Method optimisation for UHPLC–FLD analysis of human milk oligosaccharides

Significant analytical challenges remain due to the structural diversity of HMOs, difficulties in isolation, and the lack of commercially available standards. This study develops and validates an adaptable ultra-high-performance liquid chromatography with fluorescence detection (UHPLC-FLD) for the quantification of eight specific HMOs: 2′-Fucosyllactose (2′-FL), 6′-Sialyllactose (6′-SL), 3-Fucosyllactose (3-FL), 3′-Galactosyllactose (3′-GL), 6′-Galactosyllactose (6′-GL), Lacto-N-difucohexaose I (LNDFH I), Lacto-N-difucohexaose II (LNDFH II), and Lacto-N-neotetraose (LNnT) and testing 74 human milk samples. The method for quantifying HMOs was successfully validated regarding linearity (r ≥ 0.98), recoveries rate of spiked samples ranging from 71% to 119%. For all investigated oligosaccharides, LOD ranged from 5.698 – 7.101 µg mL -1 , and LOQ values ranged between 17.267 – 21.517 μg mL −1 . This method demonstrates great performance compared to existing techniques, providing a reliable tool for the analysis of HMOs in milk. • The developed UHPLC-FLD method was successfully applied to determine 8 commonly presented oligosaccharides in human milk. • High analytical accuracy was demonstrated, with recovery rates of 71–119% for spiked milk samples. • Analytical column temperature optimisation was determinant for effective HMO separation.