A Derivatization-Free GC-MS Method for Reliable Quantification of Short-Chain Fatty Acids in Sparus aurata

Background/Objectives: This study presents a gas chromatography-mass spectrometry (GC-MS) method for the derivatization-free quantification of the three major short-chain fatty acids (SCFAs)-acetate, propionate, and butyrate-in gilthead sea bream (Sparus aurata) fecal samples. SCFAs are key metabolites involved in intestinal physiology and are widely recognized as indicators of gut microbiota activity and host health across animal species. In aquaculture research, SCFA profiling can provide valuable insight into diet-microbiota interactions and gut functional status. Methods: The method was developed and optimized using gut-content samples collected from 105 gilthead sea bream at the end of two independent feeding trials, yielding 42 pooled samples (18 from FT1 and 24 from FT2). An extraction protocol based on acidified aqueous extraction with water followed by liquid-liquid extraction using methyl tert-butyl ether was developed (ExA) and subsequently optimized (ExB) to improve extraction consistency and efficiency. SCFA separation and quantification were performed by GC-MS using external calibration. Method validation included evaluation of linearity, matrix effects, and partition coefficients between water and methyl tert-butyl ether. Results: The optimized extraction method (ExB) demonstrated high analytical performance, with linearity coefficients exceeding 0.992 and matrix effects below 17.4%. Conclusions: The proposed method represents a practical and reliable approach for routine SCFA analysis in fish fecal samples. It provides a useful analytical tool for studies investigating diet-microbiota interactions, gut health, and functional responses to nutritional interventions in aquaculture species.