Development of NMR Methods for the Characterization of Heparin and its Impurities
- Author(s): Limtiaco, John Francis Kuper
- Advisor(s): Larive, Cynthia K
- et al.
The goal of this dissertation is to develop and apply NMR methods for the analysis of heparin and its impurities. As a result of the intentional contamination of heparin with oversulfated chondroitin sulfate (OSCS), significant efforts have been placed on the development of analytical methods to evaluate heparin purity. To advance the suite of analytical methods available for heparin analysis, HPLC-NMR and diffusion NMR methods were developed for its characterization. Our HPLC-NMR method focuses on the WAX separation of intact glycosaminoglycans (GAGs) coupled to on-line UV and NMR spectroscopy for detection and identification of heparin contaminants. The weak anion exchange (WAX) method developed allows for the timely separation of heparin and its impurities, OSCS, dermatan sulfate, and chondroitin sulfate A. In addition to the study of heparin impurities using WAX-HPLC, the characterization of intact and digested heparin samples was investigated using diffusion NMR. Because the diffusion behavior of the intact biopolymers can be dominated by viscosity and molecular crowding effects even in dilute GAG solutions, NMR diffusion results must only be interpreted qualitatively. These limitations can be largely overcome by digesting heparin to low molecular weight oligosaccharides, significantly improving the resolution of heparin and its biopolymer impurities.
The second part of this dissertation is on the characterization of heparin-derived oligosaccharides. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, extensive or partial digestion of the full length polysaccharides is a common practice. Size-exclusion chromatography was used to separate digested heparin oligosaccharides into size-based fractions while SAX-HPLC was used to further purify individual oligosaccharides for NMR characterization. Although, NMR spectroscopy is a powerful tool for structure determination, it trails other analytical techniques in sensitivity. Microcoil NMR is an inexpensive method for enhancing the mass sensitivity of NMR allowing structure elucidation with 5-10 μg of isolated material. Results are presented showing the utility of microcoil NMR for the structure characterization of microgram amounts of heparin-derived tetrasaccharides, reducing the NMR sample requirements by 2-fold when compared to analysis in a Shigemi tube.
In Chapter 6, an IMPACT-HNMBC experiment was used in the detection of 15N in amino sugars through long-range couplings to carbon-bound protons of the sugar ring. Comparison of the ge-HMBC, IMPACT-HMBC and a modified IMPACT-HNMBC pulse sequence revealed improved sensitivity, resolution and reduced F1 noise with the IMPACT-HNMBC experiment.