1884
Anal. Chem. 1085, 57, 1864-1868
Liquid Chromatographic Determination of Low Molecular Weight Alkylthiols in Air via Derivatization with 7-Chloro-4-nitro-2,1,3-benzoxadiazole Yoshinori Nishikawa* and K a z u h i r o K u w a t a Environmental Pollution Control Center, 62-3, 1 Chome, Nakamichi, Higashinari-ku, Osaka City 537,J a p a n
Traces of C1-C4 alkylthiols In air were collected by using a SepPAK Florisll cartridge Impregnated with 7-chioro-4-nitro2,1,3-benzoxadlazole, and the adsorbed substances were eluted with methanol. The derivatives of the thiols produced were detemlndd by reversed-phase hlgh-performance liquid chromatbgraphy. The C1-C4 thiol vapors except for 2methyl-2-propanethioi could be determlned with 0.7-2.8% relative standard deviation in the range 1.3-6.7 nmoi/L in air sample. The detection limits of the thiols were 3-139 pmol/L for 100 L of air sample. The thiol vapors from pollution sources could be successfully determlned.
Low-molecular-weight aliphatic thiols are important substances as odors,flavors, or metabolites in environmental, food, and biological studies. As the thiols may be reactive and decompose with time, it is preferable that the thiols are derivatized to a stable state and analyzed for the derivatization products with increased sensitivity. In the previous work ( I ) , a high-performance liquid chromatographic (I-PLC) technique was used to determine traces of C1-C7 alkylthiols via derivatization with 5,5’-dithiobis(2-nitrobenzoicacid) (Ellman’s reagent). In the method, the thiols in vapor phase should be absorbed in aqueous medium by bubbling them and derivatized with the reagent for the HPLC analysis. The absorption efficiency of the thiol vapors was however somewhat poor so that two fritted bubblers with the sampling solution in series were required to sample the thiols from air. Besides, the sampling system was inconvenient to sample simultaneously a number of samples in field works. 7-Fluoro-4-nitro-2,1,3-benzoxadiazole and 7-chloro-4nitro-2,1,3-benzoxadiazole(NBD-Cl), which are known as a fluorescence labeling agents for amino groups (2-7), have been used to determine sensitive thiol groups in biological samples (8-12). When a thiol compound is allowed to react with NBD-CI in a weakly alkaline organic medium, a NBD derivative is formed RS-H
/ \
+ NO,
Thiol
NED-C1
O & f
NO,
Fluorescent Product
The reaction has been found to be rapid. However, there are few published methods for determination of traces of alkylthiols in environmental samples by HPLC via the derivatization with NBD-C1. On the other hand, convenient cartridges such as Sep-PAK Cu (SP-18) have been used to sample traces of aliphatic aldehydes (13) and amines ( 7 , 1 4 ) in air samples with consid0003-2700/85/0357-1864$01.50/0
erable savings of time and labor. However, no such cartridges have so far been used to sample alkylthiols in air sample. In this paper, a Sep-PAK Florisil (SP-F) cartridge impregnated with NBD-C1 is presented to sample C1-CI alkylthiols in air or gas samples. The thiols trapped on the cartridge were removed by treatment with methanol and derivatized with NBD-C1 by catalytic effects of the Florisil. The derivatives of the thiols produced were determined by HPLC. The method resulted in a simple and easy determination of traces of the alkylthiols in air and gas samples. EXPERIMENTAL SECTION Reagents and Materials. Methanethiol standard solution (at 1 pg/pL in benzene) sealed in a 2-mL ampule and Cz-C4 alkylthiols used as standards were of special grade from Wako (Osaka, Japan). 7-Chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and other reagents used were special grade from Wako and Tokyo Kasei (Tokyo, Japan). Methanol, acetonitrile, and water were of liquid chromatographic grade. Sep-PAK CIS(SP-18) cartridge and Sep-PAK Florisil (SP-F) cartridge were from Waters Associates (Milford, MA). The stock solutions containing 500 Mmol/mL of the Cz-C4 thiols were made with methanol. Standards of lower concentrations were made by appropriately diluting the stock solutions with methanol. Apparatus. A Hewlett-Packard (Avondale,PA) 1084B liquid chromatograph with an absorbance detector adjusted to 425 nm and a Waters Associates ALC/GPC 244 liquid chromatograph equipped with a U6K injector and a Shimazu (Kyoto, Japan) RF-530 fluorescence