Simultaneous GC-MS Determination of Unsubstituted, Hydroxy-, Amino-, and Hydroxy-amino Short-Chain Fatty Acids Together with Amino Acids in Biological and Food Samples Following Derivatization.

The simultaneous determination of short-chain fatty acids (SCFAs) and amino acids is of increasing interest, as these metabolite classes are important indicators of host metabolism and gut microbiota activity. They also serve as biomarkers for disease diagnosis and indicators of food composition. In this study, a gas chromatography-mass spectrometry method was developed for the simultaneous determination of both substituted (hydroxy-, amino- and hydroxy-amino-) and unsubstituted SCFAs, alongside amino acids, following derivatization and liquid-liquid extraction. Isobutyl chloroformate was employed as the derivatization reagent. The proposed method demonstrated excellent linearity, with coefficients of determination (R2) ranging from 0.9840 to 0.9992. Method limits of detection ranged from 0.011 to 7.2 μg/mL, while method limits of quantification ranged from 0.033 to 8.0 μg/mL. Intra-day precision (%RSD) ranged from 1.0% to 6.5% for biological samples and from 1.1% to 4.5% for eggs. Inter-day precision (%RSD) ranged from 1.0% to 5.6% for biological samples and from 2.0% to 6.7% for eggs. Matrix effects ranged from 98% to 108% for biological samples and from 96% to 113% for egg, while recoveries ranged from 90% to 111% and from 95% to 108%, respectively. To the best of our knowledge, this is the first GC-MS method enabling the simultaneous determination of substituted and unsubstituted SCFAs together with amino acids in a single analytical procedure. The proposed approach provides a reliable and versatile platform for metabolomics, microbiome research, clinical investigations, and food analysis, and can readily be extended to a broader range of target metabolites.