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Comparison of HPTLC and HPLC procedures for the determination of certain xenobiotic residues in apples and pears a
a
a
P. Corti , E. Dreassi , N. Politi & C. Aprea
a
a
Dipartimento Farmaco Chimico Tecnologico , University of Siena , Banchi di Sotto 55, Siena, Italy Published online: 10 Jan 2009.
To cite this article: P. Corti , E. Dreassi , N. Politi & C. Aprea (1992) Comparison of HPTLC and HPLC procedures for the determination of certain xenobiotic residues in apples and pears, Food Additives & Contaminants, 9:3, 243-251, DOI: 10.1080/02652039209374068 To link to this article: http://dx.doi.org/10.1080/02652039209374068
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, sublicensing, 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, 1992, VOL. 9, NO. 3, 2 4 3 - 2 5 1
Comparison of HPTLC and HPLC procedures for the determination of certain xenobiotic residues in apples and pears P. CORTI, E. DREASSI, N. POLITI and C. APREA Dipartimento Farmaco Chimico Tecnologico, Banchi di Sotto 55, University of Siena, Siena, Italy
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(Received 21 January 1992; revised 22 April 1992; accepted 12 May 1992) HPTLC was used to check for residues of benomyl, carbendazim, ethoxyquin and thiabendazole in apples and pears. The method used showed good precision with a percentage coefficient of variation of less than 5%; recoveries were always greater than 90%. The limits of determination using HPTLC were always at least four times lower than Italian statutory limits. Selectivity with respect to other matrix components was excellent for all fruit varieties tested. Comparison with HPLC confirmed the validity of the results. Keywords: HPTLC, HPLC, benomyl, carbendazim, ethoxyquin, thiabendazole
Introduction
High performance thin layer chromatography (HPTLC) has been successfully applied in our laboratory to the analysis of chemical residues in vegetables (Corti et al. 1991) . The success of modern TLC is due to the specificity, sensitivity, accuracy and precision of the plates available today. On this basis we studied the application of HPTLC to the control of residues used to treat apples and pears in storage. The results obtained were compared with HPLC data. The compounds studied, the statutory limits and safety intervals of which are reported in table 1, were as follows: l-[(butylamino)carbonyl]-lH-benzimidazol-2yl]carbamic acid methyl ester or benomyl (BYL), lH-benzimidazol-2-yl carbamic acid methyl ester or carbendazim (BCM), 6-ethoxy-l,2-dihydro-2,2,4 trimethylquinoline or ethoxyquin (EMQ) and 2-(4-thiazolyl)-lH-benzimidazole or
Table 1. Legal limits for the compounds in food and the safety intervals between treatment and distribution for consumption (Italian Health Ministry Legislative Ordinance of 6-6-1985). Compounds
Permissible Suspension residue (jig/g) period (days)
Product
Benomyl
Apples, pears
1
15
Carbendazim
Apples, pears
1
15
Ethoxyquin Apples Thiabendazole Apples, pears
3 3
90 30
Note As the sum of benomyl and carbendazim As the sum of benomyl and carbendazim
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P. Corti et al.
thiabendazole (TBZ). Although EMQ is not used to treat pears, we included it in the analysis in the interests of homogeneity. Among existing methods, HPLC predominates (Cano et al. 1987, Miles and Moye 1988, Bicchi et al. 1989, Lopez et al. 1989). There are a few reports of the use of TLC (Baldi et al. 1980, Kuz'min 1985), GC (Agnihotzi and Jain 1985, Gyorfi et al. 1987), spectrophotometry (Camoni et al. 1987, Rangaswamy et al. 1987, Garcia-Sanchez and Cruces-Blanco 1988) and electrochemical methods (Xu et al. 1983).
