Determination of diazepam and its pharmacologically active metabolites in blood by bond Elut column extraction and reversed-phase high-performance liquid chromatography.

A rapid and quantitative analytical micro method for the determination of diazepam and its major pharmacologically active metabolites utilizing high-performance liquid chromatography (HPLC) is reported. The drug and its metabolites were extracted from 50-100 microliter samples of whole blood, serum or plasma using Bond Elut C 18 column and quantitated by high-performance liquid chromatography, using Technician Fast-LC-C-8 (RP 5 micrometers) bonded column and a mobile phase consisting of 53% methanol, 1% acetonitrile in KH2PO4 buffer and 10 microliters/l triethylamine. Methyl nitrazepam and medazepam were used as internal and external standards respectively. The extraction and recovery of diazepam and its major pharmacologically active metabolites, i.e., 3-hydroxydiazepam, desmethyldiazepam and oxazepam from blood were higher than 88% for all compounds. The minimum detection range of each compound was approximately 2.5 ng per 100-microliter sample. This micro method of simultaneous quantitation of diazepam and its major pharmacologically active metabolites provides a valuable technique for the study of diazepam pharmacokinetics in a small animal model without disturbance of normal hemodynamics from excess blood loss, as well as in clinical evaluation of pediatric patients.