Application of the Blind Assay to Biological Activity and Tobacco Smoke Terpenes C. H. Ho," W. H. Griest, and M. R. Guerin Tobacco Smoke Research Program, Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830
The blind assay technique was applled to the chromatographic data generated from GLC profiles of terpene-enriched fractions from the total particulate matter (TPM) of elght experimental cigarettes. Correlation of the entire terpene fraction peak area and 16 individual peaks with data from three skin-painting bioassay parameters indicated that the terpene fraction as a whole and at least 12 of its constituents correlate well wlth biological activity. Six Correlating peaks were tentatively idendified as d-limonene, damascenone, norphytene, neophytadiene,phytol, and squalene. High correlations were also observed between the TPM concentrationsof these constituents and the total terpene fraction. Of the terpenes visualized, d-limonene would act as the best indicator of the tobacco smoke biological activity.
T h e traditional approach t o the identification of biologically active constituents of complex mixtures has entailed extensive subfractionation and biological testing of specific compounds thought t o possess biological activity. This method suffers from at least two major disadvantages: a tremendous time and manpower requirement, and a tendency t o overlook unexpected constituents. In contrast, the blind assay method overcomes these disadvantages. High resolution gas-liquid chromatographic (GLC) profiling of a class fraction eliminates t h e need for extensive subfractionation. The term "blind" refers t o t h e advantage that t h e identity of each component visualized in t h e profile does not have to be known-those constituents correlating well with biological activity can be subjected t o intensive structural identification, while those failing t o correlate are disregarded. This method has been successfully applied in this group to t h e location of three to eight biologically-related compounds in tobacco smoke gas phase ( I ) . Tobacco smoke is known t o possess a wide range of terpene compounds (2-5), and these are found t o constitute a significant portion of the carcinogenic neutral fraction of tobacco smoke condensate ( 3 ) .Significantly, the known polynuclear aromatic hydrocarbon content of this fraction is not sufficient in itself to account for t h e carcinogenicity of t h e fraction, suggesting that synergistic effects and/or complete carcinogenicity is contributed by other constituents. The possible contribution of t h e terpenes t o such activity is implied by separate experiments demonstrating good correlations between both gas phase isoprene (6) and condensate neophytadienes (7) with mouse skin-painting carcinogenesis. The purpose of this paper is t o report on the successful application of t h e blind assay method to efficiently elucidate those constituents of a complex isolate related in some manner t o biological response. Terpenes in tobacco smoke are considered.
EXPERIMENTAL Materials. The following terpenes were obtained from the indicated sources: d-limonene (Pfaltz and Bauer), phytol (Aldrich), squalene (Applied Science Laboratories). Neophytadiene and damascenone were kindly provided by Donald Roberts of R. J. Reynolds
Tobacco Co. Norphytene was synthesized in-house by a published method (8).All solvents were analytical reagent grade, and were used directly. GLC supplies were purchased from Applied Science Laboratories and Supelco; the Cambridge filters and pads were bought from Phipps and Bird Company. The eight experimental cigarettes (Series 1code numbers lR1,6,7,10,14,15,19,22)were obtained from the National Cancer Institute through the Smoking and Health Program. Blind Assay Method. Preparation of Terpene-Enriched Fraction. The separation scheme for a terpene-enriched fraction from tobacco smoke total particulate matter (TPM) is shown in Figure 1. About 2 g of TPM (collected on the Cambridge filters under standard smoking conditions of one 35-cm3puff of 2-9 duration, per min), were soaked overnight in 100 ml of methanol/water (4/1 by volume) and 100 ml of cyclohexane. The mixture was stirred 2 h, filtered, and washed with 50 ml of cyclohexaneand 50 ml of methanol/water (4/1). The methanol/water phase was back-extracted with 3 X 75 ml of cyclohexane, and the pooled cyclohexane layers were extracted with 2 X 75 ml of nitromethane. The nitromethane phase was back-extracted with 2 X 50 ml cyclohexane, and the combined cyclohexane layers were extracted again with 1 X 30 ml of nitromethane. The cyclohexane was evaporated under reduced pressure (-60 Torr). The residue was dissolved in 15 ml acetone, cooled to about -70 "C (dry ice powder) for 5 min, and filtered. The resulting acetone filtrate is a terpene enriched fraction, the concentration of which was adjusted to 160 mg TPM/ml by addition or evaporation of acetone. This extract was analyzed directly by GLC. Gas Chromatographic Profiling. A 5-kl sample of the terpene fraction was injected into a silanized 10 f t X l/s in. 0.d. glass column packed with 3% Dexsil400 on 80/100 