Rapid determination of benzodiazepines, zolpidem and their metabolites in urine using direct injection liquid chromatography–tandem mass spectrometry. (December 2015)

• Record Type: Journal Article
• Authors: Jeong, Yu-Dong; Kim, Min Kyung; Suh, Sung Ill; In, Moon Kyo; Kim, Jin Young; Paeng, Ki-Jung
• Is Part Of: Forensic science international. Volume 257(2015)
• Page Start: 84
• Page End: 92
• Publication Date: 2015-12
• Subjects: Benzodiazepines -- Zolpidem -- Metabolite -- Urine -- Direct injection -- LC–MS/MS; Medical jurisprudence -- Periodicals; Chemistry, Forensic -- Periodicals; Forensic Medicine -- Periodicals; Médecine légale -- Périodiques; Chimie légale -- Périodiques; Gerechtelijke geneeskunde; Gerechtelijke chemie; Gerechtelijke psychiatrie; Chemistry, Forensic; Medical jurisprudence; Electronic journals; Periodicals; Electronic journals; 614.1
• DOI: 10.1016/j.forsciint.2015.07.043
• Languages: English
• ISSNs: 0379-0738

Abstract

Graphical abstract: Highlights: A rapid and simple LC–MS/MS method was developed for benzodiazepines, zolpidem and their metabolites. An aliquot (5 μL) of the sample solution was directly injected into the LC–MS/MS. The separation and detection of 18 analytes were achieved within 10 min with good precision and low detection limits. The applicability of this method was successfully verified to human urine samples from drug users. Abstract: Benzodiazepines and zolpidem are generally prescribed as sedative, hypnotics, anxiolytics or anticonvulsants. These drugs, however, are frequently misused in drug-facilitated crime. Therefore, a rapid and simple liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for identification and quantification of benzodiazepines, zolpidem and their metabolites in urine using deuterium labeled internal standards (IS). Urine samples (120 μL) mixed with 80 μL of the IS solution were centrifuged. An aliquot (5 μL) of the sample solution was directly injected into the LC–MS/MS system for analysis. The mobile phases consisted of water and acetonitrile containing 2 mM ammonium trifluoroacetate and 0.2% acetic acid. The analytical column was a Zorbax SB-C18 (100 mm × 2.1 mm i.d., 3.5 μm, Agilent). The separation and detection of 18 analytes were achieved within 10 min. Calibration curves were linear over the concentration ranges of 0.5–20 ng/mL (zolpidem), 1.0–40 ng/mL (flurazepam and temazepam), 2.5–100 ng/mL (7-aminoclonazepam, 1-hydroxymidazolam, midazolam, flunitrazepam and alprazolam), 5.0–200 ng/mL (zolpidem phenyl-4-carboxylic acid, α-hydroxyalprazolam, oxazepam, nordiazepam, triazolam, diazepam and α-hydroxytriazolam), 10–400 ng/mL (lorazepam and desalkylflurazepam) and 10–100 ng/mL (N-desmethylflunitrazepam) with the coefficients of determination ( r 2 ) above 0.9971. The dilution integrity of the analytes was examined for supplementation of short linear range. Dilution precision and accuracy were tested using two, four and ten-folds dilutions and they ranged from 3.7 to 14.4% and −12.8 to 12.5%, respectively. The process efficiency for this method was 63.0–104.6%. Intra- and inter-day precisions were less than 11.8% and 9.1%, while intra- and inter-day accuracies were less than −10.0 to 8.2%, respectively. The lower limits of quantification were lower than 10 ng/mL for each analyte. The applicability of the developed method was successfully verified with human urine samples from drug users ( n = 21). Direct urine sample injection and optimized mobile phases were introduced for simple sample preparation and high-sensitivity with the desired separation.