A Rapid Solid Phase Extraction Solution

Solid Phase Extraction Polymeric SPE

Solid Phase
Extraction for
Clinical
Research
Clean | Quick | Accurate
Revision: 1
PHEN-RUO-00203

www.phenomenex.com/SPE
 Contents

A Rapid Solid Phase Extraction Solution

Solid Phase Extraction 3

Opiates from Urine 4

Synthetic Benzodiazepines 6

Testosterone from Human Serum 9

Pain Panel from Oral Fluid 12

Comprehensive Drug Research Panel from Oral Fluid Collection Devices 17

Extracting Pain Relievers from Plasma 19

GHB from Whole Blood 21

Enzymatic Markers from Human Serum 24

25-Hydroxyvitamin D3 from Serum 30

2
 Solid Phase Extraction
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on Advantages
• • • • • • • •
simplified liquid extraction

A separation process that is used to remove compounds from a mixture, based on their physical
and chemical properties. Analytical laboratories use solid phase extraction to concentrate and
se purify samples for analysis from a wide variety of matrices.

n
3 Unique Sorbent Platforms

A Rapid Solid Phase Extraction Solution

Solid Phase Extraction

Polymeric SPE
Silica-based SPE sorbent
Underline reversed phase Polymeric sorbent available
provides a reliable and clean
polymer with matrix removal in reversed phase and
extracts with high recoveries
technology offers a faster, ion-exchange capabilities for
for target analytes across all
cleaner way to perform SP. wide range of applications.
sample matrices.

RAPID ER
EXPR LIABL LIABL
RE RE

3 2 5 5
E

E
SUP

ES
S

STEPS STEPS STEPS STEPS

Identify Your SPE Retention Mechanism

Aqueous Organic Organic

Add Water miscible, Add
Sample Water, Urine, Plasma, H2O Hexane Not water miscible,
Blood, Aqueous Methanol, Hexane,
Matrix Acetonitrile, Acetone
Tissue Prep. Dichloromethane

For ionic
species only

Reversed Phase
Strata-X PRO, Strata-X, Ion-Exchange Normal Phase
XL, C18, Phenyl, C8,
Strata-X-C, X-CW, X-A,
PAH, EPH, Melamine, Strata NH2 , CN, Si-1,
X-AW, XL-C, XL-CW,
Suggested SDB-L, GCB,
XL-A, XL-AW, SAX, FL-PR, EPH, AL-N
Retention Strata Activated Carbon
SCX, WCX, NH2, ABW,
Mechanism Ion-Exchange Screen-A, Screen-C,Basic
Strata-X-C, X-CW, X-A Screen, GCB
X-AW, XL-C, XL-CW, XL-A,
XL-AW, SAX, SCX, WCX, NH2,
ABW, Screen-A, Screen-C,
Basic Screen, GCB, Strata
PFAS (WAX/GCB)

3
 Opiates from Urine

Solid Phase Extraction

SPE is used in combination with LC-MS/MS to analyze a panel of

opiates from urine sample containing β-glucuronidase that needs to
be cleaned up prior to sample injection. The SPE method utilizes a
100% organic wash to maximize sample cleaniless, recovery values,
and relative standard deviations. Large Reservoir Cartridges (LRC)
are used in applications that allow for a large load or wash volumes,
but still utilize a small sorbent volume for a concentrated elution
volume, leading to more sensitive extractions.

4
Opiates from Urine

Solid Phase Extraction

Pre-treatment
In a single tube, combine 2 mL 0.1% Formic acid in Water. Then add 100 L of Campbell β-glucu-
ronidase Enzyme and 10 µL of 1 g/mL Internal Standard and vortex for 3-5 seconds.

SPE Protocol LC-MS/MS Conditions

Cartridge: Strata Basic Screen 200 mg/10 mL Column: Kinetex™ 2.6 µm Biphenyl
Dimensions: 50 x 3.0 mm
Part No.: 8B-S327-FTH Part No.: 00B-4622-Y0
Condition: 2.4 mL Methanol SecurtyGuard™ ULTRA: AJ0-9208
Mobile Phase: A: 0.1 % Formic acid in Water
Equilibrate: 2.4 mL 0.1 % Formic acid in Water B: 0.1 % Formic acid in Acetonitrile
Load: 4.1 mL Pre-treated Urine Sample Gradient: Time (min) % B
0 5
Wash 1: 2.4 mL 0.1% Formic acid in Water 4 100
Wash 2: 2.4 mL Methanol 5 100
5.01 5
Elute: 2x 1.2 mL Ethyl acetate/IPA/Ammonium hydroxide (75:20:5) 7.01 5
Dry: Vacuum at 10’ Hg for 5 minutes Flow Rate: 0.6 mL/min
Injection Volume: 1 µL
Reconstitute: 200 µL 0.1 % Formic acid/Methanol (4:1) with internal standard Detection: MS/MS (SCIEX® API 4000™), ESI+

Recovery Values and % CVs for compounds tested

TIC of Extracted Analytes
Analyte % Recovery % CVs (n=6) 2.9e6
2.8e6
6-MAM 99 15
Amphetamine 99 9 2.6e6

Benzoylecgonine 60 10 2.4e6

Buprenorphine 104 7
2.2e6
Codeine 89 12
2.0e6
Diazepam 96 7
1.8e6
Fentanyl 101 4
Intensity, cps

Hydrocodone 103 11 1.6e6

Hydromorphone 98 10 1.4e6

Methamphetamine 110 8 1.2e6

Morphine 106 10
1.0e6
Norbuprenorphine 91 8
8.0e5
Nordiazepam 97 2
6.0e5
Oxycodone 109 8
App ID 24639

PCP 101 6 4.0e5

Temazepam 88 7 2.0e5
Oxazepam 87 18
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min 5.0

5
 Separation of Synthetic Benzodiazepines by
SPE and LC-MS/MS
Polymeric SPE
Drug B

In this application note, we quantify 14 compounds from a urine

matrix with high sensitivity using fast LC analysis in less than
4 minutes. 13 of the analytes are “synthetic” benzodiazepines,
and Alprazolam was added to the panel because it is an isomer
of Deschloroetizolam, and therefore differentiation is necessary.
Strata-X-Drug B Plus was chosen as an ideal SPE plate to maximize
recovery, eliminate matrix interferences, and optimize the workflow
by combining the hydrolysis and SPE within the same platform.
The high efficiency Kinetex™ Biphenyl LC column was used for
chromatographic separation.

