Bioanalytical RP-HPLC Method for Simultaneous Quantification of Rivaroxaban and Ticagrelor in Spiked Human Plasma: Validation and Stability

Abstract

For the effective assessment of preclinical, biopharmaceutical and clinical pharmacological investigations, selective and sensitive analytical methods for the quantitative evaluation of drugs and their metabolites (analytes) are essential. Using Rilpivirine as an internal standard (IS), a simple, fast, sensitive, precise and accurate high performance liquid chromatographic method for simultaneous determination of Rivaroxaban and Ticagrelor in human plasma was developed. The analytes were extracted using a direct protein precipitation method with acetonitrile from 500μL aliquots of human plasma. The content of the drugs was assessed using a mobile phase consisting of acetonitrile and 0.1% orthophosphoric acid buffer in a 40:60 v/v ratio and with a flow rate of 1.0ml/min, and an injection volume of 10μL. A Kromasil C18 (250×4.6mm; 5µm) analytical column was used for chromatographic separation and the effluents were monitored at 265nm using a photo diode array (PDA) detector.Rivaroxaban, Ticagrelor and Rilpivirine had retention times of 4.716min, 6.396min and 5.405 min, respectively. Rivaroxaban was shown to be linear at concentrations ranging from 0.018µg/mL - 1.80µg/mL, whereas Ticagrelor was found to be linear between 0.020µg/Ml - 2.00µg/mL. The method was validated according to US-FDA guidelines, and the findings satisfied the acceptance criteria. It was successfully used to determine Rivaroxaban and Ticagrelor in human plasma at the same time and this method is applicable for the pre-clinical and clinical estimation of drugs in biological fluids.