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Experimental
Equipment Densitometric reading of the Chromatographie plates was performed with a Camag Scanner photodensitometer connected to a Perkin-Elmer R100 recorder. The plates used for quantitative analysis were Merck (Darmstadt, Germany) HPTLC NH 2 , 10 X 10 cm, thickness 0-2 mm. Spots were deposited with Minicapstype calibrated capillaries from Hirschmann Laborgerate, Germany. For HPLC, Perkin-Elmer equipment consisting of a Series 410 Chromatograph, a variable wavelength LC85B UV detector and an R100 recorder were used. The column was a 15 cm Supelcosil LCig, 4*6 mm internal diameter, 5 μην particle size (Supelco, Bellefonte, PA, USA). Materials Merck Lichrosolv solvents were used for chromatography; Merck RP chloroform was used for extraction of the active principles from the apples and pears. Standards of the active principles (99% purity) were obtained from LabService-Analytica (Bologna, Italy). The fruit varieties investigated were Golden Delicious, Renetta del Canada and Stayman apples, William, Coscia and Abbe Fetel pears. Extraction of active principles from apples and pears Apples or pears (1000 g) were minced to a homogeneous pulp. About 25 g, exactly weighed, were placed in a 100 ml centrifuge tube and 20 ml saturated NaCl solution added; the sample was then extracted with three 15 ml aliquots of chloroform. Each time the tube was shaken in a Vortex apparatus for 5 min and centrifuged for 20 min. The organic phases (chloroform phase 1) were pooled and extracted with two 15 ml aliquots of 0·1 M HCl solution. Each time the tube was kept in a Vortex apparatus for 2 min and centrifuged for 20 min. Each aqueous fraction was collected and immediately treated with Na2CO3 until alkaline and extracted with two 10 ml aliquots of chloroform which were pooled and kept in the dark under nitrogen (chloroform phase 2). Chloroform phase 1 was placed in contact with 15 ml 0· 1 M HCl in aqueous solution and kept at 50°C under agitation for 30 min. Heating was suspended and the mixture centrifuged for 20 min. The chloroform was evaporated and the acid aqueous phase extracted again with a 15 ml aliquot of chloroform, kept in a Vortex for 2 min and centrifuged for 20 min (chloroform phase 3). Chloroform phases 2 and 3 containing BCM, EMQ and TBZ were combined, dried over anhydrous Na2SC>4 and evaporated at ambient temperature; the residue
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was first made up with 500 μ\ methanol, 2 μ\ of which was deposited on HPTLC NH 2 plates. It was then diluted to 5 ml and 6 μ\ analysed by HPLC on Cig columns. Recovery and repeatability evaluation of the extraction After the preparation of apple and pear homogenates consisting of equal parts of the different varieties, the different active principles were added and ten runs were made for each active principle, at each of the chosen concentrations (0-5, 1*5 and 4 ;tg/g of matrix). Recovery was evaluated quantitatively by HPTLC as described below.
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Chromatographie conditions Thin layer chromatography. HPTLC NH2 plates derivatized with propylamine were used. Two μ\ of methanol extracts were deposited 0·9 mm from each other with calibrated Minicaps-type capillaries. The deposition line was 1 ·0 cm from the lower edge of the plate and was eluted up to 0·5 cm from the upper edge. Development was performed first with a mobile phase consisting of cyclohexane/chloroform (1·0:0·3) (MPI) to elute ethoxyquin which was read at 235 nm. The plate was eluted again with a mobile phase consisting of chloroform/cyclohexane/methanol (6·0:1·0:0·1) (ΜΡ2) and carbendazim and thiabendazole read at 285 nm. Densitometric reading was performed at sensitivity 10, span 8, plate speed 0-5 mm/s, recorder baseline 200 mV, paper speed 15 mm/min. The plate was scanned for 80 mm starting 5 mm below the line of deposition. To check the hydrolysis of benomyl, the same conditions were used with MP2 only. HPLC. For the compounds (BCM, EMQ and TBZ) a Supelco L d 8 column was used at a flow rate of 1-5 ml/min. For BCM and TBZ, the mobile phase was methanol/phosphate buffer (0-01 M, pH 6-6) (56:44) MP3 and the analytical wavelength 280 nm. For EMQ, a mobile phase consisting of acetonitrile/phosphate buffer (0-01 M, pH 6-6)(60:40) MP4 was used and the absorbance read at 235 nm . Calibration of the active principles Appropriate quantities of methanol solutions of benomyl (1 mg/ml), carbendazim (1 mg/ml), ethoxyquin (4 mg/ml) and thiabendazole (4 mg/ml) were added to 25 g of apple or pear homogenate. Results and discussion Figures 1 and 2 show the HPTLC resolutions of the compounds analysed with various mobile phases. Product I was the most intense and constant, and is derived from the breakdown of ethoxyquin as a result of exposure to air, UV radiation and heat. We checked that ethoxyquin did not break down during extraction. Compound I did not appear when 10 μ\ of extract, obtained from homogenate supplemented to a concentration of 200 /ig/g, was run on the plates. Treatment for 30 min with 0-1 M HC1 at 50°C caused complete hydrolysis of benomyl to carbendazim and this was verified by the disappearance of the first compound from the mixture as
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TBZ
0
EMQ
0 min
min
Figure 1. HPTLC resolution of the extract of apples with the addition of BCM, EMQ and TBZ with MPI (cyclohexane/chloroform (1-0:0-3)) and MP2 (chloroform/cyclohexane/methanol (6-0:1-0:0·1).
