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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Confirmatory Methods for the Thermal Energy Determination of N-Nitroso Compounds at Trace Levels I. S. Krull," E. U. Goff, G. G. Hoffman, and D. H. Fine New England Institute for Life Sciences, 725 Second Avenue, Waltham, Massachusetts 02154
Methods are presented which allow for the rapid and reliable confirmation of N-nltroso compounds as determined by use of the nitrosyl speciflc thermal energy analyzer. These approaches utllize minor modifications in the normal operation of the analyzer, gas chromatography and high performance liquid chromatography Interfaced wlth the analyzer, ultraviolet Irradiation of the sample, and wet chemical procedures. Comparisons are made between these analyzer associated methods of confirmation, and other approaches for the determlnatlon of N-nitroso compounds at trace levels. An overall general scheme is described which allows for the conflrmation of nitrosyl analyzer responsive materials as either N-nitroso, X-nltroso ( X = C, 0 ) or X-nitro ( X = N, C, 0 ) .
T h e nitrosyl specific thermal energy analyzer (TEA), interfaced to a gas chromatograph (GC) and/or a high performance liquid chromatograph (HPLC), has become a widely used detector for the analysis of trace levels of N-nitroso compounds (1-3). Compounds eluting from a chromatograph enter the analyzer and are first pyrolyzed ( 4 ,releasing the nitrosyl radical (.NO). The effluent from the hot oven is then frozen out in cryogenic traps maintained at temperatures as low as -150 "C, with only the .NO remaining to react with ozone a t reduced pressure to generate electronically excited NO2* ( 5 ) . The excited NOz* decays to its ground state, emitting light in the near infrared region of the spectrum. The intensity of the emission is monitored with a photomultiplier tube and is a measure of the amount of nitrosyl-containing species entering the analyzer. Although the analyzer has been used extensively for the analysis of N-nitroso (N-NO) compounds, the earliest description of the detector in 1973 by Fine et al. (5),pointed out that compounds containing the 0-nitroso (0-NO) and 0-nitro (0-NOz) moiety, as well as some compounds with the C-nitro (C-N02)group, could all produce a sizable TEA analyzer response. Also, a t that time a single compound with a C-nitroso (C-NO) group was shown to produce a weak TEA analyzer response. These very early studies were confirmed by Stephany and Schuller ( 6 ) ,Fan et al. ( 7 , 8 ) ,and Lafleur e t al. (9). In addition, Hotchkiss et al. (IO) and Fiddler e t al. (11) have now shown that N-nitro (N-N02)compounds can produce a TEA analyzer response. In the routine analysis of N-nitroso compounds, possible TEA analyzer responses to compounds other than N-nitroso do not generally present a problem, since the establishment of a compound's identity can be readily accomplished by co-elution with known standards on GC-TEA and/or HPLC-TEA systems (3, 12-14). If the compound is GC amenable, then the sample can be chromatographed on both GC-TEA and HPLC-TEA, which provides additional confirmation (13). Alternatively, GC-mass spectrometry (GCMS), with appropriate methods of operation can be utilized (15). However, a sample whose chromatogram contains a sizable TEA analyzer peak of unknown origin cannot be ignored, since the majority of N-nitroso compounds have been shown to be carcinogenic in various laboratory animals (16, 17). 0003-2700/79/035 1-1706$01 .OO/O
If there is a sufficient amount of the sample, if the compound of interest can be sufficiently purified, and if the same material is amenable to gas chromatography, then GC-MS is undoubtedly the preferred method of structure identification. However, if the above requirements for GC-MS cannot be met, then isolation and structure characterization can become time-consuming, laborious, and quite expensive. Thus, considerable effort was expended in our laboratory on the isolation and identification of a weakly TEA analyzer responsive material, ethylene glycol dinitrate, from drinking water in Washington, D.C. (7). Similarly, we isolated and (bronopol), a identified 2-bromo-2-nitro-1,3-propanediol bactericide, from a facial cosmetic (18). In both of these cases, because of the relatively large amounts of the TEA analyzer responsive compounds present in the final extracts, and because their molar TEA analyzer responses were small when compared with typical N-nitroso compounds, the first unknown TEA analyzer peaks were still suggestive of the presence of authentic N-nitroso materials. In order to maximize efforts directed toward the isolation and identification of the environmentally important N-nitroso compounds, we have now developed an analytical scheme for the confirmation of these materials. When these methods are used in conjunction with GC-TEA and/or HPLC-TEA systems, it is possible to distinguish N-nitroso compounds from most other materials which produce a TEA analyzer response. This current analytical scheme combines certain well established literature methods with various new approaches.
