Suresh Ralapati, batf/National Laboratory Center

Regulatory: The presence, not the amount, of nicotine is the primary determinant of what constitutes a tobacco product.

Abundance: Nicotine is the most abundant tobacco alkaloid, comprising about 98% of the total alkaloid content.

Nicotine is one of the few liquid alkaloids. This principal tobacco alkaloid

contains pyridine and pyrrolidine base rings [C₁₀N₁₄N₂; 3-(1-methyl)-2-pyrrolidinyl)-pyridine]. First isolated from smoke in 1809 by Vauquelin (79), and its properties described in 1828 (80), nicotine is a colorless to pale yellow liquid, found in tobacco leaves and smoke, and which turns brown when exposed to light and air. Nicotine is a molecule that is strongly chromophoric in the ultraviolet region and is directly detected at about 260 nm (81). Nicotine has been analyzed using spectrophotometry (15), gas chromatography (82), high-performance liquid chromatography (83,84) and more recently capillary electrophoresis (26,85).


Historical Development of Analytical Methods for Analysis of Nicotine in ATF Regulated Tobacco Products. Advances in the technology applied for the analysis of nicotine in ATF regulated tobacco products have occurred with time. The analytical methods for the analysis of nicotine historically practiced at ATF include spectrophotometry and gas chromatography. These two techniques will be discussed briefly in this section followed by a review of the current analytical method, the technique of capillary electrophoresis (CE).

Spectrophotometric Analysis of Nicotine. Historically, nicotine was first routinely analyzed in tobacco products at the ATF National Laboratory Center using spectrophotometry. The advantages of spectrophometry include ease of use, rapid analysis capability and well established, reliable instrumentation. Unfortunately, there are inherent disadvantages. The main disadvantage with using spectrophotometry, despite the simplicity of technique as well as instrumentation, is that structurally related tobacco alkaloids, for example nornicotine, and other UV absorbing compounds also absorb in the 260 nm region, thus interfering with the absorbance of nicotine. Nevertheless, prior analysis of nicotine in ATF regulated tobacco products reported total alkaloids as nicotine.

When working with pure nicotine (for example, nicotine standards), UV absorbance measurements can be very useful, providing valuable information regarding the chemistry of the nicotine molecule. Nicotine absorbs strongly in the UV at about 260 nm, the maximum UV absorbance of nicotine (Figure 17). This UV absorbance is dependent on the pH of the solvent used. Figure 18 shows the UV spectra of nicotine in distilled, deionized water, (B), sodium phosphate buffer, pH 6.9 (A), and sodium phosphate buffer, pH 2.5 (C). It can be seen that the UV absorbance of nicotine at 260 nm in sodium phosphate buffer, is different at pH 2.5 and pH 6.9. This can be explained as follows.

When nicotine is dissolved in water, it can exist in one of three forms depending on the pH: unprotonated (free base), monoprotonated and diprotonated (85,86). Figure 19 taken from Morie (86) shows the fraction of these nicotine species as a function of pH. At pH 2.5, nicotine exists as a diprotonated species, while it is monoprotonated at pH 6.9. The UV absorbance of nicotine has been reported to vary as a function of pH (15,85). As a diprotonated species (pH 2.5), the magnitude of UV absorbance of nicotine at 260 nm is approximately twice that for nicotine as a monoprotonated species (pH 6.9). From the known molar extinction coefficient of nicotine (depending on the solvent used), one can calculate the concentration of nicotine and determine the amount in a tobacco product. This pH dependence has a definite impact on the CE performance of nicotine as will be seen later.

The main features of the historical spectrophotometric method can be described as follows: