Nicotine and four minor alkaloids (nornicotine, anabasine, myosmine and anatabine) are compounds synthesized within the tobacco plant and are precursors of the tobacco-specific N-nitrosamines (TSNAs). The accurate quantitation of these compounds is important in evaluating tobacco leaf quality and final product composition. Products on the market today consist of a wide variety of tobacco blends and widely differing levels of these compounds. Also, many new smokeless tobacco products (STPs) contain flavors that have been shown to compromise quantitation in currently published methods (e.g. CDC method for nicotine).

The objective of this work was to develop a simple, robust and efficient method to analyze nicotine and the minor alkaloids in a wide range of tobacco products/matrices without requiring standard addition. A variety of published methods were evaluated with the focus on extraction efficiency and quantitation. It was observed that the most difficult alkaloid to extract was nornicotine. The methods examined required up to 3 hours to reach an extraction plateau, which is often (erroneously) equated with complete extraction.

Due to the poor extraction efficiency of the methods evaluated, a new method was developed that extracts the alkaloids within 30 minutes using 5N NaOH (aqueous) to pretreat the matrix and diluting with methanol to make the extract suitable for GC/MS. By using GC/MS as the analytical system, the alkaloid levels in Quest 3 tobacco and a variety of flavored STPs were determined with excellent precision and no observed interferences. The levels of nornicotine from this method ranged from 15 to 80% higher than the other methods tested, while the nicotine was not significantly different.