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Chapter ~ CONTROL, OCCURRENCE, AND IDENTIFICATION . Although concern for the health ef feats of aromatic amines has focused pr imari ly on industr ial use of these substances, it is becoming increasingly evident that there are many sources of exposure to these compounds and to the ir precursors . me facile biochemical reduction of arylnitro compounds by both mammal fan and microbial organ isms necessitates that ache ir precursors be identif fed so that the distribution of potentially hazardous aromatic amines can be surveyed. Similarly, azo dyes are readily reduced to free amines by a variety of enzymes. Given the metabolic capacities likely to be involved, it is prudent to regard any N-substituted aromatic compound as a potential aromatic amine. In general, aromatic amine derivatives to which humans might be exposed are either synthesized intentionally for some specif ic commercial use or produced by enzymic reduction of aromatic nitro or azo compounds or are formed inadver ten tly as byproducts in processes apparently d irectly or ind irectly related to combustion . Commercial Products The development of the synthetic dye industry in Europe during the latter half of the 19th century led to the f irst recognition of arylamine-induced bladder cancer in humans. Since that time, industr ial organ ic chemistry has become more sophisticated and 23
it is not surprising that aromatic amines are now widely marketed in dyes and as compounds to be used in var ious manufacturing processes. The synthesis of these substances can cause occupational health hazards, and inadequate control of wastes associated with the ir production can result in contamination of the environment. Furthermore, the practical use of the products can expose both workers and consumers to their dangers. Because of the var ious uses intended for the aromatic amines and the ingenuity of the chemist, commercial products have var ious and ever~hanging compositions. Amines are integral to the following compounds: dyes o antioxida nts 0 polymers 0 explos Ives 0 pesticides pharmacologic Byproducts and Combustion Processes In contrast to the often large-scale industrial synthesis, aromatic amines are also produced inadvertently in low concentra Lions a s byproducts of processes that expose organic mater ials to elevated atmospher ic temperatures . The combustion of organic materials can generate aromatic amine derivatives by two dif ferent mechanisms. The partial combustion or pyrolysis of n i trogen-conta in ing or gan ic ma ter ial can produce both 24
azoheterocyclics and arylamine compounds, as exemplified by the detection of 22 p9 of 2-naphthylamine in the smoke of 100 cigarettes (Hoffmann and Wynder, 1976), amino-substituted carbolines in amino acid pyrolysates (Kosuge et al., 1978), and indirect mutagens believed to be primary aromatic amines in synthetic fuels {Epler et al., 19801. ~ The presentation by Guer in (1980) and subsequent discussion by several authors agreed that most of the observed mutagenic activity resulting from many different samples derived from coal, shale-derived oil, and petroleum crudes, could be attributed to an alkaline isolate fraction constituting only a fraction of a percent of the sample mass. These samples contained (among other things) identif table polynuclear aromatic amines. A second, more ind irect, mechanism that produces amines is seen in the formation of nitroaromatic compounds as a consequence of combustion processes. Polycyclic aromatic hydrocarbons can be nitrated by nitrogen oxides formed at high temperatures from atmospheric nitrogen. These compounds may be formed during or immediately after the combustion process, but the possibility of their subsequent photochemical formation has not been excluded. Indirect evidence for the formation of nitrated aromatics as byproducts of the combustion process comes from two sources. The model studies of Pitts and his collaborators (1980 ~ demonstrated that polycyclic aromatic hydrocarbons were readily nitrated by levels of nitrogen oxides found in the atmosphere. me second line of evidence comes from analysis of the mutagenicity of particulates 25
