Reverse phase high-performance liquid chromatography method for the analysis of amprenavir, efavirenz, indinavir, lopinavir, nelfinavir and its active metabolite (M8), ritonavir, and saquinavir in heparinized human plasma

The increasing interest in applying therapeutic drug monitoring (TDM) to antiretroviral therapy is related to the observed interindividual variation in antiretroviral pharmacokinetics that results in a wide range of drug exposure from fixed-dosing regimens and the rapid evolution in the availability of phenotypic assays that generate a target 50% inhibitory concentration (e.g., IC₅₀) as a basis for adjusting individual antiretroviral dosages. To facilitate the application of TDM, a method for the simultaneous determination of eight species has been developed. This method is used to quantitate efavirenz and the following protease inhibitors: amprenavir, indinavir, lopinavir, nelfinavir and its active metabolite (M8), ritonavir, and saquinavir. The method using reversed-phase high-performance liquid chromatography (RP-HPLC) was validated. Detection is effected using a photodiode-array detector (PDA) scanning at four different wavelengths. This method allows for detection of all analytes to a lower limit of quantitation of 0.1 to 0.2 μg/mL with an interday variation in CV ranging from 3.5% to 10.4%. The method is being applied to a TDM program that is currently being implemented in the authors' laboratory.