Extending the detection window of diazepam by directly analyzing its glucuronide metabolites in human urine using liquid chromatography–tandem mass spectrometry

A method for the simultaneous direct analysis of diazepam oxazepam glucuronide, temazepam glucuronide, oxazepam, nordiazepam, and temazepam in human urine was developed and validated. Urine sample was purified by solid phase extraction (SPE), and the analysis was achieved using a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system equipped with an electrospray ionization source (ESI). Multiple reaction monitoring (MRM) mode was used to analyze the target compounds. Extraction recoveries were 65–122% for all the analytes. The method showed acceptable intra–assay and inter–assay precision (both relative standard deviation (RSD) ≤ 11.2%) for quality control (QC) samples. The limits of detections (LODs) were in the range of 0.1–2 ng/mL. The present assay was applied to analyze the urine obtained from three volunteers after oral administration of a single dose 5 mg of diazepam. The results showed that, the detection periods of oxazepam glucuronide and temazepam glucuronide were much longer than diazepam and other metabolites.

► A LC–MS/MS method was developed for analyzing diazepam and its metabolites in urine.
► Glucuronides of diazepam in urine were analyzed without enzymatic or acid hydrolysis.
► LODs (0.1–2 ng/mL) of diazepam and its metabolites demonstrated high sensitivity.
► Urine of three volunteers after orally administrated 5 mg of diazepam was analyzed.
► Detection window of diazepam in urine was extended by detecting its glucuronides.