Determination of Low Levels of Dimethylcarbamoyl Chloride in Air George M. Rusch,* Stephen L. LaMendola, Gary V. Katz, and Sidney Laskin New York University Medical Center, Institute of Environmental Medicine, 550 First Avenue, New York, N. Y. 10016
The reaction of dimethylcarbamoyl chloride with 4-(p-nitrobenzyl)pyrldine,a general reagent for alkylating and acylating agents, produces a bright red 1,4-dihydropyridlne product. Through spectroscopic determlnations of the concentration of this product in samples taken from air, levels as low as 17 parts per billion of dimethylcarbamoyl chloride have been detected quantitatively.
Dimethylcarbamoyl chloride (DMCC) has been reported to be carcinogenic in mice by skin painting and subcutaneous injection ( I ) , Recently, inhalation studies with rats have demonstrated that this material is an extremely potent carcinogen ( 2 ) .Chemically, DMCC is a derivative of carbamic acid and is related to the urethanes, a class of compounds whose carcinogenic activity has been known for many years ( 3 , 4 ) .DMCC has had, and may be considered for future, industrial applications in the manufacture of herbicides, pesticides, anthelmintics, and specialized pharmaceuticals. Interest in this material is suggested by some 50 patents issued for its use in 1975 alone. The procedure described below involves the reaction of DMCC with 4-(p-nitrobenzyl)pyridine(NBP) and subsequent determination of the concentration of the derivative formed using a simple spectrophotometer. This method is capable of determining DMCC concentrations in air as low as 17 parts per billion. The reagent NBP was chosen because of its wide application in the determination of concentrations of alkylating and acylating agents (5-8). In general, the alkylating or acylating agent attacks the nitrogen of the pyridine ring thereby forming a pyridine salt. Addition of a base, such as sodium carbonate, diethylamine, or piperidine, to this salt results in the loss of one of the benzylic protons and rearrangement of the double bonds to give a 1,4-dihydropyridine. As a result of the extended conjugation in the dihydropyridines, a red shift occurs in the absorption spectra and they become highly colored.
EXPERIMENTAL
One ml of this solution wasthen added to acetone in a second 100-ml volumetric flask which was brought to volume. This yielded a final standard with a concentration of 117 pg/ml. The dilute standard was prepared in the same manner starting with 0.10 ml of DMCC. This yielded a standard solution of 11.7 pg/ml. Various amounts of the standard solutions (from 0.10 to 1.20 ml) were added to 10 ml of the reagent solution in a large test tube. The solution was then evaporated in a boiling water bath until only a small red residue remained at the bottom (about 10 min). The residue was then dissolved in a small amount of acetone, transferred to a 5- or 10-ml volumetric flask and brought to volume. The absorbancy of the resulting orange to red solution was measured on a spectrophotometer a t 475 nm. This color is stable for several hours. The range of concentrations prepared in this manner was from 0.12 to 14.0 pg/ml in the final reagent solution. A plot of optical density vs. concentration was linear up to a concentration of 5.8 pg/ml of DMCC with a slope of 0.143 and a zero intercept. From 5 pg/ml up to 10 pg/ml, a deviation from linearity was observed; however, the concentrations of samples could be determined directly from the standard curve. Concentrations above 10 pg/ml could not accurately be measured by this method. Air Flaw
1
Pump
Charcoal Trap
Figure 1. Sample collection assembly
Instrumentation. The initial visible spectra were measured on a Perkin-Elmer Model 402 UV-Visible Spectrophotometer, all subsequent spectra were measured on a Coleman Jr. Model 620 visible spectrophotometer. The GC/MS spectra were obtained using a Varian MAT CH4 mass spectrometer interfaced with a Hewlett-Packard Model 5710A gas chromatograph using a Watson-Biemann type two-stage separator. A Hewlett-Packard Model 5750 Gas Chromatograph with a flame ionization detector was used in the gas chromatographic analysis. Chemicals. 4-(p-Nitrobenzyl)pyridine,98% pure, was obtained from Aldrich Chemical Company, Milwaukee, Wis. 53233. Acetone, reagent grade, was obtained from J. T. Baker Chemical Company, 222 Red School Lane, Phillipsburg, N.J. 08865. Dimethylcarbamoyl chloride, practical grade, was obtained from M.C.B. Manufacturing Chemists, 2909 Highland Avenue, Norwood, Ohio 45212. Preparation of Derivative Reagent. NBP, 2.5 g, was dissolved in 250 ml of acetone. The reagent is stable for 14 days. Calibration. Two standard solutions of DMCC were prepared in acetone. For the concentrated standard, 1.00 ml(l.17 g) of DMCC was added to acetone in a 100-ml volumetric flask and brought to volume.
