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Food Additives & Contaminants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac19
Comparison of an HPTLC and an HPLC procedure for the determination of chlorpropham, propham and thiabendazole residues in potatoes a
a
a
P. Corti , E. Dreassi , N. Politi & C. Aprea
b
a
Dipartimento Farmaco Chimico Tecnologico , Università degli Studi di Siena , Banchi di Sotto 55 b
Istituto di Medicina del Lavoro , Università degli Studi di Siena , Siena, Italy Published online: 10 Jan 2009.
To cite this article: P. Corti , E. Dreassi , N. Politi & C. Aprea (1991) Comparison of an HPTLC and an HPLC procedure for the determination of chlorpropham, propham and thiabendazole residues in potatoes, Food Additives & Contaminants, 8:5, 607-615, DOI: 10.1080/02652039109374014 To link to this article: http://dx.doi.org/10.1080/02652039109374014
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
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forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
FOOD ADDITIVES AND CONTAMINANTS, 1991, VOL. 8, NO. 5, 6 0 7 - 6 1 6
Comparison of an HPTLC and an HPLC procedure for the determination of chlorpropham, propham and thiabendazole residues in potatoes P. CORTI†, E. DREASSI†, N. POLITI† and C. APREA‡
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† Dipartimento Farmaco Chimico Tecnologico, Banchi di Sotto 55, Università degli Studi di Siena; ‡ Istituto di Medicina del Lavoro, Università degli Studi di Siena, Siena, Italy (Received 21 March 1991; revised 12 June 1991; accepted 2 August 1991) HPTLC was used to check for residues of chlorpropham, propham (sprout inhibitors) and thiabendazole (fungicide) applied to potatoes. The method used gave good precision and analytical sensitivity. The sample preparation method developed by the authors was simple and gave good recovery and selectivity as far as other components of the matrix were concerned. Comparison with liquid chromatography confirmed the validity of the results. Keywords: HPTLC, propham, chlorpropham, thiabendazole, residues
Introduction
The commercial availability of thin layer Chromatographie (TLC) plates with layers of very different polarity and of high performance (HP) plates are two good reasons for reconsidering the use of TLC methods for qualitative and quantitative analysis. The performance of TLC is improved giving increased selectivity as a function of silica derivatization and greater analytical precision and sensitivity for HP plates. If the considerable advantages of thin layer chromatography are also considered (simplicity, economy, speed, recovery of single components, direct visualization in most cases of all the components of the sample), it is logical to think of a re-evaluation of this method. In the present note we report the results of an investigation into the use of HPTLC to check for residues of chlorpropham [isopropyl-3-chlorophenylcarbamate (CIPC)], propham [isopropyl-phenylcarbamate (IPC)] and thiabendazole [2-(thiazol-4-yl)benzimidazole (TBZ)] in potatoes. A comparison was also made between densitometric methods and the HPLC method developed, in order to give a better evaluation of the results. Table 1 summarizes the statutory limits for residues of the three compounds in potatoes and the safety period stipulated between the last treatment and marketing, according to Italian law (Health Ministry regulation of 6/6/1985). The literature abounds with methods for the quantitative analysis of these three compounds. There are a large number of recent HPLC methods (Voyksner et al. 1984, Pena and Sanchez 1986, Thomas and Sturrock 1986, Miles and Moye 1987, Sidhu et al. 1987) and GC methods (Andersson and Ohlin 1986, Buben and Karmazin 1986, Gerasimenko and Vaskobainikov 1987, Lafuente and Tadeo 1987, 0265-203X/91 $3.00 © 1991 Taylor & Francis Ltd.
608
P. Corti et al. Table 1. Maximum permissible levels of residues and suspension periods for chlorpropham, propham and thiabendazole in potatoes according to Italian legislation and Ministerial regulation of 6/6/1985. Active principle
Condition of the sample to be analysed
Maximum permissible residue (mg/kg)
Suspension period (days)
Chlorpropham Propham Thiabendazole
Peeled potatoes Peeled potatoes Washed potatoes
0-5 a 0-5 a 4-0
30 30 30
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a
As the sum of propham and chlorpropham.
