Experimental Conditions (continued)

Method 1 Method 2 Method 3

SPE Cartridge: Strata-X-Drug B 60mg/6 mL

Load: Pre-treated urine sample Wash

1: 2 mL 100 mM Sodium acetate buffer (pH 5.0)

Wash 2:

2 mL Methanol

Dry: 10 min under full vacuumElute: 2 mL Ethyl acetate:Isopropanol:Ammonium

hydroxide mixture (7:2:1)

PO

950

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A Simplified and Streamlined Approach to Solid Phase Extraction for Drugs of AbuseMichael Rummel, Matthew Trass, Seyed Sadjadi, Ngoc Nguyen, Carl Sanchez, Jeff Layne, and Sky Countryman Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501 USA

TrademarksKinetex is a registered trademark of Phenomenex, Inc. Zebron and Strata-X are trademarks of Phenomenex, Inc.

Strata-X is patented by Phenomenex, Inc. U.S. Patent No. 7, 119, 145 © 2012 Phenomenex, Inc. All rights reserved.

Introduction

Experimental Conditions

Tightened drug testing guidelines are placing additional stress on toxicology labs. As such, there is an increased need for a streamlined procedure for sample cleanup and analysis. Recent examples of revised guidelines for drugs of abuse testing from urine samples include testing of additional compound classes such as MDMA and 6-monoacetylmorphine (6-MAM) requiring labs to include additional analyses and extractions in their drug screening protocols. Another trend is lowering the cutoff limits (Table 1) for several drug classes could result in a potential increase in positive samples that will need further confirmation by GC/MS or LC/MS analysis.1

The goal of this work was to develop

a minimum number of extraction methods on a single SPE sorbent for all required analytes followed by analysis by GC/MS or LC/MS. While developing SPE methods, it was also important to minimize the number of steps to save time and solvent, thus increasing sample throughput, while maintaining high standards for linearity, recovery, and reproducibility. To demonstrate the performance of the extraction methodology, urine samples were prepared for each drug class and spiked at 40, 100, and 125 % of the cutoff level. For those samples requiring hydrolysis, both ß-glucuronidase and acid hydrolysis were used; depending on analyte class.

Conclusion• TheSPEextraction for the common

drugs was effectively reduced to a load step, two wash steps, a dry, and an elution step. No conditioning was required.

• The SPEmethodswere streamlinedto three methods overall with minimal changing of solvents between meth-ods drastically reducing processing time and user error.

• GC/MSandLC/MS/MSwere shownto both work under the same extrac-tion methodology.

• The LC/MS procedures resulted invery short analysis times increasing sample throughput.

1. Mandatory Guidelines for Federal Workplace Drug Testing Programs Federal Register / Vol. 73, No. 228 / Tuesday, November 25, 2008 / Notices

References

The majority of existing drugs of abuse extraction procedures require multiple variations of solvents and steps. This unnecessarily exacerbates an already labor intensive step, sample preparation. Therefore, this study was completed in order to simplify and streamline the extraction using one sorbent and a minimal amount of methods. This was done under the assumption that optimal results would not be sacrificed.

2 mL urine samples (spiked at 40 %, 100 %, and 125 % of the cutoff) after appropriate pre-treatments, were extracted using the Strata-X-Drug B SPE cartridge in five steps: load, 2 x wash, drying, and elution (Figure 1). Conditioning and equilibration steps were eliminated using the new Strata-X-Drug B sorbent. Minimal solvent combinations were also achieved with only methanol, sodium acetate buffer, dilute hydrochloric acid, acetonitrile, ethyl acetate, and isopropanol for extraction of all drug classes.

Post extraction, samples were diverted

to either LC/MS or GC/MS for analysis to confirm extraction performance (Figures 3-5). Visually, excellent clean- up was achieved (Figure 2) and also, chromatographically, a nice, clean baseline free of major interferences was found, albeit in SIM and MRM modes.

Extraction performance, measured by recovery, RSD, and linearity over the 40 %, 100 %, and 125 % of the cutoff range, confirmed great overall results. Linearity correlation figures ranged from 0.995 to 0.999, recovery values ranged from 96.6 % to 102.2 %, and RSD values ranged from 0.43 to 3.03 % (Figure 6 and Table 2).

The study resulted in an elegant extraction solution that eases the workflow for drugs of abuse analyses. With simplicity, comes fewer mistakes and quicker sample turn around times. These advantages become even more important in a high sample volume environment such as the toxicology industry.

