Optimization and validation of direct gas chromatography-mass spectrometry method for simultaneous quantification of ten short-chain fatty acids in rat feces

Short-chain fatty acids (SCFAs) play key roles in maintaining health and treating disease. Quantification of important fecal SCFAs is necessary to facilitate the clarification of their biological roles. However, the existing quantifying methods mainly depend on complicated precolumn derivatization, and/or are unable to determine formic acid, a SCFA commonly associated with toxicity. In this study, a direct gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of ten SCFAs including formic acid in rat feces was developed. The approach was optimized in terms of chromatographic and spectrometric conditions as well as sample preparation. DB-FFAP capillary column with temperature programming was used to get baseline separation and symmetrical peak shape of SCFAs without precolumn derivatization in a relatively short running time (8 min). Multiple reaction monitoring (MRM) scan mode was employed to enhance the sensitivity and selectivity of SCFAs. Acidification with 50% HCl and immediate extraction with diethyl ether were utilized to achieve sample preparation of ten SCFAs from feces. Furthermore, the developed method was validated with wide linear range, high sensitivity and precision, low matrix effect and acceptable accuracy. The established method was successfully applied to compare the contents of fecal SCFAs between normal and immunosuppressed animal models.