Author ORCID Identifier


Defense Date


Document Type


Degree Name

Doctor of Philosophy


Pharmaceutical Sciences

First Advisor

Dr. Matthew Halquist, Ph.D.


The main focus of this study was the development of various quantitative bioanalytical techniques for the determination of plasma, vitreous, and aqueous humor ranibizumab (fab therapeutic) and bevacizumab (mAb therapeutic) concentrations after intravitreal administration. It’s vital to access both vitreous/aqueous humor concentrations (efficacy evaluations), as well as systemic plasma concentrations (toxicity and life cycle management evaluations). First, we focused on the development of a novel bottom-up hybrid-IA-LC-MS/MS method for quantitative analysis in human plasma, which was able to achieve more clinically relevant ranges compared to the gold standard LBA, which suffered from sensitivity issues related to selectivity interferences affecting low concentration samples. Then additional methods were developed, including a bottom-up HRMS method and a LBA (to represent the gold standard). It was determined that all three methods were in agreement statistically with the LC-MS/MS methods showing higher sensitivity, and reaching more clinically relevant ranges. In addition to value achieved for these specific analytes, these techniques can also be modified slightly to obtain highly selective methods for other mAbs and fabs with minimal development time. Lastly, a novel technique was explored, in which these therapeutic proteins were quantitated intact in human vitreous and aqueous humor via LC-HRMS and deconvolution data processing. From this proof-of-concept study, the clinically relevant range was achieved, and the possibility of reduced extraction time with increased post-lab information was demonstrated for this and future methods. All of the methods were validated using the criteria or modified criteria based on the 2018 FDA guidance for industry.


© Catherine Del Guidice

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Date of Submission


Available for download on Monday, December 14, 2026