mesh HP Chromosorb G. The flame ionization detector-equipped Perkin-Elmer model 3920 gas chromatograph was adjusted to the following conditions: column temperature programmed from 100 "C (8-min isothermal hold) to 320 " C at 2"/min; inlet temperature, 330 "C; detector temperature, 300 "C; helium carrier gas flow of 26 cm3/minute at 100 "C. Correlation Coefficient. The peak areas per milligram of TPM for 16 peaks from each of 8 experimental cigarettes as well as the total profile peak area (estimated by a cut-and-weigh technique) were correlated with 3 parameters of skin-painting biological activity: T75 (time in days to 75% of the animals without tumors) and 2 Pfparameters (probability in % of survival without tumor after 18 months of low dose treatment, with visual and histopathological data). The bioassay data were measured elsewhere and were obtained through the National Cancer Institute (6). A programmable model 9100A Hewlett-Packard calculator with a least squares program was used to calculate simple correlation coefficients. Identification and Quantitatiue Estimations. The GLC terpene profile peaks correlating highly with biological activity were tentatively identified by co-chromatography.Some chemical identifications were confirmed by gas chromatography-mass spectrometry (GC-MS). The GC-MS instrumentation consists of a Perkin-Elmer Model 3920 gas chromatograph interfaced via a glass jet separator to a magnetic deflection type mass spectrometer designed and fabricated at Oak Ridge National Laboratory. Quantitative estimations were made from the integration of peak areas; working curves were obtained from injections of standard solutions.
RESULTS AND DISCUSSION The procedure for preparing the terpene-enriched fraction is modified from that of Hoffmann (9) and Stedman (IO). Advantages of the modified procedure include (a) the required working time (exclusive of the soaking time) is only 4-5 h; (b) n o acidic or basic aqueous solutions contact the samples, thus minimizing the decomposition of some unstable compounds in the samples; and (c) no chromatographic column separation
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
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I
1. 100 ml methanol/water (4/1) t 100 ml cyclohexane 2. Stirred 3. Filtered 4. Washed
Cyclohexane
Methanol/water (4/1) 3 x 75 ml Cyclohexane
I
Methanol/water (4/1)
Cyclohexane
2 x 75 ml Nitromethane
' i
Nitromethane
Cyclohexane
2 x 50 ml Cyclohexane
'I
Cyclohexane
Nitromethane
Nitromethane
I
Nitromethane
?'
evaporated
Figure 1. Separation scheme of terpene-enriched fraction from TPM
technique is used. Such purification steps can cause terpene isomerization ( 4 ) . Examination of a trimethylsilylated terpene fraction (prepared by reacting a n aliquot of this terpene fraction overnight a t room temperature with one half the volume of BSTFA) revealed the presence of non-terpenoids such as glycerol and several free fatty acids, mainly palmitic acid. However, these constituents did not interfere with the analysis of non-derivatized terpene fractions, and their presence was disregarded. No phenols or polynuclear aromatic hydrocarbons were detected in the terpene-enriched fraction. These were removed during the procedure. In the first step of the blind assay method, terpene-enriched fractions were obtained by the scheme outlined above. These fractions were next separated under GLC conditions adjusted for a maximum resolution of terpene components. More than 2224
100 major and minor constituents were visualized in these profiles. T h e high resolution GLC profiles of the terpeneenriched fraction for three of the eight experimental cigarettes are shown in Figure 2. T h e t o p profile corresponds to a cigarette possessing high skin-painting biological activity, the middle profile is from a cigarette of intermediate activity, and the bottom profile is from a low activity cigarette. From just this preliminary visual evaluation of the terpene profiles, it is immediately evident t h a t the concentration of the TPM terpene fraction as a whole correlates well with the biological activity of the tobacco smoke condensate implying a group reiationship of the terpenes t o such activity. Next, the peak areas of 16 peaks visually selected from t h e terpene profiles for the eight experimental cigarettes were individually correlated with three parameters of biological activity. These simple correlation coefficients are presented
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
-0.89 -0.89 -0.89 0.70 -0.87
i2 (phytol) 13 14 15
16 (squalene)
-0.85 -0.86 -0.83 0.69 -0.84
1 I
-0.95 -0.86 -0.94 0.53 -0.83
LOW ACTIVITY (CODE 141
NORPHYTEN:]
NEOPHYTADIENE7 '0
SQUALENE,
,4
16
From Figure 2 (The number is counted from right to left). Peak area/mg TPM vs. biological activity parameter. " Correbased on visual data (Probability in % of survival lated with Pf, without tumor after 18 months with low dose treatment). Y Corbased on histopathology data (Probability in % related with Pf, of survival without tumor after 18 months with low dose treatment). * Correlated with T7j (Time in days to 75% without tumor).
