Document B54rb1VYGNVZq46Yqy9bvk45o

R&S 107703 JfcB$0~*IDZC$L ftzSEARCH 3^0CU>2KT E>iSCR?TION FORM fi3 68 69 76 Duplicate in all cards:--> y year as-1961- Pile number [Eight justify [Numeric only] Author (s), as Last Kane PS (No Punctuation) and coden for journal as JAMA, preceeded-by. one blank spacs 77 78 Sub-Index Code Title of Report; end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with -coma-space. Avoid other punctuation; do not abbreviate. 1? ' . v V 61 62 [ ... 2T C __ -- ^re-^K,c. (J 7 ^h,shr- r '"n \ nj4 / of tJtoy/ t *- s dh 22 23 /-' ** 24 / Source (Journal, Vol., Number, Pages,bate ) 1 2________________________ ..................... ........... : '_________________ ___ ____________________ ' & fit., tv 4- f[it-M tvic^ , 7, & 1^7^ S3 -JcLy). 7? 7C, ~T '------------------------ ---------------- ^7------- ------- ^ TJu--------------------- 7-------------------------------- 61 62.31 32 Brief Sunnary' 61 62 61 62 63 64 R&S 107704 0 10 io 30 *0 TIME, OATS figure 5. GC retention and resolution data for a mixture of c/s- and trans-0 -octadecenes on a 12.5% EGS polyester on silanized silver sulfobertzyi Porasil C column using 99.9999% helium carrier at a (low of PC cm3/min. Column temperature, 180 C ence of FL in the carrier gas. Zero Gas grade helium con- tains about 1 ppm of H; (12) which reduced the Ag+ to Ag in 10 days of operation at ISO C under the conditions of chromatography. The packing was removed from the col umn, extracted with CFLCla and Ag removed with 5 N HN03. About 70% of initial capacity of the ionogenic 1 groups remained. In order to determine the effects of H> in j the carrier, Airco No. 6 grade helium was utilized. Each day f for 44 days, over a total period of 10 weeks, two samples of a mixture of the as- and trans-9-octadecenes were injected into the column. Figure 5 summarizes the experiment and depicts an initial decrease in retention time, relative reten tion, and resolution although an adequate separation was achieved throughout the course of the study. When the col umn was operated above 180 C, alkene retention times de creased, but bleeding of the EGS polyester increased rapid ly with temperature and loss of resolution was accelerated. ' this '* of hecodi6a excessive. the column I^^Backing may be removed and regenerated as the Ag+ form. The extent of interaction of solutes with the surface of the supposedly inert support in GLC is often debated. On face does interact. ,i.> cvicleucctiiTv^THe ei.vcr^rTTTTnTTTMTM upon retention, hi addition, ihe difference in interaction of AgSP with respect to rhe hydrogen-form support indicates that solutes penetrate the liquid coating and approach near to the surface. The surprising extent of coating penetration was apparent when cis- and rran.s-9-octadecenes were sep arated in 30 min at 1S3 C with R -- 1.45 on HMDS-treated AgSP at 15% loading of EGS. When a column with 20% EGS was used, a resolution of 0.57 at 180 C was obtained. Thus, the approach described here has utility for analytical separations, provides a probe for the study of the migration of a solute through a liquid phase, and may be used to mea- ~~ sure interactions of volatile solutes with surface-bonded groups. ACKNOWLEDGMENT " The authors thank T. Foglia of our Center for the sam ples of cis- and rans-9-octadecenes, and M. A. Kaiser, M, O'Brien, and R. L. Grob, Villanova University, for deter mining the surface areas reported herein. LITERATURE CITED (1) O. K. Guha and J, Jand'x, J. Chromatogr.. 