烟草和烟气中酚类物质的分析方法研究及应用
摘要
建立了固相萃取-高效液相色谱-质谱法测定烟草样品中多酚类物质的新方法。用80％的甲醇溶液回流提取烟叶中的多酚物质，然后用Waters Sep-Park-C_(18)固相萃取柱分离脱脂；以Waters Nova-Pak-C_(18)色谱柱(3.9×150mm，5μm)为固定相，甲醇-1％醋酸溶液为流动相；流速为0.5mLmin~(-1)；线性递增(减)的梯度洗脱，进样体积10μL，并用电喷雾质谱对其进行辅助定性；标样和烟草样品在345nm波长处提取色谱图计算峰面积。此方法的优点主要是采用固相萃取柱对样品进行前处理，使干扰组分保留在小柱上而酚类物质不保留，而且对环境不造成污染。它比传统的方法脱脂时间缩短约2小时，相对标准偏差为1.31％-2.36％，回收率为94％-103％。在该方法的应用中，研究了烟叶部位、烤烟品种、施肥量、烟叶种类以及调制对多酚类物质的影响。结果表明：同一烤烟，上部烟叶多酚含量比下部高；不同烤烟品种中，红大品种绿原酸含量高；中等程度施肥量对多酚积累最有利；烤烟中多酚含量最高、白肋烟中最低；调制对多酚类物质的影响不大。
对于卷烟烟气中酚类物质的测定，综合了国内外的研究进展，主要采取了两种方法。自动水汽蒸馏分光光度法测定卷烟烟气中的总酚，检测波长为460nm，回收率为96％-105％，相对标准偏差为1.1％-1.4％。与普通水蒸汽分光光度法相比，此方法蒸馏时间缩短约55分钟，整个系统密闭性好，气流稳定，从而使测定结果更准确、精度更高。其次，用高效液相色谱荧光检测法测定了卷烟烟气中的七种酚类物质即邻苯二酚、间苯二酚、对苯二酚、苯酚、邻甲酚、间甲酚和对甲酚。实验条件为：色谱柱为Waters公司的Symmetry(?) C_(18)反相键合硅胶柱，流动相为甲醇-磷酸二氢钾缓冲液，流速为0.5mLmin~(-1)，分析时间为30min，回收率为96％-101％，相对标准偏差为1.2％-2.6％。此方法的优点是无需衍生化，样品处理时间缩短3.5小时，分析时间缩短一半，便于批量处理，而采用甲醇作为流动相比乙腈在成本上降低了一半。
另外，本文对酚类物质与感官评吸的相关性进行了研究。首先采用GC-MS联用仪测定47种卷烟烟气中的225种成分，筛选出其中的35种酚类物质作为分析数据，然后采用SPSS统计分析软件进行多元线性回归分析并建立相关性数学模型。感官评吸指标与酚类物质基本呈负相关，只有少量酚类物质呈正相关。均负相关的是2，5-二甲基苯酚和3-乙基苯酚；除刺激性外，其他5项均与4-吡啶酚、4-乙烯基苯酚负相关；除刺激性和和香气外，均与2，4-二甲基苯酚负相关；谐调、刺激
性和综合得分均与苯酚、4一乙基苯酚负相关:香气、刺激性和余味均与4一乙烯基一2-
甲氧基一苯酚负相关:香气、刺激性与对苯二酚负相关:刺激性和余味与1，2一苯二
酚负相关。均正相关的是4一甲基苯酚、2，3一二甲基苯酚;除谐调外，均与2一甲基
苯酚正相关:除刺激性和谐调外.均与2.3，5一三甲基一l，4一苯二酚正相关、余味和谐
调均与2，6一二甲基苯酚正相关;香气和余味均与香兰素正相关;而2，4一二甲基
苯酚只与刺激性正相关.这些相关性对感官评吸人员和实际生产应用有指导作用.
根据酚类物质的危害及其与感官评吸的相关性结果，本文首先合成了降低卷烟
烟气中酚类物质的CW系列添加剂，即由聚乙二醇、自制催化剂和化合物A合成
的聚乙二醇衍生物(Cw系列嘴棒添加剂)。反应条件为:反应温度120℃，催化剂
用量为0.1%一0.3%，反应配比1:1。把筛选出的CW4和CW6添加剂应用到省内
3家卷烟厂四种牌号的不同等级卷烟上，并采用己建立的两种方法测定出上述四种
卷烟烟气中酚类物质的含量，并计算其下降的百分比。CW添加剂能使苯酚下降
10%一45%，邻苯二酚则不超过20%。除样烟B的CW4外，其它样烟的间苯二酚
均呈下降趋势，为66%一99%，对苯二酚在样烟D中均下降，以CW4效果最好，
为10.92%。毒性较大的邻、间、对甲酚在样烟A中呈上升趋势，而在其它三种品
牌中均有不同程度的下降，为30%一56%。总酚下降为6一26%。为了保证卷烟的总
体指标合格，对加入添加剂后制成的卷烟进行了一系列检测.感官评吸是通过人体
的感觉来评价卷烟的香吃味，判断刺激性的强弱，进而知道烟气中酚类物质含量的
多少。采用前面建立的方法测定烟气中总酚和七种酚，并分别计算酚类物质下降的
百分比。以上五种检测结果均表明:合成的CW系列添加剂能降低上述卷烟烟气
中的酚类物质，CW6使中低档样烟A、B、C中的酚类物质下降幅度最大，效果最
显著。而对优质的样烟D而言，CW4系列添加剂比CW6系列的效果要好。此添
加剂优点是环保性添加剂，易合成且工艺条件易控制。为此类添加剂的工业化生产
和在烟草中的应用提供了有力的理论依据。
A new method for determination of polyphenols in tobacco leaf with solid phase extraction-high performance liquid chromatograpgy-mass spectrum (SPE-HPLC-MS) were studied. Polyphenols can be extracted from tobacco leaf with 80% methanol by heating circumfluence, then subjected to degrease by Waters Sep-Park-C18 solid phase extraction cartridge. Main polyphenols can be separated on a Waters Nova-Pak-C18 chromatogram column (3.9
×150mm),by methanol and 1% acetic acid solution as mobile phase at flow rate of 0.5mL min-1 with injection volume of 10 u L. Qualitative analysis electrospray ionization mass spectrometry (ESI/MS) method was used. Standard sample and samples were determinated at the maxmum absorption wavelength of 345nm,and their peak areas were calculated. Compared with traditional method, this method shortened the time of preparation sample greatly. The relative standard deviation and recovery were 1.31%-2.36% and 94%-103% respectively. The regulations of main polyphenols in tobacco between the variety tobacco, the stalk position of tobacco leaf, the amount of nitrogen-containing fertilizer and the modulation progress of tobacco were investigated by use of advanced SPE-HPLC-MS. The results were as follow:the contents of total polyphenols in Hongda breed are higher than other breeds; for the different stalk position of the same breed, the content of total polyphenols in top leaves in higher than in middle and bottom; prop
er amount of appliled nitrogen-containing fertilizer (such as 6-8kg/mu) is benefited to the accumulation of polyphenols; in the different types of tobacco, the content of polyphenols in flue-cured tobacco is highest, but burley tobacco is lowest; the content of total polyphenols is not obviously changed during the course of fore-and-aft tobacco accumulation.
