohydroxylated metabolites of JWH-018 and AM2201 in
human urine and blood (Figures 2 and 3). The chromatography of
standards, QC samples, and unknown urine specimens is similar
for all matrices evaluated. Retention times established for each
analyte internal standard remained constant (±0.1 min). All cali-
bration curves were linear over the tested analytical range, where
r2 values were ≥0.99. The lower limits of quantification (LLOQ) for
each analyte are comparable to previous LLOQ measurements
reported with similar methods and mass spectra are consistent
with reference libraries previously reported. 2,3
App ID 22142

Figures 4a and 4b show representative LC-MS/MS chromato- JWH-018 and AM2201 are both subjected to cytochrome-P450
grams of human samples which tested positive for the (ω-1)-mo- mediated oxidation as well as uridine diphosphate glucuro-
nohydroxylated metabolite of JWH-018 (Figure 1, Metabolites nyltransferase (UGT) conjugation during the metabolism process.
1a/1b) and for the (ω-1)-monohydroxylated metabolite of AM2201 Cytochrome-P450 metabolizes JWH-018 and AM2201 in the lung
(Figure 1, Metabolites 2a/2b). As shown in Figure 1, the (ω-1)-mo- and liver while UGT is thought to be responsible for conjugating
nohydroxylated metabolites are unique biomarkers for each res- each metabolite with glucuronic acid. The pie chart inset compa-
pective synthetic cannabinoid. res the total relative percentage of free cytochrome P450 metabo-
lites versus the total relative percentage of glucuronic acid conju-
Figure 4. gates. The conjugation percentage was determined by measuring
Representative LC-MS/MS chromatograms produced from (A) a human metabolite concentrations pre- and post-ω-glucuronidase treat-
sample positive for the (ω-1)-monohydroxylated metabolite of JWH-018, ment (see Reference 1 for full details). These results show that
and (B) a human urine sample positive for the (ω-1)-monohydroxylated when research subjects are exposed to only JWH-018 (Figure 5),
metabolite of AM2201 the JWH-018 (ω-1)-monohydroxylated metabolite was excreted in
a much higher concentration as compared to the other JWH-018
metabolites studied. In contrast, AM2201 (ω-1)-monohydroxyla-
ted enantiomers were not preferentially excreted. This indicates
that UGTs may exhibit stereospecificity toward chiral synthetic
cannabinoid metabolites.
App ID 22143
App ID 22144
Conclusion
The LC-MS/MS method described in this technical note is capab-
le of fully resolving and quantifying chiral metabolites of JWH-018
and AM2201 as well as parent drugs. The precision and accuracy
measurements are similar to previously developed assays which
make this method easily transferrable to clinical research, foren-
sic, and toxicology labs for analytical testing. Moreover, this chiral
method can help researchers in the understanding and evaluation
In Figure 5, we demonstrate how this method was used to gene- of the clinical toxicity, pharmacodynamics and pharmacokinetics
rate the metabolic profile of a human urine specimen which tested of achiral and chiral synthetic cannabinoid metabolites produced
positive for JWH-018/AM2201 metabolites. The relative percenta- from JWH-018 and AM2201.
ge of each metabolite is represented and the relative percentage
of S or R enantiomers is provided above the bar of the correspon- References
ding (ω-1)-monohydroxylated metabolite. 1. Moran, J. H. et al. Anal. Chem. 2013, 85, 9390− 9399.
2. Moran, J. H. et al. Anal. Chem. 2011, 83, 6381− 6388.

Figure 5.
Metabolic profile generated from a human urine sample which tested 3. Moran, J. H. et al. Anal. Chem. 2011, 83, 4228− 4236.
positive forJWH-018/AM2201 metabolites
Parent Cannabinoids
Cytochrome P450 Metabolites
UDP-UGT Metabolites

THC-COOH and Barbiturates from Urine
Matthew Brusius and Daniel Spurgin
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
Overview SLE Protocol

In this technical note we develop a simplified liquid extraction
96-Well Plate: Novum™ SLE MAX
(SLE) application for barbiturates and THC-COOH from a urine
matrix containing β-glucuronidase followed by LC-MS/MS meth- Part No.: 8E-S138-5GA
od using Kinetex® EVO C18 LC column and SCIEX API 4000™ in Pretreated sample and pulse vacuum
negative mode electrospray ionization (ESI-). Load: (~5” Hg) for 5-10 seconds or until sample
has complete entered the sorbent
Materials 2 x 900 µL aliquots of 7 test solvents (see list below)
Secobarbital, Amobarbital, Phenobarbital, Butabital, Pento- followed by a short pulse 5” Hg to initiate flow. Allow
barbital, Pentobarbital-D5. 11-nor-9-Carboxy-Δ9-THC (COOH- Elute: the remaining solvent to flow via gravity. At the
THC) and 11-nor-9-Carboxy-Δ9-THC-D3 (COOH-THC-D3) completion of the second aliquot, apply vacuum at
5” Hg for 15 seconds to complete the extraction
standards were purchased from Cerilliant ® (Round Rock, TX).
Campbell Beta Glucuronidase Enzyme was purchased through Dry down:
Evaporate eluate to dryness @ room temperature
Campbell Science Product, 100,000 units/mL (Rockford, IL). under a gentle stream of nitrogen
Formic acid was purchased from Sigma-Aldrich ® (St. Lou- In 200 µL of methanol/water (50:50) containing
is, MO). HPLC-grade acetonitrile, methanol, methyl tert-butyl Reconstitute: 1 % NH4OH and 100 ng/mL of COOH-THC-D3
and 250 ng/mL of Pentobarbital-D5100 ng/mL
ether, ethyl acetate and hexane were purchased from Honey-
well™ (Morris Plains, NJ).
Tested Elution Solvents
For all elution solvents, the following sample pretreatment, SLE, 1. MTBE (Methyl Tert-Butyl Ether)
2. Ethyl Acetate
and LC conditions were followed, with the elution solvent being 3. Hexane/Ethyl acetate (1:1)
the only variant. 4. Hexane/Ethyl acetate (1:3)
5. Hexane/MTBE (1:1)
Sample Pretreatment 6. Hexane/MTBE (1:3)
Each sample was comprised of 150 µL of urine, 25 µL β-glucuro- 7. Hexane/Ethyl acetate (3:1)
nidase with 73 µL of 0.1M Ammonium Acetate buffer, pH 4 and
2 µL of barbiturate standards with THC-COOH. This 250 µL was LC-MS/MS Conditions
then combined with 200 µL of 1 % formic acid in water solution.
Column: Kinetex 2.6 µm EVO C18 100Å
Dimension: 50 x 2.1 mm
Mobile Phase: A: 10 mM Ammonium bicarbonate, pH 9
B: Acetonitrile
0 5
2 15
5 20
5.01 60
6 60
6.1 5
7.5 5
Temperature: Room temperature
Instrument: Agilent® 1200 LC System
Detection: MS/MS (SCIEX API 4000), ESI+

Results and Discussion Figure 2. Extracted samples post dry down and reconstitution in 200 µL of
While all seven solvents were screened for the SLE protocol, re- solvent for visual comparison. Solvents shown are: A) MTBE, B) EtOAc, C)
covery data was only generated for the two samples that were Hexane/MTBE (1:3), D)Hexane/EtOAC (3:1).
visually the cleanest after dry down and reconstitution. Figure 1
shows the sample vat before it is extracted using Novum™ SLE.
Figure 2 shows the reconstituted samples using the following ex- A B C D
tracting solvents: MTBE (Vial A), EtOAc (Vial B), Hexane/MTBE
(1:3) (Vial C) and the Hexane/EtOAc (Vial D). Vials C and D appear
clearer, therefore presumptively cleaner than Vials A and B. This
effect becomes more apparent with dirtier and more concentrated
urine samples.
Figure 1. Sample prior to extraction
Table 1. Recovery values of Barbiturates and THC-COOH for the two visu-
al clean solvents: Hexane/MTBE (1:3) and Hexane/EtOAc (3:1).
Hexane/ Hexane/
MTBE (1:3) EtOAc (3:1)
Average %CV Average %CV
Recovery (N=4) Recovery (N=4)
Amobarbital 105 % 8 93 % 4
Pentobarbital 95 % 9 87 % 4
Butabital 98 % 11 93 % 7
Phenobarbital 95 % 6 85 % 8
Secobarbital 103 % 12 88 % 13
THC-COOH 89 % 6 81 % 12

Hexane/MTBE (1:3)
Historically, MTBE provides acceptable recoveries for these class-

Basic mobile phase was selected in order to provide a stronger
es of compounds, but often times provides mediocre clean up. By
source of ionization in negative mode. We selected Kinetex EVO C18
cutting the MTBE with a 25 % volume of hexane, we are able to
for stable separation at pH 9. While the THC-COOH represented
drastically improve the cleanup of the sample while maintaining
here was spiked at 100 ng – and 10 ng spike of THC-COOH was
the recovery values that are shown in Table 1. This extracting
also performed; unfortunately due to system limitations, the peaks
solvent is the best choice to achieve the highest recovery.
for this were merely qualitative, and since they did not meet the
Hexane/EtOAc (3:1) required threshold for quantitation (10:1 signal-to-noise ratio),
integration was not performed and recovery was not calculated.
It has been discussed in previous technical notes that using However, relative approximations between pre and post spike
straight hexane, or any non-oxygen containing solvent by itself responses, coupled with the results presented herein, indicate
with Novvum SLE plates does not usually produce acceptable that the method should scale to a lower concentration of THC-
results.The addition of an oxygen containing solvent is usually re- COOH. An example of the neat standards are show in Figure 4,
quired to help solvate the sorbent in order to promote an efficient which illustrates the required separation of the pentobarbital and
extraction. Moreover, hexane is often so non-polar that many an- amobarbital isomers.
alytes are not soluble in it, which can be good for maintaining
sample cleanliness, but not for recovery. Therefore, the addition
of ethyl acetate is used to reduce the solvent’s hydrophobicity to
allow polar compounds (i.e. barbiturates) to be extracted while
also functioning as an oxygen containing modifier to further im-
prove extraction efficiency.
Additionally, this solvent mixture dries down faster than ethyl ac-
etate, while providing the acceptable recovery values (Table 1).
This solvent is the best choice for those who are interested in
good recovery with fast dry down. Figure 3 shows a chromato-
gram for this extract using this solvent.

Figure 3. Representative chromatogram for analytes extracted using Hexane/EtOAc (3:1). Peaks in order of elution: Phenobarbital
(1.15 min), Butabital (2.04 min), Pentobarbital (3.21 min), Amobarbital (3.48 min), Secobarbital (4.12 min), THC-COOH (6.05 min)
5.5e4
5.0e4
4.5e4
4.0e4
3.5e4
Intensity, cps
3.0e4
2.5e4
2.0e4
1.5e4
App ID 24126
1.0e4
5000.0
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 min
Figure 4. Representative chromatogram of neat standards of barbiturates and THC-COOH. Peaks in order of elution: Phenobarbital
(1.15 min), Butabital (2.04 min), Pentobarbital (3.21 min), Amobarbital (3.48 min), Secobarbital (4.12 min), THC-COOH (6.05 min)
4.8e4
4.6e4
4.4e4
4.2e4
4.0e4
3.8e4
3.6e4
3.4e4
3.2e4
3.0e4
2.8e4
2.6e4
Intensity, cps
2.4e4
2.2e4
2.0e4
1.8e4
1.6e4
1.4e4
1.2e4
App ID 24127
1.0e4
8000.0
6000.0
4000.0
2000.0
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 min
Conclusion
In this technical note we identified the best elution solvents for
the simultaneous extraction of barbiturates and THC-COOH from
urine. Using Novum SLE, coupled with Hexane/MTBE extracting
solvent provided a visually clean extract and good recovery for
all analytes. Hexane/EtOAc provides slightly less recovery with
the same visual cleanliness, but with the added benefit of fast
dry down.

Nicotine and Metabolites from Urine
Xianrong (Jenny) Wei, Ann Montross, and Sean Orlowicz
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
This method has been validated and shown acceptable accuracy SLE Protocol
and precision using a small volume of human urine in the analy- 96-Well Plate: Novum™ SLE MAX
sis of nicotine and its metabolites (anabasine, nornicotine, cotinine
and 3-hydroxycotinine). The modified method employed uses a Part No.: 8E-S138-5GA
Novum™ SLE MAX 96-well plate with pH adjusted organic elution Load:
Pretreated sample) and apply 5” Hg of vacuum to
solvent, and a Kinetex® EVO C18 column with high pH mobile phase initiate loading. Stop vacuum and wait 5 minutes
to increase analyte recoveries and sensitivity using LC-MS/MS. 3x 600 μL of 1 % Formic acid in DCM/IPA (95:5).
Elute:
Collect eluents in a collection plate (2 mL)
Overview Dry down:
under a slow stream of N2 at 45 °C for 60 minutes
Analysis of nicotine in urine has become more widespread with
In 300 μL of 20 mM Ammonium Bicarbonate,
the increased use of tobacco-less products, such as nicotine Reconstitute:
pH 8.2/Methanol (90:10) and vortex
lozenges, vaporizers, and gums. Analyzing nicotine metabolites
(anabasine, nornicotine, cotinine and 3-hydroxycotinine) allows
researchers to distinguish between an active smoker and some- LC-MS/MS Conditions
one using tobacco-less products. The presence of tobacco me- Column: Kinetex® 2.6 µm EVO C18
tabolites, anabasine and nornicotine, in urine is an indication of Dimensions: 100 x 3.0 mm
an active smoker. Part No.: 00D-4725-Y0
The goals of this study were to simplify the extraction process Mobile Phase: A: 20 mM Ammonium Bicarbonate, pH 8.2
using a Simplified Liquid Extraction (SLE) method, increase re- B: Methanol
coveries, and demonstrate the assay accuracy, precision and lin- 0 10
earity for all compounds under (GLP) Good Laboratory Pratice 3 90
guidance. The endogenous level of nicotine and its metabolites 5 90
5.01 10
were monitored during the method development for the deter- 6. 10
mination of LLOQ quantification level purpose. The method uses Flow Rate: 0.75 mL/min
higher pH mobile phase at pH 8.2 to ensure the separations of Temperature: Ambient
all compounds, and therefore the Kinetex EVO C18 column was Detection: MS/MS (SCIEX QTRAP 4500 Turbo™)
chosen for its widely stable pH range and chromatography. Sample: 1. 3-Hydroxycotinine
2. Nornicotine
3. Cotinine
Experimental Conditions 4. Anabasine
Reagents and Chemicals 5. Nicotine
All solvents and reagents were HPLC or analytical grade. HPLC ESI Ionization Source Parameters
Grade methanol was purchased from Honeywell Burdick & Curtain Gas (CUR): 20
Jackson® (Muskegon, MI), Milli-Q® water was used for sample Collision Gas (CAD): 7
preparation. HPLC Grade water was purchased from Honeywell Temperature (TEM): 600
Burdick & Jackson and used to prepare the LC mobile phase. Gas 1 (GS1): 50
Gas 2 (GS2): 50
Standards IS: 4500
Reference standards were purchased from Cerilliant® Corporation. Entrance Potential (EP): 10
Equipment and Materials Collision Potential (CXP): 12
Agilent® 1260 pumps and autosampler were used along with a

SCIEX QTRAP® 4500, positive polarity, ESI for detection.
Sample Pretreatment
200 µL of 2 % NH4OH in water and 50 µL of mixed deuterated in-
ternal standards (200 ng/mL each in water) were added to 100 µL
of human urine (spiked with analytes and/or blank) and mixed.

