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 (CC) 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 C-13 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-[C-13]acetate.