Article Title

Determination of cotinine in human urine by high-performance liquid chromatography


A reversed-phase liquid chromatography with ultraviolet detection is described for the determination of cotinine. A mobile phase consisted of an acetate buffer with sodium octanesulfonate as an ion pair reagent and methanol was used to resolve cotinine from theobromine, theophylline, arecoline and nicotine. Using an octadecyl-type column, a good separation was achieved in 12 min. The wavelength of UV detector was set at 260 nm. With p-nitroaniline as internal standard, human urine samples were cleaned up by liquid-liquid extraction. The regression equations obtained from both the standard and the biological samples were linear between 0.1 - 10.0 μg/mL (r > 0.999). The detection limit for cotinine is 30.0 ng/mL. The R.S.D. and R.E. of intraday and interday are less than 3.93% and 3.07%, respectively (n = 6). The recoveries are all greater than 96.44%. This method has been successfully applied to the determination of urinary cotinine content in several heavy smokers.

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