Table 1. Mass Transitions
Table 2. Novum Recovery
Q1 Mass Q3 Mass Dwell Time
Compounds DP CE
(amu) (amu) (amu)
Anabasine Nicotine Nornicotine Cotinine 3-OH-Cotinine
Anabasine 1* 163 80 25 80 27
QCL 71.1 82.8 62.4 96.2 73.0
Anabasine 2 163 120.1 25 80 22
QCM 79.5 83.9 64.3 95.6 77.5
Anabasine D4 1* 167 84 25 80 27
QCH 83.5 89.6 70.4 91.0 76.7
Anabasine D4 2 167 124 25 80 22
Cotinine 1 177 80 25 80 29
Cotinine 2* 177 98 25 70 28
Cotinine D3 1* 180 80 25 80 28
Cotinine D3 2 180 101 25 80 28
3-Hydroxycotinine 1* 193 80 25 60 34
3-Hydroxycotinine 2 193 134 25 60 25
3-Hydorxycotinine D3 1* 196 80 25 60 34
3-Hydroxycotinine D3 2 196 134 25 60 25
Nicotine 1 163 130 25 60 26
Nicotine 2* 163 132 25 60 20
Nicotine D4 1* 167 134 25 60 26
Nicotine D4 2 167 136 25 60 20
Nornicotine 1 149 80 25 50 25
Nornicotine 2* 149 130 25 50 21
Nornicotine D4 1 153 84 25 55 29
Nornicotine D4 2* 153 134 25 55 18
Anabasine 1* 163 80 25 80 27
* Quantitation mass
Figure 1. Representative chromatograms of QCM at 200 ng/mL in human urine

3
4.0E6
3.8E6
3.6E6
3.4E6 1. 3-Hydroxycotinine
3.2E6 5 2. Nornicotine
3.0E6 3. Cotinine
2.8E6 4. Anabasine
2.6E6 5. Nicotine
4
2.4E6
INTENSITY, CPS
2.2E6 1
2.0E6
1.8E6
1.6E6
2
1.4E6
1.2E6
1.0E6
8.0E5
App ID 23787
6.0E5
4.0E5
2.0E5
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min

Table 3. Accuracy and Precision
Anabasine Nicotine Nornicotine Cotinine 3-OH-Cotinine

Sample ID Conc. Found (ng/mL) - LLOQ
Nominal Conc. (ng/mL) 1 5 1 1 1
LLOQ_1 0.946 5.14 1.15 1.05 0.979
LLOQ_2 0.950 4.45 0.994 0.900 0.900
LLOQ_3 0.993 4.27 1.04 0.941 0.987
LLOQ_4 0.986 6.56 0.942 0.971 0.988
LLOQ_5 0.962 5.29 1.02 1.08 1.18
LLOQ_6 1.09 5.32 1.15 1.04 1.10
Mean Conc. Found (ng/mL) 0.988 5.172 1.05 0.997 1.02
STDV 0.054 0.812 0.085 0.070 0.10
CV% 5.42 15.7 8.06 7.06 9.80
Accuracy (%) 98.8 103 105 99.7 102
n= 6 6 6 6 6
Sample ID Conc. Found (ng/mL) - QCL
QCL_1 5.71 17.0 5.12 5.49 5.53
QCL_2 5.20 17.2 4.90 4.82 5.06
QCL_3 4.70 15.8 4.91 5.32 5.75
QCL_4 5.43 17.1 5.27 5.38 4.90
QCL_5 5.47 15.4 5.08 5.56 4.57
QCL_6 5.50 16.8 5.22 5.57 5.04
Mean Conc. Found (ng/mL) 5.34 16.55 5.08 5.36 5.14
STDV 0.351 0.758 0.154 0.281 0.430
CV% 6.58 4.58 3.03 5.24 8.36
Accuracy (%) 107 110 102 107 103
n= 6 6 6 6 6
Sample ID Conc. Found (ng/mL) - QCM
QCM_1 213 210 213 197 222
QCM_2 221 211 202 198 194
QCM_3 225 203 207 222 215
QCM_4 212 214 206 224 213
QCM_5 214 224 222 209 217
QCM_6 234 222 204 231 218
Mean Conc. Found (ng/mL) 220 214 209 214 213
STDV 8.61 7.87 7.38 14.29 9.87
CV% 3.92 3.68 3.53 6.69 4.63
Accuracy (%) 110 107 105 107 107
n= 6 6 6 6 6
Sample ID Conc. Found (ng/mL) - QCH
QCH_1 413 414 409 395 393
QCH_2 370 396 411 367 399
QCH_3 402 397 385 378 387
QCH_4 421 438 390 395 413
QCH_5 381 392 407 417 396
QCH_6 378 377 410 371 391
Mean Conc. Found (ng/mL) 394 402 402 387 397
STDV 20.8 21.1 11.4 18.8 9.1
CV% 5.27 5.24 2.84 4.85 2.29
Accuracy (%) 98.5 101 101 96.8 99.1
n= 6 6 6 6 6

Figure 2. Representative chromatograms of blank human urine
6720
8500 2500
a) Anabasine b) Nicotine c) Nornicotine

6500
2400
8000
6000 2300
7500 2200
5500 7000 2100
2000
5000 6500 1900
6000 1800
4500 1700
5500
1600
4000
5000 1500
Intensity, cps
Intensity, cps
Intensity, cps
1400
3500 4500
1300
4000 1200
3000
1100
3500
2500
1000
3000 900
2000 2500 800
700
2000 600
1500
1500 500
1000 400
1000
300
500 500 200
100
0 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 6.0 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min 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 6.0 min
1.24e4 1.8e4
d) Cotinine e) 3-Hydroxycotinine
1.20e4
1.7e4
1.15e4
1.10e4 1.6e4
1.05e4
1.5e4
1.00e4
9500.00 1.4e4
9000.00 1.3e4
8500.00
1.2e4
8000.00
7500.00 1.1e4
7000.00
Intensity, cps
Intensity, cps
1.0e4
6500.00
6000.00 9000.0
5500.00 8000.0
App ID 23784
5000.00
7000.0
4500.00
4000.00 6000.0
3500.00
5000.0
3000.00
2500.00
4000.0
2000.00 3000.0
1500.00
2000.0
1000.00
500.00 1000.0
0.00 0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min
Figure 3. Representative chromatogram of LLOQ at 1.0 ng/mL in human urine

2.6e4
3.5e4
2.4e4 a) Anabasine 3.4e4
3.2e4
b) Nicotine 4.0e4
c) Nornicotine
3.8e4
2.2e4 3.0e4 3.6e4
3.4e4
2.8e4
2.0e4
3.2e4
2.6e4
1.8e4 3.0e4
2.4e4
2.8e4
1.6e4 2.2e4 2.6e4
2.0e4 2.4e4
Intensity, cps
Intensity, cps
1.4e4
Intensity, cps
1.8e4 2.2e4
1.2e4 2.0e4
1.6e4
1.8e4
1.0e4 1.4e4
1.6e4
1.2e4
8000.0 1.4e4
1.0e4 1.2e4
6000.0
8000.0 1.0e4
8000.0
4000.0 6000.0
6000.0
4000.0
2000.0 4000.0
2000.0
2000.0
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min 0.0 0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min
Max. 3.0e4 cps.
3.2e4
4.0e4
3.8e4
d) Cotinine 3.0e4 e) 3-Hydroxycotinine
3.6e4
2.8e4
3.4e4
2.6e4
3.2e4
3.0e4 2.4e4
2.8e4 2.2e4
2.6e4
2.0e4
2.4e4
Intensity, cps
Intensity, cps
1.8e4
2.2e4
2.0e4 1.6e4
1.8e4 1.4e4
App ID 23785
1.6e4
1.2e4
1.4e4
1.0e4
1.2e4
1.0e4 8000.0
8000.0
6000.0
6000.0
4000.0
4000.0
2000.0 2000.0
0.0 0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min

Figure 4. Representative chromatogram of ULOQ at 500 ng/mL in human urine
4.1e6
4.0e6 5.4e6 2.6e6
3.8e6 a) Anabasine 5.0e6

b) Nicotine 2.4e6
c) Nornicotine
3.6e6
3.4e6 4.5e6 2.2e6
3.2e6
2.0e6
3.0e6 4.0e6
2.8e6 1.8e6
2.6e6 3.5e6
1.6e6
2.4e6
Intensity, cps
Intensity, cps
3.0e6
Intensity, cps
2.2e6 1.4e6
2.0e6
2.5e6 1.2e6
1.8e6
1.6e6 1.0e6
2.0e6
1.4e6
1.2e6 8.0e5
1.5e6
1.0e6
6.0e5
8.0e5
1.0e6
6.0e5 4.0e5
4.0e5
5.0e5 2.0e5
2.0e5
0.0 0.0 0.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min
6.5e6 3.9e6
d) Cotinine e) 3-Hydroxycotinine
3.8e6
6.0e6
3.6e6
3.4e6
5.5e6
3.2e6
5.0e6 3.0e6
2.8e6
4.5e6
2.6e6
4.0e6 2.4e6
Intensity, cps
Intensity, cps
2.2e6
3.5e6
2.0e6
3.0e6 1.8e6
App ID: 23786

1.6e6
2.5e6
1.4e6
2.0e6 1.2e6
1.0e6
1.5e6
8.0e5
1.0e6 6.0e5
4.0e5
5.0e5
2.0e5
0.0 0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min
Figure 5. Representative calibration curves in human urine (n=2)
7.5 7.5 7.5

7.0 7.0
Anabasine:1 - 500 ng/mL 7.0
Nornicotine:1 - 500 ng/mL 3-Hydroxycotinine:1 - 500 ng/mL
6.5 6.5 6.5
6.0 r = 0.9988 6.0 r = 0.9996 6.0 r = 0.9991
5.5 5.5 5.5
5.0 5.0 5.0

4.5 4.5 4.5

4.0 4.0 4.0
3.5 3.5 3.5
3.0 3.0 3.0
2.5 2.5 2.5
2.0 2.0 2.0
1.5 1.5 1.5
1.0 1.0 1.0
0.5 0.5 0.5
0.0 0.0 0.0
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Analyte Conc. / IS Conc.
7.5 7.5
7.0 Nicotine:5 - 500 ng/mL 7.0 Cotinine:1 - 500 ng/mL

6.5 6.5
r = 0.9990 r = 0.9993
6.0 6.0
5.5 5.5
5.0 5.0
4.5 4.5
4.0 4.0
3.5 3.5
3.0 3.0
2.5 2.5
2.0 2.0
1.5 1.5
1.0 1.0
0.5 0.5
0.0 0.0
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Analyte Conc. / IS Conc. Analyte Conc. / IS Conc.

Figure 6. Representative Internal Standard Plots separation and peak shape of analysis. A representative total ion
chromatogram at medium QC level (200 ng/mL) is shown in Fig-
3.1e6 ure 1. A representative chromatogram of a blank, Lowest Limit of
3.0e6
Quantitation (LLOQ), and Upper Limit of Quantitation (ULOQ) are
2.8e6 seen in Figures 2, 3, and 4 respectively.
2.6e6
2.4e6
The results of the accuracy and precision from the LLOQ, Quality
2.2e6
Control Low (QCL), Quality Control Medium (QCM), and Quality
Control High (QCH) meet the GLP acceptance criteria (Table 3).
2.0e6
± 20 % Mean of all samples

The assay accuracy at above four QC levels over all compounds
1.8e6
was between 96.8 and 110 %. The Coefficient of Variation (%
1.6e6 Cotinine CV) for the LLOQ ranged between 5.42 % and 15.7 %; the QCL
1.4e6
analytes had a % CV that ranged between 3.03 % and 8.36 %;
1.2e6 the QCM analytes had a % CV that ranged between 3.53 % and
1.0e6 6.69 %; the QCH analytes had a % CV that ranged between
8.0e5 2.29 % and 5.27 %, respectively. The linearity of each calibration
App ID 23788
6.0e5 curve had the following R2 values from 0.9988 to 0.9996 for all
4.0e5 analytes. The dynamic curve ranges are at 1-500 ng/mL for all
2.0e5
compounds except Nicotine at 5-500 ng/mL due to the endoge-
0.0
nous level of nicotine in human body (Figure 2 and 5).
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
Index
The internal standard responses were also evaluated. The assay
showed the consistent response of internal standard, which was
Results and Discussion within ±20 % of mean of standards and QCs in the run for all com-
The assay of nicotine and metabolites (anabasine, nornicotine, pounds (Figure 6).
cotinine and 3-hydroxycotinine) was validated for linearity, accu-
racy and precision using five levels of calibration standards for all Conclusion
compounds. This method was able to obtain the LLOQ at 1 ng/mL The simplified quantitation method of analysis of nicotine, anab-
(5 ng/mL for Nicotine) using SCIEX QTRAP® 4500. Also with the asine, nornicotine, cotinine and 3-hydroxycotinine in human urine
modifications to the SLE method with improved recovery of ana- shows acceptable, accurate, and precise results over the wide
lytes, the need of sample volume was reduced to 100 µL making calibration range. The modified SLE method was optimized to
this method ideal for any laboratory environment. provide higher recovery across all compounds. The Kinetex® EVO
C18 column coupled with high pH mobile phase showed better
There were several modifications made to the SLE extraction that peak shape, resolution and sensitivity in the analysis of nicotine
produced the higher recoveries over the entire calibration range. and its metabolites in human urine. The lower sample volume
The addition of 2 % ammonium hydroxide neutralized the analytes and optimized simplified liquid extraction method can be easily
before the sample loading, and the 1 % formic acid added to the transfer into various laboratory settings. The assay is also auto-
elution step were the two major modifications that increased re- mation friendly which allows for high throughput laboratories to
coveries as seen in Table 2. The mass transitions of the analytes utilize this method.
were listed in Table 1, respectively. The optimal mobile phase re-
quired 20 mM ammonium bicarbonate with a pH of 8.2 to ensure

Nicotine and Metabolites from Oral Fluid
Overview Sample Collection and Pretreatment

Nicotine is the active ingredient in tobacco and vape products that Oral fluid specimens, calibrators, and QC samples were collected
is highly addictive. Nicotine, its metabolite cotinine, and anabasine, on the cellulose pad (on a plastic stick from Intercept i2 devices)
a tobacco alkaloid, are often used to detect tobacco exposure. For until the indicator window turns blue. The saturated pad on the
tests like this, oral fluid collection has emerged as an alternative to stick was then placed into the transport tube containing the buffer
other biological matrices. The reason for its popularity is due to its solution and left overnight to represent transit time. The plastic
low chance of adulteration. In addition, oral fluid collection is easy nipple from the transport tube was removed and cellulose tab was
and non-invasive. As a test matrix, oral fluid shows great promise for placed in a centrifuge tube, which was centrifuged at 600 g for 15
detection of recent drug use, its disposition, and detection times.1 mins. The supernatant was collected for sample preparation.
However, it is not free of challenges. The additives and preservative
buffer (critical for preservation of the chemical integrity of the oral
fluid sample) present in the collection devices, must be removed for SPE Method
the proper upkeep of the mass spec detector.
In this technical note, we present a solid phase extraction (SPE) Step Basic analyte extraction
method for simultaneous detection and quantitation of nicotine,
96-Well Plate: Strata-X-C, 30 mg
cotinine, and anabasine from oral fluid. We employ Intercept i2®,
a commercially available oral fluid collection device for sample Part No.: 8E-S029-TGB
collection and transport. A polymeric, strong cation-exchange
sorbent, Strata®-X-C was utilized for solid phase extraction (SPE). pH Condition: 1 mL Methanol
stable Kinetex® 2.6 µm EVO C18 column was used for the analysis
purposes to obtain the best selectivity between the two isomeric Equilibrate: 1 mL DI Water
compounds nicotine and anabasine under an ESI mode in LC-MS/
Combine 0.5 mL of pretreated sample with 1 mL 1%
MS analysis. Formic acid, add 40 µL working internal standard
Load:
Materials and Methods solution (0.5 µg/mL). Mix/vortex 5-10 sec and load on
Strata-X-C.
Analytical reference standards were purchased from Cerilliant®
Weak Wash: 1 mL DI Water
Corporation (Round Rock, TX). Negative calibrator oral fluid and
Intercept i2 collection device were obtained from Orasure Technol- Strong Wash: 1 mL Acetone/Water (50:50)
ogies (Bethlehem, PA). All other chemicals, were obtained from the
Sigma-Aldrich Company (St. Louis, MO). Ultrapure DI water was Dry: 3-4 minutes at maximum vacuum (15” Hg or higher)
obtained from Sartorius® arium® comfort II, courtesy of Sartorius
Corporation (Bohemia, NY) Elute:
2 x 500 µL Ethyl acetate / Isopropanol / Ammonium
hydroxide (7:2:1)
Experimental Conditions Evaporate to dryness under gentle stream N2 at
Calibrators for the 7-point linearity curve were prepared by serial Dry down:
45-50 °C
dilution of the negative oral fluid control. The curve spans a total of
Reconstitute: With 200 µL initial mobile phase
seven concentration. level, covering a wide range. The QC samples
for extraction were prepared at three concentration (low, medium
and high) level.

LC-MS/MS Conditions 1.4
1.3
1.2
Column: Kinetex® 2.6 μm EVO C18

1.1
Dimensions: 100 x 3.0 mm 1.0
Part No.: 00D-4725-Y0 0.9
0.8
Recommended Guard: AJ0-9297 0.7
Mobile Phase: A: 20 mM Ammonium bicarbonate (pH 8.2) 0.6
B: Methanol 0.5
Gradient: Time (min) %B 0.4
0.3
0 10 0.2
3 90 0.1
5 90 0.0
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
5.01 10
6 10 Analyte Conc. / IS Conc.
Figure 3.
Linearity curve for nicotine (1 to 500 ng/mL) from oral fluid extracted
samples. R=0.9978.
Detection: MS/MS (SCIEX Triple Quad™ 4500), ESI+
Detection Mode: ESI+
Sample: 1. Cotinine
2. Cotinine-D3
3. Anabasine
4. Anabasine-D4
5. Nicotine
6. Nicotine-D4
0.80
0.75
0.70
0.65
N N 0.60
0.55
N H 0.50
N O N 0.45
N 0.40
0.35
H 0.30
0.25
0.20
0.15
Nicotine Cotinine Anabasine 0.10
logP=1.17 logP=0.21 logP=0.97 0.05
0.00
pKa=8.86 pKa=4.79 pKa=9.29
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Figure 1. Figure 4.
Structure, pKa and logP Value of Nicotine, Cotinine, and Anabasine Linearity curve for cotinine (1 to 500 ng/mL) from oral fluid extracted
samples. R=0.9996.
2.9e6 1,2
2.8e6
2.6e6
2.4e6 3,4
2.2e6
Intensity, cps
2.0e6 5,6
1.8e6
1.6e6
1.4e6
1.2e6
1.0e6
8.0e5 0.85
0.80
6.0e5 0.75
4.0e5
0.70
2.0e5 0.65
0.0 0.60
0.55
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 min 0.50
0.45
0.40
0.35
0.30
Figure 2. 0.25
0.20
Representative chromatogram of cotinine, anabasine, and nicotine from 0.15
oral fluid extracted samples. 0.10
0.05
0.00
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Figure 5.
Linearity curve for anabasine (1 to 500 ng/mL) from oral fluid extracted
samples. R=0.9983.