collected f rom internal combustion engines (Claxton and Huis ingh, 1980) and from the atmosphere (Wang et al., 1980~. The mutagenicity of these materials in Salmonella typhimurium are decreased if tested in strains that are deficient in the ability to be reverted to prototrophy by arylnitro compounds. Emus, the population may be exposed to arylamine precursors from several noncommercial sources (Lofroth, 1978~: o tobacco smoke, 0 food pyrolysates, 0 synthetic fuels, o internal combustion engines, o atmospher ic particulates, and o fossil fuel-f ired power plants. The last three sources contain direct-acting mutagens to bacteria that contain nitroreductase which suggests the presence of n itroaromatics . Unlike the long-recognized potential hazards of the industrially produced aromatic amines, the widespread distribution of these compounds in prepared food and the environment has only recently been demonstrated. Thus, the analytic methodology required for the ir study is only now be ing developed. When the appropr late techniques are available, it will be possible to evaluate the compounds involved, the levels of exposure, and the biolog ic consequences of contamination. 26
RECOMMENDATION The anticipated changes in the use of fossil fuel, implicit in the promotion of diesel engines and coal, may raise existing environmental levels of aromatic amines. Accordingly, the committee recommends that steps be taken to evaluate both the qualitative and quantitative factors that attend these changes in practice. GENERAL ANALYTICAL PROCEDURES Aniline · E~-cresidine, 2, 4-diaminotoluene, and MOCA [4,4 '-methylene-bis (2-chloroaniline) ~ are primary aromatic amines and should respond to the following general analytic procedures. Trifluralin, a tertiary aromatic amine, and furazolidone, a nitrofuran, do not respond to these procedures and must be analyzed by other means as discussed at the end of this chapter. General Procedures for Pr imary Aromatic Amines Color imetr ic Me thods . A spectrophotometr ic method for amides, amino acids, and a peptide using 2,4,6-trinitrobenzenesulfonic acid (TNBS) as the chromogen ic agent was reported by Satake et al . (1960 ~ . Rinde and Troll (1975 ~ Waif fed this method to determine free benzidine in the urine from monkeys dosed with a benzidine-based dye. Urine samples were extracted with chloroform and back-extracted with 0.01 M hydrochlor ic acid. The acid extract (pH 5 ~ was reacted with lNBS and the yellow color extracted into chloroform for analys is . Extraction of the reaction products into 27
an organic solvent was required to prevent excess reagent in the aqueous phase from interfering with the analysis. TNBS was also used as a spray to render the spots visible on thin-layer chromatographic (TLC) plates. Although the procedure is sensitive in the nanomole range, it lacks the specif icity usually required of analytic methods . Never theless, the procedure is currently used by the National Institute for Occupational Safety and Health (NIOSH) to monitor urine from industrial workers (Lowry et al., in press) . The reagent fluorescamine (Fluram) was introduced by Udenfriend _ al. (1972) to quantify aliphatic amines in a sensitive fluorescent assay. Aromatic atones form fluorescent products just as the aliphatics do, but the aromatic products are unstable. However, the parent compounds do form stable yellow derivatives. Rinde and Troll (19 76 ) used Fluram to develop a color imetr ic procedure to identify several carcinogenic aromatic amines. Dry residues of the amines were reacted with 50 y 1 of Fluram solution (1 mg/ml in glacial acetic acid) for 10 minutes. me reaction was stopped by adding 0.5 ml of methanol. Optical density readings of the yellow color were made in 0 .5 ml cove ttes In a spectrophotometer set at the approximate wavelength for maximum absorption. Fluram is sa id to react only with aromatic amines in a glacial acetic acid medium. The reaction can be performed directly on TLC plates to add specif icity to the measurement, and the yellow product can be quanti ta Lively eluted from the TLC plates . Fluram is colorless, so blanks appear as zero, a distinct advantage over procedures employing a TNBS reagent. The sensitivity of the procedure ranges from 2 to 10 nanomoles for compounds such as benzidine and 28