EXPERIMENTAL Apparatus. For gas chromatography (GC), a TEA analyzer
(Thermo Electron Corp., Waltham, Mass., Model 502LC) was used, interfaced t o an isothermal gas chromatograph (Thermo Electron Corp., model 661), or t o a temperature programmable instrument (Varian Corp., model 3700). The most widely utilized inch 0.d. stainless steel GC column was made from a 14 ft X tube packed with 10% Carbowax 20M plus 0.5% KOH on Chromosorb W/HP, 80/100 mesh (SupelcoCo., Bellefonte, Pa.). This was operated isothermally at temperatures varying from 130-185 "C, depending on the N-nitroso compounds of interest. Alternatively,this and the other GC columns were operated with temperature programming, from 130 to 200 "C at a rate of 4-6 "C/min. A second GC column which was used was a 6 ft X 'Ig inch 0.d. stainless steel tube packed with Chromosorb 102,100/120 mesh (Supelco Co.), operated isothermally at temperatures between 180 and 220 "C or with temperature programming. A third set of GC conditions was an 8 f t X inch Teflon-lined stainless steel or all glass column packed with 4% Carbowax 20M plus 0.8% KOH on Carbopack B operated from 1OC-180 "C with temperature programming at 4 "C/min. Argon was used as the carrier gas, at a flow rate of 10-30 mL/min, depending on the column being used. The TEA analyzer was operated in the GC mode, with the stainless steel cold trap maintained at -110 "C, and a furnace temperature of 450-500 "C. The high performance liquid chromatograph (HPLC) was constructed by serially connecting a high pressure pump (Varian Corp., Palo Alto, Calif., model 8500 or Altex Corp., Berkeley, Calif., model 110),an injector (Rheodyne, Altex Corp., model 905-42), an HPLC column, and a TEA analyzer (Thermo Electron, model 502). The HPLC column used was made from a 30-cm long, 4-mm i.d., stainless steel tube. Silica gel type packings, such as pPorasil C 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
(Waters Associates, Milford, Mass.) or 10 p Lichrosorb Si60 (EM Labs, Elmsford, N.Y.) were generally used, with the solvent flow rate between 1.0 and 2.0 mL/min. The solvent system was made from various mixtures of acetone/hexane, acetone/methylene chloride, and methanol/methylene chloride. Specific HPLC conditions for the compounds studied here have been published elsewhere (14, 19). Ultraviolet (UV) irradiations were performed using a 275-W sunlamp (General Electric, Schenectady, N.Y.) with the samples placed in Pyrex culture tubes (13 mm X 100 mm) having Teflon-lined caps. Materials. All solvents utilized for HPLC analyses, as well as for sample preparation and confirmatory tests, were obtained from Burdick & Jackson Co. (Muskegon, Mich.), and had been Distilled-in-Glass. Technical grade solvents, maintained at their freezing points with liquid nitrogen, were used for maintaining the temperatures of the low temperature TEA analyzer cold traps. Glacial acetic acid (HOAc) was obtained from Fisher Scientific Co. (Medford, Mass.) and the hydrobromic acid in glacial acetic acid (HBr/HOAc) (30-32%) was purchased from J. T. Baker Chemical Co. (Phillipsburg, N.J.). All dilutions were performed with glacial acetic acid. Standard solutions of N-nitroso compounds were obtained from the Analytical Services Laboratory of Thermo Electron Corporation. All other organic compounds were obtained from commercial suppliers, and were used as received, without further purification. GC-TEA and/or HPLC-TEA responses for such materials were assumed to arise from the major constituent. Warning. Utmost caution must be used in handling N-nitroso compounds, as well as other potentially carcinogenic materials. Adverse effects of exposure to such materials may not become apparent for many years. N-nitramines (N-nitro amines) may also be carcinogenic in laboratory animals (20, 21). Procedures. All solutions of N-nitroso compounds and other TEA analyzer responsive materials were used at concentrations known to be present in environmental samples. Thus, solutions in the low ppm concentration range were