Analysis of Certain Nitrogenous Compounds in Tobacco. Part 1: Adenosine, 2,5- and 2,6-Deoxyfructosazines, Mannosamine and Glucosamine

Nitrogenous compounds such as amino acids and proteins are frequently analyzed in tobacco since they are considered precursors of toxicants in cigarette smoke. However, much less attention is given to other nitrogenous compounds such as amino sugars and deoxyfructosazines, although their concentration in tobacco can be equal to or even higher than that of most free amino acids. These nitrogenous compounds may contribute to the formation of toxicants in smoke, or may contribute to the sensory properties of cigarette smoke, reasons for which their analysis is important. This study describes a procedure for the analysis of adenosine, 2,5- and 2,6-deoxyfructosazines (DFs), mannosamine and glucosamine in tobacco. The analysis uses a liquid chromatographytandem mass spectrometry (LC/MS/MS) technique. Sample preparation for analysis consists of the extraction of the tobacco with a solution of 90% water and 10% methanol, followed by filtration. The separation of the analytes was done on a hydrophilic interaction liquid chromatography HILIC column using an isocratic procedure with a solvent consisting of 78% CH₃CN, 22% H₂O, that also contained 0.1 % HCOOH and 0.143 g/L CH₃COONH₄. The measurements were done using electrospray positive ionization mass spectrometric detection. The analytical procedure was validated and was proven very reliable. A number of tobaccos were analyzed, including several fluecured and Burley USA tobaccos, off-shore tobaccos, two

Oriental tobaccos, two green tobaccos, as well as tobaccos from commercial and Kentucky reference cigarettes. The ranges for the analytes per g tobacco were found between 0.4 and 20.3 µg/g for adenosine, between 0.0 and 608.5 µg/g for 2,5-DF, between 0.0 and 424.5 µg/g for 2,6-DF, between 12.5 and 415.5 µg/g for mannosamine and between 25.9 and 1885.7 µg/g for glucosamine. The study also indicated that the levels of DFs and that of the amino sugars in tobacco show a very good correlation. This correlation can be explained by the same source of the two classes of compounds, namely the reaction of (reducing) sugars and ammonia.