6
Separation of Synthetic Benzodiazepines by
SPE and LC-MS/MS
Polymeric SPE
Drug B

Sample Preparation LC Conditions

Column: Kinetex™ 2.6 µm Biphenyl
Sample Pre-treatment: Combine 200 µL of urine sample spiked with 40 µL mix of internal Dimensions: 50 x 3.0 mm
standards (IS) (250 ng/mL), 60 µL hydrolysis buffer, and 20 µL of Part No.: 00B 4622 Y0
IMCSzyme® RT enzyme on the Strata-X-Drug B Plus, 30 mg plate (Part Mobile Phase: A: 0.1 % formic acid in water
No.: 8E-S128-TGB-P). Incubate at room temperature for 15 minutes. B: 0.1 % formic acid in methanol
Gradient: Time (min) % B
200 µL 0.1 % formic acid in water. Shake/vortex for 1 min, then apply 0 60
Load:
vacuum. 0.5 95
Wash 1: 1 mL 0.1 % formic acid in water. 2.5
2.51
95
60 MS Conditions
Wash 2: 1 mL 30 % methanol. 4 60
Dry: 5 min at high vacuum (15-20 in Hg). Flow Rate: 500 µL/min Ion Source: Positive
Injection Volume: 5 µL Source Temperature: 550 °C
Dry Down: Sample under gentle stream of nitrogen at 40-45 °C. Temperature: 40 °C GS1: 40
LC System: Agilent® 1260 Infinity GS2: 60
Reconstitute: Dried samples in 200 µL initial mobile phase.
Detection: MS/MS CUR: 20
Detector: SCIEX® Triple Quad™ 4500 IS: 4500

Table 1. MRM Transitions

Analyte Q1 (m/z) Q2 (m/z) Analyte Q1 (m/z) Q2 (m/z)

Adinazolam 352.1 58 Flunitrazolam 338 292

Bromazolam 352.9 324.8 Methylclonazepam 329.8 283.9
Clonazolam 354 308 Nitrazolam 320 246.2
Deschloroetizolam 309.1 280.1 Phenazepam 351.09 185.8
Diclazepam 319.01 154 Alprazolam 309.1 205.2
Etizolam 343.05 314.1 Diclazepam-D4 (IS) 323 231
Flualprazolam 327.01 299 Etizolam-D3 (IS) 346.01 317
Flubromazepam 333.03 226 Phenazepam-D4 (IS) 355 183
Flubromazolam 371 292.02 Clonazolam-D4 (IS) 358 312.02

Figure 1. Separation of 14 Synthetic Benzodiazepines and Internal Standards Using

the Kinetex 2.6 µm Biphenyl LC Column

App ID 26969

7
 Separation of Synthetic Benzodiazepines by
SPE and LC-MS/MS
Polymeric SPE
Drug B

Figure 2. Separation of Isomeric Alprazolam and Deschloroetizolam

Intensity, cps

App ID 26973
Intensity, cps

App ID 26973
Figure 3. Absolute Recovery and % CV for 14 Benzodiazepines
App ID 26973

8
Analyzing Testosterone in Human Serum with
SPE using Strata-X-A
Polymeric SPE
Drug A

Testosterone was extracted from human serum by Strata-X-A strong

anion-exchange polymeric Solid Phase Extraction (SPE)
and analyzed using a Kinetex™ C18, 30 x 2.1 mm, 1.7 µm LC column
and positive polarity ESI LC/MS/MS system. Kinetex sub-2 µm
core-shell technology offers higher efficiencies than traditional
sub-2 µm columns, producing greater chromatographic resolution,
sensitivity, and higher peak capacities.

9
 Analyzing Testosterone in Human Serum with
SPE using Strata-X-A
Polymeric SPE
Polymeric SPE
Drug A

Materials and Methods

Sample Preparation

The sample preparation is based on a simple solid phase extraction method using strong
anion-exchange to produce a clean extract from human serum.

Solid Phase Extraction Method

Cartridge: Strata-X-A, 30 mg/3 mL
Part No.: 8B-S123-TBJ
Condition: 2 mL Methanol
Equilibrate:2 mL Water
Load: Into individual labeled test tubes, combine
0.25 mL serum sample (or calibrator or QC
sample), 1 mL DI water and 0.1 mL working
internal standard solution (2 ng/mL).
Wash: 0.6 mL Methanol/Water (1:1)
Dry: 5 minutes under high vacuum
Elute: 2 x 0.3 mL Methanol

Sample Preparation HPLC Conditions

Following evaporation of elution solvent @ 50-55 °C under Column: Kinetex™ 1.7 μm C18 100 Å
gentle nitrogen stream. Add 50 µL 25% hydroxylamine solution Dimensions: 30 x 2.1 mm
Part No.: 00A-4475-AN
and heat at 60-65 °C for 5-10 min, then add 200 µL 5% aque- Mobile Phase: 0.1% Formic Acid +1 mM Ammonium Formate in Water
ous formic acid and vortex the tubes. Transfer the solution to 0.1% Formic Acid +1 mM Ammonium Formate in Acetonitrile
autosampler vials and inject 25 µL on column. Inject 20 µL on Gradient: Time (min) % B
0 10
HPLC / Mass Spectrometer (MS) @ amu (ambient). 2.5 90
3.5 90
3.6 10
Flow Rate: 0.4 mL/min
Table 1. LC Gradient Program Temperature: 55 °C
Detection: LC/MS/MS ESI+
Analyte: 1. Testosterone
Step Total Time (min) Flow Rate (µL/min) B (%) 2. Testosterone – D3 (IS)
3. Epitestosterone
0 0 400 10
1 2.5 400 90
60 3.5 400 90
104 3.6 400 10
89 5 400 10

Sample Preparation

SCIEX® API 5000™ triple-quadrupole tandem mass spectrometer is used for analysis equipped with an ESI probe operating in pos-
itive polarity mode. Under an MRM mode, two channels were monitored for Testosterone and Testosterone-D3 (Table 2).