detector adjusted to 425 nm of excitation maximum (Aex) and 510 nm of emission maximum (Aem) were employed. The analytical column used was a 10 cm X 4.0 mm i.d. stainless steel tube packed with Develosil ODs-3 (3 pm) (Nomura Kagaku, Aichi, Japan). The column temperature was 30 OC. The mobile phase was 45/55 acetonitrile/water and the flow rate was 1.0 mL/min. Preparation of Sampling Tube. A SP-F cartridge was washed with 5 mL of diethyl ether. A 3-mL volume of 20 pmol/mL NBD-Cl in diethyl ether was forced through the cartridge. The cartridge was dried for 30 min under reduced pressure in a nitrogen stream and then by passing pure nitrogen at 100 mL/min for 30 min. The cartridge was closed with glass stoppers, sealed in a vial, and stored in a cool place in the dark until use. A SP-18 cartridge coated with phosphoric acid, which excluded basic compounds and other interfering substances in sample, was made as previously described (7, 14). Generation and Trapping of Standard Thiol Vapors. Figure 1 shows a simple device to generate thiol vapors for calibration or examination of the analytical method. Nitrogen gas was introduced into the T-shaped glass tube at 0.1-0.2 L/min from inlet A and diluting gas (pure air or nitrogen) was passed at 0.8-0.9 L/min from inlet B. A SP-F cartridge was attached to the exit of the tube. A 2-10 pL volume of a thiol standard solution was injected onto the quartz wool in the heated zone (120 OC) by using a microsyringe. The vaporized thiols were moved with the stream of the carriers and trapped with the coated SP-F cartridge. Calibration. Amounts of 40-200 nmol of C1-CI alkylthiols in a standard solution were vaporized and trapped for 10 min on the coated SP-F at 1.0 L/min of nitrogen stream (Figure I). The adsorbed substances on the cartridge were eluted with methanol 0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985 1865 1
3
4
5 Corrier gos (N 2 ) 0.1 -O.ZL/rnin
7
Table 11. Formation of the NBD Derivatives with Time in Eluate from the Coated SP-F Cartridge thiol
B Dilution gos ( a i r o r N z ) 0.8-0.9L/rnin
methanethiol ethanethiol 2-propanethiol 1-propanethiol 2-methyl-2-propanethiol 2-butanethiol 2-methyl-1-propanethiol 1-butanethiol
Figure 1. Generation and trapping of standard thiol vapors: (1)injector nut, (2) septum, (3)T-shaped glass tube, (4) quartz wool, (5)heating block (120 "C), (6) nut, (7) SP-F cartridge.
Table I. Formation of the NBD Derivatives with Time in a Methanol Medium Suspended with Florisil Particles thiol
yield," % 5min 10min 15min 60min
yield,' % 5min 10min 15min 60min
66 76 70
84 92 87 92 67 84 89 90
71
44 69 74 74
90 96 98 99 100 93 98 95
100 100 100 100 100 100
100 100
The percent yield was fluorescence intensity relative to that in 60 rnin of reaction time. ~~
methanethiol ethanethiol 2-propanethiol 1-propanethiol 2-methyl-2-propanethiol 2-butanethiol 2-methyl-1-propanethiol 1-butanqthiol
61 70 64 72
42 63 68 70
81 89 86 91 75 88 91 90
85 93 89 95 71
90 94 94
100 100 100 100 100 100 100 100
"he reaction occurred in methanol medium in which 50 mg/ mL of Florisil was suspended. The perceht yield was fluorescence intensity relative to that in 60 min of reaction time. in the direction contrary to that of the sampling, and an initial 2-mL volume of eluate was collected. The eluate was allowed to stand for 15 min or more at room temperature. Then, a 2-10-rL aliquot of the eluate was analyzed by HPLC. Calibrations were made with the amounts of the thiols against their peak heights in absorbance or fluorescence intensity. Sampling and Analytical Procedure. A 1-100-L volume of air sample was sampled at 0.8-1.2 L/min with the SP-18 cartridge and the SP-F cartridge in series. For cigarette smoke, a 30-mL volume of the smoke was puffed for 2 s at every 30-5 interval (15). The smoke was filtered with a Toyo Roshi (Tokyo, Japan) GB-100R glass fiber filter to stop the particulates. A 120-mL volume of gas phase of smoke from four puffs was sampled with the SP-F cartridge. The adsorbed substances on the SP-F cartridge were analyzed in the same way as described in the calibration. The thiols were identified by retention time and quantified by peak height. The blank test was made with a coated SP-F cartridge. The cartridge was eluted with 2 mL of methanol and the eluate was analyzed in a similar way.