68, 325 (1972). (2) J* J. Duffield and L. 8, Rogers, Anal. Cnem.. 34, 1193(1952). (3) R. F. Hirscfi, H. C. Stober M. KowOfansky, F. N. Huoner, and A. W, O'Connell, Anal. Chcm.. 45, 2101 (1973), (4) j. J. Voltmer and 3, A. Gordon, Chemistry, 47, 6 (1974). (5) R, A. Barford, L. T, Olszewski, D. M. Saunders, P. Magidman, and H, L Roibbart, J. Chromatogr. Set., 12, 555 (1974), (6) K. Sistdo, Y. Takeba, and Z. Kmugawa, J. Am, Ch&m. Soc., 83, 538 (1961). (7) D. H. Saunders, R. A. Barford, P. Magidman, L. T. Olszewski, and H. L Rothbart, Anal. Chem., 46, 834 (1974). (8) O. Sey'erth, R. Suzuki, C. J. Murphy, and C. R. Sabet, J. Organcmet. Chem,, 2, 431 (1964), (9) M. A, Kaiser, M, O'Brien, and R. L. Grob, Amer. Lab., June-Juty (1975). (10) K, Unger, Angew, Chem., Int. Ed. Engl,. 11, 267 (1972), (11) V. Y. Davydov, L. T. Zhuralev, and A. V. Kieselev, J. Phys. Chem., USSR. 38, 7103 (1964). (12) Private communication from Liquid Carbonic Corp.. Duty's Lane. Bur lington, N.J. (1374). RECEIVED for review June 27, 1975. Accepted October 2, 1975. Mention of commercial products does not constitute an endorsement by the United States Department of Agri culture over others of a similar nature not mentioned. OOOP/.47 Chromatographic Determination of Vinyl Chloride in Tobacco Smoke Dietrich Hoffmann,* Constantin Patrianakos, and Klaus D. Brunnemann Division of Environmental Carcinogenesis. American Health Foundation. Valhalla. N. Y. 10595 Gio B. Gori Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Kid 20014 A chemical-analytical method has been developed for the quantitative determination of vinyl chloride (VC) in tobacco smoke. VC Irom the mainstream smoke is trapped on char coal, extracted, and subsequently converted to 1,2-dlbromo-1-chioroethane (DB-VC). The latter is enriched by column chromatography and determined by gas liquid chro matography using an electron caoture detector with a high r.sltlvlty for DB-VC. From the mainstream smoke ol a polar 85-mm cigaratte without fitter tip, we Isolated 12.2 ng of VC per cigarette. The VC content In the smoke of some domestic and foreign cigarettes and little cigars ranged from 5 to 27 ng, and that of a marijuana cigarette was 5.4 ng. The analytical data suggest that ttie total inor ganic chloride in tobacco is a determining (actor (or tho amount of VC in the smoke. VC may also be released into our respiratory environment during tho burning of othor chlorine-containing organic matter. Vinyl chloride (VC) :s causally associated with angiosar coma of the liver in VC workers (/, 2), Exposure of mice, [_ ' ANALYTICAL CHEMISTRY, VOL. 48. NO. 1. JANUARY 1976 . 47  f t 1 i i R&S 107705 t i i* figure 1. Gas chromatogram of a concentrate of vinyl chloride as 1,2-dibromo-1-chloroethane (DB-VC) from cigarette smoke Peak 1 " a dibromopropane; ? a dibrpmoprcpene; 3 = a dibromobutane: 4 = 2,3-dibromo-/>-butane: a dibromobutane and a dibromabutene: 6 (shou/derj = fl dibrpmobutene; 7 (last peak) = 1.2-dibromt>1-chio.-oethane (DB-VC) rats, and Syrian golden hamsters to VC in concentrations as low as 50 ppm leads to the induction ot various types or' tumors, including angiosarcoma of the liver (1~3). Until now, VC has been identified only in environments and ma terials related to vinyl chloride and poly(vinyl chloride) (1, 2). The relatively high reactivity of VC, as well as its pre sumably low concentration in non-occupational respiratory environments, requires special analytical methods and sen sitive detection techniques. This study reports a quantita tive method for the determination of nanogram amounts of vinyl chloride in tobacco smoke, EXPERIMENTAL Apparatus. A Hewlett-Packard gas chromatograph Model 7260A with s-1N'i-ECO was used for the analysis. Cigarettes were smoked individually with a 30-port, automatic smoker (Heinrich Borgwaldt, Hamburg, Germany). Mass spectra were determined on a Hitachi/Perkin-Elmer RMU-GD instrument by the Mass Spectroscopy Laboratory of the Massachusetts Institute of Technology and on a Hewlett-Packard 5710-59S0A GC/MS instrument. Conditions during analysis were: ion source, 1 So "C; interface, 60 *C. NMR spectra were obtained with u Hitachi/Perkin-Elmer Model R-2-1 instrument. The elemental chlorine analysis was car ried out by Galbraith Laboratories. Knoxville, Tenn. Reagents, n-Hexane (spectrugrade) was freshly distilled over Na-jSOj (unhydrous) prior to use. Bromine wa> extracted with con centrated H.