Table 2. MRM Transitions Used for Data Analysis

Peak Name MRM Channel

Testo (1) 304.3 124.0

Testo (2) 304.3 112.0
IS (Testo-D3 1) 307.3 124.0
IS (Testo-D3 2) 307.3 112.0

10
Analyzing Testosterone in Human Serum with
SPE using Strata-X-A
Polymeric SPE
Drug A

Results and Discussion

As demonstrated in Figure 1, the Kinetex™ 1.7 µm 30 x 2.1 mm UHPLC column efficiently separates testosterone from its isomeric
form epitestosterone. This column provides a very high degree of selectivity, even in a short dimension, resulting in superior
chromatographic separation in a short run time.

19761
Figure 1. Separation of Testosterone and Epitestosterone by LC/MS/MS
4.0e4 1
3.8e4
3.6e4
3.4e4
3.2e4
3.0e4
2.8e4
2.6e4
2.4e4
2.2e4
2.0e4
1.8e4
2
1.6e4
1.4e4
1.2e4 3
1.0e4
8000.0

App ID 19761
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 min
49 98 146 194 243 291 340 388 436

11
 Quantitation of Pain Panel Analytes from Oral
Fluid Utilizing Microelution Solid Phase
Extraction Coupled with LC-MS/MS
Polymeric SPE

Oral fluid has emerged as a popular biological matrix for analysis

due to its non-invasive nature and ease of sample collection. It has
wide applicability for drug testing and screening in clinical research.
However, the analysis of these compounds in oral fluid becomes
challenging due to the presence of the excipients, surfactants, and
preservatives in the collection buffer of commercially available oral
fluid collection (OFC) devices. These additives are necessary to
ensure the stability and authenticity of the sample during collection
and transport. However, the presence of these additives can foul the
optics of the mass spectrometer and diminish the signal response if
the samples are not cleaned up adequately before injection. In this
communication, we present an effective sample cleanup method
for oral fluid analysis that targets 32 pain panel analytes, utilizing a
mixed mode strong cation exchange Strata-X-C microelution
96-well plate. A Kinetex™ core-shell 2.6 µm Biphenyl, 50 x 4.6 mm
LC column was employed for fast chromatographic separation.

12
Quantitation of Pain Panel Analytes from Oral
Fluid Utilizing Microelution Solid Phase
Extraction Coupled with LC-MS/MS
Core-Shell
Polymeric Technology
SPE

Sample Preparation LC Conditions -

Quantitative
Pre-treatment: Drug free human saliva was spiked (conc. used as per Table 1 & 2)
with standards. 1 mL of oral fluid was pipetted onto the cellulose pad Column: Kinetex™ 2.6 µm Biphenyl
and allowed to absorb until the indicator window turned blue. The sat- Dimensions: 50 x 4.6 mm
urated pad was placed into a transport tube containing buffer solution Part No.: 00B-4622-E0
and allowed to sit overnight. The plastic nipple at the end of transport Mobile Phase: A: 10 mM Ammonium Formate
tube was removed, and the tube was placed in a centrifuge at 6000 rpm B: Methanol
for 10 minutes. The supernatant was collected. Gradient: Time (min) % B
Condition: Strata-X-C, 2 mg/well 96-well microelution plate (8M-S029-4GA) with 0 15
200 µL Methanol 1 70
3 95
Equilibrate: Plate with 200 µL Water 5.5 85
5.51 15
Load: 150 µL supernatant diluted with 150 µL 1 % Formic Acid in Water
7 15
Wash 1: With 200 µL Water Flow Rate: 0.6 mL/min
Injection Volume: 5 µL
Wash 2: With 200 µL 50 % Acetone in 1 % Formic Acid Temperature: Ambient
Dry down 1: For 30 sec at high vacuum (15-20 in. Hg) LC System: Agilent® 1260 Infinity
Detection: MS/MS
Elute: With 2 washes of 50 µL Methanol / Acetonitrile / Ammonium Hydroxide Detector: SCIEX® Triple Quad™ 4500
(5:5:2, v/v/v)
Dry down 2: Evaporate to dryness under a gentle stream of nitrogen at 40-45 °C
Elute: With 100 µL initial mobile phase

Table 1. MRM Transitions and Linearity Data for 32 Pain Panel Analytes Extracted from
Oral Fluid Using the Strata-X-C 96-well Microelution Plate
Reference conc. Q1 Q3 Linearity Range Linear
Analyte Name RT (min)
(ng/mL) (m/z) (m/z) (ng/mL) regression (R2)

Hydroxyalprazolam 4.1 100 325.1 297 1-300 0.998

Amphetamine 2.8 500 136.1 91.1 5-1500 0.999
Benzoylecgonine 2.8 150 290.1 168.1 1.5-450 0.999
Codeine 3.8 100 300.2 152.1 1-300 0.999
Diazepam 4.6 100 285 193.2 1-300 0.998
Methylenedioxy-methamphetamine (MDMA) 3.2 250 194.1 105.1 2.5-750 0.999
Methamphetamine 2.99 500 150.1 91 5-1500 0.998
Oxymorphone 3.3 100 302.1 227 1-300 0.997
Phencyclidine (PCP) 4.9 25 244.3 91 0.25-75 0.999
Sufentanil 4.9 3 387.2 238.1 0.03-9 0.995
6-Monoacetylmorphine 3.5 10 328.1 165.1 0.1-30 0.998
Clonazepam 3.8 100 316.1 270.1 1-300 0.995
2-Ethylidene-1,5-dimethyl-3,3-dipehnylpyrrolidine (EDDP) 5.2 100 278.2 234.2 1-300 0.997
Fentanyl 4.6 3 337.3 105.1 0.03-9 0.998
Flunitrazepam 4.3 100 314.1 268.2 1-300 0.998
Flurazepam 4.7 100 388.2 315.2 1-300 0.996
Hydrocodone 4.2 100 300.2 199 1-300 0.999
Hydromorphone 3.4 100 286.1 185.1 1-300 0.999
3,4-Methylenedioxymethamphetaminen (MDA) 2.9 250 180.1 133 2.5-750 0.999
Methyl diethanolamine (MDEA) 3.2 250 208.2 163 2.5-750 0.998
Meperidine 3.9 250 248.2 220.2 2.5-750 0.999
Methadone 5.4 100 310 265 1-300 0.999
Midazolam 4.5 100 326.1 291.1 1-300 0.999
Morphine 3.1 100 286.1 152.1 1-300 0.997
Naloxone 4.17 100 328.2 212 1-300 0.995
Naltrexone 4.19 100 342.2 267.1 1-300 0.996
Nordiazepam 4.15 100 271 140 1-300 0.997
Normeperidine 3.5 100 234.1 160.1 1-300 0.995
Oxycodone 4.2 100 316.1 241.2 1-300 0.999
Temazepam 4.3 100 301.1 255.1 1-300 0.996
Tramadol 3.5 100 264.1 58.1 1-300 0.999
Cocaine 4.2 100 304.2 150 1-300 0.998

13
 Quantitation of Pain Panel Analytes from Oral
Fluid Utilizing Microelution Solid Phase
Extraction Coupled with LC-MS/MS
Polymeric SPE

Figure 1. Representative Chromatogram of 32 Pain Panel Analytes Extracted from Oral

Fluid Matrix Utilizing a Kinetex™ 2.6 µm Biphenyl LC Column
Intensity, cps

App ID 26999
min.

