Determination of clonazepam and flunitrazepam in blood and urine by electron-capture GLC.

A sensitive and specific electron-capture GLC assay was developed for the determination of clonazepam and flunitrazepam (two members of the 7-nitro-1,4-benzodiazepin-2-one class of compounds) in blood and urine. The assay for clonazepam uses flunitrazepam as a reference standard and vice versa. The assay for both compounds involves selective extraction into ether from blood or urine at pH 9.0 followed by acid hydrolysis to their respective benzophenones, which are extracted and quantitated by electron-capture GLC. Recovery from blood or urine is quantitative, and the sensitivity limits of the assay are 0.5–1.0 ng/ml of specimen analyzed. The assay was successfully applied to the disposition of clonazepam in man following single 2-mg oral doses and of flunitrazepam in the dog following intravenous and oral doses of 2 mg/kg. A differential pulse polarographic assay for the determination of the major urinary metabolites of clonazepam is also presented.