Development of a HPLC-UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation.
A high performance liquid chromatography (HPLC) method was developed for the determination of seven short-chain fatty acids in equine caecal liquor. Samples were cleaned up on a Sep-pak (C18) cartridge, and the analyte was eluted from the extraction cartridge and filtered through a 0.45 micron cellulose nitrate filter. The analyte was chromatographed by ion exchange HPLC. Detection was by UV at 210 nm. Recovery from phosphate buffer (0.05 M, pH 7.0) and equine caecal liquor was 76.95% (lactic), 76.76% (valeric). The limit of (propionic), 89.35% (isobutyric), 88.73% (butyric), 80.33% (isovaleric) and 72.61% (valeric). The limit of detection of the short-chain fatty acids in phosphate buffer was 0.00006 M (lactic), 0.0001 M (acetic), 0.0002 M (propionic), 0.0001 M (isobutyric), 0.0002 M (butyric), 0.0002 M (isovaleric) and 0.0003 M (valeric). The specificity and sensitivity of this method was sufficiently high to allow the characterization of the pattern of these short-chain fatty acids in equine caecal liquor following intravenous administration of oxytetracycline at the recommended dose rate in a pony.