Figure 2. Representative Q1 Scan Comparison for Extracted Oral Fluid Sample Before
and After SPE Cleanup
Intensity, cps

App ID 26998
min.

Figure 3. Representative Chromatogram Obtained From Post Column Infusion of

Codeine, for Extracted Oral Fluid Sample Before and After SPE Cleanup
Intensity, cps

App ID 26999

min.

14
Quantitation of Pain Panel Analytes from Oral
Fluid Utilizing Microelution Solid Phase
Extraction Coupled with LC-MS/MS
Core-Shell
Polymeric Technology
SPE

Table 2. Precision and Accuracy Data for 32 Pain Panel Analytes Extracted from Oral
Fluid Using the Strata-X-C 96-well Microelution Plate
QC-1 QC-2 QC-3
Analyte Name (5% of Reference) (40% of Reference) (2x Reference)
(ng/mL) (ng/mL) (ng/mL)

% % CV % % CV % % CV
Accuracy (N=4) Accuracy (N=4) Accuracy (N=4)
Hydroxyalprazolam 104.2 6.4 105.9 7.4 108.4 13.1
Amphetamine 103.7 13.5 99.3 5.2 94 6.2
Benzoylecgonine 111.3 11.3 114.3 13.4 105.5 2.9
Codeine 108.9 12.3 108.6 6.2 103.2 3.3
Diazepam 101.3 6.2 105.3 9.9 104.9 9.9
Methylenedioxy-methamphetamine (MDMA) 109.4 10.1 99.8 4.7 99.9 2.2
Methamphetamine 99.1 6.4 95.6 3.7 93.4 3.6
Oxymorphone 106.8 5.3 109 7 100.6 4.8
Phencyclidine (PCP) 95 4.8 100.3 7.6 97 1.5
Sufentanil 103.9 18.1 118.1 10.9 89.6 11.3
6-Monoacetylmorphine 99.3 16.7 104.9 4.3 103.8 4.2
Clonazepam 108.3 16.4 99.6 11 95.2 7
2-Ethylidene-1,5-dimethyl-3,3-dipehnylpyrrolidine (EDDP) 114.3 15.6 99.8 12.8 105.4 10.9
Fentanyl 107.4 14.9 117 8.8 98.8 1.9
Flunitrazepam 107.3 18.1 113 15.6 115 10.2
Flurazepam 98.5 20.3 114.4 14.9 117.8 1.9
Hydrocodone 112.4 8.8 107.4 7.4 95.7 1.8
Hydromorphone 116.3 13.5 112.9 3.4 108.7 1.6
3,4-Methylenedioxymethamphetaminen (MDA) 100 6.7 109.4 4.9 94.9 5.3
Methyl diethanolamine (MDEA) 110.8 10.6 102.3 4.5 101.1 5.4
Meperidine 91.6 15.3 98.6 3.4 91.3 3.2
Methadone 86.5 9.3 89.5 9 92.5 5.9
Midazolam 12.2 13 111.1 7.6 105.7 8.5
Morphine 102.6 7.1 102.5 4.3 93.9 5.3
Naloxone 113.5 19.3 102.7 14.9 98.7 6
Naltrexone 105.2 8.5 109 10.2 97.6 6.5
Nordiazepam 93.6 14.5 118 15.5 97.1 14.4
Normeperidine 80.8 18.3 90.2 13.2 95.2 11.2
Oxycodone 115.5 5.1 112.1 8.1 97.9 1.4
Temazepam 96.1 10.7 116.9 11.2 109.3 12.5
Tramadol 92.3 9.6 109.1 10.3 85 3
Cocaine 102.7 5.3 114.8 7.7 91 15.1

15
 Quantitation of Pain Panel Analytes from Oral
Fluid Utilizing Microelution Solid Phase
Extraction Coupled with LC-MS/MS
Polymeric SPE

Figure 4. Linearity Curves for Selected Analytes in Oral Fluid Sample Extracted Using a
Strata-X-C, 96-well Microelution Plate over a 300-fold Dynamic Concentration Range

Fentanyl (0.03-9 ng/mL) Amphetamine (5-1500 ng/mL)

Analyte Area / IS Area

Analyte Area / IS Area

App ID 27002

App ID 27003
Analyte Conc. / IS Conc. Analyte Conc. / IS Conc.

Codeine (1-300 ng/mL) 6-Monoacetylmorphine (0.1-30 ng/mL)

Analyte Area / IS Area

Analyte Area / IS Area

App ID 27004

App ID 27005
Analyte Conc. / IS Conc. Analyte Conc. / IS Conc.

Phencyclidine (0.25-75 ng/mL) Diazepam (1-300 ng/mL)

Analyte Area / IS Area

Analyte Area / IS Area

App ID 27006

App ID 27007

Analyte Conc. / IS Conc. Analyte Conc. / IS Conc.

Conclusion
The prescribed sample prep method utilizing microelution SPE resulted in a simple, rapid extraction for identification and quantitation
of 32 pain panel analytes from oral fluid which is cost effective and can efficiently be incorporated in clinical workflow analysis.

16
Comprehensive Drug Research Panel from
Oral Fluid Collection Devices

The oral fluid collection device provides a buffer solution that

contains a number of antibacterial agents and surfactants that act
to prevent bacterial growth and increase the analytes stability.
The buffer solution poses many chromatography challenges, such
as ion supression. Here, we present a fast sample preparation
procedure to reduce the effects of the device’s buffer solution while
maintaining good recovery of analytes using Strata-X-CW SPE
method for different classes of analyes from a comprehensive
drug research panel.