read by HPLC and HPTLC. Figure 3 shows the HPTLC resolution of the BYL-BCM mixture after 15 mins of hydrolytic treatment of BYL. The overall preparation procedure of samples for analysis was satisfactory in terms of mean recovery and analytical precision, as shown in table 2. Finally, as regards possible interference of matrix components in the determination of all compounds, the extraction process was found to be sufficiently selective towards matrix components; among the different varieties of fruit there were no significant Chromatographie differences in the various extracts. In order to verify the first aspect, 10 μ\ (for HPTLC) and 100 μΐ (for HPLC) of ten extracts of different lots of each variety not treated with chemical agents, were deposited or injected respectively. No peaks were found in the areas of the substances in question. Later all the compounds (BYL, BCM, EMQ and TBZ) were added to the same extracts. Comparing the Chromatographie responses of the supplemented extracts and the solutions of active principles alone, it was found that for a given content, there were no statistically significant differences (for ρ = 0-05) in peak height or area in either system (HPTLC or HPLC). As far as the second aspect is concerned, the non-significant differences between Chromatographie resolutions of the different varieties of apples and pears led us to conclude that the calibrations could be constructed using mixtures of equal amounts of the different variety of each fruit as matrix.
HPTLC in pesticide residue control
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BCM
A. 10
12
It
16
18 mln
Β EMQ
Ί
0
2
4
6
8
10 rain
Figure 2. HPLC resolution of the extract of apples with the addition of BCM, EMQ and TBZ with MP3 (methanol/phosphate buffer 0-01 M, pH 6-6 (56:44)) (A) and MP4 (acetonitrile/phosphate buffer 0-01 M pH 6-6 (60:40)) (Β).
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BCM
BYL
0
mm Figure 3. HPTLC resolution of the BYL-BCM mixture.
Table 2. Evaluation of the recovery and repeatability.
Product Benomyl Carbendazim Ethoxyquin Thiabendazole
Mean percentage recovery ± SD (CV%) Apples
Mean percentage recovery ± SD (CV%) Pears
92-2 + 3-2 (3-5) 93-114-2 (4-5) 95-5 ±3-7 (3-8) 94-9 ± 3-6 (3-7)
91-8 ±2.1 (2.2) 94-0±3-l (3-3) 95-1 ± 3-7 (3-9) 94-0 ±4-0 (4-3)
The analysis was performed using HPTLC.
The investigation performed on apples and pears did not show any significant qualitative differences in Chromatographie profiles or quantitative parameters. It was thus considered unnecessary, except in table 2, to report the figures and parameters for both biological matrices; the results reported in the present paper therefore refer to the data of apples. HPTLC method The best results were obtained with HPTLC NH2 plates. In order to evaluate the reproducibility of the Chromatographie and inter-plate homogeneity, extracts of
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homogenates of the three varieties of apples and pears were prepared, and spiked with quantities of the active principles to give the maximum allowable concentration under Italian law. Significant differences were not found between single plates (nine plates from three different batches, each containing deposits of homogenates of the different types of apples) as far as Rf of single spots evaluated at the peaks was concerned. The percentage coefficient of variation (CV%) ranged from 0·51 for a plate with ethoxyquin and 0-15 for a plate with carbendazim; comparison between plates did not reveal any significant differences (ρ = 0·05). For quantitative measurements, the CV% ranged from 2-35 for a series with ethoxyquin and 1 -41 for a series with thiabendazole. Comparison between plates did not reveal significant differences (p = 0-05). HPLC method Methanol/phosphate buffer (0-01 M, pH 6-6) (56:44), the first mobile phase used, gave excellent separation of BCM, EMQ and TBZ. The peak of ethoxyquin could not be optimised in the presence of methanol, and for low doses, resolution was poor. Different elution conditions were therefore used for the quantification of ethoxyquin. None of the peaks of the degradation products of EMQ interfered with BCM or TBZ (figure 2(A)). With the mobile phase used for EMQ, there was good separation of the degradation products. Injection precision was verified by injecting an extract of apples and one of pears, ten times (the content of active principle was close to the maximum allowable by Italian law). The same extract, stored under nitrogen in the refrigerator in the dark, was analysed again on the following six days. In analysis of different combined extracts performed each day, there were never CV% values exceeding the value of 2· 12 once obtained for TBZ. Comparison between the different combined extracts on successive days did not reveal significant differences (ρ = 0·05), indicating that under these conditions of storage, there was no visible degradation of the active principles. Comparison of the results obtained with samples of apple and pear extracts (twenty of each) supplemented with various quantities of the different active principles showed that the coefficient of correlation (r = 0-9838 for carbendazim, r = 0-9986 for ethoxyquin, r = 0-9982 for thiabendazole) was excellent in all cases, showing good agreement between the experimental values obtained with both methods. The intercepts and the slopes of the curves constructed with experimental points were not significantly different from zero and one respectively, confirming that the two methods compare well over the range of concentrations investigated. Conclusions The analytical method was valid both in the sample preparation stage and for the measurement of active principles by HPTLC and HPLC. The lower sensitivity of the former method, which was to be expected, was not a problem since the levels allowable by law are much higher than the quantification limits of the plates. On the whole, HPTLC had good operating characteristics and is a valid method of comparison for HPLC. HPTLC can also be used as a preliminary approach to HPLC. In fact, using HPTLC NH2 plates with the mobile phases described it is possible to establish
Table 3. Qualitative parameters, calibration ranges and detection limits of benomyl, carbendazim, ethoxyquin and thiabendazole with HPTLC and HPLC. HPLC
HPTLC
Detection limits
Detection limits Compounds Benomyl Carbendazim Ethoxyquin Thiabendazole
Qualitative (/*g/g)
Quantitative
Rt value
Regression range (/ig/g)
0-31 0.31 0.26 0-52
0.12-4-00 0-12-4-00 0-25-4-00 0-25-4-00
0-08 0-06 0-05 0-05
0-26 0-20 0-17 0-16
V-%\%
Retention time (min)
Regression range (pg/g)
Qualitative (/ig/g)
Quantitative (/»g/g)
2-3 2-3 4-9 2-9
0-1-1-6 0-1-1-6 0-2-4-0 0-2-4-0
0-91 0-74 0-43 0-57
3-00 2-44 1-42 1-90
The calibration range, detection and quantification limits are evaluated as /tg/g on the biological matrix.
