Establishment Of UPC~2-Q-TOF-MS Technology For Detecting Triglyceride And Its Application In Human Milk Fat Research

Human milk is the most ideal natural food source for infants,it can provide energy and essential nutrients for growth and development for infants.The fat in human milk is one of the most important energy-supplying nutrients for infants,its main constituents are triglycerides(TAGs)composed of fatty acids(FAs)and glycerol.The composition of human milk fat is affected by various factors,there are many studies on FAs,and the infant formula on the market is generally produced according to the type and content of FAs in human milk.However,in recent years,the study showed that compared with FAs,TAGs in human milk fat has a more direct effect on the growth and development of infant.It is necessary to strengthen the research on human milk fat TAGs.TAGs in human milk fat is one of the most complex lipids in nature,and the method on TAGs analysis is still a challenging work.On the basis of previous studies,this study improved the TAGs analysis method of human milk fat.Then,this method was used to analyze the composition of human milk fat between dietary patterns in the same area different and different lactation periods,and compared with that of infant formulas and raw materials.Firstly,ultra-high performance combined-phase chromatography combined with quadrupole time-of-flight mass spectrometry(UPC~2-Q-TOF-MS)was used to separate and identify the TAGs using the improved mode of the mass spectrometry(survey mode).The results showed that the types of human milk fat TAGs were more comprehensive,and a qualitative and quantitative analysis was performed for a parent ion corresponding to different fatty acid isomers.A total of 95 human milk fat TAGs were identified,twenty-four TAGs with a content of more than 1% were found,with the highest content being O-P-L.The contents of the three measured human milk samples were 18.08±0.63%,19.15±0.20% and 13.11±0.51%,respectively,followed by O-P-O(14.42±1.09%,16.59±0.47%,8.86±0.88%),and the content of O-O-L,O-L-L,and L-P-L is also 5% or more.This result is different from the O-P-O reported by other countries as the highest content of human milk fat TAGs,and has individual differences.Subsequently,the composition and content of different lactation periods milk fat TAGs in Wuxi,and mature milk fat TAGs in Shandong and Shanghai were determined by established UPC~2-Q-TOF-MS method.The total FAs and sn-2 FAs were also compared and analyzed.The results show that the major TAGs in human milk were composed of O-P-L,O-P-O,L-P-L,OO-L and O-L-L,and the content was above 5%.The differences of O-P-L and O-P-O are not significant between different lactation periods and different regions.The content of L-P-L,OO-L and O-L-L increased with the prolongation of lactation period.The content of TAGs containing oleic acid(O,C18:1)is more in Shandong than in Wuxi and Shanghai(such as OO-O),and the TAGs content of linoleic acid(L,C18:2)is less than that in the other two regions(such as L-L-L).With the prolongation of lactation,medium-sized carbon chain fatty acids(MCFAs,C8:0-C12:0)in FAs decreased,while long chain fatty acids(LCFAs,C16:0-C20:0)gradually increase.The total amount of saturated fatty acids(SFAs)of mature milk in Wuxi was lower than that in Shandong and Shanghai,and there were significant differences,and the total PUFAs were significantly higher than those in the other two regions.The difference of sn-2 FAs between different lactation periods and different regions is small,they were mainly SFAs,and the content is above 70%.Palmitic acid(P,C16:0)is the most important FAs,and the average content range is 54%-58%.Finally,the fat ingredients in commercially available infant formula,its raw materials,milk,goat milk,donkey milk,and yak milk were analyzed and compared with human milk fat ingredients.The results show that infant formulas and their raw oil usedhad similar composition of TAGs and FAs.Howvere they differed significantly from that of human milk,the main reason is that the total amount of SFAs,MUFAs and PUFAs in infant formula milk powder is close to human milk,but the specific types and location distribution of FAs are different.For example,the sn-2 FAs in infant formula milk is mainly C18:1 ?-9 and C18:2 ?-6,and the proportion of C16:0 is low,resulting in a different TAGs profile from that of human milk.The most prominent feature of several animal milk fats is that the types and contents of TAGs composed of FAs with short and medium carbon chains.Among them,donkey milk is the one most closely resembles human milk fat,which is mainly reflected in the content of O-P-O and O-P-L and the distribution of sn-2 FAs.However,the TAGs which long carbon chain saturated fatty acids(LCSFAs)in combination with the sn-1/3 position of several kinds of animal fat occupy a certain proportion,such as O-P-P and O-O-P,so it is not suitable as a long-term source of fat for infants.