A new isopropyl esterification method for quantitative profiling of short-chain fatty acids in human and cow milk by gas chromatograph-mass spectrometer

Short-chain fatty acids (SCFA) content in milk may have been underestimated due to the neglect of the esterified SCFA content and the lack of an accurate detection method, especially for C1:0, C2:0, and C3:0 SCFA. In this study, an accurate GC-MS profiling method was established for 10 SCFA. A 2-step esterification, including alkaline saponification (60°C for 30 min) and acid-catalyzed esterification (80°C for 150 min) in water/isopropyl/hexane (1:2:1, volume ratio), was found to be the most suitable for the quantification of esterified and nonesterified SCFA analysis. The validation results demonstrate satisfactory linearity, sensitivity, matrix effects, precision, and accuracy. The recoveries of nonesterified and esterified SCFA ranged from 82.78% to 112.49%, respectively. Human milk is distinguished from cow milk by its higher C1:0 and C2:0 content and lower C4:0 and C6:0 content. This method successfully accomplished qualitative and quantitative estimation of all 10 SCFA in milk, including both nonesterified and esterified SCFA. Furthermore, whether our method is applicable for the determination of SCFA in serum, rumen fluid, and feces remains to be explored.