Simultaneous determination of fifteen low-dosed benzodiazepines in human urine by solid-phase extraction and gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method was developed for the simul taneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, alpha -hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7 -aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylfluraz epam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, loraz epam, triazolam and alpha -hydroxytriazolam. Nitrogen-methylclonazepam is u sed as the internal standard. The urine sample preparation involves enzymat ic hydrolysis of the conjugated metabolites with Helix pomatia beta -glucur onidase for 1 h at 56 degreesC followed by solid-phase extraction on a phen yl-type column. The extracted benzodiazepines are subsequently analyzed on a polydimethylsiloxane column using on-column injection to enhance sensitiv ity. The extraction efficiency exceeded 80% for all compounds except for ox azepam, lorazepam and 4-hydroxyalprazolam which had recoveries of about 60% . The LODs ranged from 13 to 30 ng/ml in the scan mode and from 1.0 to 1.7 ng/ml in the selected ion monitoring (SIM) mode. Linear calibration curves were obtained in the concentration ranges from 50 to 1000 ng/ml in the scan mode and from 5 to 100 ng/ml in the SLM mode. The within-day and day-to-da y relative standard deviations at three different concentrations never exce eded 15%. (C) 2001 Elsevier Science B.V. All rights reserved.