Abstract: Objective · To establish a quantification method for understanding the varieties and concentrational changes of fatty acids under physiological and pathophysiological conditions. Methods · Based on isobutyl esterification using (-)-menthyl chloroformate and isobutanol, 27 typical fatty acids were qualitatively and quantitatively analyzedgas chromatography-flame ionization detector/mass spectrometry (GC-FID/MS). The sensitivity and stability of the method were detectedlimit of detection (LOD), limit of quantification (LOQ), and intra-day and inter-day relative standard deviation (RSD). The method was applied to four typical biological samples (human serum, urine, feces and rat liver) to verify the universality in different substrates. Results · Isobutyl esters of 27 fatty acids were effectively separated with an HP-5MS column. The results showed nice linearity within 2-3 orders of magnitude in terms of concentration with R2>0.99 for all 27 tested fatty acids. The LODs of the method were between 0.03 and 2.96 pmol on column whereas the LOQs were between 0.09 and 9.86 pmol. The results of method validation showed that the intra-day and inter-day RSDs were all under 10% in the range of high, medium and low concentrations. With this method, moreover, 10, 7, 14 and 9 fatty acids were detected in human serum, urine, feces and rat liver, respectively. Conclusion · A quantitative analytical method for fatty acids with short, media and long chains (C1-C24) is established based on isobutyl estrification in aqueous media. This method has the advantages of mild condition, high sensitivity, good stability, and simple and quick operation, and can be directly used in the detection of serum, urine, feces, liver and other biological samples, which may provide a new idea for high-throughput metabolome analysis.

Key words: fatty acid, isobutyl esterification, metabonomics, gas chromatography-flame ionization detector/mass spectrometry (GC-FID/MS)