Determination of nicotine and cotinine in tobacco harvesters' urine by solid-phase extraction and liquid chromatography.

A solid-phase extraction method using Drug Test-1 column containing chemically modified silica as a solid support for sample clean up and reversed phase ion-paired high-pressure liquid chromatography method have been developed for the simultaneous determination of nicotine and its metabolite cotinine from the urine samples. Mobile phase was consisted of acetate buffer (containing 0.03 M sodium acetate and 0.1 M acetic acid) pH 3.1 and acetonitrile (78:22% (v/v)) containing 0.02 M sodium octanosulfonate as an ion pair agent. pH of the mobile phase was adjusted to 3.6 with triethylamine for better resolution and to prevent peak tailing. The linearity was obtained in the range of 0.5-10 microg/ml concentrations of nicotine and cotinine standards. The correlation coefficients were 0.998 for cotinine and 0.999 for nicotine. The recoveries were obtained in the range of 79-97% with average value of 85% for nicotine and in the range of 82-98% with average value of 88% for cotinine. The limit of detection was 2 ng/ml for cotinine and 5 ng/ml for nicotine with 2 ml urine for extraction, calculated by taking signal to noise ratio 10:3. The intra-day co-efficient of variation (CV) were <4 and 7% and inter-day CV were <9 and 7% for nicotine and cotinine, respectively. The method was applied to the urine samples of tobacco harvesters, who suffer from green tobacco sickness (GTS) to check the absorption of nicotine through dermal route during the various processes of tobacco cultivation due to its good reproducibility and sensitivity.