Document Type

Dissertation - Open Access

Award Date

2019

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Brian A. Logue

Keywords

Cobinamide, Gas Chromatography, High Performance Liquid Chromatography Tandem Mass spectrometry, Ice Concentration Linked with Extractive Stirrer (ICECLES), Nitrosoamines, Cyanide, Sorptive Stir bar Extraction

Abstract

Cyanide and nitrosoamines (NAs) are ubiquitous compounds, found in our food and water, either by natural process or through man-made activities. The toxicity of cyanide is exerted by its ability to inhibit metalloenzymes such as cytochrome c oxidase, causing concomitant cascades of biochemical effects such as lactic acidosis, inhibition of ATP production, respiratory seizure, and potential death. Nitrosoamines, on the other hand, undergo biotransformation (metabolic activation in cytochrome P450) in the body to produce unstable intermediates that alkylate DNA, causing mutations, and leading to carcinogenesis. In order to further the advancement of a promising cyanide, cobinamide (Cbi), an LC-MS-MS method was developed to analyze cyanide-complex Cbi, while an ICECLES-GC-EI-MS procedure was developed to detect nitrosodipropylamine (NDPA) at low concentrations in drinking water via a flexible, facile, relative easy performed, and green method. Cbi has shown promise as a therapeutic for cyanide poisoning. While current analysis techniques only measure total Cbi, methods to elucidate the behavior of cyanidebound Cbi, total Cbi, and available Cbi (i.e., the difference between cyanoCbi and total Cbi) would be valuable for biomedical and pharmacokinetic studies. Therefore, a method was developed for the analysis of cyanoCbi in plasma via liquid chromatography-tandem mass spectrometry (LC-MS-MS). Plasma samples were prepared by denaturing proteins with 10% ammonium hydroxide in acetonitrile. The resulting mixture was centrifuged, and the supernatant was removed, dried, and reconstituted. CyanoCbi was then analyzed via LC-MS-MS. The limit of detection was 0.2 μM, and the linear range was between 1- 200 μM. The accuracy was 100±17% and the precision, measured by relative standard deviation (%RSD), was ≤18.5%. Carryover, a severe problem when analyzing Cbi via liquid chromatography, was eliminated using a polymeric-based stationary phase (PLRPS) and a controlled washing protocol. The method allowed evaluation of the cyanidebound and ‘available’ Cbi from treated animals and, when paired with a method for total Cbi analysis. This method allowed for estimation of Cbi utilization when treating cyanide poisoning and verified for the first time, the hypothesized mechanism of treatment of cyanide poisoning by Cbi (direct binding of cyanide). To overcome the challenges associated with the analysis of NAs at ultratrace levels (i.e., difficult extraction protocols, laborious sample preparation techniques, and requirement for sophisticated/expensive instrumentation), an advanced sample preparation technique, ICE Concentration Linked with Extractive Stirrer (ICECLES), coupled to an inexpensive low-resolution gas-chromatography electron ionization massspectrometry instrument was used to analyze NDPA (MRL = 7 ng/L (ppt)). An LOD of 0.2 ng/L was obtained for NDPA, along with linear range of 2 to 50 ng/L was produced (using NDPA-d14 internal standard). Both inter- and intraassay precision were ≤13%RSD, while the method accuracy was 100±17.5%. The ICECLES method was applied to screen for possible NA contamination in selected drinking water sources. The concentration of NDPA in one drinking water source was 2.38±0.34 ng/L. Moreover, NDPA was detected in the two other municipalities tested (i.e., concentrations > 0.2 ng/L), but it was not quantifiable.

Library of Congress Subject Headings

Cyanides -- Toxicology.
Nitrosoamines -- Toxicology.

Publisher

South Dakota State University

Rights

In Copyright - Non-Commercial Use Permitted
http://rightsstatements.org/vocab/InC-NC/1.0/

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