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Development of Analytical Methods for Trace Impurity Analysis and Structure Determination of Heparin/Heparan Sulfate-Derived Oligosaccharides



Development of Analytical Methods for Trace Impurity Analysis and Structure Determination of Heparin/Heparan Sulfate-Derived Oligosaccharides


Stacie Liane Eldridge

Doctor of Philosophy, Graduate Program in Chemistry

University of California, Riverside, August 2009

Dr. Cynthia K. Larive, Chairperson

The goal of this dissertation is to develop improved analytical methods and approaches that address the difficulties in separating and detecting pharmaceutical impurities and heparin oligosaccharides. The specific methods used were capillary electrophoresis with ultraviolet detection (CE-UV), liquid chromatography-mass spectrometry (LC-MS), with special emphasis given to capillary isotachophoresis-nuclear magnetic resonance (cITP-NMR).

The FDA requires structural identification of all pharmaceutical impurities and degradants present at levels ≥ 0.1% of the parent compound. This poses a significant analytical challenge since these impurities are typically present in trace amounts along with high levels of the parent drug and matrix components. Greater complications arise in the analysis of heparin and heparan sulfate (HS). These highly sulfated linear polysaccharides display a wide range of biological activities through interaction with proteins. The enzymatic modifications that occur during biosynthesis for the purposes of cell adaptation and regulation, contribute to the heterogeneity of heparin and HS, making them a challenge to characterize.

Progress in the areas of impurity and heparin/HS substructure analysis requires the use of NMR for molecular characterization. NMR is a powerful analytical tool despite its intrinsically poor sensitivity when compared to other analytical methods. This limitation becomes magnified when the structure elucidation of concentration or mass-limited compounds is required. This problem is addressed in this work by coupling microcoil NMR to the pre-concentration method cITP. Microcoil NMR is an inexpensive way to increase the intrinsic sensitivity of NMR, and cITP can concentrate analytes up to 2 to 3 orders of magnitude. This makes cITP-NMR ideal for studying analytes that are mass- or volume-limited.

Results are presented illustrating the ability of cITP-NMR to separate and detect charged impurities in the presence of 1000-fold excess of the parent compound. Also, cITP-NMR was used to develop a disaccharide chemical shift database and probe intracapillary pD, providing insight into processes that drive cITP separations. NMR, together with CE-UV and LC-MS, facilitated the characterization of several physico-chemical properties of heparin di- and tetrasaccharides, including functional group pKa values, with the goal of advancing the methods for the separation and structural characterization of heparin and HS oligosaccharides.


Video file for Figure 5.7. Cationic cITP focusing of 100 μM methyl green dye

Video file for Figure 5.8. Anionic cITP focusing of 100 μM bromophenol blue dye

Video file for Figure 5.9. Counter ion migration in cationic cITP

Video file for Figure 5.14. Anionic cITP of bromophenol blue using the TE buffer, ACES

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