According to the domestic and foreign review, two new methods were appilied in the determination of phenolic compounds in smoke. The total phenolic contents were determined through auto-vapor distillation and spectrophotometer at 460nm wavelength. Recovery and ralative standard deviation of this method were 96%-105% and 1.1%-1.4% separately. Contrary on the way of common water vapor, it is an
exacter result, higher precision, simple operation and shorter time. In addition, seven phenolic compounds (catechol, hydroquinone, resorcinol, phenol, o-cresol, m-cresol and p-cresol) tested by the way of high performance liquid chromatograph at the same time. Experimental condition is as follow : Waters Symmetry?C18 reversed phase bonded silica gel chromatogram column (3.9 X 150mm), methanol and 0.05molL-1 potassium dihydrogen phosphate solution gradient eluting as mobile phase at flow rate of 0.5mL min-1 with injection volume of 10 u L. Contrary on the reported method, its operation of simple treatment is simpler, analytical time is shorter, the accordance is better and the price of flow phase is lower.
Test thirty-five phenolic compounds in forty-seven brands cigarette smoke by GS-MS. The correlations between phenolic compounds and sensory properties were studied by SPSS(Statistics Package for Social Science) in the computer. The mathematics models were set up by means of correlation and multi-variable linear regression analysis. The results showed that most phenolic compounds are indirect to sensor quality and only a few ones are direct. So some measurements must be taken to reduce phenolic compounds which are harmful to human body.
Acording to the corelation analytical results, polyethonolisomers as an additives of reducing phenolics was synthesized by reaction between polyethonol, catalyzer made by us and compound A. reaction conditions are 120 degree in reaction temperature, 0.1 to 0.3 precent in the catalyzer does and the rate of one to one. This serial additives were applied in four brand cigarettes from three companies of Yunnan privence. The two above mentioned methods of determinating phenolics in smoke were appilied in four types of cigarettes and the reduced percentage of the contents of phenolic compounds was calculated. Sensor
对于卷烟烟气中酚类物质的测定，综合了国内外的研究进展，主要采取了两种方法。自动水汽蒸馏分光光度法测定卷烟烟气中的总酚，检测波长为460nm，回收率为96％-105％，相对标准偏差为1.1％-1.4％。与普通水蒸汽分光光度法相比，此方法蒸馏时间缩短约55分钟，整个系统密闭性好，气流稳定，从而使测定结果更准确、精度更高。其次，用高效液相色谱荧光检测法测定了卷烟烟气中的七种酚类物质即邻苯二酚、间苯二酚、对苯二酚、苯酚、邻甲酚、间甲酚和对甲酚。实验条件为：色谱柱为Waters公司的Symmetry(?) C_(18)反相键合硅胶柱，流动相为甲醇-磷酸二氢钾缓冲液，流速为0.5mLmin~(-1)，分析时间为30min，回收率为96％-101％，相对标准偏差为1.2％-2.6％。此方法的优点是无需衍生化，样品处理时间缩短3.5小时，分析时间缩短一半，便于批量处理，而采用甲醇作为流动相比乙腈在成本上降低了一半。
另外，本文对酚类物质与感官评吸的相关性进行了研究。首先采用GC-MS联用仪测定47种卷烟烟气中的225种成分，筛选出其中的35种酚类物质作为分析数据，然后采用SPSS统计分析软件进行多元线性回归分析并建立相关性数学模型。感官评吸指标与酚类物质基本呈负相关，只有少量酚类物质呈正相关。均负相关的是2，5-二甲基苯酚和3-乙基苯酚；除刺激性外，其他5项均与4-吡啶酚、4-乙烯基苯酚负相关；除刺激性和和香气外，均与2，4-二甲基苯酚负相关；谐调、刺激
性和综合得分均与苯酚、4一乙基苯酚负相关:香气、刺激性和余味均与4一乙烯基一2-
甲氧基一苯酚负相关:香气、刺激性与对苯二酚负相关:刺激性和余味与1，2一苯二
酚负相关。均正相关的是4一甲基苯酚、2，3一二甲基苯酚;除谐调外，均与2一甲基
苯酚正相关:除刺激性和谐调外.均与2.3，5一三甲基一l，4一苯二酚正相关、余味和谐
调均与2，6一二甲基苯酚正相关;香气和余味均与香兰素正相关;而2，4一二甲基
苯酚只与刺激性正相关.这些相关性对感官评吸人员和实际生产应用有指导作用.