17
 Comprehensive Drug Research Panel from
Oral Fluid Collection Devices

Pre-treatment
Transfer 1 mL of oral fluid collected on an applicator tip in its preservative buffer. Leave it for 2 hours followed by centrifugation for
15 minutes at 600 g. Remove 0.5 mL of supernatant and combine with 1 mL of 1% Formic acid. Vortex for 5-10 seconds.

%
Absolute % CV
SPE Protocol Analyte panel recovery (N=4) Ionization
6-MAM 79 7.2 ESI+
96-Well Plate: Strata-X-CW 30 mg/well
a-Hydroxyalprazolam 88 6.8 ESI+
Part No.: 8E-S035-TGB
Condition: 1mL Methanol Alprazolam 79 7.7 ESI+

Equilibrate: 1 mL DI Water Amitriptyline 57 11.6 ESI+

Load: Pre-treated Sample Amphetamine 94 9.8 ESI+
Wash 1: 1 mL 1% Formic acid in DI Water Benzoylecgonine 89 3.9 ESI+
Wash 2: 1 mL DI Water Buprenorphine 63 5.5 ESI+
Dry: 5-6 minutes at maximum vacuum (20”Hg or higher)
Carisoprodol 95 4.8 ESI+
Elution: 2x 500 µL (2 aliquots of 500uL) Methylene chloride/Isopropanol/30% Ammo-
nium hydroxide in Water (80:18:2) Citalopram 81 3 ESI+
Dry Down: Evaporate to dryness under nitrogen at 45-50 °C Cocaine 84 11.2 ESI+
Reconstitute: 200 µL of 0.1% Formic acid in Water/0.1% Formic acid in Methanol (90:10) Codeine 89 5 ESI+
Diazepam 74 5.7 ESI+
EDDP 72 5.7 ESI+
Fentanyl 74 4.8 ESI+
Representative LC-MS Chromatogram of Fluoxetine 42 7.7 ESI+
See the
Buffer Solution (Quantisal®) reduction in
Hydrocodone 102 10 ESI+
buffer Hydromorphone 98 6.6 ESI+
interferences! Imipramine 64 10.5 ESI+
Lorazepam 69 3.8 ESI+
6.5e9
MDMA 67 16.1 ESI+
6.0e9
5.5e9 Meperidine 84 8.9 ESI+
5.0e9
4.5e9
Mephedrone 84 8.9 ESI+
4.0e9 Meprobamate 92 13 ESI+
Intensity, cps

3.5e9
3.0e9 Methadone 72 6.6 ESI+
2.5e9
Methamphetamine 81 5.4 ESI+
2.0e9
1.5e9 Morphine 56 15.5 ESI+
1.0e9
Naloxone 83 6.7 ESI+
5.0e8
0.0 Norbuprenorphine 87 11 ESI+
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 min
4.1e6
Nordiazepam 75 5.5 ESI+
4.0e6 6.5e6
3.8e6
3.6e6
3.4e6
6.0e6
5.5e6
Norfentanyl 85 7 ESI+
3.2e6
3.0e6 5.0e6
Norhydrocodone 89 3 ESI+
Intensity, cps

2.8e6 4.5e6
2.6e6
Intensity, cps

2.4e6 4.0e6
2.2e6 3.5e6
2.0e6
1.8e6
1.6e6
3.0e6 Noroxycodone 85 4.6 ESI+
2.5e6
1.4e6

Nortriptyline
1.2e6 2.0e6
1.0e6
8.0e5 1.5e6 48 1.6 ESI+
6.0e5 1.0e6
4.0e5
2.0e5
0
5.0e5
0
Oxycodone 84 7.1 ESI+
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 Oxymorphone 89 17.1 ESI+
Paroxetine 42 8.8 ESI+
PCP 84 3.6 ESI+
Ritalinic Acid 47 19.9 ESI+
Tapentadol 83 7.5 ESI+
Temazepam 86 2.8 ESI+
Tramadol 87 0.8 ESI+
Zolpidem 64 1 ESI+
Butalbital 71 6 ESI-
Secobarbital 82 5 ESI-
Phenobarbital 83 3.4 ESI-
THC-COOH 53 5.7 ESI-

18
Extracting Pain Relievers from Plasma

A Rapid Solid Phase Extraction Solution

Implementing Strata-X PRO offers a fast and effective alternative that

provides high recoveries and reduces matrix effects. Strata-X PRO
uses a 3-step method, eliminating the conditioning and equilibration
steps, yet still yields high recovery and removes phospholipids,
equivalently to Waters® Oasis® PRiME HLB SPE. This application
displays that for a range of pain relievers, an easy SPE method can
be implemented and produce acceptable results.

19
 Extracting Pain Relievers from Plasma

A Rapid Solid Phase Extraction Solution

Implementing Strata-X PRO offers a fast and effective alternative that provides high
recoveries and reduces matrix effects. Strata-X PRO uses a 3-step method, eliminating
the conditioning and equilibration steps, yet still yields high recovery and removes
phospholipids, equivalently to Waters® Oasis® PRiME HLB SPE. This application displays
that for a range of pain relievers, an easy SPE method can be implemented and produce
acceptable results.

SPE Protocol LC-MS/MS Conditions

Column: Kinetex™ 5 µm EVO C18 100Å
Strata-X PRO, 30 mg/well
96-Well Plates: Dimensions: 100 x 2.1 mm
Waters Oasis PRiME HLB
Part No.: 00D-4633-AN
Part No.: 8E-S536-TGA (for Strata-X PRO) SecurityGuard™ ULTRA: AJ0-9298
Load: 500 µL Plasma/0.1 % Formic acid in Water (1:1) Mobile Phase: A: 0.4 % v/v Ammonium Hydroxide in Water
B: Methanol
Wash: 1 mL 5 % Methanol Gradient: Time (min) %B
0 90
Dry: 1 minute at 5” Hg
3.1 20
Elute: 1 mL 0.1 % Formic acid in Acetonitrile/Methanol (90:10) 4.5 20
6.1 90
Dry Down: 1 minute at 5” Hg
11 90
14 90
Flow Rate: 0.3 mL/min
Temperature: Ambient
Detector: TSQ Quantum™ Ultra QQQ (MS/MS)
HPLC System: Accela® 1250

Recoveries of Pain Relievers From Plasma

Waters Oasis PRiME HLB Strata-X PRO
130%
120%
110%
100%
Percent Recovery

90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
ac

ne
e

e
en

te
e
lo
lin

on
in

a
ro
en

so
ox

yl

et
ty

tis
op

pt
of

ni
r

Ac
ip
ap

or
ed
cl

tri
et
itr

C
N
Di

e
or
M

Pr
Am

on
N

tis
or
C

Save over 40% of your method time using Strata-X PRO and still achieve
high recoveries comparable to Waters Oasis PRiME!

