Development of analytical methods for the quantification and enhanced detection of unsaturated short-chain FAHFAs

Short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs) comprise a novel class of endogenous lipids predominantly localized in the gut and play essential roles in metabolic and inflammatory regulation. Previous studies have enabled chemical synthesis and quantitative analysis of saturated SFAHFAs using validated analytical methods. However, the detection and quantification of unsaturated SFAHFAs remains challenging because of their synthetic complexity and inherently low abundance in biological matrices. In this study, a comprehensive in-house library comprising thirty isomeric unsaturated SFAHFAs was synthesized to facilitate the development of two LC-MS/MS based analytical approaches: (i) direct analysis without derivatization for reliable quantification and (ii) a sensitivity-enhanced method based on 2-(2-pyridyl) ethylamine derivatization. Liquid chromatography-tandem mass spectrometry was optimized for sensitive and selective quantification, demonstrating excellent linearity with limits of detection between 0.5 and 5 fmol and limits of quantification between 5 and 50 fmol. The non-derivatized method was used to profile fecal SFAHFAs in murine and human samples. In mice, exercise increased the levels of both saturated and unsaturated SFAHFAs, whereas an obesogenic diet selectively reduced saturated SFAHFA levels. By contrast, analysis of fecal samples from humans with mild cognitive impairment revealed no significant differences from those of healthy controls, suggesting potential disease- and matrix-specific variability. A chemical derivatization approach using 2-(2-pyridyl) ethylamine enhanced detection sensitivity by approximately 1000-fold with LOD between 0.0005 and 0.001 fmol and LOQ 0.005 and 0.01 fmol. This study presents an analytical approach for the synthesis, quantification, and improved detection of unsaturated SFAHFAs, establishing a foundation for the further exploration of their roles in metabolic disorders associated with gut dysbiosis.