Assay of the concentration and stable isotope enrichment of short-chain fatty acids by gas chromatography/mass spectrometry

Isotope dilution gas chromatographic mass spectrometric assays were developed for the concentration of shortchain fatty acids and of lactate in plasma. The assays involve spiking the samples with 2H‐ and/or 13‐C‐labeled internal standards and direct derivatization with 2,4‐difluoroaniline, using 1,3‐dicyclohexylcarbodiimide as a coupling agent. The assay can be used to determine the isotopic enrichment or mass isotopomer distribution of the fatty acids and of lactate. Blanks by ubiquitous formate, acetate and lactate are minimized by the one‐step derivatization procedure without deproteinization, using reagents dissolved in hydrocarbons. The assays were applied to experiments conducted in live dogs, pigs and monkeys, and in perfused rat livers. These techniques, in conjunction with appropriate stable isotope models, can be used to study carbohydrate fermentation in humans, ruminants and non‐ruminant mammals.