Determination of methyl-, 2-hydroxyethyl- and 2-cyanoethylmercapturic acids as biomarkers of exposure to alkylating agents in cigarette smoke☆
Introduction
Alkylating agents such as N-nitrosamines, ethylene oxide and acrylonitrile are toxicologically important chemicals, because of their carcinogenic potential [1], [2], [3], [4]. These agents can covalently bind to nucleophiles [5] which may account for their toxic, mutagenic and carcinogenic effects. Reaction products of alkylating agents with DNA, proteins and glutathione have been used as biomarkers of exposure to these chemicals [6], [7], [8], [9], [10]. With the advent of more sensitive and specific detection methods including mass spectrometry coupled with gas or liquid chromatography, the quantification of protein and DNA adducts as well as mercapturic acids related to individual alkylating agents became possible [9], [10], [11]. Assessing the exposure to these compounds by suitable biomonitoring methods, therefore, might be relevant for understanding the potential biological effect from these compounds, which would need further investigation with risk markers.
Tobacco smoke contains both direct alkylating agents (alkyl halides, acrolein, crotonaldehyde, ethylene oxide, propylene oxide, acrylonitrile and acrylamide) as well as indirect alkylating agents which require metabolic transformation to reactive species (tobacco-specific nitrosamines such as NNK, volatile dialkylnitrosamines such as NDMA, and ethylene) [12], [13]. We have developed and validated a non-invasive method for measuring biomarkers of exposure to methylating (NDMA, NNK), hydroxyethylating (ethylene, ethylene oxide) and cyanoethylating (acrylonitrile) agents in tobacco smoke resulting from the conjugation of these compounds with glutathione and excretion as mercapturic acid metabolites in urine. For this purpose, methyl- (MMA), 2-hydroxyethyl- (HEMA) and 2-cyanoethylmercapturic acid (CEMA) were regarded to be most suitable. Fig. 1 shows the chemical structures of these three mercapturic acids.
Methylating chemicals in tobacco smoke comprise methyl halides (e.g., methyl chloride, mainstream smoke yield: 150–840 μg/cigarette [14], N-nitrosodimethylamine (NDMA, 0.1–180 ng/cigarette [12]) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 110–133 ng/cigarette [12]). NDMA and NNK are capable of methylation after metabolic activation. Ethylene oxide (7 μg/cigarette [12]) is a potential 2-hydroxyethylating agent in tobacco smoke. Ethylene (400–700 μg/cigarette [14]) is also a potential 2-hydroxyethylating agent because a small fraction of it can convert to ethylene oxide in the body. N-Nitrosodiethanolamine (NDELA, up to 36 ng/cigarette [12]) has also been shown to form 2-hydroxyethyl adducts [12]. Hydroxyethylation is also effected by vinyl chloride, ethylene dibromide and other electrophilic compounds with a two-carbon backbone [6], [15]. Acrylonitrile is the major cyanoethylating agent. Its yields in mainstream smoke of cigarettes amount to 3–15 μg/cigarette [12].
We describe here an LC–MS/MS method for the simultaneous determination of MMA, HEMA and CEMA in human urine. This method was applied to urine samples from two clinical studies.
Section snippets
Standards and chemicals
N-Acetyl-S-methyl-l-cysteine (methylmercapturic acid, MMA), [D3]-N-acetyl-S-methyl-l-cysteine (MMA-D3), N-acetyl-S-(2-hydroxyethyl)-l-cysteine (2-hydroxyethylmercapturic acid, HEMA), N-acetyl-S-([D4]-2-hydroxyethyl)-l-cysteine (HEMA-D4), N-acetyl-S-(2-cyanoethyl)-l-cysteine (2-cyanoethylmercapturic acid, CEMA), [D3]-N-acetyl-S-(2-cyanoethyl)-l-cysteine (CEMA-D3) were purchased from Toronto Research Chemicals, North York, Canada. The supplier stated a purity of 98% or greater for all reference
Chromatography and mass-selective detection
Product ion mass spectra of the PFBBr derivates of MMA, HEMA, and CEMA and of the deuterated internal standards together with the suggested structures of the ion fragments are shown in Fig. 2. The [M+H]+ ion of MMA (m/z 358), MMA-D3 (m/z 361), HEMA (m/z 388), HEMA-D4 (m/z 392), CEMA (m/z 397) and CEMA-D3 (m/z 400) are formed from all compounds. The mercapturic acid-specific ion fragment of m/z 130 (133 for CEMA-D3) is formed from each analyte and internal standard derivative. The same is true
Discussion
We have developed and validated a LC–MS/MS method for the simultaneous determination of three major urinary metabolites of these compounds, namely, MMA, HEMA and CEMA. Orginally, ethylmercapturic acid (EMA, a potential biomarker of exposure to ethylating agents) was also included in this method. The method comprises liquid/liquid extraction of the urine sample, solid phase extraction on anion exchange cartridges, derivatization with PFBBr, liquid/liquid extraction of the reaction mixture and
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This paper is part of the special issue “Biological Monitoring and Analytical Toxicology in Occupational and Environmental Medicine”, Michael Bader and Thomas Göen (Guest Editors).
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