employed, and 10-20 pL injections of such solutions were made onto GC-TEA and/or HPLC-TEA. Absolute amounts of the N-nitroso compounds and other TEA analyzer responsive organic compounds were in the range of 10-200 ng per injection. Glacial Acetic Acid Method. The compound to be studied was dissolved in an organic solvent such as acetone, ethyl acetate, or methylene chloride, which is fully miscible with HOAc. It was important that the solvents be of the highest purity, and free of water or alcohol contaminants. A known amount of a standard N-nitroso compound was added as an internal control. A small amount of this total solution (approximately1CC-250 FL) was then diluted with HOAc, so that the final ratio of HOAc to organic solution was about 1:4 or 1:5. An aliquot of this mixture was removed, and kept in the dark a t room temperature as an additional control. The remainder was then heated in a sealed, Teflon capped, glass vial for about 30 min a t 40 OC, after which time all three solutions were analyzed by HPLC-TEA. Peak heights for the compound of interest and the internal standard were compared. The method could not be used routinely with GC-TEA analysis since the GC column packing material was degraded by HOAc. Hydrobromic Acid (HBr) in Glacial Acetic Acid (HBrIHOAc) Methods. This procedure was identical to the HOAc method above, except that a 10% HBr/HOAc solution was used in place of HOAc alone. The dilute HBr/HOAc reagent was prepared fresh by dilution of the concentrate (3&32%) with HOAc or by bubbling gaseous HBr through HOAc to produce a final 10% (w/w) solution. Analyses of the three solutions (the original organic solution, the control with HBr/HOAc added and kept in the dark at room temperature, and the reaction mixture heated to 40 OC for 30 min) were performed using HPLC-TEA. The peak height for the compound of interest was then compared to that of the internal standard. Ultraviolet Irradiation. The test sample was dissolved in a solvent such as hexane, isooctane or methanol, and a known amount of a standard N-nitroso compound was added as an internal control. A portion of the test solution was set aside in the dark for comparison purposes. Irradiation tubes were set approximately 8-10 inches from the G.E. sunlamp, with cooling
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Table I. Reactivities of Organic Nitroso and Nitro Compounds to HBr/HOAc and HOAc Alone compound studied
loss of TEA analyzer signal, % HBr/ HOAc HOAc alone
N-nitroso compounds N-nitrosodipropylamine N-nitrosodimethylamine N-nitrosopiperidine N-nitrosodiphenylamine N-nitrosocarbazole N-nitrosodimethylurea N-methyl-N-nitroso-ptoluenesulfonamide N-methyl-N-nitros0-N'nitroguanidine
100
N.R.a N.R. N.R. N.R. N.R. N.R. N.R.
100
N. R.
N-methyl-N-nitroso-1-naphthyl
100
carbamate (N-nitrosocarbaryl) 2-chloro-4-(N-nitroso-N-
N.R.
100
N.R.
100 100 100
N. R. N.R. N.R. N.R.
100
100
100
50 100
100
100 1OOb 60
100 100
ethylamine)-6-isopropylamino-
s-triazene (N-nitrosoatrazine) C-nitroso compounds 2-methyl-2-nitrosopropane p-nitrosodiphenylamine nitrosobenzene p-nitrosodimethylaniline
75
0-nit roso compounds
isopentyl nitrite tert-butyl nitrite n-butyl nitrite
100
C-nitro compounds 2-nitro-1-propanol N,R.a 1,5-difluoro- 2,4-dinitrobenzene N.R. 2,3-dimethyl-2,3-dinitrobutane N. R.
N.R. N.R. N. R.
N-nitro compoundsC N-nitromorpholine N-nitrodimethy lamine N-nitrodiethylamine
N.R. N.R. N.R.
N.R. N.R. N.R.
0-nitro compounds n-propyl nitrate n-butyl nitrate amyl nitrate
N.R. N.R. N.R. N.R. N.R. N.R. a N.R. = N o reaction detected by HPLC-TEA. Run in 1%HBr/acetone a t room temperature for less than 5 min. N-Nitramines and N-nitramides (one each) have been reported unreactive in the HBr/HOAc test ( 2 4 ) . by a small fan, where necessary. Aliquots of the irradiated solutions were removed a t regular intervals, and analyzed immediately by either GC-TEA or HPLC-TEA, along with the control. Changes in peak height for the compound under study were then compared with corresponding changes for the internal standard.