Cord e t al.
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Ο
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qualitatively which compounds (BYL, BCM, EMQ and TBZ) are present, before planning suitable conditions for HPLC. Table 3 gives the qualitative parameters, calibration ranges and detection limits of the HPTLC and HPLC methods. References AONIHOTZI, N. P., and JAIN, H. K., 1985, Persistence of thiophanate methyl residues in soil, water, sediment and plant. Pesticides, 19, 30a-30d. BALDI, M., ANGIULI, G., BOVOLENTA, Α., and ZANONI, L., 1980, A TLC-Spectrophotometric method
for determining MBC (methyl esters of 2-benzimidazocarbamic acid) in benomyl-treated fruit: studies on the fruit products of Ferrara Province. Rivista della Società Italiana di Scienza dell' Alimentazione, 9, 103-108.
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BICCHI, C., BELLIARDO, F., CANTAMESSA, L., GASPARINI, G., ICARDI, M., and SESIA, E., 1989,
Simultaneous determination of benzimidazole fungicides by HPLC on apples, pears and their pulps. Pesticide Science, 25, 355-360. CAMONI, I., AUSILI, Α., DI MuccIo, Α., DOMMARCO, R., and CITTI, P., 1987, Analytical methodology
used for the detection and determination of residues of benomyl, carbendazim, vinclozolin, azinphosmethyl, phosalone, pirimicarb, and dithiocarbamates in peaches. Rapporto ISTISAN ISTISAN 87/38, 20 pp.
CANO, P., DE LA PLAZA, J. L., and MUNOZ-DELGADO, L., 1987, Determination and persistence of
several fungícides in postharvest-treated apples during their cold storage. Journal of Agricultural and Food Chemistry, 35, 144-148. CORTI, P., DREASSI, E., POLITI, N., and APREA, C., 1991, Comparison of an HPTLC and an HPLC procedure for the determination of chlorpropham, propham and thiabendazole residues in potatoes. Food Additives and Contaminants, 8, 607-616. GARCIA-SANCHEZ, F., and CRUCES-BLANCO, C , 1988, Spectrofluorometric determination of pesticide residue mixtures by isodifferential derivative spectroscopy. Analytical Chemistry, 60, 323-328. GYORFI, L., AMBRUS, Α., and BOLYGO, E., 1987, Optimization of determination and clean-up
parameters for sensitive multiresidue analysis of pesticides. Pesticide Science and Biotechnology, Proc. Int. Congr. Pestic. Chem. 6th 1986, 353-356. KUZ'MIN, Α. Α., 1985, Determination of benzimidazole carbamate derivatives in products of animal origin. Veterinariya (Moscow), 9, 62-64.
LOPEZ, L. F., LOPEZ, A. G., and RIBA, M. V., 1989, HPLC method for simultaneous determination
of fungicides: carbendazim, metalaxyl, folpet, and propiconazole in must and wine. Journal of Agricultural and Food Chemistry, 37, 674-677. MILES, C. J., and ΜΟΥΕ Η. Α., 1988, Postcolumn photolysis of pesticides for fluorometric determination by high-performance liquid chromatography. Analytical Chemistry, 60, 220-226. RANGASWAMY, J. R., VIJAYASHANKAR, Y. N., and PRAKASH, S. R., 1987, Colorimetric method for
the determination of carbendazim (MBC), benomyl and their degradative product 2-aminobenzimidazole. Journal of Food Science and Technology, 24, 309-311. Xu, Z., FENG, Y., HE, Z. and Hou, Y., 1983, Polarographic analysis of carbendazol. Fenxi Huaxue, 11, 63-65.