Table 1. References
Precision and accuracy data for 3 different levels of QC.
1. N. Robson, A. J. Bond and K. Wolff; “Salivary nicotine and cotinine concentrations
in unstimulated and stimulated saliva”; African Journal of Pharmacy and Pharmacol-
a) Nicotine
ogy, Vol. 4(2), 061-065, 2010.
Conc. (ng/mL) Sample Name Replicate % CV Accuracy
4 QC-Low 4 8.4 97.6
40 QC-Med 4 3.9 92.9 2. S. Sadjadi, S. Huq, L. Snow; “An Investigation into Removing the Excipients from
150 QC-High 4 5.1 94.0 Selected Oral Fluids Collection Devices by SPE and LC-MS Detection”; Mass Spec
Application for Clinical Laboratory Conference, 2016.
b) Cotinine
4 QC-Low 4 10.8 84.1
40 QC-Med 4 4.1 97.7
150 QC-High 4 4.6 95.2
c) Anabasine
4 QC-Low 4 3.2 95.3
40 QC-Med 4 3.7 94.1
150 QC-High 4 2.3 98.0

Kinetex® EVO C18 column was chosen because it is robust and
provided the best selectivity. The isomeric species nicotine and
anabasine were well resolved, as shown in Figure 2.
For SPE, a strong cation-exchanger sorbent, Strata®-X-C, was
used to allow the use a 50% acetone wash. This strong organ-
ic wash effectively removed the excipients from the sample and
transport buffer.2 An elution solvent composing of ethyl acetate,
isopropanol, and ammonium hydroxide eluted the analytes selec-
tively, leaving any residual impurities behind.
Calibration curves were constructed from spiked saliva samples
ranging from 1 to 500 ng/mL, using seven points for all three an-
alytes. The linearity curves with a quadratic fit and 1/x weighting
factor showed the correlation coefficient value (R) for all analytes
more than 0.997 (Figures 3-5) over a wide dynamic range.
Three levels of QC samples (low, medium and high) obtained a pre-
cision and accuracy ranging from 2-10% and 84-98% respectively,
for 4 replicate extraction at each concentration level (Table 1).
Conclusion
In this technical note we demonstrated an effective sample prepa-
ration technique for quantitation of nicotine, cotinine, and anabasine
from oral fluid. This is a reliable and reproducible assay with good
separation of isomeric analytes and demonstrates linear regression
values (R) more than 0.997 for all analytes that reflects the robust-
ness of the assay over a wide dynamic range.

TN-0103
Analyze 11 Synthetic Cannabinoids

in Under 10 Minutes!
Save Time and Solvent
with Strata-X-Drug B SPE
Condition: Not Required ! Polymeric SPE
Equilibrate: Not Required !
Load: Pre-treated Urine Sample
Wash 1: 2 mL 100 mM Sodium acetate buffer pH 5.0
Wash 2: 2 mL Acetonitrile/100 mM Sodium acetate buffer
pH 5.0 (30:70)
Dry: 15 minutes under 10” - 15” Hg vacuum
Elute: 2 mL Ethyl acetate / Isopropanol (85:15)
High Performance LC-MS/MS Analysis

with Kinetex 2.6 µm Core-Shell Column
20925
1.20e6
9
1.15e6
1.10e6 &
1.05e6 10 11 12
1.00e6 Dimensions: 150 x 3.0 mm
9.50e5 Part No.:
Mobile Phase:
00F-4462-Y0
A: 10 mM Ammonium formate
9.00e5 B: Acetonitrile
8.50e5 Gradient: Time (min) B (%)
0 45
8.00e5 7 50
7.50e5 7.01 95
10 95
7.00e5
Achieve separation of Flow Rate: 0.6 mL/min
Intensity, cps
6.50e5 Injection Volume: 10 µL

6.00e5
5.50e5
critical isomers Detection:
Sample:
MS/MS (SCIEX API 4000™), ESI+
1. JWH073-butanoic acid metabolite
2. JWH018-pentanoic acid metabolite
5.00e5 3. JWH073-4-hydroxybutyl metabolite
4.50e5 4. JWH073-3-hydroxybutyl metabolite
5. JWH018-5-hydroxypentyl metabolit
4.00e5 3 6. JWH018-4-hydroxypentyl metabolite
3.50e5 7. AM2201-4-hydroxypentyl metabolite
3.00e5 1 4 8. AM694
5 9. AM2201
2.50e5 2 6 10. D5-AM2201
2.00e5 & 11. JWH073
App ID 20925
12. JWH018
1.50e5 7
8
1.00e5
5.00e4
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 min
Scale your method with Kinetex
1.3 1.7
µm
2.6
µm
3.5
µm
5
µm
µm
™ ™ ™ ™
™

Chiral Analysis of Amphetamines and
Substituted Amphetamines
In this technical note, we demonstrate that the use of higher 1200 binary HPLC equipped with a multiple wavelength UV/Vis de-
pH (>9) under Reversed Phase mode can dramatically improve tector was used for the chromatographic separation and data acqui-
the chiral separation of various amphetamine derivatives sition. The HPLC column used in the successful separation of the
such as Methamphetamine, Ephedrine, Amphetamine, and components was an amphetamine selective phase, Lux 3 µm AMP
3,4-Methylenedioxymethamphetamine. 150 x 4.6 mm. Mobile phases used in this analysis were made using
DI water generated from a Sartorius® arium® water system, and other
Overview solvents were purchased at HPLC grade (>99.5 %) from Honeywell®,
When it comes to most chemical and physical interactions of enantio- all other reagents and additives were purchased at HPLC grade from
mers, separation can be difficult due to the fact that both enantiomers Sigma-Aldrich®.
share the same characteristics. Chiral compounds have historically
been separated via derivatization followed by chromatography. More
recently, enantioselective chromatographic techniques have been de-
veloped. Typical chiral chromatography is done via HPLC in normal Structures (pH Range, pKa’s)
or reversed phase conditions by a stationary phase functionalized
with a chiral selector. These stationary phases range from chiral-func-
tionalized silica, metal-ligand exchange, to polysaccharide phases.
H H H H
Recently, polysaccharide coated silica stationary phases have prov- H
N
H
N N N
H
N
en to be the most efficient in separating the widest variety of chiral O O

H
compounds. They are versatile in their compatibility and are stable

in acidic and basic conditions (pH 2-9) and can be run in normal, re-
versed, and polar organic solvents. While polysaccharide coated silica
stationary phases are the most popular and the most widely success-
ful columns in separating chiral compounds, there are still some com- Ephedrine (- , +) Methamphetamine (-, +) Phentermine
pounds that prove difficult to separate, especially when limited to a pKa = 9.52, 13.89 pKa= 10.28 pKa = 10.25
specific pH range.
Historically, chiral amphetamines were separated from one another
using chiral derivatizing agents to form diastereomers that could be H H H H
H H
separated by traditional means (reversed/normal phase HPLC, GC, N N
N
O
N
O
etc.) However, by derivatizing the analytes, they become subject to a
reaction yield, both for the diastereomer formation, and in the case of O O
preparative studies, the reverse reaction as well. While this may not
be as large of an issue on an analytical scale, analysis of the parent
compound is preferable to that of the derivativized product. Specif-
ically, for amphetamine analysis in toxicological assays, the parent Amphetamine (-, +) MDMA (-, +)
pKa = 9.90 pKa = 10.14
compound, in its unaltered form, is the primary component for the
qualitative and quantitative analysis for amphetamines in blood and
urine. By eliminating a derivatization, analyzing the parent compound
in its native form provides a more wholesome and precise means of
analysis.

Ephedrine (+ and -), Amphetamine (-), and Phentermine were added
in equal quantities to a 2 mL HPLC vial with a 200 µL glass insert.
Methamphetamine (+ and -) was added in a 1:10 ratio relative to
the other compounds in order to freely identify peaks of interest for
method development purposes. Optimized separation was able to
be achieved through manipulating a combination of high pH and
temperature on a Lux® 3 µm AMP stationary phase.
Analytical standards for Methamphetamine, Ephedrine, Amphet-
amine, 3, 4-Methylenedioxymethamphetamine (MDMA), and Phen-
termine were purchased from Cerilliant® in 1.0 mg/mL concentrations
and were further diluted to concentration in methanol. An Agilent®

Figure 1. Figure 3.
Low pH and High pH Comparison of Methamphetamine Enantiomers Temperature Effects on the Retention and Peak Shape of Chiral
Amphetamines
Column: Lux® 3 µm AMP Column: Lux 3 µm AMP
Dimensions: 150 x 4.6 mm Dimensions: 150 x 4.6 mm
Part No.: 00F-4751-E0 Part No.: 00F-4751-E0
Recommended Guard: AJ0-8476 Recommended Guard: AJ0-8476
Mobile Phase: 5 mM Ammonium Bicarbonate pH Mobile Phase: 5 mM Ammonium Bicarbonate pH 11.0
Low pH (3.0 or 11.0)/Methanol (45:55) adjusted with NH4OH/Methanol (45:55)
Flow Rate: 1.0 mL/min Flow Rate: 1.0 mL/min
Detection: UV/Vis @ 218 nm Detection: UV/Vis @ 218 nm (ambient)
Temperature: 45 °C Temperature: Top to Bottom (20 °C, 35 °C, 50 °C)
App ID 23957
mAU
App ID 23954
80
20 ºC
60
mAU
40 100
80
20 60
40
20
0 0
2 4 6 8 10 12 14 16 min
2 4 6 8 10 min
High pH 35 ºC
App ID 23955
App ID 23952
mAU
30 mAU
25 150
20 125
15 100
10 75
50
5 25
0 0
-5
-10 2 4 6 8 10 min
2 4 6 8 10 12 14 16 min
App ID 23956
mAU 50 ºC
200
150
Figure 2. 100
High pH Separation of Chiral Amphetamines 50
0
2 4 6 8 10 min
Column: Lux 3 µm AMP
Part No.: 00F-4751-E0
Mobile Phase: 5 mM Ammonium Bicarbonate pH 11.0
adjusted with NH4OH/Methanol (45:55) Figure 4.
Flow Rate: 1.0 mL/min Separation of MDMA Enantiomers
mAU 1
Detection: UV/Vis @ 218 nm
Column: Lux 3 µm AMP
175 2 Temperature: 45 °C
Sample: 1. (+) Ephedrine Part No.: 00F-4751-E0
150 2. (-) Amphetamine Recommended Guard: AJ0-8476
3. (-) Ephedrine Mobile Phase: 5 mM Ammonium Bicarbonate pH 11.0
125 4. (+) Methamphetamine
3 adjusted with NH4OH/Acetonitrile (40:60)
5. (-) Methamphetamine Flow Rate: 1.0 mL/min
100
6. Phentermine
App ID 23953
6 mAU
Detection: UV/Vis @ 218 nm
75 Temperature: Ambient
50 200
25
4 5
150
0
App ID 23674
100
4 6 8 10 min
50
0 1 2 3 4 5 6 7 8 min

Results and Discussion Conclusion
The advantages of high pH modes of separation are demonstrated Temperature and pH are important factors when optimizing a chiral
in Figure 1 where a low pH separation is compared to that of an separation. High pH conditions are especially useful when analyzing
appropriately adjusted high pH separation. The low pH mode lacks basic compounds like amphetamines. At low pH, amphetamines are
the selectivity to separate the enantiomers of methamphetamine. positively charged and in turn may interact readily with any exposed
Typically, when dealing with chiral separations on polysaccharide silanol sites and can instigate peak tailing and possibly overshadow
phases, it is advised to limit analysis to compounds that exhib- any chiral selectivity the stationary phase may otherwise demon-
it two of the following traits; aromaticity, charged groups, polar strate. Traditional polysaccharide HPLC columns do not typically fa-
groups, hydrophobic groups, or conjugation. Although in many cilitate a high pH mode of separation due to their decreased stability
cases, a molecule that displays more than two of these charac- in highly basic conditions. The Lux 3 µm AMP stationary phase does
teristics simultaneously in solution may cause additional modes of not suffer from the same instability as other polysaccharide phases.
interaction that may inhibit the chiral aspect of separation. Temperature can be a useful tool in speeding up analysis time,
increasing peak sharpness, reducing tailing, and in turn, increasing
When looking at the low pH example, the nitrogenous base of the peak symmetry. When used in combination with one another, pH and
methamphetamine molecule is protonated and charged, thus add- temperature have demonstrated to be useful tools in optimizing the
ing an additional mechanism under which the molecule can inter- separation of amphetamines and substituted amphetamines on a
act with the stationary phase and is likely the cause for peak tailing robust polysaccharide stationary phase.
and decreased selectivity. For the high pH example (pH 11), the
predominant species is deprotonated, and therefore relieves the
charge from the nitrogenous base and greatly reduces peak tailing,
resulting in successful enantiomeric separation. This correlation
holds true for many of the compounds in this analysis.
After defining the effective pH range for the analysis of these com-
pounds, a mixture of amphetamines was created to develop a Go to www.phenomenex.com/ClinicalResources
method for the selective separation of amphetamines and their en- to view technical resources
antiomers (Figure 2). This separation demonstrates how operating
at an optimized pH range, and with an optimized temperature set-
ting, can be beneficial to the speed and efficiency of a separation,
although, an optimized temperature setting would not have been
intuitive if not for the temperature comparison study shown in
Figure 3. This three chromatogram overlay shows the separation
of the amphetamine mixture at varying temperatures. At around
20 °C the mixture does show selectivity for the compounds of in-
terest, though, the peak tailing and broadening is substantial and
the late eluting peaks are heavily retained, thus leading to a longer
analysis time. As the temperature increases, each peak’s retention
time decreases along with a substantial increase in peak efficien-
cy and symmetry. As the temperature approaches 50 °C, the peak
shape continues to improve and run time continues to decrease,
though the threshold for resolution becomes compromised. After
further method development, the optimal temperature for maxi-
mizing separation, analysis time, and peak shape was found to be
45 °C. Investigation of more complex substituted amphetamines
was performed in the high pH medium shown in Figure 4. The
enantiomers of MDMA were able to be quickly and efficiently sep-
arated from one another on this phase, thus further reinforcing the
sentiment that this phase, when analyzed under appropriate pH
conditions, can demonstrate selectivity across numerous different
amphetamines and amphetamine-like compounds, and their en-
antiomers. Maximizing the positive effects of pH and temperature
can facilitate a fast, wholesome separation of amphetamines and
their enantiomers on this Lux® 3 µm AMP stationary phase.

novum
simplified liquid extraction
PATENT PENDING
PATENT PENDING
Liquid-Liquid Extraction is Not

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Try Novum, a NEW synthetic SLE sorbent
• Consistent cleanup and recovery from lot-to-lot

• Readily available
• Simplified method development
Recovery of 18 Pain Management Drugs from Urine using a single

extraction method on Novum SLE
120 %
100 %
80 %
Recovery (%)
60 %
40 %
20 %
0%
e
ne
e
ne
am
ne
am
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e
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P
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in
at
in
an
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PC
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A Simplified Procedure
1 Load diluted urine (diluted 1:1 with 0.5 M ammonium hydroxide) onto Novum 400 µL SLE
96-well plate, apply vacuum for 2-15 seconds.
2 Allow sample to soak into Novum SLE sorbent for 5 minutes.
3 Elute with ethyl acetate.
Disclaimer
Novum is patent pending.
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Sample
TN-0083
Preparation

