Suresh Ralapati, batf/National Laboratory Center

Capillary Gas Chromatography Method for the Simultaneous Determination of Nicotine, Nornicotine, Myosmine, Anabasine and Anatabine in Tobacco

In 1958, a cooperative effort between tobacco growers, export tobacco leaf dealers, cigarette manufacturers, seed companies, USDA scientists, university researchers and agricultural extension personnel began to evaluate new flue-cured tobacco cultivars before the new varieties are extensively planted and become part of an annual crop. The program is known as “The Regional Minimum Standards Program for the release of Flue-Cured Tobacco Varieties in the United States” (35). The minimum standards program was initiated in response to the release of undesirable varieties in the mid-1950s. Minimum standards for release of a new flue-cured tobacco cultivar originally included chemical, physical and smoke taste evaluations. Currently, emphasis is placed on chemical evaluation of tobacco samples because it was found that cultivars that were rejected based on taste were also rejected based on the chemical evaluation parameters.

The determination of individual alkaloids in tobacco is one requirement of the minimum standards program. Because significant levels of secondary alkaloids in flue-cured tobacco (i.e., nornicotine, myosmine, anabasine and anatabine) impart undesirable taste characteristics in smoke, there is a minimum standard for the relative amounts of secondary alkaloids in a tobacco cultivar. Total secondary alkaloids may not exceed 13% of the total alkaloids found in the tobacco if a new cultivar is to be accepted.

For many years nicotine, nornicotine, myosmine, anabasine and anatabine have been simultaneously determined by capillary gas chromatography in both flue-cured and burley tobacco samples to support the development of new tobacco varieties. The main features of the capillary gas chromatography method can be described as follows:

Method Scope. Ground tobacco samples are extracted with aqueous sodium hydroxide and partitioned into chloroform. The separation and quantitation of individual alkaloids is achieved by capillary gas chromatography with anethole as internal standard.