RESULTS AND DISCUSSION TEA Analyzer Procedures. Utilization of a -150 "C cold trap after t h e TEA analyzer pyrolyzer ( 4 ) , freezes out most organic compounds, apart from a few low molecular weight olefins. If a substituted olefin is able to survive this cold trap, it can still be trapped out on a short column of Tenax-GC, placed between the cold trap and the chemiluminescent reaction chamber (22). Thus, in principle, only compounds with labile nitro and nitroso groups can elicit a response on the TEA analyzer. Glacial Acetic Acid Alone. The results for all the organic nitro and nitroso compounds which were studied are shown in Table I. Only the alkyl nitrites (0-NO) suffer loss of their
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979 Part 2 . Chemical confirmatory techniques
Part 1. TEA analyzer parameters
N - N O , C-NO,0 - N O
[
I
-150°C TEA-bE&,O,fsTEA+
Most Organics
1
TEA Respokse Unchanged
eR p o n s-e-
Cold T r a p Plus Tenax-GC In-Line T r a p
Unchanged
Loss of TEA Response
i-1
Loss of TEA
Loss of TEA Response
Unsaturated
TEA Responsk Unchanged
TEA Responke Unchanged
Loss of TEA Response
1Figure 1. Analysis scheme to distinguish N-NO compounds from C-NO, 0-NO, N-N02, C-NOp, and 0-NOp compounds, utilizing the TEA analyzer
HPLC-TEA signals under the test conditions, while the HPLC-TEA responses for all the remaining classes of compounds remain unchanged. Thus, alkyl nitrites as a class, can be readily differentiated from N-nitroso compounds by means of this method alone. The acid catalyzed cleavage of alkyl nitrites has been used for many years as a method for the nitrosation of secondary amines (13). Hydrobromic Acid in Glacial Acetic Acid. From the data in Table I, it is apparent that the organic nitro compounds (N-N02,O-NOz, and C-N02)are stable under the test conditions. By comparison, the organic nitroso compounds (N-NO), 0 - N O , and C-NO) decompose under the test conditions. Thus, this test can be used to distinguish organic nitroso compounds from organic nitro compounds. T h e use of hydrobromic acid (HBr) in glacial acetic acid (HOAc) as a denitrosation reagent (HBr/HOAc) for N-nitroso compounds was f i s t applied as a method of detection for these compounds by Eisenbrand and Preussmann (23). In their original work, as well as that of Johnson and Walters (24), nitrite released from an N-nitroso compound was determined colorimetrically with the Griess reagent. Johnson and Walters described the reactions of some organic nitroso and organic nitro compounds using this method, but the number of compounds studied was limited. More recent work by Walters et al. (25, 26) and by Drescher and Frank (27), has utilized a chemiluminescent detector to determine the nitric oxide released from N-nitroso derivatives by this denitrosation method. All of these studies involved the formation of either nitrite or nitric oxide from the N-nitroso compounds, rather than the disappearance of the original material. Relatively little work has been reported for other classes of organic nitro and nitroso compounds (25-27). The methods of Walters et al. and Drescher and Frank can be used to determine the total N-nitroso content of a crude sample; however, alkyl nitrites, and possibly other compounds, may interfere. UV Irradiation Test. Doerr and Fiddler (28) and Fiddler et al. (11) have described the application of this procedure as a test for whether a TEA analyzer responsive compound was N-nitroso. ‘IJnfortunately, compounds other than N nitroso also suffer a loss of their TEA analyzer response under the conditions of this test (29),so that the result, by itself, is inconclusive. Because the HBr/HOAc and the HOAc methods were not capable of distinguishing C-nitroso and N-nitroso compounds, we evaluated only these two classes of compounds in the UV irradiation test. All of the N-nitroso compounds in Table I1 underwent loss of their HPLC-TEA response within 60 min. However, in the case of the C-nitroso compounds, some
Table 11. Photolysis of C-Nitroso and N-Nitroso Compounds
compound studied
time of irradiation, min
results
60 36 60 60
loss of TEA signal loss of TEA signal stable to Photolysis stable to Photolysis
