Simultaneous determination of benzodiazepines and their metabolites in human serum by liquid chromatography-tandem mass spectrometry using a high-resolution octadecyl silica column compatible with aqueous compounds

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a high-resolution octadecyl silica column compatible with aqueous compounds was developed for the simultaneous determination of benzodiazepines and their metabolites in human serum. This method enabled us to determine multiple benzodiazepines, including flurazepam, bromazepam, chlordiazepoxide, nitrazepam, clonazepam, flunitrazepam, estazolam, clobazam, lorazepam, alprazolam, triazolam, brotizolam, fludiazepam, diazepam, quazepam, prazepam and their metabolites such as 7-aminonitrazepam, 7-aminoclonazepam, 7-acetamidonitrazepam, N-desmethylclobazam and N-desmethyldiazepam. The analytes spiked into human serum were subjected to solid-phase extraction followed by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The running time was within 25 min for the measurement of 22 benzodiazepines and their metabolites. The recovery rates exceeded 58.1% for those compounds except for quazepam, which showed a recovery of 45.8%. The limit of detection ranged from 0.3 to 11.4 ng/mL. Linearity was satisfactory for all compounds. These data suggest that the present method can be applicable to routine assay for benzodiazepines in the clinical setting.