Food Additives & Contaminants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac19
Comparison of HPTLC and HPLC procedures for the determination of certain xenobiotic residues in apples and pears a
a
a
P. Corti , E. Dreassi , N. Politi & C. Aprea
a
a
Dipartimento Farmaco Chimico Tecnologico , University of Siena , Banchi di Sotto 55, Siena, Italy Published online: 10 Jan 2009.
To cite this article: P. Corti , E. Dreassi , N. Politi & C. Aprea (1992) Comparison of HPTLC and HPLC procedures for the determination of certain xenobiotic residues in apples and pears, Food Additives & Contaminants, 9:3, 243-251, DOI: 10.1080/02652039209374068 To link to this article: http://dx.doi.org/10.1080/02652039209374068
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, sublicensing, 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, 1992, VOL. 9, NO. 3, 2 4 3 - 2 5 1
Comparison of HPTLC and HPLC procedures for the determination of certain xenobiotic residues in apples and pears P. CORTI, E. DREASSI, N. POLITI and C. APREA Dipartimento Farmaco Chimico Tecnologico, Banchi di Sotto 55, University of Siena, Siena, Italy
Downloaded by [Monash University Library] at 02:54 07 December 2014
(Received 21 January 1992; revised 22 April 1992; accepted 12 May 1992) HPTLC was used to check for residues of benomyl, carbendazim, ethoxyquin and thiabendazole in apples and pears. The method used showed good precision with a percentage coefficient of variation of less than 5%; recoveries were always greater than 90%. The limits of determination using HPTLC were always at least four times lower than Italian statutory limits. Selectivity with respect to other matrix components was excellent for all fruit varieties tested. Comparison with HPLC confirmed the validity of the results. Keywords: HPTLC, HPLC, benomyl, carbendazim, ethoxyquin, thiabendazole
Introduction
High performance thin layer chromatography (HPTLC) has been successfully applied in our laboratory to the analysis of chemical residues in vegetables (Corti et al. 1991) . The success of modern TLC is due to the specificity, sensitivity, accuracy and precision of the plates available today. On this basis we studied the application of HPTLC to the control of residues used to treat apples and pears in storage. The results obtained were compared with HPLC data. The compounds studied, the statutory limits and safety intervals of which are reported in table 1, were as follows: l-[(butylamino)carbonyl]-lH-benzimidazol-2yl]carbamic acid methyl ester or benomyl (BYL), lH-benzimidazol-2-yl carbamic acid methyl ester or carbendazim (BCM), 6-ethoxy-l,2-dihydro-2,2,4 trimethylquinoline or ethoxyquin (EMQ) and 2-(4-thiazolyl)-lH-benzimidazole or
Table 1. Legal limits for the compounds in food and the safety intervals between treatment and distribution for consumption (Italian Health Ministry Legislative Ordinance of 6-6-1985). Compounds
Permissible Suspension residue (jig/g) period (days)
Product
Benomyl
Apples, pears
1
15
Carbendazim
Apples, pears
1
15
Ethoxyquin Apples Thiabendazole Apples, pears
3 3
90 30
Note As the sum of benomyl and carbendazim As the sum of benomyl and carbendazim
0265-203X/92 $3.00 © 1992 Taylor & Francis Ltd.
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P. Corti et al.
thiabendazole (TBZ). Although EMQ is not used to treat pears, we included it in the analysis in the interests of homogeneity. Among existing methods, HPLC predominates (Cano et al. 1987, Miles and Moye 1988, Bicchi et al. 1989, Lopez et al. 1989). There are a few reports of the use of TLC (Baldi et al. 1980, Kuz'min 1985), GC (Agnihotzi and Jain 1985, Gyorfi et al. 1987), spectrophotometry (Camoni et al. 1987, Rangaswamy et al. 1987, Garcia-Sanchez and Cruces-Blanco 1988) and electrochemical methods (Xu et al. 1983).
