Quantitative analysis of the major linkage region tetrasaccharides in heparin

Heparin is a polysaccharide based anticoagulant drug composed of a complex mixture of glycosaminoglycan chains and peptidoglycosaminoglycan chains. In an effort to better characterize this important polysaccharide based drug, we examined the peptide components of the minor peptidoglycosaminoglycan chains comprising heparin. Three different the glycan-peptide linkage regions tetrasaccharide fragments were isolated from pharmaceutical heparin using heparin lyase II and characterized the structure of these tetrasaccharides using nuclear magnetic resonance spectroscopy and mass spectrometry. A sensitive and quantitative assay was developed for these linkage regions using multiple reaction-monitoring tandem mass spectrometry. These three different linkage regions were found in heparins coming from porcine intestine and bovine lung. Two of these were also present in the low molecular weight heparin, enoxaparin.