title: 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.
abstract: 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.
authors.affiliation: Shanghai Key Laboratory of Forensic Medicine, Department of Forensic Toxicology, Institute of Forensic Science, Ministry of Justice, Guangfu Xi Road 1347, Shanghai 200063, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Shi
authors.forename: Ping
authors.initials: P
authors.lastname: Xiang
authors.forename: Baohua
authors.initials: B
authors.lastname: Shen
authors.forename: Min
authors.initials: M
authors.lastname: Shen
mesh_terms: D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D053593:Forensic Toxicology; D006801:Humans; D008297:Male; D011725:Pyridines / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics / Q000652:urine; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000077334:Zolpidem
chemical_list: D011725:Pyridines; D000077334:Zolpidem; C521325:zolpidem carboxylic acid