Journal of Analytical Toxicology, Vol. 16, March/April 1992
Measurementof EthylenethioureaUsingThermospray Liquid Chromatography-MassSpectrometry Piiivi Kurttio, Terttu Vartiainen, and Kai S a v o l a i n e n *
National Public Health Institute, Department of Environmental Hygiene and Toxicology, P.O.B. 95, SF-70701, Kuopio, Finland S e p p o Auriola
University of Kuopio, Department of Pharmaceutical Chemistry, P.O.B. 6, SF-70211, Kuopio, Finland
,
i Abstract
Experimental
i
Reliable measurement of ethylenethiourea (ETU) is important because ETU is a potent thyroid carcinogen. A method for the separation and identification of ethylenethiourea (ETU) by applying reversed-phase high-pressure liquid chromatography (HPLC) followed by thermospray (TSP) mass spectrometry (MS) detection is described9 Single ion recording detection applying HPLC-MS of ETU appeared to be highly selective and equally sensitive as an HPLC method applying UV detection reported in our earlier study (1). The detection limit for ETU was 100 pg per injection9
Introduction Ethylenethiourea (ETU) is a toxic byproduct of manufacturing and degradation of ethylenebisdithiocarbamate (EBDC) fungicides. ETU as such is widely used in the rubber industry as an accelerator. The most significant toxic effect of ETU is its potential to produce thyroid carcinomas at low doses in rodents (2). Thus, reliable identification of ETU is important. ETU has been detected in air and in urine samples by gas-liquid chromatography (GC) with electron capture detection (ECD), flame ionization detection (FID), or nitrogen-phosphorus selective detection (3,4). Several other methods for analysis of ETU apply high-pressure liquid chromatography (HPLC) with ultraviolet (UV), polarographic, or electrochemical detection (1,5,6). Reversed-phase column separation appears to be suitable for the analysis of ETU because the compound is soluble in water. Some reports that apply mass spectrometric (MS) detection without chromatography of ETU have been published (4,7). Derivatized ETU has also been analyzed by MS and GC/MS techniques (8,9). The thermospray HPLC-MS technique offers a sensitive and selective method for the analysis of polar and nonvolatile compounds, such as pesticides, without derivatization (10,11). In this study the suitability of reversed-phase HPLC with thermospray MS detection was evaluated for the analysis of ETU.
9Author to whom correspondence should be addressed
Chemicals and sample preparation. The purity of N,N'ethylenethiourea was greater than 98% (Fluka AG, Switzerland). Methanol and dichloromethane were HPLC grade. The urine samples were prepared as described earlier (1). Briefly, a urine sample (10 mL) was evaporated to dryness, after which the sample was transferred with methanol and silica gel on an aluminum oxide column and eluted with 2% methanol in dichloromethane. The eluate was evaporated to dryness, and the sample was dissolved in water and filtered. HPLC conditions. The HPLC system consisted of an LC Perkin-Elmer Model 601 pump and a Rheodyne 7125 injector with a 20-~L loop. A N u c l e o s i l Cj8 (250 x 8 • 4 mm, Chrompack) reversed-phase HPLC column was used. The isocratic elution was carried out with 5% methanol in 0.05M ammonium acetate (pH 6.5) with a flow rate of 1.3 mL/min. Mass spectrometry. The HPLC-MS system used was a VG t h e r m o s p r a y - p l a s m a s p r a y probe coupled to a VG Trio-2 quadrupole mass spectrometer (Manchester, UK). The instrument was in the TSP mode. The instrument parameters were as follows: source temperature 200~ repeller voltage 80 V, TSP capillary temperature 211~ multiplier 800 V. The TSP mass spectrum of ETU was obtained by injecting I0 ng of standard via the column. The instrument was scanned from m/z 90 to m/z 180. Determination of ETU was based on selected ion recording (SIR) of the protonated molecule ion at m/z 103. A four-point calibration curve was created for the range of 0.1 to 10 ng of ETU per injection.
Results a n d D i s c u s s i o n The thermospray mass spectrum of ETU exhibits a protonated molecule ion (M+H) + at m/z 103 as the base peak (Figure 1). The ion at m/z 162 (M+60) + may correspond to loss of water from an adduct of the molecule with protonated ammonium acetate (12) or it may be the adduct of ETU with acetic acid (13). The identity of the ion at m/z 121 (M+I9) + is not known, but it may correspond to an adduct of the molecule with protonated water (M+H30) +.
Reproduction (photocopying) of editorial content of this joumal is prohibited without publisher's permission.
