Keywords: 3-O-Sulfo group

RPIP–LC–MS Heparin lyase 2 a b s t r a c t Complete heparin digestion with heparin lyase 2 affords a mixture of disaccharides and resistant tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Quantita-tive online liquid chromatography–mass spectrometric analysis of these resistant tetrasaccharides is described in this article. The disaccharide and tetrasaccharide compositions of seven porcine intestinal heparins and five low-molecular-weight heparins were analyzed by this method. These resistant tetra-saccharides account for from 5.3 to 7.3 wt % of heparin and from 6.2 to 8.3 wt % of low-molecular-weight heparin. Because these tetrasaccharides are derived from heparin’s antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. The content of 3-O-sulfo group-containing tetrasaccharides in a heparin correlated positively (r = 0.8294) to heparin’s anticoagulant activity.! 2014 Elsevier Inc. All rights reserved. Heparin, a sulfated linear polysaccharide, and its low-molecu-lar-weight derivatives are widely used as clinical anticoagulant