Suresh Ralapati, batf/National Laboratory Center

Sample Preparation. Nicotine in ATF regulated tobacco products is extracted using a method described by Ralapati (26,89). A sample of tobacco (50-100 mg), used as is or ground, is extracted with occasional shaking in 10 mL of distilled, deionized water at room temperature for 1 h. The tobacco extracts obtained are used fresh, usually in less than 4 h. The aqueous tobacco extracts are diluted 1:10 or 1:20 with water to give a working sample. An aliquot of working sample is filtered directly into an autosampler vial for CE analysis.
End Determination. CE is typically performed on a SpectraPHORESIS 500 System equipped with a UV absorbance detector, an autosampler and a data system using PC1000 System Software version 3.0.1A from Thermo Separation Products. The capillary is preconditioned prior to its first use and thereafter at the beginning of each day as follows: 10 min wash with 1.0 N NaOH at 60^oC, 5 min wash with 0.1 N NaOH at 60^oC, 5 min wash with distilled, deionized water at 60^oC and 5 min wash with run buffer at 20^oC. Electrolyte solutions are degassed under vacuum for 2 min prior to each run. Two buffer blank runs and two sample runs are performed prior to starting the actual analysis to allow the system to stabilize, and for the electrolyte solutions, nicotine standards and tobacco sample extracts to equilibrate to the run conditions. The capillary is rinsed with 0.1 N NaOH and then with distilled, deionized water each for 1 min between runs in a postrun capillary wash. The capillary is rinsed and filled with the run buffer in a prerun capillary wash for 1 min. Nicotine is analyzed by CE using the separation conditions described in Table 18.
Nicotine Reporting Requirements. Current ATF regulatory classifications are based on qualitative information. As such, declaration of the presence or absence of nicotine in a particular sample is the only reporting requirement with this method. However, quantitative determination of nicotine in tobacco is possible with the method as described below.
Before establishing standard CE analysis conditions at the ATF National Laboratory, research was conducted to optimize CE analysis conditions. As mentioned above, nicotine in an aqueous solution can exist in three different ionic forms depending on the pH. This pH dependence has definite impact on the CE performance when analyzing nicotine (26). CE analysis of nicotine in sodium phosphate buffer at pH 6.9 and pH 2.5 demonstrate two important pH effects, (1) impact on the net mobility of nicotine and (2) detection sensitivity. As a monoprotonated species at pH 6.9, nicotine has a high electrophoretic mobility (migration time of 3.276 min) compared to nicotine as a diprotonated species at pH 2.5 (migration time of 6.200 min)(Figure 21). Second, as a diprotonated species (pH 2.5), the molar extinction coefficient of nicotine at 260 nm is greater than that observed for nicotine as a monoprotonated species (pH 6.9) (Figure 18). These experimental observations are in agreement with similar observations reported by others (15,85). Based on these results, a pH 2.5 sodium phosphate buffer was established as the standard analysis condition for CE analysis of nicotine at the National Laboratory.

Method precision (repeatability) has been evaluated for the standard conditions used at the National Laboratory (26). Repeated injections of nicotine standards typically yield coefficients of variation of 8 or less for corrected peak area response and 2.5 or less for migration times. Corrected peak area takes into account that in CE, analytes with high electrophoretic mobility pass the detection window faster than low mobility analytes. Identical concentrations of highly mobile analytes, therefore, will generate smaller peak areas when compared to less mobile analytes. Corrected peak area is calculated as the peak area of nicotine divided by the migration time of nicotine.

Many types of products are analyzed for classification purposes at the National Laboratory. Evaluation of a beedi provides an example of the classification process employing CE analysis. According to the Dictionary of Tobacco Terminology, a beedi (also known as bidi, biri) is “a form of cigarette found in India; it consists of granulated tobacco rolled in a section of Indian ebony leaf and tied with thread” (79) (Figure 22). Prior records have indicated that neither was this product analyzed for nicotine, nor a determination made to properly classify it for tax purposes according to the Code of Federal Regulations (78). According to the Dictionary of Economic Plants, the species of Indian ebony leaf used for wrapping beedies in India is Diospyros melanoxylon Rox (91,92). Furthermore, according to Hegnauer’s Chemotaxonomie der Pflanzen, this and the other Diospyros species contain no alkaloids (93, Ravindranath B, personal communication). Based on this information, evaluation of beedies provide both an example of the regulatory classification process for tobacco products and a challenging tobacco product substrate.