Whole Blood Pretreatment Procedures
Due to their nature, bioanalytical samples often require a pre-treat- Note: A comparison of the above pre-treatment techniques for
ment step prior to further cleanup by solid phase extraction (SPE). whole blood was performed for acidic, basic, and neutral drugs.
Each sample matrix poses its own unique challenges such as the Recoveries were generally the highest when the whole blood
removal of proteins from plasma and serum, the disruption of red sample was diluted with buffer and subjected to physical dena-
blood cells in whole blood, hydrolysis of glucuronidated analytes turing (sonication) rather than chemical means. In fact, the son-
in urine, and homogenization of tissue samples. This technical ication process disrupts the cell membranes to the extent that
note outlines common sample pre-treatment procedures for bio- no clogging was observed when the procedure listed above was
analytical samples. followed.1
Plasma/Serum Urine
Plasma and serum pre-treatments are analyte dependent. If the Enzymatic hydrolysis is necessary in case of conjugated forms
analyte of interest is an acid, 2 % phosphoric acid can be used (sulfated or glucuronide form) of the analye present. Enzymatic
(20 µL 85 % H3PO4 to 1 mL of plasma or serum) to disrupt the hydrolysis requires specific pH (pH 4-5) and temperature ranges.
drug-protein interaction. If the analyte of interest is basic, 0.1 M An acid or base hydrolysis can be performed as well, depending
sodium hydroxide can be used to disrupt the drug-protein interac- on the stability of the compound.
tion. After addition of acid or base, the sample should be vortexed
for 20-30 seconds followed by centrifugation. The supernatant is a. Enzymatic hydrolysis: To 500 µL sample (spiked with analyte
now ready for further analysis. and internal standard) add 100 µL acidic buffer (see below)
and 20 µL beta-glucuronidase. Vortex 5-6 seconds. Incubate
Whole Blood in a water bath at 63 °C for 30 minutes. Transfer sample to a
There are several pre-treatment strategies that can be followed 96-well collection plate or autosampler vial. Seal and
for whole blood. If the target analyte is present in red blood cells, centrifuge for 10 minutes at 2,000 rpm.
a hemolysis step is necessary.
Preparation of acidic buffer (1.0 M acetate buffer, pH 4.0):
a. Hemolysis: To 0.2 mL whole blood (spiked with analytes and Dissolve 3.0 g of glacial acetic acid and 4.1 g of sodium
internal standard) in a 1.2 mL centrifuge tube, add 400 µL of acetate in a 1 L volumetric flask.
2 % zinc sulfate/80 % methanol. Vortex for 10-20 seconds
followed by centrifugation at 14,000 rpm for 10 minutes. Collect b. Base hydrolysis: To 1 mL urine (spiked with analyte and
the supernatant for further analysis. internal standard) add 100 µL 10 N KOH. Mix, vortex, and
hydrolyze for 20 minutes at 60 ˚C. Cool and adjust pH to 3.5-
Preparation of zinc sulfate/methanol: Into a 100 mL volumetric 4.0 (by adding 200 µL glacial acetic acid).
flask add 20 mL water and 3.6 g ZnSO4, 7H2O. After the
solution is clear and the salt crystals have dissolved, add c. Acid hydrolysis: To 1 mL urine add 0.25 mL HCl in a screw
100 % methanol. Refrigerate the solution at 2-8 °C for 7 days. capped test tube. Screw the tube top on loosely and heat in a
boiling water bath for 60 minutes. Adjust to pH 7 (or as
b. Osmotic breakdown: To 1 mL of whole blood add internal stan- needed) with 1.0 N NaOH.
dard and 4 mL of distilled water. Mix/vortex and let stand for 5
minutes. Centrifuge at 670 g for 10 minutes and discard the Saliva
pellet. Adjust the pH of the supernatant accordingly with the No hydrolysis is required for oral fluids and the generic protocol
addition of a buffer solution. used for plasma/serum pre-treatment may be followed.
c.
Sonication: Sonicate 1 mL whole blood for 15 minutes at
Tissue
room temperature. Add 3-6 mL of an appropriate pH buffer
Homogenize with organic or aqueous solvent depending upon
(such as potassium phosphate buffer). Mix/vortex. Let stand
analyte solubility. Settle, decant, centrifuge or filter supernatant.
for 5 minutes. Centrifuge at 670 g for 15 minutes. Analyze
Perform direct Matrix Solid Phase Dispersion (MSPD) extraction
supernatant.
on tissue.
References:
1. Chen et al., J. Anal. Toxicol. 1992, v18, pages 352-355.

Whole Blood Pretreatments for Basic Drugs
Overview Table 1. List of pain panel drugs

Drug analysis from whole blood is a challenge due to the com-
plex matrix and the presence of erythrocytes, the concentration Class Analyte Class Analyte
of which can vary from sample to sample.
Alprazolam Methadone
Any drug analysis in whole blood generally requires some form of Clonazepam EDDP
a pretreatment procedure that simplifies the blood matrix before
Diazepam Fentanyl
the actual analyte extraction. However, many procedures can ef-
ficiently be applied to hemolyze the erythrocytes. Likewise, there Flunitrazepam Norfentanyl
are equally many capable methods to precipitate the plasma pro- Lorazepam Meperidine
teins. Ultimately, a successful pretreatment method should pro- Synthetic Opioids
Midazolam Normeperidine
duce a high degree of recovery for all analytes in the sample. Benzodiazepines
Nordiazepam Naloxone
Here, we evaluated several common pretreatment procedures1,2
that both lyse the cells and precipitate the plasma proteins. These Oxazepam Norpropoxyphene
include acidic reagents (10 % TCA and 6 % HClO4), organic sol- Temazepam Propoxyphene
vent mixtures (MeOH and ACN) and a combination of zinc sulfate
α-Hydroxyalprazolam Sufentanil
and an organic solvent (Table 2). Subsequently, the clarified su-
pernatant was processed through a polymeric cation-exchange Alprazolam Naltrexone
solid phase extraction (SPE) tube (Strata®-X-C) to extract the ba-
Codeine Amphetamine
sic drugs. The list of the class of compounds that were targeted
for this work includes amphetamines (amphetamine, metham- Hydrocodone Methamphetamine
phetamine, MDA, MDEA, etc), natural and synthetic opiates (mor- Hydromorphone Amphetamines MDMA
phine, codeine, hydromorphone, hydrocodone, etc) illicit drugs Opiates
(PCP, benzoylecgonine), benzodiazepines (alprazolam, loraze- Morphine MDA
pam, etc) and analgesics (tramadol) (Table 1). 6-Acetylmorphine
MDEA
(6-MAM)
After the initial evaluation, we constructed a calibration curve us-
Oxymorphone Tramadol
ing whole blood as a matrix. Replicate analysis of 20 and 200 ng/
mL spiked whole blood samples were used for the precision and Phencyclidine Analgesics Carisoprodol
accuracy study.
Illicit Drugs Buprenorphine
Benzoylecgonine Norbuprenorphine
Table 2. Evaluated pretreatment methods
10 % TCA
Acidic Reagents
6 % HClO4
90:10 ACN/MeOH
50:50 ACN/MeOH
Organic Solvents 10:90 ACN/MeOH
100 % MeOH
100 % ACN
100 % ACN
Organic Solvent + ZnSO4 90:10 ACN/MeOH
100 % MeOH

Final Sample Preparation Method Discussion
It appeared that not all pretreatment procedures produced the best
Pretreatment: results. In general, the acidic pretreatment methods produced the
• Add 0.5 mL whole blood (with EDTA preservative) into a glass poorest overall responses despite a very clear pretreated sample
tube (Figures 1 and 5), with some exceptions. This might be due to
the hydrophobic nature of many of the compounds used here that
• Add 100 μL 5 % (w/v) ZnSO4 and vortex 3-5 sec are soluble (or stable) in the pretreated acidic solutions (Figures
6 and 7). Use of MeOH alone was not adequate to achieve a clear
• Add 1.5 mL of chilled (~0 °C) 90:10 ACN/MeOH while vortexing
enough supernatant from whole blood. Acetonitrile with a small
• Centrifuge samples at 6000 rpm for 10 min and transfer amount of MeOH produced better than expected recoveries for
supernatant some classes of compounds such as opiates (Figures 2 and 6).
• To supernatant, add 4 mL of aqueous 0.1 % formic acid to The pretreatment procedure with ZnSO4 and an organic solvent
acidify and dilute the mixture (acetonitrile or 90:10 ACN/MeOH) produced the most consistent
results for many compounds (Figure 3 and Table 3).
SPE Protocol
Cartridge: Strata®-X-C, 30 mg, mL with adapter cap (Part No. AH0-7191) and a 12 mL Reservoir
(Part No. AH0-7003)
Figure 1. Acidic supernatant
Part No.: 8B-S029-TBJ
Condition: 1 mL Methanol Addition of Acidic Reagent
Equilibrate: 1 mL Water • Both 10 % trichloroacetic acid and 6 % perchloric acid produced very
Wash 1: 1 mL 0.1 % Formic acid in water clear, colorless supernatants, even after dilution.
Wash 2: 1 mL 30 % Methanol in water
Dry: 3 to 4 mins at high vacuum (~10” of Hg)
Elute: 2x 500 μL (2 aliquots of 500 μL) Ethyl acetate/Isopropanol/Ammonium hydroxide
(70:20:10)
Dry down: Evaporate to dryness under nitrogen at 40-45 ºC
Reconstitute: With 500 μL of 85:15 (A/B) of LC mobile phase
Final LC-MS Method

Part No.: 00B-4622-Y0
Mobile Phase: A: 0.1 % Formic acid in water
B: 0.1 % Formic acid in methanol 6 % HCIO4 10 % TCA
0 10
2.5 100
3.5 100
3.6 10
5 10
System: Shimadzu® Nexera® UFLC with LC-30AD pumps
Injection: 10 μL
Key to Abbreviations:
ZnSO4 = Zinc sulfate
HClO4 = Perchloric acid
MeOH = Methanol
ACN = Acetonitrile
TCA = Trichloroacetic acid

Figure 2. Supernatant from organic solvents
Addition of Organic Solvents
• MeOH and/or mostly methanol solvent produced a supernatant with a slight hazy yellow tint
• Acetonitrile and/or mostly acetonitrile solvent produced a more clear and colorless supernatant. However,
the supernatant turned cloudy when diluted.
Supernatant Supernatant post dilution
(A)
90:10 MeOH/ACN
(B)
50:50 MeOH/ACN
(C)
10:90 MeOH/ACN
Figure 3. Supernatant from ZnSO4 and ACN post dilution

Addition of ZnSO4 and an Organic Solvent
• When added to whole blood, zinc sulfate produced a bright red cloudy solution.
• Upon addition of the organic solvent, a brown precipitate appeared which lead to a clear and colorless
supernatant.
• Upon dilution, the solution showed slight turbidity (ZnSO4 and ACN supernatant is shown below).

Figure 4. Representative chromatogram of the basic compounds
5.0e6
4.5e6
4.0e6
3.5e6
Intensity, cps
3.0e6
2.5e6
2.0e6
1.5e6
App ID 22773
1.0e6
5.0e5
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min
Figure 5. Comparison of the effects of various pretreatment options on amphetamine. Chromatograms

are overlaid with time shift to provide clarity.
3.5e5
3.4e5
3.2e5
ZnSO4 + ACN
3.0e5 10:90 MeOH/ACN
2.8e5
2.4e5
2.2e5
6 % HClO4
Intensity, cps

1.8e5
1.6e5 10 % TCA
1.4e5
1.2e5
1.0e5
App ID 22775
8.0e4
6.0e4
4.0e4
2.0e4
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min

Figure 6. Comparison of the effects of various pretreatment options on Codeine (peak 1) and Hydrocodone
(peak 2). Chromatograms are overlaid with time shift to provide clarity.
2
2
5.5e4
10:90 MeOH/ACN
5.0e4 1
1 ZnSO4+ACN
4.5e4 2
6% HClO4
1
Intensity, cps
4.0e4
3.5e4 10% TCA

3.0e4
1
1 1
2.5e4
2 2
2
2.0e4
App ID 23033
1.5e4
1.0e4
5000.0
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min
Figure 7. Comparison of the effects of various pretreatment options on Benzoylecgonine. Chromatograms are
overlaid with time shift to provide clarity.
4.5e5
4.0e5
ZnSO4 + ACN
3.5e5
10:90 MeOH/ACN
3.0e5
Intensity, cps
90:10 MeOH/ACN
2.5e5
2.0e5 6 % HClO4
1.0e5
App ID 22776
10 % TCA
5.0e4
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min

Table 3. Method Precision and Accuracy Data Based on Replicate Quality Control Samples
Expected Conc, %RSD % Accuracy Expected Conc, %RSD % Accuracy

Analyte Class
ng/mL (Low) (Low) (Low) ng/mL (High) (High) (High)
Alprazolam 20 10 108 200 12 104

Clonazepam 20 9 114 200 11 107
Diazepam 20 10 97 200 12 103
Flunitrazepam 20 7 112 200 7 105
Lorazepam Benzodiazepines 20 15 108 200 10 111
Midazolam 20 7 115 200 4 88
Nordiazepam 20 11 101 200 13 103
Oxazepam 20 6 108 200 12 105
Temazepam 20 7 105 200 9 99
α-Hydroxyalprazolam 20 6 88 200 11 91
Codeine 20 10 92 200 9 87
Oxycodone 20 4 95 200 2 93
Opiates
Hydromorphone 20 6 85 200 14 97
Hydrocodone 20 7 105 200 9 99
Morphine 20 8 91 200 10 86
Methadone 20 10 110 200 5 105
EDDP 20 10 98 200 2 94
6-MAM 20 7 100 200 7 100
Fentanyl 20 9 115 200 5 90
Norfentanyl 20 12 95 200 4 100
Meperidine Synthetic Opioids 20 7 105 200 7 103
Normeperidine 20 9 103 200 10 102
Naloxone 20 7 118 200 3 111
Norpropoxyphene 20 9 100 200 14 90
Propoxyphene 20 12 111 200 5 101
Sufentanil 20 8 98 200 7 89
Naltrexone 20 4 113 200 11 108
Amphetamine 20 9 107 200 11 107
Methamphetamine 20 10 115 200 3 96
Amphetamines
MDMA 20 13 111 200 8 92
MDA 20 8 102 200 7 101
MDEA 20 16 107 200 3 105
Tramadol 20 4 105 200 3 96
Carisoprodol Analgesics 20 8 106 200 9 100
Buprenorphine 20 12 104 200 11 101
Norbuprenorphine 20 6 105 200 13 106
Phencyclidine 20 7 110 200 4 92
Illicit Drugs
Benzoylecgonine 20 10 104 200 5 101

Conclusion
We have developed an effective pretreatment and SPE cleanup
method for whole blood followed by targeted LC-MS/MS anal-
ysis. Zinc sulfate with an acetonitrile and methanol combination
provided the best response for the majority of analytes tested.
Further sample cleanup was successfully accomplished by us-
ing a cationic exchange SPE, Strata®-X-C, sorbent. This combi-
nation can greatly improve the column longevity and maintain a
clean LC-MS/MS system. The combination of the pretreatment
and SPE method can sufficiently be employed for a wide range
of basic compounds generally encountered in existing pain panel
methods.
Acknowledgement
The authors are grateful for Mrs. Amanda Kaspick’s gracious con-
tributions and efforts to this project.
References
1. Polson C, Sarkar P, Incledon B, Raguvaran V, Grant R.; Optimization of pro-
tein precipitation based upon effectiveness of protein removal and ionization
effect in liquid chromatography-tandem mass spectrometry; Chromatogr B
Analyt Technol Biomed Life Sci. 2003 Mar 5;785(2):263-75
2. Wang, P (Ed); High throughput analysis in pharmaceutical industry, 2008,
CRC Press
3. S Huq, S Sadjadi, and S Countryman, “Quantitative Bio Analysis of the Most
Commonly Used Pain Medications in Urine Using a Reliable Sample Prepa-
ration Technique in Combination With an API 5000 LC-MS-MS”, Mass Spec
Application for Clinical Laboratory Conference, 2013
4. Dalsgaard et al,Quantitative Analysis of 30 Drugs in Whole Blood by SPE and
UHPLC-TOF-MS. (2013) J of Forensic Science and Criminology 1(1):101

Maximizing Recoveries on Novum™ SLE
Overview Sample Pretreatment

Supported liquid extraction (SLE) has been developed as an alter- 100 µL of plasma was diluted with 100 µL of water.
native sample preparation method to remedy some of the draw-
backs of liquid-liquid extraction (LLE) including the formation
SLE Protocol
of emulsions, the use of large amounts of hazardous solvents, 96-Well Plate: Novum SLE MINI
and difficulty in automating. Traditional SLE implements a solid Part No.: 8E-S138-FGA
support of diatomaceous earth that absorbs an aqueous-based
200 µL of pretreated sample and pulse vacuum
sample to increase the surface area between it and the extracting Load: (~5” Hg) for 10 seconds or until sample has
solvent, yielding an extremely efficient liquid-liquid extraction with complete entered the sorbent. Wait 5 minutes.
no emulsions and no need to manually separate liquids. Although Dispense 1000 µL of elution solvent onto the Novum SLE
efficient, traditional diatomaceous earth-based SLE products can media and allow the solvent to elute by gravity
be prone to sorbent inconsistencies and limited availability due (~5 min elution time) and collect the eluant. Apply vacuum
to the fact that diatomaceous earth is a natural resource. Recent Elute: at 5” Hg for 10 secs to complete the extraction.
advances have introduced Novum Simplified Liquid Extraction Note: Prolonged application of vacuum will
(SLE) products which are packed with a unique, synthetic SLE result in elution of plasma from the Novum SLE
sorbent that alleviates the challenges associated with a natural media and into the final extracted solvent.
resource. The novel, synthetic Novum SLE sorbent also provides Evaporate the final extract to complete dryness
additional benefits including a higher loading capacity and con- Dry down: under a slow stream of N 2 at 40°C. Initially at
12 psi (for 20 minutes), then at 30 psi.
sistently more in-well headroom. For example, Novum MAX 96-
well plates have a loading capacity of 450 µL and a headroom of Reconstitute:
In 100 µL Acetonitrile/Water (20:80) by vortexing
1.15 mL while the Biotage® Isolute® SLE+ 400 µL 96-well plate the plate at 1200 rpm for 5 minutes.
has a headroom of 1.05 mL.
Sample Pretreatment
In an effort to transition methods away from traditional diato-
100 μL of plasma was diluted with 200 μL of water.
maceous earth SLE materials, we performed a case study on a
steroid panel with a specific focus on betamethasone to demon- SLE Protocol
strate two effective ways to increase recovery using the novel 96-Well Plate: Novum SLE MINI
Novum synthetic SLE material.
Part No.: 8E-S138-FGA
Materials and Methods 300 μL of pretreated sample water/plasma (2:1) and
All reagents and solvents were HPLC or analytical grade. Analy- Load: pulse vacuum (~5” Hg) for 10 seconds or until sample
ses were performed using an API 3000™ LC-MS/MS (AB SCIEX, has complete entered the sorbent. Wait 5 minutes.
Framingham, MA). Dispense 1000 μL of elution solvent onto the Novum SLE
media and allow the solvent to elute by gravity
(~5 min elution time) and collect the eluant. Apply
Elute: vacuum at 5” Hg for 10 secs to complete the extraction.
result in elution of plasma from the Novum SLE
Dry down: under a slow stream of N 2 at 40°C. Initially at
12 psi (for 20 minutes), then at 30 psi.
In 100 μL Acetonitrile/Water (20:80) by vortexing
Reconstitute:
the plate at 1200 rpm for 5 minutes.
HPLC Conditions
HPLC analysis was performed with a Kinetex® 2.6 µm C18, 50 x
2.0 mm column packed with core-shell particle media, providing
high resolving power and fast analysis time.

