Direct Determination of Free Nicotine Content in Tobacco

[Image: see text] The accurate determination of the free nicotine content in tobacco is of great importance for the quality assessment of tobacco leaves and cigarettes, as well as for the study of tar reduction and cigarette formulation. A method based on solvent extraction and gas chromatography–mass spectrometry (GC–MS) was established for direct determination of free nicotine content in tobacco. Free nicotine extraction from tobacco was optimized, using different solvents and physical techniques (shaking, ultrasonic oscillation, and static extraction). Ultimately, a 24 h static extraction with cyclohexane, followed by GC–MS analysis, gave superior results. The standard addition recovery was 98.0–104.7%, with a limit of detection of 5.3 μg/g and a relative standard deviation between 1.3 and 4.1% (n = 5). Quadratic regression of the standard curve was excellent (R(2) ≥ 0.9994). The free nicotine content was determined in 67 tobacco samples, with parallel samples showing relative deviations of 0.1–3.1%. To evaluate the effect of nicotine salts present in tobacco, malic acid, citric acid, acetic acid, and nicotine citrate were spiked into samples prior to extraction and analysis. The results show no interference from bound nicotine compounds on the method. The experimental results show that direct determination of free nicotine in tobacco was achieved using a simple static cyclohexane extraction. Moreover, the high extraction efficiency of free nicotine was also achieved with ease of operation and good repeatability.