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RJ Reynolds

The Tobacco Chemists' Research Conference: a Half Century Forum Advances in Analytical Methodology of Tobacco and Its Products.

Date: Oct 1996
Length: 183 pages
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REPORT
Alias
R 11117
Site
R&D
Biochem Biobehavioral
Rodgman Ar
Sr Staff Scientist
Rodgman Post '89 Docs
Named Organization
American Health Foundation
List of Philip Morris Awardees
Assn of Official Analytical Chemist
Univ of Ky Tobacco & Health Researc
List of Smoking & Health Authors
Intl Ag for Research on Cancer
Stanford Research Institute
American Machine & Foundry
List of Organizations
Dept of Agronomy
Univ of Ky
Natl Cancer Institute
Eastern Regional Research Laborator
Tobacco Science
Sloan Kettering Institute for Cance
Medical College of Va
List of Researchers
List of Affiliations
Japan Tobacco Salt
Natl Institute of Occupational Safe
Registry of Toxic Effects of Chemic
Plenum Press
Academic Press
Request
Mitchell Int27
Burton by Agreement
US Priority Request 2
Wallace 1rfp14
Wallace 1rfp12
Wallace 1rfp10
Wallace 1rfp11
Wallace 1rfp9
Wallace 1rfp7
US Comprehensive Request 447
US Comprehensive Request 130
US Comprehensive Request 38
US Comprehensive Request 103
US Comprehensive Request 174
US Comprehensive Request 80
US Comprehensive Request 46
US Comprehensive Request 326
US Comprehensive Request 37
US Comprehensive Request 47
US Comprehensive Request 264
US Comprehensive Request 86
US Comprehensive Request 425
US Comprehensive Request 251
US Comprehensive Request 263
US Comprehensive Request 42
US Comprehensive Request 241
US Comprehensive Request 249
US Comprehensive Request 201
US Comprehensive Request 253
Wallace Court Order 20010629
Longden Int3
Longden Int2
Longden Int7
Willard Brown 1rfp15
Willard Brown 1rfp22
Willard Brown 1rfp19
Willard Brown 1rfp54
Willard Brown 1rfp56
Willard Brown 1rfp58
Wallace 8rfp40
Named Person
Tcrc
Parmele, H.B.
Jeffrey, R.N.
Rjr
Kosak
American
Ftc
Philip Morris
Iso
Coresta
Usda
Lindsey
Hicks, G.W.
Atf Natl Laboratory
American Chemical Society
Grob, K.
Ishiguro
Sugawara
Guerin
Fieser, L.F.
Newman, M.S.
Cook, J.W.
Lacassagne, A.
Buuhoi, N.P.
Clar, E.
Surgeon General
Snook
Willard, R.L.
Furman
Kennaway
Lamphier, D.R.
Roffo
Groffith
Dalhamn
Hiller
Rodgman, A.
Keith
Procter
Epa
Harke
Maskarinec
Neurath
Esquier
Hee
Risner
Jenkins
Mait
Fischer, K.
Deluca
Demain
Brahms
Beethoven
Bach
Benner
Burdick
Burton
Touey
Mumpower
Information Resources
Brice
Willits
Analytical Chemistry
Crowder
Bates
Shell Chemical
Martin
Williams
Schumacher
Wolfrom, M.L.
Oh, S.T. Univ
Severson
Adams
Rayburn
Davis
George
Kensler
Resnik, F.
Spears, A.
Lorillard
Carpenter
Holmes
Seligman
Wolff
Bgsm
Hoffmann
Newsome
Liggett
Stedman
Schlotzhauer
Schmeltz
Cook, L.C.
Mckennis
Bowman
Cundiff
Glock
Griffith
Celanese
B.&W.
Nc, S.T. Univ
Ornl
Chung, H.
Kelly, S.
Oldaker, G.B. III
Tabakforsch Intl
Hhs
Chem Comm
Japan Monopoly
Wake Forest Univ
Characteristic
Marginalia
Box
Rjr7010
Author
Green, C.R.
Rodgman, A.
