Separation, isolation and identification of optical isomers of 1,4-benzodiazepine glucuronides from biological fluids by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of four separate 1,4-benzodiazepine glucuronides in urine, plasma and bile is presented. We succeeded not only in determining the single glucuronides but also in separating the enantiomers (optical isomers) of the 1,4-benzodiazepine glucuronides. The optical isomers of the glucuronides of oxazepam and cinolazepam and of two other glucuronides of benzodiazepine metabolites could be well separated. The ratio of the isomers could be evaluated. An octadecyl reversed phase was used with a mobile phase of acetonitrile and 0.01 M orthophosphoric acid. After the initial separation, the isomers were fractionated by HPLC. After treatment with beta-glucuronidase to yield the aglycone, the separated fractions were hydrolysed to the corresponding benzophenones whose identity was confirmed by HPLC. Gas chromatography and gas chromatography-mass spectrometry demonstrated that the separated glucuronides corresponded to the enantiomeric benzodiazepines. Human urine and plasma as well as rabbit urine, plasma and bile were examined.