Validated LC‐MS/MS Method for Vibegron Quantification in Human Plasma via a Selective Fragment Ion ( <i>m</i> / <i>z</i> 135.1): Addressing Matrix Interference and Sample Instability

A sensitive and robust LC-MS/MS method was developed and validated for the quantification of vibegron in human plasma, employing electrospray ionization in positive mode and multiple reaction monitoring (MRM). The precursor ion m/z 445.5 yielded two product ions: m/z 427.4 and m/z 135.1. Despite higher intensity, m/z 427.4 was excluded due to chromatographic interference, while m/z 135.1, previously unreported for vibegron, was selected for its superior selectivity and specificity. A stable isotope-labeled internal standard, vibegron ¹³C₅ (m/z 450.3 → 139.1), was used to enhance precision and minimize matrix effects. Chromatographic separation was achieved on a Kromasil C18 column with a mobile phase of acetonitrile and 10-mM ammonium formate (pH 5.8), yielding a retention time of 1.33 min and total runtime of 3.00 min. Liquid-liquid extraction with ethyl acetate provided consistent recovery (87.7%), and sodium fluoride was added to inhibit enzymatic degradation. The method demonstrated excellent linearity (0.500-400.223 ng/mL), precision (0.58%-3.34%), and accuracy (98.26%-102.29%), using only 100 μL of plasma. Fully compliant with ICH M10 guidelines, the method is well suited for future pharmacokinetic, bioavailability, and bioequivalence studies.