A simple HPLC method for simultaneous determination of lopinavir, ritonavir and efavirenz

We developed a simple HPLC method for the simultaneous determination of lopinavir (LPV), ritonavir (RTV) and efavirenz (EFV) to evaluate the efficiency of co-administration of LPV/RTV and EFV in Japanese patients enrolled in a clinical study. The monitoring of LPV plasma concentration is important because co-administration of LPV/RTV with EFV sometimes decreases plasma concentrations of LPV caused by EFV activation of cytochrome P-450 3A. A solution of acetonitrile, methanol and tetramethylammonium perchlorate (TMAP) in dilute aqueous trifluoroacetic acid (TFA) has been used as the mobile phase in a HPLC method to elute LPV and RTV. We found that a solvent ratio of 45 : 5 : 50 (v/v/v) of acetonitrile/methanol/0.02 M TMAP in 0.2% TFA optimized separation of LPV, RTV and EFV. A column temperature of 30 degrees C was necessary for the reproducibility of the analyses. Standard curves were linear in the range 0.060 to 24.06 micro g/ml for LPV, 0.010 to 4.16 micro g/ml for RTV, and 0.047 to 37.44 micro g/ml for EFV. Coefficients of variation (CVs) of LPV, RTV and EFV in intraday and interday assays ranged from 1.5 to 4.0%, 2.5 to 16.8% and 1.0 to 7.7%, respectively. Accuracies ranged from 100 to 110%, 101 to 116% and 99 to 106% for LPV, RTV and EFV, respectively. The extraction recoveries were 77-87, 77-83 and 81-91% for LPV, RTV and EFV, respectively.