LC–MS/MS Method for Simultaneous Quantification of Zolpidem, Zopiclone, and Their Metabolites in Human Urine and Its Pharmacokinetic Application

ABSTRACT Zolpidem (Z1) and zopiclone (Z2) are widely prescribed for insomnia; but their misuse in drug‐facilitated crimes presents significant forensic and clinical challenges. This study aimed to develop and validate a simple and sensitive LC–MS/MS method, coupled with dispersive liquid–liquid microextraction (DLLME), for the simultaneous determination of Z1, Z2, and 18 phase I and phase II metabolites (20 species) in human urine. DLLME employed 1 mL of dichloromethane as extractant and 2 mL of acetonitrile as disperser per 2 mL of urine, reducing halogenated‐solvent use compared with conventional liquid–liquid extraction. Chromatographic separation was achieved on a HyPURITY C18 column with water–acetonitrile (70:30, v/v) containing 0.2% formic acid. Z1 and Z2 were validated according to ICH guidelines over 0.1–200 ng/mL, showing linearity ( R 2 > 0.999), accuracy (96.29%–99.56%), precision (RSD ≤ 3.12%), and recovery (96.12%–98.74%). Because authentic metabolite standards were unavailable, metabolite concentrations were estimated using parent‐drug calibration curves and are considered semi‐quantitative. In five healthy volunteers, hydroxylated and carboxylated metabolites peaked at 2–12 h and remained detectable up to 84 h. These findings support the method's potential for retrospective urinary screening of Z‐drug exposure in forensic and clinical toxicology.