Anal. Chem. 1998, 70, 4800-4804
Analysis of the Dithiocarbamate Fungicides Ziram, Maneb, and Zineb and the Flotation Agent Ethylxanthogenate by Ion-Pair Reversed-Phase HPLC Kenneth W. Weissmahr, Christie L. Houghton, and David L. Sedlak*
Department of Civil and Environmental Engineering, University of California at Berkeley, Berkeley, California 94720
Although large quantities of dithiocarbamate fungicides and ethylxanthogenate mineral flotation agents are released routinely to the environment, little is known about their fate and effects because sensitive analytical techniques are not available. A simple and selective HPLC method for trace level analysis of dithiocarbamate fungicides and xanthogenates (dithiocarbonates) in natural waters has been developed. The method is based on the in situ formation of a 1:1 Cu(II)-dithioligand complex and its separation as an ion pair with hexanesulfonate on a C-18 reversed-phase column. The compounds are detected at the wavelength of maximum absorption of the Cu(II)-ligand complex (260-287 nm). Method detection limits are 20 nM dimethyldithiocarbamate (i.e., 3 µg/L Ziram), 33 nM ethylenebis(dithiocarbamate) (i.e., 9 µg/L Maneb), and 23 nM ethylxanthogenate (i.e., 4 µg/ L). An on-line preconcentration step can be used to lower the detection limits by a factor of 5-10. Complexing agents with a dithio functional group are widely used in industry and agriculture (Figure 1). Large quantities of dithiocarbamates are used in agriculture as fungicides on almond trees, stone fruits, and vegetables. Dithiocarbamates also are used as rodent repellents, vulcanization additives in rubber manufacturing, and additives in lubricants. As fungicides, the most commonly used compound is dimethyldithiocarbamate (DMDC), which is applied as Zn(DMDC)2 (i.e., Ziram) and to a lesser extent as Fe(DMDC)3 (i.e., Ferbam). Other common dithiocarbamate fungicides include the ethylenebisdithiocarbamates (EBDCs) Nabam, Zineb, Maneb, and Mancozeb (disodium, zinc, manganese, and mixed Mn/Zn complexes of EBDC, respectively).1,2 Xanthogenates (dithiocarbonates), such as ethylxanthogenate (EX), are used extensively by the mining industry as collectors in sulfide mineral flotation.3 To prevent fungal growth, large quantities of DMDCs usually are applied in early spring. On a mass basis, Zn(DMDC)2 is one of the 10 most common pesticides used in California, with an * Corresponding author: (e-mail)
[email protected]. (Fax) 510 6427483. (1) Ludwig, R. A.; Thorn, G. D. Adv. Pest Control Res. 1960, 3, 219-252. (2) Thorn, G. D.; Ludwig, R. A. The Dithiocarbamates and Related Compounds; Elsevier Publishing: Amsterdam, 1962. (3) Rao, S. R. Xanthates and Related Compounds; M. Dekker: New York, 1971.
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Figure 1. Structures of dithiocarbamates (DMDC, DEDC, EBDC) and a dithiocarbonate (EX).
