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ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
Characterization of Pyrolysis Conditions and Interference by Other Compounds in the Chemiluminescence Detection of Nitrosamines Thomsen J. Hansen, Michael C. Archer,"' and Steven R. Tannenbaum Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139
Facile analysis of N-nitroso compounds has been made possible by the development of chemiluminescence detectors. Respmse of one such detector as a function of pyrolysis chamber temperature has been characterized for several nitrosamines and for an aliphatic C-nitro compound, an aromatic C-nitro compound, a nitramine, an alkylnitrite, and a nitrosourea. The response-temperature profiles are valuable in distinguishing among the various compounds and in optimizing the sensitivity of the detector for use in chromatography. Other tests, including photolysis and stability toward nitrite-scavenging reagents, further aid in distinguishing among the various compounds.
Nitrosamines may be analyzed in environmental samples by use of sensitive and selective chemiluminescence detectors such as the Thermal Energy Analyzer (TEA, Thermo Electron Corp., Waltham, Mass.) (1-5). This detector utilizes an initial pyrolysis reaction that cleaves nitrosamines at the N-NO bond t o produce nitric oxide. [Earlier papers describe the use of a catalytic pyrolysis chamber ( I , 3 , 4 ) ;in current instruments, including the one used in this study, pyrolysis takes place in a heated quartz tube without a catalyst.] T h e nitric oxide is then detected by its chemiluminescent reaction with ozone. Samples are introduced into the pyrolysis chamber by direct injection or by interfacing the detector with a gas chromatograph (GC, 6) or a liquid chromatograph (LC, 7). Chemiluminescence detectors have considerable selectivity for nitrosamines because the light emitted from the NO-ozone reaction is in the near infrared region, whereas other known chemiluminescent reactions with ozone emit light in the visible or near UV region (3,8). Use of a n optical filter eliminates response t o emissions occurring below 600 nm. Selectivity is also provided by a cold trap between the pyrolysis chamber and the NO-ozone reaction chamber. T h e trap removes all but the most volatile compounds eluting from the pyrolysis chamber. Although chemiluminescence detectors have considerable selectivity for nitrosamines, the possibility exists that any compound that can produce NO during pyrolysis will produce a signal. T E A responses have been observed from organic nitrites, C-nitro and C-nitroso compounds (3, 9, I O ) , and nitramines (11). Pyrrole (12)and a n unidentified breakdown product of a benzyltrialkylammonium salt (13) have also been shown to give T E A responses, but the mechanisms leading to their responses are unknown. In our studies on the interaction of food materials with nitrous acid, we required a method of distinguishing nitrosamines from other compounds that give a TEA response. One important variable in the operation of the T E A is the temperature of the pyrolysis chamber. This temperature 'Present address, Department of Medical Biophysics, University of Toronto, 500 Sherborne Street, Toronto, Canada, M4X 1K9. 0003-2700/79/0351-1526$01 .OO/O
largely determines pyrolytic fragmentation pathways and possibly whether or not a compound breaks down to yield NO. We have therefore systematically examined the variation in TEA response as a function of pyrolysis chamber temperature for a number of different types of compounds. T h e study included the TEA interfaced both with a gas chromatograph (GC-TEA) and liquid chromatograph (LC-TEA). We also report methods involving photolytic and chemical degradation for further characterizing TEA-positive compounds.
EXPERIMENTAL Thirteen compounds were chosen for this study on the basis of availability, presence of an N-0 bond, or previous reports of a positive response on the TEA. N-Nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, and N-nitrosodibutylamine were obtained from Eastman Organic Chemicals, Rochester, N.Y.; N-nitrosopyrrolidine and acetohydroxamic acid were obtained from Aldrich Chemical Co., Milwaukee, Wis.; N-nitrosomorpholine and 1-nitrohexane were obtained from ICN Life Sciences, Plainview, N.Y.; 2-nitrotoluene was obtained from J. T. Baker Chemical Co., Phillipsburg, N.J.; acetone oxime was obtained from Matheson, Coleman and Bell, Norwood, Ohio. N-Nitroso-n-propylurea was prepared from n-propylurea as described by Arndt (14),hexylnitrite was prepared by nitrosation of 1-hexanol (151, and dipropylnitramine was prepared by oxidation of N-nitrosodipropylamine with peroxytrifluoroacetic acid (16).
