Comprehensive Validation of Rosuvastatin and Apixaban Quantification in Plasma Using LC-MS/MS: A Novel Analytical Method for Bioequivalence Studies

Abstract

This study presents the development and comprehensive validation of a highly sensitive and selective LC-MS/MS method for the simultaneous quantification of rosuvastatin and apixaban in human plasma, aimed at bioequivalence and pharmacokinetic studies. Analytical method validation was conducted following EMEA and ICH M10 guidelines. The method employed a Zorbax SB-C18 column with a mobile phase comprising 0.2% formic acid in water (Phase A) and methanol (Phase B) under gradient elution. Rosuvastatin and apixaban were quantified using multiple reaction monitoring (MRM) in positive electrospray ionization mode. The lower limit of quantification (LLOQ) was established at 0.625 ppb for rosuvastatin, ensuring a signal-to-noise ratio exceeding 10.

Validation parameters included specificity, carry-over, calibration curve linearity, accuracy, precision (within-run and between-run), matrix effects, and stability under various conditions. Specificity tests revealed no interference from blank plasma, with placebo interference below 1%. Calibration curves exhibited excellent linearity (R² > 0.999) over the concentration range of 0.625–60 ppb. Accuracy and precision were within acceptable limits, with relative standard deviation (RSD) values below 10% for all quality control levels. Matrix effects were minimal, with matrix effect factors (MEF) consistently meeting validation criteria. Stability assessments, including short-term, freeze-thaw, and long-term evaluations, demonstrated negligible degradation under simulated bioanalytical conditions.

Application of the validated method to plasma samples from study volunteers demonstrated its robustness and reproducibility in real-world scenarios. These results confirm the method's suitability for bioequivalence and pharmacokinetic analyses of rosuvastatin and apixaban, contributing to enhanced therapeutic monitoring and optimization.