annual application rate of ∼500 tons.4 The overall annual use in the United States is ∼1400 tons.5 After it is released into the environment, DMDC can form strong complexes with trace elements such as Cu(II).6 The pH, type, and concentration of trace metals and ligand-to-metal ratio will determine the fraction of DMDC that is present as free ligand, positively charged 1:1 metalDMDC complexes, or uncharged and hydrophobic metal(DMDC)2 complexes.7 Because each of these DMDC species will behave differently, environmental conditions will affect the fate, transport, and toxicity of the DMDCs. Recently, it was suggested that Zn(DMDC)2 may be responsible for toxic effects of water collected from the Sacramento River in early spring.8 Hydrophobic metal-(DMDC)2, metal-(EX)2, or metal-EBDC complexes have been shown to significantly increase trace metal uptake by aquatic organisms.9-11 Furthermore, there is growing concern about the potential estrogenic effects and chronic toxicity of these compounds on other animal species.12-14 (4) EPA Pesticide Use Report; State of California Departement of Pesticide Regulation, Sacramento, CA, 1994. (5) USGS http://water.wr.usgs.gov/pnsp/use92, 1992. (6) Goksoyr, J. Physiol. Plant. 1955, 8, 719-755. (7) Weissmahr, K. W.; Sedlak, D. L. Environ. Sci. Technol., in preparation. (8) Miller, J. L. AQUA-Science, Davis, CA, 1998. (9) Phinney, J. T.; Bruland, K. W. Environ. Sci. Technol. 1994, 28, 1781-1790. (10) Phinney, J.; Bruland, K. Environ. Toxicol. Chem. 1997, 16, 2046-2053. (11) Block, M.; Glynn, A. W.; Part, P. Aquat. Toxicol. 1991, 20, 267-283. (12) Goldman, J.; Parrish, M.; Cooper, R.; McElroy, W. Reprod. Toxicol. 1997, 11, 185-190. 10.1021/ac980626w CCC: $15.00
© 1998 American Chemical Society Published on Web 10/10/1998
Table 1. Method Detection Limits and Wavelengths of Detection for Several Dithiocarbamates and Ethylxanthogenate in River Watera compound
abbrev
λmax of Cu(II) 1:1 complex
MDL (nM)
MDL (µg/L)
RSD (%)
dimethyldithiocarbamate diethyldithiocarbamate ethylxanthogenate ethylenebis(dithiocarbamate)
DMDC DEDC EX EBDC
260 264 287 280
20 18 23 33
3 (as Ziram) 3 (as Na salt) 4 (as K salt) 9 (as Maneb)
10 5 15 3
a Sample volume, 250 µL; eluent composition, hexanesulfonate 2.5 mM, acetate buffer 2 mM pH 4, Cu(II) 100 µM with 30% methanol (for DMDC) or 45% methanol (for other compounds).
Despite the widespread use of DMDC, EBDCs, and EX and concerns over their environmental effects, few studies of their environmental fate have been conducted because convenient analytical techniques have not been developed (see ref 15 for a review of analytical techniques for dithiocarbamates). The standard U.S. EPA approved method for measuring these compounds consists of acid hydrolysis of the dithiocarbamates to CS2 and dimethylamine followed by quantification of CS2.16 However, this method does not discriminate between individual dithiocarbamate ligands and suffers from possible interference due to CS2 evolved from hydrolysis of other compounds, such as dithiocarbamate metabolites and amino acids. The method detection limits (MDLs) for the CS2 technique are about 12 and 58 nM for DMDC and EBDC, respectively. These concentrations are above levels at which toxic effects have been reported for certain species.8 Alternate analytical methods, such as voltammetry,17,18 spectrophotometry,19 and atomic absorption spectroscopy,20 are less sensitive than the CS2 technique. Furthermore, they rely on the detection of Zn(II) and are, therefore, prone to errors due to contamination of samples with zinc. In addition, the voltammetric methods require extraction with organic solvents, which is timeconsuming and prone to errors due to incomplete extraction of dissociated metal-ligand complexes. Several methods of direct analysis of dithiocarbamates by HPLC have been proposed; however, most are labor-intensive or have not been tested at trace levels.15,21-23 Furthermore, the measurements are complicated by the fact that the dithio functional group is reactive