This article was downloaded by: [Universite Laval] On: 14 June 2014, At: 08:14 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Monitoring dithiocarbamate fungicide residues in greenhouse and non-greenhouse tomatoes in Iran by HPLC-UV A. Jafari
a b
b
c
b
b
a
, Sh. Shoeibi , M. Amini , M. Amirahmadi , H. Rastegar , A. Ghaffarian &
M. Ghazi-Khansari
a
a
Department of Pharmacology , School of Medicine, Tehran University of Medical Sciences , Tehran , Iran b
Food and Drug Laboratory Research Center (FDLRC), Ministry of Health , Tehran , Iran
c
Department of Medicinal Chemistry , School of Pharmacy, Tehran University of Medical Sciences , Tehran , Iran Accepted author version posted online: 13 Jan 2012.Published online: 09 Mar 2012.
To cite this article: A. Jafari , Sh. Shoeibi , M. Amini , M. Amirahmadi , H. Rastegar , A. Ghaffarian & M. Ghazi-Khansari (2012) Monitoring dithiocarbamate fungicide residues in greenhouse and non-greenhouse tomatoes in Iran by HPLC-UV, Food Additives & Contaminants: Part B: Surveillance, 5:2, 87-92, DOI: 10.1080/19393210.2012.657693 To link to this article: http://dx.doi.org/10.1080/19393210.2012.657693
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives and Contaminants: Part B Vol. 5, No. 2, June 2012, 87–92
Monitoring dithiocarbamate fungicide residues in greenhouse and non-greenhouse tomatoes in Iran by HPLC-UV A. Jafariab, Sh. Shoeibib, M. Aminic, M. Amirahmadib, H. Rastegarb, A. Ghaffariana and M. Ghazi-Khansaria* a Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; bFood and Drug Laboratory Research Center (FDLRC), Ministry of Health, Tehran, Iran; cDepartment of Medicinal Chemistry, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Downloaded by [Universite Laval] at 08:14 14 June 2014
(Received 13 April 2011; final version received 11 January 2012) The simultaneous analysis of dithiocarbamate fungicide residues on greenhouse and non-greenhouse tomatoes was performed by high-performance liquid chromatography with UV detection. A calibration curve with spiked samples was plotted to determine dithiocarbamate residues in tomato samples. Limits of detection and quantification were approximately 0.05 and 0.2 mg g1, respectively. The ranges of mean recoveries at five spiking levels for all dithiocarbamates of interest were between 88.2%–95.7% and 99.5%–100.8% in standards and spiked samples, respectively. In this study, 40 greenhouse and 40 non-greenhouse tomato samples were analysed. None of the samples analysed contained dithiocarbamates in excess of the maximum residue levels established by the Codex Committee on Pesticide Residues, except for one greenhouse sample, with ethylenebisdithiocarbamates at 3.2 mg g1. Keywords: dithiocarbamate; fungicide; tomato; HPLC
Introduction Dithiocarbamates (DTCs) are nonsystemic fungicides that are widely used in Iran and other countries because of their broad spectrum of activity against plant pathogens, low mammal toxicity and low production costs. In addition, DTCs are used in the rubber industry as vulcanisation accelerators and antioxidants (Malik and Faubel 1999). DTCs are divided into three subclasses, that is, dimethyldithiocarbamates (DMDs), such as thiram, ziram and ferbam; ethylenebisdithiocarbamates (EBDs), such as mancozeb, zineb and maneb; and propylenebisdithiocarbamates (PBDs), such as propineb. Because of toxicologic differences (Crnogorac et al. 2008), an analytical method is required that can distinguish between DTC subclasses. The most common methods that are used for analysis of DTC residues are based on the decomposition to carbon disulfide (CS2) in hot acid, followed by spectrometry and headspace gas chromatography (Keppel 1971; Malik and Faubel 1999), but these methods are difficult, time consuming and, especially, they cannot differentiate between DTC subclasses. Also, CS2 can release from some plant matrices, thus leading to false-positive results (Crnogorac et al. 2008). Several methods are available to directly determine intact DTCs and to distinguish between *Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/19393210.2012.657693 http://www.tandfonline.com
their subclasses. They include liquid chromatography/ electrospray ionisation mass spectrometry (LC/ESI-MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (Crnogorac and Schwack 2007; Crnogorac et al. 2008), liquid chromatography–atmospheric pressure chemical ionisation mass spectroscopy (LC-APCI-MS) for simultaneous determination of dithiocarbamates and their metabolites in plant (Blasco et al. 2004), liquid chromatography with tandem mass spectrometry for determination of ethylenebisdithiocarbamate fungicides in fruit and vegetables (Hayama and Takada 2008), liquid chromatography with ultraviolet detection (Gustafsson and Fahlgren 1983; Brandsteterova et al. 1986; Aulakh et al. 2004; Garcinuno et al. 2004; Codex Alimentarius 2007), liquid chromatography and atomic absorption method (Lo et al. 1996) and liquid chromatography with spectrometry and electrochemical detection (Bardarov and Zaikov 1989). Although any LC/MS method is more sensitive and selective when compared to other methods, it is a facility which is not easily accessible for most laboratories. GC methods are sensitive but cannot identify the exact source of the detected CS2. In this study, a simple and inexpensive liquid chromatographic method with ultraviolet detection at 272 nm (HPLC-UV) for the simultaneous determination of
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each of the subclasses was validated and used to determine DTCs in tomato. Because of different production conditions of greenhouse and non-greenhouse tomatoes, most people consider greenhouse tomatoes (like other greenhouse products) to have fungicide residues (such as DTCs) more than non-greenhouse tomatoes. Therefore, a comparison was made between these two types of products.
