HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF AN HIV-1 NONNUCLEOSIDE 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 1 non-nucleoside r everse transcriptase inhibitor, and its hydroxy metabolites (II and II I) in plasma samples. The procedure involves the addition of a constan t known quantity of internal standard to the biological specimen follo wed by extraction of the compounds of interest into methyl tert.-butyl ether. The organic phase is evaporated to dryness under a gentle stre am of nitrogen. The residue is then dissolved in methanol and water an d injected onto a reversed-phase HPLC column. A gradient HPLC method i s used to elute the compounds which are monitored using UV detection a t 319 nm. Absolute calibration factors (from the standard curve) were calculated by analysing standards, and these factors were used to dete rmine the concentration of drug (I) and its hydroxy metabolites (II an d III) in the samples using the internal standard method. The method w as linear using a standard concentration range of 50 to 20 000 ng/ml. The limit of quantitation was 50 ng/ml using 200 mu l plasma. The proc edure was utilized to monitor plasma levels of I, II and III in acute and chronic toxicity studies in several animal species.