1202
Anal. Chem. 1981, 53, 1202-1205
Discharges"; EPA 560178-009; Environmental Protection Agency: 1978. (4) Janardan, K. G.;Tigwell, D. C.; Schaeffer, D. J. Eleventh Annual Pittsburgh Conference on Modeling and Simulation. Instrument Society of America. Research Triangle Park, N C Modeling Composite Sampling with Application to Trace Organics Monitoring, 11, Part 3, pp 985-989. (5) Garrison, A. W.; Pope, J. D.; Alford, A. L.; Doll, C. K. NBS Spec. Pub/ ( U . S . ) 1979, NO. 519, 65-78. (6) Stephan, S.F.; Smith, J. F.; Flego, U. WaterRes. 1978, 12, 447-449. (7) Lauch, R. P. "A Survey of Commercially Available Automatic Wastewater Samplers", EPA 60014-76-051; US. Environmental Protection Agency: Cincinnati, OH, 1976. (8) Huibergise, K. R.; Moser. J. H. "Handbook for Sampling and Sample Preservation of Water and Wastewater", EPA 600/4-76/049; US. Envlronmental Protection Agency: Washington, DC, 1976; National Technical Information Service, 1976, PC-259 946. (9) Shelly, P. E. "Sampling of Water and Wastewater", EPA 600/4-77/ 039; U.S. Environmental Protection Agency: Cincinnati, OH, 1977; National Technical Information Service, 1977, PB-272 664. (IO) Devera, E. R.; Simmons, B. P.; Stephens, R. D.; Storm, D. L. Samples and Procedures for Hazardous Waste Streams", EPA-BOO/ 2-80-018: US. Environmental Protection Agency: 1980. (11) Schaeffer, D. J.; Janardan, K. G. Blom. J. 1978, 20, 215-227. (12) Janardan, K. G.; Schaeffer, D. J. Anal. Chem. 1979, 57, 1024-1026. (13) Schaeffer, D. J.; Kerster, H. W.; Janardan, K. G., Environ. Manage. ( N . Y . )1980, 4 , 157-163. (14) Barkley, J. J. "Water Pollution Sampler Evaluation"; Army Medical Bloengineering Research and Development Laboratory: Fort Detrich, MD, 1975.
(15) Lin, P. C. L. "Thermal Analysis of the ISCO 1680 Portable Wastewater Sampler", EPA-600/4-80-033; U.S. Environmental Protection Agency: 1980. (16) Chian, E. S.K.; DeWalle, F. B. "Analytical Methods for Priority Pollutants in Municipal Sewage and Sludge", Water Pollution Control Federation: 1979; Abstracts 52nd Annual Conference of the Water Pollution Control Federation Oct 7-12, Session 25. (17) DeWalle, F. B.; Kalman, D. A.; Perera, C.; Chian, E. S. K. "Priority Pollutant Removal Efficiencies in POTW's as Related to their PhysicalChemical Properties", Water Pollution Control Federation: 1979; Abstracts 52nd Annual Conference of the Water Pollution Control Federation Oct 7-12, Session 8. (18) DeWalle, F.; Chian, E. "Presence of Priority Pollutants in Sewage and Their Removal in Sewage Treatment Plants First Annual Report to U S . Environmental Protection Agency", University of Washington: Seattle, WA, 1979. (19) Janardan, K. G.; Schaeffer, D. J.; Sornani, S.M. Bull. Envlron. Contamin. roxicol. 1980, 24, 145-151. (20) Tigwell, D. C.; Schaeffer, D. J. Patent Application, 1979. (21) Thurman, E. M.; Malcolm, R. L.; Aiken, G. R. Anal. Chem. 1979, 51, 1799-1803. (22) Schaeffer, D. J.; Glave, W. B.; Somani, S. M.; Janardan, K. G. Bull. Environ. Contamln. Toxlcol. 1980, 25, 569-573. (23) May, W. E.; Wasik, S. P.; Freeman, D. H. Anal. Chem. 1978, 50, 997-1000.
