Mass Spectrometric Quantitation of N′-Nitrosonornicotine-1N-oxide in the Urine of Cigarette Smokers and Smokeless Tobacco Users

Abstract

N′-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which always occur together and are present exclusively in tobacco products, are classified as “carcinogenic to humans” (Group 1) by the International Agency for Research on Cancer. While 4-(methyl­nitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) serves as an excellent biomarker for NNK exposure, the currently available biomarker for NNN exposure is urinary “total NNN” (free NNN plus its N-glucuronide). Quantitation of urinary NNN requires extensive precautions to prevent artifactual formation of NNN resulting from nitrosation of nornicotine during analysis. NNN itself can also be formed endogenously by the same nitrosation reaction, which may sometimes cause an overestimation of exposure to preformed NNN. It is thus important to develop an alternative biomarker to specifically reflect NNN metabolic fate and facilitate relevant cancer etiology studies. In this study, we report the first detection of N′-nitrosonornicotine-1N-oxide (NNN-N-oxide) in human urine. Using a highly specific and sensitive MS3 transition-based method, NNN-N-oxide was quantified with a mean level of 8.40 ± 6.04 fmol/mL in the urine of 10 out of 32 cigarette smokers. It occurred in a substantially higher level in the urine of 13 out of 14 smokeless tobacco users, amounting to a mean concentration of 85.2 ± 96.3 fmol/mL urine. No NNN-N-oxide was detected in any of the nonsmoker urine samples analyzed (n = 20). The possible artifactual formation of NNN-N-oxide during sample preparation steps was excluded by experiments using added ammonium sulfamate. The low levels of NNN-N-oxide in the urine of tobacco users indicate that the pyridine N-oxidation pathway represents a minor detoxification pathway of NNN, which further supports the importance of the α-hydroxylation pathway of NNN metabolic activation in humans.

Mass Spectrometry-Based Metabolic Profiling of Urinary Metabolites of N′-Nitrosonornicotine (NNN) in the Rat

“…Racemic [pyridine- d 4 ]­NNN was purchased from Toronto Research Chemicals (Ontario, Canada). The other isotopically labeled standards including [CH 2 - d 4 ]­HPB, [ 13 C 6 ]­NNN, [pyridine- d 4 ]­NNN- N -oxide, [pyridine- d 4 ]­keto acid, and [ 13 C 6 ]­hydroxy acid were synthesized during our previous studies. ,,, [Pyridine- d 4 ]­norcotinine was generously provided by Dr. Sharon Murphy’s laboratory. Phosphate buffered saline (PBS, 10X) was purchased from Invitrogen (Grand Island, NY) and diluted to 1X before use.…”

“…However, it was not observed in the urine and serum of the patas monkey . We recently reported the first identification of NNN- N -oxide in the urine of cigarette smokers and smokeless tobacco users . The levels of NNN- N -oxide were near the quantitation limit in the urine of cigarette smokers, amounting to a mean of 8.40 ± 6.04 fmol/mL urine with a detection rate of 31.2%.…”