Determination Of Nicotine And Cotinine In Human Urine With Membrane Assisted Three Phase Microextraction And Aqueous Reversed-Phase Liquid Chromatography

Three phase microextraction with the automated movement of the acceptor and the donor phase (TPME/AMADP) technique is described for the extraction of nicotine (NT) and cotinine (CT) from non-smokers' urine. Separation and quantitative analyses were performed using aqueous reversed-phase liquid chromatography (LC) equipped with ultra-violet (UV) detector. The target compounds were extracted from a basified sample solution (donor phase) into a few microliters of the organic solvent impregnated in the pores of the hollow fiber and then back extracted into an acidic solution (acceptor phase) inside the lumen of the hollow fiber. The fiber was held by a conventional 10 mu l LC syringe. Five microliter acidic aqueous acceptor phase was sand-witched between the plunger and another 2 mu l of an organic solvent (microcap). The microcap separated the acceptor phase and the donor phase; in addition, it was partially responsible for mass transfer of the analytes from the donor solution to the acceptor solution. The acceptor solution was repeatedly moved in and out of the hollow fiber using a syringe pump. This movement provided a fresh acceptor phase to come in contact with the organic phase and improved in enrichment of the analytes. Additionally, fresh donor phase entered through the open end of the hollow fiber also contributed to the mass transfer. Various parameters affecting the extraction efficiency, viz, type of organic solvent, extraction time, stirring speed, effect of sodium chloride, and concentration of donor and acceptor phases were studied. Precision (RSD=3.23 and 3.51% respectively for nicotine and cotinine: tested at three concentration levels), linearity (correlation coefficient, r(2)=0.9964 and 0.9985 respectively for nicotine and cotinine), detection limit (9.7 and 194.5 ng/ml respectively for nicotine and cotinine), enrichment factor (158 and 6 respectively for nicotine and cotinine), relative recovery (83 and 85% respectively for nicotine and cotinine) and absolute recovery (9.6 and 12.0 % respectively for nicotine and cotinine) have also been obtained. The developed method was applied for the analysis of non-smokers' urine.