Downloaded by [Monash University Library] at 02:54 07 December 2014
Experimental
Equipment Densitometric reading of the Chromatographie plates was performed with a Camag Scanner photodensitometer connected to a Perkin-Elmer R100 recorder. The plates used for quantitative analysis were Merck (Darmstadt, Germany) HPTLC NH 2 , 10 X 10 cm, thickness 0-2 mm. Spots were deposited with Minicapstype calibrated capillaries from Hirschmann Laborgerate, Germany. For HPLC, Perkin-Elmer equipment consisting of a Series 410 Chromatograph, a variable wavelength LC85B UV detector and an R100 recorder were used. The column was a 15 cm Supelcosil LCig, 4*6 mm internal diameter, 5 μην particle size (Supelco, Bellefonte, PA, USA). Materials Merck Lichrosolv solvents were used for chromatography; Merck RP chloroform was used for extraction of the active principles from the apples and pears. Standards of the active principles (99% purity) were obtained from LabService-Analytica (Bologna, Italy). The fruit varieties investigated were Golden Delicious, Renetta del Canada and Stayman apples, William, Coscia and Abbe Fetel pears. Extraction of active principles from apples and pears Apples or pears (1000 g) were minced to a homogeneous pulp. About 25 g, exactly weighed, were placed in a 100 ml centrifuge tube and 20 ml saturated NaCl solution added; the sample was then extracted with three 15 ml aliquots of chloroform. Each time the tube was shaken in a Vortex apparatus for 5 min and centrifuged for 20 min. The organic phases (chloroform phase 1) were pooled and extracted with two 15 ml aliquots of 0·1 M HCl solution. Each time the tube was kept in a Vortex apparatus for 2 min and centrifuged for 20 min. Each aqueous fraction was collected and immediately treated with Na2CO3 until alkaline and extracted with two 10 ml aliquots of chloroform which were pooled and kept in the dark under nitrogen (chloroform phase 2). Chloroform phase 1 was placed in contact with 15 ml 0· 1 M HCl in aqueous solution and kept at 50°C under agitation for 30 min. Heating was suspended and the mixture centrifuged for 20 min. The chloroform was evaporated and the acid aqueous phase extracted again with a 15 ml aliquot of chloroform, kept in a Vortex for 2 min and centrifuged for 20 min (chloroform phase 3). Chloroform phases 2 and 3 containing BCM, EMQ and TBZ were combined, dried over anhydrous Na2SC>4 and evaporated at ambient temperature; the residue
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245
was first made up with 500 μ\ methanol, 2 μ\ of which was deposited on HPTLC NH 2 plates. It was then diluted to 5 ml and 6 μ\ analysed by HPLC on Cig columns. Recovery and repeatability evaluation of the extraction After the preparation of apple and pear homogenates consisting of equal parts of the different varieties, the different active principles were added and ten runs were made for each active principle, at each of the chosen concentrations (0-5, 1*5 and 4 ;tg/g of matrix). Recovery was evaluated quantitatively by HPTLC as described below.
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Chromatographie conditions Thin layer chromatography. HPTLC NH2 plates derivatized with propylamine were used. Two μ\ of methanol extracts were deposited 0·9 mm from each other with calibrated Minicaps-type capillaries. The deposition line was 1 ·0 cm from the lower edge of the plate and was eluted up to 0·5 cm from the upper edge. Development was performed first with a mobile phase consisting of cyclohexane/chloroform (1·0:0·3) (MPI) to elute ethoxyquin which was read at 235 nm. The plate was eluted again with a mobile phase consisting of chloroform/cyclohexane/methanol (6·0:1·0:0·1) (ΜΡ2) and carbendazim and thiabendazole read at 285 nm. Densitometric reading was performed at sensitivity 10, span 8, plate speed 0-5 mm/s, recorder baseline 200 mV, paper speed 15 mm/min. The plate was scanned for 80 mm starting 5 mm below the line of deposition. To check the hydrolysis of benomyl, the same conditions were used with MP2 only. HPLC. For the compounds (BCM, EMQ and TBZ) a Supelco L d 8 column was used at a flow rate of 1-5 ml/min. For BCM and TBZ, the mobile phase was methanol/phosphate buffer (0-01 M, pH 6-6) (56:44) MP3 and the analytical wavelength 280 nm. For EMQ, a mobile phase consisting of acetonitrile/phosphate buffer (0-01 M, pH 6-6)(60:40) MP4 was used and the absorbance read at 235 nm . Calibration of the active principles Appropriate quantities of methanol solutions of benomyl (1 mg/ml), carbendazim (1 mg/ml), ethoxyquin (4 mg/ml) and thiabendazole (4 mg/ml) were added to 25 g of apple or pear homogenate. Results and discussion Figures 1 and 2 show the HPTLC resolutions of the compounds analysed with various mobile phases. Product I was the most intense and constant, and is derived from the breakdown of ethoxyquin as a result of exposure to air, UV radiation and heat. We checked that ethoxyquin did not break down during extraction. Compound I did not appear when 10 μ\ of extract, obtained from homogenate supplemented to a concentration of 200 /ig/g, was run on the plates. Treatment for 30 min with 0-1 M HC1 at 50°C caused complete hydrolysis of benomyl to carbendazim and this was verified by the disappearance of the first compound from the mixture as
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TBZ
0
EMQ
0 min
min
Figure 1. HPTLC resolution of the extract of apples with the addition of BCM, EMQ and TBZ with MPI (cyclohexane/chloroform (1-0:0-3)) and MP2 (chloroform/cyclohexane/methanol (6-0:1-0:0·1).
