Determination of nicotine and four metabolites in the serum of smokers by high-performance liquid chromatography with ultraviolet detection.

A simple and reliable reversed-phase high-performance liquid chromatographic method with ultraviolet detection is described for the quantitation of nicotine and its metabolites cotinine, trans-3'-hydroxycotinine, norcotinine and cotinine N-oxide in human serum. The analytes and the internal standard, N-ethylnorcotinine, were extracted by solid-phase extraction before chromatography. Two different columns and mobile phases with gradient systems were used. The detection limit of the assay was 10 ng/ml for nicotine, 3 ng/ml for cotinine N-oxide and 5 ng/ml for cotinine, trans-3'-hydroxycotinine and norcotinine. The concentrations of nicotine and its metabolites in the serum of 12 cigarette smokers are reported.