Rapid Determination of Triacylglycerols in Cow Milk and Goat Milk Using Supercritical Fluid Chromatography-Quadruple Time-of-Flight Mass Spectrometry

Triacylglycerol (TAG) is the main component in milk fat, which determines the quality of milk fat. TAG composition in milk is varied among different species, diet and lactation stages. In this work, method of supercritical fluid chromatography combined with quadruple time-of-flight mass spectrometry (SFC-Q-TOF-MS) was developed for determination of complex acylglycerols in cow milk and goat milk. Milk fat was extracted with n-hexane. The instrumental conditions was as follows: the stationary phase was BEH 2-EP column, the mixture of methanol/acetonitrile/formic acid (50∶50∶0.1, V/V/V) was chosen as the mobile phase additive, the flow rate was 1.0 m/L, the column temperature was 50 ℃, automatic back pressure regulator was 1.1×10⁴ kPa, the injection volume was 1 μL. The separation method was utilized to analyze commercial cow milk samples and goat milk samples. Further identification of TAG compounds was based on the exact mass information of quasi-molecular ions, and the fragment ions were provided by Q-TOF-MS. A total of 55 triacylglycerols (TAGs) and 16 diacylglycerols (DAGs) were separated and identified within 25 min, including some pairs of TAG isomers, such as P-M-Co and P-P-Bu, showing the fast and excellent separation of SFC. The SFC-MS data showed that there were four important TAG series existed in cow and goat milk fat, including P(O)-X-Bu, P-P-X, O-X-O and O-P-X, indicating the high amount of saturated fatty acid in cow and goat milk fat. Although there were similar main TAG series in cow milk and goat milk samples, the concentration of unsaturated fatty acid in goat milk was relatively higher than cow milk, showing that goat milk may be more suitable for manufacture infant formula. Furthermore, principal component analysis (PCA) was applied to process the multidimensional data in order to better differentiating cow milk and goat milk, and cow milk samples and goat milk samples were classified clearly in the score plot of PCA. Major differences were obtained, including some long chain TAGs and medium chain TAGs. The SFC-MS method is high-throughput and environment friendly, providing an alternative approach besides LC/MS and GC/MS for TAGs analysis in food or biological samples, and might has a prospective future in the lipidomics. Since pressure drop in SFC system is much smaller than liquid chromatography, which is beneficial for combining several columns in series, more attention might be concentrated on the development of 2-dimentional SFC system in the future in order to obtain a better separation.