Conventional HPLC method used for simultaneous determination of the seven HIV protease inhibitors and nonnucleoside reverse transcription inhibitor efavirenz in human plasma.

We developed a simple HPLC method for the simultaneous quantitative determination of seven HIV protease inhibitors: amprenavir (APV), atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), and a nonnucleoside reverse transcription inhibitor, efavirenz (EFV). This method involves a rapid liquid-liquid drug extraction from plasma, the use of an isocratic elution on a reversed-phase C18 column, and an ultraviolet detection at a single wavelength (205 nm). The mobile phase consisted of 39% 50 mM phosphate buffer (pH 5.9), 22% methanol and 39% acetonitrile. Forty-eight samples could be measured in one day since the runtime of one sample is 30 min. The assay has been validated over a concentration range of 0.05 to 12.20 microg/ml for APV, 0.09 to 12.05 microg/ml for ATV, 0.05 to 12.01 microg/ml for IDV, 0.12 to 12.36 microg/ml for LPV, 0.18 to 12.20 microg/ml for NFV, 0.12 to 12.33 microg/ml for RTV, 0.12 to 12.06 microg/ml for SQV, and 0.05 to 12.17 microg/ml for EFV. Calibration curves were linear in the described concentration ranges. The average accuracy ranged from 97.2 to 106.8%. Both the interday and intraday coefficients of variation for all drugs tested were less than 8.5%. This method provides a simple, accurate, and precise method for the therapeutic drug monitoring of the seven protease inhibitors and EFV in clinical routine use.

Pharmacokinetics of lopinavir after administration of Kaletra in healthy Japanese volunteers.

The pharmacokinetic parameters of lopinavir (LPV) were examined by administering Kaletra (LPV+ritonavir) to 8 healthy Japanese volunteers both in the fasting and postprandial conditions. LPV showed a biphasic decline, which was slower in the initial phase and became more rapid in the later phase. The behavior of LPV in the initial phase could be modeled using a one-compartment model with first-order absorption. In the fasting study, calculations based on the pharmacokinetic model revealed that the time to reach the maximum concentration (T(max)), maximum concentration (C(max)), half-life (T(1/2)), lag time, apparent volume of distribution (Vd/F) and oral clearance (Cl/F) were 3.2+/-1.0 h, 6.9+/-1.9 microg/ml, 10.0+/-3.7 h, 0.71+/-0.32 h, 51.0+/-12.4 l and 4.2+/-2.6 l/h, respectively. On the other hand, in the postprandial study, the calculated T(max), C(max), T(1/2), lag time, Vd/F and Cl/F were 5.6+/-2.0 h, 7.6+/-1.8 microg/ml, 16.7+/-7.0 h, 2.35+/-0.78 h, 48.0+/-15.9 l and 2.1+/-0.6 l/h, respectively. The values for the area under the curve for data collected over a 24-h period (AUC(24 h)) in the fasting and postprandial studies were 86.0+/-27.7 and 102.1+/-31.0 microg.h/ml, respectively. The T(1/2) had a tendency to be prolonged after food intake, but there were 2 cases with shortened T(1/2). Food intake prolonged the lag time 3-fold and as a result, the postprandial T(max) was 2 times longer.

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.