Analyst, February, 1979, Vol. 104, PP. 111-116 111 Determination of Ethylenethiourea in Ethylenebisdithiocarbamate Fungicides: Comparison of High-performance Liquid Chromatography and Gas - Liquid Chromatography D. S. Farrington and R. G. Hopkins Department of Industry, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, SE1 9NQ A rapid, sensitive method is described for the determination of ethylene- thiourea (imidazolidine-2-thione) in ethylenebisdithiocarbamate fungicides. High-performance liquid chromatography is used with an ultraviolet spectro- photometric detector. The results are compared with those obtained using gas - liquid chromatography. All fungicide samples assayed contained ethylenethiourea, and gas - liquid chromatography indicated higher con- centrations than high-performance liquid chromatography.Keywords : Ethylenethiourea determination ; ethylenebisdithiocarbamate fungi- cides ; high-performance liquid chromatography ; gas - liquid chromato- graphy Ethylenethiourea (ETU) (imidazolidine-2-thione) may occur in formulations both as a manufacturing impurity and as a product of degradation during storage.132 The presence of ETU in commercial formulations is of concern because of its possible carcinogenic and teratogenic proper tie^.^^^ However, deficiencies in available data have inhibited any firm conclusions being reached in respect of these properties. The routine method of analysis for the active ingredient in ethylenebisdithiocarbamate (EBDC) formulations is based on the determination of carbon disulphide after acid hydro- lysis.536 This technique is not applicable in the presence of copper and the Dubosq method' is then used, The latter method is based on hydrolytic cleavage using hydriodic acid but ETU interferes. As ETU may be present in formulations to the order of several per cent., this interference can be significant and a correction must be made for the ETU content. Johnson and Tyler* determined ETU in aqueous extracts from a number of EBDC fungicides using paper chromatography.All fungicides based on ethylenebisdithiocarbamic acid were reported to contain ETU. Thin-layer chromatography was used by Czegledi- Janko and Hollo9 to examine the degradation of zineb and maneb. Fishbein and FawkeslO also reported a thin- layer chromatographic method for the determination of the degradation products of EBDC fungicides.Thin-layer chromatography was used by Bontoyan and Looker2 as a screening method for detecting ETU prior to gas - liquid chromatographic analysis of EBDC fungicide formulations maintained under conditions of elevated temperature and humidity. It was thought that high-performance liquid chromatography would give high specificity, speed and sensitivity, and form the basis of a method by which the results obtained by gas - liquid chromatography could be critically assessed. A number of workers have reported methods for the assay of ETU. Experiment a1 Liquid Chromatograph A Waters Associates, Model 6000, constant-volume solvent-delivery system was used. A variable-wavelength ultraviolet monitor (Cecil Instruments, Model CE 212), fitted with a 10-pl flow cell and set at 240 nm, was used as a detector.Preparation of Column A stainless-steel column tube, 180 x 4.6 mm i.d., was washed with chloroform and Crown Copyright.112 FARRINGTON AND HOPIUWS : DETERMINATION OF ETHYLENETHIOUREA Analyst, VoZ. 104 methanol and polished on the inner surface. One end was fitted with a 3 x & in Crawford Patent column end fitting, and the other end was coupled to a 400-mm pre-column through a Spherisorb CN, 5 p m (Phase Separations Ltd.), was packed into the column from a slurry in ethanol under a pressure of 5000 lb k2. The pre-column was removed and the analytical column was prepared for stop-flow injection by removing the top few millimetres of packing and inserting a disc of