,
I-LIMONEN:]
1
DAMASCENONE
,,
a
in Table 1.The peak numbers in Figure 2 correspond to those peaks correlated with biological activity in Table I. A correlation coefficient of 1 indicates a perfect correlation with biological activity. A negative sign refers to a correlation in the same direction as biological activity; i.e., increasing concentration of that compound correlates with increasing biological activity. We observed that the entire terpene fraction and also 12 of the 16 selected peaks produced correlations of greater than about 0.80. Those individual constituents correlating most highly with the activity would be selected for priority attention in structural identification or biotesting. An indication of the reliability of these correlations is the agreement with a separately determined correlation for neophytadiene (peak 10). Measurements of neophytadiene T P M concentration by a separate standard analytical method indicated a correlation coefficient of -0.88 with T'jg ( 7 ) , which is in excellent agreement with the -0.90 calculated from our blind assay measurements. A separate measurement of d limonene (peak 1) in the gas phase of tobacco smoke yielded a correlation of -0.75 with T75 ( I ) . A correlation of -0.83 with T7; was calculated by our blind assay of T P M terpenes. Considering the variation possible in bioassay data and smoke
(
50
50
100
0
TIME (minl
Figure 2. High-resolution GLC profiles of the terpene-enriched fraction from three NCI experimental cigarettes
composition, the difference between these two correlation measurements is probably not significant. Interestingly, the non-terpene nicotine in our profiles (peak 5 ) also produced data equivalent to t h a t achieved by independent methods. Our correlation of -0.88 with low-dose Pf agrees fairly well with the -0.77 correlation determined for smoke condensate nicotine. Seven of the 12 profile peaks correlating with biological activity were tentatively identified by co-chromatography as d-limonene, damascenone, norphytene, neophytadiene, phytol, squalene, and nicotine. d-Limonene, neophytadiene, and nicotine were confirmed-by GC-MS analysis. Nicotine, though not a terpene, is a major constituent of tobacco smoke T P M (&lo%), and apparently was carried through our procedure. All of the above compounds previously have been isolated and identified in tobacco smoke ( 4 , I I ) , and they constitute about one third of this terpene-enriched fraction. Work is continuing on the confirmation of these peak identities as well as identification of the other terpene-enriched fraction constituents correlating well with biological activity. Quantitative estimates of the T P M concentration of the six
Table 11. Quantitative Estimation of Terpenes in T P M from Different Series I Experiment Cigarettes Terpene, mg/g Code 10 lR1 6 7 15 22 19 14
d-Limonene
Damascenone
Norphytene
Neophytadiene
Phytol
Squalene
1.36
0.211 0.165 0.156 0.128 0.045 0.039 0.030 0.027
0.307 0.253 0.292 0.196 0.127 0.095 0.061 0.042
8.20 6.04 7.46 8.20 2.20 7.31 2.20
0.161 0.128 0.141 0.161 0.104 0.128 0.044 0.044
0.390 0.270 0.310 0.250 0.092 0.060 0.049
1.25
1.19 0.90 0.54 0.51 0.30 0.21
0.22
0.003
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Table 111. Correlation Coefficients between Concentration of Individual Terpenes= and Total Terpene Fraction* Total terpene fraction Total terpene fraction d-Limonene Damascenone Norphytene Neophytadiene Phytol Squalene
0.95 0.92 0.94 0.88 0.92 0.95
d -Limonene
Damascenone
Norphytene
Neophytadiene
Phytol
Squalene
0.95
0.92 0.98
0.94 0.99 0.97
0.88 0.78 0.75 0.76
0.92 0.83 0.77 0.82 0.78
0.95 0.98 0.99 0.98 0.73 0.81
0.98 0.99 0.78 0.83 0.98
0.97 0.75 0.77 0.99
0.76 0.82 0.98
0.78 0.75
0.81
From Table 11. Estimated from the weight of total terpene fraction profile peak area/g TPM.
