Development and validation of a LC-MS/MS method for quantitative determination of Tenofovir, Emtricitabine, and Lamivudine in Human Plasma, and Its Application to a Bioequivalence Study of Tenofovir

HUNZICKER, GABRIEL A; HEIN, GUSTAVO J; VALIN, A; HERNANDEZ, S; ALTAMIRANO, J

Córdoba

Congreso; 3era Reunión Internacional de Ciencias Farmacéuticas RICIFA 2014; 2014

Colegio Farmaceúticos de Córdoba

A SPE-LC-MS/MS method has been developed and validated for simultaneous analysis in human plasma of one nucleotide tenofovir disoproxil fumarate (TDF) and two nucleosides emtricitabine (FTC) and lamivudine (3TC) reverse transcriptase inhibitors. Plasma samples were prepared by solid-phase extraction (SPE) using tenofovir-d8 as internal standard, 0.7 mL plasma sample and Waters Oasis® MCX cartridges. Chromatography was performed on a C-18 analytical column, the injection volume was 30 µL and the run time was 4 min. The proposed method was specific, intra- and inter-day precisions were <10.8% with an accuracy within 87-108%. A linear dynamic range of 13.2?396.8 ng/mL, 10.8?5434.3 ng/mL and 10.9?5452.2 ng/mL was established for TDF, FTC and 3TC, respectively. The limits of quantitation were consistent with trough plasma concentrations, allowing its application for therapeutic drug monitoring and clinical pharmacokinetic study. The validated method was applied to a bioequivalence study between a new pharmaceutical equivalent tablet formulation containing 300 mg of TDF and the innovator product. A randomized, single-center, open-label, single-dose, two-way crossover bioequivalence study in 24 healthy adult subjects was conducted. Dosing was separated by a wash-out period of 7 days. All subjects signed an informed consent form. In each study period, 17 blood samples were collected in VacutainersTM containing EDTA over 48 h. Rate and extent of absorption were similar between products. The 90% confidence interval (CI) of the ratio of the geometric means for log-transformed C(max), AUC(last) and AUC(inf) values were used to assess bioequivalence between the two formulations using the equivalence interval of 80 and 125%. In healthy subjects, the point estimate and 90% CI of the ratios of Ln(Cmax), Ln(AUC last) and Ln(AUC inf) values for TDF were 90.9% (85.0-97.2%), 92.30% (87.2-97.7%) and 91.6% (85.5-98.1%), respectively. It was concluded that the new pharmaceutical formulation was bioequivalent to the innovator.