Table 111. Detection Limits, Extinction Coefficients, and Relative Fluorescence for the NBD Derivatives of the Alkylthiols
thiol
absorbance fluorescent detection detection DL,' 6425,b L DL," re1 pmol mol-' cm-l pmol fluorescencec
3.1 methanethiol 3.6 ethanethiol 5.6 2-propanethiol 1-propanethiol 5.5 2-methyl-2-propanethiol 12.1 2-butanethiol 8.5 2-methyl-1-propanethiol 8.7 9.6 1-butanethiol
10 780 12 760 12 300
14 190 7 120 13500 14 440 13850
1.6 3.8 5.9 3.3 69.0 5.5 3.9 5.3
1.00 0.43 0.28 0.51 0.02 0.30 0.43 0.32
aDetection limits (a response to twice the noise levels). Extinction coefficients (td25) of each derivative were calculated from the separate peak in the chromatogram as follows: 64426 = (Ab/L,,)(W1$/M), where Ah was absorbance in peak height, L, was path length of the cell (1.0 cm), M was amount of the thiol injected (mol), W,,, was peak width at half-height (min), and R was flow rate of the mobile phase (0.001 L/min). cFluorescence intensity relative to that of the methanethiol derivative.
40 T
J!
I
RESULTS AND DISCUSSION The C1-C4 alkylthiols except for 2-methyl-2-propanethiol were quantitatively derivatized with NBD-C1 in a methanol medium containing 0.5-2.5 kmol/mL potassium hydroxide. The quantitative reaction of 2-methyl-2-propanethiol with NBD-C1 occurred in the presence of 1.5-2.5 pmol/mL potassium hydroxide in methanol. The thiols were also quantitatively derivatized with NBD-C1 in a methanol medium suspended with more than 30 mg/mL of Florisil. Table I reports the percent yield of the NBD derivatives with time in a methanol medium with 50 mg/mL Florisil particles suspended. In these cases, magnesium concentration, determined by atomic absorption spectrometry after filtration of the reaction medium with a Fluoropore filter (Sumitomo, Osaka, Japan), was 0.15 ccg/mL in the methanol solution. These catalytic effects may be due to magnesium oxide in Florisil (which consists of 16% magnesium oxide and 84% silica geU because the reaction did not completely proceed in the presence of silica gel only. On the other hand, &lo0 nmol amounts of the thiol vapors were trapped with the coated SP-F cartridge, and t h e adsorbed substances were eluted with methanol. The eluate was analyzed by HPLC at 5,10,15, and 60 rnin after the elution was finished. Table I1 reports the
W 0
5 20 0
v,
2 300 4 00 WAVELENGTH (nm)
200
500
600
Figure 2. Absorption spectra and fluorescence spectra of the reaction products: (2) ethanethiol derivative, (4) 1-propanethiol derlvative, (8) 1-butanethiol derivative, (0)NBDCI; absorption spectrum (-), emisslon spectrum (- -).
