Combined analysis of N′-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in the urine of cigarette smokers and e-cigarette users

• Urinary total NNN and total NNAL are important human carcinogen biomarkers.
• An LC–ESI+–MS/MS method was developed for their combined analysis in human urine.
• Use of monitor amine [pyridine-D4]nornicotine assessed artifact formation.
• Smokers had significantly higher levels of total NNN and NNAL than e-cigarette users.

A liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI+–MS/MS) method for the analysis of the tobacco-specific carcinogens N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and their glucuronides (total NNN and total NNAL) in human urine was developed. The method has excellent accuracy and intra-day and inter-day precision, and limits of quantitation of 0.015 and 0.075 pmol/mL urine, respectively, for total NNN and total NNAL. A unique aspect of this method is internal assessment of possible artifactual formation of NNN by inclusion of the monitor amine [pyridine-D4]nornicotine. We found that artifactual formation of NNN comprised only 2.5% of the measured amounts of total NNN in urine of cigarette smokers, under our conditions using ammonium sulfamate as an inhibitor of nitrosation. The method was applied to urine samples from cigarette smokers and e-cigarette users. Levels of total NNN and total NNAL in the urine of cigarette smokers averaged 0.060 ± 0.035 pmol/mL and 2.41 ± 1.41 pmol/mL urine, (N = 38), respectively, which were both significantly greater than in the urine of 27 e-cigarette users.