C-nitroso compounds 2-methyl-2-nitrosopropane ni trosobenzene 4-nitrosodipheny lamine
4-nitrosodimethylaniline N-nitroso compoundsQ
N-nitrosodiphenylamine 40 loss of TEA signal iV-nitrosocarbazole 15 loss of TEA signal N-nitrosopiperidine 60 loss of TEA signal N-nitrosopyrrolidine 60 loss of TEA signal N-nitrosodimethylamine 60 loss of TEA signal N-nitrosodiethanolamine 60 loss of TEA signal a Doerr and Fiddler have reported on the photolysis of several volatile N-nitrosamines using GC-TEA for analysis of their reactions (28). N-Nitrosamino acids also photolyze readily under their conditions ( 3 1 ) . underwent loss of their HPLC-TEA response, while others did not. Thus, if the TEA analyzer response for an unknown material does not disappear, when compared with the internal standard N-nitroso compound, then the material is not an N-nitroso derivative. With regard to N-nitramines (N-NOJ, Hotchkiss et al. have studied the relative photochemical behavior of N-nitrosodipropylamine (NDPA) and N-nitrodipropylamine (NODPA), using artificial sunlight as the irradiation source (10). The N-nitroso compound lost its TEA analyzer signal at least 10 times faster than the corresponding N-nitro compound, indicating that N-nitramines are considerably more stable to irradiation performed under mild conditions (30). We have compared the stabilities of N-nitromorpholine vs. N nitrosomorpholine under our irradiation conditions, and have found that here too, the N-nitro compound is at least 10 times more stable than the corresponding N-nitrosamine. Fiddler et al. have also found that a GC-TEA peak, assumed to be due to N-nitrodiethylamine, did not disappear following UV irradiation, whereas under the same conditions, the corresponding N-nitrosodiethylamine signal was lost ( 11 ) . Assignment of the N-Nitroso Structure. By a judicious use of the tests described above, it is possible to decide whether a compound giving rise to a GC-TEA or HPLC-TEA peak is an N-nitroso compound or not. The first step (see Figure
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
1) is the proper use of the TEA analyzer, which eliminates all compounds except for those with the N-NO, C-NO, 0-NO, N-NO2, C-N02,and 0-NOz functional groups. The next step is application of the HBr/HOAc test, whereby loss of the TEA analyzer response eliminates the N-NOZ,C-NOZ,and 0-NOz compounds from consideration. Application of the HOAc test will then distinguish N-NO and C-NO from 0-NO compounds. Finally, retention of the TEA analyzer response after UV irradiation, distinguishes C-NO from N-NO compounds. However, the reverse situation is not true, that is, loss of the TEA response after the UV test does not distinguish C-NO from N-NO. Thus, it is only the C-NO and N-NO compounds that cannot always be distinguished from each other. This may not present a serious problem, since C-NO compounds are so unstable in most cases that they have not yet been reported to be present in the environment.
CONCLUSION Using a combination of the above methods, it should be possible to be reasonably secure in the assignment of an N-nitroso structure to a known or unknown material, with the possible exception of C-nitroso compounds. We would expect S-nitroso and S-nitro compounds to behave in an identical manner to their oxygen analogues (0-NO and 0-NOz),and they too should be distinguishable from an N-nitroso compound. The procedures described here have been successfully applied in our laboratory on a wide variety of samples, including drugs, cosmetics, pesticides, air extracts, and industrial chemicals. ACKNOWLEDGMENT The N-nitromorpholine sample was kindly supplied by Brian Challis of Imperial College, London, and samples of N-nitrodimethylamine and N-nitrcdiethylamine were provided by John Cucco and Phyllis Brown of the University of Rhode Island, Kingston, R.I. We acknowledge many helpful discussions in the above work with Brian Challis and Cliff Walters of The British Food Manufacturing Industries Research Association, Surrey, England. LITERATURE CITED "Environmental Aspects of N-nitroso Compounds"; Walker, E. A,, Castegnaro, M., Griciute, L.; Lyle, R. E., Eds.; International Agency for Research on Cancer, Lyon, France, 1978. IARC Scientific Publications No. 19. "Environmental Carcinogens: Selected Methods of Analysis. Analysis of Vdatile Nitrosamines in Food", Reussmann, R.. Castegnaro, M., Walker, E. A., Wassernman, A. E.. Eds.; International Agency for Research on Cancer, Lyon. France, 1978. Voi. I. IARC Scientific Publications No. 18. Castegnaro, M., Walker, E. A. I n "Environmental Aspects of N-nitroso Compounds", Walker, E. A.. Castegnaro, M., Griciute. L., Lyle, R. E., Eds.;