Results and Discussion

Figure 2. Cleanup of matrix with SPE procedure for Phencyclidine

Figure 1. Only 3 SPE methods on one sorbent for six drug classes

1. Original Urine Matrix

2. Final Eluent of Extraction

Figure 4. 6-MAM extracted from 2 mL of urine at the cutoff concentration

Figure 6. Three point calibration curves (40 %, 100 %, 125 % of cutoff concentration)

Figure 5. Carboxy-THC extracted from 2 mL of urine at the cutoff concentration

Table 1. Drugs of Abuse Panel 1

Class Analyte Cutoff

Marijuana Metabolites Delta-9-tetrahydrocannabinol-9-carboxylic acid (THCA)

15 ng/mL

Cocaine Metabolites Benzoylecgonine 100 ng/mL

Opiate Metabolites Codeine 2,000 ng/mL

Morphine 2,000 ng/mL

6-MAM 6-Acetylmorphine 10 ng/mL

PCP Phencyclidine 25 ng/mL

Amphetamines Amphetamine 250 ng/mL

Methamphetamine 250 ng/mL

MDMA Methylenedioxymethamphetamine (MDMA) 250 ng/mL

Methylenedioxyamphetamine (MDA) 250 ng/mL

Methylenedioxyethylamphetamine (MDEA) 250 ng/mL

Table 2. Relative recovery, RSD, and linearity values

Class Relative Recovery RSD Cutoff Linearity

Carboxy-THC 99.4 % 0.95 % 0.999

Benzoylecgonine 99.4 % 0.95 % 0.999

Phencyclidine 99.3 % 0.63% 0.999

Codeine 99.8 % 0.43 % 0.999

Morphine 99.8 % 0.59 % 0.999

6-MAM 100.5 % 0.63 % 0.999

Amphetamine 101.8 % 1.59 % 0.997

Methamphetamine 97.7 % 1.99 % 0.998

MDA 96.6 % 3.03 % 0.995

MDMA 100 % 1.20 % 0.997

MDEA 102.2 % 1.93 % 0.998

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Marijuana Metabolites:

To each 2 mL urine sample, add 100 µL of 11.8 N (conc) KOH. Vortex. Incubate at 60°C for 20 minutes. Cool and add ~450 μL glacial acetic acid, followed by vortex. Verify that pH is between 4.0 – 6.0.

Opiates:

To each 2 mL urine sample, add 500 µL conc. hydrochloric acid.Heat at 90 °C for 2 hours . Add 2 mL of 200 mM sodium acetate buffer (pH 4.0). Add 1 mL 6 N KOH. Vor tex. Centrifuge for 5 min at 5000 rpm. Verify pH of sample is between 4.0 - 6.0.

6-MAM:

To 2 mL u r i ne s a mp le , add 1000 µL ß-glucuronidase solution. ß-glucuronidase solution contains: 5,000 F units/mL patella vulgata in 100 mM acetate buf fer (pH=5.0). Vortex. Hydrolyze for 3 hours at 60 °C. Let cool and add 1000 µL of 100 mM phosphate buffer (pH 6.0). Verify that pH is between 5.5 – 6.5. Centrifuge for 5 minutes at 5000 rpm and discard pellet.

PCP:

To each 2 mL urine sample, add 2 mL 100 mM sodium acetate buffer (pH 5.0) then vortex. Verify that pH is between 4.0 – 6.0.

Amphetamines:

To 2 mL urine sample, add 1000 µL 100 mM phosphate buffer (pH 6.0) and 1000 µL 0.35 M sodium periodate. Vortex and incubate at room temperature for 25 minutes. The pH should be approximately 5.5 - 6.5.

Cocaine Metabolites:

To each 2 mL urine sample, add 2 mL 100 mM Sodium acetate buffer (pH 5.0) Vortex. Verify that pH is between 4.0 – 6.0.Pre–

Treatment

SPE Cartridge: Strata-X-Drug B 60 mg/6 mL

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) mixture (30:70)

Dry: 15 min under full vacuum Elute: 2 mL Ethyl acetate:Isopropanol

(85:15)

SPE Cartridge: Strata-X-Drug B 60 mg/6 mL

Load: Pre-treated sample Wash 1: 2 mL 0.1 N Hydrochloric acid Wash 2: 2 mL Methanol

Dry: 10 min under full vacuum Elute: 2 mL Ethyl acetate:Isopropanol:Ammonium

hydroxide mixture (7:2:1)

Figure 3. Amphetamines extracted from 2 mL of urine at the cutoff concentration

1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75 1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15 2.20 m i n0.0

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Materials Drug of Abuse Standards: All standards purchased from Cerrilliant Corporation, Round Rock, TX.

Negative Control Urine: Purchased from UTAK Laboratories, Valencia, CA

SPE Column: Strata™-X-Drug B, 60 mg/6 mL

GC Column: Zebron™ ZB-Drug-1, 10 meter x 0.18 mm x 0.18 µm

HPLC Columns: Kinetex® C18, 2.6 µm, 50 x 2.1 mm Kinetex PFP, 2.6 µm, 50 x 2.1 mm

Analytes (MRMs):1: D11-Amphetamine 147.1/98.12: Amphetamine 136.1/91.13: D14-Methamphetamine 164.1/98.14: Methamphetamine 150.1/91.15: D5-MDA 185.3/168.06: MDA 180.2/105.17: D5-MDMA 199.3/165.08: MDMA 194.2/105.09: D5-MDEA 213.3/163.0