-
formation aspects of the NBD derivatives in the eluate. The absorbance or the fluorescence intensity for the thiol derivatives almost reached the maximum in 15 rnin and was at the same level as those obtained from the derivatization in the methanol medium suspended with Florisil. The magnesium contents were a t 0.15-0.20 pg/mL levels in the eluates. The magnesium oxide eluted from the SP-F cartridge gave apparently catalytic effect8 on the formation of the NBD derivatives. Thus, no basic substances were used as catalysts
1866
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
Table IV. Retention Times and Analytical Precision of the NBD Derivatives
retention time. min a P f std devb RSD,"%
thiol methanethiol ethanethiol 2-propanethiol 1-propanethiol 2-methyl-2-propanethiol 2-butanethiol 2-methyl-1-propanethiol 1-butanethiol
4.19 f 0.006 6.66 f 0.008 10.38 f 0.011 11.16 f 0.013
13.46 f 0.021 16.77 f 0.029 17.98 f 0.044 19.49 f 0.040
amt of thiol, pmol
0.14 0.12 0.11 0.12 0.15 0.17 0.25
62.5 125 125 125 125 125 125 125
0.21
analytical precision absorbance, x10-3 a P f std devC 3.97 f 0.068 6.87 f 0.133 4.46 f 0.098
4.58 f 0.077 2.06 f 0.040 2.94 f 0.049
2.87 f 0.055 2.60 f 0.032
RSD," % 1.72 1.93 2.21 1.68
1.97 1.67 1.94 1.22
"Average in 13 runs. Standard deviation. cRelativestandard deviation. U
Table V. Recovery of the NBD Derivatives of Thiols from the SP-FCartridge
thiol
I
1
I
0 5 10 15 20 RETENTION TIME ( m i n 1 Figure 3. Typlcal liquid chromatogram of the NBD derivatives of the alkylthiols in the absorbance detectlon: amounts of thiols, 62.5-125 pmol; mobile phase, 45/55 acetonltrlle/water; flow rate, 1.0 mL/mln; (1) methanethlol, (2) ethanethiol, (3)2-propanethlol, (41 I-propanethlol, (5)2-methyC2-propanethio1, (6) 2-butanethiol, (7) 2-methyl-I-propanethiol, (8) I-butanethiol, (0) NBD-CI.
other than Florisil in the derivatization. Figure 2 shows the absorbance spectra and the emmision spectra of typical alkylthiol derivatives. The maximum absorbances were obtained at 425 nm, and the exciting maxima A,, and the emission maxima A, were at 425 nm and 510 nm, respectively. Table I11 reports the detection limits (a response to twice the noise level), the extinction coefficients a t 425 nm, and the relative fluorescence at A,, 425 nm and A,, 510 nm for the C1-CI alkylthiols. The detection limits of the thiols except for 2-methyl-2-propanethiol were 1.69.6 pmol and were a t the same levels both in the absorbance detection and in the fluorescent detection. The detection limit of 2-methyl2-propanethiol was 12.1 pmol in the absorbance detection and 69.0 pmol in the fluorescent detection. The extinction coefficients of the thiol except for 2-methyl-Bpropanethiol were larger than those of the 5,5'-dithiobis(2-nitrobenzoic acid) derivatives (8480-9000 L mol-' cm-l) earlier reported (1). These aspects may indicate that a reasonable sensitivity is obtained by use of NBD-C1 for ultratrace determination of the alkylthiols. The NBD derivatives of the thiols were very stable in the presence of excess NBD-C1 in methanol. The derivatives are stable for 3 days at room temperature without a protective cover against light. When the sample solutions were stored a t 3 OC in the dark, the derivatives were stable for a month. Thus, standards after derivatization could be usable for analysis over a long period. HPLC separation of the NBD derivatives of the C1-Cd alkylthiols was investigated by changing the ratio of solvent components in the mobile phase on a Develosil ODs-3 (3 pm) column (10 cm X 4.0 i.d. mm). Excellent separations were obtained for the derivatives in the mobile phase of 45/55 acetonitrile/water. Figure 3 shows a typical liquid chroma-
methanethiol ethanethiol 2-propanethiol 1-propanethiol 2-methyl-2-propanethiol 2-butanethiol 2-methyl-1-propanethiol 1-butanethiol
NBD derivatives of thiol amt amcfoundb injected," f std dev," nmol nmol 40 100
34.7 f 2.68 92.8 f 4.26
recovery, %
87 93
100
94.9 f 3.87
95
100 100 100 100 100
104.1 f 1.48 97.6 f 13.9
104
103.7 f 1.84
104
104.5 f 1.50 104.4 f 1.34
105
98 104