and undergoes transformation reactions during analysis. For example, Gustafsson15 described a specific HPLC method for alkyldithiocarbamates and EBDCs requiring phase-transfer methylation. The MDLs for this labor-intensive method are approximately 66 and 180 nM for DMDC and EBDC, respectively. Another HPLC method23 uses a micellar mobile phase containing a hydrophobic surfactant, but the applicability of the method at trace concentrations has not been demonstrated. For the analysis of Thiram (tetramethyl thiuram disulfide) and EBDCs, an effective HPLC method with postcolumn derivatization and selective preconcentration steps has been proposed24 with a detection limit of 390 nM EBDC. In this paper, we describe a simple and specific HPLC method for the quantification of dithiocarbamate and dithiocarbonate ligands based on the separation and detection by in situ formation of a positively charged 1:1 Cu(II)-ligand complex using an alkylsulfonate ion pair reagent. Formation of CuDMDC+ complexes and ion pair formation between CuDMDC+ and 1-hexanesulfonate (HSA) is accomplished by using an eluent containing dissolved Cu(II) and HSA. EBDCs are analyzed with the same
eluent as uncharged CuEBDC0 complexes. A similar approach of Cu(II) complexation coupled with ion-pairing has already been used for the analysis of hydroxy acids in water;25 however, this approach has never been applied to the analysis of compounds with reactive dithio functional groups. As the result of in situ formation of CuDMDC+ or CuEBDC0 complexes, the total amount of each dithioligand is quantified, irrespective of whether it was originally present as a sodium, manganese, or zinc complex. The MDL of our method corresponds to approximately 20, 33, and 23 nM for DMDC, EBDC, and EX (see Table 1). The MDL can be lowered by a factor of 5 for DMDC and up to 10 for the other compounds using a simple on-line preconcentration step. EXPERIMENTAL SECTION Apparatus. High-performance liquid chromatography was performed with a Gynkotek HPLC system consisting of a model 480 G gradient pump, Gina 50 autosampler with 1-250-µL injection volume, and UVD 320 S diode array detector with a range of 200355 nm. The identity of the analyzed compounds was confirmed by comparing the UV spectra acquired with the diode array detector with spectra obtained using a UV-visible spectrophotometer (Perkin-Elmer Lambda 14, using 1-cm quartz cuvettes). The eluent was degassed with an ERC-3315 on-line degasser (ERC Inc.). For good peak shape it is necessary to use a stationary phase without active silanol groups. We obtained good results with the double-end-capped Alltima C-18 column (Alltech Inc., 9% (13) Maita, K.; Enomoto, A.; Nakashima, N.; Yoshida, T.; Sugimoto, K.; Kuwahara, M.; Harada, T. J. Pestic. Sci. 1997, 22, 193-207. (14) Ema, M.; Itami, T.; Ogawa, Y.; Kawasaki, H. Bull. Environ. Contam. Toxicol. 1994, 53, 930-936. (15) Gustafsson, K. H.; Thompson, R. A. J. Agric. Food Chem. 1981, 29, 729732. (16) EPA. Methods for the Determination of Nonconventional Pesticides in Municipal and Industrial Wastewater; Office of Water Engineering and Analysis Division: Washington, DC, 1993; Vol. 1, pp 299-324. (17) Ulakhovich, N. A.; Shaidarova, L. G.; Budnikov, G. K.; Un, U. A.; Saveleva, N. I. J. Anal. Chem. USSR 1991, 46, 256-260. (18) Mathew, L.; Reddy, M. L. P.; Rao, T. P.; Iyer, C. S. P.; Damodaran, A. D. Talanta 1996, 43, 73-76. (19) Mathew, L.; Rao, T. P.; Iyer, C. S. P.; Damodaran, A. D. Talanta 1995, 42, 41-43. (20) de Blas, O. J.; de Paz, J. L. P.; Mendez, J. H. J. Anal. At. Spectrom. 1990, 5, 693-696. (21) Smith, R. M.; Morarji, R. L.; Salt, W. G. Analyst 1981, 106, 129-134. (22) Brandsteterova, E.; Lehotay, J.; Liska, O.; Garaj, J. J. Chromatogr. 1984, 291, 439-444. (23) Kirkbright, G. F.; Mullins, F. G. P. Analyst 1984, 109, 493-496. (24) Irth, H.; De Jong, G. J.; Frei, R. W.; Brinkman, U. A. T. Int. J. Environ. Anal. Chem. 1990, 39, 129-139. (25) Lu, D. S.; Feng, W. Y.; Ling, D. H.; Hua, W. Z. J. Chromatogr. 1992, 623, 55-62.