2-naph thylamine . Spot Tests. Strict procedural guidelines imposed by the Occupational Safety and Bealth Administration (05RA) concerning 14 potentially carcinogenic chemicals {including some aromatic airlines) prompted Weeks et al. (1976) to investigate various spot-test procedures for the compounds. Chromogenic agents, 'such AS "rlich 's reage n t, .E ad ie thylaminobenza ldebyde, EN - ime thylaminoc innama ldehyde, chloranil, and chloramine T. and fluorogenic agents, such as Fluram and isomer ic phthaldehydes, were evaluated. The limit of detection values in terms of grams of analyte per 'square centimeter of surface being examined ranged from the low nanogram to the 5-pg level, depend ing on the compound, sampl ing techn igue, and sur face involved . Thin-Layer Chromatography. There are numerous procedures for separating and detecting aromatic amines. In recent years, reaction chromatography has been found to identify amines most successfully. In this technique, a derivative is prepared, and then both the derivative and the parent compound are subjected to separation. Some of the der ivatives of aromatic smines that have been prepared for this purpose are 3 ,5~dinitrobenzamides, 4 - imethylamino- benzeneezo-4 -benzamides, 2, 4-din itrophenylamines, ~toluene- sulfonamides, and arylazo-2-naphthols . Isomer ic toluidines and aniline, which are difficult to separate as salts, can be converted into bromine derivatives; daneyl chloride has also been used. Ore recently, Franc and Xoudelkova (1979) investigated TLC separations of 128 dif ferently substituted aromatic amines and their 2, 4-dinitrophenyl der ivatives . Tree solvent systems were used and 29
in certain cases, assays were completed by paper electrophoresis. The der ivatives were prepared by reacting the amines with 1-fluoro-2,4~dinitrobenzene. The three-solvent system used with Silufol W-254 were diethyl ether-cyclohexane (4 :3 ~ and ethyl acetate-N-propanol-ammonia (5:4:1) and (2:1:2~. The aromatic amines were detected by spraying with a 19 solution of Ehrlich 's reagent in 1 M hydrochlor ic ac id . The der ivatives were detected by spraying with 59 stannous chlor ide to reduce the nitro groups to amino groups. After the chromatogram dried, Ebrlich's reagent was used. Lepri et al. (1978) in experiments with soap TLC studied the behavior of 35 primary aromatic amines on layers of silanized silica gel alone or impregnated with 2% or 4% triethanolamine dodecylbenzenesulfonate solutions. Eluents such as a mixture of 1 M acetic acid and 30% methanol were employed. Ehrlich's reagent was used to detect the amines. Several separations that could not be made either by ion exchange or reverse-phase chromatography were carried out. Additonal TLC procedures for specif ic compounds are discussed later in this chapter. Gas Chromatography. Many of the aromatic amines are amenable to the conditions imposed by gas chromatography (GC) and can be measured by using the nonspecific flame ionization detector (FID}, where extremely high sensitivity (e.g., low nanogram or picogram range) is not required. Also, the rubidium-sensitized thermionic-type nitrogen/phosphorus (N/P) detector responds to the 30
compounds with various degrees of sensitivity and specificity. Nevertheless, most of the work in GC me Mods development for the aromatic amines has centered around the formation of halogenated derivatives with subsequent analysis by electron-capture (EC} GC. Such procedures greatly enhance sensitivity and often improve stability and GC characteristics. Francis _ al. (1978) demonstrated that flophemesyl (pentafluorophenyl-dimethylsilyl ) derivatives of amines and other classes of organ ic -compounds could be prepared and analyzed wi th high sensitivity by EC-GC. The amine (10 me or less) dissolved in toluene (30 pi) was reacted with 30 pi of flopheme~ylamine in a sealed vial at 60°C for approximately IS minutes. Aniline and 2-phenylethylamine were among the amines evaluated . The ga s chromatography was accomplished by using a 1. S-m glass column packed with 10% SE-30 on Chromosorb P AW DMCS and a 63Ni EC detector; sensitivities were reported in the picogram range. Nony and Bowman (1978 , 1980 ~ adapted the method of Walle and Ehrsson (1970) to assay several aromatic amides as their pentafluoropropionyl (PFP) or heptafluorobutyryl {HFB) derivatives. The amine (10 fig or less) in 1. S m' of benzene is added to a tube con Lain ing O . S ml of tr imethylamine catalyst (0 . OS M in benzene ), then 50 pi of heptofluorobutyric ac id anhydride is added. The sealed system is heated at 50°C for 20 minutes, the reaction is terminated, and the benzene phase is extracted by us ing 2 m1 of phosphate buffer (pH 6~. The benzene phase, dried over sodium sulfate, is analyzed on a 1.8-M glass column packed with 5% Dexsil 31
