Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system

A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid–liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14 min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30–1000 ng for nicotine and 0.06–1000 ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8–15 ng/mL and 156–372 ng/mL, respectively.