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Development and validation of a LC–MS/MS assay for tenofovir and tenofovir alafenamide in human plasma: Application in a bioequivalence study

Introduction: Tenofovir alafenamide (TAF), a prodrug of tenofovir (TFV), is currently used for treatment of chronic hepatitis B as first line recommendation. The Vietnamese market is currently circulating a tenofovir alafenamide generic. This formulation has not been assessed for in vivo bioequivalence. This study has been performed with the aim of development, validation, and application of LC-MS/MS procedure for quantitation of TAF and TFV in human plasma.

Methods: Internal standard (IS), and analytical parameters were investigated to find out the suitable IS and conditions. Chromatographic conditions were optimized by considering the column type, mobile phase component, concentration of the buffer solutions and strength, oven temperature, flow rate, and injection volume. Human plasma samples were treated by protein precipitation with acetonitrile. The assay was validated in compliance with US-FDA and EMA guidelines. This assay was applied to evaluate the bioequivalence of the generic and reference products of 25 mg TAF in the healthy Vietnamese subjects under fed conditions.

Results: TAF, TFV, IS were ionized using ESI and detected by MRM mode to obtain molecular and fragment ions for quantification. The recovery of all analytes from human plasma was above 70%. The chromatographic conditions contained a C6-Phenyl column and mobile phase including acetonitrile and 0.5% formic acid. The specificity, precision, accuracy, matrix effect of all the analytes were in the acceptable range. Thirty six subjects finished the fed trial. The two products’ geometric mean ratios for AUC0-t, AUC0-∞, and Cmax (80.00% to 125.00%) met the bioequivalence acceptance criteria.

Conclusions: A LC-MS/MS procedure for simultaneous quantitation of TAF and TFV in human plasma was developed, validated, applied.