title: A fast liquid chromatography-tandem mass spectrometry method for determining benzodiazepines and analogues in urine. Validation and application to real cases of forensic interest.
abstract: A fast liquid chromatographic/tandem mass spectrometric method was developed for the simultaneous determination in human urine of seventeen benzodiazepines, four relevant metabolites together plus zolpidem and zopiclone. The sample preparation, optimized to take into account the matrix effect, was based on enzymatic hydrolysis and liquid-liquid extraction. The separation of the twenty-three analytes was achieved in less than eight minutes. The whole methodology was fully validated according to UNI EN ISO/IEC 17025:2005 rules and 2006 SOFT/AAFS guidelines. Selectivity, linearity range, identification (LOD) and quantitation (LOQ) limits, precision, accuracy and recovery were evaluated. For all the species the signal/concentration linearity was satisfactory in the 50-1000 ng/mL concentration range. The limits of detection ranged from 0.5 to 30 ng/mL and LOQs from 1.7 to 100.0 ng/mL. Precisions were in the ranges 5.0-11.8%, 1.5-11.0% and 1.1-4.4% for low (100 ng/mL), medium (300 ng/mL) and high (1000 ng/mL) concentration, respectively. The accuracy, expressed as bias% was within ± 25 % for all the analytes. The recovery values, evaluated at 300 ng/mL concentration, ranged from 56.2% to 98.8%. The present method for the determination of several benzodiazepines, zolpidem and zopiclone in human urine proved to be simple, fast, specific and sensitive. The quantification by LC-MS/MS was successfully applied to 329 forensic cases among driving re-licensing, car accidents and alleged sexual violence cases.
authors: Centro Regionale Antidoping A. Bertinaria, Regione Gonzole 10, 10043 Orbassano, Turin, Italy. alberto.salomone@antidoping.piemonte.it
authors: Alberto
authors: A
authors: Salomone
authors: Enrico
authors: E
authors: Gerace
authors: Paola
authors: P
authors: Brizio
authors: M Carla
authors: MC
authors: Gennaro
authors: Marco
authors: M
authors: Vincenti
mesh_terms: D053961:Azabicyclo Compounds / Q000652:urine; D001569:Benzodiazepines / Q000652:urine*; D002853:Chromatography, Liquid / Q000379:methods*; D005554:Forensic Medicine / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D059625:Liquid-Liquid Extraction / Q000379:methods; D010879:Piperazines / Q000652:urine; D011725:Pyridines / Q000652:urine; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D059349:Urine Specimen Collection / Q000379:methods; D000077334:Zolpidem
chemical_list: D053961:Azabicyclo Compounds; D010879:Piperazines; D011725:Pyridines; C515050:zopiclone; D001569:Benzodiazepines; D000077334:Zolpidem