Approximately 10 mM solutions of the nitrosamines, dipropylnitramine, N-nitroso-n-propylurea,acetohydroxamic acid, and acetone oxime were prepared in dichloromethane, nitrotoluene, nitrohexane, and hexylnitrite in isooctane. AU compounds except N-nitroso-n-propylurea were analyzed by GC-TEA. GC conditions were first established with a flame ionization detector. The nitrosamines and dipropylnitramine were analyzed using a 2 m x 2.1 mm stainless-steel column packed with 10% Carbowax 2OM-TPA + 3% KOH on Gas Chrom A, at 140 "C. The other compounds were analyzed using a 2 m X 2.1 mm nickel column packed with 3% OV-17 on Chromasorb G-HP at temperature ranging from 70 O C (for hexylnitrite) to 170 O C (for nitrotoluene). A glass-lined injection port (200 "C) was used for all analyses. Injection volumes were 1.5 pL. After passing through the TEA pyrolysis chamber, the GC column effluent flowed through a dry-ice/acetone trap and a 6-inch column of Tenax before entering the chemiluminescence analyzer. Nitrosodimethylamine and N-nitroso-n-propylurea were analyzed by LC-TEA using a 30 cm X 4 mm pPorasil column (Waten Associates, Milford, Mass.) with a mobile phase of 1"h methanol in dichloromethane. Injection volumes were 10 pL. After passing through the pyrolysis chamber, the LC effluent flowed through a dry-ice/isopropanol trap and a 6-inch column of Tenax before it reached the reaction chamber. The column effluent was also monitored by a UV detector (254 nm) placed in series between the column and the TEA to ensure that the TEA response was not caused by impurities or decomposition products. Compounds giving a TEA response were subjected to photolysis. The samples were sealed in capillary tubes and irradiated by three long-wavelength UV lamps (Sylvania Model F15TS-BLB) for 16 h. The same compounds were also treated with methanol saturated with ascorbic acid (approximately 4 g/100 mL) and methanol containing phenol (5 g/100 mL). Reactions were carried C 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
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Table I. LC-TEA ResponsesQ of Nitrosodimethylamine and N-Nitroso-n-propylurea Oven temperature, "C flow rate, compound mL/min 300 35 0 400 450 nitrosodimet hylamine 0.25 31 0.50 34 53 53 65 0.75 39 1.00 2 14 18 29 N-nit roso-n-propylurea 0.25 0.1 0.50 0 0.05 0.6 3.0
-
0.75
0
1.00
0.05 0.05
-
-500 60
71
100
46 41
23
5 5 4.7 2.8
-
0.65
600
1.6
-
3.4
-
2.3
Reported as a percentage of the maximum observed response for nitrosodimethylamine (at 6 0 0 'C, 0.5 mL/min).
c ZOC
300
400 500 TEMPERATURE ( ' C )
i
600
response vs. pyrolytic chamber temperature for: (0) nitrosodimethylamine, (0) nitrosodiethylamine, (m) nitrosodipropylamine, ( 0 )nitrosodibutylamine, (A)nitrosopyrrolidine, (A)nltrosomorpholine Figure 1. TEA
out by mixing sample and methanol solution (1:l) for 1 h.
RESULTS A N D D I S C U S S I O N Figure 1 shows the GC-TEA responses of six nitrosamines as a function of the pyrolysis chamber temperature. (Responses are given as the percentage of the measured response compared with the maximum response for nitrosodimethylamine on a molar basis.) The low response at chamber temperatures between 200 and 300 "C was probably caused by incomplete pyrolysis of the nitrosamines, resulting in diminished formation of NO. At high pyrolysis chamber temperatures, the decreased response may have been caused by fragmentation of molecules a t bonds other than the N-NO bond. GC-TEA responses to an aliphatic nitro compound, an aromatic nitro compound, a nitramine, and an alkylnitrite are shown in Figure 2. Variation in the responses with pyrolysis chamber temperature differed noticeably between these compounds and the nitrosamines. Hexylnitrite pyrolysed to yield NO a t relatively low temperatures (20&300 "C), but the response decreased when the temperature exceeded 300 "C. The nitramine was also susceptible to pyrolysis to NO at rather low temperatures. Hotchkiss e t al. (11) found t h a t dipropylnitramine gave a molar response of 50%, compared with nitrosodipropylamine, a t a pyrolysis chamber temperature of 400 "C. In our study, dipropylnitramine exhibited a response of 96% at the same temperature. This discrepancy may have been caused by differences in instrument parameters which were not investigated here, such as carrier gas-flow rate or chemiluminescence reaction-chamber pressure. Nitrohexane and nitrotoluene gave measurable GC-TEA responses only a t temperatures above 350 and 500 "C, respectively. The responses of the nitramine and the nitro compounds may be caused either by the direct release of NO (preceded by a molecular rearrangement) or, more likely, by
0
200 L
700-
4 0 C-
500
-1
6iO
TEMPERATURE 1 ° C )
Figure 2. TEA response vs. pyrolytic chamber temperature for: ( 0 ) nitrohexane, ( H ) nitrotoluene, (0)hexylnitrite, (0)dipropylnitramine
the release of N O p followed by the conversion of' NO2 to NO according to the reaction 2N02
2N0
+ 02
(1)