根据酚类物质的危害及其与感官评吸的相关性结果，本文首先合成了降低卷烟
烟气中酚类物质的CW系列添加剂，即由聚乙二醇、自制催化剂和化合物A合成
的聚乙二醇衍生物(Cw系列嘴棒添加剂)。反应条件为:反应温度120℃，催化剂
用量为0.1%一0.3%，反应配比1:1。把筛选出的CW4和CW6添加剂应用到省内
3家卷烟厂四种牌号的不同等级卷烟上，并采用己建立的两种方法测定出上述四种
卷烟烟气中酚类物质的含量，并计算其下降的百分比。CW添加剂能使苯酚下降
10%一45%，邻苯二酚则不超过20%。除样烟B的CW4外，其它样烟的间苯二酚
均呈下降趋势，为66%一99%，对苯二酚在样烟D中均下降，以CW4效果最好，
为10.92%。毒性较大的邻、间、对甲酚在样烟A中呈上升趋势，而在其它三种品
牌中均有不同程度的下降，为30%一56%。总酚下降为6一26%。为了保证卷烟的总
体指标合格，对加入添加剂后制成的卷烟进行了一系列检测.感官评吸是通过人体
的感觉来评价卷烟的香吃味，判断刺激性的强弱，进而知道烟气中酚类物质含量的
多少。采用前面建立的方法测定烟气中总酚和七种酚，并分别计算酚类物质下降的
百分比。以上五种检测结果均表明:合成的CW系列添加剂能降低上述卷烟烟气
中的酚类物质，CW6使中低档样烟A、B、C中的酚类物质下降幅度最大，效果最
显著。而对优质的样烟D而言，CW4系列添加剂比CW6系列的效果要好。此添
加剂优点是环保性添加剂，易合成且工艺条件易控制。为此类添加剂的工业化生产
和在烟草中的应用提供了有力的理论依据。
A new method for determination of polyphenols in tobacco leaf with solid phase extraction-high performance liquid chromatograpgy-mass spectrum (SPE-HPLC-MS) were studied. Polyphenols can be extracted from tobacco leaf with 80% methanol by heating circumfluence, then subjected to degrease by Waters Sep-Park-C18 solid phase extraction cartridge. Main polyphenols can be separated on a Waters Nova-Pak-C18 chromatogram column (3.9
×150mm),by methanol and 1% acetic acid solution as mobile phase at flow rate of 0.5mL min-1 with injection volume of 10 u L. Qualitative analysis electrospray ionization mass spectrometry (ESI/MS) method was used. Standard sample and samples were determinated at the maxmum absorption wavelength of 345nm,and their peak areas were calculated. Compared with traditional method, this method shortened the time of preparation sample greatly. The relative standard deviation and recovery were 1.31%-2.36% and 94%-103% respectively. The regulations of main polyphenols in tobacco between the variety tobacco, the stalk position of tobacco leaf, the amount of nitrogen-containing fertilizer and the modulation progress of tobacco were investigated by use of advanced SPE-HPLC-MS. The results were as follow:the contents of total polyphenols in Hongda breed are higher than other breeds; for the different stalk position of the same breed, the content of total polyphenols in top leaves in higher than in middle and bottom; prop
er amount of appliled nitrogen-containing fertilizer (such as 6-8kg/mu) is benefited to the accumulation of polyphenols; in the different types of tobacco, the content of polyphenols in flue-cured tobacco is highest, but burley tobacco is lowest; the content of total polyphenols is not obviously changed during the course of fore-and-aft tobacco accumulation.
According to the domestic and foreign review, two new methods were appilied in the determination of phenolic compounds in smoke. The total phenolic contents were determined through auto-vapor distillation and spectrophotometer at 460nm wavelength. Recovery and ralative standard deviation of this method were 96%-105% and 1.1%-1.4% separately. Contrary on the way of common water vapor, it is an
exacter result, higher precision, simple operation and shorter time. In addition, seven phenolic compounds (catechol, hydroquinone, resorcinol, phenol, o-cresol, m-cresol and p-cresol) tested by the way of high performance liquid chromatograph at the same time. Experimental condition is as follow : Waters Symmetry?C18 reversed phase bonded silica gel chromatogram column (3.9 X 150mm), methanol and 0.05molL-1 potassium dihydrogen phosphate solution gradient eluting as mobile phase at flow rate of 0.5mL min-1 with injection volume of 10 u L. Contrary on the reported method, its operation of simple treatment is simpler, analytical time is shorter, the accordance is better and the price of flow phase is lower.
Test thirty-five phenolic compounds in forty-seven brands cigarette smoke by GS-MS. The correlations between phenolic compounds and sensory properties were studied by SPSS(Statistics Package for Social Science) in the computer. The mathematics models were set up by means of correlation and multi-variable linear regression analysis. The results showed that most phenolic compounds are indirect to sensor quality and only a few ones are direct. So some measurements must be taken to reduce phenolic compounds which are harmful to human body.
