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Title:Development of liquid chromatography-mass spectrometry protocols for quantification of imidacloprid in pig urine and for analysis of 5α-cyprinol sulfate metabolism in zebrafish
Author(s):Volland, Alyssa Emaly
Advisor(s):Ridlon, Jason M
Department / Program:Animal Sciences
Discipline:Animal Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Liquid chromatography-mass spectrometry
imidacloprid quantification
cyprinol sulfate
zebrafish bile alcohol metabolism
Abstract:Liquid chromatography-mass spectrometry (LC/MS) is a robust and sensitive technique used for the separation, identification, and quantification of small molecules within a mixed biological sample. It is commonly used in metabolomic analyses, which complement genomic, transcriptomic, and proteomic data when investigating complex systems. LC/MS is the gold standard for resolving metabolites and end-products, but the results obtained from LC/MS are not necessarily standardized and reproducible; individual LC/MS protocols must be fine-tuned for every laboratory and for every unique experiment. Depending on the research question being asked, LC/MS can be used in an untargeted strategy to investigate relative metabolite shifts and discover new biomarkers or a targeted strategy for quantitative analyses of one or a few metabolites. Because of its broad range of applications, LC/MS protocols can be created to support a number of different studies that are investigating seemingly unrelated areas of science. In one study, we quantified the levels of a neonicotinoid, imidacloprid (IMI), in pig urine using a spike of internal standard D4-Imidacloprid (D4-IMI) at known concentration. The urine samples were prepared using a modified solid phase extraction (SPE). This experiment is an example of a targeted LC/MS approach since we achieved precise quantification of trace amount of imidacloprid within 6 urine samples. The LC/MS protocol we developed also included tandem MS/MS and multiple reaction monitoring (MRM) mode that created specific channels to monitor previously identified daughter ions of IMI and the internal standard D4-IMI. The subsequent LC/MS utilization first required purification of 5α-cyprinol sulfate, the main bile alcohol present in both carp and zebrafish, which is not available for commercial purchase. Crude carp bile was purified using thin layer chromatography and flash column chromatography. The identity of the major bile alcohol, 5α-cyprinol sulfate, was determined by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The purified 5α-cyprinol sulfate was used as an LC/MS standard to investigate bile composition in carp and zebrafish. 5α-cyprinol sulfate was also used as a substrate to investigate metabolism by bacterial cultures of zebrafish intestinal communities. After SPE, the cultures were injected on to LC/MS in an untargeted approach to identify potential bile metabolites.
Issue Date:2019-04-16
Rights Information:Copyright 2019 Alyssa Volland
Date Available in IDEALS:2019-08-23
Date Deposited:2019-05

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