Mobile Phase: A: Water with 0.1 % Formic acid
B: Acetonitrile with 0.1 % Formic acid
0 20
3 95
3.50 95
3.51 20
6 20

Results and Discussion Figure 2. Elution Solvent Optimization Study
Maximizing Recoveries by Diluting to the Maximum Aqueous 120%

Holding Capacity of the Plate
100%
Figure 1 compares recoveries of 9 steroids from plasma using
two different loading volumes on the Novum™ SLE MINI 96-well 80%
Recovery (%)
plate while using dichloromentane (DCM) as the extraction sol-
vent. It was determined that loading a total volume of 300 µL 60%
(100 µL plasma plus 200 µL water) produced higher recoveries as
compared to loading a total volume of 200 µL. By diluting a sam- 40%
ple to the total water holding capacity of the plate (300 µL on the 20%
Novum SLE MINI plate and 450 µL on the Novum SLE MAX plate),
there is a higher surface area for interaction with the extraction 0%
solvent, improving the rate at which analytes partition into the or-
e
e
e
e
ne
ne
id
n
noen
at
n
on
lo
so
ro
ro
on
ganic elution solvent. A similar effect can be achieved when using
et
saos
tis
no
te
te
ni
Ac
et
htah
or
ci
os
es
ed
Ac
ete
m
e
the Novum SLE MAX 96-well plate by diluting the sample to the
tic
Pr
mam
on
ia
ro
ne
or
Tr
tis
-P
etat
lo
C
plate’s full aqueous holding capacity of 450 µL.
BeB
or
H
no
O
ci
α-
m
11
DCM 10% EtOAc in DCM EtOAc
ia
Tr
Figure 1. Analyte Recoveries under Various Loading Volumes
120% Table 1. Final Optimized Method
100%
Novum SLE Optimized DCM Extraction
80%
(Novum SLE MINI 96-well plate, Part No.8E-S138-FGA)
Recovery (%)
1. Dilute 100 µL plasma with 200 µL water

60%
2. Load 300 µL of sample onto Novum SLE MINI 96-Well Plate
40% 3. Apply 5" Hg of vacuum for 10 seconds
20% 4. Wait for five minutes

5. Add 1 mL DCM:EtOAc (90:10) and elute by gravity
0%
6. Apply 5" Hg for 10 seconds to complete elution
e
ne
e
ne
e
e
id
e
e
on
at
on
n
n
lo
on
so
ro
ro
et
no
is
tis
7. Blow down with N2 (40 °C for 60 minutes). Initially at 12 psi

et
te
te
ha
Ac
ed
ci
or
Ac
os
es
et
m
Pr
C
e
g
tic
(for 20 minutes), then at 30 psi.

m
ia
on
ne
ro
ta
Tr
or
tis
lo
-P
Be
no
or
H
O
ci
8. Reconstitute with 100 µL Acetonitrile/Water (20:80)

α-
m
ia
11
by vortexing the plate at 1200 rpm for 5 minutes

Tr
300 µL Load 200 µL Load
Conclusion
Maximizing Recoveries Using a Binary Solvent System This work shows the broad versatility of Novum SLE 96-well
plates which are able to implement a variety of extraction solvents
Another way to increase analyte recovery using the Novum SLE depending on analyst and analyte preference. By performing a
plate is to implement a binary solvent system using ethyl acetate. case study using betamethasone from plasma, we present helpful
Figure 2 compares the recoveries between an extraction using optimization tips to boost analyte recovery with Novum SLE 96-
DCM, EtOAc, and 10 % ethyl acetate (EtOAc) in DCM. While the well plates. While using ethyl acetate as an extraction solvent
EtOAc provides the best recoveries for the steroid panel, Figure provides the best recoveries in most cases, this study shows that
2 shows the marked improvement in recovery of betamethasone diluting the sample up to a total volume of 300 µL on the Novum
that can be obtained by adding 10 % EtOAc to the DCM extraction SLE MINI 96-well plate and 450 µL on the Novum SLE MAX 96-
(optimized method in Table 1). This trend is consistent across oth- well plate will maximize the area of partition between the aqueous
er extraction solvents as well, indicating that an improvement in sample and the organic solvent, thus improving recovery. More-
recovery can be achieved by adding 5-10 % EtOAc to any water over, adding 5-10 % ethyl acetate to any organic extraction sol-
immiscible extraction solvent (including but not limited to Hexane, vent will improve its ability to wet the sorbent which also yields
DCM, MTBE, etc.). improved recovery. However with very polar compounds, it may
While we were able to improve recovery by adding 10 % EtOAc to be necessary to use 100 % ethyl acetate as an extraction solvent
our DCM elution, Triamcinolone still displayed poor recovery under to obtain acceptable recoveries.
both the original and the optimized methods. Because Triamcin-
olone is very polar (Log P ~0.25), it simply requires a more polar
extraction solvent to remove it from the aqueous sample, which is
why only EtOAc gives acceptable recovery.

Simple, Fast Sample Clean Up with Phree™
Phospholipid Removal Products
Ion suppression/enhancement, a reduction in analyte sensitiv- Materials and Instrumentation

ity, lowered precision, shifting of analyte retention times, and a • SCIEX API 5000™ triple quadrupole mass spectro-
decrease in HPLC/UHPLC column lifetime are just a few prob- meter, TurboIonSpray™ ionization source (AB SCIEX,
lems that can arise in LC-MS/MS analysis. These matrix effects Framingham, MA)
are often due to endogenous phospholipids within bioanlaytical • Agilent 1260 SL consisting of binary bumps and
samples. This technical note explores a common sample prepa- autosampler (Agilent, Santa Clara, CA)
ration technique, solid phase extraction (SPE), and compares it
• TurboVap 96 dryer (Biotage, Charlotte, NC)
to a simpler, faster technique using a phospholipid removal prod-
uct, Phree™. Our work demonstrates that Phree Phospholipid • Phree Phospholipid Removal 96-well plates
Removal products can successfully remove more phospholipids (Phenomenex, Torrance, CA)
as compared to a generic reversed phase SPE procedure without • Waters Oasis HLB SPE 96-well plate (Waters, Milford,
negatively impacting analyte recovery for acidic, basic, and neu- MA)
tral target compounds. • Biotage Evolute ABN Express 96-well plate
(Biotage, Charlotte, NC)
Introduction
• Kinetex C18, 2.6 µm core-shell HPLC/UHPLC column,
A primary need in bioanalysis is to increase assay throughput and 50 x 2.1 mm (Phenomenex, Torrance, CA)
sensitivity. Common chromatography methods result in co-elu-
tion of both analytes and matrix components, forming a matrix Chemicals and Reagents
effect. The primary cause of these matrix effects is endogenous • Human plasma EDTA (Bioreclamation Inc, Hicksville, NY)
phospholipids and lysophospholipids which result in ionization • Milli-Q Water (In-house)
suppression or enhancement, thus altering the sensitivity of the
LC-MS/MS analysis. Problems associated with the phospholipids • Acetonitrile (Fisher Scientific, Pittsburgh, PA)
include reduced analyte sensitivity, lowered precision, shifting of • Methanol (Fisher Scientific, Pittsburgh, PA)
analyte retention time, decreased column life expectancy and in- • Formic Acid (Fisher Scientific, Pittsburgh, PA)
creased mass spectrometry maintenance.
• Amitriptyline, m.w. 277 (Sigma Aldrich, St. Louis, MO)
Correcting matrix effects requires an improvement in sample • Acetaminophen, m.w. 151 (Sigma Aldrich, St. Louis, MO)
cleanup procedures in order to remove the aforementioned phos-
• Diclofenac, m.w. 296 (Sigma Aldrich, St. Louis, MO)
pholipids. However, this process generally sacrifices sample
throughput due to long or complicated extraction procedures, • Metoprolol, m.w. 267 (Sigma Aldrich, St. Louis, MO)
such as solid phase extraction (SPE), contributing to increased • Naproxen, m.w. 230 (Sigma Aldrich, St. Louis, MO)
cost and time. This technical note demonstrates that Phree Phos- • Prednisone, m.w. 358 (Sigma Aldrich, St. Louis, MO)
pholipid Removal 96-well plates provide a simpler and faster set-
up to remove phospholipids and minimize matrix effects without • Amitriptyline-D6, m.w. 283 (CDN Isotopes, Pointe-Claire,
QC)
compromising analyte recovery when compared to Waters® Oa-
sis® HLB and Biotage® Evolute® ABN SPE. • Acetaminophen-D7, m.w. 158 (CDN Isotopes,
Pointe-Claire, QC)
The Phree plate contains a unique frit system which holds the • Diclofenac-D4, m.w. 300 (CDN Isotopes, Pointe-Claire,
solvent and plasma until pressure is applied, allowing for a pro- QC)
tein precipitation to be performed within the wells of the plate. • Metoprolol-D7, m.w. 274 (CDN Isotopes, Pointe-Claire,
After precipitation, the sample is pulled through the Phree™ sor- QC)
bent by vacuum, centrifugation, or positive pressure, retaining the
• Naproxen-D3, m.w. 233 (CDN Isotopes, Pointe-Claire,
proteins behind on the frit. As sample passes through the Phree QC)
sorbent, phospholipids are selectively removed and clean eluent
is collected (Figure 1). • Prednisolone, m.w. 360 (CDN Isotopes, Pointe-Claire,
QC)
Experimental Conditions Analyte Structures
Sample cleanup and analyte recoveries were compared using ge-
neric procedures provided by the Phree 96-well plate, the Waters Acetaminophen Amitriptyline Diclofenac
Oasis HLB SPE plate and the Biotage Evolute ABN SPE plate. A
combination of acidic, basic, and neutral compounds were ana-
lyzed to represent a wide variety of target compounds. To assess
the cleanup abilities of each technique, five major phospholipids
were monitored during the sample injections and matrix effects H3CO
N
CH3
were investigated through post column infusion experiments. H
In addition to SPE, protein precipitation was also performed O

Metoprolol
N
H
CH3
Naproxen Prednisone
OH
alongside the Phree phospholipid removal procedure so that we
OH
could compare the original concentration of phospholipids to the O
H
N CH3
amount of phospholipids that were depleted using the Phree and H
CH3
SPE procedures. H3CO

SPE and Phree™ Extraction Procedures
SPE Method using SPE Method using Phree™ Phospholipid

Waters® Oasis® HLB Biotage® Evolute® ABN Removal Method
6-10 minutes 6-10 minutes 3 minutes

per sample per sample per sample
Plus 10 Minutes Plus 15 Minutes Plus 5 Minutes

to Prepare Solvents to Prepare Solvents to Prepare Solvents
Condition Condition Dispense

1 mL Methanol 1 mL Methanol 100 µL Plasma
Equilibrate
Equilibrate Dispense
1 mL 0.1 % Formic Acid in Water
1 mL Water 300 µL 1 % Formic Acid in Acetonitrile
Load
Load 100 µL Plasma diluted with 300 µL 2 % Formic Mix
100 µL Plasma diluted with 300 µL Water Acid in Water
Wash Wash Filter

1 mL 5 % Methanol in Water 1 mL of 5 % Methanol in Water via vacuum, positive pressure, or centrifugation
Elute Elute
500 µL Methanol 500 µL Methanol
Chromatographic Conditions
Gradient: Time (min) B (%) without compromising results. Phree exhibited a 72-108 % range
Column: Kinetex® 2.6 µm C18 100 Å
Dimensions: 50 x 2.1 mm 0 5 in recovery with an average CV % of only 7.24 % (n=5 for each
Part No.: 00B-4462-AN 2 95 compound) across all samples tested. (Figure 2)
Mobile Phase: A: 0.1 % Formic Acid in Water 3 95
B: 0.1 % Formic Acid in Acetonitrile 3.01 5 After analyte recovery was determined, we monitored for the
5 5
presence of phospholipids to determine the extent of cleanup
provided by each technique as well as to monitor for matrix ef-
Temperature: 50 °C fects due to the endogenous phospholipids. Phree Phospholip-
Detection: MS/MS (SCIEX API 5000™) id Removal products selectively removed more than 99 % of all
Mass Transitions phospholipids from the plasma samples including phosphatidyl
ID Q1 Q3 Dwell DP CE CXP cholines and lysophosphatidyl cholines. As compared to generic
reversed phase SPE procedures, which left a significant amount
Acetaminophen 152.1 110.1 25 73 25 11 of phospholipids in the cleaned up sample, Phree was superior at
Acetaminophen-D7 159.1 115.1 25 76 62 11 removing the five major phospholipids that we monitored during
analysis. This minimized matrix effects while reducing the cost
Amitriptyline 278.3 233.3 25 90 70 13 and time required for sample analysis (Figure 3).
Amitriptyline-D6 284.3 233.3 25 90 70 13
Figure 1.
Diclofenac 296 278.1 25 100 50 14 Protein and phospholipid removal using PhreeEliminate Phospholipids
Remove Proteins
Diclofenac-d4 300 255.3 25 100 50 13 The Phree sorbent selectively
Solvent Shielding Technology™ prevents
dripping of organic solvent, allowing for removes phospholipids from
Metoprolol 268.3 116.3 25 100 45 11 precipitated plasma samples.
protein precipitation within the Phree
Phospholipid Removal Product.
Metoprolol-D7 275.8 123.2 25 100 60 15
Naproxen 231.1 185.1 25 100 30 13
Naproxen-D3 234.1 188.2 25 100 30 13
Prednisone 359.3 341.2 25 120 68 13
Prednisolone 361.2 343.2 25 100 50 13

Based upon the data, phospholipid removal using Phree provided
a simple and generic method that resulted in consistent recover-
ies for acids, bases, and neutrals. When extracted using generic
reversed phase SPE procedures, the same analytes did not exhib-
it reproducible recoveries indicating that it may be necessary to
develop separate SPE methods for each compound class. Phree
allowed us to analyze a broader range of compounds in a single Proteins
cleanup step without the need to develop new methods for each
Phospholipids
compound. By reducing or eliminating the time spent developing
Target Analyte
methods and preparing samples, Phree optimizes cost efficiency

6400
Figure 2.
6000 PC-1
Acidic, basic and neutral analyte absolute recoveries using Phree™ Phos-
5500 Blue – Phree
pholipid Removal (blue), Waters® Oasis® HLB SPE (red) and Biotage® Red – Waters
Evolute® ABN SPE (yellow) 5000
Green – Biotage
4500 Burgundy – PPT
180
Intensity, cps
4000
160 3500
3000
140
Phree 2500
Absolute Recovery
120 Waters Oasis® HLB 2000
App ID 21996
100 Biotage Evolute® ABN 1500
1000
80
70% 500
60 0 3.13 3.62
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
51 101 151 201 251 301 351 401 451 min
40
20 1560
1500
PC-2
0 1400
Metoprolol
Naproxen
Acetaminophen
Diclofenac
Prednisone
Blue – Phree
Amitriptyline
1300
Red – Waters
1200 Green – Biotage
1000
Intensity, cps
Basic Neutral Acidic 900
Compounds Compounds Compounds 800
700
N=5 for all cleanup techniques 600
500
App ID 21997
400
Figure 3. 300
Profile of five major phospholipids in Phree extracted plasma, SPE extract- 200
ed plasma, and protein precipitated plasma 100
0
1.28e4 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
1.20e4 Lyso-1 Blue – Phree
51 101 151 201 251 301 351 401 451
Red – Waters
1.10e4
Green – Biotage 2720
Burgundy – PPT 2600
1.00e4
PC-3 Blue – Phree
9000.00 2400
Red – Waters
8000.00 2200 Green – Biotage
Intensity, cps

7000.00
6000.00 1800
1600
Intensity, cps
5000.00
4000.00 1400
App ID 21994
3000.00
1200
1000
2000.00
800
1000.00
App ID 21998
0 .00 600
0.35 1.59 2.41 2.52
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 400
51 101 151 201 251 301 351 401 451 min
200
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
1800 51 101 151 201 251 301 351 401 451
1700
Lyso-2
1600 Blue – Phree Lysophosphatidyl cholines:
Red – Waters
1500
Green – Biotage Lyso-1: 1-Palmitoyl-2-OH-sn-glycero-phosphocholine, 496>184 m/z
1400
1300
Burgundy – PPT Lyso-2: 1-Oleoyl-2-OH-glycero-phosphocholine, 522>184 m/z
1200
1100
Phosphatidyl cholines:
Intensity, cps
1000 PC-1: 1-Palmitoyl-2-Oleoyl-sn-glycerol-phosphocholine, 760>184 m/z

900 PC-2: 1-Stearoly-2-Linoleoyl-sn-glycerol-phosphocholine, 786>184 m/z
800
PC-3: 1-Oleyol-2-Linoleoyl-sn-glycerol-phosphocholine, 784>184 m/
700
600
500
Conclusion
App ID 21995
400
300 This project demonstrated recoveries of acids, bases and neutrals
200
100
and their respective matrix effects caused by phospholipids and
0 lysophospholipids using Phree Phospholipid Removal 96-well
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
51 101 151 201 251 301 351 401 451 plates, Waters Oasis HLB and Biotage Evolute ABN SPE 96-well
plates. The data concludes that Phree selectively removed both
phospholipids and lysophospholipids better than SPE when ac-
ids, bases, and neutrals were extracted using a generic reversed
phase procedure. The Phree extraction method provided a rap-
id, simple, and transferable platform to achieve cleaner samples,
saving time on method development and sample preparation. In
addition, analyte recovery was uncompromised using Phree in all
cases yielding the same or better results than SPE.

