Suresh Ralapati, batf/National Laboratory Center

Chapter 9. Determination of Nicotine in Tobacco, Tobacco Processing Environments and Tobacco Products

Michael F. Borgerding, R. J. Reynolds Tobacco Company
Thomas A. Perfetti, R. J. Reynolds Tobacco Company

Suresh Ralapati, BATF/National Laboratory Center

I. Introduction

From the viewpoint of the analyst, nicotine is a wonderful molecule. Its physical and molecular properties provide the analyst with a host of opportunities for qualitative and quantitative analysis. For example, the vapor pressure characteristics of nicotine are appropriate for analysis by gas chromatography and the presence of heteroatoms in the molecular structure permits selective detection with element specific detectors. Both reverse phase and normal phase liquid chromatography of nicotine are possible because of the solubility characteristics of nicotine and the fact that nicotine can be protonated. Nicotine’s electronic and molecular structures make spectroscopic techniques such as ultraviolet spectroscopy and near infrared spectroscopy viable analysis techniques. While chromatographic and spectroscopic analyses are widely used in analytical chemistry, these techniques are just a few examples of the many approaches to nicotine analysis that are possible. In addition to unique molecular and physical properties that provide many different qualitative analysis opportunities, nicotine is an ideal molecule for quantitative analysis. It is a significant component of the tobacco leaf, often representing more than 98% of the total alkaloid content in tobacco.

Nicotine analysis of tobacco has been conducted for many purposes. The chemical composition of tobacco crops varies from growing season to growing season and from one geographical region to the next. Tobacco nicotine analysis provides one means of characterizing a new tobacco crop and understanding the raw material that will be included in final products. The research and development of new tobacco breeding lines also requires tobacco nicotine analysis. The smoking quality of different tobacco varieties has been related to the alkaloid distribution found in the leaf. Nicotine and other (secondary) alkaloids have been analyzed for many years as new tobacco breeding lines are developed in order to provide tobacco varieties that are palatable to smokers. Another area in which the determination of tobacco nicotine is of critical importance is the classification of tobacco products for subsequent taxation. The presence and location of nicotine within the components of a smoking article are critical to whether the smoking article is classified as a cigarette, cigar or non-tobacco product, and to the amount of tax that is levied by the federal government.

Since the determination of nicotine in tobacco can be undertaken for many different reasons, there are several fundamental questions the analyst must consider before implementing a tobacco nicotine analysis. For example, is a qualitative analysis that determines the presence or absence of nicotine in a sample necessary, or is a quantitative analysis required to accurately determine the amount of nicotine in a sample? If a quantitative analysis is the goal, is it important to measure nicotine specifically, or is it desirable to measure the total alkaloid content of tobacco with the summary response expressed as nicotine? Will nicotine concentration in tobacco be expressed on a dry weight basis or on an “as is” basis? While the purpose for conducting a particular test will largely determine the answers to the types of questions just posed, practical considerations will also shape the implementation of a tobacco nicotine analysis. The current availability of analytical instruments in the laboratory, the cost of any new instruments needed, sample throughput requirements, the level of analyst training required, sample preparation constraints, instrument sensitivity, the limit of detection required and safety considerations, among others, are practical considerations that will help determine what type of tobacco nicotine determination is conducted to address a particular need for analysis.

The legislative and regulatory history of tobacco control efforts is a substantial and varied topic that has been considered by all levels of government, e.g., at the federal, state and local levels in the United States (1). Historically, laws have been enacted to regulate and control the potential fire hazard created by smoking, to increase revenues through taxation and to address the estimated health costs and risks of tobacco use. More recently, tobacco control efforts have included limitations on tobacco use in public places and strengthened restrictions on youth access to tobacco products. Thus, tobacco control efforts have generally affected consumers in a much more direct way than analytical chemists. Since 1996, however, the determination of nicotine in tobacco has become a specific requirement in some state regulations. With this new requirement, the scope and limitations of analytical methods that have been used for tobacco nicotine analysis must be carefully considered.

