Development and validation of simultaneous assay of simvastatin, beta-hydroxy simvastatin as metabolite in human plasma using liquid chromatography-tandem mass spectrometry

Abstract

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. 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. 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. The method can be applied for quantification of these compounds in human plasma for in vivo bioavailability and bioequivalence studies.