A novel C6-phenyl liquid chromatographic technique for rapid and simultaneous measurements of urinary cotinine and nicotine

Abstract

A simple and rapid liquid chromatographic assay using diode array detection for simultaneous measurements of urinary concentrations of cotinine and nicotine was described. Each urine sample in a volume of 100 μL was extracted with 500 μL of dichloroethane at 60 °C for 5 min. 20 μL of the dried and reconstituted extract was eluted at 1 mL min⁻¹ with a mobile phase at pH 7.6. The urinary cotinine, nicotine and internal standard (indole-3-acetamide) were separated from matrix interferences within 4.2 min on a C₆-phenyl column maintained at 50 °C. The calibration curve was linear from 10 to 5000 μg L⁻¹ for cotinine, and from 20 to 7500 μg L⁻¹ for nicotine. The recovery rates of spiked concentrations were close to or about 100% and reproducibility generally greater than 90%. It was observed that the extraction yield of nicotine was very much affected by the extraction temperature. Among various extraction solvents evaluated, dichloroethane offered the highest yield, especially for nicotine. Thus, the urinary nicotine levels determined were generally higher than those reported earlier. This method was used on urine samples from 245 subjects, which included 188 non-smokers, 17 light smokers and 40 heavy smokers. The mean values of cotinine and nicotine were 50 and 96 μg g⁻¹ creatinine among non-smokers, 771 and 679 μg g⁻¹ creatinine among light smokers, and 1422 and 1104 μg g⁻¹ creatinine among heavy smokers.