High-performance liquid chromatographic method for the determination of an HIV-1 non-nucleoside reverse transcriptase inhibitor (L-696,229) in plasma samples from animals.

A sensitive high-performance liquid chromatographic (HPLC) method was developed and validated to separate and quantitate the levels of L-696,229 (I), a novel human immunodeficiency virus type I non-nucleoside reverse transcriptase inhibitor, and its hydroxy metabolites (II and III) in plasma samples. The procedure involves the addition of a constant known quantity of internal standard to the biological specimen followed by extraction of the compounds of interest into methyl tert.-butyl ether. The organic phase is evaporated to dryness under a gentle stream of nitrogen. The residue is then dissolved in methanol and water and injected onto a reversed-phase HPLC column. A gradient HPLC method is used to elute the compounds which are monitored using UV detection at 319 nm. Absolute calibration factors (from the standard curve) were calculated by analyzing standards, and these factors were used to determine the concentration of drug (I) and its hydroxy metabolites (II and III) in the samples using the internal standard method. The method was linear using a standard concentration range of 50 to 20,000 ng/ml. The limit of quantitation was 50 ng/ml using 200 microliters plasma. The procedure was utilized to monitor plasma levels of I, II and III in acute and chronic toxicity studies in several animal species.