Development, Validation, and Application for Simultaneous Assay of Amlodipine, Atorvastatin, and Ortho- and Para Hydroxy Atorvastatin as Metabolites in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry

Background: The objective of this study was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify amounts of amlodipine, atorvastatin, and two metabolites of atorvastatin (ortho- and para-hydroxy atorvastatin) in human plasma and to apply this method for a bioequivalence study. Methods: An LC-MS/MS method was developed, validated, and applied to quantify amlodipine, atorvastatin and two metabolites of atorvastatin (ortho- and para-hydroxy atorvastatin) in human plasma. Rosuvastatin was used as an internal standard. LC-MS/MS with electrospray ionization (ESI) in positive ion mode, performed under the multiple reaction monitoring (MRM) mode, was used to analyze the analytes. Results: The method was developed and fully validated with respect to selectivity, carryover, dilution integrity, and intra- and inter-day accuracy and precision according to US Food and Drug Administration (FDA) guidelines. Analytes were extracted from human plasma via a simple liquid-liquid extraction technique using a mixture of methyl tert-buthyl ether and ethyl acetate (1:1) after acidification with phosphoric acid. Mean extraction recovery for the analytes was between 70% and 99.95%, and matrix effects had only minor influence on precision. Conclusion: The validated method was applied for a clinical bioequivalence study to evaluate the in vivo bioequivalence of two commercial products containing 5 mg amlodipine and 10 mg atorvastatin. 36 healthy subjects participated in this randomized, two-period, two-treatment, open label, crossover design study. Standard pharmacokinetic parameters were calculated to compare a test product to the CADUET® reference product. it is concluded that the two formulations are bioequivalent.