-SOj. Activated charcoal was obtained from the Pitts burgh Activated Carbon Co. (PCB -- 12 X 301, silica gel (mesh 40140) from J. T. Baker Chemical Co., OV-17 on Gas Chrom Q (mesh 80-100) from Applied Science Laboratories, pure vinyl chloride gas from Mathcson Gas Products, and vinyl chloride mixture (49.0 ppm in air) from Scott Research Laboratories (PlumsUiidville, Pa.). (All experiments with VC references were performed under well ventilated hoods|. Cigarettes. Domestic and foreign cigarettes were purchased on the open market in New York City and Westchester County, N.Y., during 1974-75. The cigarettes were selected by weight (20 mg of t-L * A t* orirv . dvetage *cigitv _____ 1 u,,, J______ tr cft. wwo, uf u.u. ~ -* average value of 50 weight selected cigarettes). Before smoking, the cigarettes were stored at 22 2 C for at least 24 hr in a cham ber with a relative humidity of 60 3%. l-Chloro-l,2-Dibromoethane (reference compound). Twenty ml of bromine were dissolved in 100 ml of n-hexane. Under magnetic stirring at -5 aC. pure vinyl chloride gas was bub bled slowly through the solution until the color disappeared. Most of the solvent was distilled at atmospheric pressure, and the re mainder under reduced pressure. l-Chloro-1.2-dibromoeihane (di- bromovinyl chloride, DB-VC) was distilled at 52 C under 12 mm Hg pressure. It was sealed under dry nitrogen, covered with dark paper, and stored at 0 "C. Under these conditions, DB-VC remains unchanged for up to two weeks. The known DB-VC was character ized by NMR: 4.0 ppm. d, 2H; 5.6 ppm, t, 1H; and bv MB (m/t; re!, int.): 145 (25), 143, (100), 141 (50). 105, 107; 79, 81 (The parent peaks (220, 222, 224, 226) of DB-VC are not observed under our conditions: see Figure 2j; the ratius of the major fragments are con sistent with the natural abundance of Br and Cl isotopes. The pu rity of the compound was ascertained by gas chromatography. Gas Chromatography. The most satisfactory separation of DB-VC from a cigarette smoke concentrate was obtained at 60 C on a 12-ft by 2-mm i.d. glass column tilled with 10% OV-17 on Gas Chrom Q (mesh 80-100). 5% Methane in argon was used as the car rier and purge gas with a (low rate of 22 mi/inin. The retention time for DB-VC was 32.5 min. Using a l,:,Ni-ECD at 200 C. the de tection limit was 15 pg (10"'*g) per injection. The detector re sponse was linear in the range of 5fi-30%pg. Model Studies. In model tests, it could be demonstrated that 1.0 g of activated charcoal packed in a 12-cm X 6-mrn i.d. glass tube (ends closed with glass wool) retains VC quantitatively, at least up to 200 iig of VC from a stream of air (20 ml/sec) containing 49.5 ppm of VC. (VC remains unchanged on charcoal for at least 6 hr.) The sepaiation method was developed by blowing an air stream (flow rate 20 ml/sec) of 4 I. containing 200 mg of"VC through a Cambridge filter (CM-113A) loaded with tar of 30 cigarettes (8U0 mg), and through 1.0 g of charcoal with the gas phase de posits from the cigarettes. After londmg with VC, the charcoal was /B ANALYTICAL CHEMISTRY. VOL. 48, NO. 1, JANUARY 1976 ( f R&S 107706 Table I. Vinyl Chloride in Cigarette Smoke, ng/cig.a 100- B. ISOLATED 60- 60- 40- ,U,L J,20i i i i iiirii1 40 60 60 100 120 MO 160 m/e No, of anatysij Vinyl