This chapter will deal with three topics, the determinations of nicotine in tobacco, nicotine in tobacco processing environments (concentrating primarily with processes in the cigarette industry), and regulatory aspects dealing with nicotine in tobacco products. The first topic will be presented as an historical perspective of the progress of researchers in tobacco science and achievements in analytical chemistry made in the determination of alkaloids in tobacco (particularly nicotine) from 1826 to present. The second part of this chapter, on nicotine in tobacco processing environments, will describe tobacco processing that occur within the tobacco industry, mainly the cigarette industry, and the determinations of nicotine that have been made in the individual process streams. This section will describe a typical tobacco manufacturing operation and present reasons why precise and accurate measurements of tobacco nicotine are necessary for quality assurance / quality control determinations. The third part of this chapter will highlight the current legislative and regulatory issues concerning the determinations of nicotine in tobacco and tobacco products.
II. Historical Aspects

Tobacco is one of the most widely used commodities in the world. It has been studied extensively because of its scientific uniqueness, its economic importance in society, the health consequences of tobacco use, the economic and political importance of the industry it produced, because of its ability to generate massive revenues and due to governmental regulation (2,3). Although, thousands of chemicals have been isolated from tobacco, nicotine has received a great deal of attention from the time it was first identified and determined. Nicotine is the principal alkaloid in all tobaccos grown and used commercially today (4).

The history associated with the discovery and use of “oil” derived from tobacco dates back to about 1571 (3). This “oil” prepared by the French chemist Gohory undoubtedly contained some level of nicotine and was used as a remedy for diseases of the skin. In 1660 another French chemist, LeFevre, described means to steam distill tobacco to obtain oil that had medicinal uses (4). In 1807 Cerioli discovered what he called the “olio essenziale” of tobacco (5). In 1809 Vauquelin apparently made the same discovery. Both researchers described the oil as a volatile and colorless substance. Vauquelin recognized the basic nature of the material but failed to recognize its alkaloidal properties. He attributed the basicity of the material to the presence of ammonia.

In 1822, Hermbstadt confirmed Vauquelin’s results of the presence of the oil described in sixteen different species of what is now known as Nicotiana (5). It was not until 1828 that Posselt and Reimann succeeded in isolating a pure sample of the oil and recognized it as an alkaloid. They characterized it as a water-clear liquid, boiling at 246°C, under atmospheric pressure and miscible with water, alcohol, and ether. They named the pure compound nicotine after Jean Nicot who introduced tobacco into the French court in about 1560 (6). In 1826 Unverdorben isolated a water-soluble base from a dry distillation of tobacco (7). The base contained nicotine. Melsens, in 1843, succeeded in isolating nicotine from the smoke of pipe tobacco and assigning the empirical formula, C₁₂ H₁₄ N₂ (8). In 1893 Pinner reported on the final clarification of the constitution of nicotine, determined via degradation studies. Pinner’s structural formula for nicotine was confirmed by Pictet’s classical synthesis of nicotine in 1895. For many years nicotine was believed to be the only alkaloid in tobacco (9). It was not until 1928 that Ehrenstein reported finding nornicotine in tobacco (10). Anabasine was isolated in tobacco by Smith in 1935. Spath and Kesztler isolated anatabine in tobacco in 1937 (10). A. Gautier and G. LeBon in 1892 were the first chemists to clearly recognize that additional alkaloids accompanied nicotine in tobacco smoke. These additional alkaloids were the secondary alkaloids and decomposition products of alkaloids. Unfortunately, Gautier and LeBon did not report further on their observations (11). Wensuch and Scholler, in the early 1930s, worked diligently to separate the secondary alkaloids in tobacco smoke. Though they were not wholly successful they did discover and determine the formula for myosmine in cigar smoke in 1936 with the collaboration of Spath (11). By 1936 Wensuch and Scholler had distinguished a large number of tobacco smoke bases in cigar and cigarette smoke by their behavior during steam distillation and extraction procedures. By preparation of derivatives (picrates and picrolonates) and by qualitative organic reactions many assertions concerning the structure of the smoke bases were made. Until the study by Kuffner, Schick and Buhn in 1959, there was much confusion concerning the correct identity of the secondary alkaloids of tobacco smoke. By the use of paper chromatography Kuffner and co-workers unraveled the confusion. Kuffner and co-workers analytically separated, by two-dimensional chromatograms, and determined, through derivatization, the presence of nineteen tobacco alkaloids in cigar tobaccos and cigar smoke (11).

III. Determination of Nicotine in Tobacco
The initial discoveries of the major alkaloids of tobacco (nicotine, nornicotine, anabasine and anatabine) illustrated the creativity and high degree of chemical knowledge of such notable chemists as Posselt, Reimann, Pictet, Pinner, Spath and Kuffner, to mention only a few. It is amazing that these chemists had the perseverance to diligently work long hours and complete the difficult tasks of separation, isolation, determination and quantitation without the aid of the modern analytical techniques. After the initial identification and determination of nicotine, relatively few techniques or improvements to the chemistry of nicotine alkaloids were made, with the exceptions of the discoveries of the secondary alkaloids in 1928,1935 and 1937. Technology had not advanced at this point in time such that new methods and analytical techniques were available to chemists.