A 4-pL volume of thiol standard solution was injected by using the apparatus in Figure 1. bAverage in five runs. 'Standard deviation. togram of the NBD derivatives where the column offered more than loo00 theoretical plates. Table IV reports the retention times and analytical precision of these derivatives. The same-day relative standard deviation of the retention times was less than 0.25%. The thiols were determined at 62.5-125 pmol levels with 1.22-2.21 % relative standard deviation by the HPLC technique. The derivatives were completely eluted with less than 1.5 mL of the eluent from the SP-F cartridge, and no thiol derivatives were detected in the later eluate. Accordingly, 2 mL of the eluate was collected for the analysis. The collection efficiency was investigated by using two coated SP-F cartridges in series. Amounts of 200 nmol of the thiols were vaporized and 100 L of air was passed a t 1.5 L/min through the cartridge. The thiols were recovered on the first cartridge only and no thiols were detected on the second. The breakthrough volumes of the thiols were estimated to be more than 100 L. One cartridge may be thus useful to sample the thiol vapors. The recovery test was conducted by vaporizing 40-100 nmol of the thiols, trapping them on the cartridge, and passing 30 L of air through the cartridge. Table V reports that a satisfactory recovery was obtained for the thiol vapors at the trace levels. In complex air samples from anaerobic pollution sources, there may be basic compounds such as ammonia and alkylamines and other organic substances that could exhaust NBD-C1 on the SP-F cartridge during the sampling or produce ghost peaks in the HPLC analysis. T o remove such compounds, a SP-18cartridge coated with phosphoric acid, which was used to sample alkylamines in the earlier reports (7,141, was connected before the coated SP-F cartridge in series. Sampling of an air sample was conducted by use of the two cartridges in series. The C1-CI alkylthiols were recovered on the SP-F cartridge only while no thiols were detected on the SP-18 cartridge. Table VI reports that small effects of the
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
1887
Table VI. Effect of the SP-18 Cartridge on Collection of the Thiol Vapors in the Sampling System
amt of thiol injected, nmol
thiol
40
methanethiol
80
100 200 100 2-propanethiol 200 100 1-propanethiol 200 100 2-methyl-2-propane200 thiol 100 2-butanethiol 200 100 2-methvl-l-~ro~ane- thioi 200 1-butanethiol 100 200 ethanethiol
fluorescence intensity (SP-18+ SP-Fb SP-F)c 74 151 96.5 196 57 113
120.5 244 6 12 70.5 144 96.5 198 71.5 146.5
74.5 145 92.5 186 56.5 108 123.5 245 6.5 13 71 146 97.5 196 71 144.5
(Sp.18 + SP-F)/ SP-F, % 100.6 96.0 95.9 94.9 99.1 95.6 102.4 100.2 108.3 108.3 100.7 101.3 101.0 98.6 99.3 98.6
Average peak height in two runs, arbitary units. b A SP-F cartridge only was used. e A SP-F cartridge attached with a sp-18 cartridge at the inlet was used.
SP-18 cartridge were observed on the collection of the thiol vapors in the sampling system. As C1-C4 alkylamines trapped on the first cartridge could be determined by the earlier the sampling system may be useful for colmethods (7,14), lection of both the alkylthiols and the alkylamhes. Interfering organic compounds were not present in most cases because few organic peaks that overlapped with those of the NBD derivatives were found in the absorbance detection at 425 nm or in the fluorescent detection at X,425 nm and X,510 nm. Table VI1 reports the analytical precision and the calibration data of the C1-C4 alkylthiols at 0.2-1.0 nmol levels through the analytical procedure. No blank peaks were observed in the chromatogram. The alkylthiols except for 2-
0
4
O
8
L
8
RETENTION TIME ( M I N I
Flgure 4. Analysis of thiol vapors from polluted sources (fluorometric detector): (A) emission gas from a night sdl container, sample vdume 69 L, (1) mthamthbl (120 pmol) 347 pmolll; (6)gas from a sdubllizer In a night soil treatment plant, sample volume 3 L, (1) methanethiol(l25 pmol) 8.33nmollL; (C) gas phase of tobacco smoke (Japan HOPE; 60 mm X 8 mm cigarette, 10 mm X 8 mm filter), (1) methanethiol (380 pmol) 2.6 Nmollcigarette.
methyl-2-propanethiolwere determined with 0.7-2.8 9% relative standard deviation in the range 1.3-6.7 nmol/L in air samples where excellent linearity was obtained in calibration. The analytical Precision for 2-methyl-2-propanethiolwas somewhat poor compared to those of the other thiols, but was within a permissible limit. The estimated detection limits of these thiols in vapor phase were 3-139 pmol/L for 100 L of air sample without concentration of the eluates. Alkylthiols in emission gases from night soil treatment plants and in tobacco smoke were determined. Figure 4 shows typical liquid chromatograms of the NDB derivatives from the sources. Methanethiol was detected a t picomole-perliter levels with several unknown peaks. The proposed HPLC method may have advantages over conventional methods in terms of rapid and simple procedure, high analytical sensitivity, and low background effects. Determination of the alkylthiols by use of the HPLC technique could probably be applicable to many other types of samples after an appropriate modification.