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carbon load, 5-µm particles, 250 mm × 4.6 mm). In preliminary experiments, an Econosphere C-18 column (Alltech Inc.) also was tested but not used further because it resulted in poor peak shape with strong tailing. As the preconcentration stationary phase, an Alltima C-18 precolumn cartridge was used (Alltech Inc., 16% carbon load, 7 mm × 4.6 mm). The cartridge could be switched on- and off-line with a motorized switching valve (Rheodyne). Samples were pumped through the cartridge with an auxiliary Rainin B-100S one-piston pump. Reagents. A Nanopure II apparatus (Barnstead) purified all water used. Methanol was HPLC grade from Fisher Scientific. 1-Hexanesulfonic acid sodium salt (HSA), sodium dimethyldithiocarbamate (DMDC), sodium diethyldithiocarbamate (DEDC), and potassium ethylxanthogenate (EX) were obtained from Fluka. Tetramethyl thiuram disulfide (Thiram), and ethylenebis(dithiocarbamate) disodium salt (EBDC) were obtained from Chem Service Inc. (West Chester, PA). All other chemicals were analytical reagent grade from Fisher Scientific. Zn(DMDC)2 was prepared by dissolving 1.8 g of DMDC (10 mmol) in 200 mL of water and adding 310 mg of ZnSO4‚7H2O (1.1 mmol). To prevent oxidation of the ligand, 250 mg of ascorbic acid was added to the solution.26 After 15 min of vigorous stirring at room temperature, the white precipitate was filtered, washed once with cold water, and dried. Aqueous solutions of Zn(DMDC)2 and DMDC hydrolyze via a pH-dependent reaction.27,28 The half-lives for the reaction range from 1 to 10 days at pH 7 and 8, respectively.7 Therefore, stock solutions were prepared in methanol (25 mM DMDC, 1 mM Zn(DMDC)2). Diluted aqueous standards were prepared each day. Stock solutions of EX, EBDC, and Thiram were prepared immediately before use. The methanolic stock solutions were stored in a refrigerator at 4 °C and checked regularly for degradation by UV-visible spectroscopy. The aqueous solutions prepared in Nanopure water were buffered at pH 8 with 1 mM HEPES buffer containing 10 mM NaClO4. Samples of 0.45-µm filtered river water were amended with stock solutions of analytes. Chromatography. For the analysis of DMDC, an aqueous buffer solution and methanol (70:30) were used as the eluent at a flow rate of 1 mL/min. For the analysis of the EBDCs, DEDC, and EX, a similar eluent was used, except the buffer solution/ methanol ratio was changed to 55:45. The buffer typically contained 2-3 mM sodium acetate (pH adjusted to a value between 4 and 5), 100-200 µM CuSO4, and 2-4 mM HSA ion pair reagent. As mentioned before, the hydrolysis of most dithiocarbamates is fast at low pH (i.e., t1/2 is 1.5 min at pH 4 28). However, the CuDMDC+ complex is very stable, and complexation of DMDC with Cu(II) present in the mobile phase prevents the degradation of DMDC during the analysis.7 Before analysis, the column was equilibrated for at least 30 min with mobile phase. Equilibration was complete when a breakthrough of Cu(II) species was detected at 260 nm. To remove Cu(II) from the column after each day of analysis, the HPLC system was flushed with 30 mL of water/methanol (70:30), 20 mL of 10 mM EDTA/methanol (80: 20), and again followed by 30 mL of water/methanol (70:30).
Preconcentration. The precolumn cartridge was conditioned off-line with 1-2 mL of buffer solution containing Cu(II) and HSA. Following conditioning, it was loaded with 2-3 mL of sample and washed with 0.5 mL of water. The flow rate during the loading step was ∼1 mL/min. The exact volumes delivered were determined by measuring the volume of effluent from the precolumn. After loading the cartridge, the analyte was introduced into the separation column by switching the valve to allow the mobile phase to flow through the cartridge. Sample Treatment. River water samples were collected from the Sacramento River and the Napa River (CA). The samples were collected in glass bottles, filtered through a 0.45-µm PTFE filter, and stored on ice until they were analyzed in 1-2 days. The water samples used to determine the MDLs of our method contained no detectable amounts of dithiocarbamates.