Table I. Mass Spectrum of Dimethylcarbamoyl Chloride Relative Fragment
m/e
percent
C3H6NOC137
109
c3H6Noc135 CNOC137 CNOCP5
107
59 96
79 77 72 65 63 66 51 49 44 43 42
18 100 10 31 29 7 22 24 22 49
C3H6NO
coc137 coc135 C2H2NO CH&137 CHZCI~~ C2H6N C2H5N C2H4N or CNO
6
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Table 11. Determination of Air Concentrations of Dimethylcarbamoyl C h l o r i d e Volume of air sampled,
Final volume of test soln, ml
1. 20 20 20 15 15 15 5 5 5 5 5 5 5
Concn of DMCC in test soln, d m l ,
OD of test s o h
5 5 5
0.040 0.062 0.080
0.29 0.45
5
0.100
25
0.046 0.072 0.069 0.118 0.093
0.72 0.33 0.52 0.495 0.846 0.667 0.774 0.867 1.376 2.61
25 10 10 25 25 25 25 25
Concn of DMCC in air sample, ppba
17 26 34 56 128 202 230 393 776 900
0.58
0.108 0.121 0.192 0.366
1000 1600 3040
For DMCC, 1.0 p p b is equivalent t o 4.5 gg/m3 a t 20 "C a n d standard pressure.
CH3 \
0
N-C-CIIt
+
-
/
CH3
N > C H ~ ~ N O ~
Table 111. Replicate D e t e r m i n a t i o n s of the C o n c e n t r a t i o n of Dimethylcarbamoyl Chloride with 4-(p-Nitrobenzyl) pyridine
I
Sample
No. 1 2 3 4 5
Optical density 0.60 0.62 0.63 0.60 0.61
Sample
No. 6 7
8 9 10
Optical density 0.62 0.60 0.60 0.62 0.63
Table IV. Replicate D e t e r m i n a t i o n s of the C o n c e n t r a t i o n of Dimethylcarbamoyl Chloride with 4-(p-Nitrobenzyl) pyridine in t h e Presence of A i r Figure 2. Reaction of dimethylcarbamoyl chloride with 4-(pnitrobenzy1)pyridine
Air Level Monitoring. A DMCC test atmosphere was generated in a dynamic 1.0 m3 inhalation chamber (9) by passing a controlled air stream over the liquid DMCC in a sealed flask, and subsequently blending this air stream with the chamber intake air. For sampling, 10 ml of the reagent solution was placed in a gas bubbler and air samples varying from 2.5 to 20 1. were drawn through the bubbler a t a rate of 1 l./min usfng a vacuum pump (Figure 1).Although this procedure was demonstrated to be highly efficient, residual traces of DMCC were removed by passing the effluent gas from the bubbler through a 6-14 mesh activated coconut charcoal trap. Sample volumes were measured using a Model 63126 Precision wet test meter. When the desired volume had been collected, the sample was removed from the bubbler. The bubbler was washed with a small amount of acetone, which was then added to the original solution. The resulting solutions were treated as described above for the calibration. Chromatographic Conditions. The column used was 4 feet long, constructed of YB-in. stainless steel tubing and packed with 60/80 mesh Chromosorb W coated with 10% by weight UC-W98. The flow rate of the pre-purified helium carrier gas was 30 ml/min and for the hydrocarbon free air was 340 ml/min. The temperature of the injection port was 150 "C and that of the detector was 155 "C. The oven was operated isothermally at 80 "C. Under these conditions DMCC had a retention time of 6 min and tetramethylurea (TMU) had a retention time of 14 min. Gas Chromatography-Mass Spectrometry. The gas chromatographic column was the same as that described above. The flow rate of the pre-purified helium carrier gas, measured at ambient pressure with the separator vacuum pumps off, was 30 ml/min. The temperatures of the various components of the GC/MS system were: oven temperature 120 "C; injection port 150 "C; transfer line to separator 150 "C; separator 180 "C; and, transfer line to mass spectrometer 190 2260
Sample No.
Optical density
1
0.59 0.62 0.59 0.63 0.62
2 3 4 5
Sample
No. 6
7 8 9 10
Optical density 0.62 0.62 0.63 0.62 0.62
"C. Under these conditions, DMCC had a retention time of 1.1 min and TMU of 2.0 min. The mass spectrometric conditions were: source temperature 210 "C, accelerating voltage 2.9 kV;ionizing voltage 70 eV; filament current 40 PA; and resolving power 1500. During GC/MS runs, the parent peaks for DMCC - 107 (C3H,jNOCP5) and 109 (C3HeNOC137) and for TMU - 116 (CjH12N20) were monitored by scanning from 104 to 118 mass units. With the exception of the molecular ion peaks, the major peaks for DMCC and TMU were the same. The first bond cleaved in DMCC is the carbon-chlorine bond and in TMU it is one of the carbon-nitrogen bonds. Both yield the same fragment (C3HsNO). The mass spectrum of a sample of DMCC was determined with the mass spectrometer conditions described above. The major fragments and their assignments are given in Table I.