Saxton 1987, Tonagai etal. 1987, Voznakova et al. 1988); the number of TLC procedures is much smaller (Ambrus et al. 1981, Korsos and Lantos 1984, Lawerenz et al. 1986, Osselton and Snelling 1986, Wachalz et al. 1988). Experimental
Apparatus The plates used were Merck C8 HPTLC, 10 x 10 cm, 0-2 mm thick (Merck, Darmstadt, Germany). Spotting was performed using Hirschmann Laborgerate (Germany) Minicaps calibrated capillaries. Densitometric scanning of the Chromatographie plates was performed with a CAMAG Scanner connected to a Perkin-Elmer R 100 A recorder. For the HPLC investigation a Perkin-Elmer series 410 Chromatograph with variable wavelength LC 85B spectrophotometric detector and R 100 A recorder were used. The column was a Supelco Supelcosil LCis (particle size 5 pm, internal diameter 4-6 mm, length 15 cm). For the preliminary Chromatographie separation, Merck Si6o TLC plates (20x20 cm, 0*25 mm thick) were used. Spotting was made with a Desaga 80 Microdoser with a graduated syringe. Materials Merck Lichrosolv solvents were used for chromatography and Merck RP dichloromethane to extract the residues from the potatoes. The analytical standards were purchased from Labo Service (Bologna, Italy). Extraction of residues from potatoes Potato (100 g) was carefully washed or peeled as stipulated by Italian law in relation to analysis for residues (table 1) and minced in a meat-mincer to give a homogeneous mixture. An exact weight of around 25 g was placed in a separating funnel, 20 ml of a saturated aqueous solution of NaCl added and the mixture extracted with three 50 ml portions of dichloromethane. The pooled organic phases were dried over Na2SC>4 and evaporated to dryness on the rotavapor. The residue was redissolved in 1 ml dichloromethane and 400 p.\ of this solution was spotted on Silica 60 TLC plates with an automatic doser under the conditions described below. Chromatographie conditions Initial TLC clean up of the samples extracts. Four 400 ^1 portions of potato
HPTLC in the control of pesticide residues
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Table 2. Preparation of the samples for analysis. Elution of single compounds (cm)
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1st plate elution 2nd plate elution Band removed for analysis
Mobile phase
CIPC
IPC
TBZ
Total plate elution (cm) Total plate elution (cm)
CHC13 :triethylamine 1:0-05 CHC13:CH3OH 1:0-05
15-3
15-3
9-3
19-0
—
—
11-8
14-0
14-5-16-0
14-5-16 •0
11-1-12-2
extract were spotted on to a plate along with a fifth, similar spot containing only the standards. The spots were spaced at a distance of 0-7 cm along a 3-cm line. Deposition was performed so as to have a margin of 1 cm from the edges of the plate and 2 cm from the side immersed in the mobile phase. After development using the two eluents shown in table 2, the layers corresponding to the compounds CIPC and IPC and that corresponding to the compound TBZ were removed by scraping with a metal spatula. The compounds were eluted from the silica by shaking with 400 /x\ methanol and then separated from the solid phase by centrifuging. The solution was spotted directly on HPTLC plates for quantitative analysis. For HPLC the residual solution was diluted to 800 /tl for compounds CIPC and IPC and to 2-5 ml for compound TBZ; the solutions were filtered through a 0-45 fiva membrane before injection. Quantitative analysis on HPTLC plates. On the RP8 HPTLC plates the spots were placed 0*9 cm apart and 1-5 cm from the upper edge of the plate. Development was carried to within 0-5 cm of the upper edge of the plate. The Rt values were 0-35 for CIPC, 0-48 for IPC and 0-33 for TBZ. The plates were read at 248 nm and the spots were of 5 /tl of the methanol fraction containing the CIPC and IPC and 2 fA of the fraction containing TBZ, with sensitivity at 10, span 8, plate speed 30 mm/min and baseline 200 mV. In order to optimize elution performance, the mobile phase consisting of a methanol/water mixture (1:0-3) was placed in the Chromatographie chamber 12 h before elution. The position of the plate was kept constant and the internal temperature of the chamber (20°C ±1-5) was kept constant by a thermostat. HPLC analysis. The analysis was carried out with detection at 248 nm with a flow of 2 ml/min, volume of injection 6/J, mobile phase methanol/0-1 M phosphate buffer pH 6-6 (3:2). Calibration curve for the three compounds Different volumes of a methanol standard solution containing chlorpropham (1 mg/ml), propham (1 mg/ml) and thiabendazole (4 mg/ml) were added to 25 g of homogenate of washed or peeled potatoes (as specified by the legislation). The range of calibration is reported in table 3.