Table VII. Analytical Precision and Calibration Data of the Thiols
thiol methanethiol
amt of thiol, nmol
1-propanethiol 2-methyl-2-propanethiol
0.5 1.0
130 f 2.6 247 f 1.7
2.0 0.7
84 f 2.1 163 f 2.3
2.5 1.4
150 f 2.6
1.7
292 f 3.6
1.2
0
0.5 1.0 0
0
0.5 2-methyl-1-propanethiol
2.4 0.7
0
0.5 1.0 2-butanethiol
127 f 3.0 229 i 1.6
0
0.5 1.0 2-propanethiol
RSD,d %
*
0
0.2 0.4 ethanethiol
analytical precision fluorometric intenso avb std de?
1.0 0
0.5 1.0 0
7.2 f 0.75 14 f 1.0
10.3 7.2
90 2.5 175 f 2.7
*
2.8
*
1.9
127 2.4 247 f 2.0
1.5 0.8
regression linee and corr coefg
Y = 563X + 4.0 r = 0.997 Y = 247X + 2.2 r = 0.999 Y = 163X + 1.0 r = 0.999
Y = 292X + 1.5
r = 0.999
Y = 14X + 0.06
r = 0.991
Y = 175X r = 0.999
+ 0.9
Y = 247X + 1.0 r = 0.999
Y = 183X + 0.8 94 2.2 2.4 r = 0.999 1.0 183 f 1.7 0.9 a Peak height, arbitary units. bAveragein four runs. Standard deviation. dRelative standard deviation. ‘X = amount of thiol, nmol; Y = fluorometric intensity. fThe number of samples was 12. 1-butanethiol
0.5
*
1868
Anal. Chem. 1985, 57, 1868-1871
Registry No. NBD-Cl, 10199-89-0; methanethiol, 74-93-1; ethanethiol, 75-08-1; 2-propanethiol, 75-33-2; 1-propanethiol, 107-03-9; 2-methyl-2-propanethiol,75-66-1; 2-buhethio1, 513-53-1; 2-methyl-1-propanethiol, 513-44-0; 1-butanethiol, 109-79-5.
LITERATURE CITED (1) Kuwata, K.; Uebori, M.; Yamada, K.; Yarnazaki, Y. Anal. Cbem. 1082, 54, 1082-1087. (2) Watanabe, Y.; Imai, K. Anal. Blocbem. 1081, 116, 471-472, (3) Toyooka, T.; Watanabe, Y.; Imai, K. Anal. Cblm. Acta 1083, 149, 305-312. (4) Wolfram, J. H.; Feinberg, J. I.; Doerr, R. C.; Fiddler, W. J. ChromatOgr. 1077, 132, 37-43. (5) Kroi. G. J.; Banovsky, J. M.; Mannan, C. A.; Pickering, R. E.; Kho, B. T. J . CbrOmatOgr. 1070, 163, 383-389.
(6)
Ahnoff, M.; Grundevik, I.; Arfwidsson, A.; Fonseiius, J.; Persson, B.
(7)
Anal. Cbem. 1081, 53, 485-489. Nishikawa, Y.; Kuwata, K. Anal. Cbem. 1084, 56, 1790-1793.
(8) (9)
Watanabe. Y.; Imai, K. Anal. Cbem. 1083, 55, 1786-1791. Whitehouse, M. W.; Ghosh, P. B. Blocbem. Pbarmacol. 1088. 158-161.
17,
(10) Birk&, D, J.; Price, N. C.; Rad&, (3, K.; Salmon,A, G, FEBS ~ ~ 1070, 6 , 346-348. (11) Stuchbury, T.; Shipton, M.; Norris, R.; Malthouse, J. P. G.; Brockiehurst, K.; Herbert, J. A. L.; Suschitzky, H. Biocbem. J. 1075, 151, 417-432. (12) Shipton, M.; Stuchbury, T.; Brocklehurst, K. Biochem. J . 1078, 159, 235-244. (13) Kuwata, K.; Ueborl, M.; Yamasaki, H.; Kuge, Y.; Kiso, Y. Anal. Cbem. 1083, 55, 2013-2016. (14) Kuwata, K.; Akiyama, E.; Yamazaki, Y.; Yamasakl, H.; Kuge, Y.; Kiso, Y. Anal. Cbem. 1983, 55, 2199-2201. (15) Evans, D. J.; Mayfield, R. J. Analyst (London) 1075, 100, 540-543.
