Gas Chromatography–Mass Spectrometry-Based Analyses of Fecal Short-Chain Fatty Acids (SCFAs): A Summary Review and Own Experience

Abstract

The gut microbiome, crucial to human health, changes with age and disease, and influences metabolic profiles. Gut bacteria produce short-chain fatty acids (SCFAs), essential for maintaining homeostasis and modulating inflammation. Dysbiosis, commonly due to poor diet or lifestyle, disrupts the integrity of the intestinal barrier and may contribute to conditions such as obesity, diabetes, and non-alcoholic fatty liver disease (NAFLD). Analytical methods such as gas chromatography–mass spectrometry (GC/MS) are vital for SCFA analysis, with various preparation and storage techniques improving the accuracy. Advances in these methods have improved the reliability and sensitivity of SCFA quantification, which is crucial for the identification of disease biomarkers. Evidence from GC/MS-based studies has revealed that accurate SCFA quantification requires meticulous sample preparation and handling. The process begins with the extraction of SCFAs from biological samples using methods such as direct solvent extraction or solid-phase microextraction (SPME), both of which require optimization for maximum recovery. Derivatization, which chemically modifies SCFAs to enhance volatility and detectability, is a crucial step, typically involving esterification or silylation. Following this, the cleanup process removes impurities that might interfere with the analysis. Although recent advances in GC/MS technology have significantly improved SCFA-detection sensitivity and specificity, proper sample storage, with acid preservatives and the avoidance of repeated thawing, is essential for maintaining SCFA integrity.