Acording to the corelation analytical results, polyethonolisomers as an additives of reducing phenolics was synthesized by reaction between polyethonol, catalyzer made by us and compound A. reaction conditions are 120 degree in reaction temperature, 0.1 to 0.3 precent in the catalyzer does and the rate of one to one. This serial additives were applied in four brand cigarettes from three companies of Yunnan privence. The two above mentioned methods of determinating phenolics in smoke were appilied in four types of cigarettes and the reduced percentage of the contents of phenolic compounds was calculated. Sensor
引文
1 胡群．卷烟铺料研究．昆明：云南科技出版社，2001
2 李汉超，王淑娴．烟草·烟气化学及分析．郑州：河南科学技术出版社，1995
3 王晶晶．卷烟配方与烟支设计．北京：中国科学技术出版社，2000
4 张保振．中国烟草大辞典．北京：中国经济出版社，1992
5 石碧，狄莹．植物多酚．北京：科学出版社，2000
6 Viart P. Phenolic index oftobbaco, Tobacco science, 1976, 11: 857
7 韩富根．用福林法测定烟叶中总酚含量的探讨．郑州：河南农业大学学报，1993，1：95-98
8 张槐苓，葛翠英．烟草分析与检验．郑州：河南科技出版社，1994
9 Rayburn C H, Harlan W R, Hanmer H R. Determination of volatile phenols in cigarette smoke.Anal.Chem. 1953, 25: 1419
10 胡旺云．烟草化学家研讨会-半个世纪以来讨论烟草及其制品分析方法发展的论坛．国外烟草科技文选，1998，5：19
11 Shigeo A. A method of ion change chromatography. Hatano Taboko Shikensh Hokoku. 1966,57:5-15, CA, 68:27581f
12 Shigeo A. A method of ion change chromatography, Hatano Taboko Shikensh Hokoku, 1966,57:16-26, CA, 68:27582g
13 Lattanzio, Vincenzo. Silica gel method of determination in poylphenols J.Food Sci..1981, 46 (6): 1907-1917
14 Anderson R A.Scpoletin determination of tobacco in GC, Phytochemistry. 1972,11 (8): 2593-2595
15 Sakaki, Takeshi.Kennyu Hokoku-Nippon Senbai Kosha Chou Kenkyusho.1982,(124): 33-6, CA, 99: 85360j
16 Court, William A.J.Polyphenol determination by HPLC, J Chromatogr, 1977, 130:287-291
17 Nisseror S. Liquid chromatography separation of polyphenols application to tobacco extract analysis.Analusis.Dec 1984,12 (10): 523-524
18 Ishguro S, Sugawara S. Studies on the composition of smoke components of lamina and midrib cigarettes. Ⅳ. Comparisons of smoke components from lamina and midrib cigarettes of flue-crued tobacco leaves by the trimethylsilylation method. Agric. biol. Chem.. 1978, 42:407-417.
19 Mcwilliams D, Deutsch L J, Gladden J D. Simple gas chromagraphic determination of
phenol and cresols in Cambridge pad extracts. TCRC. 1986
20 Tomkins B A. Studies on determining of phenolic compounds of tobacco and cigarette smoke. IARC Sci. Publ.,1987, 81:279-89
21 Nanni E J. Lovette M E, Hicks R.D. A method of reducing phenlics in tobacco and smoke. J Chromatogry. 1990, May 505:65-74
22 赵明月，周富臣．卷烟主流烟气中酚类物质的毛细管气相色谱分析．中国烟草学报，1992．2：1-14
23 Clark J, Bunch J E. Solid phase microextraction/gas chromatograph/selected ion mode mass spectrometry analysis of phenolics in cigarette smoke condedsate. Tob. Chem. Res. Conf..1995, 49:57
24 毛多斌，马宇平，张俊松，等．国内外混合型卷烟主流烟气中酚类物质的分析．烟草科技．2002，4 1：7-20
25 苏国岁．卷烟烟气中对、间甲酚总量的气相色谱分析．烟草科技．2003，8：16-18
26 Tomkins B A, Jenkins R A, Griest W H, Reagan R R, Holladay S K J. A liquid chromatography method of determination of phenol and cresol in cigarette smoke. Assoc Off Anal Chem.. 1967,5:19-23
27 Kallianos A G, Means RE, Mold J D.Alkyi catechols in cigarette smoke .TCRC.1967
28 Kuwata, Kazuhiro, Michiko.Revered-phase liquid chromatographic determination of phenols in auto echaust and tobacco smoke as p-nitrobenzeneazophenoi derivatives. Anal. Chem.. 1981, 53:1531-1534
29 Kuwata, Kazuhiro, Uebori,et al. The new method of determinting phenolics of cigarette smoke. Anal. Chem. 1980, 52:857-860
30 吴帼英．用反相高效液相色谱对卷烟烟气冷凝物中单酚的定暈分析．中国烟草科学．1986，3：45-48
31 Risner C H, Cash S L.The determination of major phenolic compounds in tobacco smoke. J Chromatogr Sci..1990, May 28:39-44
32 Prakasha A, Ireland M S.A high performance liquid chromatograohic method to quantitate phenols in isopropanl extract of mainstream cigarette smoke. TCRC. 1992
33 Ricner C H.An improved method for the quantification of selected phenolic compounds in ambient air. TCRC. 1992
34 欧阳清．高校液相色谱法定量分析主流烟气中的酚类物质．上海烟业，1994，3：48-49
35 Rayburn C H.Chemical analysis of tobacco and tobacco smoke: determination of smoke volatile phenols in cigarette smoke . TCRC.1952
36 Lindsey A J. Burrows I E, Smith Gal et al.Thin layer chromatography: smoke applications in tobacco chemistry. TCRC. 1963
37 Anderson I G M.Comparision of methods for the determination of phenols in tobacco smoke highlighting a new approach using capillary electromatography. CORESTA.1998, sport2:194
38 Munteanu, Florentina-Daniela, Lindgren et al. A method of determining the phenolics in tobacco and smoke.Anal.Chem.. 1998, 13:2596-2600
39 李忠，施红林，王岚，等．新型自动水蒸汽蒸馏仪蒸馏测定烟草及其制品中总挥发酚．理化检验-化学分册．2002，12：628-630
40 NCI, National Cancer Institute. Smoking and Health program: Toward less hazardous cigarettes: Report No.1. The first set of experimental cigarettes. DHEW Publication No.(NIH).1976, 76-905.
41 Crosthwaite L, Sheen S J, Burton H R. Alkylating activity in the extract and pyrolyzate of tobacco leaves varying ingenotype and chemical treatment;Tob. Sci..1979, 23:35-37.
42 Bentley H R and Burgan J G.Polynuclear hydrocarbons in tobacco and tobacco smoke. Part Ⅲ. The inhibition of the formation of 3,4-benzopyrene in cigarette smoke; Analyst, 1960, 85:727-730.
43 Kaburaki Y H, Shigematsu Y M, Kusakabe H. Studies on the composition of tobacco smoke (Ⅺ). Relatioms between vapor phase components of smoke and constituents of tobacco. Kenkyo Hokoku-Nippon Sembai Kosha Chuo Kenkyusho. 1969, 111:143-149.