RECEIVED for review February 14, 1980. Resubmitted February 17, 1981. Accepted April 15, 1981.
Trace Level Determination of Selected Nitroaromatic Compounds by Gas Chromatography with Pyrolysis/Chemiluminescent Detection Arthur L. Lafleur"' and Kevin M. Mills Therm0 Electron Corporation, Analytical Instruments Department, 125 Second A venue, Waitham, Massachusetts 02254
A novel method for the trace level identification and determination of nttroaromatic compounds is described. It consists of a gas chromatograph coupled with a nitric oxide selective pyroiysis/chemilumlnescence detector (TEA Analyzer). The method gives linear response over 4 orders of magnitude and precision of 11% or better (relative standard deviation, N = 10) at the picomole level. Compounds studied include the nitrotoiuenes, five dinitrotoiuenes, and 2,4,6-trinitrotoluene. Pyrolyzer temperatures above 800 OC were required to produce optimum yields of nitric oxide from the compounds studied.
Recently, it was demonstrated that the coupling of a high-performance liquid chromatograph (HPLC) with a nitric oxide selective pyrolysis/chemiluminescence detector (TEA Analyzer) yielded a selective and sensitive method for the detection and determination of explosives and related compounds at trace levels (1-4). The types of explosive compounds studied were limited to nitrate esters and nitramines, although, in theory, any compound capable of releasing NO upon pyrolysis will produce a response. The detector is principally used for the determination of nitrosamines in a wide variety of matrices because of the property of most N-nitrosamines to liberate NO quantitatively when pyrolyzed. The advantages of the TEA Analyzer for the analysis of Present address: Massachusetts Institute of Technology, 77 Massachusetts Av., Cambridge, MA 02139.
N-nitrosamines in complex matrices have been comprehensively reviewed (5). However, when aromatic nitro compounds were investigated by using the HPLC/TEA approach, it was found that under conditions ideal for the determination of nitrosamines, nitrate esters, and nitramines, the nitroaromatics gave a relatively poor molar response (2,6). Because nitroaromatic compounds are very prominent in terms of commerce, national security, and environmental toxicology, a study was initiated to determine the feasibility of the selective determination of these compounds using some other approach that would still incorporate the advantages of highly selective NO-specific detection. The development of analytical methodology focused on the nitrotoluenes because they are among the foremost of the many nitroaromatics produced in commercial quantities. Moreover, because there is a large body of data showing the speed and simplicity of gas chromatography (GC) for their analytical separation (7), this technique was also used in the present investigation.
EXPERIMENTAL SECTION Experimental Conditions and Apparatus. Identical systems were used for determining TEA Analyzer response as a function of pyrolyzer temperature and for obtaining optimum gas chromatographic separation of the nitrated toluene derivatives: The gas chromatograph used was a Model 5840A (Hewlett-Packard Corp., Palo Alto, CA). It had a glass column, 1.8 m in length, 2.0 mm inside diameter, packed with 3% OV-225 on Chromosorb W-HP 100/120 mesh. The carrier gas was argon flowing at a rate of 30 mL/min. The injection temperature was 240 "C. The column temperature was programmed from 100 to 240 O C at 8 OC/min.
0003-2700/81/0353-1202$01.25/00 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
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T
OZONE
l
SAMPLE INJECT I ON
=-
PYROLYSIS 800'- 900' C
'
HEATED INTERFACE
VACUUM
--
I
FILTER
COLD TRAP
CHROMATOQRAPH
II
-130°C
H V . SUPPLY AMPLIFIER
ELECTRONICS
RECORDER
Figure 1. Instrumental configuration.