chloride isolated* 1 2 3 4 5Average Std dev Dev coefficient 12.6 11.4 12.9 10.8 13.1 12.2 0.90 7,4 % 0 Commercial, 85-mm, nonfilter cigarette. * Not corrected for losses. Table II. Vinyl Chloride in Cigarette Smoke Figure 2. Mass spectra of 1,2-dibromo-1-chloroethane (DB-VC) (parent ions 220, 222, 224, 226 are unstable and not detected) ' poured into a flask which was kept at --30 C and which contained 0.5 ml of bromine and 11 ml of n-hexane. The determination of DB-VC by GLC from the solution after column chromatography gave recovery rates of more than 95%. Two control runs (as out lined under Procedure), one involving extraction with n-hexane of . charcoal with smoke gas-phase deposits (no bromination) and the other involving bromination of charcoal without smoke (blank test), produced no signals at the retention time of DB-VC. (All equipment used was the same as under Procedure). Procedure, isolation of Vinyl Chloride, An amount of the con centrate from cigarette smoke sufficient ( = 20 ag) for mass spec tral identification of the 1,2-dibromo derivative of VC was collect ed by several runs following the procedure for quantitative analy sis of VC (see below). Each successive run was quantitatively and qualitatively monitored by gas chromatography. Quantitative Analysis. Thirty cigarettes (selected by weight and draw resistance) were smoked with a 30-port automatic smoking machine at a rate of one puff of 2-sec duration per min, a puff vol ume of 35 ml, to a butt length of 23 mm. The mainstream smoke drav.-r. through ?, Cambridge filter (CM-113A; 92-mm diameHr) and subsequently through a glass tube (I2-cm X 6-mm l.d.J Containing 1.0_g of activated charcoal (glass wool at both ends of the tube). Immediately after smoking, the contents of two such tubes were poured into a flask [Reacti-flask with screw cap and Teflon-faced disk (Pierce Chemical Co.)] which was kept at --30 C and contained 0.5 m! of bromine in 11 ml of n-hexane. The flask was sealed and covered with dark paper, and the contents were magnetically stirred for 7 min. An aliquot of the reaction mix ture was chromatographed with n-hexane on silica gel (mesh 50-- 80) 1.0 X 60-cm at a column temperature of 0 C. Fractions (aver age 7.3 ml) were collected every 2 min. Fractions 13-15 were com bined and concentrated slowly under nitrogen at 45 C to a 7-ml volume. This solution was rechromatographed with n-hexane on silica .gel mesh 60-80 (column 1.3 X 75-cm) at room temperature: 7.3-ml fractions were collected every 1.25 min. An aliquot of fraction 24 was analyzed by gas chromatography. (To ensure maximal recov ery, neighboring fractions were also analyzed for DB-VC and, if needed, combined with fraction 24). Quantitative Analysis of Inorganic Chloride in Tobacco. The technique employed was the Caldwell and Mover'modificntion of the Volhard method (4), with the further change of filtering the sample solution with Norite prior to titration. Cl" was extracted from tobacco samples according to Yamazaki and Yamazaki (5). RESULTS AND DISCUSSION A gas chromatogram of a vinyl chloride (VC) concentrate from cigarette smoke as 1,2-dibromo derivative (DB-VC) is shown in Figure 1. The column effluent, which by retention time corresponded with DB-VC, was identified by its mass spectrum (Figure 2). The main peaks in the gas chromato ram (Figure 1) with retention times lower than that of IB-VC were identified by mass spectrometry as dibromopropancs and dihromohutanes; however, because of a lack of reference compounds, we were unable to assign struc tures with the exception of one case. Five analyses ol GO product3 Length, mm Butt length, mm Vinyl chloride, ng/cig. Reference cigarette IRI Cigarette A NF 85 23 12.4 Commercial