Sensitive and high-throughput isomer-specific analysis of human milk oligosaccharides using UPLC-MS/MS and its...

Human milk oligosaccharides (HMOs) are now the principal component of the latest infant formula generation. However, it is challenging to separate and quantify highly heterogeneous isomers when analyzing HMOs. Here, we developed a high-throughput isomer-resolved quantification method for 21 native HMOs based on ultrahigh-performance liquid chromatography-mass spectrometry-multiple reaction monitoring (UPLC-MS-MRM) technology. High-resolution cyclic ion mobility-mass spectrometry technology was applied to distinguish HMO isomers and identify specific ion pairs for quantification to solve the measurement challenge of HMOs without available standards. Simultaneously, nine HMO standards were used to create universal calibration curves to quantify those without standards. This MRM approach features wide isomer coverage (seven series of isomers), short analysis time (12 min), high sensitivity (ng/L level detection limit), and a broad 5-order-of-magnitude quantitation range. After that, the technique was used on 58 Chinese human milk samples, which showed that the three genotype milk groups had different HMO profiles at the isomer level. Four crucial fucosylated HMOs were identified as markers to rapidly assign secretor status. Moreover, correlations analysis unveiled a co-regulatory relationship among Fuc-(α1-3/4), Fuc-(α1-2), and sialylated glycanforms. Our research offers a comprehensive solution for isomeric glycome profiling, and provides a crucial reference for designing precise nutrition formula for infants.