Accurate and reliable quantitation of short chain fatty acids from human feces by ultra high-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS).

Short chain fatty acids (SCFAs), generated from microbial fermentation of dietary fibers, can regulate weight, appetite and energy homeostasis. Therefore, measuring SCFAs in fecal samples is important to understand the relationship between dietary patterns, gut microbial metabolism, and their impact on host metabolism homeostasis. However, due to the chemical complexity of fecal samples and the volatility of these SCFAs, the quantitative measurements of SCFAs remain challenging. In this study, we developed an absolute quantitation method for accurate and reliable analysis of SCFAs using an UPLC-Q Exactive HRMS system. Nine C2-C6 SCFAs were first derivatized and then separated on a reversed-phase CSH C18 column, and quantitated by UPLC-HRMS with targeted-selected ion monitoring (t-SIM) mode. Our calibration plots showed high linearity (R2>0.99) with high quantitation accuracy (from 91.24%-118.42%); additional analyses showed excellent precisions ranging from 1.12 % to 6.13 %, and accurate recoveries between 92.38 % and 109.65 % with relative standard deviations of 0.31 %-6.44 %. Meanwhile, the short-term stability, freeze and thaw stability, and 168 h storage stability were tested and reported from 85.07%-106.44% with RSDs 0.44%-20.00%, 98.99%-128.84% with RSDs 0.77%-19.79%, and 77.53%-104.42% with RSDs 0.92%-18.65%, respectively. Lastly, this quantitative method was applied to determine the SCFA concentrations and compositions in forty fecal samples from a group of study subjects participating in an obesity prevention trial, and a broad range of concentrations was noted for the detected SCFAs.