Standard-Free Bioanalytical Approach for Absolute Quantitation of Drug Metabolites Utilizing Biosynthesis of...

The following work describes a combined enzymatic and bioanalytical method that permits absolute quantitation of metabolites in biological samples without the requirement for reference metabolite standards. This technique was exemplified using a radio (¹⁴C) isotopologue and a stable (¹³C₆) isotopologue of acetaminophen as substrates for in vitro biosynthesis of the corresponding radio and stable isotope labeled metabolites, namely, ¹⁴C- and ¹³C₆-glucuronides and sulfates. By supplanting the use of authentic metabolite standards, traditionally used to calibrate ¹³C₆-metabolites via liquid chromatography-tandem mass spectrometry (LC-MS/MS), ¹³C₆-metabolites were radiocalibrated by their ¹⁴C-isotopologues via liquid chromatography coupled with radioactivity detection and mass spectrometry (LC-RAD/MS). The radiocalibrated ¹³C₆-isotopologues were in turn used to quantitate acetaminophen and its corresponding metabolites in rat plasma samples by LC-MS/MS. Variation between this and a conventional LC-MS/MS method using authentic standards for calibration was within ±17%, permitting its use in preclinical and clinical applications. Since authentic metabolite standards are not required under the concept of radio and stable isotopologues using adapted LC-RAD/MS protocols, significantly fewer resources are required to support accurate metabolite quantitation which in turn enables efficient analysis of simple and complex metabolite profiles.