44 Garcia Roche M O, Cabrera A, Becquer A et al. Health risk due to the use of nitrate in the manufacture of Cuban golden cigarettes; Rev, Cubana Hig. Epidemiol. 1986, 24:487-492.
45 Pyriki C, Moldenhauer W , Knappe T. Zur Frage der Behandlung des Zigarettenpapiers und des Tabaks zwecks Verminderung von polycyklischen Kohlenwasserstonffen in deren RAUCH: Ber. Inst. Tabakforsch. Dresden. 1965, 12:35-37.
46 Rathkamp G, Hoffmann D. Chemical studies on tobacco smoke ⅩⅢ. Inhibition of the pyrosyntheses of several selective smoke constituents Beitr. Tabakforsch, 1970, 5:302-306.
47 Kallianos A G,. Means R E and Mold J D.Effect of nitrates in tobacco on the catechol yield in cigarette smoke: Tob. Sci..1968, 12:125-129.
48 Brunnenmann K D , Posset L.Routine high-performance liquid chromatographic method for assessing sorbate in tobacco. Jourmal of Chromatography. 1980, 202:494-499.
49 Budick D, benner J F, Burton H R. Thermal decomposition of tobacco Ⅳ. Apparent correlations between thermogravimetric datd and certain constituents in smoke from chemically-treated tobaccos. Tob. Sci..1969, 13:33-37.
50 Burton H R. Thermal analysis of chemically-treated tobacco. Proc. Tob.. Health Workshop. 1969, 33-37.
51 Burton H R, Benner J F.Investigations on the effect of chemical modifiers on tobacco smoke: Proceedings of the University of Kentucky. Tobacco and Health Workshop Conference. Lexington. 1972,50:341-363.
52 Wynder E L, D Hoffmann Ein. experimenteller Beitrag zur Tabakrauchkanzerogenese Dtsch. Med. Wschr. 1963, 88:623-628.
53 Wynder E L, Hoffmann D. A study of tobacco carcinogenesis. Ⅹ. Tumor promoting activity. Cancer. 1969, 24:289-301.
54 Dontenwill W, chevalier H J, Harke H P et al. Klimischungen über die tumorerzeugende Wirkung von Zigarerrenrauch-Kondensaten an der Mausehaut. Ⅳ. Einzelvergleiche yon Kondensaten aus verschiedenen Tabakfolien, Einflub eines Zusatzes von NaNo3 zum Tabak Oder zu Tabakfolien, Wirkung Fluchtiger Bestandteile des Rauches, Einflub einer Initiabenhendlung mit Dimethylbenzanthracen. Z. Krebsforsch. 1972, 78:236-264.
55 Dontenwill W, Chevalier H J , Harke H P et al. Experimentelle Untersuchungenüber die tumorerzeugende Wirkung von Zigarerrenrauch-Kondensaten an der Mausehaut. Ⅴ. Mitteilung: modifizierter Zigaretten.Z. Krebsforsch. 1976, 85:141-153.
56 Klimisch H J. Vergleichende chemisch-analysche Untersuchungen an Kondensaten modifizierter Cigaretten. Diskussion einer Korrelation zur biologischen Wirksamkeit; Zeitschrift fur LebensmitteI-Untersuchung und-Forsch.1972, 150:93-99.
57 Halter H M. Reconstituted tobacco smoking and health possibilities.J. Natl. Cancer Inst.. 1972, 48:1869-1883.
58 Lyerly L A. Direct vapor chromatographic determination of menthol, propylene glycol, nicotine and triacetin in cigarette smoke; Tob. Sci.,1967, 11:49-51.
59 Laurene A H, Cundiff R. H. Greene. Determination of glycerol and propylene glycol in cigarette smoke; Tob. Sci.. 1965, 9:1-4.
60 Kallianos A G, Means R E, Smoking and smoke quality. Mold J D, LIGGETT & MYERS, 1967,12:17-20.
61 Tomkins B A, Jenkins R A, Hoiladay S K. J Assoc Off .Anal Chem.. 1967,59:19-23
62 Carmines E L. Evaluation of the potential effects of ingredients added to cigarettes. Part 1: Cigarette design, testing approach, and review of results. Food Chem. Toxicol. 2002, 40:77-91.
