High sensitivity separation and detection of heparan sulfate disaccharides

Eight Δ-disaccharide standards from heparan sulfate/heparin were derivatized with the fluorophore 4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, hydrazide (BODIPY) via formation of a Schiff's base and separated using HPAEC on a Propac PA1 column with a linear salt gradient and isocratic 150 mM NaOH. Detection was with an in-line fluorescence detector. The standard deviation (σn−1) in retention times were 0.7–2% over nine runs. The limit of detection, was 100 fmol (100 × 10−15 mol) of BODIPY labeled Δ-disaccharides, representing considerably improved detection compared to other fluorophore labeled derivatives and, unlike these, required no further purification steps. Separation and improved detection of BODIPY-Δ-disaccharide conjugates will assist the structural analysis of HS and the development of improved sequencing methodologies.