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This work describes an on-line molecularly imprinted solid-phase extraction (MISPE) method for spectrophotometric determination of nicotine in urine samples of smokers. This method is based on manganese (VII) to manganese (VI) reduction in an alkaline medium, promoted by nicotine. Two wash solutions (1:4 (v/v) acetonitrile:sodium hydroxide – pH 11.4, and nitric acid – pH 2.5) were employed to circumvent interferences. Aqueous solutions containing nicotine plus different possible concomitants (cotinine, anabasine, norcotinine and caffeine) were tested individually. The analytical calibration curve was prepared in urine samples collected from non-smokers and spiked with nicotine standard from 1.1 to 60μmolL⁻¹ (r²>0.998). The limit of quantification and the analytical frequency were 1.1μmolL⁻¹ and 11h⁻¹, respectively. The precision, evaluated using 3, 10 and 30μmolL⁻¹ nicotine in urine, was 10, 10 and 4 …