Rjr
Brand
Premier
UCSF Legacy ID
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I 1 I I I 1 I a I first question than to answer the second. For instance, the 2nd and 14t1i volumes of Recent Advances in Tobacco Science are dedicated to discussions of "Leaf composition and physical properties in relation to smoking quality and aroma" and "Chemical and sensory aspects of tobacco flavor," respectively. For example, scientists from American Tobacco presented papers documenting the detrimental character of nornicotine towards acceptable smoke flavor (11/03,16/22). Much of the analytical work relating chemical composition to consumer preference may be proprietary and not available in publication. The second major goal in tobacco science analytical methodology has been the search for components of tobacco and its smoke that may be responsible for the human health risks associated with tobacco usage. TCRC's record is filled with papers attempting to isolate the "culprit du jour," e.g., benzo[a]pyrene (BaP), other polycyclic aromatic hydrocarbons, aza-arenes, aldehydes and ketones, nitrogen oxides, N- nitrosamines, aromatic amines, chlorinated hydrocarbons, free radicals, etc. We have the so called "List of 43" tumorigenic agents in tobacco and smoke prepared by Hoffmann and Hecht (47). In terms of human lung cancer, this list has its limitations because most of the components have not been shown to be 1) tumorigenic to any human tissue or 2) tumorigenic to lung tissue in laboratory animals (48). Only 5 of the 43 compounds, benzo[a]pyrene, N-nitrosodimethylamine, N-nitrosodiethylamine, cadmium, and polonium- 21Q, have produced lung tumors in animal experiments but these studies were conducted at massive dose levels relative to smoking or ETS exposure. Results of the BaP, N- nitrosodimethylamine, and N-nitrosodiethylamine inhalation studies are rated as "equivocal" by RTECS (49). At least two points need to be made about this search for toxic agents: 1) the levels of reported toxic agents and 2) the presence of constituents with anti-toxic properties. About the first, Wakeham (46) remarked, ...Some analysts with sophisticated methods are no, w finding chemicals in fraetional nanogram quantities, o, ften ignoring the fact that toxicity is a fanction of concentration. They are looking for the needle in the haystack, as it were, to prevent the cow Jrom eating it. 14 52118 5214
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l I I I I . ..~- • Tobaceo •.• -Smoks A B SO 55 60 65 70 75 80 85 90 95 Legaro Year eDM)&Mte Qsttt eoehticaLietboft x .. . A pria to 19Si B 19631960 C 19641970 l D 1970 b mid- E 1970s mid-1970s to dats I I 1 The tremendous escalation in the number of identified tobacco smoke (and tobacco) components from 1954 to-date was achieved by successive advances in analytical methodology, particularly the technologies concerned with the separation and identification of individual components in complex mixtures. In Fig. I is shown the increase in the number of identified tobacco smoke components from 1950 to date. Also noted in Fig. I are the approximate dates when the major analytical advances were implemented in the study of tobacco smoke composition. Figure 1 Number of Identified Tobacco Smoke Components Reported since Kosak (40): Accumulative by Year 'alsssleal' dwnical tactniques adumn chramatopraphy gas chromampraphy plass cap5lary pa eMomatopaphy aapbd with maea speckametry derkatives ta hiph readuion pas chromatopraphy (HR6C), HPLC, - masa spedrom.try Prior to 1950, the major part of the isolation and characterization of individual components from tobacco smoke involved so-called "classical" chemical techniques, i.e., the fractionation of tobacco extracts or cigarette smoke condensate (CSC) into neutral (aliphatic hydrocarbons, PAHs (polycyclic aromatic hydrocarbons), and esters), acidic (acids and phenols), and basic fractions (amines, nicotine-related alkaloids), followed by crystallization and/or distillation of these subfractions. In the early 1950s, liquid column chromatography on alumina, silicic acid, or Florosil® of the neutral or acidic fractions permitted further separation of components 10 1 52118 5210
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I I I l .l I I I I I Examination of, Fig. I indicates that, as each analytical, separation, and identification technology was developed and utilized, the number of smoke components identified per year increased. It is realized that various investigators who pioneered an emerging analytical technology were often involved with the development and/or use of the technology prior to the period indicated in Fig. 1. No slight of their noteworthy contributions is intended. The periods indicated in Fig. I are those when the analytical technology in question was sufficiently advanced and used by almost all investigators involved, in the analysis of tobacco, tobacco smoke and/or definition of their composition. The next dramatic break in the number vs. year plot occurred in the late-1970s when the glass capillary' gas chromatograph was coupled directly to the mass spectrometer. We recall that at this time there was a good-hearted argument between mass spectroscopists and chromatographers as to whether the mass spectrometer was just another GC detector or the gas chromatograph was a sophisticated inlet device for the mass spectrometer. Present day mass selective detectors appear to support the cluomatographers' position. Regardless, this