indinavir-sulfate and acetonitrile

A comparative study with three conventional extraction techniques namely protein precipitation (PP), liquid-liquid extraction (LLE) and solid phase extraction (SPE) has been demonstrated to assess the magnitude of matrix interference by post-column analyte infusion and post extraction analyte spiking for the determination of atazanavir from human plasma. Severe ion suppression observed in PP and to a lesser extent in LLE was circumvented by SPE on LiChrosep Sequence extraction cartridge. Based on these observations a selective, rugged and high throughput SPE-LC-MS/MS method has been developed for reliable determination of atazanavir in human plasma. The chromatographic separation was achieved on a Hypersil Gold C18 (50mm×4.6mm, 5μm) analytical column using 5mM ammonium formate in water:methanol (10:90, v/v) as the mobile phase under isocratic conditions. The method was validated over a wide dynamic concentration range of 10-6000ng/mL. The mean relative recovery and absolute matrix effect across quality controls were 84.9 and 93.2%, respectively. The precision value for relative matrix effect between eight different lots of plasma, expressed as %CV of the slopes of the calibration lines was 2.41. The stability of atazanavir under different storage conditions varied from -8.4 to 5.4%. The method was successfully applied to a bioequivalence study of 300mg atazanavir capsule formulation in 24 healthy Indian males under fasting condition.

Topics: Acetonitriles; Atazanavir Sulfate; Chromatography, Liquid; Cross-Over Studies; Drug Stability; Humans; Indinavir; Male; Methanol; Oligopeptides; Pyridines; Reproducibility of Results; Solid Phase Extraction; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Therapeutic Equivalency

A sensitive high-perforrmance liquid chromatographic assay has been developed to determine the concentrations of the HIV-protease inhibitor indinavir in human plasma. The sample pretreatment involved a protein precipitation procedure using 100 microl of human plasma and 400 microl of acetonitrile. Chromatography was carried out on an Octadecyl column using a mobile phase of acetonitrile-water (40:60, v/v). The water phase contained 50 mM phosphate buffer pH 6 and 4 g/l tetramethylammoniumchloride. Ultraviolet detection at 210 nm was used. The method has been validated with regard to specificity, detection limit, lower and upper limit of quantitation, recovery, accuracy, and inter- and intra-assay precision. Stability tests under various conditions were performed. The bioanalytical assay is now in use for the determination of indinavir in several clinical pharmacokinetic studies in HIV-infected patients.

Topics: Acetonitriles; Administration, Oral; Adult; Chromatography, High Pressure Liquid; Circadian Rhythm; Drug Stability; HIV Infections; HIV Protease Inhibitors; Humans; Indinavir; Male; Middle Aged; Osmolar Concentration; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Time Factors