Regioisopecific analysis of triacylglycerols in mammalian milk with ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC− ESI− MS/MS)

Mammalian milk fats are composed of over 95% triacylglycerols (TAGs). TAG regioisomers, which refer to the different stereochemical numbering positions (sn-1/3 or sn-2) of fatty acids (FAs) on the glycerol backbone of TAG molecules, influence the biochemical properties of milk fat. Human milk fat is especially rich in TAGs with palmitic acid in sn-2 position, which contributes to the growth and wellbeing of infants. For the optimal absorption of fat and calcium, the molecular structure of fats in infant formulas should be similar to human milk fat. However, TAG species vary among fat sources. There is a lack of thorough understanding of the complex profile of TAG regioisomers in milk of human and other mammalian species. Therefore, creating comprehensive profile of TAG regioisomers in mammalian milk fats would contribute to a better understanding of the similarities and differences in TAG composition among different species. Such knowledge is crucial for developing the next generation human milk fat substitute.

In this study, TAG regiosiomers of milk samples of seven mammalian species including human, ruminants: cow, camel, goat, sheep, non-ruminants: horse and pig were analyzed with a previously developed ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry method (UHPLC−ESI−MS/MS) combined with a calculation algorithm. Totally 68 TAG molecular species including 1038 TAGs and 395 TAG regioisomer pairs or triplets were detected, with equivalent carbon number ranging from 26 to 54. Pig milk had the most similar TAG composition to human milk, while ruminants’ milk was different. The regioisomer compositions of horse milk were quite comparable with human milk, even though the molecular species was similar to ruminants. Interestingly, palmitic acid preferred sn-2 in horse, human and pig milk compared with ruminants. The in-depth molecular-level knowledge of TAG regioisomers provides a constructive reference for further investigations to optimize novel human milk fat substitutes.