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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.

Introduction: Several generic products containing simvastatin are circulating on the Vietnamese market at a more inexpensive price than that of a brand-name one. These formulations, however, have not been assessed for in vivo bioequivalence to the reference product. After oral administration, simvastatin (SIM) is extensively converted into an active metabolite, beta-hydroxy simvastatin acid (SIM-A) and a very low concentration of simvastatin can be found in plasma. Therefore, a method for quantification of simvastatin and its metabolite needs to be developed with a high specificity and sensitivity to detect these analytes in human plasma at such low concentrations. Our purpose was to develop a reliable LC-MS/MS (liquid chromatography-tandem mass spectrometry) method for simultaneous determination of simvastatin and metabolite of simvastatin, beta-hydroxy simvastatin acid, in human plasma and to apply this method to evaluate the bioequivalence of a test product in comparison with the reference product. Methods: Mass spectrometry, internal standard (IS), and chromatographic conditions were investigated to find out the suitable IS and conditions. Human plasma samples were treated by liquid-liquid extraction (LLE). The assay was validated in compliance with US-FDA (United States-Food and Drug Administration), and EMA (European Medicines Agency) guidelines. Results: LC-MS/MS with electrospray ionization interface in positive (for SIM and lovastatin as IS) and negative (for SIM-A) ionization mode performed under the multiple reaction monitoring mode was used for detection of the analytes. The transition of m/z is 436.00 → 285.15, 435.10 → 319.15, and 404.95 → 199.10 for SIM, SIM-A, and IS, respectively. Tert-buthyl methyl ether was used for extraction of analytes from human plasma by a simple LLE followed by addition of an ammonium acetate buffer. The developed method was fully validated with acceptable selectivity, linearity and linear range, matrix effect, lower limit of quantitation (LLOQ), carryover, dilution integrity, and intra- and inter-day accuracy and precision, free-thaw stability. Conclusions: The method can be applied for quantification of these compounds in human plasma for in vivo bioavailability and bioequivalence studies.