Quantitative method for intestinal short chain fatty acids based on stable isotope labeling combined with liquid chromatography-mass spectrometry

Abstract

Short chain fatty acids (SCFAs) are produced from the breakdown of dietary proteins and fiber by gut microbes, and they have a close relationship with the health and diseases of the host. However, due to the similar structures of SCFAs, the abundance of active molecules, the wide concentration range in biological samples, and the characteristics such as high polarity, poor chromatographic separation, and ionization performance, it is challenging to comprehensively and accurately quantify SCFAs. This study utilized a stable isotope-labeled carboxyl derivatization reagent d₀-/d₆-2,4-dimethoxy-6-piperazin-1-yl pyrimidine (d₀-/d₆-DMPP) to establish a new method for the wide-coverage quantification analysis of SCFAs using ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS), capable of detecting the content of 16 SCFAs. The method demonstrated low limits of detection (LODs) of 0.05–0.5 nmol/L and limits of quantification (LOQs) of 0.1–1.0 nmol/L, with excellent linearity (R² > 0.99), intra-day precision (RSD < 8.5 %), and inter-day precision (RSD < 7.8 %). Using this quantitative analysis method, we successfully quantified 16 SCFAs from the colonic contents of rats with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ulcerative colitis (UC) and Sini decoction (SND) intervention. It was found that after the interventional treatment with SND, the levels of 7 SCFAs in the colonic contents of rats with UC were significantly up regulated, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, 2-methylbutyric acid, and hexanoic acid, while 4 SCFAs were significantly down regulated, including 3-hydroxyisovaleric, 3-methyl-2-oxobutanoic acid, 3-methyl-2-oxovaleric acid, and 4-methyl-2-oxovaleric acid. These findings suggested that SND might exert its therapeutic effect on UC by regulating the metabolism of SCFAs. Overall, this study not only provides a new method for the analysis of SCFAs with high sensitivity and wide-coverage but also offers important scientific evidence for understanding the mechanism of SND against UC.