Although Fine et al. ( 4 ) suggested t h a t this reaction is unlikely to occur in air at a pressure of 1 atm, the conditions of high temperature and low partial pressure of oxygen t h a t exist in the pyrolysis chamber would tend to drive the equilibrium toward the formation of NO ( 1 7 ) . It is interesting to note that, while the rate of the forward reaction in Equation 1 increased in the normal manner with increasing temperature, the rate of the reverse reaction decreased with increasing temperature (17). Fine et al. (4) also suggested that NOp might be reduced to NO by particles of active carbon that build up in the pyrolysis chamber from organic compounds. We showed, however, that the temperature-response profile of nitrohexane is identical in new and used pyrolysis chambers. Thus, the TEA response of nitro compounds does not appear to depend on the presence of carbon. No GC-TEA response was observed at temperatures up to 600 "C for either acetohydroxamic acid or acetone oxime (the preferred tautomer of 2-nitrosopropane). Table I shows the LC-TEA response of N-nitrosodimethylamine and N-nitroso-rz-propylurea at several different solvent flow rates and pyrolysis chamber temperatures. Variation in the LC-TEA response of N-nitrosodimethylamine at different temperatures at a fixed flow rate followed the same general pattern as the GC-TEA response. Maximum response, however, was observed a t higher temperatures in LC-TEA. This difference was probably caused by introduction of solvent into the pyrolysis chamber. The temperature was measured by a thermocouple positioned a t the center of the chamber. Introduction of solvent at one end of the chamber undoubtedly causes a temperature gradient along its length. Evaporation of solvent in the pyrolysis chamber may also markedly increase the gas flow rate in this region of the TEA and hence decrease
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
the residence time of the sample. Variations in the LC-TEA response of N-nitrosodimethylamine at different solvent flow rates a t a constant chamber temperature indicated that the response reaches a maximum at about 0.5 mL/min. The decrease in response as the flow rate was increased from 0.5 to 1 mL/min may be explained by a decreased sample residence time in the pyrolysis chamber. At still higher flow rates, the background signal and sample response became somewhat erratic. This may have been caused by incomplete condensation of the hot solvent vapors in the cold trap. We cannot a t this stage explain the increase in response that occurred when the flow rate was increased from 0.25 to 0.5 mL/min. Table I shows that nitroso-n-propylurea gave a TEA response, hut the maximum signal was only 5% of the maximum response of .V-nitrosodimethylamine. The maximum occurred at about 500 "C at solvent flow rates of 0.5 and 1.0 mL/min, and a further increase in temperature caused a diminution in response. For nitroso-n-propylurea, then, formation of NO apparently represents only a minor pathway in its pyrolytic fragmentrltion. Similarities have been noted between the molecular fragmentation pathways in electron-impact mass spectrometry and in decomposition by pyrolysis (18). Whereas the nitrosamines generally undergo an N-NO bond cleavage during MS fragmentation to yield NO, nitrosoureas fragment during ills mainly by cleavage of the N-CO bond followed by cleavage of the N-0 bond of the resulting monoalkylnitrosamine ion to yield the diazonium ion (19). Since the yield of NO during pyrolysis of the nitrosourea was very low, the electron impact and pyrolytic fragmentation pathways are probably similar. Following our characterization of TEA response as a function of pyrolysis temperature for a number of different compounds, we were interested in devising other methods that might be uscful in analyzing environmental samples in order to distinguish among the various classes of TEA-positive compounds. Photolysis of nitrosamines has previously been used by Doerr and Fiddler as an aid to confirmation during analysis (20). With our compounds, UV irradiation destroyed all the nitrosamines and the nitrosourea, and in addition the alkylnitrite and nitramine. Only the two C-nitro compounds were stable to irradiation. Alkylnitrites have been reported to decompose in methanol (21). This reaction is an equilibrium which we thought could be driven toward the destruction of the alkylnitrites by addition of a reagent, such as ascorbic acid (22) or phenol (231, that would react rapidly to remove nitrite from the system. IVe found t h a t both ascorbic acid and phenol in methanol completely destroyed the alkyl nitrite without affecting any other compound. On the basis of the photolysis and chemical test then, an N-nitroso compound could be distinguished from a C-nitro compound and an alkyl nitrite, but not from a nitramine. Treatment of an N-nitroso compound with
peroxytrifluoroacetic acid would convert it to the corresponding nitramine, whereas the nitramine itself would not react further. Nitrosamines and nitrosoureas may be distinguished by treatment with a base, since nitrosamines are stable under basic conditions whereas nitrosoureas and other N-nitroso compounds-such as nitrosamides, nitrosoguanidines, and nitrosourethans-readily decompose ( 2 4 ) . We have shown that the profile of TEA response vs. pyrolysis temperature can be useful in distinguishing among various TEA-positive compounds, and may be indicative of compound class. The profile is also useful in optimizing the sensitivity of the TEA as a chromatographic detector for various compounds. Further study is clearly needed to understand the mechanisms underlying some of the observed responses.