mating of technologies permitted separation of the components of a particular tobacco or smoke fraction or subffaction and determination of the molecular weight and/or fracture pattern of each component or mixture of closely related components immediately after they exited the chromatograph. Interpretation of the data thus obtained, occasionally in concert with findings from W, IIt, and/or NMR studies, permitted very rapid and unequivocal identification of smoke components. Even with extremely low amounts of a particular smoke fraction or subfraction, definitive chromatograms and MS data may be generated on several hundred chromatographic peaks, each of which may represent an individual compound or a mixture of several compounds. An outstanding example of the use of GC-mass spectrometry in the definition of tobacco smoke composition is the study by Snook et al. (28/40, 45) on the PAHs in cigarette smoke. Nearly 1,000 individual PAHs, their homologs, and oxygen analogs were ' The term glass capillary is used in tlx generic sense to include Posed silica columns. 12 52118 5212
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I .I I I I .1 1 f J J reported (Table II), but many of the identifications were subsequently shown to be incorrect. By use of various analytical methodologies, the identities of many components cataloged by Kosak in 1954 (Table II) were resolved. Of the approximately 80 entries, the identities of 33 (over 40°/a) of the components were questioned by Kosak because he did not "consider the evidence cited in the literature to be definitive proof' of their identities. Two of the listed items (benzopyrene, "condensed aromatics") were reported by Roffo (36) who did not study tobacco smoke generated by a process approximating human smoking but studied a"desttvctive distillate" from tobacco. Several of the components (a-, (i-, and y-socratine,;obelin, lohitam, anodmin, Iathraein, poikiline, and gudham) first reported by Wenusch and Schbller (37) and listed under "Alkaloids" were subsequently demonstrated to be mixtures or a component Gsted elsewhere in Table 11. Foe;example, Kuffher et al. (38) demonstrated that obelin is a salt of ammonia, a- and Q- socratine are mixtures of nicotyrine and 2,3'-bipyridine, and y-socratine is !-nornicotine. Poikiline is identified as 4-amino-l-(3-pyridyl)-butanone. And finally, the complexity of either tobacco or its smoke presents a professional challenge to the analytical chemist. Analytical chemists who can master the difficulties of separating, identifying, and quantifying components of tobacco and smoke are masters of analytical technology. Whatever the reason for our intense scrutiny of tobacco and smoke, there appears to be a relationship between the techniques applied and the numbers of compounds either quantified or merely identified. In 1958, RJRT R&D personnel cataloged from in-house research and literature reports 230 identified compounds in tobacco and 202 identified components in cigarette mainstream smoke; 54 components were common to both 6sts. The RJRT R&D compilation of tobacco smoke components has been revised periodically, more recently in a computerized catalog: The number of identified and cataloged tobacco smoke components is about 3,900; the number of identified tobacco components is about 3,800. Relative to tobacco composition, about 2,800 components are unique to tobacco smoke. For the purposes of Fig. 1, we estimate that there are about 4,800 known components in tobacco smoke (39). 8 52118 5208
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I I deteated; and many were identified unequivocally. These results should be compared with the ridiculous controversy that raged in the early 1950s as to whether PAHs were indeed present in cigarette mainstream smoke. The early- to mid•1970s also saw the emergence of high performance liquid chromatography (HPLC), a highly efficient and effective variation of liquid column chromatography. See reviews by Bell (4), Haeberer and Chortyk (5), Green et al. (8), and Court (14). HPLC made possible the identification and quantification of many compounds, e.g., heat sensitive and ionic substances, that are difficult to analyze by other means. From the existing research on tobacco and smoke. composition, one can only conclude that both are very complex mixtures requiring the most advanced analytical technology to completely characterize their makeup. There are currently about 6,700 chemical substances known to be present in tobacco and smoke. Wakeham (46) as early ac•"1971 opined that as many as a hundred thousand chemical components may exist in tobacco smoke alone. Numerous authors have implied that this is a unique property of the tobacco plant and its smoke. However, this known complexity is more likely not unique but due to the intense analytieal scrutiny to which this valuable and controversial commodity has been subjected. "HOLY GRAII S" OF TOBACCO RESEARCH When 50 years of TCRC analytical research on tobacco and smoke are viewed, it is obvious that the objective of this work is an attempt to answer one or both of the following questions: 1. What components of tobacco and its smoke are important to the flavor, sensory experience, and consumer acceptance of tobacco products? 2. What components of tobacco and its smoke may be responsible for the human health risks ass6ciated with tobacco usage? Unfortunately, the brief answer to both of these questions is that we still do not know. . J. More has been written and discussed within the TCRC forum in an attempt to answer the t+ S.-.• 13 . ~ 52118 5213 a
