Simultaneous determination of cotinine and trans-3'-hydroxycotinine in human serum by high-performance liquid chromatography.

A high-performance liquid chromatographic method with ultraviolet photometric detection has been developed for the quantitation of cotinine and trans-3'-hydroxycotinine in human serum. A solid-phase extraction procedure was performed for the analytes and the internal standard, N-ethylnorcotinine, before chromatography. The use of a 30-cm reversed-phase column and a mobile phase of water-methanol-0.1 M sodium acetate-acetonitrile (67:24.5:6.5:2, v/v), pH 4.3, prevented the co-elution of caffeine with cotinine. The limit of quantitation observed with this method was 5 ng/ml for both cotinine and trans-3'-hydroxycotinine. The present method proved useful for the determination of serum levels of these metabolites, correlating with nicotine daily intake.