These solutions were freshly prepared before use. Every day a mixed standard solution with concentrations of 40 mg mL1 for EBDs (mancozeb) and PBDs (propineb) and 20 mgmL1 for DMDs (thiram) was methylated, based on the method of Gustafsson and Fahlgren (1983), then diluted in the range 1, 2, 4, 8, 16 and 40 mgmL1 for mancozeb and propineb and 0.5, 1, 2, 4, 8 and 20 mgmL1 for thiram by adding methanol.
Sample extraction
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Materials and methods
One hundred millilitres of EDTA solution (0.25 M) and 0.5 g of L-cysteine were added to 20 g of each tomato sample that was previously cut into 15–20 pieces and shaken for 10 min in a closed glass container. The extract was centrifuged for 5 min at 3400 rpm and then filtered through a glass-fibre filter. The container and the filter were rinsed with 20 mL of the EDTA solution and combined with the extract. Five millilitres of tetrabutylammonium hydrogen sulfate (0.41 M) and 10 g of sodium chloride was added into the extracts, while stirring. The pH of the extracts was adjusted to 7.0–8.0 by adding 2 M hydrochloric acid. Forty millilitres of 0.05 M iodomethane in dichloromethane-hexane (1:1) was added to the extracts. The mixture was strongly shaken for 10 min and the organic phase was separated. Five millilitres of 1,2-propanediol in dichloromethane (20%) was added to 30 mL of organic phase and well mixed. The solvent was removed at 30 C under reduced pressure in a rotary evaporator. Methanol (500 mL) was added to the dried residue and 20 mL was injected into the HPLC and analysed at 272 nm. Eighty tomato samples (1–2 kg) were collected from Tehran Central Fruits and Vegetables Market in 2010. Tomato samples from four different cities were prepared. Half of them were greenhouse products and the other half were non-greenhouse tomatoes. In the same way, DTC residues in tomatoes were monitored, in order that a comparison could be made between DTC residues in greenhouse and non-greenhouse tomatoes.
Reagents Mancozeb 72%, propineb 78% and thiram 99% were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Ethylenediaminetetraacetic acid, tetrasodium salt dihydrate 99%, tetrabutyl ammonium hydrogen sulfate 97% and L-cysteine hydrochloride anhydrous 98% were obtained from Sigma-Aldrich (St. Louis, MO, USA). Iodomethane 99%, 1,2propanediol 99%, dichloromethane 99%, hexane 99.9%, hydrochloric acid 37%, sodium hydroxide 99%, sodium chloride 99%, methanol (HPLC grade), acetonitrile (HPLC grade) and sulfuric acid 99% were purchased from Merck (Darmstadt, Germany). All chemicals were analytical grade.
Apparatus The HPLC system consisted of a Waters 600 controller equipped with a UV detector (2487, Dual Absorbance Detector) set at a wavelength of 272 nm (Waters, Milford, MA, USA) and a C18 reversed-phase 250 4-mm column. The mobile phase was acetonitrile–water–methanol (25:65:35) with a flow rate of 1.0 mL min1. A centrifuge (Heraeus Biofuge stratos, Germany), an orbital shaker (Gallen-kamp & Co. Ltd., Widnes, UK) and a rotatory evaporator (Bu¨chi B-490 Heating Baths, Germany) were used. Extracts were filtered through glass-fibre filters.
Standards Stock solutions of DTCs were prepared at 0.4 mg mL1 (mancozeb and propineb) and 0.2 mg mL1 (thiram) in an alkaline solution of EDTA (0.25 M).
Method validation For calibration purposes, a set of six standard solutions was prepared in quadruplicate ranging from
Table 1. Calibration curves of thiram, mancozeb and propineb obtained from standard and spiked tomatoes. Analyte
Calibration
Thiram
Standard Spike Standard Spike Standard Spike
Mancozeb Propineb
Range 0.5–20.0 0.125–1.5 1.0–40.0 0.25–3.0 1.0–40.0 0.25–3.0
(mg (mg (mg (mg (mg (mg
mL1) g1) mL1) g1) mL1) g1)
Regression equation
R2
y ¼ 2648.9x þ 5986.3 y ¼ 2553.3x þ 6872 y ¼ 2041.9x – 3518.2 y ¼ 1869.8x – 16815 y ¼ 1433.9x þ 6867.8 y ¼ 1302.1x – 482.76
0.9998 0.9992 0.9999 0.9976 0.9997 0.9998
89
Food Additives and Contaminants: Part B 1 to 40 mgmL1 for mancozeb and propineb and 0.5 to 20 mgmL1 for thiram. Limits of detection (LODs) and quantification (LOQs) for each DTC were calculated from spiked tomato blanks. LOD being three times baseline noise and LOQ in turn three times the LOD value. These experiments were also performed in quadruplicate. To determine recovery, blank tomatoes were spiked at five levels by adding dropwise a mixed fungicide solution with an Eppendorf pipette. These samples were maintained at room temperature for 1 hour before extraction to allow the solution to penetrate the test tomatoes. The spiked blanks were
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Table 2. Accuracy and precision data (n ¼ 4).