The detector was a TEA Model 543 Analyzer (Thermo Electron Corp., Analytical Instruments, Waltham, MA). The interface temperature was 250 "C and the pyrolyzer temperature was 900 "C. The ozonator used air at a flow rate of 15 mL/min. The chamber pressure was 1.0 torr, and the cold trap temperature was -78 "C. The ozonator used air at a flow rate of 15 mL/min. The chamber pressure was 0.7 torr and the cold trap temperature was -78 "c. For determination of molar response, the same gas chromatograph was used (Model 58406); however, a less polar column was used to obtain lower elution temperatures for a given compound with the intention of minimizing any possible decomposition on the column. The column was a 1.8 m by 2.0 mm i.d. glass column packed with 3% UCW-98 on Chromosorb W-HP 80/100 mesh. The carrier gas was argon at a flow rate of 45 mL/min. The column temperature was 160 "C (isothermal),and the injector temperature was maintained at 200 "C. The detector was also the same in this case (TEA Model 543 Analyzer). The interface temperature was maintained at 250 "C, and the pyrolyzer was held at 900 "C. The chamber pressure was 1.0 torr, and the cold trap temperature was -78 "C. The ozonator used air at a flow rate of 15 mL/min. To obtain the calibration curves in Figure 4, we used the following system. The gas chromatograph was a Model 5720A (Hewlett-Packard Corp., Palo Alto, CA). It had a glass column 1.8 m in length by 2.0 mm i.d. packed with 3% UCW-98 on Chromosorb W-HP 80/100 mesh. The carrier gas was argon at a flow rate of 40 mL/min. The injector temperature was maintained at 200 "C. The instrument was operated isothermally as follows: For 2-nitrotoluene, the temperature was 120 "C; for 2,4-dinitrotoluene, it was 175 "C; and for 2,4,64rinitrotoluene, it was 200 "C. The detector used was a TEA Model 502A Analyzer with Explosives Analysis Package (Thermo Electron Corp., Analytical Instruments, Waltham, MA). The pyrolyzer temperature was held at 800 "C, and the interface temperature was maintained at the temperature of the column. The chamber pressure was 1.7 torr, and the cold trap temperature was -90 "C. The ozonator used oxygen at a flow rate of 10 mL/min. Chemicals. Two nitrotoluene isomers (ortho, para) were obtained from Eastman Kodak Corp., Rochester, NY; m-nitrotoluene, 2,4dinitrotoluene, and 2,4,6-trinitrot~luenewere obtained from ChemService, West Chester, PA. The remaining dinitrotoluene isomers (2,3-;2,5-; 2,6-; 3,4-) were obtained from Aldrich Chemical Co., Milwaukee, WI. The solvent used for standard solutions was Distilled-in-Glass grade acetone obtained from Burdick and Jackson Laboratories, Muskegon, MI. The Calibration gas used was nominally 100 ppm nitric oxide in nitrogen. Two separate standards were used: a 103 ppm mixture supplied by Matheson Gas Co., East Rutherford, NJ, and a 104 ppm mixture supplied by Scientific Gas Products, Inc., South Plainfield, NJ.
RESULTS AND DISCUSSION The basic instrumental configuration used is shown in Figure 1. Samples of interest are injected onto the GC column where they separate into their components. Each component subsequently passes through a heated interface and then into a pyrolyzer where appropriate compounds decompose to liberate NO and other pyrolysis products. These pyrolysis products are removed from the gas stream through the use of either a cold trap or a solid sorbent device. The NO then passes into a reaction chamber where i t is allowed to react with ozone to produce electronically