Development and Validation of HPLC-MS/MS Method for Rivaroxaban Quantitation in Human Plasma using Solid Phase Extraction Procedure

摘要

A bioanalytical method was developed and validated using High Performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) technique for the determination of Rivaroxaban in human plasma. The samples were extracted using solid-phase extraction (SPE) technique wherein Rivaroxaban D4 has been used as the internal standard. The use of isocratic Liquid chromatography (LC) method has enabled to achieve 2.0 minutes along with their respective internal standard using a Phenomenex Gemini C18, 50*4.6mm, 5μ column.The developed method was specific and sensitive having no interfering peaks in the drug free plasma. The method was validated for a linear range of 2.00-500.93 ng/mL for Rivaroxaban with a correlation coefficient e" 0.99. The limit of detection (LOD) of 2 ng/mL for Rivaroxaban a signal-to noise (S/N) >10 was achieved, Electrospray ionization source in positive mode was used for the detections of Rivaroxaban and IS. Precursor to product ion transition of m/z 436.20 >144.80 for rivaroxaban and m/z 440.20 >144.70 for IS were used in multiple reaction monitoring mode. Intra-run precision (%CV) ranged from 3.8% to 0.9% for Rivaroxaban. Inter-run accuracy(%Bias) ranged from -3.1% to -1.9% for Rivaroxaban The overall recoveries for Rivaroxaban, Rivaroxaban D4 were found to be >96%. Rivaroxaban was found to be stable at various temperatures and for about 5 freeze-thaw cycles and reconstituted samples were stable up to 72 hours post to extraction. The developed and validated method was found to be precise, reproducible and a high throughput of analyzing more than 400 samples per day could be achieved with a shorter run time of 2.0 minutes. The developed method is useful to measuring Rivaroxaban plasmatic concentrations in pharmacokinetics studies and in therapeutic drug monitoring.