We developed a method to quantify tobacco specific N-nitrosamines (TSNAs): N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in leaf tobacco.

We compared GC analysis by CI/MS/MS and EI/MS/MSn, different extraction solvents for TSNAs in tobacco: methanol, dichoromethane, buffer pH 4.3 and ethyl acetate with and without the addition of MgSO₄ or NaOH. We tried different purification media: glass wool + MgSO₄, MgSO₄ + Na₂SO₄ and SupelMIP™SPE-TSNAs; and tested Nitrosoguvacoline (NGV), N-nitrosopentyl-3-picolylamine (NNPA) and diphenylamine (DFA) as internal standards.

Although TSNAs could be detected by EI/MS/MSn, the specificity and sensitivity by CI/MS/MS was better due to the instability of N-nitroso compounds. TSNA extraction from one gram of milled leaf tobacco with 10 ml of basic dichloromethane was selected as the best extraction solvent with purification with MgSO4 + Na₂SO₄ and no other treatment before injecting two microliters of the sample in the GC 3900/ Varian Saturn 2100 GC/MS (ion trap). The column was a VF-1ms in CI/MS/MS mode using DFA as an internal standard. The method showed a linear range between 2 and 5000 ng/mL with detection limits (3σ) of 0.01 - 0.07 µg/g of tobacco.

45 different tobacco types were analysed for TSNAs; 34 Virginias from Argentina, Brazil, Italy, Paraguay and Uruguay and 11 different Burleys from Argentina, Paraguay, regional blends and Italy. Total content of TSNAs was almost double in Burley compared with Virginia, having a range from 2 to 12 µg/g of tobacco, with NNN and NAT being the highest, while in Virginia the total TSNAs range was from 1 to 4 µg/g of tobacco, with very similar content for each TSNAs except for NAB which was the smaller. Burley tobacco from Argentina, Paraguay and regional blends showed almost double the content of TSNAs compared with Burley tobacco from Italy.