read by HPLC and HPTLC. Figure 3 shows the HPTLC resolution of the BYL-BCM mixture after 15 mins of hydrolytic treatment of BYL. The overall preparation procedure of samples for analysis was satisfactory in terms of mean recovery and analytical precision, as shown in table 2. Finally, as regards possible interference of matrix components in the determination of all compounds, the extraction process was found to be sufficiently selective towards matrix components; among the different varieties of fruit there were no significant Chromatographie differences in the various extracts. In order to verify the first aspect, 10 μ\ (for HPTLC) and 100 μΐ (for HPLC) of ten extracts of different lots of each variety not treated with chemical agents, were deposited or injected respectively. No peaks were found in the areas of the substances in question. Later all the compounds (BYL, BCM, EMQ and TBZ) were added to the same extracts. Comparing the Chromatographie responses of the supplemented extracts and the solutions of active principles alone, it was found that for a given content, there were no statistically significant differences (for ρ = 0-05) in peak height or area in either system (HPTLC or HPLC). As far as the second aspect is concerned, the non-significant differences between Chromatographie resolutions of the different varieties of apples and pears led us to conclude that the calibrations could be constructed using mixtures of equal amounts of the different variety of each fruit as matrix.
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BCM
A. 10
12
It
16
18 mln
Β EMQ
Ί
0
2
4
6
8
10 rain
Figure 2. HPLC resolution of the extract of apples with the addition of BCM, EMQ and TBZ with MP3 (methanol/phosphate buffer 0-01 M, pH 6-6 (56:44)) (A) and MP4 (acetonitrile/phosphate buffer 0-01 M pH 6-6 (60:40)) (Β).
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BCM
BYL
0
mm Figure 3. HPTLC resolution of the BYL-BCM mixture.
Table 2. Evaluation of the recovery and repeatability.
Product Benomyl Carbendazim Ethoxyquin Thiabendazole
Mean percentage recovery ± SD (CV%) Apples
Mean percentage recovery ± SD (CV%) Pears
92-2 + 3-2 (3-5) 93-114-2 (4-5) 95-5 ±3-7 (3-8) 94-9 ± 3-6 (3-7)
91-8 ±2.1 (2.2) 94-0±3-l (3-3) 95-1 ± 3-7 (3-9) 94-0 ±4-0 (4-3)
The analysis was performed using HPTLC.
The investigation performed on apples and pears did not show any significant qualitative differences in Chromatographie profiles or quantitative parameters. It was thus considered unnecessary, except in table 2, to report the figures and parameters for both biological matrices; the results reported in the present paper therefore refer to the data of apples. HPTLC method The best results were obtained with HPTLC NH2 plates. In order to evaluate the reproducibility of the Chromatographie and inter-plate homogeneity, extracts of
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homogenates of the three varieties of apples and pears were prepared, and spiked with quantities of the active principles to give the maximum allowable concentration under Italian law. Significant differences were not found between single plates (nine plates from three different batches, each containing deposits of homogenates of the different types of apples) as far as Rf of single spots evaluated at the peaks was concerned. The percentage coefficient of variation (CV%) ranged from 0·51 for a plate with ethoxyquin and 0-15 for a plate with carbendazim; comparison between plates did not reveal any significant differences (ρ = 0·05). For quantitative measurements, the CV% ranged from 2-35 for a series with ethoxyquin and 1 -41 for a series with thiabendazole. Comparison between plates did not reveal significant differences (p = 0-05). HPLC method Methanol/phosphate buffer (0-01 M, pH 6-6) (56:44), the first mobile phase used, gave excellent separation of BCM, EMQ and TBZ. The peak of ethoxyquin could not be optimised in the presence of methanol, and for low doses, resolution was poor. Different elution conditions were therefore used for the quantification of ethoxyquin. None of the peaks of the degradation products of EMQ interfered with BCM or TBZ (figure 2(A)). With the mobile phase used for EMQ, there was good separation of the degradation products. Injection precision was verified by injecting an extract of apples and one of pears, ten times (the content of active principle was close to the maximum allowable by Italian law). The same extract, stored under nitrogen in the refrigerator in the dark, was analysed again on the following six days. In analysis of different combined extracts performed each day, there were never CV% values exceeding the value of 2· 12 once obtained for TBZ. Comparison between the different combined extracts on successive days did not reveal