Determination of nicotine and its metabolites by capillary electrophoresis and mass spectrometry.

In England an estimated, 284,000 patients are admitted to NHS hospitals each year due to disease caused by smoking. It is estimated that half of all teenagers who are currently smoking will die from diseases caused by tobacco if they continue to smoke. An estimated one quarter of smokers will die after 70 years of age and one quarter before, with those dying before 70 losing on average 23 years of life. It is the addictive nature of nicotine that exacerbates the toxicities of the other components of cigarette smoke. The Royal College of Physicians has affirmed that the way in which nicotine causes addiction is similar to drugs such as heroin and cocaine. Thus studies of nicotine metabolism are of great importance since they determine the extent of which the addictive nature of nicotine can influence smoking behaviour and hence the onset of smoking related illnesses. In this area of research capillary electrophoresis (CE) is still in its infancy. But with its high resolution and high number of theoretical plates achieved, CE makes for an attractive separating device for coupling with a mass spectrometer (MS). The overall aims of this work were to produce a sensitive, highly selective, and simple CE-sample stacking/MS assay for the measurement of nicotine and its metabolites in urine, to develop a highly sensitive transient isotachophoretic/MS method, that yields detection limits comparable to that observed by HPLC/MS and with a separation efficiency to match that of CE-sample stacking/MS, and finally to characterise the metabolic activity of cytochrome P450 (e.g. CYP2D6) in the placenta, with respect to nicotine, from a representative in-vitro human trophoblast-like cell line, BeWo, via HPLC/MS and CE/UV. Analysis of urine samples was accompanied by sample clean up via SPE to ensure the appropriate removal of inorganic salts. An optimised hydrodynamic and electrokinetic injection method (HE injection) was used for CE-sample stacking/MS. HE-sample stacking/MS brought about lower detection limits (LODs of nicotine and cotinine, by CE-sample stacking/ MS (via HE injection), were found to be 0.11 and 2.25 mug/ mL, respectively) when compared to sample stacking/MS via hydrodynamic or electrokinetic injection alone. The added selectivity that the selected ion monitoring mode of MS provided ensured the clear identification of nicotine and its metabolites in urine. A counterflow transient isotachophoresis (tITP) method was developed, with MS detection, for the analysis of even lower analyte concentrations. Limits of detection for both nicotine (0.03 mug/ mL) and cotinine (0.34 mug/ mL), via HE-tlTP/MS, were considerably lower than those obtained by HE injection-sample stacking/MS, suggesting that HE-tlTP/MS could be used complementary to HE injection-sample stacking/MS. When HPLC/MS and CE/UV were used to analyse cytochrome p450 activity in the human trophoblast-like BeWo cell line, it was clear from our data that nicotine metabolism was observed. Thus it is probable that CYP mediated processes play an important role in nicotine metabolism in the placenta. When compared to HPLC/MS both HE-sample stacking/MS and HE-tlTP/MS exhibited higher resolutions and peak efficiencies; with HE-tITP/MS exhibiting comparable limits of detection and quantitation to those obtained by HPLC/MS with respect to nicotine, but not cotinine. The major improvements to the coaxial interface performance and sensitivity enhancements, has made CE/MS an attractive alternative to HPLC/MS for the determination of nicotine and its' metabolites from biological matrices.