RECEIVED for review January 14, 1985. Accepted April 19, 1985.
Determination of Aminodibenzothiophenes in a Coal Liquid Masaharu Nishioka, Robert M. Campbell, W. Raymond West, Philip A. Smith, Gary M. Booth, and Milton L. Lee* Departments of Chemistry and Zoology, Brigham Young University, Prouo, Utah 84602
Hirotaka Kudo and Raymond N. Castle Department of Chemistry, University of South Florida, Tampa, Florida 33620
Polycycllc aromatic compounds contalnlng both nltrogen and sulfur heteroatoms In a coal llquld (SRC I1 HD) were determined by caplllary column gas chromatography (GC) and gas chromatography/mase spectrometry (GCIMS). Gas chromatography wHh a flame photometrlc detector and a 25% biphenyl polyslioxane statlonary phase was applied to the nltrogen-contalnlng polycyclic aromatic compound fraction. Ail amlnodibenzothlopheneisomers were posltlvely ldentlfled by comparison wlth the retentlon times of newly syntheslzed standard compounds. The amlnodlbenzothlophenes were the maJor nltrogen/sulfur-containIng heterocycles in the SRC I I HD. The four amlnodlbenzothlophenes were assayed for gsnotoxktty by two methods: the Ames test and unscheduled DNA synthesls. The 2- and 8-amlnodlbenzothlophenesdemonstrated slgnlflcant genotoxlcltles In both assays.
Coal-derived liquids are extremely complex mixtures of organic chemicals, the majority of which are polycyclic aromatic compounds (PAC). While the polycyclic aromatic hydrocarbons (PAH) comprise the largest chemical class fraction of most coal liquids, there are substantial amounts of nitrogen-, sulfur-, and oxygen-containing PAC. All of these fractions have been extensively characterized using a variety of techniques (1-3) including gas chromatography (GC), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). The major objective for much of the previous work on detailed characterization of coal liquids, and the main motivation for this work, was to provide the necessary base line data for proper evaluation of the health effects of synthetic fuel production from coal. In this respect, chemical class separation of coal-derived products, followed by microbial mutagenicity and mouse skin tumorigenicity assays revealed that the PAH were mainly responsible for the observed tumorigenicity ( 4 , 5 ) ,while the nitrogen-containing PAC contributed to the overall tumorigenic activity and were the 0003-2700/85/0357-1868$01.50/0
principal microbial mutagens (3). While the tumorigenicity of the sulfur heterocycles (PASH) is presently unknown, the PASH were found generally to be more toxic and to bioconcentrate to a greater extent in various aquatic organisms than their analogous PAH (6, 7). After fractionation of the nitrogen-containing PAC fraction, it was found that the amino polycyclic aromatic hydrocarbons (APAH), which are present in low concentrations in coal liquids (usually less than l % ) , were responsible for the major part of the mutagenicity demonstrated by the nitrogen-containing PAC (8-11). The nitrogen heterocycles were generally much less mutagenic than the APAH. This result has prompted the investigation of other possible trace components in coal liquids, particularly the PAC that contain two heteroatoms, that may be responsible for some of the observed biological activity. In this paper, the analysis of a solvent-refined coal liquid for PAC which contain both nitrogen and sulfur heteroatoms is described. Capillary column gas chromatography with sulfur-selective flame photometric detection and mass spectrometry was used to identify several new compounds which were previously unreported. Pure reference compounds were also synthesized and tested for genotoxicity.
EXPERIMENTAL SECTION Materials. The solvent-refined coal heavy distillate (SRC I1 HD: 260-450 "C boiling point range) was collected during the processing of a West Virginia coal from the Pittsburgh Seam, and obtained from the Fort Lewis, WA, pilot plant which was operated by the Pittsburgh & Midway Coal Mining Co. This material is of pilot plant origin and should not necessarily be considered as representative of products that may eventually be produced on a commercial scale. The four aminodibenzothiophene isomers used in this study were not commercially available and, therefore, had to be synthesized in our own laboratories. The synthetic procedures are reported elsewhere (12). Fractionation and Derivatization Procedures. The SRC I1 HD material was fractionated into chemical classes by adsorption chromatography on neutral alumina and silicic acid 0 1985 American Chemlcal Society
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