excited nitrogen dioxide (NO2*). The NO2* decays to its ground state with the characteristic emission of light. The light is detected with a suitable photomultiplier. The filter is used to block possible light emission resulting from sources other than NO chemiluminescence. A detailed description of the detector and its principle of operation can be found in the literature (8, 9). The first step in evaluating the pyrolysis/chemiluminscence method for detecting and determining nitrotoluenes was to ascertain whether it was possible to obtain conditions where a molar or nearly molar yield of nitric oxide could be derived from these compounds. It is well-known that most explosives containing the nitrosyl moiety liberate nitric oxide when standing at storage temperatures for extended periods or when heated to elevated temperatures for shorter periods of time. Although much data are available on the thermal decomposition and release of NO in nitrate esters such as methyl nitrate (IO), nitroglycerine ( I I ) , and pentaerythritol tetranitrate (PETN) ( B )little , data are available concerning pyrolysis of nitroaromatic compounds. Therefore, a study of pyrolytic release of nitric oxide as a function of temperature was undertaken. The results are shown in Figure 2. It can be seen that the maximum response occws at temperatures above 800 "C and that the response will be diminished by almost 3 orders of magnitude at temperatures below 500 "C. Although results are shown for only five nitrotoluenes, all of the nine compounds shown chromatographed in Figure 3 were tested. The others were omitted for the sake of clarity because several compounds produced overlapping response curves. For example, 3,4-dinitrotoluene produced nearly the same response curve as 2,3-dinitrotoluepe (2 in Figure 2); similarly, 2,5- and 2,4-dinitrotoluene produced nearly identical results and in turn matched closely those of 2,6-dinitrotoluene (3 in Figure 2). Other compounds producing similar results were 3-nitrotoluene and 4-nitrotoluene. TOobtain analytical results in the most efficient manner, it was decided to investigate the gas chromatographic sepa-
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
Table I. Determination of Molar Response areab response re1 molar toluene derivative amounta injected raw data (SY (~,)d molare response responsef 2-nitro 5x 3.15 x 104 4.61 x l o 2 1.4 6.3 x 1 0 1 3 0.59 3-nitro 5x 5.33 x 104 7.36 x lo2 1.4 1.06 X 10l4 1.00 4-nitro 5 x 10-1° 4-66 x 1 0 4 5.44 x 102 1.2 9.1 x 1013 0.86 2,6-dinitro 5x 7.33 x 104 1.31 x 103 1.8 1.46 X 10l4 1.38 2,5-dinitro 5 x 10-'0 7.72 x 104 1.82 x 103 2.4 0.54 x 1014 1.45 2,4-dinitro 5 x 10-'0 8.29 x 104 1.33 x 103 1.6 1.65 x 10l4 1.56 2,3-dini tro 5 x 10-'0 9.31 x 1 0 4 1.17 x 103 1.3 1.86 x 1014 1.75 3,4-dinitro 5 x 10-'0 9.90 x 104 8.71 x l o 2 0.9 1.98 x 1014 1.87 2,4,6-trinitro 5 x 10-1° 1.20 x 105 3.27 x 103 2.7 2.39 x 1014 2.25 nitric oxide 2.30 X lo-'' 2.48 X l o 4 8.85 x l o 2 3.6 1.06 x 10l4 1.00 a Moles. Arbitrary units. Standard deviation, N = 5. Relative standard deviation expressed as a percentage. e Molar response = raw area response/mol. f Relative molar response = molar response of compound/molar response of NO,
-
I
I
I
J
500 550 600 650 700 750 800 850 900 PYROLYZER TEMPERATURE
PC)
Flgure 2. Plot of response vs. pyrolyzer temperature for (1) 2,4,6trinitrotoluene, (2) 2,3dinitrotoluene, (3) 2,6dinRrotoluene, (4) 3-nitrotoluene, and (5) 2-nitrotoluene obtained with the TEA Model 543 Analyzer.
