Separation of N′-nitrosonornicotine isomers and enantiomers by supercritical fluid chromatography tandem mass spectrometry

Abstract N′-nitrosonornicotine (NNN) is one of the most prevalent and toxic tobacco-specific nitrosoamines. A chiral center at its 2′-position results in R and S enantiomers, the partial double bond character of the N-N=O group also results in E and Z isomers, therefore, NNN can form a total of four absolute configurations (E-(R)-NNN, E-(S)-NNN, Z-(R)-NNN, and Z-(S)-NNN). This study investigated the resolution of R/S enantiomers and E/Z isomers of NNN by supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS). The baseline separation of E/Z-(R,S)-NNN isomers/enantiomers was accomplished through the optimization of chiral columns and co-solvents. Due to the lack of single standard of E/Z isomers, only R-NNN (sum of E-(R)-NNN and Z-(R)-NNN) and S-NNN (sum of E-(S)-NNN and Z-(S)-NNN) were further examined. Through the comprehensive optimization of SFC-MS/MS conditions, R-NNN and S-NNN were separated with a run time of 5 min, the developed method was validated, and its applicability to the determination of NNN enantiomers in burley tobacco samples was demonstrated. This study could be applied to preparative separation of single enantiomer and/or isomer of NNN, and could provide potential benefits to biologic activity studies on these enantiomers and isomers.