A rapid and accurate UPLC/MS/MS method for the simultaneous determination of zolpidem and its main metabolites in biological fluids and its application in a forensic context.

Zolpidem (ZPD) is an imidazopyridine derivative used as a new type of hypnotic and is commonly used in drug-facilitated crimes. A rapid, sensitive, and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) assay method for the simultaneous determination of zolpidem and its main metabolites zolpidem 6-carboxylic acid (ZCA) and zolpidem phenyl-4-carboxylic acid (ZPCA) in biological fluids was developed and validated. Aliquots of 0.1mL blood or urine specimens were used for the analysis, and zolpidem and its metabolites were extracted in a single step using acetonitrile (containing 0.1% formic acid) precipitation. The supernatant was then dried, and 100μL methanol was added. The separation was performed on an Acquity™ UPLC HSS T3 (100mm×2.1mm, 1.8μm) analytical column by API 4000Q ultra-performance liquid chromatography-tandem mass spectrometry. Positive ionisation tandem MS detection in the multiple reaction monitoring (MRM) mode was used. The mobile phases consisted of either acetonitrile or water containing 20mmol/L ammonium acetate and 0.1% formic acid, and the flow rate was 0.5mL/min. The chromatographic separation time was 4min, and calibration curves for human blood were generated over the range of 0.1-300ng/mL for ZPD, 0.1-500ng/mL for ZPCA and 0.1-200ng/mL for ZCA. For urine, the linear range was 0.1-600ng/mL for ZPD and ZPCA, and 0.1-300ng/mL for ZCA. The limit of detection was 0.05ng/mL and the limit of quantitation was 0.1ng/mL for ZPD, ZCA and ZPCA. The linear correlation coefficients were greater than 0.9995. Both the inter-day and intra-day precisions were less than 15%, the recoveries were in the range of 70.0-98.3%, the matrix effects were approximately 79.5-99.0%, and the process efficiency was between 60.7% and 94.4%. This method allowed for the determination of zolpidem and its metabolites in human blood and urine and may be applied to forensic toxicological analyses.