Quantification of Cotinine in Plasma and Urine by HPLC‐UV Detection

Abstract

Cotinine, the main metabolite of nicotine in man, is widely used as an abstention marker to Nicotiana tabacum smoke, as well as to evaluate passive inhalation of tobacco smoke by non‐smokers. Development and validation of a reversed‐phase high performance liquid chromatography (RP‐HPLC) method with ultraviolet (UV) detection for identification and quantification of cotinine in human plasma and urine are described. After diluting plasma with distilled water (1/6), NaOH 5 M (1:1) was added to both matrices and cotinine extracted using a RP 18 solid phase extraction column (SPE). Extracts were resuspended in mobile phase phosphate buffer pH 6.8:acetonitrile (90:10) and injected into a Lichrospher 100 RP‐18 column (5 µm). The UV detector was set to 260 nm. Linear calibration curves in the range of 10–1000 ng/mL of cotinine with correlation coefficients greater than 0.999 were obtained. Within‐run and between‐run accuracy was less than 5%. Cotinine detection limit was 20 ng/mL. The proposed chromatographic system allows cotinine to be separated from caffeine.