Analyte Thiram
Mancozeb
Propineb
Spiking level (mg g1)
Found SD (mg g1)
RSD%
Accuracy
0.125 0.25 0.5 1.0 1.5 0.25 0.5 1.0 2.0 3.0 0.25 0.5 1.0 2.0 3.0
0.122 0.01 0.24 0.01 0.50 0.01 1.02 0.06 1.48 0.08 0.26 0.04 0.50 0.06 0.92 0.09 2.07 0.13 2.96 0.15 0.26 0.01 0.51 0.02 0.97 0.05 2.0 0.10 3.0 0.09
8.2 4.2 2.0 5.9 5.4 15 12 9.7 6.2 5.0 3.8 3.9 5.2 5.0 3.0
97.6 96.0 100 102 98.7 100.4 100 92 96.5 98.7 104 102 97 100 100
analysed as described, and thus recoveries at five spiking levels for all of the DTCs were obtained.
Results and discussion The obtained calibration curves for pure standards of EBDs (mancozeb), PBDs (propineb) and DMDs (thiram) were linear, with correlation coefficients above 0.999 (n ¼ 4). In addition, calibration curves of spiked tomato blanks in the range 0.25–3.0 mg g1 showed good linearity with correlation coefficients above 0.997 (Table 1). Results of precision and accuracy are presented in Table 2. The relative standard deviations (RSDs) were lower than 15% in all items, which is in compliance with the recommendations as defined by the European Community (EC 2009). Recoveries were measured in a range of 0.25– 3.0 mg g1 for mancozeb and propineb and 0.125– 1.5 mg g1 for thiram. As shown in Table 3, recovery rates for all dithiocarbamates were between 88.2– 95.7% and 99.5–100.8% for standard and spike calibration curves, respectively. LOQ is defined as the lowest analyte concentration that can be determined with an accuracy of 70%–120% and a precision better than 20% (EC 2009). LOD was 0.05 mg g1 for mancozeb and propineb and 0.01 mg g1 for thiram. LOQ in spiked tomato samples was 0.2 mg g1 for mancozeb and propineb and 0.05 mg g1 for thiram (Table 4). Table 5 shows the analytical results of levels for each of the DTC subclasses in the investigated tomato samples. DMDs were detected in 76.2% of the 80 samples and showed not much difference between DMD residues in greenhouse (72.5%) and non-greenhouse tomatoes (80%). EBD residues were detected in
Table 3. Mean recovery data (n ¼ 4) obtained by comparing standards and spiked tomato samples. Mean recovery (n ¼ 4)
RSD%
Analyte
Spiking levels (mg g1)
Standard sample calibration curve
Spiked sample calibration curve
Standard sample calibration curve
Spiked sample calibration curve
Thiram Mancozeb Propineb
0.125–1.5 0.25–3.0 0.25–3.0
95.7 88.3 88.3
99.6 100.8 100.8
6.7 4.2 4.3
7.6 5.8 4.6
Table 4. Limits of detection (LODs) and quantiEcation (LOQs) as determined in tomatoes. Analyte (mg g1) LOD LOQ
Thiram
Mancozeb
Propineb
0.01 0.05
0.05 0.20
0.05 0.20
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A. Jafari et al.
Table 5. Analytical results for the investigated 80 tomato samples, Iran, 2010. No. of samples containing DBDs (thiram)
Samples (n) Greenhouse tomatoes (40) Non-greenhouse tomatoes (40) All tomatoes (80)
No. of samples containing EBDs (mancozeb)
LOD
LOQ
Meanb of DMD residues
29
–
0.025
1
2
1
0.20
LOD
0.20
32
1
0.025
3
0
0
0.10
LOD
0.09
60
1
0.025
4
2
1
0.15
LOD
0.14
LOQ
LOQ
MRLa
Meanb of EBD residues
PBDs (propineb)
Meanb of DTC residues
a
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Notes: MRLs issued by the Codex Committee of Pesticide Residues. b To calculate the mean, samples at 5LOQ were considered as 1/2 LOQ.
Figure 1. HPLC chromatograms (UV detector set at 272 nm): (a) Standards (16 mg mL1 for EBDs and PBDs and 8 mg mL1 for DMDs). DMD (dimethyldithiocarbamate) such as thiram, ziram and ferbam (retention time 9 0.2 min), EBD (ethylenebisdithiocarbamate) such as mancozeb, maneb and zineb (retention time 13 0.3 min) and PBD (propylenebisdithiocarbamate) such as propineb (retention time 16 0.3 min); (b) Tomato sample contaminated with EBD (3.2 mg g1).