Sensitivity Gains Using Strata®-X Microelution
Solid Phase Extraction (SPE)
Overview in volumes as low as 25 µL. These low elution volumes result in

Solid phase extraction (SPE) is an excellent cleanup solution for ultra-concentrated samples that do not need to be dried down.
bioanalytical samples because it is selective, reproducible, and In this study, we compared the microelution 96-well SPE plate
results in ultra-clean and concentrated samples. While the tech- to a traditional 10 mg 96-well SPE plate to determine the amount
nique is effective, traditional formats such as 10 mg 96-well plates of sensitivity gain that can be achieved by moving a 10 mg SPE
or tubes pose challenges for small or limited sample volumes and method to the microelution format.
can result in dilute eluents if the sample is not dried down and
reconstituted after the extraction procedure. This dry down step Experimental Conditions
can require 30 or more minutes, adding a significant amount of Extraction Procedures
time to the procedure. To overcome these challenges, the micro- Timolol and procaine were extracted from serum using a tradition-
elution 96-well SPE plate format was developed. The microelu- al 10 mg 96-well SPE plate and a 2 mg microelution 96-well SPE
tion SPE plates contain significantly less sorbent as compared to plate, each of which was packed with the same polymeric strong
a traditional 10 mg 96-well SPE plate, allowing analysts to elute cation-exchange chemistry, Strata®-X-C. The same volume of
sample was loaded onto each SPE plate and the condition, equil-
ibration, wash, and elution solvents were the same across both
extractions to standardize the extraction protocols.
Table 1 .
SPE Extraction of Timolol and Procaine from Serum
Strata-X-C 96-Well SPE Plate, 10 mg/well Strata-X-C Microelution 96-Well SPE Plate, 2 mg/well
Part No.: 8E-S029-AGB 8M-S029-4GA
Condition: 500 µL Methanol 200 µL Methanol
Equilibrate: 500 µL Water 200 µL Water
750 µL diluted serum (375 µL serum diuted 1:1 with 4 % Phosphoric 750 µL diluted serum (375 µL serum diuted 1:1 with 4 % Phosphoric
Load:
acid in water) acid in water)
Wash 1: 500 µL 2 % Formic acid in water 200 µL 2 % Formic acid in water
Wash 2: 500 µL Methanol 200 µL Methanol
375 µL (3x 125 µL) 5 % Ammonium hydroxide in acetonitrile/methanol

Elute: 25 µL 5 % Ammonium hydroxide in acetonitrile/methanol (60:40)
(60:40)
Inject: 1 µL 1 µL

LC-MS/MS Conditions Figure 2. Procaine (1ng/mL) Extracted from Serum Resulted in 15x More
After cleanup by SPE, 1 µL of each extraction was injected onto a Concentrated Samples using the Microelution Format
22983
Kinetex® 2.6 µm Biphenyl core-shell HPLC column and the result- 9.5e5
ing concentrations were determined by LC-MS/MS. 9.0e5

8.5e5
Procaine Microelution
8.0e5 SPE 15x More
Column: Kinetex 2.6 µm Biphenyl 7.5e5 Concentrated
7.0e5
App ID 22983 and 22985

Dimensions: 50 x 2.1 mm 6.5e5
Part No.: 00B-4462-AN 6.0e5

5.5e5
Recommended Guard:
Intensity, cps
AJ0-8782 5.0e5
4.5e5
Mobile Phase: A: 0.1 % Formic acid in Water 4.0e5
B: 0.1 % Formic acid in Acetonitrile 3.5e5
3.0e5
Gradient: Time (min) B (%) 2.5e5
0 5 2.0e5 10 mg
0.5 5 1.5e5 SPE
3 95 1.0e5
5.0e4
3.5 95 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
3.51 5
5.5 5
Conclusion
While both the 10 mg 96-well SPE plate and the 2 mg microelution
96-well SPE plate were both extremely effective at cleaning up our
serum samples, the microelution format allowed us to produce
Both timolol and procaine were extracted from serum using two
ultra-concentrated samples without the need to dry down and
different Strata®-X SPE formats; a traditional 10 mg 96-well SPE
reconstitute. By skipping this step we were able to save 30-45
plate and a 2 mg microelution 96-well SPE plate. The microelu-
minutes without losing any sensitivity. These time savings essen-
tion format is designed to allow analysts to elute in small sample
tially doubled our productivity as the total cleanup time using the
volumes which results in ultra-concentrated samples without the
microelution format took approximately 30 minutes. In addition
need to perform a dry down step. To standardize our comparison,
to the time savings, the microelution format is also amenable to
the same polymeric strong cation-exchange sorbent, Strata®-X-C,
small or limited sample volumes (as low as 10 µL) which provides
was packed in each 96-well SPE plate and the same method was
an excellent solution in the event that our sample size decreases.
performed however the solvent volumes were reduced for the mi-
croelution method (Table 1). While both extraction methods were
extremely effective and resulted in clean samples, the microelu-
tion format resulted in 15x more concentrated samples (Figures 1
and 2) when 1 µL of the eluent was injected onto the LC-MS/MS.
Figure 1. Timolol (1ng/mL) Extracted from Serum Resulted in 15x More

Concentrated Samples using the Microelution Format
22984
5.5e5
5.0e5
Timolol
Microelution
4.5e5
SPE 15x More
App ID 22982 and 22984
4.0e5 Concentrated
3.5e5
3.0e5
Intensity, cps
2.5e5
2.0e5
1.5e5
10 mg
1.0e5 SPE
5.0e4
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

Improved Sensitivity of Hydrolyzed Urine
Samples Using β-Gone™ β-Glucuronidase

During metabolism, drugs are tagged with a glucuronic acid Column: Kinetex® 2.6 µm Biphenyl
that helps change the polarity of the drug compound and aids in Dimensions: 50 x 3.0mm
Part No.: 00B-4622-YO
absorption into the kidneys. When the drugs exit the body through Recommended Guard: AJ0-9208
urine, they are still in their glucuronide form and before chromato- Mobile Phase: A: 0.1% Formic acid in Water
graphic analysis can occur, the glucuronide bond must be cleaved B: 0.1% Formic acid in Methanol
through hydrolysis. Enzymatic hydrolysis, using β-glucuronidase, is Gradient: Time (min) % B
preferred over acid hydrolysis because the bond is cleaved without 1 10
introducing harsh solvents into the sample. Now the sample con- 4 100
tains drug compounds and residual β-glucuronidase enzyme, which 5 100
if the enzyme is not removed can precipitate out in the LC column 5.01 10
6 10
during the run. The column’s selectivity and lifetime is negatively Detection: MS/MS (SCIEX API 4000)
affected and can result in buildup in the mass spectrometer (MS).
“Dilute-and-shoot” is a common method that is used to prepare hy-
drolyzed urine samples for LC-MS analysis. This method can cause
issues with the sensitivity because it dilutes the sample 10x up to
30x before injection onto the column. In this application note, we
In Figure 1 the signal response for buprenorphine is shown for a
will demonstrate how using β-Gone™ β-Glucuronidase Removal
sample that has been diluted 10x (blue peak) and one that has been
Products helps to improve sensitivity in comparison to the “dilute-
filtered through the β-Gone 96-Well Plate (red peak).
and-shoot” method. Focusing on two notoriously low responding
compounds, norbuprenorphine and buprenorphine, the differences Figure 2 shows the same comparison for norbuprenorphine. In both
in methods are displayed. cases, β-Gone indicates a gain in sensitivity almost 10x more than
the dilute-and-shoot prepared sample.
In addition, Table 1 shows the average recovery values (n=8) using
Experimental Conditions β-Gone for a large panel of drug compounds. All % CVs are below
All reagents and solvents were HPLC or analytical grade. Analyses 6% and most recoveries are near 100%.
were performed using an API 4000™ LC-MS/MS (SCIEX, Framing-
ham, MA).
Sample Preparation: Conclusion
Prepare Urine Hydrolysate as follows: This work shows that by utilizing β-Gone β-Glucuronidase Removal
1) Add 10 µL of analyte spike (1 µg/mL) to 200 µL of urine Products, laboratories can expect to significantly improve sensitivity
while adding little variability to the results (%CV) in comparison to
2) Dilute with 100 µL of 0.1 M ammonium acetate buffer
dilute-and-shoot protocols.
3) Add 40 µL of Campbell Science β-Glucuronidase Enzyme
Solution (Part No.: DR2102)
4) Add 400 µL of 0.1 % formic acid in water to mixture and Table 1.
vortex for 15 seconds
Analyte Average Recovery % % CV
β-Gone Protocol: Benzoylecognine 109 3

1) Dilute 200 µL of Urine Hydrolysate with 133 µL of 0.1 % Buprenorphine 93 6
Formic acid in Methanol
Codeine 109 4
2) Load diluted sample onto β-Gone 96-Well Plate (Part No.:
Lorazepam 79 5
8E-S322-DGA) and apply 2-5 psi using a positive pressure
manifold or a vacuum manifold Methamphetamine 106 3
3) Collect eluent and inject 10 µL for analysis Norbuprenorphine 109 5
PCP 102 3
Dilute-and-Shoot Protocol:
1) Transfer 100 µL of Urine Hydrolysate to vial
2) Dilute sample by adding 900 µL of 0.1 % Formic acid in
Water
3) Vortex and inject 10 µL for analysis

Figure 1.
Buprenorphine: β-Gone™ vs Dilute-and-Shoot
App ID 23778
1.4e4
1.3e4
1.2e4
1.1e4
1.0e4
β-Gone
9000.0 Dilute-and-Shoot
Intensity, cps
8000.0
7000.0
6000.0
5000.0
4000.0
3000.0
App ID 23778
2000.0
1000.0
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
Figure 2.
Norbuprenorphine: β-Gone vs Dilute-and-Shoot
23779
4.0e4
3.8e4
3.6e4
3.4e4
3.2e4
3.0e4
2.8e4 β-Gone
2.6e4
Dilute-and-Shoot
2.4e4
Intensity, cps
2.2e4
2.0e4
1.8e4
1.6e4
1.4e4
1.2e4
1.0e4
App ID 23779
8000.0
6000.0
4000.0
2000.0
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
For additional technical notes, visit Dilute-and-Shoot

Buprenorphine: www.phenomenex.com
vs. β-Gone

Extend UHPLC Column Lifetime and Performance
with Column Protection
UHPLC columns can significantly improve chromatographic Scanning electron microscopy of column inlet frit
separations, but they also present unique challenges. Once the
UHPLC system components are optimized, perhaps your greatest With SecurityGuard ULTRA
concern is protecting the column from the damaging effects of
microparticulates and sample contaminants. GUARD Column
An ultra-high performance column protection system, specifically

designed for UHPLC systems using sub-2 µm and core-shell
particle columns, can be used to extend column lifetime (saving Inlet Frit Column Media
both money and time through less frequent column replacement),
while minimizing system troubleshooting and downtime.

It is well known that UHPLC systems and columns require higher
levels of care and attention than traditional HPLC in order to reap
their full ultra-high chromatographic performance benefits. Once
system components are optimized, chromatographic cleanliness Without SecurityGuard ULTRA
is vital to maintain UHPLC performance, and column protection
is compulsory. Unprotected columns may suffer from reduced
performance and lifetime, and may lead to an increased need for
system troubleshooting and/or downtime. Inlet Frit Column Media
UHPLC columns packed with sub-2 µm particles tend to clog much

more rapidly than traditional HPLC columns packed with larger 3 µm
and 5 µm particles. This may be due to the fact that, not only is the
interstitial space between the particles much smaller, but columns
packed with sub-2 µm particles also use frits with a much smaller
Figure 1.
porosity compared to conventional HPLC columns. With a “tighter”, Scanning electron microscopy of non-contaminated and contaminated
more restricted flow path, any undissolved matter or particulates column inlet frit.
from the sample, the mobile phase, or the system (such as micro-
particulates shedding from piston seals and injection valve rotors),
will quickly and irreversibly foul your UHPLC column.
Accelerated Column Lifetime Test
An easy way to extend the performance and lifetime of your UHPLC
columns (either sub-2 µm fully-porous or core-shell media) is to 300
prevent any contaminants from reaching the column by using the
SecurityGuard™ ULTRA guard cartridge system (Figure 1). 280
Presented in Figure 2 is an accelerated lifetime test using an 260

Backpressure (Bar)
endogenous biological matrix injected onto a core-shell column Cartridge Replacement Cartridge Replacement
240
(Kinetex 2.6 µm C18 50 × 4.6 mm column Phenomenex, Inc.
Torrance CA, USA). With the unprotected column (grey dots), 220
sequential injections of the matrix lead to a steady and irreversible
increase in backpressure. Without SecurityGuard ULTRA column 200
protection, the increase in backpressure becomes exponential.
180
This increase in backpressure will eventually lead to degraded
chromatography, including band broadening and possibly peak 160
splitting. As a result, method sensitivity, quantitation and peak Injection No.
identification may also be adversely affected.
No Guard
However, column lifetime is greatly extended by using the SecurityGuard™ ULTRA
SecurityGuard ULTRA (black boxes). In this case, sequential
injections of the matrix will still lead to an increase in pressure, but
this is due to the particulates being captured in the SecurityGuard Figure 2.
ULTRA itself, rather than in the UHPLC/HPLC column. Thus, by SecurityGuard ULTRA cartridges protect UHPLC, sub-2 μm and core-
simply replacing the SecurityGuard ULTRA cartridge at regular shell columns from microparticulates and chemical contamination, as
well as sample matrix fouling. The result is increased column lifetime,
intervals, backpressure returns to starting levels and effective
longer performance, and more reproducible chromatography.
column lifetime can be greatly extended.

PhenoLogix SM
Custom
Method Development and
Optimization Services
A majority of our services are free of charge!