300 on Gas Cbrom Q by EC-GC employing a 63Ni detector. Sensitivities are generally in the low picogram range; however, the procedure requires Edification to preclude low recoveries of volatile compounds such as aniline. No cleanup procedures were used in the assays . In another study us ing PFP der ivatives, Bowman and Rushing (1977) used both EC and N/P detectors to assay for trace levels of 3, 3 ' -dichlorobenzidine in animal feed, wastewater, and human urine. The use of cleanup procedures using XAD 2, silica gel, and/or liquid-liquid partitioning permitted the detection of low ppb levels of the compound in feed and low ppt levels in wastewater and human urine. mese procedures could probably be adapted to assay all four pr imary aromatic amines discussed in this report. High Pressure Liquid Chromatography. Although few high-pressure liquid chromatography (HPLC) methods for aromatic amines have been described in the literature, the technique appears promising when the compounds are not amenable to Go or der ivatization techniques or when optimum sensitivities are not required. Popl et al. (1978) measured retention data for polar-substituted benzenes and naphthalenes, including several aromatic amines, by using reverse-phase, liquid-liquid chromatography on macroporous polystyrene gel with methanol-water and acetonitrile-water as the eluents. Riggin and Howard (1979) employed electrochemical detection (thin-layer glassy carbon electrode) for EPLC assays of benzidine and related compounds at ppb levels. Rice and Kissinger (1979) described a specific method using HPLC with amperometric detection (carbon paste electrode vs silver/silver chloride reference electrode) for benzidine and its mono- and diacetyl 32
metabolites in human urine. A detection limit of 10 ppb based on twice noise was reported for benzidine. Nony and Ian (1980) reported HPLC Cathode for ~ variety of aromatic airlines and related oompounde in hamster and human urine using ~ acndepak Call column, Fixtures of "t~hanol-phosphate buffer {pE 6) as the mobile phase, and a variable ultra~roilet absorption detector set at the appropriate wavelength for maximum response. The detection limit for bend idine in the ur ine by th is procedure was approximately 180 ppb, based on twice background . Where applicable, the use of 8 fluorescence detector generally enhances the sensitivity and specificity of assays by HPLC. Other Aroma t ic Amines Considerable research has been conducted on methodologies for detecting trifluralin because of its widespread use as a herbicide. Because the compound captures electrons well and is amenable to GO, the EC-GC procedures (with minor modifications) should meet most of the requirements. Alternative methods include TLC, HPLC, and mass spectrometry (MS). There are adequate procedures (e.g., colorimetry, TLC, and TALC) for analyzing relatively high concentrations of furazolidone. However, assays for the compound at low ppb levels in various substrates appear almost intractable. Although HPLC is probably the best procedure available, deficiencies in cleanup prior to injection and in the inherent sensitivity lignite of the system may preclude 33
its use at low ppb levels. Research in the areas of sample cleanup and mass spectroreetry methodology is the next step to improve the stateoftheart CONCLUSIONS - . Techn iques such as color imetry, TLC, GO, HPLC, and MS have been used to analyze pr imary aromatic amines . Modif ications of sampling and cleanup procedures may be required for different substrates, and techniques must be instituted to ensure adequate measurements of the more volatile compounds, such as aniline. Nevertheless , existing procedures with appropriate modifications should satisfy most analytic requirements. me use of EC der ivatives, HI?LC, and MS are a ttract ive prospects . 34
