Analysis of volatile fatty acids in biological specimens by capillary column gas chromatography

A method was developed to analyze and quantify volatile fatty acids (VFA) such as acetic, propionic, butyric, isobutyric, valeric and isovaleric acids in biological specimens. To obtain good sample transfer into the chromatographic system an organic solvent had to be used together with an aqueous milieu, thus improving wetting properties of the liquid sample plug introduced into the column. Sample preparation was carried out under alkaline conditions in order to exclude or minimize sample losses due to sample transfer during the extraction and work-up procedure. A cold on-column injection was applied to avoid irregular discrimination of the various acids due to sample splitting and an automatic injection technique was used to accommodate the large number of samples generated from biological origin. Connection of a pre-column of wide internal diameter (0.53 mm) to the analytical column (0.32 m) was optimized and adapted to the nature of the injection solvent mixture consisting of acetonitrile, water and hydrochloric acid. To obtain well-separated and correctly quantifiable gas chromatographic peaks, it was essential to perform the chromatography under acidic aqueous conditions. Standard resolution conditions and response factors were evaluated. The chromatographic results of applying this method to biological specimens from both rats and humans are provided.