An Analysis of Nicotine and its Metabolites in Plasma and Urine Samples of Tobacco Smokers and Nonsmokers by High Performance Liquid Chromatographic Method

Park, Junggyeong; Park, Joowon; Rheem, Insoo; Song, Junghan; Park, Hyosoon; Kim, Jongwan

Abstract

BACKGROUND: Smoking has been suggested to invoke many health problems. Nicotine, one of the effective major components of tobacco smoke, has a half-life less than 2 hours and is oxidized to its major metabolite, cotinine. This study was conducted to establish the measurement system of nicotine and cotinine by high performance liquid chromatographic method (HPLC) and to set reference range in Korean population.
METHODS
Fifteen nonsmokers (25-66 years old, 37.9 average) and 30 smokers (22-63 years old, 27.9 average) were investigated. We modified the methods from Kyerenmaten, et al. and from Hariharan, et al. Urine and heparinized plasma samples were pretreated. Pretreated samples were injected into Waters u-Bondapak C18 reverse column (3.9 mm 30 cm) of Waters HPLC system unit with flow rate of 2 mL/min. Absorbance was monitored at 254 nm of wavelength.
RESULTS
The retention times of the NNX (nicotine-1'-N-oxide), cotinine, and nicotine peaks were 2.9, 3.7, 5.1 min, respectively, and readily delayed with increase of pH in the mobile phase. Nicotine and cotinine levels in plasma and urine samples by a modified HPLC method showed high linearity from 0 to 1000 ng/mL for both compounds. Intra- and inter-assay coefficients of variation were 7.49% and 6.54%, respectively for nicotine assay and 5.71% and 14.20%, respectively for cotinine assay. The averages and standard deviations for plasma cotinine, nicotine, urine cotinine, and nicotine in nonsmokers (N=15) were 277.8+/-313.9, 0.7+/-2.4, 382.0+/-273.7, and 17.2+/-27.5 ng/mL, respectively, and in smokers (N=30) were 312.9+/-267.1, 26.3+/-50.1, 1,049.2+/-556.2, and 555.7+/-895.1 ng/mL, respectively (P=0.351, 0.009, 0.0026, 0.000004).
CONCLUSIONS
A modified HPLC method for nicotine and cotinine measurement showed a high precision and accuracy. Nicotine and cotinine levels in plasma and urine samples of smokers were significantly higher than those of non-smokers, except for plasma cotinine in passive smokers of nonsmoker group. And this method can be used as a routine test for detection of passive smoking and managing of smoking habit. Reference values of nicotine and cotinine measured in Korean nonsmokers and smokers were suggested.