GC-MS confirmation of urinary benzodiazepine metabolites

A confirmatory method using gas chromatography-mass spectrometry (GC-MS) is described for benzodiazepine metabolites commonly encountered in urine. The targeted drugs are diazepam, nordiazepam, temazepam, oxazepam, lorazepam, alpha-hydroxyalprazolam, alpha-hydroxytriazolam, 2-hydroxyethyl-flurazepam, N-desalkyl-3-hydroxyflurazepam, and N-desalkylflurazepam. After enzymatic hydrolysis, the free benzodiazepines are recovered from the urine by liquid-liquid extraction. The organic solvent is evaporated to dryness. The residue is subjected to sequential derivatization involving propylation of the secondary amine in the lactam ring followed by propionylation of hydroxyl groups. The derivatives are separated on a methyl silicone capillary column and then identified and quantitated by full-scan GC-MS. Characteristic ion clusters are selected for quantitation. Four internal standards are used: oxazepam-d5, lorazepam-d4, alpha-hydroxyalprazolam-d5, and alpha-hydroxytriazolam-d4.