Assessment of tobacco specific nitrosamines (TSNAs) in oral fluid as biomarkers of cancer risk: A population-based study

Environ Res. 2016 Nov;151:635-641. doi: 10.1016/j.envres.2016.08.036. Epub 2016 Sep 10.


Background: Smoke-free laws are expected to reduce smoking habits and exposure to secondhand smoke. The objective of this study was the measurement of tobacco specific carcinogens (TSNAs) in oral fluid to assess the most suitable biomarker of cancer risk associated with tobacco smoke.

Methods: TSNAs, N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), as well as nicotine and cotinine were measured in oral fluid samples from 166 smokers and 532 non-smokers of the adult population of Barcelona, Spain. A simple method with an alkaline single liquid-liquid extraction with dichloromethane/isopropanol was used and lower limits of quantification for cotinine, NNN, NNK and NNAL were set at 0.10ng/mL, 1.0, 2.0 and 0.50pg/mL respectively. The NNN/cotinine ratio was also calculated.

Results: NNN was the most abundant TSNA present in oral fluid with a significant difference between smokers and non-smokers (mean concentrations of 118 and 5.3pg/mL, respectively, p<0.001). NNK and NNAL were detectable in fewer samples. NNN and cotinine concentrations had a moderate correlation within both groups (Spearman's rank correlation coefficient of 0.312, p<0.001 in smokers and 0.279, p=0.022 in non-smokers). NNN/cotinine ratio was significantly higher (p<0.001) in non-smokers than in smokers, in line with equivalent findings for the NNAL/cotinine ratio in urine.

Conclusions: TSNAs are detectable in oral fluid of smokers and non-smokers. NNN is the most abundant, in line with its association with esophageal and oral cavity cancers. The NNN/cotinine ratio confirms the relative NNN increase in second hand smoke. Findings provide a new oral fluid biomarker of cancer risk associated with exposure to tobacco smoke.

Keywords: NNN; Saliva; TSNA; Tobacco biomarker; Tobacco carcinogen.

MeSH terms

  • Adult
  • Biomarkers / metabolism
  • Carcinogens / metabolism*
  • Carcinogens / toxicity
  • Case-Control Studies
  • Chromatography, Liquid
  • Female
  • Humans
  • Male
  • Middle Aged
  • Neoplasms / etiology*
  • Neoplasms / metabolism
  • Nitrosamines / analysis
  • Nitrosamines / metabolism*
  • Risk
  • Saliva / chemistry*
  • Smoking / adverse effects
  • Smoking / metabolism*
  • Spain
  • Surveys and Questionnaires
  • Tandem Mass Spectrometry
  • Tobacco*


  • Biomarkers
  • Carcinogens
  • Nitrosamines