Screening with Quantification for 64 Drugs and Metabolites in Human Urine using UPLC–MS-MS Analysis and a Threshold Accurate Calibration

• Published in: Journal of analytical toxicology Vol. 41; no. 6; pp. 536 - 546
• Main Authors: Rosano, Thomas G., Ohouo, Patrice Y., Wood, Michelle
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-07-2017
• Subjects: Calibration; Humans; Limit of Detection; Quality Control; Reference Standards; Reproducibility of Results; Street Drugs - urine; Substance Abuse Detection - methods
• ISSN: 0146-4760; 1945-2403
• DOI: 10.1093/jat/bkx035

Abstract
Drug and metabolite (analytes) identification together with quantification is an important analytical tool in forensic and clinical toxicology. We report the development and validation of a definitive detection and quantification method (UPLC–MS-MS) for initial screening of 64 analytes in urine. The principle of the method is a quantitative extension of a recently reported threshold accurate calibration (TAC) technique which employs a rapid dual-specimen analysis i.e., with and without addition of a reference-analyte standard for normalization of matrix effects. Analytes include pharmaceutical and illicit agents from opiate and opioid agonist, opiate-antagonist, benzodiazepine, amphetamine, designer amphetamine, cathinone, cocaine, hallucinogen, gabapentinoid and sedative drug classes. Using a 96-well plate format, the protocol employs glucuronidase hydrolysis, 27-fold urine dilution and a 3 min UPLC–MS-MS acquisition. Subsequent data management includes calculation of a normalized TAC ratio response and weighted least squares calibration. The method utilizes analyte-specific calibration ranges from 2.5 to 1,500 ng/mL with quality control (QC) monitoring of transition-ion ratio, calibrator re-analysis, injection precision and multi-level QC analysis. Method precision, bias, calibration linearity, detection limit, carryover, crossover studies and external proficiency performance were evaluated based on pre-established criteria. The validated method provides an alternative to stable-isotope internal standardization methods of quantification and is applicable to screening with quantification in routine toxicology practice.