20
Quantitative Analysis of Gamma
Hydroxybutyrate (GHB) in Whole Blood
Using Fast SPE and LC-MS/MS A Rapid Solid Phase Extraction Solution

As a drug of abuse, Gamma-hydroxybutyric Acid (GHB) is often

over looked in forensic toxicological analysis unless it is specifically
requested in the screen. Testing for GHB can be difficult due to
inaccurate/unreliable response in immunoassays or chromatographic
screens and due to the stability of the compound. The time sensitive
nature of this assay demands for an analytical procedure that
is quick, efficient, and one that preserves the authenticity of the
biological specimen. A quick, two-step sample preparation protocol
combining protein precipitation (PP) with SPE was employed. The
SPE method did not require any conditioning or equilibration of the
SPE cartridge, thus allowing for significant time savings. A Luna™ 3
μm 150 x 2.0 mm HILIC LC column was utilized for chromatographic
analysis, enabling direct injection of the extracted sample in a MS
friendly 75% organic. The prescribed protocol circumvents the need
for a dry down or reconstitution step and makes it quick and easy
to implement in a laboratory workflow, all while providing accurate
results.

21
 Quantitative Analysis of Gamma
Hydroxybutyrate (GHB) in Whole Blood
Using Fast SPE and LC-MS/MS A Rapid Solid Phase Extraction Solution

SPE Protocol
Sample Preparation

To 500 μL whole blood add 100 μL 5 % (w/v) ZnSO4 and, vortex 3-5 seconds. Add 1.5 mL of chilled (~0 °C) 90:10 Acetonitrile/Meth-
anol while vortexing. Centrifuge samples at 6000 rpm for 10 minutes and then collect supernatant.

SPE Conditions LC Conditions LC Conditions

Quantitative Analysis for GHB Qualitative Analysis for Phospholipids
96-Well Plate: Strata-X PRO, 30mg/well
Part No.: 8E-S536-TGA
Column: Luna 3 μm HILIC
™
Column: Kinetex™ 2.6 μm C18
Load: Pass the supernatant collected from pre-
Dimensions: 150 x 2.0 mm Dimensions: 50 x 2.1 mm
treatment, apply vacuum and collect extract.
Part No.: 00F-4449-B0 Part No.: 00B-4462-AN
Direct Injection: 5 µL of the above sample was injected
Mobile Phase: A: Acetonitrile Mobile Phase: A: 0.1 % Formic acid in Water
directly on LC-MS/MS (bypass dry-down
B: 100 mM Ammonium Formate B: 0.1 % Formic acid in Methanol
and reconstitution).
Gradient: Time (min) % B Gradient: Time (min) % B
Elute: 2 x 0.3 mL Methanol
0 20 0 40
1 20 0.5 95
1.5 50 11.5 95
2 50 11.51 40
2.01 20 13.5 40
7 20 Flow Rate: 0.4 mL/min
Flow Rate: 0.4 mL/min Injection Volume: 1 µL
Injection Volume: 5 µL IHPLC Instrument: Agilent 1260
IHPLC Instrument: Agilent® 1260 MS/MS Instrument: SCIEX API Triple Quad 4500, ESI Source (+)
MS/MS Instrument: SCIEX® API Triple Quad 4500™, ESI Source (+)

Table 1. Retention Time (RT) and MRM Transition for Analytes

Analyte RT (min) Q1 Q3

86.9
GHB 1.7 104.9
68.9

GHB-d6 1.7 111 93

Lyso PC 2.4 496.4 184.2

PC-1 6.7 760.7 184.2

PC-2 7.2 786.8 184.2

PC-2 5 400 10

Table 2. % Absolute Recovery of GHB From Extracted Whole Blood Sample (N=4) Using
Strata-X PRO SPE
Spiked Conc. % Recovery % CV

10.0 µg/mL 98 % 1.7 %

Figure 1.Representative Chromatogram of GHB Extracted Whole Blood Analyzed by a Luna

3 µm 150 x 2.0 mm HILIC LC column
1.70
6.9e4

6.5e4

6.0e4

5.5e4

5.0e4

4.5e4

4.0e4
Intensity, cps

3.5e4

3.0e4

2.5e4
App ID 25616

2.0e4

1.5e4

1.0e4

5000.0

0.0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5

22
 Quantitative Analysis of Gamma
Hydroxybutyrate (GHB) in Whole Blood
Using Fast SPE and LC-MS/MS A Rapid Solid Phase Extraction Solution

Figure 2. Representative Chromatogram Showing Phospholipid Trace of Blood Matrix

in Pre (A) and Post (B) SPE (Strata-X PRO) Extracted Sample

Table 3. Precision and Accuracy Data for

2.40
9.0e6

8.5e6
A
8.0e6

7.5e6
QC Samples
7.0e6
Phospholipid
6.5e6 presence before Expected Replicates
SPE extraction Sample % CV Accuracy
6.0e6
Conc. (µg/mL) (N)
5.5e6

5.0e6 15 QC 1 4 11.4 104.2

Intensity, cps

4.5e6

4.0e6 75 QC 2 4 4.5 94.5

3.5e6

3.0e6

2.5e6

2.0e6

1.5e6

1.0e6

5.0e5

0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 min

9.0e6

8.5e6

8.0e6

7.5e6

7.0e6

B
6.5e6

6.0e6

5.5e6

5.0e6
In te n s ity , c p s

Significant
4.5e6
reduction of
4.0e6 Phospholipids
3.5e6 with
Strata-X PRO SPE
App ID 25526

3.0e6

2.5e6

2.0e6

1.5e6

1.0e6

5.0e5

0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 min

Figure 3. Linearity Curve for GHB Extracted Blood Matrix Over 300-fold Dynamic
Concentration Range
17.8

17.0

16.0

(R=0.9994)
15.0

14.0

13.0

12.0

11.0
Analyte Area / IS Area

10.0

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
Analyte Conc. / IS Conc.