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I ~ I _1 .1 I I ~ I J "mellowing" effect on the smokability of the tobacco. TCRC audiences have heard many reports on the chemical changes that occur during aging. Filling power of purchased or processed tobaccos has great impact on the economics of cigarette production because cigarettes are sold by unit rather than by weight. The seemingly simple analytical methodology of determining the volume occupied by a known mass of tobacco under a defined pressure has led to numerous TCRC presentations, cf. 12/04, 25/04, 30/3 t. Manufacturers and suppliers to the tobacco industry use analytical technology to ensure that they provide a consistent product to their consumers. Likewise they have a product stewardship responsibility to know the ingredients in their products and that these meet all regulatory standards. Manufacturers need to understand how their products work so that new, improved products that are more economical or meet market-place demands can; be developed. Learning based upon analytical methodology is the key to this understanding. Additionally, manufacturers and suppliers use analytical results to defend themselves i"n litigation and support advertising claims for their products. • Regulators utilize analytical methodology to assess the safety of products, inform consumers and the public, and classify various tobacco products for taxation purposes. The FTC "tar" and nicotine smoking methods are examples of analytical methodology useAj)y regulators (22). jjISTORICAL REVIEW A review of the TCRC literature reveals that 2,008 titles are recorded for presentation.a Aside from the reviews, nearly every paper given at the TCRC either presents new or uses existing analytical methodology. Our count indicates that at least 779 papers describe analytical technology directly and 307 additional papers are concerned with isolation and identification techniques. Practically everyone who has conducted a Nthough 2,008 titles are recorded, an unknown, but few, number of papers have been withdrawn at the last minute. The record is unclear on the precise number but it is believed to range from 5 to 10 papers. This is a remarkable record. 21 , 52118 5221
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I I I J I I I .l Sugar, total nitrogen, nicotine, total alkaloids, and protein levels are examples of targets for the development of new tobacco cultivars. See Mendell et al. (54) for additional factors. Once tobaccos reach the auction floor, manufacturers want to know more about the physical and chemical characteristics of their intended purchase to achieve product consistency. However, it is rarely possible to conduct analyses before tobacco purchase. For this decision, we must rely on the art of experienced buyers. Several attempts have been made to relate the color (10/25, 12/01, 13/04), feel, and odor clues used by the buyer to analytical results. Numerous studies presented at the TCRC have attempted to correlate tobacco volatiles obtained by steam distillation or headspace analysis to tobacco quality. These studies have met with only modest success. Table V Reasons for Application of Analytical Technology to the Study of Tobacco Products .y; Area Need Agronomy - • Chemical characterization and breeding to obtain the greatest profit • Agronomic chemical residues, e.g., fertilizers, pesticides, herbicides • Production research, e.g., curing methods, sucker control • Alternative uses of tobacco e. ., protein production 55 Buyers • Purity, e.g., sand • Usabilit e. ., fines stems, fillin power Maniifacturers • Quality assurance and Suppliers • Product stewardship • Product development • Product understanding • Regulatory requirements • Pest control • Product defense Regulators • Safety • Advertising • Consumer information • Taxation Once purchased, tobacco is subjected to chemical and physical tests. Among these are moisture, sand, stems, and Iamina content measurements. Green tobaccos, i.e., new crop tobaccos, are usually stored for months to years because this aging has a 20 ,, 52118 5220
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I -1 I I I I 1 adapted hot wire anemometer for measurements of the velocity and duration of an inspired puff. For the regular length unfiltered cigarettes reported in this study, average characteristics for the 120 subjects were a 1.9-sec. puff, taken twice a minute, with a 44- cm3 puff volume. Adams (20/31) reported on U.K. studies with tipped cigarettes that employed a flow measuring orifice and found values of 31-cm'volume, 1.4-sec. duration, and 1.8 puffs per minute. Gottscho (27/16) presented results of an international study on smoking parameters of cigar smokers. They took on average 20-cm' puffs. In 1982, Griffith (36/35) presented details of methodology used to characterize human smoking behavior at the University of Kentucky Tobacco and Health Research Institute. Jenkins et al. (38/49) described a variable puff parameter smoking machine to study changes in smoke composition as a function of puff shape and other parameters. McBride (39/35) reported on the construction and performance of a "smoke duplicator" system that aocyirately records and then reproduces human smoking behavior. Jenkins et al. (40/56) presented results from an ORNL project that combined a pressure transducer and a small IR backscgnering detector