Analysis of Short-Chain Fatty Acids/Organic Acids (3-NPH Derivatives) in Fecal Specimens from SPF and Antibiotic-Fed Mice-C168

Introduction

More than several hundred species of intestinal microbiota exist in the human intestines. Because they grow in colonies and have an appearance exactly like a field of flowers when observed under a microscope, they are also called intestinal flora. Intestinal microbiota produces so-called short-chain fatty acids, namely, acetic acid, propionic acid and butyric acid, by using indigestible saccharides as a main energy source. In recent years, reports have linked these short-chain fatty acids to autoimmune disease by absorption in the body, and to lifestyle-related diseases, such as obesity and diabetes. In order to understand the connection between the short-chain fatty acids and disease and gain a deeper understanding of the metabolic activity of both the intestinal microbiota and the host body, there is increasing demand for quantitative analysis of the short-chain fatty acids. Since short-chain fatty acids generally display high volatility and high hydrophilicity, LC/MS analysis by a conventional reverse-phased system is difficult. Moreover, although the derivatization method is widely used in the general GC/MS method, loss of volatile components is possible, as exsiccation of the specimen is necessary. For this reason, analysis is performed by derivatization in an aqueous solution. In this article, analysis was conducted by derivatizing carboxylic acid with 3- nitrophenylhydrazine (3- NPH), and matching the C2 to C5 short-chain fatty acids (6 components) and organic acids such as lactic acid, pyruvic acid, succinic acid (16 components). The analysis was performed by using the registered MRM transition and analysis method in the LC/MS/MS method package “Short-chain fatty acids.” Although the derivatization method using 2-nitrophenylhydrazine (2-NPH) has long been known, it was applied mainly to the analysis of fatty acids. In this study, derivatization of not only short-chain fatty acids but also organic acids was performed with high efficiency by using 3-NPH rather than 2-NPH, supporting an analysis by matching short-chain fatty acids and organic acids. The registered MRM transitions were optimized based on the 3-NPH derivative. Because 3-NPH also reacts with ketone bodies, MRM transitions derivatizing not only carboxylic acid but also the carbonyl group were set for pyruvic acid, oxaloacetic acid and other acids having a carbonyl group. Here, analysis of the short-chain fatty acids and organic acids was performed using fecal specimens from SPF (Specific Pathogen Free) mice, in which physiological intestinal microbiota existed, and mice in which the level of intestinal microbiota was remarkably reduced by administration of an antibiotic. After collecting and weighing fecal specimens from the respective mice, the specimens were suspended in ethanol and the supernatant was recovered centrifugally separation. This supernatant was subjected to derivatization by 3-NPH. The derivative was then reacted at room temperature for 30 min using pyridine as a catalyst and carbodiimide as a condensing agent. After the reaction, the product was diluted with a methanol solution containing formic acid, and simultaneous analysis was performed with a LCMS-8060.

May 14, 2018 GMT