3 (7.5%) of 40 non-greenhouse samples. In 4 (10%) greenhouse tomato samples, EBD was detected. One residue was 3.2 mg g1, which is above the maximum residue level (MRL). No PBD residues were detected. In general, the mean DTC level in the 80 tomato samples analysed was 0.14 mg g1. Typical chromatograms are presented in Figure 1. Part (a) is a
chromatogram obtained after injecting 20 mL standard solution with DTC concentrations of 16 mg mL1 EBDs and PBDs and 8 mg mL1 DMDs. Part (b) is the chromatogram from the positive sample. Most dithiocarbamate residue monitoring and survey program samples were analysed via CS2, generated after hydrolysis of DTCs (Dogheim et al. 1999;
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Food Additives and Contaminants: Part B Ripley et al. 2000; Berger and Von Holst 2001; Dogheim et al. 2001; Dogheim et al. 2002; Caldas et al. 2004; Chang et al. 2005; Basa Cesnik et al. 2006), but this method cannot identify the origin of the detected CS2, which may or may not be related to the presence of DTCs. In our study, we were able to not only validate the method for tomato but also to determine intact DTCs and to distinguish between DTC subclasses. In Iran, of dithiocarbamate fungicides only mancozeb, maneb and zineb are used for tomato production, and MRLs apply to total residues from the use of any or each of the groups of dithiocarbamates (MRL ¼ 2 mg g1). In the current study, a simple and accurate method was used to determine each separately accessible DTC. The representative chromatogram in Figure 1a illustrates good separation between DMD, EBD, PBD and also from unknown peaks related to the matrix. A comparison between greenhouse and nongreenhouse tomatoes showed that results for DMD (thiram) residues were almost similar to each other. EBD (mancozeb) residues in greenhouse tomatoes were higher than non-greenhouse. It seems that the high levels of EBD residues in greenhouse tomatoes is due to their particular product conditions and higher use of these fungicides to control pathogens in the greenhouse. One of the greenhouse tomato samples had an EBD (mancozeb) residue level above the MRL (up to 3.2 mg g1). Comparing the results obtained in Iran with those in Brazil, the percentage of tomato samples containing detectable residues (40.01 mg g1) of 81.2% in Iran was higher than those in Brazil (60.8%), but the mean DTC residue level of 0.31 mg g1 in Brazilian samples (Caldas et al. 2004) was higher than Iranian samples in this study (0.14 mg g1). Another study in Egypt also showed higher values than Iran, although DTC residues in Egyptian tomatoes decreased from 1995 to 1997 (Dogheim et al. 1999; Dogheim et al. 2001; Dogheim et al. 2002). Monitoring programs carried out in other countries, such as European Union, Canada, Taiwan and Slovenia, showed that DTCs are some of the most frequently detected pesticides, like in Iran (Ripley et al. 2000; Chang et al. 2005; Basa Cesnik et al. 2006).
Conclusion According to the studied parameters in this work, the presented method is suitable to simultaneously determine residues of the three main subclasses of dithiocarbamate fungicides in tomatoes. Although the mean DTC residues found in tomato samples were lower than the maximum level as recommended by the Codex Alimentarius, one greenhouse tomato sample exceeded the DTC residue MRL, which indicates the need for regular product monitoring.
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Acknowledgements This study was supported by the Food and Drug Laboratory Research Center (FDLRC), Iran Ministry of Health and Medical Education. The authors are grateful to the staff of the Toxicology Laboratory, FDLRC.
References Aulakh JS, Sharma VK, Mahajan RK, Malik AK. 2004. HPLC-UV Determination of tetramethylthiuram disulphide (thiram) and thiourea in wheat grains and in a commercial sample. Electron J Environ Agric Food Chem. 3:751–757. Bardarov V, Zaikov C. 1989. Application of highperformance liquid chromatography with spectrophotometric and electrochemical detection to the analysis of alkylene bis(dithiocarbamates) and their metabolites. J Chromatogr. 419:97–105. Basa Cesnik H, Gregorcic A, Velikonja Bolta S, Kmecl V. 2006. Monitoring of pesticide residues in apples, lettuce and potato of the Slovene origin, 2001–04. Food Addit Contam. 23:164–173. Berger B, Von Holst C. 2001. Pesticide residues in products of plant origin in the European Union. Environ Sci Pollut Res. 8:109–112. Blasco C, Font G, Pico Y. 2004. Determination of dithiocarbamates and metabolites in plants by liquid chromatography—mass spectrometry. J Chromatogr A. 1028:267–276. Brandsteterova E, Lehotay J, Liska O, Garaj J, Zacsik I. 1986. High-performance liquid chromatographic determination of dimethyldithiocarbamate residue in some agricultural product. J Chromatogr. 354:375–381. Caldas ED, Conceicao MH, Miranda MCC, Souza LCKR. 2004. Dithiocarbamates residues in Brazilian food and the potential risk for consumers. Food Chem Toxicol. 42:1877–1883. Chang JM, Chen TH, Fang TJ. 2005. Pesticide residue monitoring in marketed fresh vegetables and fruits in central Taiwan (1999–2004) and an introduction to the HACCP system. J Food Drug Anal. 13:368–376. Codex Alimentarius. 2007. Report of the Thirty-ninth Session of the Codex Committee on Pesticide Residues. Beijing, China. 7–12 May 2007. Crnogorac G, Schwack W. 2007. Determination of dithiocarbamate fungicide residues by liquid chromatography/ mass spectrometry and stable isotope dilution assay. Rapid Commun Mass Spectrom. 21:4009–4016. Crnogorac G, Schwack W, Schmauder S. 2008. Trace analysis of dithiocarbamate fungicide residues on fruits and vegetables by hydrophilic interaction liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 22:2539–2546. Dogheim SM, El-Marsafy AM, Salama EY, Gadalla SA, Nabil YM. 2002. Monitoring of pesticide residues in Egyptian fruits and vegetables during 1997. Food Addit Contam. 19:1015–1027. Dogheim SM, Gadalla SA, El-Marsafy AM. 1999. Monitoring pesticide residues in Egyptian fruits and vegetables in 1995. J AOAC. 82:948–955.
