Improved highly sensitive method for determination of nicotine and cotinine in human plasma by high-performance liquid chromatography

A highly sensitive and reliable method for the determination of nicotine and its metabolite cotinine in human plasma by high-performance liquid chromatography was developed. Nicotine and cotinine were extracted from alkalinized plasma with dichloromethane and the volatility of nicotine was prevented by the addition of conc. HCl to the organic solvent during evaporation. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3 to 0.2 ng/ml nicotine and 1.0 ng/ml cotinine. The method was validated over linear ranges of 0.2–25.0 ng/ml for nicotine and 1.0–80.0 ng/ml for cotinine. The intra-day precision and accuracy were ≤15.9% relative standard variation (RSD) and 89.9–103.5% for nicotine and ≤8.0% RSD and 98.7–103.0% for cotinine. The inter-day precision and accuracy were ≤17.0% RSD and 94.2–100.9% for nicotine and ≤8.2% RSD and 98.0–105.1% for cotinine.