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P. Corti et al.
Table 3. Calibration curve parameters for HPTLC and HPLC analysis of chlorpropham, propham and thiabendazole added to the biological matrix. Intercept ± p 1CT2
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Product
Slope ± P
Coefficient of correlation
Detection limits (*4g/g)
Quantification limits (fg/g)
Chlorpropham
a b
0-020 ±0-210 1-056 ±0-012 0-081 ±0-530 1-660 ±0-091
0-9982 0-9998
0-023 0-002
0-075 0-007
Propham
a b
0-024 ±0-180 1-204 ±0-014 0-072 ±0-470 1-351 ±0-080
0-9994 0-9987
0-019 0-002
0-065 0-008
Thiabendazole
a -0-011 ±0-100 0-572 ±0-070 b 0-112 ±0-720 2-140 ±0-130
0-9988 0-9991
0-100 0-0005
0-350 0-0017
The parameters were calculated from peak height (mm) and concentration (ng/dep or ng/injection). a and b refer to the curves obtained with the HPTLC and HPLC respectively. 1st confidence interval p calculated on five calibration curves (five points in the range for each) was below that determined for p = 0-05 in all cases. The calibration range was 5-300 ng/deposit or ng/injection in all cases.
Results Preparation of the sample Table 2 summarizes the conditions for the preparation of samples for analysis. Elution of the plate with a mobile phase consisting of chloroform/ triethylamine (1:0-05) enabled the fraction corresponding to compounds CIPC and IPC to be isolated in a relatively pure state. The second elution with chloroform/methanol (1:0-05) eliminated certain components from the thiabendazole fraction, which interfere in the successive HPTLC and HPLC analysis from the first elution solvent with the above mobile phases. The widths of the two bands of silica to be removed were the maximum widths obtained by repeating many spottings on many plates, as described below. The groups of untreated potatoes of the varieties Kennebec and Majestic grown in different parts of the Province of Siena, were checked for significant differences in endogenous components in the fractions of extract to be analysed. The repeatability of the widths of the silica layer to be removed was also examined. Out of ten samples from each group it was found that there were no variations in the type of components in the two silica bands removed for analysis of the three compounds. No Chromatographie signals interfering with compounds CIPC, IPC or TBZ were found by TLC or HPLC. The results of the analysis of potatoes which had either been washed or peeled, gave similar results. The reproducibility of the width of the band of silica to remove for the three standards was also satisfactory. In the ten plates analysed, also with a reference spot containing a mixture of the three standards (a total of five spots per plate), a coefficient of variation of 5-4% was obtained for thiabendazole and 3-4% for the other two components. In order to understand in detail the behaviour of the TLC plates used in this preliminary phase, spots (400 jtl distributed over 3 cm) of a solution containing CIPC (12-5 jtg/ml), IPC (12-5/tg/ml) and TBZ (100^g/ml) were also made to determine the reproducibility of the spots in quantitative terms. Deposits were made on six plates from three different lots of production and the single fractions were analysed by HPTLC.