85
Journal of Analytical Toxicology,Vol. 16, March/April 1992
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TIME Fioure 1. (a) Thermospray mass spectrum of the ethylenethiourea peak obtained after reversed-phase HPLC column separation; (b) thermospray mass spectrum of the urine sample of a potato field applicator exposed to ethylenethiourea (see Figure 2c). Identification of the ETU peak after HPLC can be supported by the UV-absorbance profile of the compound and by the retention time, as in our previous study ( I ). In the present work, the identity of the peak was confirmed by TSP HPLC-MS. The mass spectrum and chromatogram of ETU in a urine sample obtained from a potato applicator exposed to EBDC fungicides and ETU are in good agreement with the standard of ETU (Figures la, lb, 2a, and 2c). The ions at m/z 96, 97, and 119 originated from the background. The SIR chromatogram of the protonated molecule ion obtained from blank urine sample shows no interfering peaks at the corresponding retention time (Figure 2b). The detection limit of the present HPLC-MS method was 0.1 ng of ETU/injection, which equals the limit of detection of the HPLC-UV method reported earlier ( 1). The current results are in agreement with the HPLC-UV method. The linearity of the detection of ETU with the present HPLC-MS method was calculated and appeared to be excellent, between 0.1 and 10 ng of ETU/injection: 3' = bx + a, where v = peak area, b = constant (25), x = rig/injection, and a = y-intercept (-6) (re = 0.998). The detection limit of the GC methods for ETU (3,4) is not sensitive enough for detecting ETU in biological samples. HPLC methods reported (1,5,6) can be used for the analysis of ETU in several types of samples. However, none of the previous methods with sufficient sensitivity (1) provide totally reliable identifica-
86
(min)
Fioure 2. HPLC-MS selected ion recording (m/z 103) detection of ethylenethiourea (a) in water standard (0.22 mg/L), (b) in blank urine, (c) in urine sample of a potato field applicator exposed to ethylenethiourea. The concentration of ethy]enethiourea was 2.6 pg/L. The chromatograms were normalized to the highest peak in each chromatogram tion of ETU, Therefore, the major improvement of the present HPLC-MS method is that it is both sensitive and reliable for detecting ETU in biological samples. To our knowledge, this is also the first paper reporting HPLC-MS confirmation of the identity of ETU in biological samples. The drawbacks of the present method are that it is laborious to use and requires expensive equipment. The HPLC-MS method described in this work for detection of low concentrations of ETU is sensitive enough to be used for the analysis of ETU in occupational air and in urine during and after occupational exposure to EBDCs or ETU (14-16). This method may also prove valuable for detecting trace amounts of ETU in water and food samples, inflmnation that would be crucial for purposes such as setting of tolerances and acceptable daily intake values in different food items.
Acknowledgments The authors wish to thank Mr. Tuomo Korhonen and Mr. Jukka Knuutinen for skillful technical assistance. This work was financially supported by The Finnish Work Environment Fund and the North-Savo Cultural Foundation.
Journal of AnalyticalToxicology,Vol. 16, March/April1992
References 1. P. Kurttio, T. Vartiainen, and K. Savolainen. A high pressure liquid chromatographic method for the determination of ethylenethiourea in urine and on filters. Anal Chim. Acta 212: 297-301 (1988). 2. World Health Organization. (WHO Environmental Health Criteria 78). Dithiocarbamate pesticides, ethylenethiourea, and propylenethiourea: A general introduction. World Health Organization, Geneva, 1988. 3. W.H. Newsome. Determination of ethylenethiourea residues in apples. J. Agric. Food Chem. 20:967-69 (1972). 4. T. Hirvi, H. Pyysalo, and K. Savolainen. A glass capillary gasliquid chromatography method for determining ethylenethiourea without derivation. J. Agric. Food Chem. 27:194-95 (1979). 5. H.B. Hanekamp, P. Bos, and R.W. Frei. Design and selective application of a dropping mercury electrodeamperometric detector in column liquid chromatography. J. Chromatogr. 186:489-96 (1979). 6. J.R Lawrence and F. Iverson. Liquid chromatography with UV absorbance and polarographic detection of ethylenethiourea and related sulfur compounds. J. Chromatogr. 212:245-50 (1981). 7. W.R. Bontoyan, J.B. Looker, T.E. Kaires, P. Giang, and B.M. Olive. Survey of ethylenethiourea in commercial ethylenebisdithiocarbamate formulations. J. Assoc. Off. Anal Chem. 55: 923-25 (1972). 8. K. Autio. Ethylenethiourea: Metabolism, analysis and aspects of toxicity. Technical Research Centre of Finland, Research Reports 91, Espoo, Finland, 1982. 9. J. Singh, W.P. Cochrane, and J. Scott. Extractive acylation of
10.
