Mass Spectrometric Quantitation of N′-Nitrosonornicotine-1 N-oxide in the Urine of Cigarette Smokers and Smokeless Tobacco Users

Yupeng Li, Stephen S. Hecht

Research output: Contribution to journalArticlepeer-review

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N′-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which always occur together and are present exclusively in tobacco products, are classified as "carcinogenic to humans" (Group 1) by the International Agency for Research on Cancer. While 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) serves as an excellent biomarker for NNK exposure, the currently available biomarker for NNN exposure is urinary "total NNN" (free NNN plus its N-glucuronide). Quantitation of urinary NNN requires extensive precautions to prevent artifactual formation of NNN resulting from nitrosation of nornicotine during analysis. NNN itself can also be formed endogenously by the same nitrosation reaction, which may sometimes cause an overestimation of exposure to preformed NNN. It is thus important to develop an alternative biomarker to specifically reflect NNN metabolic fate and facilitate relevant cancer etiology studies. In this study, we report the first detection of N′-nitrosonornicotine-1N-oxide (NNN-N-oxide) in human urine. Using a highly specific and sensitive MS3transition-based method, NNN-N-oxide was quantified with a mean level of 8.40 ± 6.04 fmol/mL in the urine of 10 out of 32 cigarette smokers. It occurred in a substantially higher level in the urine of 13 out of 14 smokeless tobacco users, amounting to a mean concentration of 85.2 ± 96.3 fmol/mL urine. No NNN-N-oxide was detected in any of the nonsmoker urine samples analyzed (n = 20). The possible artifactual formation of NNN-N-oxide during sample preparation steps was excluded by experiments using added ammonium sulfamate. The low levels of NNN-N-oxide in the urine of tobacco users indicate that the pyridine N-oxidation pathway represents a minor detoxification pathway of NNN, which further supports the importance of the α-hydroxylation pathway of NNN metabolic activation in humans.

Original languageEnglish (US)
Pages (from-to)1579-1588
Number of pages10
JournalChemical research in toxicology
Issue number9
StatePublished - Sep 19 2022

Bibliographical note

Funding Information:
This study was supported by grant CA-81301 from the National Cancer Institute. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, supported in part by Cancer Center Support Grant CA-077598. The authors thank Dr. Laura Maertens for the biorepository management and Nicole Thomson, Steven Carmella, and Mei-Kuen Tang for their help with locating the urine samples and providing the urinary biomarker data. We thank Dr. Jiehong Guo for her help with statistical analysis. We also thank Drs. Linda von Weymarn, Yingchun Zhao, and Peter Villalta for help with the operation of the mass spectrometer. Editorial assistance from Robert (Bob) Carlson is greatly appreciated.

Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.


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