以區帶毛細管電泳定性定量菸草及尿液中尼古丁及其主要代謝物之分析研究

Qualitative and Quantitative Determination of Nicotine, Urinary Nicotine and its Major Metabolites in Tobacco and Urine by Capillary Zone Electrophoresis

許慧芬(Hui -Fen)

指導教授 ： 蔡錦蓮 ； 洪清吉

高雄醫學大學/藥學院/藥學研究所碩士在職專班/碩士(2005年)

摘要

煙草之中之尼古丁(Nicotine)及其主要代謝物可丁寧(Cotinine)皆已被証實為煙害曝露評估指標。本研究以毛細管電泳分離技術配合內標準法之應用，以提昇分析方法的準確度和精密度。以25 mM 甲酸氨 (Ammonium formate) 添加10%乙腈(Acetonitrile)有機修飾劑， pH 2.5為緩衝溶液，在管長67cm內徑75μm之毛細管，施加正向電壓25 kV，樣品注射時間10 sec，在內標準品菸醯胺 (Nicoinamide) 濃度固定為0.1 ppm之條件下，可將Nicotine、Cotinine及內標準品三者之波峯做最佳的分離。結果計算出Nicotine及Cotinine具良好之線性關係，分別為R2=0.9995及R2=0.9994，Nicotine之偵測極限為0.85 μg/ mL，Cotinine之偵測極限為0.266 μg/ mL，同日間變異分別為5.73 % 及 5.26 %，異日間變異分別為 7.76 % 及6.10 %。
煙草之溶劑萃取分析Nicotine含量亦於本研究中進行探討，以 乙醇為萃取溶劑，在約8小時之萃取後，即可達96 %之萃取效率。另外，加入超音波進行溶劑萃取，可將萃取時間縮短為4小時即可達約近100 %之萃取效率。建立煙草中Nicotine之含量測定，使誤食Nicotine造成毒害時，Nicotine之含量有所依循。
人體生物基質尿液檢體之分析，以二階段固相沖提後萃取尿液中Nicotine及Cotinine，配合內標準法進行Nicotine及Cotinine之毛細管電泳分析測定，其回收率分別為78.34 %及93.01 %，CV分別為 4.08 % 及 1.18 %。

關鍵字

區帶毛細管電泳 ； 可丁寧 ； 尼古丁 ； 菸草 ； 尿液

並列摘要

Both the nicotine and its metabolite, cotinine, of tobacco are confirmed as the evaluation index of smoking exposed damage. In this study, intermal standard method and capillary electrophoresis were combined to promote the accuracy and precision of our analysis. The buffer solution was made by 25 mM Ammonium formate (10% acetonitrile), and the pH was maintain as 2.5. The capillary with the length of 67cm and the diameter of 75μm was used. With the condition of 25 kV applying bias potential, 10 sec sample injection time, 0.1 ppm internal standard concentration, the peak of nicotinine, internal standard and cotinine can be separated appropriately. The experimental results were calculated and indicate the fine linear relationship of nicotine and cotinine with the R2 of 0.9995 and 0.9994, respectively, and with the sensitivity of measured limit of 0.85 and 0.266 μg/mL, respectively, and with the interday variation of 5.73 and 5.26 %, respectively, and with the intra day variation of 7.76 and 6.10 %, respectively.
The solvent extraction of tobacco and the content analysis of nicotine in tobacco were also discussed in our study. With ethanol extraction, the extraction efficiency can arrive 96% after 8 hours extraction. In addition, the extraction with ultrasonic was used. The result indicates the extraction efficiency can arrive 100% after 4 hours extraction with ultrasonic. The determination of nicotine content in tobacco was established. When the tobacco was eaten and cause the poison, the measurement of nicotine contaminant can be followed.
Qualitative and quantitative determination of urinary nicotine and cotinine were flow through the solid phase extraction, after two stage eluent with the combination of internal standard method and capillary electrophoresis, the recovery of nicotine and cotinine were 78.34 and 93.01%, respectively. The CV of nicotine and cotinine were 4.08 and 1.18 %, respectively.

並列關鍵字

Nicotine ； Urine ； Tobacco ； Capillary Zone Electroporesis ； Cotinine

參考文獻

陸、 參考文獻：

1. 賴大年，「台灣地區十二鄉鎮區居民吸菸對各種死因之世代追蹤研究」，國立台灣大學公共衛生研究所碩士論文。1994

2. Hoffmann D, Rivenson A, Hecht SS. The biological significance of tobacco-spedific N-nitrosamines: smoking and adenocarcinoma of the lung. Crit Rev Toxicol 1996;26:199-211.

3. McCorquodale EM, Boutrid H, Coyler CL. Enantiomeric separation of N’-nitrosonornicotine by capillary electrophoresis. Anal Chim Acta 2003;496:177-84.

4. 邵宏、牛天華、徐希平：尼古丁成癮的研究概況。中國藥物依賴性雜誌 2000;9: 9-11.