HPLC-MS/MS-Based Quantification of 25 Human Milk Oligosaccharides and their Structural Isomers: Method Development and Longitudinal Profiling in Chinese Breast Milk

Objective: This study established and validated a high-throughput HPLC-MS/MS method for the simultaneous quantification of 25 human milk oligosaccharides (HMOs), specifically targeting the separation of 8 complex isomeric groups, and characterized the longitudinal HMO evolution in human milk from Chinese lactating women. Methods: Chromatographic separation utilized an amide column with a mobile phase of 5 mM ammonium formate and 50 nM sodium formate, completing analyte elution within 17 min. Diagnostic fragments were captured using fragment-guided multiple reaction monitoring (MRM) and positive/negative ion switching to distinguish isomers. The method was applied to 160 longitudinal samples collected from 40 mothers across four lactation stages. Results: Analytical validation yielded spiked recoveries from 89.6% to 106.6%, with intra-day and interday precision (relative standard deviation, RSD) of 5.2% and 11.0%, respectively55. Calibration curves showed determination coefficients (R2) exceeding 0.996. Median total HMO concentrations decreased from 15.6 g/L in colostrum to 7.0 g/L at 12 months postpartum. Individual components showed distinct trends, with 3-FL increasing by 32% and 2'-FL decreasing by 68% over the lactation period. Conclusion: This HPLC-MS/MS method allows for the quantification of complex oligosaccharide mixtures in large-scale profiling. The results define the dynamic evolution of HMO profiles in Chinese lactating women, providing data to support research into infant health.