A rapid and easy liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed, validated, and used to quantify amlodipine besylate and metoprolol succinate in human plasma. Hydrochlorothiazide was used as the internal standard. LC–MS/MS with electrospray ionization in the positive ionization mode was performed under multiple reaction monitoring to examine analytes, which were drawn from human plasma via liquid–liquid extraction using a mixture of diethyl ether and dichloromethane (6:4). The extracts were dried under a nitrogen stream at 40°C, after which the samples were dissolved in a mixture of methanol and water (80:20). The re-dissolved samples were injected onto a C18 column and eluted using a mixture of methanol and 0.4% formic acid (80:20) as the mobile phase. Linear standard calibration was performed, and lower limits of quantification were determined for each analyte. The developed method was fully validated with respect to selectivity and carryover, as well as intra- and inter-day accuracy and precision in accordance with guidelines of the United State Food and Drug Administration and the European Medicines Agency. Mean analyte recovery was between 71.72% and 97.55%, and matrix effects exerted only a minor influence on precision. The validated method was applied in a clinical bioequivalence study to evaluate the in vivo bioequivalence of two commercial products containing 5 mg amlodipine and 50 mg metoprolol. This randomized, single-dose, two-treatment, three-period, two-sequence, open-label, crossover study involved 14 healthy subjects, and a two-week washout period between the two phases of the research was implemented. Standard pharmacokinetic parameters were calculated to compare a test product with Selomax® as the reference.
Amlodipine besylate; metoprolol succinate; LC–MS/MS, bioequivalence.