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 0.1 mL 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 100 mu L methanol was added. The separation was performed on an Acquity (TM) UPLC HSS T3 (100 mm x 2.1 mm, 1.8 mu m) 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 20 mmol/L ammonium acetate and 0.1% formic acid, and the flow rate was 0.5 mL/min. The chromatographic separation time was 4 min, and calibration curves for human blood were generated over the range of 0.1-300 ng/mL for ZPD, 0.1-500 ng/mL for ZPCA and 0.1-200 ng/mL for ZCA. For urine, the linear range was 0.1-600 ng/mL for ZPD and ZPCA, and 0.1-300 ng/mL for ZCA. The limit of detection was 0.05 ng/mL and the limit of quantitation was 0.1 ng/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. (C) 2012 Elsevier B.V. All rights reserved.