11.
12.
13.
14.
15.
16.
ethylenethiourea from water, Bull. Environ. Contam. ToxicoL 23: 470-74 (1979). T.R. Covey, J.B. Crowther, E.A. Dewey, and J.D. Henion. Thermospray liquid chromatograph/mass spectrometry determination of drugs and their metabolites in biological fluids. Anal. Chem. 57:474-81 (1985). R.D. Voyksner and C.A. Haney. Optimization and application of thermospray high-performance liquid chromatography/mass spectrometry. Anal Chem. 57:991-96 (1985). R.W. Smith, C.E. Parker, D.M. Johnson, and MM. Bursey. Eluent pH and thermospray mass spectra: Does the charge on the ion in solution influence the mass spectrum? J. Chromatogr. 394: 261-70 (1987). D. Barcel6. Effect of ammonium formate as ionizing additive in thermospray liquid chromatography-mass spectrometry for the determination of triazine, phenylurea and chlorinated phenoxyacetic acid herbicides. Organic Mass Spectrom. 24:219-24 (1989). K. Savolainen, P. Kurttio, T. Vartiainen, and J. Kangas. Ethylenethiourea as an indicator of exposure to ethylenebisdithiocarbamate fungicides. Arch. ToxicoL Suppl. 13:120-23 (1989). P. Kurttio, T. Vartiainen, and K. Savolainen. Environmental and biological monitoring of exposure to ethylenebisdithiocarbamate fungicides and ethylenethiourea. Br. J. Ind. Med. 47:203-206 (1990). P. Kurttio and K. Savolainen. Ethylenethiourea in air and in urine: implications to exposure to ethylenebisdithiocarbamate fungicides. Scand. J. Work Environ. Health 16:203-207 (1990). Manuscript received July 6, 1990; revision received October 11, 1990.
87
Measurementof EthylenethioureaUsingThermospray Liquid Chromatography-MassSpectrometry Piiivi Kurttio, Terttu Vartiainen, and Kai S a v o l a i n e n *
National Public Health Institute, Department of Environmental Hygiene and Toxicology, P.O.B. 95, SF-70701, Kuopio, Finland S e p p o Auriola
University of Kuopio, Department of Pharmaceutical Chemistry, P.O.B. 6, SF-70211, Kuopio, Finland
,
i Abstract
Experimental
i
Reliable measurement of ethylenethiourea (ETU) is important because ETU is a potent thyroid carcinogen. A method for the separation and identification of ethylenethiourea (ETU) by applying reversed-phase high-pressure liquid chromatography (HPLC) followed by thermospray (TSP) mass spectrometry (MS) detection is described9 Single ion recording detection applying HPLC-MS of ETU appeared to be highly selective and equally sensitive as an HPLC method applying UV detection reported in our earlier study (1). The detection limit for ETU was 100 pg per injection9
Introduction Ethylenethiourea (ETU) is a toxic byproduct of manufacturing and degradation of ethylenebisdithiocarbamate (EBDC) fungicides. ETU as such is widely used in the rubber industry as an accelerator. The most significant toxic effect of ETU is its potential to produce thyroid carcinomas at low doses in rodents (2). Thus, reliable identification of ETU is important. ETU has been detected in air and in urine samples by gas-liquid chromatography (GC) with electron capture detection (ECD), flame ionization detection (FID), or nitrogen-phosphorus selective detection (3,4). Several other methods for analysis of ETU apply high-pressure liquid chromatography (HPLC) with ultraviolet (UV), polarographic, or electrochemical detection (1,5,6). Reversed-phase column separation appears to be suitable for the analysis of ETU because the compound is soluble in water. Some reports that apply mass spectrometric (MS) detection without chromatography of ETU have been published (4,7). Derivatized ETU has also been analyzed by MS and GC/MS techniques (8,9). The thermospray HPLC-MS technique offers a sensitive and selective method for the analysis of polar and nonvolatile compounds, such as pesticides, without derivatization (10,11). In this study the suitability of reversed-phase HPLC with thermospray MS detection was evaluated for the analysis of ETU.