(26) Robards, K.; Starr, P. Analyst 1991, 116, 1247-1273. (27) Aspila, K. I.; Sastri, V. S.; Chakrabarti, C. L. Talanta 1969, 16, 1099-1102. (28) Aspila, K. I.; Joris, S. J.; Chakrabarti, C. L. J. Phys. Chem. 1970, 74, 36253629.
(29) Scharfe, R. R.; Sastri, V. S.; Chakrabarti, C. L. Anal. Chem. 1973, 45, 413415. (30) Wai, C. M. In Preconcentration Techniques for Trace Elements; Alfassi, Z. B., Wai, C. M., Eds.; CRC Press: Boca Raton, FL, 1992; pp 101-132.
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RESULTS AND DISCUSSION To evaluate the applicability of our analytical technique in the analysis of dialkyldithiocarbamates, EBDCs, and alkylxanthogenates, we concentrated our efforts on the analysis of DMDC. DMDC is probably the most difficult dialkyldithiocarbamate to analyze, because it is the most polar of the compounds and it forms the weakest Cu(II) complexes. After verifying the technique with DMDC, we tested the method with the other compounds. In water, Ziram dissociates to form a mixture of free ligand (DMDC), 1:1 complex (ZnDMDC+), and 2:1 complex (Zn(DMDC)2).6 Note that, at the pH used in the analysis, the free ligand DMDC (pKa ) 3.22)29 is present in its deprotonated form and carries one negative charge. The complexes between Zn(II) and DMDC are not very strong. The log K value for Zn(DMDC)2 complex has been reported to be 9.1.29 The log K for the ZnDMDC+ complex is ∼4.7 As a result of the relatively low stability constant of the zinc complexes, the presence of other trace metals may result in the formation of other metal-DMDC complexes. The stability constants for most other metal complexes of DMDC are unknown. However, it can be assumed that they are similar in magnitude to, albeit lower than, the stability constants for DEDC complexes. The complexing strength of the DEDC complexes follows the order Cu(II) > Ni(II) > Pb(II) > Cd(II) > Zn(II),30 and a similar trend is expected for DMDC. When Zn(DMDC)2 enters a natural water, it is likely that many different DMDC species will be present, depending on the composition of the water. To ensure a quantitative analysis of the DMDC ligand under these conditions, all these species have to be transformed into a single species before analysis. In our method, in situ complexation results in the formation of 1:1 complexes between Cu(II) and ligand (DMDC). Excess Cu(II) in the mobile phase forms strong complexes with DMDC that outcompete all other trace metals (including Fe(III)) likely to be present in natural water samples. CuDMDC+ shows a distinctive UV-visible spectrum (Figure 2, spectrum a) and is easily detected with an UV-visible detector at 260 or 385 nm. Detection at the charge-transfer band at 385 nm is ∼4.5 times less sensitive than detection at the UV band at 260 nm. However, it offers the advantage of selectivity due to lack of interference
Figure 2. Spectra of the 1:1 Cu(II)-ligand complexes of dithiocarbamate fungicides and ethylxanthogenate in aqueous solution: (a) DMDC 1 µM; (b) EBDC (Nabam) 1 µM; (c) EX 1 µM. All solutions in 100 µM Cu(II), 1 mM acetate pH 5.
Figure 3. Chromatograms of river water spiked with 200 nM DMDC: (a) Sacramento river at Yolo; (b) Napa river. Mobile phase: 2.5 mM hexanesulfonate, 2 mM acetate buffer pH 4, 100 µM Cu(II), 30% methanol, 1 mL/min. Detection at 260 nm.