stainless-steel fine-mesh gauze, of 8 pm nominal porosity, a plug of silanised glass-wool and a top plug of porous PTFE.Sample Injection A Varian Associates stop-flow injector was used and samples were injected on to the stainless-steel fine-mesh gauze fitted on top of the packing. A needle guide was incorporated in the injector to ensure that the samples were introduced on to the centre of the top of the column. x $in Swagelok union. Conditions for Gas - Liquid Chromatography ionisation detector, under the following operating conditions : Analyses were carried out on a Pye 104 gas - liquid chromatograph, fitted with a flame- Column . . .. .. .. . . . . 1000 x 4 mm i.d., glass Column packing . . .. .. . . Chromosorb WHP, 2% of Carbowax 20M TPA on Chromosorb W, 80-100 mesh Column oven temperature .. . . .. 210°C Detector oven temperature . . . . . . 250 "C Injection zone temperature . . .. . . 230°C Carrier gas . . . . .. .. . . Nitrogen, free from oxygen Flow-rates: nitrogen . . .. . . . . 50 cm3 min-l hydrogen . . .. .. . . 50 cm3 min-l air .. .. .. . . 600 cm3 min-l Reagents Ethylenethiourea. ETU standard solution. Ethanol, absolute. Methanol. Analytical-reagent grade. Hexane. Spectrograde (Fisons). MobiZe phase for liquid chromatografihy. Recryst allised, obtained from Robinson Brothers Lt d. Prepare a standard solution in methanol, containing 0.7 g 1-1 of ETU. Prepare a solution containing 35% VjV of absolute ethanol in hexane. Procedure Weigh accurately about 0.5 g of EBDC, or a mass equivalent to 0.5 g of active ingredient, into a 15-cm3 centrifuge tube.Add 10.0 cm3 of methanol from a pipette, stopper the tube and shake it vigorously for 15min on a wrist-action shaker. Filter the resulting mixture through a filter-paper that retains particles of at least 5 pm, for example, Whatman No. 42, and collect the filtrate. Inject 4 pl of the filtrate on to the top of the liquid chromatograph using a flow-rate of 0.8 ml min-l. Complete the analysis as soon as possible. Calculate the ETU content of the sample by comparing the peak height with that obtained from a 4-pl injection of standard solution. To ensure that the response of the detector is within the linear range, dilute the sample until the peak height obtained is equal to or less than that obtained with the standard.Gas - liquid chromatographic analyses are undertaken using the same solution and the same injection volume as are used for liquid-chromatographic determinations. Results and Discussion A number of procedures were investigated for extracting ETU from EBDC fungicides. A cold-extraction technique was adopted in order to reduce the risk of production of ETU. Optimum speed and repeatability were obtained by shaking the fungicides with solvent forFebrunvy, 1979 I N ETHYLENEBISDITHIOCARBAMATE FUNGICIDES 113 15 min. Duplicate analyses of a sample of ziram fortified with ETU (3%) gave recoveries of approximately 90%. The use of water has been reported; however, it was found that this extraction solvent resulted in un- acceptably broad peaks on the liquid Chromatograph.Methanol and methanol - chloro- form have also been used for extraction and these were investigated. Both systems resulted in good peaks on the liquid chromatograph and were shown to have similar extraction efficiencies with respect to ETU. However, methanol - chloroform co-extracted greater amounts of other substances and therefore methanol was adopted as the extraction solvent. ETU has a sharp absorbance at 240 nm and this was adopted as the wavelength setting on the UV monitoring system. ETU was readily eluted from a Spherisorb ODS system, but adequate retention could not be obtained. A bonded stationary phase exhibiting polar characteristics appeared likely to be suitable and Spherisorb CN was selected. With hexane - ethanol (65 + 35) as the mobile phase, ETU is eluted from a