Quantitative analysis of short chain fatty acids in biological sample by high performance liquid chromatography-tandem mass spectrometry

Abstract

Short chain fatty acids (SCFAs) with fewer than six carbon atoms are primary metabolites produced in large bowel by bacterial activity. SCFAs are the main fuel for the coloncytes which make them important for preventing gastrointestinal disorders. The recent studies of SCFA have shown they are related to various diseases such as cancer, obesity, and irritable bowel syndrome. More recent studies suggest SCFA affects not only metabolism but also psychological functioning via microbiota-gut-brain communication. Herein, the qualitative and quantitative analysis of SCFA in liquid chromatography-tandem mass spectrometry (LCMS/ MS) is presented. Nine SCFA were chosen as target analytes ranging from acetic acid to hexanoic acid. The pre-sample treatment utilized 3-nitrophenylhydrazine (3-NPH) as a derivatizing reagent and the catalysts for the reaction were n-(3-dimethylaminopropyl)-n′-ethylcarbodiimide and pyridine. The stable isotopic derivatization agent (13C6-3-NPH) was used as the internal standard sample for better quantification. The chromatographic separation was achieved by a C18 reverse phase column in both HPLC-Q-TOF and UPLC-LTQ instrument. The instrumental setting allowed the limit of quantification of 10 pmole for hexanoic acid and lower values for other SCFAs. The sample stability was confirmed by both intra- and inter-day tests and good linearity with a minimum of 0.99 was observed in samples between 10 pmole and 1000 pmole. By this analytical method, the SCFAs from mice fecal and bacterial cell culture were successfully quantified.