from other UV-absorbing species present in some natural water samples. The CuDMDC+ complex can be analyzed by reversed-phase HPLC by adding HSA as an ion pair reagent. The reactions involved can be described by the following equlibria,26 where Lis the negatively charged DMDC ligand, R- is the negatively charged hexanesulfonate, and SP is the stationary phase of the HPLC column:
L- + Cu(II) T CuL+
(1)
CuL+ + R- T {CuL-R}
(2)
{CuL-R} + SP T {CuL-R}-SP
(3)
Eluent Composition. Figure 3 shows a typical chromatogram of DMDC added to river water samples. Although a series of relatively large system peaks elute early in the chromatogram, the region where CuDMDC+ elutes is free from interferences in all samples. Chromatograms for samples without added DMDC looked identical, except for the CuDMDC+ peak. To determine the optimal eluent composition, we measured the effect of varying
[Cu(II)], [HSA], and pH on samples containing 50, 100, 500, and 1000 nM DMDC. From eqs 1-3, it is evident that excess Cu(II) must be present to ensure a complete in situ formation of the 1:1 complex. [Cu(II)] ranging from 100 to 500 µM were tested using 2 mM acetate pH 5 and 4 mM HSA in eluent containing 30% methanol. The response increased slightly with increased [Cu(II)] due to narrower peak shape. However, this was offset by an increase in the detection limit attributable to increased baseline noise, probably due to the low solubility of Cu(II)-acetate in methanolic solution. At [Cu(II)] over 750 µM, a light blue precipitate was observed in the eluent. The concentration of the ion-pair reagent (i.e., HSA) was varied between 2 and 10 mM. Increased [HSA] resulted in an increased retention time but had no effect on response. The pH of the acetate buffer also did not affect the response. However, eluent pH influenced the size and position of system peaks in the chromatograms. One of the peaks that elutes prior to the CuDMDC+ peak is possibly due to CuOH+ that is formed when high-pH water samples are injected. When the mobile phase was buffered at pH 4, this system peak was minimized. Solid-Phase Preconcentration. The on-line preconcentration of DMDC is based on the same concept as the chromatographic separation (eqs 1-3). The cartridge is conditioned offline with a buffer solution containing Cu(II) and HSA. Although the C-18 material used is fully end-capped, enough active silanol sites are present to retain Cu(II) on the column. Furthermore, the hydrophobic HSA may interact with the C-18 chains of the solid phase, creating additional ion-exchange sites loaded with Cu(II). When aqueous samples are pumped through the conditioned precolumn, DMDC is complexed by copper, resulting in retention of CuDMDC+ ion pairs on the precolumn. After switching the loaded cartridge on-line, the complexes are flushed into the separating column by the methanolic eluent. Recoveries calculated for 2-mL samples fortified with 40 nM DMDC ranged from 55 to 90% depending on the river water sample and the conditions used. Sample volumes over 3 mL resulted in considerably reduced recoveries and were therefore not used. With this preconcentration method, the detection limit can be decreased by a factor of 5-10. Unlike the HPLC method, the preconcentration is more susceptible to interference from system peaks and interference from species other than DMDC. These interferences are also responsible for the low recoveries found for some river water samples. For example, river water containing a relatively high concentration of carbonate exhibits pHs between 7 and 8.5. Pumping these solutions through the Cu(II)-loaded cartridge may create CuOH+ species which are then eluted onto the analytical column. As a result of the carbonate buffering and the relatively large volume of the sample, these species may persist in the eluent and create peaks that interfere with the analysis of DMDC. Adjusting pH of the river water samples to 4-5 with acetate buffer and adding Cu(II) before preconcentration did not result in any improvements. The position and shape of the system peaks also depends on [Cu(II)], [acetate], and [HSA] in the conditioning solution and on the volume used for conditioning. The use of excess conditioning solution results in a system peak that overlaps with the peak of interest and interferes with the quantification. Analytical Chemistry, Vol. 70, No. 22, November 15, 1998
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Figure 4. Chromatogram of Napa river water spiked with 200 nM ethylxanthogenate (EX), diethyldithiocarbamate (DEDC), and ethylenebis(dithiocarbamate) (EBDC). Mobile phase: 3.25 mM hexanesulfonate, 2.5 mM acetate buffer pH 4, 130 µM Cu(II), 45% methanol, 1 mL/min. Detection at λmax of Cu(II) 1:1 complex of each compound.