Conclusion
The direct injection capability of the Strata-X PRO extracted sample on the Luna™ HILIC LC column results in a simple, rapid
identification and quantitation of GHB in whole blood. The prescribed method greatly benefits the time sensitive disposition of the
assay and clean-up of whole blood matrix to provide an accurate analysis.

23
 Fast and Effective SPE Clean Up of Uracil,
5,6-Dihydrouracil and 5-Fluorouracil From
Human Serum A Rapid Solid Phase Extraction Solution

Uracil, 5-Fluorouracil (5FU), and 5,6-Dihydrouracil (UH2) offer unique

markers for enzymatic activity. In order to determine the 5FU
catabolic rate, uracil and its homologues are tested in correlation
with each other to ultimately determine if sufficient metabolization is
occurring. The primary goal of this study was to develop a sample
preparation and LC-MS/MS method for quantitative analysis of uracil
and its two homologues from human serum. This application
focuses on developing a sample preparation method and
LC-MS/ MS analysis using a Strata-X PRO solid phase extraction
(SPE) and a Kinetex™ PS C18 HPLC column respectively. The Kinetex
PS C18 column is a positively charged, surface modified C18 phase
that caters its unique selectivity to separating the very polar uracil
and its homologues in this analysis, while the novel Strata-X PRO
greatly reduces the phospholipids in the sample and provides cleaner
extracts.

24
Fast and Effective SPE Clean Up of Uracil,
5,6-Dihydrouracil and 5-Fluorouracil From
Human Serum A Rapid Solid Phase Extraction Solution

Materials and Methods

Reagents and Chemicals

Analytical reference standards and internal standards were purchased from Cerilliant®
Corporation (Round Rock, TX, USA). Doubly stripped MS grade human serum and pooled
human plasma K2EDTA were obtained from Golden West Diagnostics® and Bioreclama-
tionIVT® (Westbury, NY). All other reagents and chemicals were purchased from the Sig-
ma-Aldrich® Company (St. Louis, MO). Ultrapure D.I water was obtained from Sartorius®
Arium® Comfort I, courtesy of Sartorious Corporation (Bohemia, NY).

5,6-Dihydrouracil SPE Procedure

Molar mass: 114.104 g/mol Sample Pre-treatment: Add 100 μL Human serum (doubly
Acidic pKa: 11.73 stripped) to a tube. Add 900 μL of chilled (~0°C) Acetonitrile to
LogP: -1.21 the serum while vortexing. Centrifuge samples at 6000 rpm for
5 minutes.
96-Well Plate:Strata-X PRO, 30mg/well
Part No.: 8E-S536-TGA
Condition: 1 mL Acetonitrile
Load: Pass the supernatant (From Pre-treatment)
and apply vacuum to collect eluted extract.
Uracil Dry Down: Under Nitrogen and heat at ~ 45 °C
Reconstitute: With 100 μL initial mobile phase
Molar mass: 112.088 g/mol Note: For phospholipid analysis
(1) Human plasma K2EDTA was utilized
Acidic pKa: 8.8 (2) Direct injection (bypass dry down and
LogP: -0.5 reconstitute) of the eluted sample
was made.

Quantitative Analysis for Uracil Homologues

5-Fluorouracil Column:
Dimensions:
Kinetex™ 2.6 μm PS C18
150 x 3.0 mm
Part No.: 00F-4780-Y0
Molar mass: 130.078 g/mol Mobile Phase: A: 0.1% Formic acid in Water
Acidic pKa: 7.18 B: Methanol
Gradient: Time (min) % B
LogP: -0.66 0 7
12 7
Flow Rate: 0.2 mL/min
Injection Volume: 10 µL
Instrument: Agilent® 1260
Detection: MS/MS (SCIEX® Triple Quad™ 4500, ESI Dual Polarity)
(Positive for U and UH2, Negative for 5FU)

Qualitative Analysis for Phospholipids

Column: Kinetex 2.6 μm C18
Dimensions: 50 x 2.0 mm
Part No.: 00B-4462-AN
Mobile Phase: A: 0.1 % Formic acid in Water
B: 0.1 % Formic acid in Methanol
Gradient: Time (min) % B
0 40
0.5 95
11.5 95
11.51 40
13.5 40
Flow Rate: 0.4 mL/min
Injection Volume: 2 µL
Column Temperature: 40 °C
Instrument: Agilent 1260
Detection: MS/MS (SCIEX® Triple Quad™ 4500, ESI+)

25
 Fast and Effective SPE Clean Up of Uracil,
5,6-Dihydrouracil and 5-Fluorouracil From
Human Serum A Rapid Solid Phase Extraction Solution

Table 1. Retention Time (RT), MRM Transition, and % Recovery for Analytes
Q1 Q3 Spiked conc. % CV
Analyte RT (min) % Recovery
(m/z) (m/z) (ng/mL)

UH2 4.31 114.9 55.04 100 90 5.4

U 4.65 112.9 69.8 100 84 3.2
41.9
5FU 7.07 128.8 86.1 100 89 3.2
58.9
Uracil 1,3-15N2 (+Ve IS) 4.65 114.8 96.9 200 N/A N/A
5 CI Uracil (-Ve IS) 10.82 145.1 42.1 200 N/A N/A

Figure 1. Representative Chromatogram of Extracted Human Serum Analyzed by a

Kinetex™ 2.6 μm PS C18 LC Column (Under ESI Positive Polarity)

Uracil 1,3-15N2

Uracil

UH2

26
Fast and Effective SPE Clean Up of Uracil,
5,6-Dihydrouracil and 5-Fluorouracil From
Human Serum A Rapid Solid Phase Extraction Solution

Figure 2. Representative Chromatogram of Extracted Human Serum Analyzed by a

Kinetex™ 2.6 μm PS C18 LC Column (Under ESI Negative Polarity)

5 CI Uracil

5 FU

Figure 3. Representative Chromatogram for Qualitative Evaluation of Phospholipid in

Extracted Human Plasma Samples With Two Different Clean-up Techniques Employed
(A) Protein Precipitation (B) Strata-X PRO

27
 Fast and Effective SPE Clean Up of Uracil,
5,6-Dihydrouracil and 5-Fluorouracil From
Human Serum A Rapid Solid Phase Extraction Solution

