Determination of some benzodiazepines and metabolites in serum, urine and saliva by high-performance liquid chromatography

The performance of a number of liquid--solid systems, consisting of mixtures of buffers (0.05 M) and methanol as mobile phase and methyl-silica as stationary phase, were investigated with respect to their use in the separation of 1,4-benzodiazepines by reversed-phase high-performance liquid chromatography with UV detection at 254 nm. Phase system selectivities and column efficiencies were determined. A nomogram is presented from which the chromatographic parameters can be calculated. A complete separation of nine benzodiazepines within 12 min has been achieved, using methyl-silica as the stationary phase and 50% methanol as the eluent. The results were applied to the development of a method for the determination of therapeutic levels of diazepam and its metabolites in human serum, urine and saliva. The first step in the analysis, the extraction of diazepam and its metabolites from serum and urine, was also investigated and good recoveries were achieved. A low detection limit (0.2 ng) and high precision were obtained. The concentrations of diazepam and its metabolites in human serum, urine and saliva were determined after both single and multiple oral doses of diazepam (and oxazepam).