cigarettes Cigarette A F 85 23 5.6 Cigarette B F 85 23 14.1 Cigarette C F 85 23 11.4 Cigarette D NF 70 23 11.9 Cigarette E NF 70 23 15.8 Cigarette G F 85 23 10.9 Cigarette H NF 85 23 12.2 Commercial little cigars Little cigar A F 85 23 14.4 Little cigar B F 85 23 27.3 Charcoal filter cigarettes* Cigarette A with F 85 35 5.1 Cigarette A with F cut off 60 10 15.3 Cigarette B with F 85 35 ' 1.3 Cigarette B with F cut off 60 10 4.0 Marijuana cigarette 85 23 5.4 3 F: Filter; NF: nonfilter,* Cigarette column smoked: 50 mm. cigarettes each yielded an average of 12.2 ng VC isolated as DB-VC from the mainstream smoke of an 85-mm U.S. blended cigarette without filter tip (Table I). The recovery rate was found to be at least 85%. The minimum recovery rate was calculated by directing known amounts of VC in air through charcoal which was earlier loaded with the gas eous phase of the mainstream smoke of 10, 20, or 30 stan dard cigarettes, respectively. After completing the analysis, we calculated the recovery rate. In addition to the standard nonfilter cigarettes, we ana lyzed the mainstream smoke of some domestic and foreign cigarettes, of two little cigars, and a marijuana cigarette. The results are summarized in Table II and indicate signif icant differences in the concentration of VC in the main stream smoke of commercial cigarettes. As expected, the smoke of cigarettes with charcoal filter tips contains signif icantly lower amounts of VC than does the smoke of ciga rettes without this type of filter tip. CH radicals are long known to be generated during the burning of tobacco products (6). Since many tobacco con stituents give rise to CH radicals, a search for possible spe cific precursors for the C;H:i moiety of VC appears rather futile. On the other hand, tobacco contains traces of resi dues of chlorinated pesticides (7), some unknown, chlori nated organic compounds (6, 8), and up to 8% of inorganic chloride (9). It is, therefore, reasonable to assume that the inorganic chloride in tobacco contributes significantly to the concentralion of V'C. in the smoke. This hypothesis is supported hv the observation that the amount ol methyl chloride in cigarette smoke is determined by the conccntra- ANALYTICAL CHEMISTRY. VOL. 48. NO. 1, JANUARY 1976 49 0 1 / I I ~e~~ oo 20 fi th Q, JO fio C/7 V..e 4 / / 0/ A/ A/ / / / / /a c; o -4 -vl O -4 !II .5 10 .15 mgCI"( o ), mg Cl ( A ) per g Dry Tobacco figure 3. Correlation between inorganic chloride and total chlorine in tobacco and vinyl chloride in cigarette smoke th;- incco smoke may also contain other unsmurnff-rl. chic,.. ..ted hydrocarbons with carcinogenic activity. This may include the suspected human rarcinogen'2-chlombutadienc (chloroprcnc; II. 12) as well as chlorinated prod ucts of isoprene, the major unsaturrd hydrocarbon in to bacco smoke. Note Added in Proof. This study was presented at the 29th Tobacco Chemists' Research Conference (College Park, Md., Oct. 8-10, 1975}. Two questions arose, 1) How much VC is in ambient air?' Using the above method, at different times we passed 20 1. of ambient laboratory air through charcoal. The extract of the charcoal was each time free of VC. 2) Is VC artificially formed from the ethyl ene of the smoke (trapped on charcoal) and the trace amounts of chlorine possibly present in the bromine? To answer this question, we led 400 ml of ethylene (CP-Matheson Gas Products) through charcoal and, using the same bromine, we processed it in the usual way. We did not ob serve a signal in the glc at the retention time of DBVC using the ECD at twice the usual sensitivity. ACKNOWLEDGMENT The authors thank C. Costello of the Mass Spectroscopy Laboratory-, Massachusetts Institute of Technology, for her cooperation