Analytical method development and validation for extraction and quantification of apixaban in human plasma using HPLC

Background: Apixaban, an oral anticoagulant and direct factor Xa inhibitor, is used to reduce the risk of various thromboembolic diseases.Measuring the active substance in blood can assess its effectiveness compared to the reference drug.In this study, a rapid, new, and reliable reversed-phase liquid chromatography method was developed for measuring apixaban in human plasma.Materials and methods: The chromatographic conditions were optimized, followed by validation using spiked standard samples.To evaluate the extraction method of Apixaban from spiked blood samples, the recovery rate was calculated.Results: The chromatographic analysis was conducted on a C18 column (250 mm 4.6 mm, 5) using isocratic elution with a mobile phase consisting of a mixture of acetonitrile: ammonium acetate buffer 0.15% at pH 7.6 0.05 with a 50:50 volume ratio and a flow rate of 1 mL/min at room temperature.Apixaban detection was carried out at 276 nm.The calibration curve for apixaban in human plasma was linear in the range of 0.3 to 0.12288 g/ml with a correlation coefficient of r 2 = 0.9957.Limit of Quantitation and Limit of Detection concentrations were obtained as 0.06 and 0.02 g/ml, respectively and the average extraction recovery of the extraction method was 78.73%. Conclusion:The developed and validated HPLC-UV method presented in the study is suitable for accurate determination of apixaban levels in the pharmacokinetic studies.