identified terpenes are shown in Table 11. We refer to “estimations” rather than “determinations” because positive confirmation of all peak identities is not complete. Again, these terpenes are observed to generally parallel the biological activity of the smoke over more than an order of magnitude change in their concentrations, especially for squalene, which varied over two orders of magnitude. Independent direct measurements of the T P M concentrations of squalene, neophytadiene, and d-limonene indicated recoveries of loo%, 94%, and 80%, respectively, for these components in the terpene-enriched fraction. These recoveries appear t o parallel the terpene boiling points, but they did not affect the blind assay correlations in that these recoveries were reproducible. The high correlations with biological activity of both the entire terpene fraction and several of its constituents suggests that a measurement of the entire terpene fraction is not necessary to screen the biological activity associated with the experimental cigarettes. T o test this hypothesis, correlations were calculated between the peak areas of the entire terpene-enriched fraction GLC profiles and several of t h e identified constituents. These correlations are presented in Table 111. Fairly good correlations were obtained for all the constituents, with d-limonene appearing to offer the generally highest correlation with the entire terpene fraction and with the other identified terpenes. Significantly, d-limonene also exhibited the highest individual correlation with biological activity (Table I), and thus it appears to be the best single indicator of the terpene fraction. The exact relationship of this fraction t o the biological activity of smoke is not clear. T h e terpenes are not necessarily carcinogenic; it has been suggested that the terpenes can act as precursors of carcinogens or tumor promoters ( 3 ) .
CONCLUSIONS The present work has demonstrated the utility of the blind assay toward location of tobacco smoke terpenes associated
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in some manner-as of yet, undefined-with the biological activity of the condensed smoke. Application of this method to other tobacco smoke fractions should allow the rapid location of other constituents (e.g., phenols, bases, polynuclear aromatic hydrocarbons) associated with or contributing t o the biological activity of tobacco smoke.
ACKNOWLEDGMENT The authors are grateful t o W. T. Rainey of the Oak Ridge National Laboratory for his assistance in obtaining GC-MS data.
LITERATURE CITED (1) C. E. Higgins, J. R. Stokely, M. R. Guerin, and T. L. Roberts, “Application of Blind Assay Methodology to Organic Gas Phase Constituents in Tobacco Smoke”, 28th Tobacco Chemists’ Research Conference, Raleigh, N.C., October 28-30, 1974. (2) R. A. W. Johnstone and J. R. Plimmer, Chem. Rev., 59, 885-939
(1959). (3)E. L. Wynder and D. Hoffmann, “Tobacco and Tobacco Smoke”, Academic Press, New York and London, 1967,pp 351-362. (4)R. L. Stedman, Chem. Rev., 68, 153-207 (1968). (5)A. Rodgman, L. C. Cook, and C. K. Chappei, Tobacco Sci., 5, 1-5 (1961). (6)National Cancer Institute Smoking and Health Program Monograph, Toward Less Hazardous Cigarettes, Report No. 1, The First Set of Experimental Cigarettes, Gio B. Gori. NCI (in preparation).
(7)M. R. Guerin and G. Olerich, fnwiron. Lett., 10 (3),265-273 (1975). (8)R. A. W. Johnstone and P.W. Quan, J. Chem. SOC.,5706-5713 (1963). (9)D. Hoffmann and E. L. Wynder, National Cancer Institute Monograph, No. 28, 151-197. (10)A. P. Swain, J. E. Cooper and R. L. Stedman, Cancer Res., 29, 579-583 (1969). (11) E. Demole and D. Berthet, Helv. Cbim. Acta, 54, 681-682 (1971).
RECEIVEDfor review April 30, 1976. Accepted August 19, 1976. Research sponsored by the National Cancer Institute under Union Carbide’s contract with the U.S. Energy Research and Development Administration.
DECEMBER 1976