in obtaining and interpreting mass spectral dataonDB-VC. j ii l j ii i s; t t fF c I, . t. it LITERATURE CITED tion of inorganic chloride in tobacco (10). Figure 3 indi cates that water soluble chloride, as well as total chlorine, in tobacco may contribute to the concentration of VC in the smoke. We realize that detailed studies are needed to ensure the nature of the major precursors in tobacco for the chlorine of VC in the smoke. Nevertheless, we should not disregard the possibility that also during the burning of other plant materials, and in fact of all organic matter (1) International Agency (or Research on Cancer. fJonogr. 7, 291 (1974). (2) I. J, SeWkotf and E, C. Hammond. "Toxicity ol Vinyl Chloride-Polyvinyl Chloride", Ann. N.Y, Acad. Sc!., 246, 1 (1975). (3) C. Waltoni, G, Lefemine. P. Checo. and D, CarreUi. Ospedaiidi. Bologna. Italy, 1 (5-6), 1 (1974). (4) J. R. Caldwell and H, V. Moyer, Anal. Chant., 7, 3a (1935). (5) M Yamazaki and J. Yamazaki, Sci. Pap. Central Res. Inst. Jpn Monopo ly Ccrp., 114, 45 (1972). (6) E. L. Wynder and D. Hoffmann. "Tobacco and Toeacco Smoke, Studios in Experimental Carcinogenesis", Academic Press. New York, N Y.. IT tr ti CO v-LLl'. vuiuains chiorine, vinyi chloride can be generated and can be released into our respiratory environment, as demonstrated for marijuana smoke. This study has shown that smoke contains up to 16 ng of vinyl chloride per cigarette, and 27 ng of VC per little cigar. This amount corresponds to a concentration of about 30 ppb. Based on human data and results from animal studies, it appears to us that these minute amounts of VC will not contribute to a measurable degree to the carcinogenic ac tivity of tobacco smoke. It should be realized, however, that we have directed our analysis only toward the identifica tion and quantitative determination of vinyl chloride and (7) F. F. Guthrie and P. G. Bowery. Residue Rev., 19, 31 (1967). (8) R. L. Stedman, Cherrt. Rev., 63, 153 (1968). (9) T. C. Tso. "Physiology and Biochemistry of Tobacco Plants". Dowden, Hutchinson, and Ross. Stroudsburg, Pa.. 1972. (10) R. R. Johnson and T. E. Smith. Abstr. 24th Tobacco Chemists' Res. Coni.. 24 (1970). (11) E. A. Khackatryan. Gig, Tr. Prof. Zabol.. 18, 54 (1972). (12) E. A. Khachatryan, Pub!. Oncol. USSR., 18, 85 (1972). RECEIVED for review August 5, 1975. Accepted September 22, 1975. This is No. XXXVI of "Chemical Studies on To bacco Smoke". It was supported in part by American Can cer Society Grant BC-56T and by Public Health Service Contract ECI-SHP-74-106. Thin Layer Chromatography/Densitometry with Transferable Calibration Factors H. Bethke and FL W. Frel* Analytical Research and Development, Pharmaceutical Division, Sandoz Ltd., 4002 Basle, Switzerland The principle of a transferable calibration technique Is dis tion alter separation Is demonstrated with samples of dlgox- ter nit am iso ror hat sur go* F Me was mat 10 t chrt acid (30 over atur D. matt G.F. Boul tron: M-P dihy' In kin-1 kin-1 Bode cussed and Introduced with examples ol dihydreergotaminb. In. The reproducibility of the results obtained is comparablo The quantitation of spots Is carried out with direct UV re to Individual calibration carried out for each plate (rel std flectance spectromctflc measurements of melhylergo- dev about 2%). With the use of transferable calibration, 5 baslne on different series of chromatoplates. The applicabil ity of this same calibration transfer model to in situ fluores cence measurements which require a derivatlzatlng reac analyses can be carried out on one 20 X 20 cm plate which represents an Increase In capacity of 60% compared to j previously discussed method. Th TLC a+o 50 ANALYTICAL CHEMISTRY. VOL 48, NO. 1, JANUARY 1976