Determination of ritonavir, a new HIV protease inhibitor, in biological samples using reversed-phase high-performance liquid chromatography.

A simple, accurate and precise high-performance liquid chromatographic method has been developed for measurement of ritonavir concentrations in human plasma. Ritonavir was partitioned from the plasma using liquid-liquid extraction with a mixture of ethyl acetate and hexane at neutral pH, with an average recovery >80%. Following two sequential washings of the reconstituted sample with hexane, chromatographic separation was accomplished on a C18 analytical column with a mobile phase containing acetonitrile, methanol and 0.01 M tetramethylammonium perchlorate in 0.1% aqueous trifluoroacetic acid (40:5:55, v/v) with low wavelength UV detection at 205 nm. Standard curves were linear (r2>0.9998) over the concentration range 0.01-15 microg/ml with both inter- and intra-day coefficients of variation typically less than 5%. The stability of ritonavir in plasma was excellent, with no evidence of degradation after 5 days at room temperature or after 6 months in a freezer. Decontamination procedures for HIV-positive plasma samples showed 5.6 and 10.2% degradation following heating to 60 degrees C for 30 or 60 min, respectively.