Development of a Widely Targeted Lipidomics Method for Precise Quantification of Human Milk Phospholipids Using HILIC-Triple Quadrupole-MS

Abstract

Human milk (HM) phospholipids (PLs) are essential bioactive components that support infant nutrition and development. To comprehensively characterize the composition and structural features of HM PLs and facilitate accurate comparisons with infant formula, a widely targeted hydrophilic interaction chromatography-triple quadrupole mass spectrometry (HILIC-TQ-MS) platform was developed for high-coverage analysis and precise quantifications. Systematic optimization of chromatographic conditions enabled rapid baseline separation of 10 PL subclasses within 8 min, with significantly improved peak shapes for acidic PLs. The method resolved fatty acyl positional isomers of glycerophospholipids.and provides a novel approach for the identification of isobars. Using this platform, 150 phospholipid molecular species were quantified across a wide dynamic range, with LOQs as low as 1 μg/L and RSDs of less than 4.8%, demonstrating excellent sensitivity, robustness, and suitability for high-throughput analysis. Application to HM samples across different lactation stages provided a reliable analytical framework for investigating dynamic changes in PL composition.

Keywords: human milk, Phospholipids, Hydrophilic interaction chromatography-tandem mass spectrometry, Targeted phospholipidomics