Nicotine exposure and metabolizer phenotypes from analysis of urinary nicotine and its 15 metabolites by LC-MS

Bioanalysis. 2011 Apr;3(7):745-61. doi: 10.4155/bio.11.42.


Smokers who inhale less deeply are exposed to lower amounts of the toxic substances present in tobacco smoke. In order to more rigorously assess tobacco smoke exposure, it is necessary to have an accurate method for quantifying nicotine and all of its known metabolites.

Methods: A stable-isotope dilution LC-MRM/MS assay has been developed for quantification of urinary nicotine and the 15 possible metabolites that could arise from known metabolic pathways. Nicotine, cotinine, trans-3´-hydroxy-cotinine, nicotine-N-oxide, cotinine-N-oxide, nornicotine, norcotinine and 4-hydroxy-4-(3-pyridyl)butanoic acid were quantified by direct analysis. The corresponding glucuronide metabolites were quantified after urine hydrolysis with β-glucuronidase.

Results: Nicotine and all 15 nicotine metabolites were quantified by LC-MRM/MS in most urine samples from 61 tobacco smokers. Urinary nicotine and metabolite concentrations ranged from 7.9 to 337.8 µM (mean 75.5 ± 67.8 µM). Three nicotine metabolizer phenotypes were established as reduced metabolizers (ratio < 8), normal metabolizers (ratio 8-30), and extensive metabolizers (ratio > 30). 4-hydroxy-4-(3-pyridyl)butanoic acid, which has not been quantified previously, was an abundant metabolite in all three phenotypes.

Conclusion: Using this assay it will now be possible to determine whether there are relationships between nicotine exposure and/or metabolizer phenotype with exposure to toxic substances that are present in tobacco smoke and/or to biological response biomarkers to tobacco smoking. This will help in identifying individuals at high risk for developing smoking-related diseases as well as those amenable to smoking cessation programs.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / analysis
  • Biomarkers / urine
  • Biopharmaceutics / methods*
  • Chromatography, Liquid / methods*
  • Female
  • Glucuronides / metabolism
  • Glucuronides / urine
  • Humans
  • Male
  • Mass Spectrometry / methods*
  • Middle Aged
  • Nicotine / analysis
  • Nicotine / metabolism*
  • Nicotine / urine*
  • Phenotype
  • Smoking / metabolism
  • Smoking / urine*


  • Biomarkers
  • Glucuronides
  • Nicotine