Suresh Ralapati, batf/National Laboratory Center

Sample Preparation. Each ground tobacco sample is wetted with a small volume of water. After wetting, chloroform and 10% sodium hydroxide are each added and the tobacco sample is then extracted by agitation. Centrifugation follows the extraction step, from which an aliquot of the chloroform layer is analyzed by capillary gas chromatography.
End Determination. A capillary gas chromatograph with temperature programming capability is used to separate the individual alkaloids. Gordon and Greene described capillary column specifications and chromatographic parameters to separate nicotine, nornicotine, myosmine, anabasine and anatabine in a tobacco extract in 1981 (36). An apolar fused silica capillary column (SE-54 stationary phase) was used with flame ionization detection. Total separation time was approximately 40 minutes. The advent of “fast chromatography” has greatly reduced separation times. Separation of the individual alkaloids is now possible in less than six minutes (Figure 4) with a total separation time under ten minutes (Gordon BM, personal communication). Quantitation is based on an internal standard technique.
Nicotine Reporting Requirements. Individual alkaloid concentrations are reported as a percentage of the original tobacco weight on an “as is” basis to an accuracy level of from two to four decimal places, depending on the specific alkaloid.

Studies reported by Gordon and Greene have shown the capillary gas chromatography method to be both accurate and precise for the determination of specific alkaloids. Results from tobacco samples analyzed simultaneously by the Cundiff and Markunas method (37) and the capillary gas chromatography method were in good agreement. Precision was improved with the chromatography method.

The Determination of Nicotine in Smokeless Tobacco Products

In 1996, an initial collaborative investigation of a method for the determination of nicotine in smokeless tobacco was completed. Five corporate laboratories and one university laboratory conducted the investigation as part of a working group formed in response to federal regulations governing smokeless tobacco (Bennett CB, personal communication). The analytical method investigated for smokeless tobacco is similar in some ways to the method described earlier in this section for the determination of specific alkaloids in tobacco. The smokeless tobacco method requires that tobacco samples are ground, made basic with sodium hydroxide and partitioned with methyl t-butyl ether. The organic layer is analyzed by capillary gas chromatography to determine the nicotine concentration in the smokeless tobacco sample. Tobacco nicotine concentrations determined with the method represent nicotine response only, free of any additional response from other alkaloids such as nornicotine or anatabine. The method investigated is described in further detail in the Regulatory Aspects section of this chapter.

Results from an initial collaborative study to investigate the smokeless tobacco method are summarized in Table 3. The five tobacco types evaluated in the study (dry snuff, loose leaf, moist snuff, plug and twist) contained vastly different amounts of moisture. As measured by the oven volatiles method, “moisture contents” ranged from approximately 10% (dry snuff) to in excess of 50% (moist snuff). For all tobacco types, it is clear from the results in Table 3 that repeatable results can be obtained within a given laboratory, and that reproducible results can be obtained between laboratories when using the smokeless tobacco method. In addition to the precision results in Table 3, nicotine recovery studies were performed in two laboratories to assess accuracy (Bennett CB, personal communication). For the five smokeless tobacco types studied, one laboratory found nicotine recoveries of ~ 104 – 108%. The other laboratory found recoveries from 98% to 118%.