63 Sledmen R L. The chemical composition of tobacco and tobacco smoke.Chem. Rev..1968,
68: 153
64 周冀衡，朱小平，等．烟草生理与生物化学．北京：中国科学技术大学出版社，1996
65 Wynder E.L, Hoffmam D. Tobacco and tobacco smoke. Academic press Newyork and Londonm. 1967
66 Armstrong G, Mrohrbaugh L M, Wender S H. the component of leaf and their relationship to smoking quality and aroma Recent advances in tobacco science. 1972, 2:925-948
67 阎新甫，韩锦峰．烟草多酚类化合物的研究进展．华北农学报，1987，2：31-38
68 朱汉春．中国烟草知识大全．长沙：湖北科学技术出版社．1999
69 周正红，高孔荣，张水华．烟草中化学成分对卷烟色香味品质的影响及其研究进展．烟草科技，1997，197(2)：22-25
70 钟庆辉．烟草芳香吃味化学指标的探索．烟草科技，1981，41(4)：21-23
71 李丛民．植物多酚对烟草制品品质的影响．烟单科技，2000，140(1)：27-29
72 Snook M E, Mason P F, Sisson V A. Polyphenols in the nicotiana species. Tobacco Science. 198630(18):43
73 雷永和．云南烟草品种志，昆明：云南科技出版社，1999
74 左天觉，朱尊权．烟草生产、生理和生物化学．上海：上海远东出版社，1993
75 金闻博，戴亚．烟草化学．北京：清华大学出版社，1994
76 王岚，方瑞斌，杨光宇等．固相萃取光度法测定烟草中的挥发酚．分析实验室，2002，21(2)：14-16．
77 The determination of phenolic compounds in cigarette smoke.Lorillard Tobacco Company, 1999
78 Quin LD. Chemical study on tobacco and its smoke. Selecta Chme., 1962, 20:37-62
79 Frankenburg W G, Chemical change in the harvested tobacco leaf. Part Ⅰ. Chemical and Enzyatic Conversions During the Curing Process. Adv. Enzymol. 1947, 6:309-387
80 Stedman R L.The chemical composition of tobacco and tobacco smoke. Chem. Rev., 1968, 689:153-207
81 Phillips M, Bacot A M. The chemical coposition of certain grades of type 11, American flue-cured tobacco. Relationships of composition to grade characteristics. JAOAC. 1953, 36: 504-523
82 Stedman R L. Polyphenolic constituents of tobacco. Tob. Sci.,1957,1:155-160
83 Wright H E. The phenolics of tobacco and their significance. Proceedings of a symposium on plant phenolics and their industrial significance. Ed. V. C.Runeckes. Imperial Tob. Co. Montreal. Canada.1962
84 Tso T C.Physiology and biochemistry of tobacco plants. Dowden, Hutchinson and Ross Inc.,. 1972
85 Zane A, Steck, W, Wender, S. Identification of 4-O-caffeoyl-quinic acid and related depsides in tobacco from cigarettes.Tob.Sci.,1965, 9:85-87
86 Johnson W H, Gwynn, G R. Characterization of oxidation browning in flue-trued tobacco. I. A method for evaluating varietal discoloration potentials. Tob. Sci..1970, 14:1-6
87 Oakley E T , Millham J O. A rapid sepectrophotometric metnod for tthee determination of chlorogenic acid in leaf tobacco. 15th Tob. Chem. Res. Conf., Philadelphia Pa. 1961
88 Nystrom C W. Effect of aging on the content of chlorogenic acid, rutin and scopoletin in flue-crued tobacco. 15th Tob. Chem. Res. Conf., Philadelphia Pa,1961
89 Wilkinson F B, Phillips M , Bacot A M, Cchoiogenic and caffeic acid in certain standard grades of U. S. type 12 tobacco. JAOAC,1954, 37:1004-1012
90 Penn E T, Weybrew J A. Some factors affecting the content of the principal polyphenols in tobacco leaves. Tob. Sci., 1958, 2:68-72
91 Sledmen R. L. The chemical composition of tobacco and tobacco smoke, Chem, Rev., 1968, 65:126
92 Phillips J P. Organic electronic spectral date. lnterscience publishers.Newyork, 1953
93 Majors R E. Polyethylene glycols and methoxypolyethylene glycols. Union Carbide, 2003
2 李汉超，王淑娴．烟草·烟气化学及分析．郑州：河南科学技术出版社，1995
3 王晶晶．卷烟配方与烟支设计．北京：中国科学技术出版社，2000
4 张保振．中国烟草大辞典．北京：中国经济出版社，1992
5 石碧，狄莹．植物多酚．北京：科学出版社，2000
6 Viart P. Phenolic index oftobbaco, Tobacco science, 1976, 11: 857
7 韩富根．用福林法测定烟叶中总酚含量的探讨．郑州：河南农业大学学报，1993，1：95-98
8 张槐苓，葛翠英．烟草分析与检验．郑州：河南科技出版社，1994
9 Rayburn C H, Harlan W R, Hanmer H R. Determination of volatile phenols in cigarette smoke.Anal.Chem. 1953, 25: 1419
10 胡旺云．烟草化学家研讨会-半个世纪以来讨论烟草及其制品分析方法发展的论坛．国外烟草科技文选，1998，5：19
11 Shigeo A. A method of ion change chromatography. Hatano Taboko Shikensh Hokoku. 1966,57:5-15, CA, 68:27581f
12 Shigeo A. A method of ion change chromatography, Hatano Taboko Shikensh Hokoku, 1966,57:16-26, CA, 68:27582g
13 Lattanzio, Vincenzo. Silica gel method of determination in poylphenols J.Food Sci..1981, 46 (6): 1907-1917
14 Anderson R A.Scpoletin determination of tobacco in GC, Phytochemistry. 1972,11 (8): 2593-2595
15 Sakaki, Takeshi.Kennyu Hokoku-Nippon Senbai Kosha Chou Kenkyusho.1982,(124): 33-6, CA, 99: 85360j
16 Court, William A.J.Polyphenol determination by HPLC, J Chromatogr, 1977, 130:287-291
17 Nisseror S. Liquid chromatography separation of polyphenols application to tobacco extract analysis.Analusis.Dec 1984,12 (10): 523-524
18 Ishguro S, Sugawara S. Studies on the composition of smoke components of lamina and midrib cigarettes. Ⅳ. Comparisons of smoke components from lamina and midrib cigarettes of flue-crued tobacco leaves by the trimethylsilylation method. Agric. biol. Chem.. 1978, 42:407-417.