Spherisorb CN packed column with a good peak shape and optimum resolution.Ethylenethiuram monosulphide (ETRI) elutes shortly before ETU, but is not extracted efficiently by methanol and does not absorb strongly at 240nm. The procedure was applied to both technical samples and formulations of zineb, maneb, mancozeb and Vondozeb (a co-ordination product of EBDC with zinc and manganese ions). Figs. 1 and 2 show typical chromatographic traces obtained from analyses of these compounds. With the procedure described, 0.01% of ETU can be determined in the four active ingredients investigated. The choice of the solvent was limited by the low solubility of ETU. The results obtained are shown in Table I. TABLE I ETHYLENETHIOUREA CONTENT OF ETHYLENEBISDITHIOCARBAMATE FUNGICIDES Fungicide Maneb technical 1 ..Maneb technical 2 .. Maneb formulation 1 . . Maneb formulation 2 . . Maneb formulation 3 . . Zineb technical 1 .. Zineb technical 2 .. Zineb formulation .. Maneb - zineb technical . . Maneb - zineb formulation Mancozeb technical 1 . . Mancozeb technical 2 . . Mancozeb technical 3 . . Mancozeb technical 4 . . Mancozeb formulation . . Vondozeb technical 1 . . Vondozeb technical 2 . . Vondozeb technical 3 . . Vondozeb formulation . . Figures are expressed as percentages. Results by high-performance liquid chromatography .. 0.1’ 0.1 .. 1.2, 1.3 .. 0.7, 0.7 .. 1.1, 1.1 .. 1.0, 1.1 .. .. .. 1.1, 1.2 1.5, 1.4 2.1, 2.0 0.4, 0.4 1.0, 1.0 .. 0.1, 0.1 . . 0.2, 0.2 . . 0.2, 0.2 .. 0.2, 0.2 . . 0.2, 0.2 ..1.0 .. 1.3, 1.3 . . 1.4, 1.5 .. 0.9, 1.0 Results by gas - liquid chromatography 0.2, 0.2 2.8, 2.9 1.2, 1.3 2.0, 2.0 1.8, 1.9 1.5, 1.5 2.5, 2.4 2.1, 2.2 0.6, 0.6 1.2, 1.3 0.1, 0.1 0.4, 0.4 0.4, 0.4 0.4, 0.4 0.2, 0.3 1.6 2.1, 2.1 2.9, 2.6 1.5, 1.3 High-performance liquid chromatographic traces obtained from analyses of extracts of maneb samples showed that maneb yields greater concentrations of co-extractives than the other EBDC fungicides examined (Fig. 1). This was also observed by Czegledi- Janko and Hollo9 when analysing a number of EBDC fungicides by thin-layer chromatography. Mancozeb samples contained comparatively little ETU and less co-extractives (Fig. 1) , which agrees with the findings of Bontoyan and Looker.2 Gas - liquid chromatographic analysis of all samples gave two peaks (Fig.2), the second eluting shortly after ETU. A correlation between the area of this peak and the rate of114 FARRINGTON AND HOPKINS : DETERMINATION OF ETHYLENETHIOUREA Analyst, VoZ. 104 formation of ETU from EBDCs was noted by Bontoyan and Looker.2 They concluded that ETU may arise as a result of two decomposition routes, a direct route from the parent EBDC and formation via intermediate degradation products. A mechanism for the formation of ETU through degradation products, but not directly from the parent EBDC, was proposed by Marshall.ll This was based on studies of the thermal decomposition of EBDCs in aqueous media. a ) 1 c u 0 5 10 0 5 10 C ) It L - 0 5 10 Ti me/m i n 11 0.2 unit L 0 5 1 0 0 5 1 0 Fig. 1. Typical liquid chromatograms obtained from 4-1.11 injections of: (a) ETU standard; (b) maneb technical; (c) zineb technical ; (d) mancozeb technical ; and (e) Vondozeb technical. The analysis of an aqueous solution of nabam and an aqueous suspension of zineb, by injection on to a gas chromatograph, was investigated by Zielinski and Fishbein.12 These experiments gave rise to peaks attributable to ETU rather than peaks directly attributable to the EBDCs.They concluded that this was due to the formation of ETU on sample injection, although ETU already present in the sample could have contributed to the peaks observed. A sample of ETM, which high-performance liquid chromatographic analysis indicated contained no apparent ETU, when dissolved in chloroform - methanol and injected on to the gas chromatograph yielded a significant peak