Date of Award

12-31-2013

Document Type

Campus Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry/Physical/Analytical Chemistry

First Advisor

Jason Evans

Second Advisor

Jonathan Rochford

Third Advisor

Deyang Qu

Abstract

In 2008, United States Pharmacopeial Convention (USP) proposed a new general chapter to replace the outdated and qualitative heavy metal test specified in USP Heavy Metals <231>. In 2010, USP proposed two new general chapters, Elemental Impurities Limits <232> and Procedures <233>, specifying sixteen elements that need to be controlled in drug articles. Chapter 1 of this dissertation focuses on an ICP-MS method developed and qualified as a replacement method for the quantification of these sixteen elements. To minimize sample matrix effects, a one-point standard addition method was applied for the quantification of fifteen elements. Due to the toxicity of osmium, a semi-quantitative analysis was applied using nine non-Os standards. This method was qualified for specificity, sensitivity, accuracy, precision and linearity with satisfactory results for the release test for a soluble drug substance.

Chapter 2 of this dissertation focuses on the development of an in vitro lipase activity assay using LC-MS to quantify oleic acid. Compared to the USP pH titration assay, this method has no pH limitation with native triolein substrate. Cross-linked lipase crystal (CLLC) was bioengineered to protect its activity in the acidic environment of the stomach, while maintaining its activity in the small intestine through differences in solubility. Solubility-activity relationship is the most important attribute of CLLC, which is designed to improve its in vivo efficacy. Two CLLCs were characterized with various analytical techniques and this LC-MS method. In vivo test demonstrated that CLLC more efficiently improved patient fat digestion compared to its porcine derived lipase counterpart.

Chapter 3 summarizes preliminary results for the synthesis and characterization of a Phos-Tag linker that could potentially be used to quantify phoshphorylated peptides and proteins. Phos-Tag is an organic macro-molecule with Zn metal centers, which bind to phosphate group through electron donation from two oxygen atoms. Phos-Tag binding to different phospho-amino acids was examined with ESI-MS. The preliminary results demonstrated Phos-Tag has strong binding affinity to phospho-amino acids, which could be used for absolute quantification of phosphopeptide or phosphoprotein with ICP-MS or laser ablation ICP-MS tissue imaging analysis.

Comments

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