19 Mcwilliams D, Deutsch L J, Gladden J D. Simple gas chromagraphic determination of
phenol and cresols in Cambridge pad extracts. TCRC. 1986
20 Tomkins B A. Studies on determining of phenolic compounds of tobacco and cigarette smoke. IARC Sci. Publ.,1987, 81:279-89
21 Nanni E J. Lovette M E, Hicks R.D. A method of reducing phenlics in tobacco and smoke. J Chromatogry. 1990, May 505:65-74
22 赵明月，周富臣．卷烟主流烟气中酚类物质的毛细管气相色谱分析．中国烟草学报，1992．2：1-14
23 Clark J, Bunch J E. Solid phase microextraction/gas chromatograph/selected ion mode mass spectrometry analysis of phenolics in cigarette smoke condedsate. Tob. Chem. Res. Conf..1995, 49:57
24 毛多斌，马宇平，张俊松，等．国内外混合型卷烟主流烟气中酚类物质的分析．烟草科技．2002，4 1：7-20
25 苏国岁．卷烟烟气中对、间甲酚总量的气相色谱分析．烟草科技．2003，8：16-18
26 Tomkins B A, Jenkins R A, Griest W H, Reagan R R, Holladay S K J. A liquid chromatography method of determination of phenol and cresol in cigarette smoke. Assoc Off Anal Chem.. 1967,5:19-23
27 Kallianos A G, Means RE, Mold J D.Alkyi catechols in cigarette smoke .TCRC.1967
28 Kuwata, Kazuhiro, Michiko.Revered-phase liquid chromatographic determination of phenols in auto echaust and tobacco smoke as p-nitrobenzeneazophenoi derivatives. Anal. Chem.. 1981, 53:1531-1534
29 Kuwata, Kazuhiro, Uebori,et al. The new method of determinting phenolics of cigarette smoke. Anal. Chem. 1980, 52:857-860
30 吴帼英．用反相高效液相色谱对卷烟烟气冷凝物中单酚的定暈分析．中国烟草科学．1986，3：45-48
31 Risner C H, Cash S L.The determination of major phenolic compounds in tobacco smoke. J Chromatogr Sci..1990, May 28:39-44
32 Prakasha A, Ireland M S.A high performance liquid chromatograohic method to quantitate phenols in isopropanl extract of mainstream cigarette smoke. TCRC. 1992
33 Ricner C H.An improved method for the quantification of selected phenolic compounds in ambient air. TCRC. 1992
34 欧阳清．高校液相色谱法定量分析主流烟气中的酚类物质．上海烟业，1994，3：48-49
35 Rayburn C H.Chemical analysis of tobacco and tobacco smoke: determination of smoke volatile phenols in cigarette smoke . TCRC.1952
36 Lindsey A J. Burrows I E, Smith Gal et al.Thin layer chromatography: smoke applications in tobacco chemistry. TCRC. 1963
37 Anderson I G M.Comparision of methods for the determination of phenols in tobacco smoke highlighting a new approach using capillary electromatography. CORESTA.1998, sport2:194
38 Munteanu, Florentina-Daniela, Lindgren et al. A method of determining the phenolics in tobacco and smoke.Anal.Chem.. 1998, 13:2596-2600
39 李忠，施红林，王岚，等．新型自动水蒸汽蒸馏仪蒸馏测定烟草及其制品中总挥发酚．理化检验-化学分册．2002，12：628-630
40 NCI, National Cancer Institute. Smoking and Health program: Toward less hazardous cigarettes: Report No.1. The first set of experimental cigarettes. DHEW Publication No.(NIH).1976, 76-905.
41 Crosthwaite L, Sheen S J, Burton H R. Alkylating activity in the extract and pyrolyzate of tobacco leaves varying ingenotype and chemical treatment;Tob. Sci..1979, 23:35-37.
42 Bentley H R and Burgan J G.Polynuclear hydrocarbons in tobacco and tobacco smoke. Part Ⅲ. The inhibition of the formation of 3,4-benzopyrene in cigarette smoke; Analyst, 1960, 85:727-730.
43 Kaburaki Y H, Shigematsu Y M, Kusakabe H. Studies on the composition of tobacco smoke (Ⅺ). Relatioms between vapor phase components of smoke and constituents of tobacco. Kenkyo Hokoku-Nippon Sembai Kosha Chuo Kenkyusho. 1969, 111:143-149.
44 Garcia Roche M O, Cabrera A, Becquer A et al. Health risk due to the use of nitrate in the manufacture of Cuban golden cigarettes; Rev, Cubana Hig. Epidemiol. 1986, 24:487-492.
45 Pyriki C, Moldenhauer W , Knappe T. Zur Frage der Behandlung des Zigarettenpapiers und des Tabaks zwecks Verminderung von polycyklischen Kohlenwasserstonffen in deren RAUCH: Ber. Inst. Tabakforsch. Dresden. 1965, 12:35-37.
46 Rathkamp G, Hoffmann D. Chemical studies on tobacco smoke ⅩⅢ. Inhibition of the pyrosyntheses of several selective smoke constituents Beitr. Tabakforsch, 1970, 5:302-306.
47 Kallianos A G,. Means R E and Mold J D.Effect of nitrates in tobacco on the catechol yield in cigarette smoke: Tob. Sci..1968, 12:125-129.
48 Brunnenmann K D , Posset L.Routine high-performance liquid chromatographic method for assessing sorbate in tobacco. Jourmal of Chromatography. 1980, 202:494-499.
49 Budick D, benner J F, Burton H R. Thermal decomposition of tobacco Ⅳ. Apparent correlations between thermogravimetric datd and certain constituents in smoke from chemically-treated tobaccos. Tob. Sci..1969, 13:33-37.
50 Burton H R. Thermal analysis of chemically-treated tobacco. Proc. Tob.. Health Workshop. 1969, 33-37.
51 Burton H R, Benner J F.Investigations on the effect of chemical modifiers on tobacco smoke: Proceedings of the University of Kentucky. Tobacco and Health Workshop Conference. Lexington. 1972,50:341-363.
52 Wynder E L, D Hoffmann Ein. experimenteller Beitrag zur Tabakrauchkanzerogenese Dtsch. Med. Wschr. 1963, 88:623-628.
53 Wynder E L, Hoffmann D. A study of tobacco carcinogenesis. Ⅹ. Tumor promoting activity. Cancer. 1969, 24:289-301.
54 Dontenwill W, chevalier H J, Harke H P et al. Klimischungen über die tumorerzeugende Wirkung von Zigarerrenrauch-Kondensaten an der Mausehaut. Ⅳ. Einzelvergleiche yon Kondensaten aus verschiedenen Tabakfolien, Einflub eines Zusatzes von NaNo3 zum Tabak Oder zu Tabakfolien, Wirkung Fluchtiger Bestandteile des Rauches, Einflub einer Initiabenhendlung mit Dimethylbenzanthracen. Z. Krebsforsch. 1972, 78:236-264.