CE analysis results show the presence of nicotine in the beedi filler, indicating that the beedi is consistent with a tobacco product. On the other hand, CE analysis of the beedi wrapper demonstrates an absence of nicotine, indicating that the beedi is consistent with a cigarette and would be properly classified as such. Therefore, from CE nicotine analysis results, it is possible to show that the beedi is indeed a tobacco product, and beedies are properly classified as cigarettes. Results are shown in Figure 23. Also, as can be seen in Figure 23, the beedi wrapper did not analyze positive for nicotine which is in agreement with published reports (93, Ravindranath B, personal communication).


It may come as a surprise to some, but many of the products analyzed by the National Laboratory do not have a dominant position in the U.S. marketplace. Mandalay Cheroot is another product that has been analyzed for nicotine and subsequently classified according to ATF regulations (Figure 24). Originally manufactured and imported from Myanmar (formerly, Burma), these Mandalay Cheroots consist of cut granulated tobacco wrapped in the leaf material from the Sebesten tree (Cordia dichotoma Forst), that belongs to the family Boraginaceae (Tana leaf). Like the beedies, CE analysis of the cheroot, shows the presence of nicotine in the filler material, indicating that the cheroot is consistent with a tobacco product. CE analysis of the wrapper did not demonstrate the presence of nicotine, indicating that the cheroot is consistent with a cigarette. Based on these CE data, Mandalay cheroot is indeed a tobacco product, and would be properly classified as a cigarette.

Capillary electrophoresis has also been applied to the analysis of nicotine in other ATF regulated tobacco products, including cigarettes, cigars, pipe tobacco, roll-your-own (RYO), chewing tobacco and snuff. For the different tobacco products analyzed, minor peaks, in addition to the major nicotine peak, are also detected with virtually no interference. The electropherograms obtained are generally clean and straightforward with all peaks well resolved. Minor alkaloids such as nornicotine, anabasine and anatabine have been reported to occur in tobacco and tobacco products (94). Two of the minor alkaloids, nornicotine and anabasine, have been identified in a CE analysis of ATF regulated tobacco products (85). Table 19 shows the application of CE to the analysis of nicotine in a wide variety of ATF regulated tobacco products.

Quantitative Analysis of Nicotine by CE. Despite the quantitative capability of the analytical techniques discussed above (spectrophotometry, gas chromatography and capillary electrophoresis), attempts to quantitate the amount of nicotine in ATF regulated tobacco products have not been made hitherto. The main reason for this is that the ATF regulation as it stands today calls for regulating the tobacco product based on the presence, not the amount of, nicotine.

In 1994, the Food and Drug Administration (FDA) held discussions to consider a proposal(s) to regulate the tobacco products based on nicotine content. Even though this proposal has yet to become law, scientists at the National Laboratory Center have conducted CE studies to quantitatively analyze nicotine in ATF regulated tobacco products in anticipation of a possible FDA proposal to regulate tobacco products based on nicotine content. Method validation studies have been conducted to determine response linearity, limits of detection and limits of quantitation for nicotine when analyzing tobacco products (89). Quantitative analysis results for selected ATF regulated tobacco products are summarized in Table 20.

Quantitative analysis results of nicotine in ATF regulated tobacco products determined by CE (89) (Table 20) compare reasonably well with the ranges reported for similar products in other studies (80,82,94-96). However, some differences between CE results and values reported in the literature do exist.

As discussed earlier in this chapter, the nicotine content of tobacco is greatly influenced by the cultural practices employed. The nicotine content of the fully aged and cured tobacco leaf in the field is no indication of the nicotine content in the final commercially manufactured product. This is because in commercial tobacco products, processing parameters and conditions greatly impact final nicotine content. For example, gradual and mild curing and aging conditions, generally seen in the manufacture of cigarette products, show minor losses of nicotine. On the other hand, heavy aging and fermentation processes, commonly seen in the manufacture of cigar and smokeless tobacco products, show drastic losses of nicotine (80). Nevertheless, the nicotine content of popular blended US cigarettes is generally about 1.75%. For cigars of all classes, the nicotine content is approximately 1.5%. In case of pipe tobaccos containing the popular blended mixtures, the nicotine content is about the same as the blended cigarettes. The scrap type of chewing tobacco contains less than 1.0% nicotine while the plug type of chewing tobacco contains nicotine ranging from 1.5-2.5% (80,82,94-96). Snuff is reported to contain nicotine ranging from <1.0-3.35% (82,93).

In summary, the analysis of nicotine in tobacco is the primary regulatory determinant that drives the science and regulation performed at the ATF National Laboratory Center. ATF regulations, as they are applied today, monitor the presence (without regard to the amount) of nicotine in tobacco products. Nevertheless, nicotine can be quantitated with techniques such as capillary electrophoresis in response to any future regulation.

