Quantification of Whole Blood Short-chain Fatty Acids by Gas Chromatographic Determination of Plasma 2-chloroethyl Derivatives and Correction for Dilution Space in Erythrocytes

A precise, accurate and stable method for quantification of short-chain fatty acids (SCFA) in ovine whole blood is presented and validated. The method is based on esterification of plasma SCFA by reaction with chloroethyl chloroformate in a water/acetonitrile/2-chloroethanol solution and gas chromatographic (GC) analysis of the derivatives. Whole blood concentrations of SCFA could be obtained by correcting plasma concentrations for a 45% dilution space of SCFA in the erythrocyte fraction of the blood. The recovery of SCFA in plasma and whole blood was 96-100% independent of the haematocrit value when compared with water standards. The method avoided carry-over from sample to sample, contrary to earlier published methods. The average intra-assay and inter-assay coefficient of variation for repeated measurement of SCFA content in plasma samples was 2.5% and 3.1%, respectively. The derivative was found to be suitable for a precise and accurate determination of the ¹³C enrichment of blood acetate by gas chromatography/isotope ratio mass spectrometry (GC/IRMS). The recovery of acetate added to water, plasma or whole blood was estimated as 99±1% by isotopic dilution of 2-[¹³C]acetate.