Simultaneous analysis of stimulants, opioids, gabapentin, xylazine, benzodiazepines, cannabinoids, and novel stimulants/hallucinogens in human urine.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of 67 drugs including stimulants, opioids, gabapentin, xylazine, benzodiazepines, cannabinoids, novel stimulants/hallucinogens, and their metabolites in urine. Urine samples were deconjugated, diluted, and fortified with isotopically labelled internal standards prior to analysis. The method was linear for all analytes with regression coefficients (R-values) > 0.99. The method limits of quantification were in the range of 0.1-15 ng/mL for all analytes. The recoveries of all drugs fortified in human urine at low (0.15-6 ng/mL), medium (200-400 ng/mL), and high (400-800 ng/mL) concentrations were in the range of 75-126%. Repeated analysis of similarly fortified urine yielded intra- and inter-day variations in the range of 0.3-14% and 0.45-17%, respectively. Enzymatic deconjugation using β-glucuronidase significantly improved sensitivity and enabled positive identification of several drugs that were predominantly excreted as glucuronides. The validated method was applied in the determination of drugs in 200 urine samples collected from non-fatal overdose patients. Fentanyl, norfentanyl, hydromorphone, morphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, 4-anilino-N-phenethylpiperidine, methamphetamine, benzoylecgonine, and xylazine were detected in ≥ 50% of the 200 urine samples analyzed, with the sum concentration of all drugs ranging from 8.59 to 2840,000 ng/mL. Several drugs belonging to different chemical classes (e.g., xylazine and opioids) showed positive correlations, suggesting co-exposure to multiple drugs in overdose patients.