Document Type

Dissertation - Open Access

Award Date

2021

Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

First Advisor

Brian A. Logue

Keywords

HPLC-MS/MS, MESNA, method development, Methyl isocyanate, sulfur mustard, Toxic Inhaled Agents

Abstract

The inhalation of toxic inhaled agents (TIAs) such as methyl isocyanate and cyanide can compromise an individual’s general health due to their extremely toxic nature. Nonetheless, these gases are used extensively in the electronics, chemical, welding, electroplating industries, etc., increasing the risk of exposure to both civilians and industrial workers. It is therefore very crucial to develop analytical methods for TIA metabolites and therapeutic agents. Methyl isocyanate (MIC), an intermediate in the synthesis of carbamate pesticides is a toxic industrial chemical that causes irritation and damage to the eyes, respiratory tracts, and skin. Due to its reactivity with proteins, protein adducts can be used to confirm exposure, but this type of analysis is cumbersome, time consuming and expensive. Hence, we investigated the interaction between MIC and proteins to identify a novel marker, phenyl methyl carbamate (PMC) based on hydrolysis of tyrosine-MIC adducts. A simple base hydrolysis was used for sample preparation with sensitive HPLCMS/ MS analysis of this marker. The method produced excellent sensitivity for PMC with a detection limit of 0.02 mg/kg and calibration curve linearity extending from 0.06‒1.51 mg/kg (R2 = 0.998 and overall %RA >90). The accuracy and precision (100±9% and < 10% relative standard deviation, respectively) of the method were outstanding. The validated method was successfully able to detect elevated levels of PMC from MIC-spiked hemoglobin and further used to detect significantly elevated PMC levels from hemoglobin isolated from MIC-exposed rats. Sodium-2-mercaptoethane sulfonate (MESNA), has been suggested as a potential antidote for MIC exposure. It has previously proven to be effective in inactivating acrolein, the toxic metabolite of anti-tumor drugs (cyclophosphamide and ifosphamide). However, current methods available to analyze MESNA have limitations, including low sensitivity, poor selectivity, high degree of difficulty, and many time-consuming steps. Hence, we developed and validated a simple and selective HPLC-MS/MS method for the analysis of MESNA in plasma. The method showcases an excellent limit of detection of 20 nM with excellent linearity (R2 = 0.999 and overall %RA > 90), and a wide linear range (0.05‒200 μM). The method also produced very good accuracy and precision (100±10% and < 10% relative standard deviation, respectively). The validated method was successfully used to analyze MESNA from treated animals and will enhance pharmacological and therapeutic research on this promising antidote. Lastly, sulfur mustard (SM) is a toxic bifunctional alkylating warfare agent, which causes severe damage to skin, eyes, and respiratory tract. However, there is no antidote for treating sulfur mustard poisoning and treatment is limited to either washing the affected area with soap and water or supportive care in cases of inhalation exposure. In recent research, methimazole (METH), an antithyroid drug has proven to be an effective nucleophilic scavenger to combat the toxic effects of sulfur mustard analog, 2-Chloroethyl ethyl sulfide (CEES) but there are no studies on the underlying mechanism of the scavenging ability of methimazole for CEES in preventing cell death. In this study, we investigated the interaction between methimazole and CEES for a possible formation of the novel compound, 2-(2-(ethylthio)ethylthio)-1-methyl-1H-imidazole (ETTMI). The production of ETTMI from the reaction of CEES with methimazole was proven by the detection of ETTMI from CEES-methimazole cell lines. An HPLC-MS/MS method was developed to analyze ETTMI from HaCat cell lines and media. Using this method, we showed that ETTMI is produced from the interaction of CEES with methimazole and the formation of ETTMI could be one of the underlying pathways for the scavenging ability of methimazole for CEES.

Library of Congress Subject Headings

Metabolites.
Pulmonary toxicology.
Therapeutics.
Methyl isocyanate.
Mustard gas.
Cyanides.

Number of Pages

114

Publisher

South Dakota State University

Download

Share

COinS
 

Rights Statement

In Copyright