High-Resolution Ion Mobility Approach for Rapid Determination of Multiple Human Milk Oligosaccharide Isomer Ratios

Abstract

This study presents a high-throughput ion mobility approach that integrates flow injection analysis (FIA) with multiple stitched high-resolution ion mobility ranges to enable comprehensive analysis of human milk oligosaccharide (HMO) isomer mixtures in a single acquisition. The developed FIA-SIM²-stitching-MS approach provides rapid, reproducible, and accurate determination of multiple HMO isomer ratios (R_iso) with good performance in both positive and negative ion modes. Reliable R_iso values were obtained using seven-level regression-based calibration. Application of the method to two individual breast milk samples revealed distinct HMO compositional profiles, likely reflecting phenotypic differences, i.e., secretor versus nonsecretor. Our validated analytical method demonstrates strong potential for future applications in oligosaccharide characterization and food product quality control.