NOVEL LIQUID CHROMATOGRAPHY / TANDEM MASS SPECTROMETRY METHOD OF WARFARIN IN BUFFERED RAT PLASMA AND ANALYSIS PLASMA PROTEIN BINDING ASSAY SAMPLES AS ITS APPLICATION

Sensitive and selective liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for warfarin (WAR) in buffered SD (Sprague Dawley) rat plasma (PBS and Plasma: 50:50, v/v) using propranolol (PRP) as internal standards (IS) with the Plasma protein binding (PPB) assay samples to a determination of free and total warfarin. Plasma protein precipitation extraction technique with acetonitrile containing internal standard was used for extraction of the analyte. A generic gradient method with a short run time of 2.0 min was developed for the analysis of warfarin with a C18 analytical column (Synrgi 4 µm Fusion-RP, 50X2 mm), mobile phase composed of aqueous phase: 0.1% formic acid containing Milli-Q-water and Organic Phase 0.1% formic acid containing acetonitrile. Detection was performed on a triple quadrupole mass spectrometer that employed the Positive electrospray ionization (ESI) technique, operating in multiple reaction monitoring (MRM), with the transitions of m/z 309.2-163.1. The analysis was carried out over a linear concentration range of 1–1200 nM, where the 0.1% of the free fraction of the compound was able to quantify by this method, which resulted in successfully quantifying the tightly bound compounds; the method was validated following the FDA guidelines⁵ for bioanalytical method. All obtained recoveries were higher than 95.0%, while the accuracy was 88.14–113.05%, and the relative standard deviation was below 10.0%. Successfully developed LC-MS/MS method used to analyze the plasma protein binding assay samples. Where WAR is the standard idle compound for plasma protein binding, it shows species-specific % free fraction.