Analysis of N ′-nitrosonornicotine enantiomers in human urine by chiral stationary phase liquid chromatography–nanoelectrospray ionization–high resolution tandem mass spectrometry

Abstract

We have developed a chiral stationary phase liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method to investigate the enantiomeric composition of low parts per trillion amounts of the carcinogen N′-nitrosonornicotine (NNN) in the urine of cigarette smokers and smokeless tobacco users. (S)-NNN is the major enantiomer in tobacco and is more carcinogenic than (R)-NNN in rats, but no data are available on the enantiomeric composition of NNN in humans. The method used [13C6]NNN as an internal standard and [pyridine-D4]nornicotine to monitor possible artifactual formation of NNN, which was found to be less than 2% of the quantified NNN. The enantiomeric composition of NNN (20.5 ± 27.1 fmol/ml urine) in 20 cigarette smokers was 67 ± 5% (S)-NNN while that in 10 smokeless tobacco users (67.1 ± 56.7 fmol/ml urine) was 56 ± 3% (S)-NNN. These results demonstrate that the highly carcinogenic (S)-NNN is the major enantiomer in human urine, and that the enantiomeric composition of NNN in human urine is remarkably similar to that in cigarette smoke and smokeless tobacco. This is the first study to combine chiral stationary phase separations with nanoelectrospray ionization and high resolution tandem mass spectrometry to quantify trace levels of enantiomeric metabolites in human urine.

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

“…The relationship of urinary NNN- N -oxide levels with the smoking status of the positive smokers can also be found in Tables S1 and S2. The level of NNN- N -oxide can be compared to that of urinary total NNN in cigarette smokers from our two previous studies (60 ± 35 fmol/mL, n = 38; 20.5 ± 27.1 fmol/mL, n = 20) . Since NNN- N -oxide has been shown to be less tumorigenic than NNN in rats and Syrian golden hamsters, these results indicate that pyridine- N -oxidation is a minor detoxification pathway in NNN metabolism …”

“…Since the artifactual formation of NNN can result from nitrosation of urinary nornicotine during analysis, this is a major concern for NNN quantitation in urine. , Thus, we considered the possible artifactual formation of NNN- N -oxide in our analyses. We added ammonium sulfamate, a known inhibitor of nitrosation, to the urine solutions and quantified levels of NNN- N -oxide compared to those in untreated urine.…”

“…This biomarker has been successfully applied in the Shanghai Cohort study, providing strong evidence of its potential in predicting future esophageal cancer incidence in cigarette smokers . However, quantitation of urinary NNN requires extensive precautions to prevent artifactual formation. , Endogenous formation of NNN through facile nitrosation of nornicotine 17 , the metabolite of nicotine and also present in tobacco products, may also cause potential overestimation issues. − It is thus important to develop a new biomarker that can specifically reflect NNN metabolic fate and facilitate tobacco-associated cancer etiology studies.…”

“…Urine Treated with Ammonium Sulfamate. To investigate the possible artifactual formation of NNN-N-oxide resulting from nitrosation of its potential precursor compounds such as nornicotine-1N-oxide during sample preparation, ammonium sulfamate (∼5 mg) was added to the urine sample (0.5 mL) 14,15 with the same mixed solution of CH 3 CN/MeOH/acetone (1.0 mL) and processed as described above. The dried sample was dissolved in 1 mL of H 2 O with addition of another ∼5 mg of ammonium sulfamate.…”

Chiral Stationary Phases for Liquid Chromatography: Recent Developments

“…Along with the continuous developments of this type of CSPs, it is important to emphasize that a broad range of recent applications have also been reported [ 201 , 202 , 203 , 204 , 205 ].…”