Simultaneous gas-chromatographic analysis for diazepam and its major metabolite, desmethyldiazepam, with use of double internal standardization

Abstract

Electron-capture gas-liquid chromatography was used for simultaneous quantitation of plasma diazepam (I) and its major metabolite, desmethyldiazepam(II). Because concentrations of I can greatly exceed those of II after single doses of I, two benzodiazepine-analog internal standards were added to all samples, thereby allowing reliable quantitation of high concentrations of I and low concentrations of II. After extraction at neutral pH with benzene (containing isoamyl alcohol), the organic extract is evaporated and the residue is reconstituted in a small volume of solvent and chromatographed on a 3% OV-17 column. The sensitivity limits are 2 to 3 ng of I or II per milliliter of original sample, with a CV for identical samples of less than 5%. The applicability of the method to single-dose pharmacokinetic studies of I in humans is illustrated.