Conversion Reaction of Stable-Isotope Oxygen Labeling of Carboxylic Acids for Accurate Screening LC-MS/MS Assay: Application of Behavioral Changes of Short-Chain Fatty Acids in Sports Athletes under Exercise Loading

Short-chain fatty acids (SCFAs) have attracted considerable interest as potential biomarkers, therapeutic targets, and nutritional factors in athletic training. SCFAs are typically produced by the intestinal microbiome and exhibit various structural forms, including linear- and branched-chain types. In particular, branched-chain SCFAs have been associated with muscle metabolism during exercise loading. Consequently, accurate and efficient analytical methods are essential for identifying these biomarkers. Liquid chromatography–tandem mass spectrometry is a suitable and accurate technique for SCFA analysis; however, stable isotope calibrations are required for all analytes. Because of technological limitations, the available species are restricted to certain types of SCFAs. To address this issue, this study performed a simple conversion reaction involving the incorporation of ¹⁸O into the carboxyl group. Specifically, oxygen atoms in the carboxyl groups were substituted with ¹⁸O sourced from commercially available H₂¹⁸O. An SCFA mixture standard solution was successfully labeled under optimized conditions, and the SIL purity and amount were sufficient for isotope dilution (95.2–96.9%, 250 assays using 10 μL of H₂¹⁸O). Moreover, no reversion to ¹⁶O was observed during storage or analysis. Analytical validation was performed in human serum using the substituted isotopic standard mixture, achieving good accuracy (90–110%) and precision (<10% relative standard deviation) across three concentration levels. Finally, changes in SCFA patterns were examined in athletes during exercise loading.