significant differences (ρ = 0·05), indicating that under these conditions of storage, there was no visible degradation of the active principles. Comparison of the results obtained with samples of apple and pear extracts (twenty of each) supplemented with various quantities of the different active principles showed that the coefficient of correlation (r = 0-9838 for carbendazim, r = 0-9986 for ethoxyquin, r = 0-9982 for thiabendazole) was excellent in all cases, showing good agreement between the experimental values obtained with both methods. The intercepts and the slopes of the curves constructed with experimental points were not significantly different from zero and one respectively, confirming that the two methods compare well over the range of concentrations investigated. Conclusions The analytical method was valid both in the sample preparation stage and for the measurement of active principles by HPTLC and HPLC. The lower sensitivity of the former method, which was to be expected, was not a problem since the levels allowable by law are much higher than the quantification limits of the plates. On the whole, HPTLC had good operating characteristics and is a valid method of comparison for HPLC. HPTLC can also be used as a preliminary approach to HPLC. In fact, using HPTLC NH2 plates with the mobile phases described it is possible to establish
Table 3. Qualitative parameters, calibration ranges and detection limits of benomyl, carbendazim, ethoxyquin and thiabendazole with HPTLC and HPLC. HPLC
HPTLC
Detection limits
Detection limits Compounds Benomyl Carbendazim Ethoxyquin Thiabendazole
Qualitative (/*g/g)
Quantitative
Rt value
Regression range (/ig/g)
0-31 0.31 0.26 0-52
0.12-4-00 0-12-4-00 0-25-4-00 0-25-4-00
0-08 0-06 0-05 0-05
0-26 0-20 0-17 0-16
V-%\%
Retention time (min)
Regression range (pg/g)
Qualitative (/ig/g)
Quantitative (/»g/g)
2-3 2-3 4-9 2-9
0-1-1-6 0-1-1-6 0-2-4-0 0-2-4-0
0-91 0-74 0-43 0-57
3-00 2-44 1-42 1-90
The calibration range, detection and quantification limits are evaluated as /tg/g on the biological matrix.
Cord e t al.
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qualitatively which compounds (BYL, BCM, EMQ and TBZ) are present, before planning suitable conditions for HPLC. Table 3 gives the qualitative parameters, calibration ranges and detection limits of the HPTLC and HPLC methods. References AONIHOTZI, N. P., and JAIN, H. K., 1985, Persistence of thiophanate methyl residues in soil, water, sediment and plant. Pesticides, 19, 30a-30d. BALDI, M., ANGIULI, G., BOVOLENTA, Α., and ZANONI, L., 1980, A TLC-Spectrophotometric method
for determining MBC (methyl esters of 2-benzimidazocarbamic acid) in benomyl-treated fruit: studies on the fruit products of Ferrara Province. Rivista della Società Italiana di Scienza dell' Alimentazione, 9, 103-108.
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BICCHI, C., BELLIARDO, F., CANTAMESSA, L., GASPARINI, G., ICARDI, M., and SESIA, E., 1989,
Simultaneous determination of benzimidazole fungicides by HPLC on apples, pears and their pulps. Pesticide Science, 25, 355-360. CAMONI, I., AUSILI, Α., DI MuccIo, Α., DOMMARCO, R., and CITTI, P., 1987, Analytical methodology
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CANO, P., DE LA PLAZA, J. L., and MUNOZ-DELGADO, L., 1987, Determination and persistence of
several fungícides in postharvest-treated apples during their cold storage. Journal of Agricultural and Food Chemistry, 35, 144-148. CORTI, P., DREASSI, E., POLITI, N., and APREA, C., 1991, Comparison of an HPTLC and an HPLC procedure for the determination of chlorpropham, propham and thiabendazole residues in potatoes. Food Additives and Contaminants, 8, 607-616. GARCIA-SANCHEZ, F., and CRUCES-BLANCO, C , 1988, Spectrofluorometric determination of pesticide residue mixtures by isodifferential derivative spectroscopy. Analytical Chemistry, 60, 323-328. GYORFI, L., AMBRUS, Α., and BOLYGO, E., 1987, Optimization of determination and clean-up
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LOPEZ, L. F., LOPEZ, A. G., and RIBA, M. V., 1989, HPLC method for simultaneous determination
of fungicides: carbendazim, metalaxyl, folpet, and propiconazole in must and wine. Journal of Agricultural and Food Chemistry, 37, 674-677. MILES, C. J., and ΜΟΥΕ Η. Α., 1988, Postcolumn photolysis of pesticides for fluorometric determination by high-performance liquid chromatography. Analytical Chemistry, 60, 220-226. RANGASWAMY, J. R., VIJAYASHANKAR, Y. N., and PRAKASH, S. R., 1987, Colorimetric method for
the determination of carbendazim (MBC), benomyl and their degradative product 2-aminobenzimidazole. Journal of Food Science and Technology, 24, 309-311. Xu, Z., FENG, Y., HE, Z. and Hou, Y., 1983, Polarographic analysis of carbendazol. Fenxi Huaxue, 11, 63-65.