ration of nitrated toluene derivatives using packing materials of varying polarity and selectivity with the aim of obtaining acceptable separation of the largest number of compounds. After several possibilities were obtained from the literature (7,131and investigated, it was decided that OV-225 possessed the required properties for this use. The separation obtained using this liquid phase is shown in Figure 3. Glass columns 1.8 m long and designed for on-column injection were used because it was desired to eliminate all possible causes of sample loss. With columns of such length, it was not possible to separate 2,4-dinitrotoluene (6 in Figure 3) from 2,3-dinitrotoluene (7 in Figure 3) with other liquid phases having lower polarity than OV-225, although the search for such a phase was not exhaustive. Exact details concerning actual composition of the packing material and other pertinent data are found in the Experimental Section. When it is not necessary to obtain complete separation of all the compounds discussed in this study, a nonpolar column material such as those based on methylsilicones could be used. Therefore, a UCW-98 liquid phase was used in the molar response studies and in obtaining calibration data. This phase was selected so that the lowest practical temperatures for the elution of trinitrotoluene and the more labile dinitrotoluenes could be obtained, thereby reducing the possibility of sample loss by thermal decomposition to an absolute minimum. After the data shown in Figure 2 were obtained, it was possible to select an optimum temperature for molar response studies. To ensure maximum thermal decomposition and thus maximum release of nitric oxide, we selected a temperature of 900 OC. The data are tabulated in Table I. The major significance of the data is as follows: The relative response
Flgure 3. GC-TEA chromatogram for a mixture of nitrated toluene derivatives: (1) 2-nitrotoluene, (2)3-nitrotoluene,(3)4-nltrotoluene, (4) 2,6dlnitrotoluene, (5) 2,5dinitrotoluene, (6) 2,4dinitrotoluene, (7) 2,3dinitrotoluene, (8) 3,4dinitrotoluene,(9) 2,4,6-trinitrotoluene.
Table 11. Precision of Determination nitrotoluene responsea derivative concn, mg/L 3.43 x l o 2 2-nitro 0.137 3.66 X l o 3 1.370 5.06 X l o 4 13.700 4.64 X l o 5 137.000 2,4-dinitro 0.0182 1.23 X l o 2 1.14 X l o 3 0.182 1.42 X l o 4 1.82 1.77 X l o 5 18.20 2,4,6-trinitro 0.0227 1.00 x l o 2 0.227 1.32 x 103 2.21 X l o 4 2.27 2.84 X lo5 22.70
(x)
(SIb 0.12 0.13 0.15 0.07 0.13
(%IC
3.6 3.6 2.9 2.0 11.0 0.04 4.0 0.10 7.0 0.11 6.2 0.10 10.0 0.32 9.3 0.05 2.3 0.08 2.8
a (K) ;mean value for ten determinations, arbitrary area units. (S) = standard deviation. (S,) = relative standard deviation expressed as a percent.
for all of the compounds was highly reproducible and the release of NO was therefore also reproducible but not necessarily molar; that is, one nitro group did not necessarily yield one molecule of nitric oxide. An important factor in the
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Anal. Chem. 1981, 53, 1205-1208
Results are shown for 2-nitrotoluene (MNT), 2,4-dinitrotoluene (DNT), and 2,4,6-trinitrotoluene (TNT). The data were obtained from a series of experiments designed to give the precision of determination for the nitrotoluenes using a standard, commercially available detector (TEA Model 502A Analyzer-see Experimental Section), the simple integrator provided with it, and manual injection into an isothermally operated gas chromatograph. This is the simplest practical instrumental arrangement that can be used to obtain quantitative results. The experimental data are tabulated in Table 11. Ten determinations were obtained for each concentration level. The volume injected was 10 pL in all cases. The precision of the results range from 11% relative standard deviation for 2,4-dinitrotoluene at the picomole level (0.0182 mg/L) to 2% for 2-nitrotoluene a t the 10 nmol level (137 mg/L). When the data were plotted as in Figure 4, a linear regression analysis yielded correlation coefficients of greater than 0.999 for each of the three compounds.
e
0
;i
LITERATURE CITED
I
\
8 12 TIME (MINUTES)
4
(1) Fan, T. Y.; Ross, R.; Fine, D. H.; Keith, L. H.; Garrison, A. W. €nviron. Sci. Technoi. 1978, 12,692-695. (2) Lafleur, A. L.; Morriseau, B. D.; Fine, D. H. “Proceedings, New Con-
L 16
Figure 4. Plot of detector response vs. moles injected for GC-TEA detection. Substances tested were 2-nitrotoluene (MNT) 2,4dinitrotoluene (DNT), and 2,4,6-trinitrotoluene (TNT).