in a cigarette holder that measured not only human smoking parameters but also particulate matter yields. And finally, Borgerding and Winkler (49/47) studied the effect of alternate puffing regimes on cigarette yields. They concluded that although smoking parameters, e.g., volume, duration, and interval, affect yields, the rankings of products remain the same. Earlier work on the effects of changing smoking parameters was conducted by Butler et al. (13/06) and Frisch and Spivey (22/26). The second broad group of scientists participating in TCRCs has been those involved with increasing our knowledge about tobacco and its products. Often they conduct studies with unique equipment and difficult to reproduce methodologies. Sometimes the value of this exploratory work is so significant that more routine analytical techniques arise from their work. However, most of the knowledge-seeking studies reported at TCRCs are so unique that they are never repeated. 26 52118 5226 I
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I _l J -1 I _J giVALYTICP t. TECHNOLOGIES DISCUSSED AT TCRCe The rich 50-year history of the TCRC includes the discussion of numerous analytical methodologies. We must remember that in 1947 the state of analysis was much different than today. Major techniques for isolation and identification of constituents in mixtures included crystallization, distillation, and gravimetry. The use of chemical reactions to form crystalline substances that could be purified and weighed or colored derivatives quantifiable by spectroscopy was popular. Instrumentation depended upon vacuum tubes that were both bulky and notoriously unstable. Digital computers and microprocessors were not yet invented. Today's simple tasks of multiplying and dividing on a hand-held calculator available at any local shopping center for less than five dollars were laboriously accomplished by manual calculation, slide rules, or with the aid of log tables. Discussion about the principles and practice of each of these techniques as related to tobacco'science or even to TCRC presentations is beyond the scope of this review. We intend to provide commentary on the most important techniques, direct the reader to existing reviews, supplement earlier reviews with more recent presentations, and provide top-line information where none exists. Chiomatoeraohv Foremost among the analytical technologies applied to tobacco science is chromatography. Although the invention of chromatography, i.e., column adsorption chromatography, is attributed to M. S. Tswett in 1903 (56), the sophisticated practice of the technique as we know it today had not progressed greatly by the Ist TCRC. Because chromatography is a branch of analytical chemistry devoted to the separation of mixtures and also because of the complexity of tobacco and smoke composition, tobacco scientists have been leaders in the practice and advancement of this technique. TCRC presentations have been made on the following variations of chromatography: affinity, anion-exchange, cation-exchange, carbon column, counter current, flash, gas-liquid, gas-solid, gel 27 52118 5227
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I I .l 1 I I I I I J Although much has been made over the controversial compounds found in tobacco and smoke during the past 50 years of the TCRC, the inhibitors, anticarcinogens, and antimutagens present in tobacco and its smoke have received scant attention. Comparison of the Gst of identified components in tobacco smoke with 6sts of compounds shown to possess inhibitory or antitumorigenic action in tumorigenesis-type experiments in laboratory animals (52) reveals not only that tobacco smoke contains numerous antitumorigens but also that the levels in smoke of many of them far exceed those of the smoke components categorized by some investigators as "tumorigens." Tobacco smoke components demonstrated to be inhibitors and antitumorigens to these categorized components are listed in Table IV. Furthermore, Lee and co-workers (48/44, 53) have presented in vitro methodologies at the TCRC, demonstrating that both nicotine and cotinine inhibit the mutagenicity of several N-nitrosamine components of tobacco or tobacco smoke. USES OF ANALYTICAL TECHNOLOGY Tobacco and its products are among the most studied substances on record. There are numerous reasons for this intense scrutiny but basically it reflects the monetary value of.tobacco commerce. Tobacco and its products have been studied by various groups, including those involved with growing and harvesting the crop, the buyers and processors, manufacturers, suppliers, research institutes, universities, and government agencies. Why do all these folks use analytical methodology to study our crop and products? We have attempted to provide some answers in Table V. At the farm level, growers need to know the minimum amount of agronomic chemicals, e.g., fertilizers, herbicides, pesticides, sucker control agents, that are needed to grow a healthy, good-smoking crop. Farmers, extension agents, university scientists, government scientists, and manufacturers are also interested in the effects that the many tobacco production factors such as topping, harvesting, and curing have on tobacco's physical and chemical characteristics. Breeders are interested in knowing the constituents of tobacco that impart desirable smoking characteristics to the crop. ! J ,L-A r.1 18 (~J ~. 52118 5218

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