ACKNOWLEDGMENT We thank Kwanghee K. Park for synthesis of N-nitroson-propylurea.
LITERATURE CITED D. H. Fine, F. Rufeh, and 6. Gunther, Anal. Left.. 6, 731 (1973). D. H. Fine, F. Rufeh, and D. Lieb, Nature (London), 247, 309 (1974). D. H. Fine, F. Rufeh, D. Lieb, and D. P. Rounbehler, Anal. Chem., 47, 1188 (1975). D. H. Fine, D. Lieb, and F. Rufeh, J . Chromatogr., 107, 351 (1975). D. H. Fine, D. P. Roundbehier, and P.E. Oeninaer, Anal. Chim. Acta, 78, 383 (1975). D. H. Fine and D. P. Roundbehler, J . Chromatogr., 109, 271 (1975). P. E. Oettinger, F. Huffman, D. H. Fine, and D. Lieb. Anal. Lett., 8, 41 1 (19751. P.-N.-Cloughand 6. A. Thrush, Trans. Faraday Soc., 63, 915 (1967). R. W. Stephany and P. L. Schulier, in "Proceedings of the Second International Symposium on Nitrite in Meat Products", Zeist, 7-10 Sept., 1976; B. J. Tinbergen and 6. Krol, Eds., Centre for Agricultural Publishing and Documentation, Wageningen, The Netherlands, 1977, p 249. T. Y. Fan, R. Vita, and D. H. Fine, Toxicol. Lett., 2, 5 (1978). J. H. Hotchkiss, J. F. Barbour, L. M. Libbey, and R. A. Scanlan, J . Agric. Food Chem., 26, 884 (1978). W. Fiddler, R. C. Doerr and E. G. Piotrowski, in "Environmental Aspects of N-Nitroso Compounds"; E. A. Walker, M. Castegnaro, L. Griciute, and R. E. Lyle, Eds., International Agency for Research on Cancer (Sci. Pub/. No. 19), Lyon, 1978, p 33. T. A. Gough and K. S. Webb, J . Chromatogr., 154, 234 (1978). F. Arndt, Org. Syn., Coll. Vol., 2, 461 (1943). A. I. Vogel, J . Chem. Soc.. 1847 (1948). W. D. Emmons, J . Am. Chem. SOC.,76, 3468 (1954). J. W. Mellor, "Inorganic and Theoretical Chemistry", Vol. VIII, Suppl. 11, Longmans, London, 1967, p 158. R. C. Dogherty. Top. Curr. Chem., 45, 93 (1974). W. T. Rainey, W. H. Christie, and W. Lijinsky, Biomed. Mass Spectrom.. 5, 395 (1978). R. C. Doerr and W. Fiddler, J . Chromatogr., 140, 284 (1977). A. D. Alien, J . Chem. Soc., 1968 (1954). M. C. Archer. S. R. Tannenbaum, T. Y . Fan, and M. Weisman, J . Natl. Cancer Inst., 54, 1203 (1975). 6. C. Challis, Nature (London),244, 466 (1973). S. M. Hecht and J. W. Kozarich, J . Org. Chem., 38, 1821 (1973).
RECEIVED for review January 22,1979. Accepted May 21,1979. This investigation was supported by Public Health Service Contract N01-CP-33315 with the National Cancer Institute, and Research Career Development Award l-KO4-ES00033 (to M.C.A.) from the National Institute of Environmental Health Sciences.