The use of too little conditioning solution results in breakthrough of the DMDC species and poor recoveries. Therefore, the procedure given in the method section may need to be adjusted for alkaline waters. While the preconcentration is less robust than the HPLC method, it can detect trace amounts of DMDC species that would be overlooked by other techniques. As indicated by our experiments, slight changes in sample conditions can affect recoveries of DMDC. Therefore, recovery experiments and possibly modification of the technique should be considered whenever preconcentration is employed. In addition, it may be appropriate to use other solid phases for preconcentration of some samples. Analysis of DEDC, EBDC, and EX. Equations 1-3 also apply for other alkyldithiocarbamates, such as DEDC and the dithiocarbonate EX. These compounds can, therefore, be analyzed by the same method as DMDC. For the more hydrophobic DEDC and EX, analysis was performed using a mobile phase consisting of 45:55 methanol/buffer. The spectra of CuEX+ and CuEBDC0 complexes are shown in Figure 2. The EBDC fungicides Nabam, Maneb, and Zineb are bidentate ligands that are capable of chelation. Even with an excess of Cu(II) in the eluent, they probably form only the uncharged, hydrophobic CuEBDC0 complex. This complex can be separated by reversed-phase HPLC without ion pair formation. However, the presence of HSA does not affect the separation of CuEBDC0. Therefore, the analysis can be performed using the same eluent as for DEDC and EX. Figure 4 shows a chromatogram of river water spiked with 200 nM of these three compounds. The regions where the Cu complexes with EX, DEDC, and EBDC elute are free from interference from system peaks in all river water samples that we spiked. Chromatograms for samples without added compounds looked identical, except for the peaks attributable to the Cu complexes with EX, DEDC, and EBDC. Note that the absence of interference for the early-eluting EX can be attributed partly to the relatively high detection wavelength of 287 nm for
4804 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998
this compound. With this eluent (45:55 methanol /buffer), DMDC would elute too early to be detected. Therefore, DMDC and the three other compounds cannot be analyzed simultaneously. Due to the complicated composition of the eluent, a gradient from low to high methanol content is not feasible. The EBDC fungicides Maneb and Zineb are insoluble in water and even methanol, but they can be made soluble in water by sequestering the complexing metal with a stronger chelating agent. EBDC fungicides can be extracted from soil or food samples using dilute EDTA solutions.25 If such extracts are to be analyzed with this method, care should be taken that the EDTA concentration used is not in excess of the Cu(II) in the mobile phase. This may be accomplished by adding Cu(II) to the extract. Dithiocarbamate solutions containing ∼10 µM free EDTA can be analyzed without any adverse effects on recovery or sensitivity (data not shown). Thiram, the oxidative coupling product of DMDC, could not be quantified with this method. Thiram was reduced during the course of the chromatographic analysis resulting in a strongly tailing peak exhibiting the same UV-visible spectrum as the CuDMDC+ complex. EBDC and EX may be preconcentrated using the same method as outlined for DMDC. Since the Cu(II) complexes of these ligands are more hydrophobic than the CuDMDC+-HSA ion pair and elute later in the chromatograms, interference from earlyeluting system peaks was smaller and recoveries for EBDC and EX were better than for DMDC. Recoveries calculated for 2-mL river water samples fortified with 50 nM EBDC and EX were 90 ( 10 and 65 ( 5%, respectively. Detection Limits. River and laboratory water samples were spiked with 40-500 nM DMDC, DEDC, EBDC, and EX. Over this concentration range, the compounds exhibited linear calibration graphs with R2 values between 0.977 and 0.999. The slope of the calibration curves (response) was very similar for river waters and buffered lab water. The values for the method detection limit in laboratory water and in natural river water samples (Table 1) were calculated on the basis of a S/N of 3 using water samples fortified with 40 nM of each compound. Precision of the method was measured as relative standard deviation (RSD) based on injection of seven samples at one concentration level near the detection limit. ACKNOWLEDGMENT We are indebted to J. T. Phinney, G. Miller, and J. Miller for valuable discussions and comments. We thank W. W. Bedsworth and C.-H. Huang for reviewing the manuscript. This work was partially funded by a grant from the University of California Toxic Substances Research and Teaching Program. K.W.W. gratefully acknowledges financial support by the Swiss National Science Foundation. Received for review June 8, 1998. Accepted August 25, 1998. AC980626W