HPTLC in the control of pesticide residues
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Evaluation of the coefficient of variation for individual plates gave a maximum CV of 2-4% for TBZ; comparison of plates gave CV of 1-0% for CIPC, 0-9% for IPC and 1*3% for TBZ. Comparison with direct analysis of the solutions of the three active principles gave mean per cent recoveries in this phase of 99 • 7 for CIPC, 101-2 for IPC and 99-3 for TBZ. Overall recovery and repeatability were evaluated adding appropriate quantities of standards (0-25, 0-50, 1 -00 /ig/g for CIPC and IPC and 1-00, 2-00, 4-00 jig/g for TBZ) to homogenate of untreated potatoes. The mean recoveries and coefficient of variation were respectively 92-9% and 5-1 for CIPC, 90-7% and 4* 1 for IPC, 94-6% and 5-1 for TBZ. Assay by HPTLC The best analytical conditions for the assay of the three compounds were obtained with HPTLC RP8 plates and methanol/water (1:0-3) mobile phase. For other types of layers (silica, silica derivatized with propylamine or propionitrile) the separation of the three compounds from the coextracted matrix was not satisfactory. Figure 1 and table 3 give the Chromatographie resolutions and a summary of the calibration curve parameters for the standards extracted from the biological matrix. The limits of detection and quantification were well below those stipulated by the legislation, making the method suitable for surveillance and enforcement purposes. On this basis we decided to check certain parameters in order to validate the method. First we found that the performance of the plates was the same for all points of deposition. It is known that the lateral parts of the plate may suffer variations in the elution process as a result of a possible slight non-linearity of the solvent front. When this happens, one may have different retention distance (Rf value) and spot distribution for a given compound at a given concentration, with
TBZ
CIPC IPC
min
min
Figure 1. HPTLC resolution of the blank fractions of potato extract with the addition of propham, chlorpropham and thiabendazole. The contents of CIPC and IPC in the biological matrix were 0-12 jig/g and 0-15 jig/g respectively. The content of thiabendazole in the biological matrix was l-0pg/g.
P. Corti et al.
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612
a consequent difference in Chromatographie peak height between depositions which are central and lateral on the plate. On three plates from three different lots, five deposits of a mixture of CIPC and IPC and five of TBZ were made. The deposits for CIPC and IPC were started on one half of the plate, and those for TBZ on the other. The solutions run had a content of active principle equal to that of the highest point used for the calibrations. For the elution evaluated on the maximum Chromatographie peak, no significant differences were found between the central and lateral spots on individual plates; nor were statistically significant differences found (/? = ()• 05) when this parameter was compared between plates. As far as the quantitative evaluation is concerned (the parameter used was Chromatographie peak height), the results were completely satisfactory. Between individual plates, the percentage differences in peak height between lateral and corresponding central spots was always less than 1-95%, which was the value obtained for a deposit of propham. Comparison between plates did not give statistically significant differences (p = 0-05). On the basis of these results it was decided to perform ten deposits per plate so as to utilize the whole available width. Later we determined the variability of the method in the calibration range used for the three compounds in relation to the plates and to the system of manual deposition used. Diluting the standard solutions of CIPC, IPC and TBZ appropriately, two sets of calibration curves were deposited
IPC TBZ
J 10 mJn
min
Figure 2. HPLC resolution of the blank fractions of potato extract with the addition of propham, chlorpropham and thiabendazole. The contents of CIPC and IPC in the biological matrix were 0 • 40 pg/g and 0 • 20 /jg/g respectively. The content of TBZ in the biological matrix was 0 • 68 jig/g.
HPTLC in the control of pesticide residues
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on six plates from three different lots for the mixture of propham and chlorpropham and for thiabendazole. A similar test was performed using different operators. The slopes and intercepts of the different calibrations were not statistically different (p = 0 • 05) on individual plates, between different plates or for deposits performed by one or two operators. Assay by HPLC The Chromatographie resolutions of the fractions for the assay of the three compounds and the parameters of the regressive curve are summarized in figure 2 and table 3. The mobile phase chosen was able to satisfactorily separate the mixture of the three compounds. Again the precision of the method was tested. For each active principle, five points in the calibration range were injected five times in succession. The band of regression lines obtained, in each case, lay within the confidence interval calculated for p = 0-05. A similar result was obtained injecting five points for each active principle on successive days (a set of calibrations over the period of a week). Conclusions At the end of the study, the results obtained by the two methods were compared for the three compounds, adding the latter randomly but within the calibration range, to the biological matrix. The distribution of the results is summarized in figures 3-5 among with the coefficients of correlation (r). The coefficients of correlation between corresponding points shows good linearity and consequently similarity between the HPTLC and HPLC results. Clearly, the sensitivities are different, but although HPTLC is more limited than column chromatography, it is still quite sensitive enough for detecting residues of IPC, CIPC and TBZ in potatoes down to approximately 1/1 Oth of the Maximum
to
1 J Ü (
r
1
= 0
.9865
j
y
i
Food Additives & Contaminants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac19
Comparison of an HPTLC and an HPLC procedure for the determination of chlorpropham, propham and thiabendazole residues in potatoes a
a
a
P. Corti , E. Dreassi , N. Politi & C. Aprea
b
a
Dipartimento Farmaco Chimico Tecnologico , Università degli Studi di Siena , Banchi di Sotto 55 b
Istituto di Medicina del Lavoro , Università degli Studi di Siena , Siena, Italy Published online: 10 Jan 2009.
