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.
Majore et al. (Sun,) studied this question.