Benzoyl Chloride Derivatization Coupled With Liquid Chromatography-Mass Spectrometry for the Simultaneous Quantification of Molnupiravir and Its Metabolite β-d-N-hydroxycytidine in Human Plasma.
Journal:
Journal of separation science
PMID:
40349125
Abstract
A sensitive and efficient method for simultaneous quantifying molnupiravir and its active metabolite β-d-N-hydroxycytidine in human plasma was developed by combining chemical derivatization with liquid chromatography-tandem mass spectrometry. Through benzoyl chloride chemical derivatization, the analytes exhibited improved mass spectral responses and enhanced chromatographic retention. Besides, the fragmentation patterns were optimized to identify analyte-specific fragments, enhancing detection specificity beyond the common benzoyl fragments. These advancements enabled the method to achieve the lower limits of quantification of 0.4 ng/mL for molnupiravir and 1.0 ng/mL for β-d-N-hydroxycytidine, which represents the lowest reported quantification limits values to date while demonstrating a 30-fold sensitivity enhancement compared to the non-derivatized method under identical instrument conditions. In addition, the sample preparation protocol was streamlined, combining derivatization and sample extraction in a single step, completed within 5 min, eliminating the need for additional handling or reaction time. After undergoing comprehensive validations, the method was successfully applied to clinical samples from patients receiving molnupiravir therapy, demonstrating its practicality for pharmacokinetic monitoring. By combining operational simplicity with sensitivity, this assay provides a reliable tool for advancing research on molnupiravir metabolism and therapeutic drug monitoring.