Study On Determination Of Nicotine And Its Metabolite Cotinine And Hair Dyes In Human Hair

Objective1. To establish methods of determination of nicotine and its metabolite cotinine by gas chromatography and gas chromatography-mass spectrometry in human hair. 609 samples collected from three different areas in China were determined by GC through the laboratory quality control. GC-MS was applied to parts of the samples at the same time to validate the determined results by GC. This work will provide satisfactory results for epidemiological studies of tobacco control.2. This paper also describes a rapid analytical method to determine five hair dyes in dyed hair by gas chromatography with flame ionization detector. The five hair dyes were o-penylenediamine, p-penylenediamine, m-penylenediamine, 2-methyl-p- phenylene- diamine and diphenylamine.Method1. Hair samples were washed and dry at room temperature (25℃). The hair sample was cut into small segments of 2～3mm and 0.1mg/ml quinoline of 20μl was added as an internal standard. The hair sample was treated by ultrasonic for 30min in 1.5mol/l NaOH of 1.25 ml in the capped tube, and then was digested at 37℃for 6 hours. Nicotine and cotinine were extracted with 5 ml of mixed solvent of dichloromethane: methanol (3:1) by mechanical shaking for 5min. The dichloromethane was transferred to another_tube containing acetic acid of 20μl and was evaporated to dryness under a stream of nitrogen at 30℃. The residue was dissolved in methanol of 200μl, and centrifuged for 5min. The sample of 2μl was injected into the GC system and GC-MS simultaneously and analyzed.2. Dyed-hair samples were washed and dry at room temperature (25℃). Hair sample was cut into small segments and treated by ultrasonic for 30min in 1.25 ml of 1.0 mol/l NaOH in the capped tube, and then was digested at 37℃for 2 hours. Five hair dyes were extracted with chloroform of 5 ml by mechanical shaking for 5min. The chloroform was transferred to another tube containing 20μl acetic acid and was evaporated to dryness under a stream of nitrogen. The residue was dissolved with 95% ethanol of 200μl, and centrifuged for 5min. A sample of 2-μl was analyzed by GC with FID.Results1. The pretreatment of hair samples and parameters of GC were optimized. The linear ranges of nicotine and cotinine were 4.3 ng /ml～1.0mg/ml and 10ng/ml～2.0mg/ml, respectively. The correlation coefficients were more than 0.9996. And the detection limits of the assay were 0.013μg/g and 0.030μg/g for nicotine and cotinine in 0.1g hair. The range of RSD were 2.1%～7.0% and 4.4%～8.9% and the recoveries of spiked samples were 90.33%～113.1% and 92.92%～118.5%, respectively. 2. The linear ranges of nicotine and cotinine for GC-MS were 1.2 ng/ml～1mg/ml and 2.3ng/ml～2mg/ml respectively. The correlation coefficients were 0.9996 and 0.9997. And the detection limits of the two were 3.6ng/g and 6.9ng/g in 0.1g hair. The range of RSD were 4.8%～6.3% and 3.2%～7.3%, and the recoveries of spiked samples were 93.67%～93.92% and 102.1%～103.2%.3. The hair samples of 17 were treated and determined by GC and GC-MS simultaneously. The comparisons of the determined results by GC and GC-MS_were studied by matched t-test for dependent samples. From the results, the concentrations of nicotine and cotinine in hair samples by GC were consistent with those determined by GC-MS (t=0.793～1.903, P=0.075～0.439 and t=0.807～1.499, P=0.153～0.431).4. Hair samples collected were divided into three groups: non-smokers, passive smokers' and active smokers' by the questionnaire. Kruskal-Wallis Test was used on statistic analysis. Multiple comparisons show that the concentrations of nicotine and cotinine between the three groups have significant differences(P＜0.05). These results will provide experimental evidence for epidemiological studies of tobacco control.5. Under the optimized conditions of pretreatment of hair samples and GC separation parameters, the correlation coefficients of five hair dyes' regression equations were 0.9949～0.9985. The detection limits of the assay were 0.48μg/g～2.33μg/g in 0.1g hair. The range of RSD were 3.64%～9.93%, and the recoveries of spiked samples were 79.01%～110.6%.Conclusion The proposed method for simultaneous determination of nicotine and cotinine by GC with FID was sensitive, accurate, low expense, simple and convenient. And the method was applied to determining 609 human hair samples with satisfactory results. The analytical method for simultaneous determination of nicotine and cotinine by GC-MS was sensitive, quick, low detection limits and can provide a validating method for the GC analysis. There was good relationship between the determination results of GC and GC-MS. This paper provided laboratory data and information for further epidemiological researches. In this paper we also established a rapid and simple method for determination of five hair dyes by GC. It was successful applied to determine the hair dyes in dyed-hair. There were rare reports about determinations for hair dyes by GC. Therefore, the proposed method will useful for the research of relationship of dyed-hair and people's health.