Cationic Phenylpyridine Labeling for Improved Oligosaccharide and Glycan Analysis by Capillary Electrophoresis with LED-Induced Fluorescence Detection

Abstract Capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) is a powerful technique for analyzing glycans. However, it requires derivatization with a fluorescent compound to enhance both electrophoretic mobility and detection sensitivity simultaneously. The widely used labeling reagent 8-aminopyrene-1,3,6-trisulfonic acid (APTS) suffers from several limitations, including relatively low labeling efficiency and poor compatibility with mass spectrometry (MS), highlighting the need for improved alternatives. In this work, we designed and synthesized a novel cationic fluorescent label based on a phenylpyridine scaffold incorporating hydrazide functionality for efficient oligosaccharide and glycan derivatization via hydrazone formation. To fully exploit its fluorescence characteristics, we also optimized the capillary electrophoresis system by modifying the LED-induced fluorescence (LED-IF) detector through systematic evaluation of excitation LEDs and emission filters, resulting in substantially improved detection sensitivity. The optimized labeling, separation, and detection conditions enabled sensitive analysis of released glycans from various glycoproteins with detection limits in the nanomolar concentration range. The combination of this new phenylpyridine-based fluorescent label with the enhanced CE/LED-IF detection platform significantly improves the analytical performance of CE for glycan analysis and provides a more sensitive, robust, and versatile approach for comprehensive oligosaccharide and glycan characterization.