Mass Spectrometric Quantitation of N'-Nitrosonornicotine-1 N-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.

Figure 1.

Chromatograms and the MS² and MS³ fragmentation patterns of NNN-N-oxide and [pyridine-d₄]NNN-N-oxide under CID conditions.

Figure 2.

Calibration curves of NNN-N-oxide at (A) low and (B) high concentration ranges. A mean value of 3 replicates was used to establish each calibration curve with the standard error shown in bars.

Figure 3.

Typical MS traces of NNN-N-oxide in the urine of pooled non-smokers, pooled cigarette smokers and smokeless tobacco users.

Scheme 1.

Major urinary metabolites formed by NNN and NNK metabolism and their formation from the minor metabolic pathways of nicotine metabolism.