Simultaneous determination of amlodipine and atorvastatin with its metabolites; ortho and para hydroxy atorvastatin; in human plasma by LC–MS/MS

A simple liquid chromatography/ion trap mass spectrometry method for the quantification of amlodipine and atorvastatin with its metabolites, ortho and para hydroxy atorvastatin, simultaneously in human plasma was developed. Analytes with internal standard were extracted by protein direct precipitation with acetonitrile. Adequate chromatographic separation was achieved using Phenomenex Synergi 4u polar-RP 80A (150 mm × 4.6 mm, 4 μm) column in the isocratic elution mode and the eluent was water/methanol (14:86%, v/v) adjusted by trichloroacetic acid to pH 3.2 which was delivered isocratically at constant flow rate of 0.50 mL/min. Standard solutions for the analytes were prepared using amlodipine besylate, atorvastatin calcium, ortho-hydroxy atorvastatin dihydrate monosodium salt, para-hydroxy atorvastatin disodium salt, and pravastatin sodium as an internal standard. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to USFDA guideline. Standard calibration levels were prepared by pooled human plasma to attain final dynamic range of 0.2–20.0 ng/mL for amlodipine, 1.5–150 ng/mL for atorvastatin, 1.0–100.0 ng/mL for ortho-hydroxy atorvastatin and 0.2–20.0 ng/mL for para-hydroxy atorvastatin. Clinical bioequivalence study was successfully investigated by the application of this validated bioanalytical method in order to evaluate bioequivalence of two commercial products 10 mg amlodipine/80 mg atorvastatin in a single dose. In this study, 29 healthy volunteers were participated in randomized, two periods, double blend, open label cross over design. Pharmacokinetic parameters of Cmax, AUC0–t and AUC0–∞ were calculated to compare a test product with CADUET® reference product.