Tobacco Nicotine Analysis Conducted in Industry Laboratories to Comply with Federal and State Regulations

Current regulatory requirements that include tobacco nicotine testing are (1) federal regulations that require the determination of nicotine in smokeless tobacco samples (72) and (2) state regulations that require the determination of nicotine in cigarettes and smokeless tobacco (74-76). These regulations do not specify established standard methods for the determination of nicotine in tobacco (i.e., AOAC standard methods (97,98), CORESTA methods (99) and ISO methods (34) are not mandated). Rather, current regulations specify a single methodology that, at the time of this writing, has not been fully developed and validated for use in either federal or state regulation (100,101). The application of a single analysis methodology is notable since federal and state regulations deal with different forms of tobacco (smokeless tobacco vs. cigarette cut filler and smokeless tobacco).

While the federal and state regulations designate a common tobacco nicotine testing methodology, a fundamental point of difference between the two types of regulation is the method development and validation approach that has been applied as regulation is formed. Development of a testing method to meet the federal smokeless tobacco regulations has, in principle, incorporated several of the keys technical steps that must be followed when establishing a new standard method (102 – 104). As of early 1999, the method development and validation process initiated in support of federal regulations has not been completed. Issues of method validation and interlaboratory method comparison have not been addressed in the same way during formation of state regulations. In an effort to promulgate state regulation as expeditiously as possible and rapidly provide data to consumers, state regulators have attempted to adapt “published” testing procedures without the benefit of any experimentation to confirm the appropriateness of the “adapted” procedures. Thus, although a common tobacco nicotine testing methodology has been proposed or adopted for federal and state regulations, differences in test purpose, the type of tobacco sample(s) evaluated and the method development and validation processes followed are found when the two types of regulation are compared.

The Determination of Nicotine in Smokeless Tobacco Samples (Federal Regulation). In 1986, the 99^th Congress enacted into law the Comprehensive Smokeless Tobacco Health Education Act (72). The stated purpose of this act is “to provide for public education concerning the health consequences of using smokeless tobacco products.” The law calls upon the Department of Health and Human Services (HSS) to develop public education programs, the Federal Trade Commission to mandate smokeless tobacco warning labels and tobacco manufacturers to report ingredients added to smokeless tobacco. In addition to added ingredients, the federal regulation requires that the quantity of nicotine contained in fine cut, ground, powdered or leaf tobacco that is intended to be placed in the oral cavity be reported to the HSS Secretary on an annual basis.

Method Development and Validation Issues. To meet the annual reporting requirement, the federal government has recognized that a standard methodology to measure the quantity of nicotine contained in smokeless tobacco is necessary. The federal government has also recognized that the annual reporting requirement cannot be implemented until a standard protocol can be developed and validated according to accepted scientific practices (100,101). Further, the Department of Health and Human Services has affirmed the need for uniform testing methodology if understandable data are to be obtained. To achieve a technically sound method, the department of Health and Human Services has determined that method development and validation should be completed by tobacco industry scientists, rather than mandating analytical methodology as a matter of policy without regard for scientific process.

While the draft methodology proposed for the determination of nicotine in smokeless tobacco has received substantial technical criticism from smokeless tobacco manufacturers and others (105,106), several aspects of the process that has been followed to establish the draft methodology, as described in the Federal Register, are worth noting:

• 
  Prior to 1989 each smokeless tobacco company employed different methods for nicotine analysis.
  
• 
  The Department of Health and Human Services requested that “a standard methodology be developed to ensure the accuracy and reliability of the information on nicotine and moisture.”
  
• 
  The Department of Health and Human Services recognized that it did not have the technical expertise and resources to develop a standardized methodology.
  
• 
  A working group comprised of the ten major domestic manufacturers of smokeless tobacco was formed “to begin the development of uniform methods for analyzing smokeless tobacco products for nicotine and moisture content.”
  
• 
  The working group reached consensus on a technical alternative for subsequent method validation. The technical alternative was communicated to the Department of Health and Human Services.
  
• 
  After revising the recommended technical alternative based on comments from peer reviewers, the Centers for Disease control (CDC) published testing methodology for public comment.
  
• 
  Nicotine reporting requirements for smokeless tobacco will not be implemented until the methodology has been finalized and published.
  

Smokeless Tobacco Method Synopsis. This section will briefly summarize the main features of the draft analytical method proposed by federal regulators for the determination of nicotine in smokeless tobacco.