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Dogheim SM, Gadalla SA, El-Marsafy AM. 2001. Monitoring of pesticide residues in Egyptian fruits and vegetables during 1996. J AOAC. 84:519–531. European Commission [EC]. 2009. Method validation and quality control procedures for pesticide residues analysis in food and feed, Document No. SANCO/10684/2009. Available from: http://ec.europa.eu/food/plant/protection/resources/qualcontrol_en.pdf Garcinuno RM, Fernandez-Hernando P, Camara C. 2004. Simultaneous determination of maneb and its main metabolites in tomatoes by liquid chromatography using diode array ultraviolet absorbance detection. J Chromatogr A. 1043:225–229. Gustafsson KH, Fahlgren CH. 1983. Determination of dithiocarbamate fungicides in vegetable foodstuffs by high-performance liquid chromatography. J Agric Food Chem. 31:461–463.
Hayama T, Takada M. 2008. Simple and rapid method for the determination of ethylenebisdithiocarbamate fungicides in fruits by tandem mass spectrometry. Anal Bioanal Chem. 392:969–976. Keppel GE. 1971. Collaborative study of the determination of dithiocarbamates, residues by a modified carbon disulfide evolution method. J AOAC. 54: 528–532. Lo CC, Ho MH, Hung MD. 1996. Use of high-performance liquid chromatographic and atomic absorption methods to distinguish propineb, zineb, maneb, and mancozeb fungicides. J Agric Food Chem. 44:2720–2723. Malik AK, Faubel W. 1999. Methods of analysis of dithiocarbamate: a review. Pestic Sci. 55:965–970. Ripley BD, Lissemore LI, Leishman PD, Denomme´ MA. 2000. Pesticide residues on fruits and vegetables from Ontario, Canada, 1991–1995. J AOAC. 83:196–297.
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Monitoring dithiocarbamate fungicide residues in greenhouse and non-greenhouse tomatoes in Iran by HPLC-UV A. Jafari
a b
b
c
b
b
a
, Sh. Shoeibi , M. Amini , M. Amirahmadi , H. Rastegar , A. Ghaffarian &
M. Ghazi-Khansari
a
a
Department of Pharmacology , School of Medicine, Tehran University of Medical Sciences , Tehran , Iran b
Food and Drug Laboratory Research Center (FDLRC), Ministry of Health , Tehran , Iran
c
Department of Medicinal Chemistry , School of Pharmacy, Tehran University of Medical Sciences , Tehran , Iran Accepted author version posted online: 13 Jan 2012.Published online: 09 Mar 2012.
To cite this article: A. Jafari , Sh. Shoeibi , M. Amini , M. Amirahmadi , H. Rastegar , A. Ghaffarian & M. Ghazi-Khansari (2012) Monitoring dithiocarbamate fungicide residues in greenhouse and non-greenhouse tomatoes in Iran by HPLC-UV, Food Additives & Contaminants: Part B: Surveillance, 5:2, 87-92, DOI: 10.1080/19393210.2012.657693 To link to this article: http://dx.doi.org/10.1080/19393210.2012.657693
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives and Contaminants: Part B Vol. 5, No. 2, June 2012, 87–92
Monitoring dithiocarbamate fungicide residues in greenhouse and non-greenhouse tomatoes in Iran by HPLC-UV A. Jafariab, Sh. Shoeibib, M. Aminic, M. Amirahmadib, H. Rastegarb, A. Ghaffariana and M. Ghazi-Khansaria* a Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; bFood and Drug Laboratory Research Center (FDLRC), Ministry of Health, Tehran, Iran; cDepartment of Medicinal Chemistry, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Downloaded by [Universite Laval] at 08:14 14 June 2014
(Received 13 April 2011; final version received 11 January 2012) The simultaneous analysis of dithiocarbamate fungicide residues on greenhouse and non-greenhouse tomatoes was performed by high-performance liquid chromatography with UV detection. A calibration curve with spiked samples was plotted to determine dithiocarbamate residues in tomato samples. Limits of detection and quantification were approximately 0.05 and 0.2 mg g1, respectively. The ranges of mean recoveries at five spiking levels for all dithiocarbamates of interest were between 88.2%–95.7% and 99.5%–100.8% in standards and spiked samples, respectively. In this study, 40 greenhouse and 40 non-greenhouse tomato samples were analysed. None of the samples analysed contained dithiocarbamates in excess of the maximum residue levels established by the Codex Committee on Pesticide Residues, except for one greenhouse sample, with ethylenebisdithiocarbamates at 3.2 mg g1. Keywords: dithiocarbamate; fungicide; tomato; HPLC
Introduction Dithiocarbamates (DTCs) are nonsystemic fungicides that are widely used in Iran and other countries because of their broad spectrum of activity against plant pathogens, low mammal toxicity and low production costs. In addition, DTCs are used in the rubber industry as vulcanisation accelerators and antioxidants (Malik and Faubel 1999). DTCs are divided into three subclasses, that is, dimethyldithiocarbamates (DMDs), such as thiram, ziram and ferbam; ethylenebisdithiocarbamates (EBDs), such as mancozeb, zineb and maneb; and propylenebisdithiocarbamates (PBDs), such as propineb. Because of toxicologic differences (Crnogorac et al. 2008), an analytical method is required that can distinguish between DTC subclasses. The most common methods that are used for analysis of DTC residues are based on the decomposition to carbon disulfide (CS2) in hot acid, followed by spectrometry and headspace gas chromatography (Keppel 1971; Malik and Faubel 1999), but these methods are difficult, time consuming and, especially, they cannot differentiate between DTC subclasses. Also, CS2 can release from some plant matrices, thus leading to false-positive results (Crnogorac et al. 2008). Several methods are available to directly determine intact DTCs and to distinguish between *Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/19393210.2012.657693 http://www.tandfonline.com
