Microwave-assisted hydrolysis and HPAEC-PAD for simultaneous quantification of sialic acid and six other glycan monosaccharides in human serum: RSM optimization, validation, and proof-of-concept application to acute stroke.

A derivatization-free method for the simultaneous quantification of seven monosaccharides, including the labile sialic acid, in human serum was developed using microwave-assisted trifluoroacetic acid (TFA) hydrolysis coupled with high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Single-factor experiments and Response Surface Methodology (RSM) identified optimal hydrolysis conditions of 105 °C, 30 min, and 1.37 M TFA. These parameters achieved efficient release of fucose, galactosamine, glucosamine, galactose, glucose, and mannose while limiting loss of the labile sialic acid. The method was characterized on 10 μL fasting serum using a CarboPac PA10 column (30 °C) with a sodium acetate/sodium hydroxide gradient and quadruple-potential PAD waveform. Reproducibility was good (peak-area RSD < 5%, n = 4). Chromatographic separation resolved all seven monosaccharides, with near-baseline resolution and partial co-elution among galactose, glucose, and mannose. Proof-of-concept application to a clinical stroke cohort (47 acute ischaemic stroke [AIS], 50 intracerebral haemorrhage [ICH], and 51 matched healthy controls) confirmed distinct monosaccharide profiles: sialic acid and fucose were elevated in both AIS and ICH relative to healthy controls but disproportionately greater in AIS, while galactose, mannose, and glucosamine were elevated in both stroke subtypes. Serum mannose correlated with MRI-defined infarct volume (R2 = 0.543, P = 0.003), and retrospective analysis of 70 conventional laboratory tests confirmed that none differentiated AIS from ICH. This micro-volume, high-throughput platform overcomes longstanding limitations of existing glycan analysis methods and provides a promising basis for clinical chemistry and biomarker studies and a substrate for downstream multivariate glycomic analyses.