Document Type

Thesis - University Access Only

Award Date

2011

Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

Abstract

Cyanide is a ubiquitous, lethal and fast acting poison. It is present in some plants and foods (e.g., Cassava, almonds, etc.) and in products of combustion (fires, vehicle exhaust, cigarette smoke, etc.). It is also widely used in industries (e.g., mining, metallurgy, paints, plastics, etc.), and it may be used illegally as a poison or terrorist weapon. To determine exposure to cyanide, analysis of cyanide or its breakdown products from biological matrices is important either in case of accidental exposure or deliberate release. Verification of cyanide exposure is generally achieved either by direct analysis of cyanide (CN-), or by analysis of its major metabolites, thiocyanate (SCN-) and/or 2-amino-2-thiazoline-4-carboxylic acid (ATCA). Although there are many methods available for the determination of cyanide and its metabolite from human biological fluids, these methods are either time consuming or laboratory based. In this study, a fluorescence-based portable field sensor for rapid on-site verification of cyanide exposure was developed along with an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS MS) method for a novel fast-acting cyanide therapeutic (i.e., Sulfanegen). Prior to developing a field sensor, the relationship between cyanide and its metabolites in various biological fluids must be understood in order to target a specific marker for verification of cyanide exposure. This was achieved by analyzing various biological fluids (i.e., saliva, plasma, urine, and red-blood cells) for CN-, SCN- and ATCA from smokers and non-smokers (50 each). Three different analytical methods were used to analyze CN-, SCN- and ATCA. Cyanide was assayed by HPLC with fluorescence detection after reaction with napthlenedialdehyde and taurine. Thiocyanate and ATCA were analyzed by gas chromatography-mass spectrometry (GC-MS) after reaction with pentafluorobenzyl bromide (PFBBr) and N-methy-N-(trimethylsilyl) trifluoroacetamide (MSTFA), respectively. Significant differences in cyanide, thiocyanate, and ATCA concentrations of smokers and non-smokers were statistically evaluated. Sub-groups of smokers and non- smokers (e.g., male and female subgroups and groups divided by the number of cigarettes smoked per day) were created to evaluate correlations and differences between the groups. Biomarker concentration ratios (BCR) were also used to offer insight into the correlations between cyanide and its metabolites. Detailed statistical analysis of data for CN-, SCN- and ATCA in each biofluid and the comparison of BCRs along with other published literature indicated that SCN- cannot be used as a marker for verification of cyanide exposure due to its high and variable concentrations, which would likely show little change with low-level cyanide exposure. ATCA, on the other hand, showed potential to be a marker for verification of chronic cyanide exposure. Therefore, in cases of severe and acute cyanide poisoning, direct analysis of cyanide is the biomarker of choice in the initial few minutes post-exposure. Hence, direct cyanide analysis from saliva was chosen for development of a portable field sensor for rapid on-site detection of cyanide exposure. A portable cyanide sensor was developed based on microdiffusion followed by entrapment and analysis of cyanide in a basic solution. The captured cyanide was then derivatized by 2,3 naphthalenedialdehyde (NDA) and taurine to form a fluorescent product which was subsequently analyzed using the portable field sensor. Cyanide was successfully analyzed by the sensor at a detection limit of 10 μM and was linear in the range of 25 - 500 μM. Also, saliva samples of smokers prior to smoking and immediately after smoking were successfully tested for significantly elevated levels of salivary cyanide. Along with the development of novel cyanide diagnostics, there is also a need for more effective and fast-acting cyanide antidotes. Sulfanegen sodium (prodrug of 3-mercaptopyruvate) is one such antidote that has shown promise for treating cyanide exposure. However, lack of an adequate analytical method, partially owing to sulfanegen's instability, has hampered its development, and ultimately its approval as an antidote for cyanide exposure. Therefore, novel ultra-high performance liquid chromatography-mass spectrometric (UHPLC-MS-MS) method for the analysis of 3-mercaptopyruvate (3-MP) was developed. Initially, 3-MP was derivatized with monobromobimane to stabilize 3-MP and improve its chromatographic behavior. Although a complete validation of the method was not possible due to lack of proper internal standard (deuterated 3-MP), substantial improvement in the chromatographic behavior of 3-MP was observed.

Library of Congress Subject Headings

Cyanides

Cyanides -- Toxicology

Antidotes -- Therapeutic use

Format

application/pdf

Number of Pages

149

Publisher

South Dakota State University

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