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Solvent optimization software was used to develop a reversed-phase HPLC method for nicotine, five of its metabolites and caffeine under isocratic conditions, using UV detection and a LC8DB reversed-phase column. Starting from a methanol-buffer gradient, the software helped to find a quaternary solvent composition consisting of 2.3% methanol, 4.3% acetonitrile, 0.3% tetrahydrofuran and 93.1% buffer for the separation. The method was applied to human spiked serum and to serum of 12 cigarette smokers using a simple purification treatment before chromatography.
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