Quantitative analysis of prazepam and its metabolites by electron capture gas chromatography and selected ion monitoring : Application to diaplacental passage and fetal hepatic metabolism in early human pregnancy

Abstract

Methods have been developed for the determination of the benzodiazepine tranquilizer prazepam and its metabolites desmethyl diazepam, 3-hydroxy-prazepam and oxazepam by electron capture gas chromatography and selected ion monitoring with diazepam as the internal standard. The benzodiazepines were isolated from blood serum or homogenized tissue samples, either by extraction with ethyl acetate or on small Extrelut columns packed with porous silica. The concentrated extracts were directly injected into the gas chromatograph equipped with an electron capture detector. Following trimethylsilylation, analysis on a gas chromatography—mass spectrometry—computer system operated in the selected ion-monitoring mode was performed. Using 50–200 mg (μl) biological material, concentrations of prazepam and metabolites of 5 ng/g(ml) could be determined with signal-to-noise ratios of > 10. Using 1 g(ml) samples, the same signal-to-noise ratios were obtained with 1 ng/g(ml) concentrations. The methods developed were applied to the analysis of the displacental transfer of prazepam and desmethyl diazepam in early human pregnancy. Furthermore, prazepam metabolism in human fetal liver and cell cultures was studied.