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

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.