HPTLC Assay of Nicotine and Cotinine in Biological Samples

The study developed a high-performance thin-layer chromatography (HPTLC) method to identify and semi-quantitatively evaluate nicotine and its metabolite cotinine in human plasma and urine. The method achieved the best separation using a methanol: chloroform: ammonia solvent system and found liquid-liquid extraction with dichloromethane at alkaline pH to be more effective than solid-phase extraction. Calibration curves for nicotine and cotinine were linear in the range of 200-1000 ng/spot, with correlation coefficients over 0.99. This method was applicable for detecting nicotine and cotinine at toxic levels, potentially useful in forensic toxicology for cases of nicotine intoxication.