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International Agency for Research on Cancer, Lyon, France, 1978; p 53. IARC Scientific Publications No. 19. Fine, D. H.; Rounbehier, D. P. J . Cbromatogr. 1975, 709, 271-279. Fine, D. H.; Rufeh, F.; Gunther, B. Anal. Lett. 1973, 6, 731-733. Stephany, R.; Schuiler. P. L. I n "Proceedings of the Second International Symposium on Nitriie in Meat Products"; Tinbergen, 8. J., Kroi, B., Eds.; PUDOC: Wageningen, The Netherlands, 1977; pp 249-255. Fan, T. Y.; Ross, R.; Fine, D. H.; Keith, L. H.; Garrison, A. W. Environ. Sci. Tecbnol. 1978, 12, 692-695. Fan, T. Y.; Vita, R.; Fine, D. H. Toxicol. Lett. 1978, 2 , 5-10. Lafieur, A.; Morriseau, B. D.; Fine, D. H. I n "Proceedings of the New Concepts Symposium and Workshop on Detection and Identification of Explosives", Reston. Va, October 30, 1978. Hotchkiss, J. H.; Barbour. J. F.; Libbey, L. M.; Scanlan, R. A. J . Agric. Food Cbem. 1978, 26, 884-887. Fiddler, W.; Doerr, R . C., Piotrowski, E. G. I n "Environmental Aspects of N-nitroso Compounds", Walker, E. A,; Castegnaro, M., Griciute. L., Lyle, R. E., Eds.; International Agency for Research on Cancer, Lyon, France, 1978; p. 33-40. IARC Scientific Publications No. 19. Havery. D. C.; Fazio, T.; Howard, J. W. I n "Environmental Aspects of N-nitroso Compounds", Walker, E. A,, Castegnaro. M., Griciute, L., Lyle, R. E., Eds.; International Agency for Research on Cancer, Lyon, France, 1978; pp 41-52. Kruli, I. S.; Fine, D. H. In "Handbock of Carcinogens and Other Hazardous Substances. Chemical Trace Analysis"; Bowman, M. C., Ed.; Marcel Dekker; New York, 1979, in press; Chapter 6. Kruil, I.S.; Goff, U.; Wolf, M. H. In "Canadian Chromatography Conference, I"; Bhatnagar, V. M., Ed.; Marcel Dekker: New York, 1978, in press. Gough. T. A. Ana/yst(London) 1978, 103, 785-806. Magee. P. N.; Montesano, R.; Preussmann, R. In "Chemical Carcinogens": Searie, C. E., Ed.; ACS Monograph No. 173, American Chemical Society: Washington, D.C., 1976; Chapter 11. IARC Monographs on the "Evaluation of the Carcinogenic Risks of Chemicals to Humans. Some Knitroso Compounds"; IntematjoMl Agency for Research on Cancer, Lyon, France, 1978; Vol. 17. Fan, T. Y.; Fine, D. H. Unpublished data, 1977. Fan, T. Y.; KruY, I.S.; Ross, R. D.; Wolf, M. H.; Fine, D. H. In "Environmental Aspects of Knitroso Compounds"; Walker, E. A., Castegnaro, M., Giciute, L.. Lyle, R. E., Eds.; International Agency for Research on Cancer, Lyon, France, 1978: pp 3-18. IARC Scientific Publications No. 19. Drudtrey, H.; Reussmann, R.; Ivankovic, S.; Schmahl. D. Z.Krebsforscb. 1987, 69, 103-201. Goodaii, C. M.; Kennedy, T. H. Cancer Lett. 1976, 7 , 295-298. Thermo Electron Corporation, TEA instruction manual, 1979. Eisenbrand. G.; Preussmann, R. Arzneimittel-Forscb. 1970, 20, 1513-15 17. Johnson, E. M.; Waiters, C. L. Anal. Lett. 1971, 4 , 383-386. Walters, C. L.; Downes, M. J.; Edwards, M. W.; Smith, P. L. R . Analyst (London) 1978, 103, 1127-1133. 1978, Watters. C. L.; Hart, R. J.; Perse, S. Z. Lebensm.-Unters.-Forsch. 167, 315-319. Drescher, G. S.; Frank, C. W. Anal. Chem. 1978, 50, 2118-2121. Doerr, R. C.; Fiddler, W. J . Cbromatogr. 1977, 140, 284-287. Kruii, I.S.; Goff, U.; Wolf, M.; Heos, A. M.; Fine, D. H.; Arsenauit, G. P. Food Cosmet. Toxicol. 1978, 16, 105-110. Althorpe, J.; Goddard. D. A,; Sissons, D. J.; Telling, G. M. J Chromtcgr. 1970, 53, 371-373. Fiddler, W. Personal Communication, 1978.
RECEIVED for review March 22,1979. Accepted June 4,1979. The work was supported by the U S . National Science Foundation under Grant No. ENV75-20802. Any opinions, findings, conclusions, or recommendations expressed are those of the authors, and do not necessarily reflect the views of the NSF.
