LC-HR-MS/MS analysis of oligosaccharides in human milk

Experimental

Human milk oligosaccharides (HMO) were analyzed using the LC-MS system, which consisted of Dionex Ultimate 3000 RS HPLC coupled to a Q Exactive Plus hybrid FT mass spectrometer equipped with a heated electrospray ionization source (Thermo Fisher Scientific Inc.). The analytes were separated on HILIC-OH5 column (2.1×150mm, 2.7µm, Agilent) employing a linear gradient of acetonitrile (solvent B)/water with 20mM ammonium formate pH 4.3 (solvent A). The mass spectrometer was operated in negative ESI full scan and PRM (targeted MS/MS) modes. Ion source parameters were as follows: spray voltage 3.7 kV, capillary temperature 250⁰C, sheath gas rate (arb) 40, and aux. and sweep gas rates (arb) 10, aux. gas temp. 250⁰C. Mass spectra were acquired in the m/z 200-1500 Da range at resolving power 70,000 (scan mode) and 35,000 (PRM). The LC-MS system was controlled using Xcalibur software (Thermo Fisher Scientific Inc.).

By using the HILIC-5OH column not all HMOs were separated chromatographically. However, these isomeric compounds have distinctive MS/MS spectra and can be identified and quantified by different product ions. The nine HMOs namely, 2-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL) and disialyllacto-N-tetraose (DSLNT) were determined and quantified from the calibration curves prepared in ethanol/water (1:1) from commercially available HMOs standards at the following concentrations, 12.5, 50, 125, 250, 500, 1250, 2500, 5000 ng/ml. The lyophilized samples were dissolved before the analysis in 1 ml of the same solvent. Calibration samples were run separately for each batch of samples because calibration was done in external mode due to the absence of appropriate internal standards. Data were analyzed using TraceFinder software (Thermo Fisher Scientific Inc.). 

Structures and CID spectra of the analyzed HMOs

Results and conclusions

By using the developed LC-HR-MS/MS method nine HMOs can be reliably identified and quantified on the basis of high-resolution scan and MS/MS data.

The results of this work were published in: "Human Milk Oligosaccharide Profile across Lactation Stages in Israeli Women – a Prospective Observational Study", Nutrients, 2023, 15(11), 2548.