their subclasses. They include liquid chromatography/ electrospray ionisation mass spectrometry (LC/ESI-MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (Crnogorac and Schwack 2007; Crnogorac et al. 2008), liquid chromatography–atmospheric pressure chemical ionisation mass spectroscopy (LC-APCI-MS) for simultaneous determination of dithiocarbamates and their metabolites in plant (Blasco et al. 2004), liquid chromatography with tandem mass spectrometry for determination of ethylenebisdithiocarbamate fungicides in fruit and vegetables (Hayama and Takada 2008), liquid chromatography with ultraviolet detection (Gustafsson and Fahlgren 1983; Brandsteterova et al. 1986; Aulakh et al. 2004; Garcinuno et al. 2004; Codex Alimentarius 2007), liquid chromatography and atomic absorption method (Lo et al. 1996) and liquid chromatography with spectrometry and electrochemical detection (Bardarov and Zaikov 1989). Although any LC/MS method is more sensitive and selective when compared to other methods, it is a facility which is not easily accessible for most laboratories. GC methods are sensitive but cannot identify the exact source of the detected CS2. In this study, a simple and inexpensive liquid chromatographic method with ultraviolet detection at 272 nm (HPLC-UV) for the simultaneous determination of
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each of the subclasses was validated and used to determine DTCs in tomato. Because of different production conditions of greenhouse and non-greenhouse tomatoes, most people consider greenhouse tomatoes (like other greenhouse products) to have fungicide residues (such as DTCs) more than non-greenhouse tomatoes. Therefore, a comparison was made between these two types of products.
These solutions were freshly prepared before use. Every day a mixed standard solution with concentrations of 40 mg mL1 for EBDs (mancozeb) and PBDs (propineb) and 20 mgmL1 for DMDs (thiram) was methylated, based on the method of Gustafsson and Fahlgren (1983), then diluted in the range 1, 2, 4, 8, 16 and 40 mgmL1 for mancozeb and propineb and 0.5, 1, 2, 4, 8 and 20 mgmL1 for thiram by adding methanol.
Sample extraction
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Materials and methods
One hundred millilitres of EDTA solution (0.25 M) and 0.5 g of L-cysteine were added to 20 g of each tomato sample that was previously cut into 15–20 pieces and shaken for 10 min in a closed glass container. The extract was centrifuged for 5 min at 3400 rpm and then filtered through a glass-fibre filter. The container and the filter were rinsed with 20 mL of the EDTA solution and combined with the extract. Five millilitres of tetrabutylammonium hydrogen sulfate (0.41 M) and 10 g of sodium chloride was added into the extracts, while stirring. The pH of the extracts was adjusted to 7.0–8.0 by adding 2 M hydrochloric acid. Forty millilitres of 0.05 M iodomethane in dichloromethane-hexane (1:1) was added to the extracts. The mixture was strongly shaken for 10 min and the organic phase was separated. Five millilitres of 1,2-propanediol in dichloromethane (20%) was added to 30 mL of organic phase and well mixed. The solvent was removed at 30 C under reduced pressure in a rotary evaporator. Methanol (500 mL) was added to the dried residue and 20 mL was injected into the HPLC and analysed at 272 nm. Eighty tomato samples (1–2 kg) were collected from Tehran Central Fruits and Vegetables Market in 2010. Tomato samples from four different cities were prepared. Half of them were greenhouse products and the other half were non-greenhouse tomatoes. In the same way, DTC residues in tomatoes were monitored, in order that a comparison could be made between DTC residues in greenhouse and non-greenhouse tomatoes.
Reagents Mancozeb 72%, propineb 78% and thiram 99% were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Ethylenediaminetetraacetic acid, tetrasodium salt dihydrate 99%, tetrabutyl ammonium hydrogen sulfate 97% and L-cysteine hydrochloride anhydrous 98% were obtained from Sigma-Aldrich (St. Louis, MO, USA). Iodomethane 99%, 1,2propanediol 99%, dichloromethane 99%, hexane 99.9%, hydrochloric acid 37%, sodium hydroxide 99%, sodium chloride 99%, methanol (HPLC grade), acetonitrile (HPLC grade) and sulfuric acid 99% were purchased from Merck (Darmstadt, Germany). All chemicals were analytical grade.
