Quantification of 1,3-olein-2-palmitin (OPO) and Palmitic Acid in -2 Position of Triacylglycerols in Human Milk by Liquid Chromatography Coupled with Mass Spectrometry

This study describes the identification and quantification of fatty acids in the -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 -2 position of TAG (81.4 and 81.8 g 100 g 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.