Abstract
This study describes the identification and quantification of fatty acids in the sn-2 position of triacylglycerols (TAG) and of the most abundant TAG regioisomers in human milk by liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS). Over 300 individual TAG species were observed and 1,3-olein-2-palmitin (OPO) was identified as the most abundant TAG regioisomer. Validation of the HPLC-HRMS method showed repeatability and intermediate reproducibility values ranging from 3.1 to 16.6% and 4.0 to 20.7%, respectively, and accuracy ranging from 75 to 97%. Results obtained by the HPLC-HRMS method were comparable to results from the ISO 6800 method for the quantification of palmitic acid in the sn-2 position of TAG (81.4 and 81.8 g 100 g−1 total palmitic acid, respectively). Processing the data obtained with the HPLC-HRMS method is extremely time consuming and, therefore, a targeted method suitable for the quantification of OPO in human milk samples by ultra-performance (UP) LC coupled with triple quadrupole (QQQ) MS was developed and validated. OPO identification and quantification by UPLC-QQQ were based on nominal mass and a fragmentation pattern obtained by multiple reaction monitoring experiments. The method was validated in terms of accuracy and precision by analyzing different aliquots of the same human milk sample over time and comparing the results with values obtained by HPLC-HRMS. Intermediate reproducibility was <15% and trueness comparable to HPLC-HRMS. Quantification of OPO in human milk samples collected at 30, 60 and 120 days postpartum showed that OPO content varies between 333 ± 11.8 and 383 ± 18.0 mg 100mL−1.
Highlights
Human milk is considered the optimal form of nutrition for infants and is the main food for a healthy infant during the first 4–6 months of life [1]
The synthesis of saturated fatty acids (FA) is influenced by diet quality and it has been demonstrated that the levels of 6:0–14:0 FA in milk are increased when lactating women consume diets rich in carbohydrate [6,7,8], whereas the content of 18 carbon FA which are derived from the diet, e.g., 18:1 n-9 and 18:2 n-6, are reduced [6,7]
When palmitic acid is in the sn-2 position, it is well absorbed as 2-monoacylglycerol, but when located in the sn-1,3 positions, it is released as free FA and forms insoluble calcium soaps lowering the efficiency of fatty acid and calcium absorption and increasing the stool hardness [12,13]
Summary
Human milk is considered the optimal form of nutrition for infants and is the main food for a healthy infant during the first 4–6 months of life [1]. In TAG, FA esterified in the external positions of the glycerol are named sn-1(3) or alpha FA while FA esterified in the internal position are named sn-2 or beta FA. Among polyunsaturated FA, linoleic (LA, 18:2 n-6) and alpha linolenic acids (ALA, 18:3 n-3) are essential because they are not synthesized in the human body, and are mainly obtained through the diet. They are precursors of arachidonic (20:4 n-6) and docosahexaenoic (22:6 n-3) acids, which are associated with normal brain development especially in early life [9]
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