with the same retention time as ETU.It was observed by Marshallll that ETM can give rise to ETU at elevated temperatures and our findings are in agreement with that observation. It is apparent that ETU is formed from one or more precursors, present as degradation products of EBDCk or as impurities, on injection in methanolic solutions on to a gas chromatograph. It is significant that those samples which contained the greatest concentration of co-extractives, based on high- performance liquid chromatographic analyses, gave rise to the largest difference between the gas - liquid and high-performance liquid chromatographic results for ETU content. Samples extracted with chloroform -methanol (1 + 1) gave the same result for ETU content , when assayed by high-performance liquid chromatography, as those obtained following extraction with methanol (Table 11).However, on injection on to the gas- liquid chromatograph, higher results for ETU were observed than those obtained followingFebrimry, 1979 I N ETHYLENEBISDITHIOCARBAMATE FUNGICIDES ‘b) c i 116 9 0 5 10 15 G 5 10 15 0 5 10 1 5 0 5 10 1 5 0 5 10 15 T ime/’m i n Fig. 2. Typical gas - liquid chromatogranis obtained from 4-pl injections of: (a) ETU standard; (b) maneb technical; (G) zineb technical; ( d ) mancozeb technical ; and ( 6 ) Vondozeb technical. comparative methanol extractions. This serves to illustrate further that degradation of ETU precursors is occurring during gas - liquid chromatography, with possible contributions from unresolved extraneous peaks.TABLE I1 ETHYLENETHIOUREA CONTENT OF ETHYLENEBISDITHIOCARBAMATE FUNGICIDES USING CHLOROFORM - METHANOL AS THE EXTRACTION SOLVENT Figures are expressed as percentages. Results by high-performance Fungicide liquid chromatography chromatography Results by gas - liquid Maneb technical 1 .. .. 0.1, 0.1 0.2, 0.2 Maneb technical 2 .. .. 1.2, 1.2 4.3, 4.2 Maneb formulation 2 . . .. 1.1, 1.1 3.3, 3.1 Maneb formulation 3 . . . . 1.1, 1.1 2.9, 2.6 Zineb technical 1 .. .. 1.2, 1.2 1.4, 1.4 Maneb - zineb formulation . . 0.9, 1.0 1.4, 1.4 Mancozeb technical 1 . . . . Vondozeb technical 1 . . . . Vondozeb technical 2 . . .. 0.1, 0.1 0.9 1.2 0.1, 0.1 2.6 2.7116 FARRINGTON AND HOPKINS Conclusion If ETU analyses are carried out by gas - liquid chromatography, results will be obtained that reflect both the sample content of ETU and the ETU that may be formed by thermal decomposition from other compounds present.The use of high-performance liquid chromato- graphy offers a rapid analytical technique that yields a truer estimate of the ETU content. The authors thank Robinson Brothers Ltd. for supplying ETM and ETU. They also thank the Government Chemist for permission to publish this paper. Some of this work was carried out as part of the programme of the Dithiocarbamates Panel of the Pesticides Analysis Advisory Committee. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. References Bontoyan, W. R., Looker, J. B., Kaiser, T. E., Giang, P., and Olive, B. M., J . A s s . Off. Analyt. Bontoyan, W. R., and Looker, J. B., J . Agric. Fd Chem., 1973, 21, 338. Graham, S. L., Davis, K. J., and Hansen, W. H., Fd Cosmet. Toxicol., 1975, 13, 493. Pure Appl. Chem., 1977, 49, 675. Clarke, D. G., Baum, H., Stanley, E. L., and Hester, W. F., Analyt. Clzem., 1951, 23, 1842. Raw, C. R., Editor, “CIPAC Handbook,” Volume I, Heffer, Cambridge, for Collaborative Inter- Dubosq, F., Chim. Analyt., 1967, 49, 68. Johnson, E. I., and Tyler, J . F. C., Chemy Ind., 1962, 304. Czegledi-Janko, G.. and Hollo, A., J . Chromat., 1967, 31, 89. Fishbein, L., and Fawkes, J., J . Chromat., 1965, 19, 364. Marshall, W. D., J . Agric. Fd Chem., 1977, 25, 357. Zielinski, W. L., and Fishbein, L., J . Chromat., 1966, 23, 302. Chem., 1972, 55, 923. national Pesticides Analytical Council, 1970, p. 463. Iicceived Jzme 21st, 1!378 Accepted September 4th, 1978