LC-MS/MS Analysis of Cannabinoids, Opiates, Amphetamines, Cocaine and Phencyclidine in Urine

Transcription

1 LC-MS/MS Analysis of Cannabinoids, Opiates, Amphetamines, Cocaine and Phencyclidine in Urine 1 John McFarlane and 2 Adrian Taylor 1 AB SCIEX, Foster City, California, USA ; 2 AB SCIEX, Concord, Ontario, Canada Introduction Here we describe a method for the screening of the most common category of drugs of abuse, which include Cannabinoids, Opiates, Amphetamines, Cocaine and Phencyclidine (PCP). In urine, the target drugs and/or metabolites are amphetamine, methamphetamine, PCP, codeine, morphine, benzoylecgonine, 6-monoacetylmorphine (6- MAM) and, (±)-11-nor-9-Carboxy-Δ 9 -THC (THC-COOH). Laboratory-based drug analysis is usually performed in a two step fashion using two different detection methods 1. The first step is a screening analysis and is usually done by an immunoassay technique (ELISA, EMIT, or RIA). Any compounds detected in the samples can be confirmed using a mass spectrometry technique (commonly GC/MS). Recently, LC/MS/MS 2 has emerged as a reliable method to perform both screening and confirmation of drugs in urine without the setup requirements of immunoassays or the time-consuming sample preparation and derivatization required for GC/MS confirmation. The described method provides an effective means to detect, confirm and quantify drugs of abuse in urine at or below the typical GC/MS cutoff values. Methodology The sample preparation includes treatment of control urine and samples with CHAPS to reduce any non-specific binding. The assay incorporates a hydrolysis step to release the glucuronides to the free form for quantitation. The method uses a 9-point calibration curve covering the linear ranges for quantitation as follows: 2- ng/ml for methamphetamine and PCP; 2- ng/ml for all the other analytes. The method uses a 4µL injection volume with a 5.5 minute run time. The assay was run on a 32 QTRAP LC/MS/MS system. The acquisition method consists of two periods. The first period is positive polarity from to 3. minutes. Period two is negative polarity from 3. to 5.5 minutes. The second period is for THC-COOH only, period one is for the remaining analytes. 32 QTRAP LC/MS/MS system Analyte linear ranges covered by the calibration curve Analyte Range (ng/ml) Morphine 2- Codeine 2- Amphetamine 2- Methamphetamine 2- Benzoylecgonine 2-6-Acetylmorphine 2- PCP 2- THC-COOH 2- p 1

2 Results XICof +MRM(21 pairs): Period 1, 136.1/119.1 Da ID: Amphetamine-2 fromsample 5 (Std 5 A) of 22 Dec 9 Test.wiff (Turbo Spra... Max. 1.1e6 cps. 1.5e6 1.e6 9.5e5 9.e5 8.5e5 8.e5 7.5e5 7.e5 6.5e5 6.e5 5.5e5 5.e5 4.5e5 4.e5 3.5e5 3.e5 2.5e5 2.e5 1.5e Amphetamine Benzoylecgonine Methamphetamine Morphine Codeine PCP 6-MAM THC-COOH Time,min Chromatogram of a Spiked Urine Sample at 1 ng/ml for methamphetamine and PCP; and 1 ng/ml for the other analytes. Representative accuracy and precision in spiked samples Compound Limit of Quantitation (ng/ml) %CV at LOQ Accuracy at LOQ (%) +MRM(21pairs):Period1,244.2/159.2DaID:PCP-1fromSample64(Standard8-2)of9123.wiff Max.36.cps. XICof (TurboSpray) PCP Amphetamine Benzoylecgonine Morphine Methamphetamine Codeine Acetylmorphine THC-COOH Representative Chromatograms of PCP 2 ng/ml Spiked in Urine (at 2 % of cutoff level) p 2

3 e Representative Chromatogram of Benzoylecgonine 1ng/mL Spiked in Urine (at 4 % of cutoff level) Representative Chromatograms of Codeine 1 ng/ml Spiked in Urine (at 25 % of cutoff level) XICof +MRM(21pairs):Period1,29.1/168.1DaID:Benzoylecgonine-1fromSample69(Standard9-1)of9123.wiff (TurboSpr... Max.5.6e4cps. XICof +MRM(21 pairs): Period 1, 3.1/115.2 Da ID: Codeine-2 fromsample 44 (Std 8 A) of 22 Dec 9 Test.wiff (Turbo Spray), Smooth... Max. 2.2e4 cps. 2.2e4 5.5e e4 2.e4 5.e4 1.9e4 1.8e4 4.5e4 1.7e4 1.6e4 4.e4 1.5e4 1.4e4 3.5e4 1.3e4 1.2e4 3.e4 1.1e4 1.e4 2.5e e e e Representative Chromatograms of Morphine 1 ng/ml Spiked in Urine (at 25 % of cutoff level) Representative Chromatograms of Amphetamine 1ng/mL Spiked in Urine (at 4 % of cutoff level) e4 8 9.e e4 7 8.e e4 6 7.e e4 6.e e e e4 3.5e e4 2.5e4 2.e e4 1.e4 5 Representative Chromatograms of Methamphetamine 1 ng/ml Spiked in Urine (at 4 % of cutoff level) Representative Chromatograms of 6-Acetylmorphine 2ng/mL Spiked in Urine (2x cutoff) XIC of +MRM (21 pairs): Period 1, 328.2/165.1 Da ID: 6-MAM-1 from Sample 69 (Standard 9-1) of 9123.wiff (Turbo Spray) Max. 86. cps. 1.15e5 1.1e e e e e e e4 7.e e4 5 6.e4 5.5e4 I t it e e4 3.5e e4 2.5e4 2.e e4 1 1.e Time, min p 3

4 Representative Chromatograms of THC-COOH 1 ng/ml Spiked in Urine Standard Calibration Curves 1.15e e4 1.5e4 1.e4 Standard Calibration Curves Representative calibration curves for codeine and benzoylecgonine; showing the range and linearity expected for the assay. Representative calibration curves for morphine and 6- Acetylmorphine; showing the range and linearity expected for the assay. p 4

5 Conclusions 5 drug classes: 2 µl urine sample; 1 step protein precipitation after incubation step; LOQ based on each relevant concentration of interest. Easy to use Cliquid software that simplifies the running of LC/MS/MS samples and automatic report generation. Reports are customizable and predefined. imethod Application that can be unpacked directly in to the Cliquid software; once unpacked tests are ready to be run immediately. Documentation that describes sample preparation, MS and LC conditions for the analysis of 5 drug classes. Single method for the analysis of 5 drug classes. Ability to add new target compounds to the LC/MS/MS method. References [1] T.A. Brettell, K. Inman, N. Rudin, R. Saferstein. Forensic Science. Analytical Chemistry, 1999, 71: 235R-255R. [2] Riet Dams, Marilyn A. Huestis, Willy E. Lambert and Constance M. Murphy. Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: Influence of ionization type, sample preparation, and biofluid. Journal of the American Society for Mass Spectrometry, 23, 14-11: For Research Use Only. Not for use in diagnostic procedures. 211 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. Publication number: p 5