Quantification of Heparan Sulfate Disaccharides Using Ion-Pairing Reversed-Phase Microflow High-Performance Liquid Chromatography with Electrospray Ionization Trap Mass Spectrometry

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Abstract
The glycosaminoglycan (GAG) family of biomacromolecules is composed acidic and linear chains of repeating disaccharide units. Quantitative disaccharide composition analysis is essential for the study and characterization of GAGs. Heparan sulfate and heparin consist of multiple disaccharide units and can be well-separated by ion-pairing reversed-phase microflow high-performance liquid chromatography (IPRP-Mf-HPLC). Each disaccharide can be detected and its mass confirmed by electrospray ionization mass spectrometry (ESI-MS). Isotopically enriched disaccharides were prepared chemoenzymatically from a uniformly 13C,15N-labeled N-acetylheparosan (−GlcA(1→4)GlcNAc−) obtained from the fermentation of E. coli K5. These isotopically enriched disaccharides have identical HPLC retention times and mass spectra as their unlabeled counterparts and were used in liquid chromatography−mass spectrometry (LC−MS) as internal standards. The ratio of intensities between each pair of enriched and nonenriched disaccharides showed a linear relationship as a function of concentration. With the use of these calibration curves, the amount of each disaccharide (≥2 ng/disaccharide) could be quantified in four heparan sulfate samples analyzed by this method.