Novel analytical methods for the investigation of nicotine metabolism.

The thesis begins with a description of the history of tobacco usage, health effects related to tobacco consumption, the metabolism of nicotine and the analytical methods used for the determination of its metabolites. The work described in this current study is related to the use of the Konig reaction for the colorimetric determination of nicotine and its metabolites. Two novel non-polar condensing reagents were prepared and evaluated as to their performance with an extensive range of individual nicotine metabolites. The work extends our basic knowledge of the analytical chemistry of this reaction particularly in relation to the molar absorptivity and partitioning of the coloured derivatives formed. It was found that each metabolite has its own unique molar absorptivity, a finding which has profound implications for the interpretation of this reaction when used quantitatively as a test for "Total Nicotine Metabolites" since it is increasingly recognised that nicotine metabolism may differ between individuals, ethnic groups and during different physiological states. In addition its possible utility as a colorimetric derivatisation procedure for the determination of nicotine concentrations by high performance liquid chromatography (HPLC) may also be compromised if cotinine alone is used for standardisation purposes.The use of these novel derivatives was investigated in relation to their correlation with a specific radioimmunoassay (RIA) for cotinine and the ability of the test to discriminate between smokers and non-smokers. The use of more non-polar derivatives was found to result in a small enhancement of the discriminatory power of the reaction for the determination of smoking status. An excellent correlation was found between the RIA and the use of the Konig reaction. There has been considerable debate as to the precise nature of the derivatives formed during the Konig reaction. Part of this work has been dedicated to the elucidation of the definitive structures of these compounds. The evidence presented here suggests that the structure proposed by Rustemeier et al (1993) is the more likely of the proposed structures. The work concludes with a consideration of the possible use of surfactants to enhance the procedure and enable the use of potassium thiocyanate rather than potassium cyanide during the reaction. Several combinations of anionic, cationic and neutral surfactants are investigated and their effects on the Konig reaction discussed.