Rapid and simultaneous determination of multiple classes of abused drugs and metabolites in human urine by a robust LC‐MS/MS method – application to urine drug testing in pain clinics

Abstract

A simple LC-MS/MS method was developed and validated for quantitatively analyzing six classes of 26 abused drugs and metabolites in human urine: (1) illicit drugs; (2) opiates; (3) synthetic opioids; (4) sedative; (5) stimulants; and (6) γ-aminobutyric acid analogs. All urine samples were diluted with a mixture of isotope-labeled internal standards, hydrolyzed with β-glucuronidase and directly injected in a gradient chromatographic run. The mobile phase was composed of 0.1% formic acid in water and 0.1% of formic acid in methanol. A 4.9 min run time using the multiplexing driver and ultra-biphenyl column (50 × 2.1 mm, 5 µm, RESTEK) allowed all drugs to have sufficient resolution in a short elute time. The overlapping liquid chromatography runs and scheduled multiple reaction monitoring acquisition method resulted in a higher overall throughput for the system. The result was linear over the studied range (2-16,000 ng/mL) for all compounds with correlation coefficients r(2) ≥ 0.995. The intra-day and inter-day precisions and accuracies were within 15% and recovery was between 83 and 115% for all analytes. Freeze-thaw stability for three cycles and long-term stability (57 days, -20°C) were established for all analytes. The cross-validation between College of American Pathologists and in-house was validated (0.06% ≤ bias ≤ 12.3%). The applicability of the method was examined by analyzing urine samples from chronic pain patients (n = 610).