Comprehensive and quantitative profiling of lipid species in human milk, cow milk and a phospholipid‐enriched milk formula by GC and MS/MS^ALL

Abstract

Here we present a workflow for in-depth analysis of milk lipids that combines gas chromatography (GC) for fatty acid (FA) profiling and a shotgun lipidomics routine termed MS/MSALL for structural characterization of molecular lipid species. To evaluate the performance of the workflow we performed a comparative lipid analysis of human milk, cow milk, and Lacprodan® PL-20, a phospholipid-enriched milk protein concentrate for infant formula. The GC analysis showed that human milk and Lacprodan have a similar FA profile with higher levels of unsaturated FAs as compared to cow milk. In-depth lipidomic analysis by MS/MSALL revealed that each type of milk sample comprised distinct composition of molecular lipid species. Lipid class composition showed that the human and cow milk contain a higher proportion of triacylglycerols (TAGs) as compared to Lacprodan. Notably, the MS/MSALL analysis demonstrated that the similar FA profile of human milk and Lacprodan determined by GC analysis is attributed to the composition of individual TAG species in human milk and glycerophospholipid species in Lacprodan. Moreover, the analysis of TAG molecules in Lacprodan and cow milk showed a high proportion of short-chain FAs that could not be monitored by GC analysis. The results presented here show that complementary GC and MS/MSALL analysis is a powerful approach for characterization of molecular lipid species in milk and milk products.Practical applications: Milk lipid analysis is routinely performed using gas chromatography. This method reports the total fatty acid composition of all milk lipids, but provides no structural or quantitative information about individual lipid molecules in milk or milk products. Here we present a workflow that integrates gas chromatography for fatty acid profiling and a shotgun lipidomics routine termed MS/MSALL for structural analysis and quantification of molecular lipid species. We demonstrate the efficacy of this complementary workflow by a comparative analysis of molecular lipid species in human milk, cow milk, and a milk-based supplement used for infant formula.

In-Depth Lipidomic Analysis of Molecular Species of Triacylglycerides, Diacylglycerides, Glycerophospholipids, and Sphingolipids of Buttermilk by GC-MS/FID, HPLC-ELSD, and UPLC-QToF-MS

“…The monounsaturated FA (MUFA) (23%) content was predominantly oleic acid (C18:1c9, 17%), whereas polyunsaturated FA (PUFA) was low (~1% on total FAME) and mainly accounted for by linoleic acid (LA, C18:2 c9, c12). These results are similar to those reported in previous studies in a commercial BM samples (Sokol et al [18]; Kristensen et al [19]).…”

“…The same species of PE were identified in the negative ionization mode from [M − H] − ions. These results were comparable with those achieved by Sokol et al [18]. …”

“…The abundance of PC (14:0/16:0) species, which reaches around 12%, is also remarkable (Figure 3). Similar results were reported by Sokol et al [18] from a commercial BM where the predominant PC species were (16:0/18:1), (16:0/16:0), and (18:1/18:1), accounting for 25%, 11%, and 5%, respectively. Although the PC species are currently analyzed in positive mode due to the presence of the quaternary nitrogen atom with a positive charge in the choline head group, analysis in negative mode could also be carried out in the presence of a negative charge in the phosphate group and the loss of a methyl group.…”

“…It is also important to note that TAGs containing C16:0 but also C14:0 are diverted towards energetic pathways as these acyl chains are known to be the preferred acyl chains for mitochondrial β-oxidation, whereas those species containing C18:0 and C16:0 are the preferred substrate used by glycerol-3-P for de novo PL synthesis in the Kennedy Cycle [34,35]. Regarding the TAG polyunsaturated species, the most abundant were CN48:2 (16:0/16:1/16:1 and 16:1/18:1/14:0) and CN54:2 (18:0/18:1/18:1), whose abundances were 1.5%, 2.3%, and 1.5%, respectively, as has been also described by Sokol et al [18] in a commercial BM using MS/MS.…”

Mass Spectrometric Collisional Activation and Product Ion Mobility of Human Serum Neutral Lipid Extracts

“…11,12 In such studies ions are sequentially separated by an initial mass analyzer followed by collisional activation with measurement of all product ions for that mass. This process is repeated for the entire mass range of interest.…”