Kinetex Core-Shell LC Column Ordering Information
SecurityGuard™
5 μm Minibore Columns (mm) ULTRA Cartridges‡
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk
EVO C18 00A-4633-AN 00B-4633-AN 00D-4633-AN 00F-4633-AN AJ0-9298
Biphenyl 00A-4627-AN 00B-4627-AN 00D-4627-AN –– AJ0-9209
XB-C18 00A-4605-AN 00B-4605-AN 00D-4605-AN –– AJ0-8782
C18 00A-4601-AN 00B-4601-AN 00D-4601-AN 00F-4601-AN AJ0-8782
C8 –– 00B-4608-AN 00D-4608-AN –– AJ0-8784
Phenyl-Hexyl –– 00B-4603-AN 00D-4603-AN –– AJ0-8788
for 2.1 mm ID
SecurityGuard
5 μm MidBore™ Columns (mm) ULTRA Cartridges‡
Phases 50 x 3.0 100 x 3.0 150 x 3.0 3/pk
EVO C18 00B-4633-Y0 00D-4633-Y0 00F-4633-Y0 AJ0-9297
Biphenyl 00B-4627-Y0 00D-4627-Y0 00F-4627-Y0 AJ0-9208
XB-C18 00B-4605-Y0 00D-4605-Y0 00F-4605-Y0 AJ0-8775
C18 00B-4601-Y0 00D-4601-Y0 00F-4601-Y0 AJ0-8775
C8 00B-4608-Y0 00D-4608-Y0 –– AJ0-8777
Phenyl-Hexyl 00B-4603-Y0 00D-4603-Y0 –– AJ0-8781
for 3.0 mm ID
SecurityGuard
5 μm Analytical Columns (mm) ULTRA Cartridges‡
Phases 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 3/pk
EVO C18 00B-4633-E0 00D-4633-E0 00F-4633-E0 00G-4633-E0 AJ0-9296
F5
Biphenyl 00B-4627-E0 00D-4627-E0 00F-4627-E0 00G-4627-E0 AJ0-9207
XB-C18 00B-4605-E0 00D-4605-E0 00F-4605-E0 00G-4605-E0 AJ0-8768
C18 00B-4601-E0 00D-4601-E0 00F-4601-E0 00G-4601-E0 AJ0-8768
C8 00B-4608-E0 00D-4608-E0 00F-4608-E0 00G-4608-E0 AJ0-8770
Phenyl-Hexyl 00B-4603-E0 00D-4603-E0 00F-4603-E0 00G-4603-E0 AJ0-8774
for 4.6 mm ID
SecurityGuard
5 μm Semi-Preparative Columns (mm) SemiPrep Cartridges***
Phases 150 x 10 250 x 10 10 x 10
EVO C18 00F-4633-N0 00G-4633-N0 AJ0-9306
C18 00F-4601-N0 00G-4601-N0 AJ0-9278
Biphenyl 00F-4627-N0 00G-4627-N0 AJ0-9280
for 9-16 mm ID
SecurityGuard
5 μm Axia™ Packed Preparative Columns (mm) PREP Cartridges*
Phases 50 x 21.2 100 x 21.2 150 x 21.2 250 x 21.2 15 x 21.2
EVO C18 00B-4633-P0-AX 00D-4633-P0-AX 00F-4633-P0-AX 00G-4633-P0-AX AJ0-9304
F5
Biphenyl 00B-4627-P0-AX 00D-4627-P0-AX 00F-4627-P0-AX 00G-4627-P0-AX AJ0-9272
XB-C18 00B-4605-P0-AX 00D-4605-P0-AX 00F-4605-P0-AX 00G-4605-P0-AX AJ0-9145
C18 00B-4601-P0-AX 00D-4601-P0-AX 00F-4601-P0-AX 00G-4601-P0-AX AJ0-9145
C8 00B-4608-P0-AX 00D-4608-P0-AX 00F-4608-P0-AX 00G-4608-P0-AX AJ0-9205
Phenyl-Hexyl 00B-4603-P0-AX 00D-4603-P0-AX 00F-4603-P0-AX 00G-4603-P0-AX AJ0-9147
HILIC –– 00D-4606-P0-AX 00F-4606-P0-AX 00G-4606-P0-AX AJ0-9277
for 18-29 mm ID
SecurityGuard
5 μm Axia Packed Preparative Columns (mm) PREP Cartridges**
Phases 50 x 30 100 x 30 150 x 30 250 x 30 15 x 30
EVO C18 00B-4633-U0-AX 00D-4633-U0-AX 00F-4633-U0-AX 00G-4633-U0-AX AJ0-9305
F5
Biphenyl –– –– 00F-4627-U0-AX –– AJ0-9273
XB-C18 00B-4605-U0-AX 00D-4605-U0-AX 00F-4605-U0-AX 00G-4605-U0-AX AJ0-9204
C18 00B-4601-U0-AX 00D-4601-U0-AX 00F-4601-U0-AX 00G-4601-U0-AX AJ0-9204 ‡
SecurityGuard ULTRA Cartridges require holder, Part No.: AJ0-9000
C8 00B-4608-U0-AX 00D-4608-U0-AX 00F-4608-U0-AX 00G-4608-U0-AX AJ0-9217 * PREP SecurityGuard Cartridges require holder, Part No.: AJ0-8223
** PREP SecurityGuard Cartridges require holder, Part No.: AJ0-8277
Phenyl-Hexyl 00B-4603-U0-AX 00D-4603-U0-AX 00F-4603-U0-AX 00G-4603-U0-AX AJ0-9216 *** SemiPrep SecurityGuard Cartridges require holder, Part No.: AJ0-9281
for 30-49 mm ID

SecurityGuard™
3.5 μm Analytical Columns (mm) ULTRA Cartridges‡
Phases 100 x 4.6 150 x 4.6 3/pk
XB-C18 00D-4744-E0 00F-4744-E0 AJ0-8768
for 4.6 mm ID
SecurityGuard
2.6 μm Minibore Columns (mm) ULTRA Cartridges‡
Phases 30 x 2.1 50 x 2.1 75 x 2.1 100 x 2.1 150 x 2.1 3/pk
EVO C18 00A-4725-AN 00B-4725-AN –– 00D-4725-AN 00F-4725-AN AJ0-9298
Polar C18 00A-4759-AN 00B-4759-AN – 00D-4759-AN 00F-4759-AN AJ0-9532
F5 00A-4723-AN 00B-4723-AN –– 00D-4723-AN 00F-4723-AN AJ0-9322
Biphenyl 00A-4622-AN 00B-4622-AN –– 00D-4622-AN 00F-4622-AN AJ0-9209
XB-C18 00A-4496-AN 00B-4496-AN 00C-4496-AN 00D-4496-AN 00F-4496-AN AJ0-8782
C18 00A-4462-AN 00B-4462-AN 00C-4462-AN 00D-4462-AN 00F-4462-AN AJ0-8782
C8 00A-4497-AN 00B-4497-AN 00C-4497-AN 00D-4497-AN 00F-4497-AN AJ0-8784
HILIC 00A-4461-AN 00B-4461-AN 00C-4461-AN 00D-4461-AN 00F-4461-AN AJ0-8786
Phenyl-Hexyl 00A-4495-AN 00B-4495-AN 00C-4495-AN 00D-4495-AN 00F-4495-AN AJ0-8788
for 2.1 mm ID
SecurityGuard
2.6 μm MidBore™ Columns (mm) ULTRA Cartridges‡
Phases 30 x 3.0 50 x 3.0 75 x 3.0 100 x 3.0 150 x 3.0 3/pk
EVO C18 –– 00B-4725-Y0 –– 00D-4725-Y0 00F-4725-Y0 AJ0-9297
Polar C18 –– 00B-4759-Y0 –– 00D-4759-Y0 00F-4759-Y0 AJ0-9531
F5 –– 00B-4723-Y0 –– 00D-4723-Y0 00F-4723-Y0 AJ0-9321
Biphenyl –– 00B-4622-Y0 –– 00D-4622-Y0 00F-4622-Y0 AJ0-9208
XB-C18 00A-4496-Y0 00B-4496-Y0 00C-4496-Y0 00D-4496-Y0 00F-4496-Y0 AJ0-8775
C18 00A-4462-Y0 00B-4462-Y0 00C-4462-Y0 00D-4462-Y0 00F-4462-Y0 AJ0-8775
C8 00A-4497-Y0 00B-4497-Y0 00C-4497-Y0 00D-4497-Y0 00F-4497-Y0 AJ0-8777
HILIC 00A-4461-Y0 –– –– –– 00F-4461-Y0 AJ0-8779
Phenyl-Hexyl –– 00B-4495-Y0 –– 00D-4495-Y0 00F-4495-Y0 AJ0-8781
for 3.0 mm ID
SecurityGuard
2.6 μm Analytical Columns (mm) ULTRA Cartridges‡
Phases 30 x 4.6 50 x 4.6 75 x 4.6 100 x 4.6 150 x 4.6 3/pk
EVO C18 –– 00B-4725-E0 –– 00D-4725-E0 00F-4725-E0 AJ0-9296
Polar C18 –– 00B-4759-E0 –– 00D-4759-E0 00F-4759-E0 AJ0-9530
F5 –– 00B-4723-E0 –– 00D-4723-E0 00F-4723-E0 AJ0-9320
Biphenyl –– 00B-4622-E0 –– 00D-4622-E0 00F-4622-E0 AJ0-9207
XB-C18 –– 00B-4496-E0 00C-4496-E0 00D-4496-E0 00F-4496-E0 AJ0-8768
C18 00A-4462-E0 00B-4462-E0 00C-4462-E0 00D-4462-E0 00F-4462-E0 AJ0-8768
C8 –– 00B-4497-E0 00C-4497-E0 00D-4497-E0 00F-4497-E0 AJ0-8770
HILIC –– 00B-4461-E0 00C-4461-E0 00D-4461-E0 00F-4461-E0 AJ0-8772
Phenyl-Hexyl –– 00B-4495-E0 00C-4495-E0 00D-4495-E0 00F-4495-E0 AJ0-8774
for 4.6 mm ID
SecurityGuard
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk
EVO C18 –– 00B-4726-AN 00D-4726-AN 00F-4726-AN AJ0-9298
F5 –– 00B-4722-AN 00D-4722-AN 00F-4722-AN AJ0-9322
Biphenyl –– 00B-4628-AN 00D-4628-AN 00F-4628-AN AJ0-9209
XB-C18 00A-4498-AN 00B-4498-AN 00D-4498-AN 00F-4498-AN AJ0-8782
HILIC 00A-4474-AN 00B-4474-AN 00D-4474-AN –– AJ0-8786
Phenyl-Hexyl –– 00B-4500-AN 00D-4500-AN 00F-4500-AN AJ0-8788
for 2.1 mm ID
SecurityGuard
1.7 μm MidBore Columns (mm) ULTRA Cartridges‡
Phases 30 x 3.0 50 x 3.0 100 x 3.0 3/pk
XB-C18 00A-4498-Y0 00B-4498-Y0 00D-4498-Y0 AJ0-8775
C18 –– 00B-4475-Y0 00D-4475-Y0 AJ0-8775
C8 00A-4499-Y0 00B-4499-Y0 00D-4499-Y0 AJ0-8777
HILIC –– 00B-4474-Y0 –– AJ0-8779
for 3.0 mm ID
1.3 μm Minibore Columns (mm) 1.7 μm Microbore Columns (mm)

Phases 30 x 2.1 50 x 2.1 Phases 50 x 1.0 100 x 1.0 150 x 1.0
C18 00A-4515-AN 00B-4515-AN EVO C18 00B-4726-A0 00D-4726-A0 00F-4726-A0 ‡
SecurityGuard ULTRA Cartridges require holder, Part No.: AJ0-9000.

Luna LC Column Ordering Information
OMEGA
SecurityGuard™
5 μm Minibore Columns (mm) Cartridges (mm)
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 4 x 2.0*
Omega Polar C18 00A-4754-AN 00B-4754-AN 00D-4754-AN 00F-4754-AN AJ0-7600
Omega PS C18 00A-4753-AN 00B-4753-AN 00D-4753-AN 00F-4753-AN AJ0-7605
for ID: 2.0 - 3.0 mm
SecurityGuard
5 μm MidBore™ Columns (mm) Cartridges (mm)
Phases 50 x 3.0 100 x 3.0 150 x 3.0 4 x 2.0*
Omega Polar C18 00B-4754-Y0 00D-4754-Y0 00F-4754-Y0 AJ0-7600
Omega PS C18 00B-4753-Y0 00D-4753-Y0 00F-4753-Y0 AJ0-7605
for ID: 2.0 - 3.0 mm
SecurityGuard
5 μm Analytical Columns (mm) Cartridges (mm)
Phases 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 2.0*
Omega Polar C18 00B-4754-E0 00D-4754-E0 00F-4754-E0 00G-4754-E0 AJ0-7601
Omega PS C18 00B-4753-E0 00D-4753-E0 00F-4753-E0 00G-4753-E0 AJ0-7606
for ID: 3.2-8.0 mm
SecurityGuard
5 μm Axia™ Packed Preparative Columns (mm) Cartridges (mm)
Phases 150 x 21.2 250 x 21.2 150 x 30 250 x 30 250 x 50 15 x 21.2** 15 x 30.0♦
Omega Polar C18 00F-4754-P0-AX 00G-4754-P0-AX 00F-4754-U0-AX 00G-4754-U0-AX 00G-4754-V0-AX AJ0-7603 AJ0-7604
Omega PS C18 00F-4753-P0-AX 00G-4753-P0-AX 00F-4753-U0-AX 00G-4753-U0-AX 00G-4753-V0-AX AJ0-7608 AJ0-7609
for ID: 18-29 mm for ID: 30-49 mm
SecurityGuard
3 μm Minibore Columns (mm) Cartridges (mm)
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 4 x 2.0*
for ID: 2.0 - 3.0 mm
SecurityGuard
3 μm MidBore™ Columns (mm) Cartridges (mm)
Phases 30 x 3.0 50 x 3.0 100 x 3.0 150 x 3.0 4 x 2.0* 4 x 3.0*
Omega Polar C18 –– 00B-4760-Y0 00D-4760-Y0 00F-4760-Y0 AJ0-7600 ––
Omega PS C18 –– 00B-4758-Y0 00D-4758-Y0 00F-4758-Y0 AJ0-7605 ––
C18(2) 00A-4251-Y0 00B-4251-Y0 –– 00F-4251-Y0 AJ0-4286 AJ0-4287
for ID: 2.0 - 3.0 mm for ID: 2.0 - 3.0 mm
SecurityGuard
Phases 30 x 4.6 50 x 4.6 75 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 2.0* 4 x 3.0*
Omega Polar C18 –– 00B-4760-E0 –– 00D-4760-E0 00F-4760-E0 00G-4760-E0 AJ0-7601 ––
Omega PS C18 –– 00B-4758-E0 –– 00D-4758-E0 00F-4758-E0 00G-4758-E0 AJ0-7606 ––
C18(2) 00A-4251-E0 00B-4251-E0 00C-4251-E0 00D-4251-E0 00F-4251-E0 –– AJ0-4286 AJ0-4287
for ID: 3.2-8.0 mm for ID: 3.2-8.0 mm
SecurityGuard™
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk
Omega C18 00A-4742-AN 00B-4742-AN 00D-4742-AN 00F-4742-AN AJ0-9502
for 2.1 mm ID
* SecurityGuard Analytical Cartridges require holder, Part No.: KJ0-4282
♦ PREP SecurityGuard Cartridges require holder, Part No.: AJ0-8277
‡
SecurityGuard ULTRA Cartridges require holder, Part No.: AJ0-9000

Lux Chiral LC Column Ordering Information
Chiral LC Columns
3 µm Analytical Columns SecurityGuard Cartridges*

Phase 150 x 3.0 150 x 4.6 10/pk 10/pk
AMP 00F-4751-Y0 00F-4751-E0 AJ0-8475 AJ0-8476
For ID: for ID: 2.0-3.0 mm for ID: 3.2-8.0 mm
* SecurityGuard Analytical cartridges require holder, Part No.: KJ0-4282
3 µm Minibore, MidBore™, and Analytical Columns (mm) SecurityGuard™ Cartridges (mm)
Phase 50 x 2.0 150 x 2.0 100 x 3.0 150 x 3.0 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 2.0* 4 x 3.0*
Cellulose-3 00B-4492-B0 00F-4492-B0 00D-4492-Y0 00F-4492-Y0 00B-4492-E0 00D-4492-E0 00F-4492-E0 00G-4492-E0 AJ0-8621 AJ0-8622
for ID: 2.0 –3.0 mm 3.2–8.0 mm
5 µm Minibore and Analytical Columns (mm) SecurityGuard™ Cartridges (mm)

Phases 50 x 2.0 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 2.0* 4 x 3.0*
Cellulose-3 00B-4493-B0 00B-4493-E0 00D-4493-E0 00F-4493-E0 00G-4493-E0 AJ0-8621 AJ0-8622
for ID: 2.0–3.0 mm 3.2–8.0 mm
SecurityGuard™ Column Performance Check Standard

5 µm Semi-Prep Columns (mm) Cartridges (mm) Part No. Description Unit
Phases 250 x 10.0 10 x 10.0‡ AL0-8412 Chiral Test Mix No. 5 (Lux) ea
Cellulose-3 00G-4493-N0 AJ0-8623
for ID: 9–16 mm
Lux Chiral Method Screening Kits are
available. Please contact your Phenomenex
representative for more information.
5 µm Axia™ Packed Preparative Columns (mm) SecurityGuard™ Cartridges (mm)

Phase 150 x 21.2 250 x 21.2 250 x 30 250 x 50 15 x 21.2** 15 x 30.0♦
Cellulose-3 00F-4493-P0-AX 00G-4493-P0-AX 00G-4493-U0-AX 00G-4493-V0-AX AJ0-8624 AJ0-8625
for ID: 18 –29 mm 30–49 mm
* SecurityGuard Analytical Cartridges require holder, Part No. : KJ0-4282
‡
SemiPrep SecurityGuard™ Cartridges require holder, Part No.: AJ0-9281 **HPLC PREP SecurityGuard Cartridges require holder, Part No. : AJ0-8223
SFC PREP SecurityGuard Cartridges require holder, Part No. : AJ0-8617
♦
HPLC PREP SecurityGuard Cartridges require holder, Part No. : AJ0-8277
SFC PREP SecurityGuard Cartridges require holder, Part No. : AJ0-8618
Bulk Media
Phases 100 g 1 kg
10 µm
Cellulose-3 04G-4624 04K-4624
20 µm
Cellulose-3 04G-4504 04K-4504

