title: Definitive Drug and Metabolite Screening in Urine by UPLC-MS-MS Using a Novel Calibration Technique.
abstract: Drug screening is an essential analytical tool for detection of therapeutic, illicit and emerging drug use. Presumptive immunoassay screening is widely used, while initial definitive testing by chromatography-coupled mass spectrometry is hampered due to complex pre-analysis steps, long chromatography time and matrix effects. The aim of this study is to develop and validate a definitive test for rapid and threshold accurate screening of 33 drugs or metabolites (analytes) in urine. Sample preparation in a 96-well plate format involves rapid glucuronidase hydrolysis followed by dilution, filtration and ultra-performance liquid chromatography-MS-MS analysis. Chromatographic separation, on an ACQUITY UPLC®BEH phenyl column is optimized for a 3-min MS-MS ion acquisition. Matrix effect was normalized by an innovative technique called threshold accurate calibration employing an additional analysis with an analyte spike as an internal standard undergoing the same matrix effect as an analyte in a drug-positive donor specimen. Accuracy and precision, at above and below threshold concentrations, were determined by replicate analysis of control urine pools containing 50, 75, 125 and 150% of threshold concentrations. Accuracy and selectivity were further demonstrated by concordant findings in proficiency and confirmatory testing. The study shows the applicability of definitive testing as an alternative to immunoassay screening and demonstrates a new approach to normalization of matrix effect.
authors: Department of Pathology and Laboratory Medicine, Forensic Toxicology Laboratory, Albany Medical Center Hospital and College, 43 New Scotland Avenue, Albany, NY, USA Clinical and Forensic Toxicology Laboratory, National Toxicology Center, Center for Medical Science, 150 New Scotland Avenue, Albany, NY, USA rosanot@mail.amc.edu.
authors: Thomas G
authors: TG
authors: Rosano
authors: Clinical and Forensic Toxicology Laboratory, National Toxicology Center, Center for Medical Science, 150 New Scotland Avenue, Albany, NY, USA.
authors: Patrice Y
authors: PY
authors: Ohouo
authors: Clinical and Forensic Toxicology Laboratory, National Toxicology Center, Center for Medical Science, 150 New Scotland Avenue, Albany, NY, USA.
authors: John J
authors: JJ
authors: LeQue
authors: Waters Corporation, 100 Cummings Center, Suite 407N Beverly, MA, USA.
authors: Scott M
authors: SM
authors: Freeto
authors: Waters Corporation, Health Sciences Organisation, Altrincham Road, Wilmslow, UK.
authors: Michelle
authors: M
authors: Wood
mesh_terms: D000661:Amphetamine / Q000652:urine; D001569:Benzodiazepines / Q000652:urine; D002047:Buprenorphine / Q000652:urine; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D003042:Cocaine / Q000652:urine; D006801:Humans; D057230:Limit of Detection; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000652:urine; D053610:Opiate Alkaloids / Q000652:urine; D010098:Oxycodone / Q000652:urine; D010622:Phencyclidine / Q000652:urine; D015203:Reproducibility of Results; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D053610:Opiate Alkaloids; D001569:Benzodiazepines; D002047:Buprenorphine; D010098:Oxycodone; D000661:Amphetamine; D003042:Cocaine; D010622:Phencyclidine; D018817:N-Methyl-3,4-methylenedioxyamphetamine