N'-Nitrosonornicotine (NNN), a tobacco-specific nitrosamine, is a powerful carcinogen in laboratory animals and is considered, together with the related compound NNK, 'carcinogenic to humans' by the International Agency for Research on Cancer. The only available biomarkers for human uptake of NNN are NNN itself and NNN-glucuronide, both of which can be quantified in urine. However, these biomarkers represent only a small percentage of the NNN dose. In a previous study in patas monkeys, we identified trans-3'-hydroxynorcotinine (3-OHNC) as a unique and major urinary NNN metabolite. We are not aware of any previous studies which have reported 3-OHNC in human urine. Therefore, in the study reported here, we developed a method for the analysis of 3-OHNC in human urine as a potential biomarker of NNN uptake. [13C]3-OHNC was added to urine as internal standard and the mixture was treated with �-glucuronidase. After solid-phase extraction, the fraction containing 3-OHNC was analyzed by LC-ESI-MS/MS with selected reaction monitoring for 3-OHNC, m/z 179 - m/z 134 and m/z 179 - m/z 80; and for [13C]3-OHNC, m/z 183 -136 and m/z 183 - m/z 80. Levels of 3-OHNC in the urine of 48 smokers ranged from 32 - 1158 pmol/ml, with a mean of 398 pmol/ml; it was not detected in the urine of non-smokers. We also analyzed the urine of 12 non-smokers who were using the nicotine patch; levels of 3-OHNC ranged from 245 - 1687 pmol/ml, mean 658 pmol/ml. These amounts of 3-OHNC are too high to come from NNN alone. Since the nicotine patch contains also small amounts of nornicotine, we hypothesize that 3-OHNC is a previously undiscovered metabolite of nicotine or nornicotine.

Analysis of trans-3'-hydroxynorcotinine in smokers' urine by liquid chromatography-electrospray ionization-tandem mass spectrometry

248th National Meeting (2014)

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