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Direct analysis of tobacco-specific nitrosamine NNK and its metabolite NNAL in human urine by LC–MS/MS: evidence of linkage to methylated DNA lesions

  • Analytical Toxicology
  • Published:
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Abstract

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its urinary metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are the most investigated carcinogenic biomarkers of tobacco-specific nitrosamines. Here, we report the development of a sensitive and selective assay based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) to simultaneously measure urinary NNK and NNAL. With the use of isotope internal standards and online solid-phase extraction, urine samples were directly analyzed without prior sample purification. The detection limits of this method were 0.13 and 0.19 pg on column for NNK and NNAL, respectively. Inter- and intra-day imprecision was <10 %. Mean recovery of NNK and NNAL in urine was 99–100 %. This method was applied to measure urinary NNK and NNAL in 101 smokers and 40 nonsmokers to assess tobacco exposure. Urinary nicotine, cotinine, N3-methyladenine (N3-MeA), and N7-methylguanine (N7-MeG) were also measured by isotope-dilution LC–MS/MS methods. The results showed that urinary NNK was not observed in all smokers. Urinary free NNAL (0.10 ± 0.09 ng/mg creatinine) and total NNAL (0.17 ± 0.14 ng/mg creatinine) were detected in all smokers. Urinary concentrations of NNAL were significantly correlated with nicotine, cotinine, N3-MeA, and N7-MeG in smokers (P < 0.001). This method enables the direct and simultaneous measurement of NNK and NNAL in urine using only 50 μL of urine. This study first demonstrated in human that urinary tobacco-specific nitrosamines metabolite (NNAL) are highly correlated with their resulting methylated DNA lesions in urine, which may help to substantiate an increased cancer risk associated with tobacco smoke exposure.

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Abbreviations

NNK:

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

NNAL:

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

SPE:

Solid-phase extraction

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

TSNA:

Tobacco-specific nitrosamine

N3-MeA:

N3-methyladenine

N7-MeG:

N7-methylguanine

ESI:

Electrospray ionization

LOD:

Limit of detection

LOQ:

Limit of quantification

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Acknowledgments

We thank the National Science Council, Taiwan, for financial support (Grants NSC 99-2314-B-040-018-MY3 and NSC 100-2628-B-040-001-MY4).

Conflict of interest

None of the authors have any conflict of interest in relation to publication of this paper.

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Authors and Affiliations

  1. Department of Public Health, Chung Shan Medical University, Taichung, 402, Taiwan

    Chiung-Wen Hu & Lai-Man Tam

  2. Department of Family and Community Medicine, Chung Shan Medical University Hospital, Taichung, 402, Taiwan

    Chiung-Wen Hu

  3. Department of Occupational Safety and Health, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N Road, Taichung, 402, Taiwan

    Yu-Wen Hsu & Mu-Rong Chao

  4. School of Pharmacy, China Medical University, Taichung, 404, Taiwan

    Jian-Lian Chen

  5. Department of Occupational Medicine, Chung Shan Medical University Hospital, Taichung, 402, Taiwan

    Mu-Rong Chao

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  1. Chiung-Wen Hu
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  2. Yu-Wen Hsu
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Correspondence to Mu-Rong Chao.

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Hu, CW., Hsu, YW., Chen, JL. et al. Direct analysis of tobacco-specific nitrosamine NNK and its metabolite NNAL in human urine by LC–MS/MS: evidence of linkage to methylated DNA lesions. Arch Toxicol 88, 291–299 (2014). https://doi.org/10.1007/s00204-013-1137-y

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  • DOI: https://doi.org/10.1007/s00204-013-1137-y

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