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

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Abstract
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.

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