Sensitive Fluorescence Quantitation and Efficient Free Radical Characterization of N‑Glycans via LC-FLR-HRMS/MS with a Novel Fluorescent Free Radical Tag

Glycans are some of the most difficult biomolecules to analyze owing to their branching tendencies as well as their regiochemical and stereochemical diversity. Yet, the correlation between various pathological states and glycan quantity or structural alterations has demonstrated the importance and urgency for the development of a more robust glycan analytical technique. Furthermore, the manufacturing and regulation of biopharmaceuticals demands a feasible and improved analytical approach toward the characterization and quantitation of glycosylations. Unfortunately, multiple commercially available glycan tags lack, in combination, liquid chromatography detection sensitivity, chemical stability and, most importantly, optimal glycan characterization capabilities. Therefore, a novel fluorescent tag coupled with a free radical approach for glycan characterization was designed and developed to help address this gap in glycan analysis. The analytical capabilities of this novel tag were assessed via hydrophilic liquid chromatography−fluorescence quantitation and ESI/MS free radical-mediated characterization by using linear glycan standards, branched isobaric glycans lacto-N-difucohexaose I and lacto-N-difucohexaose II, and N-glycans released from ribonuclease B.