Determination of tobacco specific nitrosamines (TSNAs) in tobacco and tobacco smoke by GC-MS/MS
NNN (N'-nitrosonornicotine), NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), NAB (N'-nitrosoanabasine) and NAT (N'-nitrosoanatabine) are the most common tobacco specific nitrosamines (TSNAs) measured in tobacco and tobacco smoke. While CORESTA Recommended Methods (CRMs) exist for the determination of TSNAs in tobacco (CRM No. 72) and tobacco smoke (CRM No. 75) using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the objective of this study was to develop and validate a more sensitive, selective and higher throughput method for TSNAs using gas chromatography-tandem mass spectrometry (GC-MS/MS). This new method involved extraction of the tobacco samples using an organic solvent followed by solid phase extraction (SPE) to reduce matrix interferences and to concentrate the sample extract. Samples were then analyzed by GC-MS/MS in the chemical ionization (CI) mode using multiple reaction monitoring (MRM). All requirements for method validation were met including linearity, accuracy, precision, limits of detection (LOD), limits of quantitation (LOQ), method robustness, standard and sample extract stability. For example, the linearity for the GC-MS/MS method was demonstrated with a coefficient of determination of R2>0.995. For tobacco analysis, the LOQ (based on the lowest calibration standard) for NNN, NAT, and NNK was 162 ng/g and the LOQ for NAB was 40 ng/g. For smoke analysis, the LOQ for NNN, NAT and NNK was 8 ng/cig, and the LOQ for NAB was 2 ng/cig. The analytical performance of GC-MS/MS method was compared to the conventional LC-MS/MS method based on the analysis of CORESTA tobacco reference products (CRP1, CRP2, CRP3 and CRP4) and two Kentucky reference cigarettes (3R4F and 1R5F) where both methods showed comparable quantification of TSNAs. However, this new GC-MS/MS platform provided lower LOQs, higher selectivity, and higher throughput compared to the LC-MS/MS method.