determination of molar response is the accuracy of the response for a given quantity of nitric oxide gas. It has been found that calibrated gas samples from different sources may vary in composition and that the choice of materials in handling the gas is critical. Therefore, although it is possible to obtain reproducible results to sometimes better than 1 or 2% relative standard deviation if the conditions are favorable, the absolute accuracy is suspected to be poorer than the precision. With this in mind, it can be seen that the relative response between different nitrotoluenes will be more accurate than their absolute response referred to nitric oxide, although the precision of either result will be approximately the same. Figure 4 shows a plot of detector response vs. quantity injected into the gas chromatograph expressed in moles.
cepts Symposium and Workshop on Detection and Identification of VA. ExDlosives”, National Technical Information Service: Springfield, . 1978;pp 597-598. (3) Lafleur, A. L.; Morriseau, B. D. Anal. Chem. 1960, 52, 1313-1318. (4) Spanggord, R. J.; Keck, R. G. J . Pharm. Sci. 1980, 69, 444-446. (5) Preussman, R.; Castegnaro, M.; Walker, E. A,; Wassermann, A. E. Environmental Carcinogens: Selected Methods of Analysis; Vol. l., Analysis of Volatile Nitrosamines In Food”; International Agency for Research on Cancer: Lvon. 1978. (6) Fan, T. Y.; Vita, R.; Fine, D.’ H. Toxicol. Left. 1978, 2, 5-10. (7) Yinon, J. Crk. Rev. Anal. Chem. 1977, 7(4), 1-35. (8) Fine, D. H.; Lieb, D.; Rufeh, F. J . Chromatogr. 1975, 107. 351. (9) Fine, D. H.; Rufeh, F.; Lieb, D.; Rounbehler, D. P. Anal. Chem. 1975,
47, 1188. (IO) Urbanski, T. “Chemistry and Technology of Explosives”; Pergamon Press: New York, 1965;Vol. 2, Chapter 6. (11) Reference 10,p 52. (12) Kaye, S. M. “Encyclopedia of Explosives and Related Terms”; U.S. Army Armament Research and Development Command: Dover, NJ, 1978;Vol. 8,p P86. (13) Krauss, S . A.; Glattstein, B.; Landau, D.; Almoz, J. “Proceedings, New Concepts Symposium and Workshop on Detection and Identification of Explosives”; National Technical Information Service: Sprlngfieid, VA, 1978;pp 873-676.
RECEIVED for review October 31, 1980. Accepted March 18, 1981.
Analysis for Nitrosourea Antitumor Agents by Gas Chromatography-Mass Spectrometry Ronald G. Smith,” Silas C. Blackstock, Lily K. Cheung, and Ti Li Loo The University of Texas System Cancer Center M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030
An assay for nltrosoureas applicable to the antitumor agents BCNU, CCNU, and MeCCNU has been developed by using the gas chromatography-mass spectrometric technlque of selected Ion monltorlng. The analysis follows derlvatlration by trlfluoroacetlc anhydride, which converts the 1,3-dialkyl-lnltrosoureas to 1,3-dlacyl-l,t-dlalkylureas. The lower limits for quantifying these drugs in plasma are 1-3 ng/mL. Appllcatlon Is made to determine the rates of decomposition for the three nltrosoureas In plasma and to determine the plasma clearance of MeCCNU In a dog.
Three established antitumor agents, 1,3-bis(2-chloro-
ethyl)-1-nitrosourea (BCNU), l-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1-(2-chloroethy1)-3-trans4-methylcyclohexyl)-l-nitrosourea(MeCCNU), Figure 1, are active against certain neoplastic diseases in man (1-3). Their physical and chemical properties allow them to penetrate the blood-brain barrier ( 4 , 5 ) and are thus especially useful in the pretreatment of tumors of the central nervous system (6, 7). The activity of these agents apparently comes from their rapid nonenzymatic decomposition to unstable intermediates capable of DNA alkylation (8-10). Despite the clinical usefulness of nitrosoureas for over 15 years, relatively little is known about their disposition and metabolism in vivo. Methods previously applied to the
0003-2700/81/0353-1205$01.25/0 0 1981 American Chemical Society
