New green strategies for labelling of glycoprotein N-glycans.

2,2,5,5-Tetramethyloxolane (TMO) and Cyrene are recognised as sustainable, bio-based solvents that match the analytical performance of acetonitrile (MeCN) for fluorescent labelling of N-glycans. Eight solvents from dipolar aprotic, non-polar, and polar protic classes were systematically evaluated for compatibility, labelling efficiency, and reproducibility in the fluorescent labelling of N-glycans using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) from two glycoprotein standards, namely Immunoglobulin G and Fetuin. Hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) was used for visualisation and quantification of fluorescent N-glycans. Absolute quantification was achieved using a spiked internal standard, enabling picomolar-level quantification and facilitating direct comparison across solvents. TMO and Cyrene maintained glycan profiles, resolution, and peak intensity comparable to MeCN, while offering enhanced safety and environmental benefits. The workflow demonstrates that greener solvent systems can be incorporated into glycoanalytical pipelines without compromising performance, providing a scalable, sustainable alternative for biopharmaceutical and glycoscience applications.