10: MDEA 208.3/105.0

Analytes (MRMs):1. D3-6-MAM 331/1652. 6-MAM 328/165, 328/211

Analytes (SIM ions):1. D9-Carboxy-THC 3802. Carboxy-THC 371

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Cocaine MetabolitesEvaporation and Reconstitution: Evaporate to dryness at 50 °C. Reconstitute in 1 mL methanol / 0.1 % formic acid in water (95:5).App ID: 19596

Column: Kinetex 2.6 µm PFPPart No.: 00B-4477-AN

Dimensions: 50 x 2.1 mmMobile Phase: A: Water with 0.1 % Formic acid

B: MethanolGradient: Time (min) % B

0 95 1 50 2 50 2.1 95 4 95

Flow Rate: 400 µL/minTemperature: Ambient

Detection: Mass Spectrometer (MS)

Marijuana Metabolites Evaporation and Derivatization:Evaporate to dryness at 50 °C. Reconstitute with 25 µL ethyl acetate and 50 µL BSTFA with 1 % TMCS. Heat 20 min at 70 ºC.App ID: 19597

Column: Zebron ZB-Drug-1 Dimensions: 10 meter x 0.18 mm x 0.18 µm

Part No.: 7CD-G-023-08Injection: Split 10:1 @ 280 ºC, 1 µL

Carrier gas: Helium @ 0.7 mL/minOven Program 220 ºC to 340 ºC at 30 ºC/min

OpiatesEvaporation and Derivatization: Evaporate to dryness at 50 °C. Add 50 µL propione anhydride and 50 µL acetonitrile. Heat 30 min at 60 °C. Evaporate to dryness at 50 °C. Reconstitute in 100 µL of ethyl acetate. App ID: 19600

Column: Zebron ZB-Drug-1 Dimensions: 10 meter x 0.18 mm x 0.18 µm

Part No.: 7CD-G023-08Injection: Split 20:1 @ 260 ºC, 1 µL

Carrier Gas: Helium @ 0.7 mL/minOven Program: 185 ºC to 320 ºC @ 30 ºC/min

for 3 min

6-MAMEvaporation and Reconstitution: Evaporate to dryness at 50 °C under nitrogen gas.Reconstitute in 1 mL methanol / 0.1 % formic acid in water (10:90).App ID: 19599

Column: Kinetex 2.6 µm C18Dimensions: 50 x 2.1 mm

Part No.: 00B-4462-ANMobile Phase A: 0.1 % Formic acid

B: AcetonitrileGradient: Time (min) % B

0 10 1 10 3 70 3.1 10

Flow Rate: 400 µL/minTemperature: Ambient

Detection: Mass Spectrometer (MS)

PCP Evaporation and Reconstitution: Evaporate to dryness at 50 °C.Reconstitute in 1mL methanol / 0.1 % formic acid in water (25:75).App ID: 19595

Column: Kinetex 2.6 µm C18Dimensions: 50 x 2.1 mm

Part No.: 00B-4462-ANMobile Phase A: 0.1 % Formic acid

B: Methanol with 0.1 % Formic acidGradient: Time (min) % B

0 25 1 95 2 95 2.1 25 4 25

Flow Rate: 400 µL/minTemperature: Ambient

Detection: Mass Spectrometer (MS)

AmphetaminesEvaporation and Reconstitution: Add 300 µL 0.5 N methanolic hydrochloride to each sample. Samples can then be evaporated to dryness at < 35 ºC. Reconstitute in 1 mL methanol / 0.1 % formic acid in water (10:90). Before injecting onto the LC/MS dilute the samples by a factor of 20 to bring the concentration into a suitable range for analysis.App ID: 19598

Column: Kinetex 2.6 µm C18, Dimensions: 50 x 2.1 mm

Part No.: 00B-4462-ANMobile Phase:

A: 0.1 % Formic acidB: Methanol with 0.1 % Formic acid

Gradient: Time (min) % B 0 10 1 10 3 70 3.1 10

Flow Rate: 400 µL/minTemperature: Ambient

Detection: Mass Spectrometer (MS)

SPE Method

Analytical Method

Ap

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195

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Solid phase extraction (SPE) was performed using a 60 mg/6 mL Strata™-X-Drug B strong cation exchange sorbent. Samples were loaded without conditioning the SPE sorbent by applying a light vacuum to solvate the sorbent bed and frits. Vacuum was then turned off and samples were allowed to gravity flow. Samples were washed with various buffers and organic solutions, depending on the compound class. The SPE tubes were then dried under full vacuum for 10 minutes and eluted with the appropriate organic solution. Samples were then evaporated to dryness and derivatized prior to GC/MS analysis. Samples analyzed by LC/

MS were not derivatized and simply reconstituted in mobile phase prior to injection.

GC/MS analysis was performed using a Zebron™ ZB-Drug-1 column with the MS operated in SIM mode. LC/MS analysis was performed using Kinetex® C18 and PFP core-shell technology columns with the MS operating in Positive-ESI MRM mode.

Calibration curves were generated using the average response factor for three replicate samples at each calibration level. Recovery and RSD values were measured to evaluate extraction performance.

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