Analyitical Assay Methods for Estimation of Drugs in Human Plasma by Liquid Chromatpgraphy Tandem Mass Spectrometry

Abstract

This study comprises the use of a liquid chromatography-tandem mass spectrometry (LCESI-MS/MS) assay with robust sensitivity, reliability, and selectivity for the precise quantification of febuxostat levels in human plasma. The internal standard (IS) employed in this testing is indomethacin. The analyte and IS were mined from a 200 μL sample of human plasma through the process of extraction (liquid-liquid), utilizing methyl tert-butyl ether as the solvent used for extraction. The experiment involved conducting chromatography using a Hypurity C18 column with dimensions of 100 mm × 4.6 mm and a particle having a size of 5 μm. The chromatography was carried out under isocratic circumstances. The analyte and internal standard were detected using tandem mass spectrometry, which was capable of running in a variety of negative ionization conditions and effectively monitor different modes of reaction. The observed product ion changes for indomethacin and febuxostat were m/z356.1 → 312.0 and 315.1 → 271.0, respectively, corresponding to the deprotonated precursor ions. The method exhibited a limit of detection (LOD) of 0.0025 μg/mL and a limit of quantitation (LOQ) of 0.05 μg/mL. The validated linear dynamic range for febuxostat was determined to be 0.05-6.00 μg/mL. The precision within batches and between batches, stated as the coefficient of variation (% CV), was found to be 7.1%. Additionally, the average extraction recovery of febuxostat across different quality control levels was determined to be 87%. The present study effectively employed the aforementioned method to investigate the bioequivalence of an 80 mg febuxostat tablet form in a cohort of 14 healthy male subjects of origin. Both fed and fasted circumstances were used for the experiment. Re-examining 110 incurred instances demonstrated the ability to duplicate the measurements of study results.