Development of an LC–MS/MS method for the simultaneous determination of 25 benzodiazepines and zolpidem in oral fluid and its application to authentic samples from regular drug users

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A simple and reliable analytical method was established and validated for the simultaneous determination of 25 benzodiazepines and zolpidem in oral fluid obtained using the Quantisal™ collection device. The samples were prepared by liquid–liquid extraction with ethyl acetate and analyzed using liquid chromatography–tandem mass spectrometry. The validation parameters included limits of detection and quantification (LOD and LOQ), linearity, accuracy and precision, selectivity, recovery, matrix effects and process efficiency. To investigate the variables associated with collection of oral fluid, drug stability and drug recovery in/from the collection device were also determined. The LOD ranged from 0.01ng/ml to 0.5ng/ml and the LOQ ranged from 0.1ng/ml to 0.5ng/ml. The results of the intra- and inter-day precision and accuracy were satisfactory, i.e., <10% for precision and within ±10% for accuracy at a low (LOQ of each analyte) and high concentrations (5ng/ml). In addition, all analytes were stable under the storage condition of below −20°C for 1 month. Drug recoveries from the collection device were more than 80% (81–95%) except those of clonazepam and flunitrazepam, which were unstable in oral fluid. The developed method was successfully applied to authentic oral fluid specimens obtained from psychiatric patients who take benzodiazepines or zolpidem regularly. As a result, alprazolam, clonazepam, diazepam, flunitrazepam, flurazepam, lorazepam, zolpidem and/or their metabolites were detected at 1–18h after intake of these drugs. This study will be useful for the analysis of oral fluid samples collected in forensic toxicological cases.