Detection and quantitation of N'-nitrosonornicotine in human toenails by liquid chromatography-electrospray ionization-tandem mass spectrometry

A46

Evaluation of tobacco carcinogen uptake and metabolism in individuals, prediction of potential health risks in people who are exposed to tobacco products, and development of preventive measures is possible through the use of biomarkers of tobacco carcinogen exposure. Recently, we developed a sensitive mass-spectrometric method for analysis of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of a tobacco-specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, in human toenails and, in a separate study, provided essential validation data for the use of toenail biomarkers in the investigation of the role of chronic tobacco smoke exposure in human cancer. In the present study, we report the presence of another tobacco-specific carcinogen, N'-nitrosonornicotine (NNN) in human toenails. Based on its occurrence in tobacco products and cigarette smoke and on its carcinogenic activity in laboratory animals, NNN is believed to be a cause of esophageal cancer in smokers and is classified by the International Agency for Research on Cancer as carcinogenic to humans. Our robust and highly sensitive method for NNN analysis in human toenails involves digestion in 10N NaOH, purification of the digests on solid phase extraction cartridges, and quantitation by liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring. The method is characterized by high accuracy and precision, and its limit of detection reaches into the sub-femtomol range. Toenails of 17 smokers were analyzed for total NNN. Mean total NNN level in these samples was 4.63 ± 6.48 fmol/mg toenail and correlated with previously reported total NNAL (r = 0.96, P < 0.0001), total nicotine (r = 0.48, P < 0.05), and total cotinine (r = 0.87, P < 0.0001). An interesting finding was that amounts of NNN in smokers' toenails were generally higher than those of total NNAL. The ratio of toenail NNN to NNAL averaged 2.8, while the previously reported ratio between these biomarkers in smokers' urine was 0.1. NNN was also found in toenail samples from 12 nonsmoker volunteers, averaging 0.35 ± 0.16 fmol/mg and positively correlating with toenail cotinine (r = 0.58; P = 0.05). Use of toenail biomarkers offers a number of advantages over urine analysis, including potential evaluation of long-term cumulative exposure. Investigation of the role of secondhand smoke exposure in human cancer is the major potential application of this new biomarker.