9Author to whom correspondence should be addressed
Chemicals and sample preparation. The purity of N,N'ethylenethiourea was greater than 98% (Fluka AG, Switzerland). Methanol and dichloromethane were HPLC grade. The urine samples were prepared as described earlier (1). Briefly, a urine sample (10 mL) was evaporated to dryness, after which the sample was transferred with methanol and silica gel on an aluminum oxide column and eluted with 2% methanol in dichloromethane. The eluate was evaporated to dryness, and the sample was dissolved in water and filtered. HPLC conditions. The HPLC system consisted of an LC Perkin-Elmer Model 601 pump and a Rheodyne 7125 injector with a 20-~L loop. A N u c l e o s i l Cj8 (250 x 8 • 4 mm, Chrompack) reversed-phase HPLC column was used. The isocratic elution was carried out with 5% methanol in 0.05M ammonium acetate (pH 6.5) with a flow rate of 1.3 mL/min. Mass spectrometry. The HPLC-MS system used was a VG t h e r m o s p r a y - p l a s m a s p r a y probe coupled to a VG Trio-2 quadrupole mass spectrometer (Manchester, UK). The instrument was in the TSP mode. The instrument parameters were as follows: source temperature 200~ repeller voltage 80 V, TSP capillary temperature 211~ multiplier 800 V. The TSP mass spectrum of ETU was obtained by injecting I0 ng of standard via the column. The instrument was scanned from m/z 90 to m/z 180. Determination of ETU was based on selected ion recording (SIR) of the protonated molecule ion at m/z 103. A four-point calibration curve was created for the range of 0.1 to 10 ng of ETU per injection.
Results a n d D i s c u s s i o n The thermospray mass spectrum of ETU exhibits a protonated molecule ion (M+H) + at m/z 103 as the base peak (Figure 1). The ion at m/z 162 (M+60) + may correspond to loss of water from an adduct of the molecule with protonated ammonium acetate (12) or it may be the adduct of ETU with acetic acid (13). The identity of the ion at m/z 121 (M+I9) + is not known, but it may correspond to an adduct of the molecule with protonated water (M+H30) +.
Reproduction (photocopying) of editorial content of this joumal is prohibited without publisher's permission.
85
Journal of Analytical Toxicology,Vol. 16, March/April 1992
MH §
100
A
H
HaC--N\ HZ~__NIC=S H
>-
c'J
n.-
o
,
rxJ
1
L
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120
100
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160
m/z
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,ooj._0
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_z tl.l M3
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0
I
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h
I
.
9 .,1
120
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140
A
|
I_
9 I
i60
i
1 2 3 4 5
m/z
TIME Fioure 1. (a) Thermospray mass spectrum of the ethylenethiourea peak obtained after reversed-phase HPLC column separation; (b) thermospray mass spectrum of the urine sample of a potato field applicator exposed to ethylenethiourea (see Figure 2c). Identification of the ETU peak after HPLC can be supported by the UV-absorbance profile of the compound and by the retention time, as in our previous study ( I ). In the present work, the identity of the peak was confirmed by TSP HPLC-MS. The mass spectrum and chromatogram of ETU in a urine sample obtained from a potato applicator exposed to EBDC fungicides and ETU are in good agreement with the standard of ETU (Figures la, lb, 2a, and 2c). The ions at m/z 96, 97, and 119 originated from the background. The SIR chromatogram of the protonated molecule ion obtained from blank urine sample shows no interfering peaks at the corresponding retention time (Figure 2b). The detection limit of the present HPLC-MS method was 0.1 ng of ETU/injection, which equals the limit of detection of the HPLC-UV method reported earlier ( 1). The current results are in agreement with the HPLC-UV method. The linearity of the detection of ETU with the present HPLC-MS method was calculated and appeared to be excellent, between 0.1 and 10 ng of ETU/injection: 3' = bx + a, where v = peak area, b = constant (25), x = rig/injection, and a = y-intercept (-6) (re = 0.998). The detection limit of the GC methods for ETU (3,4) is not sensitive enough for detecting ETU in biological samples. HPLC methods reported (1,5,6) can be used for the analysis of ETU in several types of samples. However, none of the previous methods with sufficient sensitivity (1) provide totally reliable identifica-
86
(min)
Fioure 2. HPLC-MS selected ion recording (m/z 103) detection of ethylenethiourea (a) in water standard (0.22 mg/L), (b) in blank urine, (c) in urine sample of a potato field applicator exposed to ethylenethiourea. The concentration of ethy]enethiourea was 2.6 pg/L. The chromatograms were normalized to the highest peak in each chromatogram tion of ETU, Therefore, the major improvement of the present HPLC-MS method is that it is both sensitive and reliable for detecting ETU in biological samples. To our knowledge, this is also the first paper reporting HPLC-MS confirmation of the identity of ETU in biological samples. The drawbacks of the present method are that it is laborious to use and requires expensive equipment. The HPLC-MS method described in this work for detection of low concentrations of ETU is sensitive enough to be used for the analysis of ETU in occupational air and in urine during and after occupational exposure to EBDCs or ETU (14-16). This method may also prove valuable for detecting trace amounts of ETU in water and food samples, inflmnation that would be crucial for purposes such as setting of tolerances and acceptable daily intake values in different food items.
