A Method for the Determination of Tobacco-specific Nitrosamines (TSNA), Nitrate and Nitrite in Tobacco Leaves and Processed Tobacco

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Abstract
A method for the simultaneous determination of tobacco-specific nitrosamines [TSNA], N'-nitrosonornicotine [NNN], 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone [NNK], N'-nitrosoanabasine [NAB] and N'-nitrosoanatabine [NAT], as well as nitrate and nitrite in tobacco leaves and processed tobacco was developed. The analytical procedure included aqueous extraction of tobacco leaves and processed tobacco respectively under alkaline conditions in the presence of an antibacterial agent and, for TSNA analysis, a further clean-up of the aqueous extract by liquid-liquid partition extraction (kieselguhr column extraction), removal of nicotine, and determination of the nitrosamines by gas chromatography In combination with chemiluminescence detection. It is demonstrated that N'-nitrosopentylpicolylamine [NPePicA] as well as N-nitrosodibenzylamine [NDBenzA] can be used for internal standardization. For clean-up two different extraction procedures can be utilized: alkaline extraction on kieselguhr / ascorbic acid extraction columns or acidic extraction on kieselguhr including prior destruction of nitrite with sodium azide. The recoveries of the internal standards were in a similar range (73-96 %) as those of the TSNA. The analysis of nitrite and nitrate was carried out according to standard analytical procedures using the alkaline aqueous extract. The determination of nitrate and nitrite showed high accuracy and reproducibility. It is demonstrated that, under the conditions described, artifact formation did not occur even when nitrite was present. The method described is specifically applicable for the simultaneous determination of TSNA and nitrate/nitrite in tobacco leaves.