This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In order to improve their pharmacological properties, heparins are partially depolymerized to produce low molecular weight heparin (LMWH) preparations. LMWHs have the advantage that they eliminate some heparin side effects by virtue of the fact that the have fewer undesired interactions with proteins in the blood stream. The pharmacological properties of LMWHs depend strongly on the method used in their depolymerization. As a result, there are more than a dozen LMWH drugs on the market world wide, each used for a specific clinical indication. Heparins and LMWH consist of a polymeric distribution of heparin chain. Chains of a given length are distribution of sulfation, acetylation and epimerization isomers. As a result, it is nearly impossible to fully characterize the structure of a heparin or LMWH preparation. Effective tools for structural analysis of LMWH are needed to facilitate development of both new LMWH drugs and generics. Generic developers must prove that their LMWH preparations are structurall identical to the innovator material. The polydispersisty of LMWH make this a very challenging task. In many ways, this is similar to the problem of establishing sameness of recombinant glycoprotein drugs. The anticoagulant properties of LMWH depend on the levels of a particular oligosaccharide sequence containing a critical GlcN-3-sulfate group. This sequence has been shown to resist degradation by heparin lyase and to remain as a tetrasaccharide. LMWH preparations were exhaustively digested using heparin lyases and then analyzed using amide-HILIC LC/MS. The results were used to compare the abundances of the most abundant di-, tri- and tetrasaccharide products in the LMWH digests. As expected, the most abundant disaccharides have two and three sulfate groups. LMWH depolymerized using nitrous acid (Dalteparins) show disaccharides with anhydromannitol on the formerly reducing end. Pentasulfated tetrasaccharides are also observed that contain the critical GlcN-3-O-sulfate group.