Elsevier

Journal of Chromatography B

Volume 878, Issue 27, 1 October 2010, Pages 2520-2528
Journal of Chromatography B

Determination of methyl-, 2-hydroxyethyl- and 2-cyanoethylmercapturic acids as biomarkers of exposure to alkylating agents in cigarette smoke

https://doi.org/10.1016/j.jchromb.2010.02.023Get rights and content

Abstract

Alkylating agents occur in the environment and are formed endogenously. Tobacco smoke contains a variety of alkylating agents or precursors including, among others, N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acrylonitrile and ethylene oxide. We developed and validated a method for the simultaneous determination of methylmercapturic acid (MMA, biomarker for methylating agents such as NDMA and NNK), 2-hydroxyethylmercapturic acid (HEMA, biomarker for ethylene oxide) and 2-cyanoethylmercapturic acid (CEMA, biomarker for acrylonitrile) in human urine using deuterated internal standards of each compound. The method involves liquid/liquid extraction of the urine sample, solid phase extraction on anion exchange cartridges, derivatization with pentafluorobenzyl bromide (PFBBr), liquid/liquid extraction of the reaction mixture and LC–MS/MS analysis with positive electrospray ionization. The method was linear in the ranges of 5.00–600, 1.00–50.0 and 1.50–900 ng/ml for MMA, HEMA and CEMA, respectively. The method was applied to two clinical studies in adult smokers of conventional cigarettes who either continued smoking conventional cigarettes, were switched to test cigarettes consisting of either an electrically heated cigarette smoking system (EHCSS) or having a highly activated carbon granule filter that were shown to have reduced exposure to specific smoke constituents, or stopped smoking. Urinary excretion of MMA was found to be unaffected by switching to the test cigarettes or stop smoking. Urinary HEMA excretion decreased by 46 to 54% after switching to test cigarettes and by approximately 74% when stopping smoking. Urinary CEMA excretion decreased by 74–77% when switching to test cigarettes and by approximately 90% when stopping smoking. This validated method for urinary alkylmercapturic acids is suitable to distinguish differences in exposure not only between smokers and nonsmokers but also between smoking of conventional and the two test cigarettes investigated in this study.

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

References (32)

  • S. Osterman-Golkar et al.

    Mutation Research

    (1976)
  • N.P.E. Vermeulen

    TiPS

    (1989)
  • K. Frost-Pineda et al.

    Regulatory Toxicology and Pharmacology

    (2008)
  • G. Scherer et al.

    Regulatory Toxicology and Pharmacology

    (2007)
  • M. Törnqvist et al.

    Mutation Research

    (1988)
  • M. Törnqvist et al.

    Chemico-Biological Interactions

    (1992)
  • International Agency for Research on Cancer

    Ethylene and Polyethylene IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Some Monomers, Plastics and Synthetic Elastomers, and Acrolein

    (1979)
  • International Agency for Research on Cancer

    Relevance to Human Cancer of N-Nitroso Compounds, Tobacco Smoke and Mycotoxins

    (1991)
  • International Agency for Research on Cancer

    IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 71 pt 1

    (1999)
  • International Agency for Research on Cancer

    IARC Monographs on the Evaluation of Carcinogenic Risks to Humans

    (1979)
  • P.D. Lawley
  • R.T.H. van Welie et al.

    Critical Reviews in Toxicology

    (1992)
  • F. Seutter-Berlage et al.

    International Archives of Occupational and Environmental Health

    (1977)
  • D.E. Shuker et al.

    Environmental Health Perspectives

    (1993)
  • J.S. Wishnok et al.

    Environmental Health Perspectives

    (1993)
  • J. Angerer et al.

    International Journal of Hygiene and Environmental Health

    (2007)
  • Cited by (28)

    • Fast and simple screening for the simultaneous analysis of seven metabolites derived from five volatile organic compounds in human urine using on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry

      2015, Talanta
      Citation Excerpt :

      Recently, liquid chromatography tandem mass spectrometry (LC–MS/MS) methods have proven to be highly selective, sensitive, accurate, and with the capacity to simultaneously quantify VOC metabolites in human urine [2,11,19,20]. Most published methods for the determination of VOC metabolites in urine involve a labor-intensive and time-consuming procedure that requires purification by solid-phase extraction (SPE) [21–25], such as molecularly imprinted polymers (MIPs) [26] and C18 [27] or liquid–liquid extraction (LLE) [28,29]. To avoid the drawbacks of the extraction method, therefore, we developed a simple, quick and cheap sample preparation method and combined it with on-line SPE-LC–MS/MS, which was rapid, automated, and required only a small sample volume with a decreased matrix effect that provided an accurate, precise and simultaneous determination of these seven metabolites derived from five VOCs (acrylamide, 1,3-butadiene, acrylonitrile, benzene, and xylene.)

    • Simultaneous determination of five mercapturic acid derived from volatile organic compounds in human urine by LC-MS/MS and its application to relationship study

      2014, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
      Citation Excerpt :

      It is important to note that the cigarettes used in our study were Chinese Virginia cigarettes, which are different from the blended cigarettes used in the other studies. Several studies reported that CEMA was a useful biomarkers for evaluation of smoke acrylonitrile exposure [18,21], which was confirmed in our previous study [10]. Urinary CEMA levels showed a clear dose–response relationship with daily cigarette consumption.

    • Rapid determination of four short-chain alkyl mercapturic acids in human urine by column-switching liquid chromatography-tandem mass spectrometry

      2014, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
      Citation Excerpt :

      Regarding EtMA, this is confirmed by Scherer et al. [26] who did not succeed in quantifying urinary background levels of EtMA in a majority of the analyzed samples, too. They concluded that EtMA is excreted in very low physiological levels of about 0.5 μg/24 h [26]. An analytical method was developed for the determination of the four short-chain mercapturic acids MeMA, EtMA, PrMA and iPrMA in human urine.

    • A survey of liquid chromatographic-mass spectrometric analysis of mercapturic acid biomarkers in occupational and environmental exposure monitoring

      2014, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
      Citation Excerpt :

      With improvements in data acquisition rate for MS systems, mostly from the advent of much more powerful personal computers, HPLC–MS methods have been more capable of determining multiple mercapturate analytes in a single chromatographic analysis. Simultaneous determination analysis is well suited to investigate complex exposures to volatile organic compounds (VOC) in occupational settings, in exposure of urban populations to air pollutants, and in cigarette smoke [57–59]. Sabatini et al. [22] developed a simultaneous determination for the mercapturates of benzene, toluene and xylene (BTX) using FDA validation guidelines [39] to measure BTX co-exposure in traffic wardens exposures to automobile exhaust and urban air pollutants examining the urine of men and women, including smokers and non-smokers (Table 3).

    View all citing articles on Scopus

    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).

    1

    Current address: Morphotek Inc., 210 Welsh Pool Road, Exton, PA 19341, USA.

    View full text