Simultaneous determination of neutral and acidic human milk oligosaccharides (HMOs) by liquid chromatography with tandem mass spectrometry (LC-MS/MS)

Human milk oligosaccharides (HMOs) are a complex category of glycans present in high abundance in human milk, with its concentration and composition closely associated with infant health. Health benefits of HMOs include growth promotion of beneficial bacteria such as Bifidobacterium and inhibition of pathogenic agent adhesion to epithelial cell receptors. Ideally, the development of a robust, sensitive, and fast quantitative test method for key HMO species in human milk is desired for human clinical studies on HMOs, to further understand their benefits, related mechanisms and synthesis in relation to maternal phenotypes. Quantitative analysis of HMOs in human milk has been challenging due to the complex sample matrix, multiple components of HMOs and structurally similar isomers such as LNT and LNnT. In this study, a method has been successfully developed for the quantification of two pairs of neutral HMOs - namely the 2’-FL, 3-FL pair and LNT, LNnT pair, as well as a pair of acidic HMOs - 3’-SL and 6’-SL. Using ultra high-performance liquid chromatography (UHPLC) system coupled with Waters XevoTM TQ-XS Tandem Mass Spectrometer (LC-MS/MS), these three pairs of HMOs can be resolved either by chromatographic separation or unique fragment ions (MRMs). This analytical method has demonstrated excellent method performance including simple and fast sample preparation, short total run time, simultaneous determination of neutral and acidic HMO species, and unique MRM to separate and quantify the structurally similar isomers such as LNT and LNnT. This developed method was successfully validated and applied for the determination of the six HMOs in eleven individual human milk samples, with further potential to be used as a reference for food product quality control.