It has been said that the pyrolysis studies of nicotine by Woodward, Eisner and Haines in 1944 began a new phase in the development of our knowledge of the chemistry of nicotine alkaloids (12). The pyrolysis studies on nicotine afforded the opportunity to study the chemical reactivity of nicotine. Several new compounds were identified, and several new analytical techniques that had been recently developed were applied to the identification of nicotine and related alkaloids. Ultraviolet spectroscopy, paper chromatography, countercurrent distribution techniques and new chemical procedures in the investigation of the chemistry of nicotine and its analogues opened the way for investigation of these materials in tobacco, processed tobaccos and tobacco smoke (12). As additional compounds were identified in tobacco, processed tobaccos, tobacco fermentation processes, tobacco smoke and metabolites of tobacco and smoke, new methods of qualitative and quantitative detection of tobacco alkaloids were developed to meet the ever-exacting desires of chemists. The following paragraphs will describe the development of qualitative and quantitative methods developed and employed in the determination of nicotine and related alkaloids.

There are numerous analytical determinations described in the literature for the determination of nicotine. The first official method for the determination of nicotine in tobacco and tobacco products was the Kissling method. The Kissling method involves two steps. First, there is an extraction with ether and, second, this is followed by distillation with steam and titration. In 1909 Bertrand and Javillier were the first to publish a method for the determination of nicotine by a precipitation technique employing silicotungstic acid. In 1911 Chapin evaluated a number of published methods and concluded that Bertrand’s method gave precise and accurate data on the quantity of nicotine in tobacco and tobacco products. This led to its adoption of a new official method for nicotine in tobacco and tobacco products by the Association of Official Agricultural Chemists (AOAC). The Kissling and siliciotungtic acid methods for the determination of nicotine were accurate but time consuming and were subject to interference by ammonia and ammonium salts as shown by Ogg, Willits and Ricciuti in 1950.

In 1950, a significant departure from gravimetry was reported. Willits, Swaim, Connelly and Brice developed a spectrophotometric method for nicotine that was compared to the official AOAC method. It was shown that the spectrophotometric method was relatively rapid, equally reliable, showed no interference from ammonia and ammonium salts and eliminated the solubility errors inherent with the official AOAC method. Since that time many refinements have been made to the official AOAC method. Table 1 presents a representative sampling of research conducted on methods for the determination of nicotine and tobacco alkaloids in a variety of matrices from ~ 1900 to 1998. This table shows a progression of proposed refinements that parallels technological improvements in analytical chemistry in this century. Table 2 lists the current standard and official methods for the determination of nicotine in various matrices.

Somewhat arbitrarily, analytical method research conducted since 1950 can be considered as the basis for current laboratory methods as they are practiced today. Beginning with the spectrophotometric method developed by Willits, et al. in 1950, there have been many analytical methodologies applied to the determination of nicotine, or nicotine and other specific alkaloids, in tobacco. Examples of some of the many different approaches that have been applied are presented chronologically in Figure 1 (13-26).

During the last fifty years, analytical methods reported for nicotine determination have employed the newest instrumental analysis techniques of the day. For example, a technical progression from colorimetry to gas chromatography, liquid chromatography, and finally capillary electrophoresis has occurred between 1950 and 1997. Regardless of the type of end-determination specified for a particular nicotine method, analytical chemists have faced similar challenges regarding accuracy, precision and practical application when developing new methods of analysis. First, quantitative measurement of nicotine in tobacco requires both the extraction of unprotonated nicotine and nicotine bound up as salts with organic acids from the tobacco matrix. Second, interfering substances must be removed from the tobacco extract or a selective detection mode (chemical or instrumental) must be applied before accurate and precise results can be obtained. Third, practical considerations such as sample throughput, simplicity and ruggedness must be optimized. The numerous avenues that tobacco chemists have successfully explored to address these challenges demonstrate their ingenuity and the versatility of the nicotine molecule.

The remainder of this section will focus on three examples of current analytical practices for the determination of nicotine. The first method is a continuous flow analysis method that is probably the most widely practiced method for the determination of nicotine today. The second is a gas chromatography method with which nicotine and other alkaloids are individually quantitated. The third method is intended for the determination of nicotine in smokeless tobacco products.