ABSTRACT
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.
Subject(s)
Antiviral Agents/blood , Benzoxazoles/blood , Chromatography, High Pressure Liquid/methods , HIV-1 , Pyridones/blood , Reverse Transcriptase Inhibitors/blood , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Benzoxazoles/administration & dosage , Benzoxazoles/chemistry , Benzoxazoles/metabolism , Circadian Rhythm , Cohort Studies , Female , HIV-1/drug effects , HIV-1/enzymology , Macaca mulatta , Male , Osmolar Concentration , Pyridones/administration & dosage , Pyridones/chemistry , Pyridones/metabolism , Rats , Reproducibility of Results , Reverse Transcriptase Inhibitors/administration & dosage , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/metabolism , Spectrophotometry, Ultraviolet , Time FactorsABSTRACT
A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid-liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50-2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.