Figure 4. Linearity Curve for Uracil Extracted Human Serum Over 200-fold
(5 to 1000 ng/mL) Dynamic Concentration Range

R = 0.9999

Figure 5. Linearity Curve for UH2 Extracted Human Serum Over 200-fold
(5 to 1000 ng/mL) Dynamic Concentration Range

R = 0.9992

Figure 6. Linearity Curve for 5FU Extracted Human Serum Over 200-fold
(5 to 1000 ng/mL) Dynamic Concentration Range

R = 0.9993

28
 Fast and Effective SPE Clean Up of Uracil,
5,6-Dihydrouracil and 5-Fluorouracil From
Human Serum A Rapid Solid Phase Extraction Solution

Table 2. Precision and Accuracy Data for QC Samples of Uracil

Replicates
Expected Conc. (ng/mL) Sample %CV Accuracy
(N = 4)

30 QC1-1 4 4.6 97.4

150 QC1-2 4 5.3 101.6
750 QC1-3 4 6.9 102

Table 3. Precision and Accuracy Data for QC Samples of UH2

Replicates
Expected Conc. (ng/mL) Sample %CV Accuracy
(N = 4)

30 QC1-1 4 11.8 89.3

150 QC1-2 4 8.1 96.2
750 QC1-3 4 5.3 94.8

Table 4. Precision and Accuracy Data for QC Samples of 5FU

Replicates
Expected Conc. (ng/mL) Sample %CV Accuracy
(N = 4)

30 QC1-1 4 2.4 106.7

150 QC1-2 4 10.1 105.0
750 QC1-3 4 7.8 108.9

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29
 Increased Sensitivity of 25-Hydroxyvitamin D3
Detection Using a Strata-X PRO SPE Plate
A Rapid Solid Phase Extraction Solution

Matrix interferences, such as phospholipids, can cause challenges

when measuring serum levels of 25-Hydroxyvitamin D3 by
LC-MS/MS. In this application note, we describe a method
optimization approach using a Strata-X PRO solid phase extraction
(SPE) plate (Part No.: 8B-S536-TAK) and a Kinetex™ 2.6 µm XB-C18
LC column. By removing the phospholipids from serum samples,
there is a reduction in ion suppression yielding higher sensitivity with
lower limits of detection, when compared to polymeric SPE.

30
Increased Sensitivity of 25-Hydroxyvitamin D3
Detection Using a Strata-X PRO SPE Plate
A Rapid Solid Phase Extraction Solution

Sample Preparation

Step Polymeric SPE Strata-X PRO

Condition: 1 wash 600 µL Methanol Not Required

Equilibrate: 1 wash 600 µL Water Not Required

600 µL serum / 0.1 % Formic Acid 600 µL serum / 0.1 % Formic Acid
Load:
in Water (1:1, v/v) in Water (1:1, v/v)
Wash: 1 wash 600 µL 30 % Methanol in Water 1 wash 600 µL 30 % Methanol in Water

1 wash 600 µL 0.1 % Formic Acid 1 wash 600 µL 0.1 % Formic Acid
Elute:
in Acetonitrile / Methanol (90:10, v/v) in Acetonitrile / Methanol (90:10, v/v)

LC Conditions MS/MS Conditions

Column: Kinetex™ 2.6 µm XB-C18 Ion Spray Voltage: 5500 V
Dimensions: 100 x 2.1 mm Polarity: Positive
Part No.: 00B-4496-AN Scan Type: MRM
Guard: SecurityGuard™ ULTRA CUR: 10
Guard Part No.: AJ0-8782 GS1: 25
Mobile Phase: A: 0.1 % Formic Acid in Water NC: 3
B: 0.1 % Formic Acid in Methanol EP: 10
Gradient: Time (min) % B CXP: 10
0 60 Temperature: 400 °C
1 100
4 100
4.01 60
7.01 60
Flow Rate: 0.45 mL/min
Injection Volume: 20 µL
Temperature: 25 °C
Instrument: Agilent®
Detection: MS/MS
Detector: SCIEX® 4500 Triple Quad™ (APCI)

MRM Transitions

Analyte Q1 (m/z) Q3 (m/z) CE

25-Hydroxyvitamin D3 383.4l 257.2 23.0

25-Hydroxyvitamin D3 383.4 159.1 36.0

Figure 1. Comparison of Sensitivity Between Polymeric SPE and Strata-X PRO for
25-Hydroxyvitamin D3
App ID 25566

31
 A Rapid Solid Phase Extraction Solution
Solid Phase Extraction Polymeric SPE

Solid Phase
Extraction for Clean | Quick | Accurate
Clinical
Research
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Terms and Conditions: Subject to Phenomenex Standard Terms & Conditions, which may be viewed at www.phenomenex.com/phx-terms-and-conditions-of-sale.
Trademarks: Strata, Kinetex, Luna, SecurityGuard, BE-HAPPY, are trademarks of Phenomenex. TSQ Quantum is a trademark and Accela is a registered trademark of Thermo Fisher
Scientific. Waters and Oasis are registered trademarks of Waters Technologies Corp. Quantisal is a registered trademark of Alere San Diego, Inc. IMCSzyme is a registered trademark
BR53080423_W

of Integrated Micro-Chromatography Systems, LLC. Sigma-Aldrich is a registered trademark of Sigma-Aldrich Co., BioreclamationIVT a registered trademark of Bioreclamation-
IVT Holdings, LLC. Sartorius and arium are registered trademarks of Sartorius AG. Golden West Diagnostics is a registered trademark of Golden West Diagnostics, LLC. SCEIX is a
registered trademark and API 5000 and API 4000 are trademarks of AB SCIEX Pte. Ltd. Phenomenex is in no way affiliated with Thermo Fisher Scientific, Waters Technologies, Inc., Alere
San Diego, Inc., Integrated Micro-Chromatography Systems, LLC., Golden West Diagnostics, LLC., Bioreclamation-IVT Holdings, LLC., Sigma-Aldrich Co. Comparative separations may
not be representative of all applications. Strata-X is patented by Phenomenex. U.S. Patent No. 7,119,145. Kinetex EVO are patented by Phenomenex. U.S. 7,563,367 and 8,658,038 and
foreign counterparts. Disclaimers: FOR RESEARCH USE ONLY. Not for use in clinical diagnostic procedures. © 2023 Phenomenex, Inc. All rights reserved.