SimultaneousGas-Chromatographic Analysis for Diazepam and Its Major Metabolite, Desmethyldiazepam, With Use of Double Internal Standardization

Electron-capture gas-liquid chromatography was used for simultaneousquantitatlon of plasma diazepam (I)and its major metabolite,desmethyldiazepam (ii).Because con-centrationsofIcan greatlyexceed those ofiiaftersingle doses ofI,two benzodlazepine-analog internalstandards were added to allsamples, thereby allowing reliable quantitatlon of high concentrationsof Iand low concen-trationsof Ii. Afterextraction atneutralpH withbenzene (containing isoamyi alcohol), the organic extract is evap-orated and the residue Is reconstituted in a small volume of solvent and chromatographed on a 3 % OV- 17 column. The sensitivity limitsare 2 to3 ng of Por iiper milliliterof original sample, with a CV for Identical samples of <5%. The applicability of the method to single-dose phar-macokinetic studiesof Pinhumans isillustrated. Additional Keyphrases: pharmacokinetics drug assay Electron-capture gas-liquid chromatography is successfully used for simultaneous determination of diazepam (DZ), and its major metabolite, desmethyldiazepam (DMDZ), in human blood or plasma (1) (Figure 1). Use of a single internal stan-dard in theseanalyses(2-10) allows most reliable quantitation of DZ and DMDZ when plasma concentrations of these two compounds are similar, as would occur during multiple-dose therapywithDZ. After singledosesofDZ, however,reliable quantitationwith use of a singleinternalstandardcan be difficult because concentrationsoftheparentcompound can exceedthoseofits metabolite,DMDZ, by more than 100-fold. Accordingly,previousapproaches (10-12) were modified to includedoubleinternalstandardization,therebyfacilitating simultaneous quantitation of DZ and DMDZ after single therapeutic doses of DZ to humans