Regioisomeric and enantiomeric analysis of primary triglycerides in human milk by silver ion and chiral HPLC atmospheric pressure chemical ionization-MS

Abstract

Triglycerides (TG) not only provide energy for infants but have important physiological functions. Understanding the composition and structure of TG in human milk is conducive to the development of infant formulas. In this study, TG species in human milk from 3 provincial capitals (Zhengzhou, Wuhan, and Harbin) in different regions of China were determined through C18 HPLC electrospray ionization tandem mass spectrometry (MS). The results showed that in human milk from these 3 regions, oleoyl-palmitoyl-linoleoylglycerol (OPL; 16.55, 19.20, and 18.67%, respectively) was more abundant than oleoyl-palmitoyl-oleoylglycerol (OPO; 10.08, 10.22, and 12.03%, respectively). Subsequently, regioisomeric and enantiomeric analysis of main TG in the human milk were performed on silver ion and chiral HPLC atmospheric pressure chemical ionization mass spectrometry (APCI)-MS, respectively. The results showed that rac-OPL (above 85%), rac-OPO (above 85%), rac-palmitoyl-oleoyl-oleoylglycerol (PPO; above 90%), and rac-OLaO (above 70%) were the main regioisomers of OPL, OPO, PPO, and lauroyl-oleoyloleoylglycerol (LaOO), respectively. The relative ratios of enantiomer pairs of rac-OPL (rac-OPL1 and rac-OPL2) were about 37 and 63%, respectively.

Bovine Milk Triacylglycerol Regioisomer Ratio Shows Remarkable Inter-Breed and Inter-Cow Variation

“…The ratio of OPO vs. OOP is not equal in biological materials. While OPO is the dominant isomer in human milk (75–93%), the proportion of OPO is much lower (<60%) in bovine milk [ 9 , 10 , 11 , 12 ]. Being the most important commercial milk worldwide, bovine milk is used as a popular food ingredient, a common infant formula ingredient, as well as directly consumed as staple drink.…”

“…For example, Kalo et al [ 16 ] reported that normal-phase HPLC was able to separate AAB/ABA regioisomers (i.e., TAGs containing two types of FAs) with one or two short acyl (C4 or C6) chain, whereas most reversed-phase columns can only resolve regioisomers containing one double bond (DB), such as POP and PPO pair, but are unable to separate OPO/OOP pair, which contain two DBs [ 17 ]. Until now, only Ag + -HPLC-ELSD, in combination with a reversed-phase LC pre-fractioning in most cases, have been used successfully for the separation and quantitation of OPO/OOP in human milk and infant formula samples [ 10 , 12 , 18 ]. A simpler and a higher throughput method is still lacking.…”

Comparative Analysis of Triglycerides From Different Regions and Mature Lactation Periods in Chinese Human Milk Project (CHMP) Study

“…On the contrary, the share of the low-/mediate-molecular-weight TAGs was higher than that in mature milk ( 8 , 9 ). In addition, the differences of the TAGs of human milk collected from different cities were also found by several groups ( 9 – 11 ). It is common knowledge that the infants were recommended to provide human milk in the first 6 months of lactation; therefore, the composition of human milk during this period will provide more comprehensive information for designing of infant formula.…”

“… a B, butanoic acid; Ca, capric acid; La, lauric acid; M, myristic acid; Pe, pentadecanoic acid; P, palmitic acid; Po, palmitoleic acid; S, stearic acid; O, oleic acid; L, linoleic acid; Ln, linolenic acid; A, arachidic acid; E, Eicosenoic acid; B′, Behenic acid; –, unknown . b References for each country: Spain: Pons et al ( 15 ); Morera et al ( 4 ); Isabel Ten-Doménech et al ( 14 ); Denmark: Zou et al ( 5 ); America: Kim et al ( 16 ); China (previous studies): Tu et al ( 10 ); Yuan et al ( 9 ); Chen et al ( 11 ). Finland: Fabritius et al ( 17 ) .…”

“… b References for each country: Spain: Pons et al ( 15 ); Morera et al ( 4 ); Isabel Ten-Doménech et al ( 14 ); Denmark: Zou et al ( 5 ); America: Kim et al ( 16 ); China (previous studies): Tu et al ( 10 ); Yuan et al ( 9 ); Chen et al ( 11 ). Finland: Fabritius et al ( 17 ) .…”