A comprehensive and accurate determination of free fatty acids (FFA) is required for fecal metabolomic investigations. The present study compares three aqueous extraction methods (1) ULTRA-TURRAX(®), (2) whirl mixing and (3) basic ULTRA-TURRAX extraction of fecal FFA with a direct derivatization approach using ethyl chloroformate as the derivatization reagent before determination by gas chromatography-mass spectrometry. The direct derivatization method resulted in significantly higher estimations (P < 0.01) of short- and long-chain fatty acids than was the case when applying the aqueous extraction methods using ULTRA-TURRAX, whirl mixing, or basic ULTRA-TURRAX extraction before the derivatization step. Thus, avoiding an aqueous extraction before derivatization reduces the loss of volatile short-chain FFA and the less water-soluble long-chain FFA.
