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, ANDRÉS; HERNANDEZ, SILVIA; ALTAMIRANO, JORGELINA C

Cordoba

Congreso; 3era Reunion Internacional de Ciencias Farmaceuticas; 2014

Colegio Farmaceutico de Cordoba

A SPE-LC-MS/MSmethod has been developed and validated for simultaneous analysis in humanplasma of one nucleotide tenofovir disoproxil fumarate (TDF) and twonucleosides emtricitabine (FTC) and lamivudine (3TC) reverse transcriptaseinhibitors. Plasma samples were prepared by solid-phase extraction (SPE) using tenofovir-d8as internal standard, 0.7 mL plasma sample and Waters Oasis® MCXcartridges. Chromatography was performed on a C-18 analytical column, theinjection volume was 30 µL and the run time was 4 min.Theproposed 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 plasmaconcentrations, allowing its application for therapeutic drug monitoring and clinicalpharmacokinetic study.Thevalidated method was applied to a bioequivalence study between a newpharmaceutical equivalent tablet formulation containing 300 mg of TDF and theinnovator product. A randomized, single-center, open-label, single-dose,two-way crossover bioequivalence study in 24 healthy adult subjects wasconducted. Dosing was separated by a wash-out period of 7 days. All subjectssigned an informed consent form. In each study period, 17 blood samples werecollected in VacutainersTM containing EDTA over 48 h. Rate andextent 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 twoformulations 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(AUCinf) 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 wasbioequivalent to the innovator.