REEI:RENCES Control Claxton , L., and J . Hutaingh . 1980 . Character ization of the mutagens associated with diesel particle emissions. Environ. Mutagenes is 2: 239 (Abstract) . Epler, J.L., T.K. Rao, and F.W. Larimer. 1980. Isolation and identif ication of mutagenic polycyclic aromatic amines in synthetic crude oils. Environ. Mutagenesis 2:238 (Abstract) . Guerin, M.R. 1980. Bioassay chemistry and the characterization of polycyclic aromatic organonitrogen compounds - New environmental analytical problems. Second Symposium on Environmental Analytical Chemistry, Provo, Utah, June 1980. Hoffmann, D., and E.L. Wynder. 1976. Environmental respiratory carcinogenes is. Pp. 324-365 in C. E. Searle, ed . Chemical Carcinogens. ACS Monograph 173. American Chemical Society, Washington, D.C. Kosuge, T., K. Tsu ji, K. Wakabayashi, T. Okamoto, K. Shudo, Y.Titaka, A. Itai, T. Sugimura, T. K=wachi, M. Nagao, T. Yahagi, and Y. Seino. 1978. Isolation and s~:r~cture studies of mutagenic pr inciples in amino acid pyrolysates. Chem. Pharm. Bull. 26:611-619. 35
Lofroth, G. 1978. Mutagenicity assay of combustion emissions. Chemospbere 7: 791-798 . Pitts, J.N., Jr., K.A. Van Cauwenberghe, D. Gros Sean, J.P. Schmid, D. R. Fi tz, W. L. Belser, Jr ., G. B. Enudson, and P. M. Bynds . 1978 . Atmospheric reactions of polycyclic aromatic hydrocarbons: Facile formation of mutagenic nitro derivatives. Science 202:515-519. Wang , C. Y., H. -S. Lee , C.M. King , and P.O. Warner . 1980 . Evidence for nitroaromatics as direct-acting mutagens of airborne par ticulates. Chemosphere 9: 83-87 . 36
General Analytic Procedures Bowman, M.C., and L.G. Rushing. 1977. Trace analysis of 3,3'~dichlorobenzidine in animal chow, wa~tewater and human urine by three gas chromatographic procedures. Arch. Environ. Contem. Toxicol. 6 :471-482. Franc, J., and V. Koudelkova' 1979. '~in-layer chromatography of aromatic amines and their derivatives after reactions with 1-fluoro-2,4-dinitrobenzene. J. Chromatogr. 170:89-97. Francis, A.J., E.D. Morgan, and C.F. Poole. 1978. Flophemesyl der ivatives of alcohols, phenols, amines and carboxylic acids and their use in gas chromatography with electron~capture detection. J. Chromatogr. 161:111-117. Lepr i , L., P. . G. Des ider i , and D. He imler . chromatography of pr imary aromatic amines . 155: 119 -12 7 . 1978. Soap thin-layer J. Chromatogr. Lowry, L.K., W.P. Halos, M.F. Boeniger , C.R. Nony, and M.C. Bowman. In press. Chemical manitoring of urine from workers potentially exposed to benz idine-der ived azo dyes . 37 Toxicol. Lett . 7: 29-36 .
Nony, C. R., and M. C. Bowman . 1978 . Carcinogens and analogs : Trace analysis of thirteen compounds in admix~cure in wastewater and human urine. Int. J. Electron. final. Chem. 5: 203-220 . Nony , C . R., and M. C. Elowman . 1980. Trace analysis of potentially carcinogenic metabolites of an azo dye and pigment in hamster and human urine as determined by two chromatographic procedures. J. Chromatogr . Sci. 18: 64-74 . Popl, M., V. Dolansky, and J. Flbnrich. 1978. Reversed-phase liquid-liquid chromatography of aromatics on macroporous polystyrene gel. J. Chromatogr . 148 :195-201. Rice, J. R., and P. T. Kissinger . 1979 . Determination of benzidine and its acetylated metabolites in ur ine by liquid chromatography. J. Anal. luxicol. 3: 64-66. Riggin, R.M., and C.C. Howard. 1979. Determination of benzidine, dichlorobenzidine, and diphenylhydrazine in aqueous media by high performance liquid chromatography. Anal. Chem. 51:210-214. Rinde, E., and W.Troll. 1975. Metabolic reduction of benzidine azo dyes to benz idine in the rhesus monkey. 55: 181-182. 38 J. Natl . Cancer Ins ~ .
Rinde, E., and W. Troll. 1976. C:olorimetric assay for aromatic amides. Anal. Cbem. 48 s542-544 . Satake , K., T. Okuye - , M. Oheshi , and T. 8hinode . 1960 . m. apectropt~otometric determination of amine, amino acid and peptide with 2 ,4 ,6-tr initrobenzene 1-sulfonic acid. 47: 654-660 . J. Biochee. Udenfriend, S., S. Stein, P. B;;hlen, W. Dairean, W. Leiagruber , and M. Weigele. 1972. Fluorescamine: A reagent for assay of amino acids, peptides, proteins and primary amines in the pinhole range. Science 178: 871-872. Walle, T., and H. Ehrsson . 1970 . Quantitative gas chromatograpic determination of picogram quantities of amino and alcoholic compounds by electron capture detection. Part I. Preparation and properties of the heptafluorobutyryl der i~ratives . Suec . 7: 3 89-40 6 . Acts Pharma. Weeks, R.W., B.J. Dean, and S.K. Yasuda. 1976. Detection limits of chemical spot tests toward certain carcinogen" on metal, painted, and concre te sur faces . Anal. Chem. 48: 2227-2233. 39