Apparatus The HPLC system consisted of a Waters 600 controller equipped with a UV detector (2487, Dual Absorbance Detector) set at a wavelength of 272 nm (Waters, Milford, MA, USA) and a C18 reversed-phase 250 4-mm column. The mobile phase was acetonitrile–water–methanol (25:65:35) with a flow rate of 1.0 mL min1. A centrifuge (Heraeus Biofuge stratos, Germany), an orbital shaker (Gallen-kamp & Co. Ltd., Widnes, UK) and a rotatory evaporator (Bu¨chi B-490 Heating Baths, Germany) were used. Extracts were filtered through glass-fibre filters.
Standards Stock solutions of DTCs were prepared at 0.4 mg mL1 (mancozeb and propineb) and 0.2 mg mL1 (thiram) in an alkaline solution of EDTA (0.25 M).
Method validation For calibration purposes, a set of six standard solutions was prepared in quadruplicate ranging from
Table 1. Calibration curves of thiram, mancozeb and propineb obtained from standard and spiked tomatoes. Analyte
Calibration
Thiram
Standard Spike Standard Spike Standard Spike
Mancozeb Propineb
Range 0.5–20.0 0.125–1.5 1.0–40.0 0.25–3.0 1.0–40.0 0.25–3.0
(mg (mg (mg (mg (mg (mg
mL1) g1) mL1) g1) mL1) g1)
Regression equation
R2
y ¼ 2648.9x þ 5986.3 y ¼ 2553.3x þ 6872 y ¼ 2041.9x – 3518.2 y ¼ 1869.8x – 16815 y ¼ 1433.9x þ 6867.8 y ¼ 1302.1x – 482.76
0.9998 0.9992 0.9999 0.9976 0.9997 0.9998
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Food Additives and Contaminants: Part B 1 to 40 mgmL1 for mancozeb and propineb and 0.5 to 20 mgmL1 for thiram. Limits of detection (LODs) and quantification (LOQs) for each DTC were calculated from spiked tomato blanks. LOD being three times baseline noise and LOQ in turn three times the LOD value. These experiments were also performed in quadruplicate. To determine recovery, blank tomatoes were spiked at five levels by adding dropwise a mixed fungicide solution with an Eppendorf pipette. These samples were maintained at room temperature for 1 hour before extraction to allow the solution to penetrate the test tomatoes. The spiked blanks were
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Table 2. Accuracy and precision data (n ¼ 4).
Analyte Thiram
Mancozeb
Propineb
Spiking level (mg g1)
Found SD (mg g1)
RSD%
Accuracy
0.125 0.25 0.5 1.0 1.5 0.25 0.5 1.0 2.0 3.0 0.25 0.5 1.0 2.0 3.0
0.122 0.01 0.24 0.01 0.50 0.01 1.02 0.06 1.48 0.08 0.26 0.04 0.50 0.06 0.92 0.09 2.07 0.13 2.96 0.15 0.26 0.01 0.51 0.02 0.97 0.05 2.0 0.10 3.0 0.09
8.2 4.2 2.0 5.9 5.4 15 12 9.7 6.2 5.0 3.8 3.9 5.2 5.0 3.0
97.6 96.0 100 102 98.7 100.4 100 92 96.5 98.7 104 102 97 100 100
analysed as described, and thus recoveries at five spiking levels for all of the DTCs were obtained.
Results and discussion The obtained calibration curves for pure standards of EBDs (mancozeb), PBDs (propineb) and DMDs (thiram) were linear, with correlation coefficients above 0.999 (n ¼ 4). In addition, calibration curves of spiked tomato blanks in the range 0.25–3.0 mg g1 showed good linearity with correlation coefficients above 0.997 (Table 1). Results of precision and accuracy are presented in Table 2. The relative standard deviations (RSDs) were lower than 15% in all items, which is in compliance with the recommendations as defined by the European Community (EC 2009). Recoveries were measured in a range of 0.25– 3.0 mg g1 for mancozeb and propineb and 0.125– 1.5 mg g1 for thiram. As shown in Table 3, recovery rates for all dithiocarbamates were between 88.2– 95.7% and 99.5–100.8% for standard and spike calibration curves, respectively. LOQ is defined as the lowest analyte concentration that can be determined with an accuracy of 70%–120% and a precision better than 20% (EC 2009). LOD was 0.05 mg g1 for mancozeb and propineb and 0.01 mg g1 for thiram. LOQ in spiked tomato samples was 0.2 mg g1 for mancozeb and propineb and 0.05 mg g1 for thiram (Table 4). Table 5 shows the analytical results of levels for each of the DTC subclasses in the investigated tomato samples. DMDs were detected in 76.2% of the 80 samples and showed not much difference between DMD residues in greenhouse (72.5%) and non-greenhouse tomatoes (80%). EBD residues were detected in
Table 3. Mean recovery data (n ¼ 4) obtained by comparing standards and spiked tomato samples. Mean recovery (n ¼ 4)
RSD%
Analyte
Spiking levels (mg g1)
Standard sample calibration curve
Spiked sample calibration curve
Standard sample calibration curve
Spiked sample calibration curve
Thiram Mancozeb Propineb
0.125–1.5 0.25–3.0 0.25–3.0
95.7 88.3 88.3
99.6 100.8 100.8
6.7 4.2 4.3
7.6 5.8 4.6
Table 4. Limits of detection (LODs) and quantiEcation (LOQs) as determined in tomatoes. Analyte (mg g1) LOD LOQ
Thiram
Mancozeb
Propineb
0.01 0.05
0.05 0.20
0.05 0.20
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Table 5. Analytical results for the investigated 80 tomato samples, Iran, 2010. No. of samples containing DBDs (thiram)
Samples (n) Greenhouse tomatoes (40) Non-greenhouse tomatoes (40) All tomatoes (80)
No. of samples containing EBDs (mancozeb)
LOD
LOQ
Meanb of DMD residues
29
–
0.025
1
2
1
0.20
LOD
0.20
32
1
0.025
3
0
0
0.10
LOD
0.09
60
1
0.025
4
2
1
0.15
LOD
0.14
LOQ
LOQ
MRLa
Meanb of EBD residues
PBDs (propineb)
Meanb of DTC residues
a
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Notes: MRLs issued by the Codex Committee of Pesticide Residues. b To calculate the mean, samples at 5LOQ were considered as 1/2 LOQ.