Gemini and Aeris LC Column Ordering Information
SecurityGuard™
3 μm Microbore‚ Minibore and MidBore™ Columns (mm) Cartridges (mm)
Phases 50 x 1.0 20 x 2.0 30 x 2.0 50 x 2.0 100 x 2.0 150 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 4 x 2.0*
/10pk
C18 00B-4439-A0 00M-4439-B0 00A-4439-B0 00B-4439-B0 00D-4439-B0 00F-4439-B0 00B-4439-Y0 00D-4439-Y0 00F-4439-Y0 AJ0-7596
C6-Phenyl 00B-4443-A0 — 00A-4443-B0 00B-4443-B0 00D-4443-B0 00F-4443-B0 00B-4443-Y0 00D-4443-Y0 00F-4443-Y0 AJ0-7914
/10pk
NX-C18 00B-4453-A0 00M-4453-B0 00A-4453-B0 00B-4453-B0 00D-4453-B0 00F-4453-B0 00B-4453-Y0 00D-4453-Y0 00F-4453-Y0 AJ0-8367
for ID: 2.0-3.0 mm
SecurityGuard
Phases 30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0*
/10pk
C18 00A-4439-E0 00B-4439-E0 00D-4439-E0 00F-4439-E0 00G-4439-E0 AJ0-7597
C6-Phenyl 00A-4443-E0 00B-4443-E0 00D-4443-E0 00F-4443-E0 00G-4443-E0 AJ0-7915
— /10pk
NX-C18 — 00B-4453-E0 00D-4453-E0 00F-4453-E0 00G-4453-E0 AJ0-8368
for ID: 3.2-8.0 mm
SecurityGuard
5 μm Minibore and MidBore Columns (mm) Cartridges (mm)
Phases 30 x 2.0 50 x 2.0 150 x 2.0 250 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 250 x 3.0 4 x 2.0*
/10pk
C18 00A-4435-B0 00B-4435-B0 00F-4435-B0 00G-4435-B0 00B-4435-Y0 00D-4435-Y0 00F-4435-Y0 00G-4435-Y0 AJ0-7596
C6-Phenyl — 00B-4444-B0 00F-4444-B0 — 00B-4444-Y0 — 00F-4444-Y0 00G-4444-Y0 AJ0-7914
/10pk
NX-C18 00A-4454-B0 00B-4454-B0 00F-4454-B0 — 00B-4454-Y0 00D-4454-Y0 00F-4454-Y0 00G-4454-Y0 AJ0-8367
for ID: 2.0-3.0 mm
SecurityGuard
Phases 30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0*
/10pk
C18 00A-4435-E0 00B-4435-E0 00D-4435-E0 00F-4435-E0 00G-4435-E0 AJ0-7597
C6-Phenyl — 00B-4444-E0 00D-4444-E0 00F-4444-E0 00G-4444-E0 AJ0-7915
— /10pk
NX-C18 — 00B-4454-E0 00D-4454-E0 00F-4454-E0 00G-4454-E0 AJ0-8368
for ID: 3.2-8.0 mm
* SecurityGuard Analytical Cartridges require holder, Part No.: KJ0-4282
‡
SemiPrep SecurityGuard Cartridges require holder, Part No.: AJ0-9281
♦
PREP SecurityGuard Cartridges require holder, Part No.: AJ0-8277
Aeris WIDEPORE 3.6 µm Minibore Columns (mm) SecurityGuard ULTRA Cartridges*

Phases 50 x 2.1 100 x 2.1 150 x 2.1 250 x 2.1 3/pk
XB-C18 00B-4482-AN 00D-4482-AN 00F-4482-AN 00G-4482-AN AJ0-8783

XB-C8 00B-4481-AN 00D-4481-AN 00F-4481-AN 00G-4481-AN AJ0-8785
Bio Core-Shell Technology
C4 00B-4486-AN 00D-4486-AN 00F-4486-AN 00G-4486-AN AJ0-8899
for 2.1 mm ID
Aeris WIDEPORE 3.6 µm Analytical Columns (mm) SecurityGuard ULTRA Cartridges*

Phases 100 x 4.6 150 x 4.6 250 x 4.6 3/pk
XB-C18 00D-4482-E0 00F-4482-E0 00G-4482-E0 AJ0-8769

XB-C8 00D-4481-E0 00F-4481-E0 00G-4481-E0 AJ0-8771
C4 00D-4486-E0 00F-4486-E0 00G-4486-E0 AJ0-8901
for 4.6 mm ID
* SecurityGuard ULTRA cartridges require holder part number AJO-9000.

Sample Preparation Ordering Information
Polymeric SPE
Strata-X Polymer-Based Strata-X Microelution

SPE 96-Well Plates SPE 96-Well Plates
96-Well Plates (2/Box) 96-Well Plates (ea)
Phase 10 mg 30 mg 60 mg Phase 2 mg
Strata-X-AW 8E-S038-AGB 8E-S038-TGB 8E-S038-UGB Strata-AW 8M-S038-4GA
Strata-X-A 8E-S123-AGB 8E-S123-TGB 8E-S123-UGB Strata-A 8M-S123-4GA
Strata-X 8E-S100-AGB 8E-S100-TGB 8E-S100-UGB Strata-X 8M-S100-4GA
Strata-X-C 8E-S029-AGB 8E-S029-TGB 8E-S029-UGB Strata-X-C 8M-S029-4GA
Strata-X-CW 8E-S035-AGB 8E-S035-TGB 8E-S035-UGB Strata-X-CW 8M-S035-4GA
Strata-XL-AW — 8E-S051-TGB —
Strata-XL-A — 8E-S053-TGB —
Strata-XL — 8E-S043-TGB —
Strata-XL-C — 8E-S044-TGB —
Strata-XL-CW — 8E-S052-TGB —
Strata-X-Drug B 8E-S128-AGB 8E-S128-TGB 8E-S128-UGB
Strata-X Drug N 8E-S129-AGB 8E-S129-TGB —
Strata-X Polymer-Based SPE Tubes

Tubes 1 mL (100/box) 3 mL (50/box) 6 mL (30/box)
Phase 30 mg 60 mg 60 mg 200 mg 500 mg 100 mg 200 mg 500 mg
Strata-X 8B-S100-TAK 8B-S100-UAK 8B-S100-UBJ 8B-S100-FBJ 8B-S100-HBJ 8B-S100-ECH 8B-S100-FCH 8B-S100-HCH
Strata-X-C 8B-S029-TAK — 8B-S029-UBJ 8B-S029-FBJ 8B-S029-HBJ 8B-S029-ECH 8B-S029-FCH 8B-S029-HCH
Strata-X-CW 8B-S035-TAK — 8B-S035-UBJ 8B-S035-FBJ 8B-S035-HBJ 8B-S035-ECH 8B-S035-FCH 8B-S035-HCH
Strata-X-A 8B-S123-TAK — 8B-S123-UBJ 8B-S123-FBJ 8B-S123-HBJ 8B-S123-ECH 8B-S123-FCH 8B-S123-HCH
Strata-X-AW 8B-S038-TAK — 8B-S038-UBJ 8B-S038-FBJ 8B-S038-HBJ 8B-S038-ECH 8B-S038-FCH 8B-S038-HCH
Strata-XL 8B-S043-TAK — 8B-S043-UBJ 8B-S043-FBJ 8B-S043-HBJ 8B-S043-ECH 8B-S043-FCH 8B-S043-HCH
Strata-XL-C 8B-S044-TAK — 8B-S044-UBJ 8B-S044-FBJ 8B-S044-HBJ 8B-S044-ECH 8B-S044-FCH 8B-S044-HCH
Strata-XL-CW 8B-S052-TAK — 8B-S052-UBJ 8B-S052-FBJ 8B-S052-HBJ 8B-S052-ECH 8B-S052-FCH 8B-S052-HCH
Strata-XL-A 8B-S053-TAK — 8B-S053-UBJ 8B-S053-FBJ 8B-S053-HBJ 8B-S053-ECH 8B-S053-FCH 8B-S053-HCH
Strata-XL-AW 8B-S051-TAK — 8B-S051-UBJ 8B-S051-FBJ 8B-S051-HBJ 8B-S051-ECH 8B-S051-FCH 8B-S051-HCH
Strata-X-Drug B 8B-S128-TAK — 8B-S128-UBJ — — — — —
Strata-X Drug N 8B-S129-TAK — 8B-S129-UBJ — — 8B-S129-ECH — —
Additional formats available, contact your local Phenomenex office for more information.
Novum SLE 96-Well Plates

Novum Simplified Liquid Extraction (SLE) Well Plates
Part No.
8E-S138-FGA
Description
Novum SLE MINI 96-Well Plate
Unit
1 / Box
novum
simplified liquid extraction
8E-S138-5GA Novum SLE MAX 96-Well Plate 1 / Box
Novum SLE Tubes

Novum Simplified Liquid Extraction (SLE) Tubes
Part No. Description Unit
8B-S138-FAK Novum SLE 1 cc tubes 100 / Box
8B-S138-5BJ Novum SLE 3 cc tubes 50 / Box
8B-S138-JCH Novum SLE 6 cc tubes 30 / Box
8B-S138-KDG Novum SLE 12 cc tubes 20 / Box

Sample Preparation Ordering Information
Phree Phospholipid Removal Products
Part No. Description Unit Phospholipid Removal Solutions
8B-S133-TAK Phree Phospholipid Removal 1 mL Tube 100/box
8E-S133-TGB Phree Phospholipid Removal 96-Well Plates 2/box
Impact Protein Precipitation Plates

Impact Precipitation Products
CE0-7565 Impact Protein Precipitation, Square Well, Filter Plate, 2 mL 2/pk
Impact Protein Precipitation, Square Well, Long Drip, Filter Plate, 2 mL Rapid Protein Precipitation
CE0-7566 2/pk
Impact Starter Kit for Protein Precipitation
CE0-8201 Impact Protein Precipitation Plate (2 ea) ea
Collection Plate 2 mL (2 ea)
Sealing Mat, Santoprene™ (AH0-8199) (2 ea)
β-Gone β-Glucuronidase Removal Products

β-Gone
8B-S139-TAK 1 mL Tubes, Recombinant Enzyme 100 / Box β-Glucuronidase Removal Products
8B-S322-DAK 1 mL Tubes, Non-Recombinant Enzyme 100 / Box

8E-S139-TGA 96-Well Plate, Recombinant Enzyme 1 / Box
8E-S322-DGA 96-Well Plate, Non-Recombinant Enzyme 1 / Box
8N-S323-TUK 2 mL Centrifuge Tubes, Recombinant and Non-Recombinant Enzyme 100 / Box
Presston 100 Positive Pressure Manifold

Presston
AH0-9334 Presston 100 Positive Pressure Manifold, 96-Well Plate ea
AH0-9342 Presston 100 Positive Pressure Manifold, 1 mL Tube Complete Assembly ea
Phenomenex warrants that for a period of 12 months following delivery, the Presston 100 Positive
Pressure Manifold you have purchased will perform in accordance with the published specifications
and will be free from defects in materials or workmanship. In the event that the Presston 100
Positive Pressure Manifold does not meet this warranty, Phenomenex will repair or replace
defective parts.Please visit www.phenomenex.com/Presston for complete warranty information.
Accessories
Collection Plates (deep well, polypropylene)
AH0-7192 96-Well Collection Plate 350 µL/well 50/pk
AH0-7193 96-Well Collection Plate 1 mL/well 50/pk
AH0-7194 96-Well Collection Plate 2 mL/well 50/pk
AH0-8635 96-Well Collection Plate, 2 mL Square/Round-Conical 50/pk
AH0-8636 96-Well Collection Plate, 2 mL Round/Round, 8 mm 50/pk
AH0-7279 96-Well Collection Plate, 1 mL/well Round, 7 mm 50/pk
Sealing Mats
AH0-8597 Sealing Mats, Pierceable, 96-Square Well, Silicone 50/pk
AH0-8598 Sealing Mats, Pre-Slit, 96-Square Well, Silicone 50/pk
AH0-8631 Sealing Mats, Pierceable, 96-Round Well 7 mm, Silicone 50/pk
AH0-8632 Sealing Mats, Pre-Slit, 96-Round Well 7 mm, Silicone 50/pk
AH0-8633 Sealing Mats, Pierceable, 96-Round Well 8 mm, Silicone 50/pk
AH0-8634 Sealing Mats, Pre-Slit, 96-Round Well 8 mm, Silicone 50/pk
AH0-7362 Sealing Tape Pad 10/pk
Vacuum Manifolds
AH0-6023* SPE 12-Position Vacuum Manifold Set, for tubes ea
AH0-6024* SPE 24-Position Vacuum Manifold Set, for tubes ea
AH0-8950 96-Well Plate Manifold, Universal with Vacuum Gauge ea
*Manifolds include: Vacuum-tight glass chamber, vacuum gauge assembly, polypropylene lid with
gasket, male and female luers and yellow end plugs, stopcock valves, collection rack assemblies, poly-
propylene needles, lid support legs. Waste container included with 12-positive manifold.

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Phenomenex products are available worldwide. For the distributor in your country,
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Trademarks
Kinetex, Strata, Luna, Lux, and Gemini are registered trademarks and Impact, β-Gone,
Phenex, SecurityGuard, Phree, Novum, roQ, Axia, Verex, Solvent Shielding Technology,
and Aeris are trademarks of Phenomenex. Agilent is a registered trademark of Agilent
Technologies, Inc. Freedom EVO is a registered trademark of Tecan Group Ltd. Shi-
madzu, Nexera, and Prominence are registered trademarks of Shimadzu Corporation.
MultiPROBE is a registered trademark of PerkinElmer, Inc. ACQUITY, Waters, Oasis,
and UPLC are registered trademarks of Waters Technologies Corporation. Cohesive is
a registered trademark of Thermo Fisher Scientific. Isolute and Evolute are registered
trademarks of Biotage AB Corp. Intercept i2 is a registered trademark of OraSure Tech-
nologies, Inc. Quantisal is a registered trademark of Alere San Diego, Inc. DBA Immu-
nalysis Corporation. Sartorius and arium are registered trademarks of Sartorius Stedim
Biotech GmbH. Cerilliant is a registered trademark of Cerilliant Corporation. Sigma-Al-
drich is a registered trademark of Sigma-Aldrich Co. LLC. Honeywell is a trademark of
Honeywell International Inc. Santoprene is a trademark of EXXON MOBIL Corporation.
BioreclamationIVT is a registered trademark of Bioreclamation-IVT Holdings, LLC.
Milli-Q is a registered trademark of MERCK KGaA, Darmstadt, Germany. Analyst and
QTRAP are registered trademarks and Triple Quad, MultiQuant, Scheduled MRM API
3000, API 3200, API 4000, API 4500, and API 5000 are trademarks of AB SCIEX Pte,
Ltd. AB SCIEX is being used under license.
Disclaimer
FOR RESEARCH PURPOSES ONLY. Not intended for diagnostic use.
Phenomenex is not affiliated with Agilent Technologies, Inc., Tecan Group Ltd.,
Shimadzu Corporation, PerkinElmer, Inc., Waters Technologies Corporation, Thermo
Fisher Scientific, Biotage AB Corp., OraSure Technologies, Inc., Alere San Diego, Inc.
DBA Immunalysis Corporation, Sartorius Stedim Biotech GmbH, Cerilliant Corporation,
Sigma-Aldrich Co. LLC., Honeywell International Inc., EXXON MOBIL Corporation,
Bioreclamation-IVT Holdings, LLC., MERCK KGaA Darmstadt Germany.
Strata-X is patented by Phenomenex. U.S. Patent No. 7,119,145.
Novum is patent pending.
SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362.
GU52250517_W
Caution: this patent only applies to the analytical-sized guard cartridge holder, and does not
apply to SemiPrep, PREP or ULTRA holders, or to any cartridges.
Kinetex EVO and Gemini are patented by Phenomenex. U.S. Patent Nos. 7,563,367 and
8,658,038 and foreign counterparts.
Axia columns and packing technology is patented by Phenomenex. U.S. Patent No.
7,674,383.
© 2017 Phenomenex, Inc. All rights reserved.

Phenomenex Clinical Aplication Notebook

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Phenomenex Clinical Aplication Notebook

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THE

Therapeutic Drug Monitoring......................................................................................... 41

Sample Preparation....................................................................................................... 109

Ordering Information..................................................................................................... 129

If Phenomenex products mentioned in this guide do not provide at least

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Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA.

Materials and Methods Dispense 300 µL of mobile phase A (or water)

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Table 1. Retention and Recovery of the Bile Acids

Analyte & IS name RT(min) % Recovery % CV (N=5)

2.0e5 DCA-D4 6.02

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Solid Phase Strata®/

Sample Preparation Selection and Users Guide

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SPE Protocol LC-MS/MS Conditions

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Overview Table 1. Recovery Values from 10 ng/mL to 63 pg/mL

Materials and Methods 0.25 99 3

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Figure 3. 3-Methoxytyramine separated interference at 63 pg/mL on Kinetex Biphenyl

Figure 4. Representative Chromatogram of Urinary Catecholamines

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Figure 6. Representative Calibration Curve for Normetanephrine

Results and Discussion

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Find Your Kinetex Column:

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Materials and Methods Peak Name MRM Channel

HPLC Conditions 1.8e4

ESI LC-MS/MS under multiple-reactions-monitoring function1. 8000.0

used for the separation.

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Overview LC-MS/MS Conditions

9.0e5 3. 17-Deoxycortisol (Corticosterone) 1.1e6

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Overview SPE Conditions

Cortisol (m.w. 362.4) Cortisone (m.w. 360.4)

Prednisone (m.w. 358.4) Prednisolone (m.w. 360.4)

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LC-MS/MS Conditions 140 QCL (30 ng/mL)

LC-MS/MS was performed using a Kinetex 2.6 μm core-shell ®

Biphenyl HPLC/UHPLC column, 50 x 3.0 mm (P/N 00B-4622-Y0) 120

on an Agilent® 1200SL LC system (Agilent Technologies, Palo

with a binary pump, autosampler and interfaced with an API

Analyte Q1 Mass (Da) Q3 Mass (Da) Analyte Retention

Cortisone 1 361 163 3.36 selected

Cortisol 1 363 121 3.31

Prednisolone 2 361 173 3.19

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LL0Q QCL QCM QCH

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4500 Prednisone 5500

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