Acknowledgments The authors wish to thank Mr. Tuomo Korhonen and Mr. Jukka Knuutinen for skillful technical assistance. This work was financially supported by The Finnish Work Environment Fund and the North-Savo Cultural Foundation.
Journal of AnalyticalToxicology,Vol. 16, March/April1992
References 1. P. Kurttio, T. Vartiainen, and K. Savolainen. A high pressure liquid chromatographic method for the determination of ethylenethiourea in urine and on filters. Anal Chim. Acta 212: 297-301 (1988). 2. World Health Organization. (WHO Environmental Health Criteria 78). Dithiocarbamate pesticides, ethylenethiourea, and propylenethiourea: A general introduction. World Health Organization, Geneva, 1988. 3. W.H. Newsome. Determination of ethylenethiourea residues in apples. J. Agric. Food Chem. 20:967-69 (1972). 4. T. Hirvi, H. Pyysalo, and K. Savolainen. A glass capillary gasliquid chromatography method for determining ethylenethiourea without derivation. J. Agric. Food Chem. 27:194-95 (1979). 5. H.B. Hanekamp, P. Bos, and R.W. Frei. Design and selective application of a dropping mercury electrodeamperometric detector in column liquid chromatography. J. Chromatogr. 186:489-96 (1979). 6. J.R Lawrence and F. Iverson. Liquid chromatography with UV absorbance and polarographic detection of ethylenethiourea and related sulfur compounds. J. Chromatogr. 212:245-50 (1981). 7. W.R. Bontoyan, J.B. Looker, T.E. Kaires, P. Giang, and B.M. Olive. Survey of ethylenethiourea in commercial ethylenebisdithiocarbamate formulations. J. Assoc. Off. Anal Chem. 55: 923-25 (1972). 8. K. Autio. Ethylenethiourea: Metabolism, analysis and aspects of toxicity. Technical Research Centre of Finland, Research Reports 91, Espoo, Finland, 1982. 9. J. Singh, W.P. Cochrane, and J. Scott. Extractive acylation of
10.
11.
12.
13.
14.
15.
16.
ethylenethiourea from water, Bull. Environ. Contam. ToxicoL 23: 470-74 (1979). T.R. Covey, J.B. Crowther, E.A. Dewey, and J.D. Henion. Thermospray liquid chromatograph/mass spectrometry determination of drugs and their metabolites in biological fluids. Anal. Chem. 57:474-81 (1985). R.D. Voyksner and C.A. Haney. Optimization and application of thermospray high-performance liquid chromatography/mass spectrometry. Anal Chem. 57:991-96 (1985). R.W. Smith, C.E. Parker, D.M. Johnson, and MM. Bursey. Eluent pH and thermospray mass spectra: Does the charge on the ion in solution influence the mass spectrum? J. Chromatogr. 394: 261-70 (1987). D. Barcel6. Effect of ammonium formate as ionizing additive in thermospray liquid chromatography-mass spectrometry for the determination of triazine, phenylurea and chlorinated phenoxyacetic acid herbicides. Organic Mass Spectrom. 24:219-24 (1989). K. Savolainen, P. Kurttio, T. Vartiainen, and J. Kangas. Ethylenethiourea as an indicator of exposure to ethylenebisdithiocarbamate fungicides. Arch. ToxicoL Suppl. 13:120-23 (1989). P. Kurttio, T. Vartiainen, and K. Savolainen. Environmental and biological monitoring of exposure to ethylenebisdithiocarbamate fungicides and ethylenethiourea. Br. J. Ind. Med. 47:203-206 (1990). P. Kurttio and K. Savolainen. Ethylenethiourea in air and in urine: implications to exposure to ethylenebisdithiocarbamate fungicides. Scand. J. Work Environ. Health 16:203-207 (1990). Manuscript received July 6, 1990; revision received October 11, 1990.
87