Figure 1. HPLC chromatograms (UV detector set at 272 nm): (a) Standards (16 mg mL1 for EBDs and PBDs and 8 mg mL1 for DMDs). DMD (dimethyldithiocarbamate) such as thiram, ziram and ferbam (retention time 9 0.2 min), EBD (ethylenebisdithiocarbamate) such as mancozeb, maneb and zineb (retention time 13 0.3 min) and PBD (propylenebisdithiocarbamate) such as propineb (retention time 16 0.3 min); (b) Tomato sample contaminated with EBD (3.2 mg g1).
3 (7.5%) of 40 non-greenhouse samples. In 4 (10%) greenhouse tomato samples, EBD was detected. One residue was 3.2 mg g1, which is above the maximum residue level (MRL). No PBD residues were detected. In general, the mean DTC level in the 80 tomato samples analysed was 0.14 mg g1. Typical chromatograms are presented in Figure 1. Part (a) is a
chromatogram obtained after injecting 20 mL standard solution with DTC concentrations of 16 mg mL1 EBDs and PBDs and 8 mg mL1 DMDs. Part (b) is the chromatogram from the positive sample. Most dithiocarbamate residue monitoring and survey program samples were analysed via CS2, generated after hydrolysis of DTCs (Dogheim et al. 1999;
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Food Additives and Contaminants: Part B Ripley et al. 2000; Berger and Von Holst 2001; Dogheim et al. 2001; Dogheim et al. 2002; Caldas et al. 2004; Chang et al. 2005; Basa Cesnik et al. 2006), but this method cannot identify the origin of the detected CS2, which may or may not be related to the presence of DTCs. In our study, we were able to not only validate the method for tomato but also to determine intact DTCs and to distinguish between DTC subclasses. In Iran, of dithiocarbamate fungicides only mancozeb, maneb and zineb are used for tomato production, and MRLs apply to total residues from the use of any or each of the groups of dithiocarbamates (MRL ¼ 2 mg g1). In the current study, a simple and accurate method was used to determine each separately accessible DTC. The representative chromatogram in Figure 1a illustrates good separation between DMD, EBD, PBD and also from unknown peaks related to the matrix. A comparison between greenhouse and nongreenhouse tomatoes showed that results for DMD (thiram) residues were almost similar to each other. EBD (mancozeb) residues in greenhouse tomatoes were higher than non-greenhouse. It seems that the high levels of EBD residues in greenhouse tomatoes is due to their particular product conditions and higher use of these fungicides to control pathogens in the greenhouse. One of the greenhouse tomato samples had an EBD (mancozeb) residue level above the MRL (up to 3.2 mg g1). Comparing the results obtained in Iran with those in Brazil, the percentage of tomato samples containing detectable residues (40.01 mg g1) of 81.2% in Iran was higher than those in Brazil (60.8%), but the mean DTC residue level of 0.31 mg g1 in Brazilian samples (Caldas et al. 2004) was higher than Iranian samples in this study (0.14 mg g1). Another study in Egypt also showed higher values than Iran, although DTC residues in Egyptian tomatoes decreased from 1995 to 1997 (Dogheim et al. 1999; Dogheim et al. 2001; Dogheim et al. 2002). Monitoring programs carried out in other countries, such as European Union, Canada, Taiwan and Slovenia, showed that DTCs are some of the most frequently detected pesticides, like in Iran (Ripley et al. 2000; Chang et al. 2005; Basa Cesnik et al. 2006).
Conclusion According to the studied parameters in this work, the presented method is suitable to simultaneously determine residues of the three main subclasses of dithiocarbamate fungicides in tomatoes. Although the mean DTC residues found in tomato samples were lower than the maximum level as recommended by the Codex Alimentarius, one greenhouse tomato sample exceeded the DTC residue MRL, which indicates the need for regular product monitoring.
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Acknowledgements This study was supported by the Food and Drug Laboratory Research Center (FDLRC), Iran Ministry of Health and Medical Education. The authors are grateful to the staff of the Toxicology Laboratory, FDLRC.
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