RESULTS AND DISCUSSION The reaction of DMCC with NBP occurs with spontaneous rearrangement t o give a bright red product, &(p-nitrobenzy1idine)-N-(dimethylcarbamoyl)-1,4-dihydropyridine as shown in Figure 2. T h e product h a s a n absorption maximum at 475 nm with
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
Table V. Efficiency of Sample Collection Assembly
OD of soln in first sampler (Total vol. 25 ml)
DMCC in first sampler,
a
OD of soln in second sampler (Total vol. 10 ml)
0.366 0.322 0.332 0.342 0.348
65.25 57.50 59.25 61.25 62.00
0.005 0.018 0.019 0.008 0.013
a molar extinction coefficient of 15 000. Neither DMCC nor NBP absorb in this region. The absorption follows Beer’s law over a concentration range of 0.12 to 5.8 wg/ml of DMCC. If a 20-1. air sample was taken and the final product was taken up in 5 ml df acetone, theoretically an air concentration of 8 parts per billion (ppb) could be accurately determined. Tests on chamber atmospheres demonstrated that levels from 17 ppb up to 3 ppm could easily be measured (Table 11).As there are fluctuations in the stability of the chamber concentrations a t these levels, the precision of the method during actual sampling was not determined. Typical variations observed for a series of 12 analyses taken during a 6-h period were less than 10%. The precision of the analytical method was determined by running a series of 10 replicate samples prepared from a standard 4.25 Fg/ml DMCC solution in acetone. The average optical density for the standard solution was 0.61 f 0.03 ( p = 0.05) (Table 111). The effect of drawing air through the test solution was determined by pulling 10-1. room air samples through a duplicate series of 10 replicate samples. The average optical density for this standard solution was 0.62 f 0.03 ( p = 0.5) (Table IV). This would indicate that the results from the air sampling procedure are directly comparable to those obtained from standard solutions. The efficiency of the collection system was determined by mounting two collectors in tandem and determining the DMCC concentration in each. In all cases, better than 97% of the material was collected in the first sampler (Table V). Since it had previously been reported that DMCC reacts slowly with NBP (7), studies were made to demonstrate that the material reacting with the NBP was, in fact, DMCC. Samples of DMCC were analyzed by flame ionization GC and by GC/MS. They were found to be over 95% DMCC with TMU as the only discernible contaminant. Furthermore, during a GC/MS analysis, a portion of the pure DMCC column effluent was reacted with NBP. This was accomplished by modifying the GC/MS interface. A valve manifold was connected to the GC/MS transfer line. When the MS spectrum indicated that pure DMCC was eluting from the column, this
DMCC in second sampler, fig
Percent in sam p1er
0.32 1.25 1.32 0.54 0.89 Mean
99.5 97.9 97.8 99.1 98.6 98.6 srl-1 0.7 2.0 95% (Student t dist.)
effluent was diverted to a bubbler containing NBP reagent in acetone. Upon workup, this product had the same absorption characteristics as previous DMCC products. An attempt was made to prepare a derivative from TMU and NBP. No product was obtained under conditions where the DMCC readily yielded a bright red product. Subsequent characterization of the reaction product of DMCC with NBP by elemental analysis and nuclear magnetic resonance spectroscopy has further confirmed the assignment of the structure given in Figure 2 above (IO).
ACKNOWLEDGMENT The authors thank the following who participated in one or more phases of this work: Joshua Gurman, G. Roger Sparling, Fredrick Blanchard, Donna Westhafer, and Dorothy Natalizio. The authors also thank the Dow Chemical Company, Midland, Mich., for the donation of the Varian MAT CH4 mass spectrometer used in these studies.
LITERATURE CITED B. L. VanDuuren, B. M. Goldschmidt, C. Katz, and I. Seidman, J. Nat. Cancer lnst., 48, 1539 (1972). S. Laskin, G. M. Rusch, G. Katz, A. Sellakumar, and M. Kuschner, A.I.H.A. Abstr., p 87, May 1976. B. K. J. Leong. H. N. Macfariand, and W. H. Reese, Arch. Environ. Health, 22, 663 (1971). S. S.Mirvish, Adv. Cancer Res., 11, 1 (1968). J. Epstein. R. W. Rosenthal, and R. J. Ess, Anal. Chem., 27, 1435 (1955). R. Preussmann, H. Schneider, acd F. Epple. Arzneim.-Forsch., 19, 1059 (1969). A. M. Agree and R. L. Meeker, Talanta, 13, 1151 (1966). E. Boyland and W. H. Down, Eur. J. Cancer, 7 , 495 (1971). R. T. Drew and S. Laskin, “Methods of Animal Experimentation”, Academic Press, New York, 1973, pp 1-47. B. M. Goldschmidt, B. L. Van Duuren, and R. C. Goldstein, J. Heterocycl. Chem., 13, 517 (1976).
RECEIVEDfor review June 23,1976. Accepted September 7, 1976. This investigation is supported by Contract No. NO1 CP 33260 from the National Cancer Institute and is part of a center program supported by Grant No. ES00260 from the National Institutes of Environmental Health Sciences and Grant No. CA 13343 from the National Cancer Institute.
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