Automated electron-capture gas chromatographic analysis of flunitrazepam in plasma

Nanogram quantities of flunitrazepam and its major metabolite, desmethylflunitrazepam, can be reliably quantitated in human plasma using electron-capture gas-liquid chromatography. After addition of methylnitrazepam as the internal standard, plasma samples are extracted with an organic solvent (benzene:isoamyl alcohol). The organic extract is separated, evaporated to dryness, reconstituted, and injected onto the chromatograph using an automatic sampling system which allows up to 100 analyses/24 h. The sensitivity limits are 0.5 ng/ml for flunitrazepam and 1-2 ng/ml for desmethylflunitrazepam. Use of the method is illustrated in a study of the pharmacokinetic properties of flunitrazepam following a single 1.0-mg intravenous dose.