Determination of saquinavir and ritonavir in human plasma by reversed-phase high-performance liquid chromatography...

Two simple and reproducible high-performance liquid chromatography methods with ultraviolet detection were developed and validated for the quantitation of two protease inhibitors, saquinavir and ritonavir, in human plasma. The same single liquid-liquid extraction procedure with ethyl acetate-hexane (50:50, v/v), reversed-phase column and mobile phase were used. The analyses were accomplished using a Luna C(18) column (150 mm x 4.6mm i.d.) with a C(18) guard column and, the mobile phase consisted of acetonitrile and 70 mM KH(2)PO(4) adjusted to pH 5 with 80 mM Na(2)HPO(4) (46:54, v/v). The wavelength was set at 240 nm for saquinavir and at 210 nm for ritonavir. The retention times were 6.4 min for saquinavir and 8.3 min for ritonavir. The methods were linear over the range of 100-2500 ng/ml for saquinavir and 200-2500 ng/ml for ritonavir. Intra and inter-day precision and accuracy were less than 10.2% for both drugs. Recovery were 90 and 87% for saquinavir and ritonavir, respectively. The drugs were stable at different relevant storage and working conditions. After the validation, their analytical error functions were established as standard deviation (S.D., ng/ml) = 4.84 + 7.14 x 10(-2)C (C is the theoretical concentration value) for saquinavir and S.D. (ng/ml) = 39.98 + 2.40 x 10(-5)C(2) for ritonavir.