55 Dontenwill W, Chevalier H J , Harke H P et al. Experimentelle Untersuchungenüber die tumorerzeugende Wirkung von Zigarerrenrauch-Kondensaten an der Mausehaut. Ⅴ. Mitteilung: modifizierter Zigaretten.Z. Krebsforsch. 1976, 85:141-153.
56 Klimisch H J. Vergleichende chemisch-analysche Untersuchungen an Kondensaten modifizierter Cigaretten. Diskussion einer Korrelation zur biologischen Wirksamkeit; Zeitschrift fur LebensmitteI-Untersuchung und-Forsch.1972, 150:93-99.
57 Halter H M. Reconstituted tobacco smoking and health possibilities.J. Natl. Cancer Inst.. 1972, 48:1869-1883.
58 Lyerly L A. Direct vapor chromatographic determination of menthol, propylene glycol, nicotine and triacetin in cigarette smoke; Tob. Sci.,1967, 11:49-51.
59 Laurene A H, Cundiff R. H. Greene. Determination of glycerol and propylene glycol in cigarette smoke; Tob. Sci.. 1965, 9:1-4.
60 Kallianos A G, Means R E, Smoking and smoke quality. Mold J D, LIGGETT & MYERS, 1967,12:17-20.
61 Tomkins B A, Jenkins R A, Hoiladay S K. J Assoc Off .Anal Chem.. 1967,59:19-23
62 Carmines E L. Evaluation of the potential effects of ingredients added to cigarettes. Part 1: Cigarette design, testing approach, and review of results. Food Chem. Toxicol. 2002, 40:77-91.
63 Sledmen R L. The chemical composition of tobacco and tobacco smoke.Chem. Rev..1968,
68: 153
64 周冀衡，朱小平，等．烟草生理与生物化学．北京：中国科学技术大学出版社，1996
65 Wynder E.L, Hoffmam D. Tobacco and tobacco smoke. Academic press Newyork and Londonm. 1967
66 Armstrong G, Mrohrbaugh L M, Wender S H. the component of leaf and their relationship to smoking quality and aroma Recent advances in tobacco science. 1972, 2:925-948
67 阎新甫，韩锦峰．烟草多酚类化合物的研究进展．华北农学报，1987，2：31-38
68 朱汉春．中国烟草知识大全．长沙：湖北科学技术出版社．1999
69 周正红，高孔荣，张水华．烟草中化学成分对卷烟色香味品质的影响及其研究进展．烟草科技，1997，197(2)：22-25
70 钟庆辉．烟草芳香吃味化学指标的探索．烟草科技，1981，41(4)：21-23
71 李丛民．植物多酚对烟草制品品质的影响．烟单科技，2000，140(1)：27-29
72 Snook M E, Mason P F, Sisson V A. Polyphenols in the nicotiana species. Tobacco Science. 198630(18):43
73 雷永和．云南烟草品种志，昆明：云南科技出版社，1999
74 左天觉，朱尊权．烟草生产、生理和生物化学．上海：上海远东出版社，1993
75 金闻博，戴亚．烟草化学．北京：清华大学出版社，1994
76 王岚，方瑞斌，杨光宇等．固相萃取光度法测定烟草中的挥发酚．分析实验室，2002，21(2)：14-16．
77 The determination of phenolic compounds in cigarette smoke.Lorillard Tobacco Company, 1999
78 Quin LD. Chemical study on tobacco and its smoke. Selecta Chme., 1962, 20:37-62
79 Frankenburg W G, Chemical change in the harvested tobacco leaf. Part Ⅰ. Chemical and Enzyatic Conversions During the Curing Process. Adv. Enzymol. 1947, 6:309-387
80 Stedman R L.The chemical composition of tobacco and tobacco smoke. Chem. Rev., 1968, 689:153-207
81 Phillips M, Bacot A M. The chemical coposition of certain grades of type 11, American flue-cured tobacco. Relationships of composition to grade characteristics. JAOAC. 1953, 36: 504-523
82 Stedman R L. Polyphenolic constituents of tobacco. Tob. Sci.,1957,1:155-160
83 Wright H E. The phenolics of tobacco and their significance. Proceedings of a symposium on plant phenolics and their industrial significance. Ed. V. C.Runeckes. Imperial Tob. Co. Montreal. Canada.1962
84 Tso T C.Physiology and biochemistry of tobacco plants. Dowden, Hutchinson and Ross Inc.,. 1972
85 Zane A, Steck, W, Wender, S. Identification of 4-O-caffeoyl-quinic acid and related depsides in tobacco from cigarettes.Tob.Sci.,1965, 9:85-87
86 Johnson W H, Gwynn, G R. Characterization of oxidation browning in flue-trued tobacco. I. A method for evaluating varietal discoloration potentials. Tob. Sci..1970, 14:1-6
87 Oakley E T , Millham J O. A rapid sepectrophotometric metnod for tthee determination of chlorogenic acid in leaf tobacco. 15th Tob. Chem. Res. Conf., Philadelphia Pa. 1961
88 Nystrom C W. Effect of aging on the content of chlorogenic acid, rutin and scopoletin in flue-crued tobacco. 15th Tob. Chem. Res. Conf., Philadelphia Pa,1961
89 Wilkinson F B, Phillips M , Bacot A M, Cchoiogenic and caffeic acid in certain standard grades of U. S. type 12 tobacco. JAOAC,1954, 37:1004-1012
90 Penn E T, Weybrew J A. Some factors affecting the content of the principal polyphenols in tobacco leaves. Tob. Sci., 1958, 2:68-72
91 Sledmen R. L. The chemical composition of tobacco and tobacco smoke, Chem, Rev., 1968, 65:126
92 Phillips J P. Organic electronic spectral date. lnterscience publishers.Newyork, 1953
93 Majors R E. Polyethylene glycols and methoxypolyethylene glycols. Union Carbide, 2003