Abstract
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 μl samples of whole blood, serum or plasma using Bond Elut™ C 15f column and quantitated by high-performance liquid chromatography, using Technicon Fast-LC-C-8 (RP 5 μm) bonded column and a mobile phase consisting of 53% methanol, 1% acetonitrile in KH 2PO 4 buffer and 10 μl/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-μl 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.
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