Determination of the new HIV-protease inhibitor atazanavir by liquid chromatography after solid-phase extraction

An HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz is proposed here for the simultaneous analysis of the new HIV protease inhibitor atazanavir (ATV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection. After viral inactivation by heat (60 °C for 60 min), plasma (600 μl) with clozapine (internal standard) is diluted 1 + 1 with phosphate buffer pH 7 and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with 2 × 500 μl of a solution of 0.1% H3PO4 neutralised with NaOH to pH 7. ATV is eluted with 3 × 500 μl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 μl MeOH/H2O 50/50. A 40 μl volume is injected onto a Nucleosil 100–5 μm C18 AB column. ATV is analysed by UV detection at 201 nm using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.14. The mobile phase also contains 0.02% sodium heptanesulfonate, enabling an excellent separation of ATV from the other HIV protease inhibitors (PIs) amprenavir, indinavir, saquinavir, ritonavir, lopinavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. The calibration curves are linear up to 10 μg/ml, with a lower limit of quantification of 0.2 μg/ml. The mean absolute recovery of ATV is 96.4 ± 3.2%. The method is precise with mean inter-day CVs within 1.1–6.1%, and accurate (range of inter-day deviations +0.3 to +2.3%). The method has been validated and is currently applied to the monitoring of ATV in HIV patients.