Quantitation of oligosaccharides using capillary electrophoresis with laser‐induced fluorescence

Human milk oligosaccharides (HMOs) are complex glycans with significant health benefits for infants, making their accurate quantitation crucial for infant nutrition research and product development. Traditional liquid chromatography (LC) based methods face challenges in resolving isomeric HMOs, prompting the exploration of capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF) as an alternative analytical technique [1]. This work aimed to develop, optimize, and semi‐validate an analytical method for separating and quantifying of oligosaccharides, focusing on HMOs using CE‐LIF. HMOs were derivatized with 8‐aminopyrene‐1,3,6‐trisulfonate (APTS), systematically varying conditions such as temperature, reaction time, pH, and reagent concentrations to maximize labelling efficiency and reproducibility. Labelling efficiency was evaluated using CE‐LIF and liquid chromatography‐mass spectrometry (LC‐MS). While CE‐LIF demonstrated strong separation capabilities, challenges related to reproducibility and migration time stability were identified. The method achieved a limit of quantification (LOQ) comparable to High‐Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC‐PAD) and outperformed Hydrophilic Interaction Liquid Chromatography with Fluorescence Detection (HILIC‐FLD) in sensitivity for infant nutrition applications. This study establishes a strong foundation for integrating CE‐LIF into routine HMO analysis, offering a selective and sensitive alternative to conventional chromatographic methods [2].