Rapid and Accurate Simultaneous Determination of Seven Short-Chain Fatty Acids in Feces by Gas Chromatography - Mass Spectrometry (GC-MS): Application in Type 2 Diabetic Rats and Drug Therapy

Short-chain fatty acids (SCFAs) are carboxylic acids that contain fewer than six carbons and are the metabolites of the intestinal flora. SCFAs participate in almost all circulatory system components and regulate the physiological functions of an organism. In addition, these compounds are recognized to be important components in gut homeostasis and metabolic disease occurrence. The comprehensive analysis of SCFAs in feces is challenging due to their large polarity differences, wide concentration range and the complex matrix. Most of the existing analytical methods require complicated derivatization operations, which require reagents that harm both humans and the environment. In this study, we have optimized pretreatment conditions and developed a simple, rapid, and safe method to accurately quantify seven common SCFAs in feces by gas chromatography - mass spectrometry (GC-MS). In brief, the feces samples were freeze-dried, extracted with saturated sodium chloride solution, acidified with sulfuric acid, and extracted with ethyl acetate. Following dehydration with anhydrous sodium sulfate, the supernatant was harvested and analyzed by GC-MS. The methodological investigation results showed that the method has excellent linearities with determination coefficients (R-2) exceeding 0.99. In addition, the recovery values, matrix effects, precision, and stability of the method meet the requirements for biological sample analysis. Seven primary SCFAs, including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid, were identified and quantified in the feces samples. In conclusion, a rapid and accurate method was developed for the simultaneous determination of seven SCFAs in feces that was successfully applied to type 2 diabetic (T2DM) rats and drug therapy.