NOVEL LIQUID CHROMATOGRAPHY / TANDEM MASS SPECTROMETRY METHOD OF WARFARIN IN BUFFERED RAT PLASMA AND ANALYSIS PLASMA PROTEIN BINDING ASSAY SAMPLES AS ITS APPLICATIONAbstract
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.
Ravi Akki Reddy *, Pratima Srivastava, Sudhir Tiwari, Sachin Arjun Atole and Prabhakara Reddy Kolli
Aragen Life Sciences Pvt. Ltd, Bommasandra Jigani Link Road, Jigani, Bangalore, Karnataka, India.
16 February 2022
28 March 2022
03 May 2022
01 October 2022