To cite this article: P. Corti , E. Dreassi , N. Politi & C. Aprea (1991) Comparison of an HPTLC and an HPLC procedure for the determination of chlorpropham, propham and thiabendazole residues in potatoes, Food Additives & Contaminants, 8:5, 607-615, DOI: 10.1080/02652039109374014 To link to this article: http://dx.doi.org/10.1080/02652039109374014
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
Downloaded by [Monash University Library] at 02:55 07 December 2014
forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
FOOD ADDITIVES AND CONTAMINANTS, 1991, VOL. 8, NO. 5, 6 0 7 - 6 1 6
Comparison of an HPTLC and an HPLC procedure for the determination of chlorpropham, propham and thiabendazole residues in potatoes P. CORTI†, E. DREASSI†, N. POLITI† and C. APREA‡
Downloaded by [Monash University Library] at 02:55 07 December 2014
† Dipartimento Farmaco Chimico Tecnologico, Banchi di Sotto 55, Università degli Studi di Siena; ‡ Istituto di Medicina del Lavoro, Università degli Studi di Siena, Siena, Italy (Received 21 March 1991; revised 12 June 1991; accepted 2 August 1991) HPTLC was used to check for residues of chlorpropham, propham (sprout inhibitors) and thiabendazole (fungicide) applied to potatoes. The method used gave good precision and analytical sensitivity. The sample preparation method developed by the authors was simple and gave good recovery and selectivity as far as other components of the matrix were concerned. Comparison with liquid chromatography confirmed the validity of the results. Keywords: HPTLC, propham, chlorpropham, thiabendazole, residues
Introduction
The commercial availability of thin layer Chromatographie (TLC) plates with layers of very different polarity and of high performance (HP) plates are two good reasons for reconsidering the use of TLC methods for qualitative and quantitative analysis. The performance of TLC is improved giving increased selectivity as a function of silica derivatization and greater analytical precision and sensitivity for HP plates. If the considerable advantages of thin layer chromatography are also considered (simplicity, economy, speed, recovery of single components, direct visualization in most cases of all the components of the sample), it is logical to think of a re-evaluation of this method. In the present note we report the results of an investigation into the use of HPTLC to check for residues of chlorpropham [isopropyl-3-chlorophenylcarbamate (CIPC)], propham [isopropyl-phenylcarbamate (IPC)] and thiabendazole [2-(thiazol-4-yl)benzimidazole (TBZ)] in potatoes. A comparison was also made between densitometric methods and the HPLC method developed, in order to give a better evaluation of the results. Table 1 summarizes the statutory limits for residues of the three compounds in potatoes and the safety period stipulated between the last treatment and marketing, according to Italian law (Health Ministry regulation of 6/6/1985). The literature abounds with methods for the quantitative analysis of these three compounds. There are a large number of recent HPLC methods (Voyksner et al. 1984, Pena and Sanchez 1986, Thomas and Sturrock 1986, Miles and Moye 1987, Sidhu et al. 1987) and GC methods (Andersson and Ohlin 1986, Buben and Karmazin 1986, Gerasimenko and Vaskobainikov 1987, Lafuente and Tadeo 1987, 0265-203X/91 $3.00 © 1991 Taylor & Francis Ltd.
608
P. Corti et al. Table 1. Maximum permissible levels of residues and suspension periods for chlorpropham, propham and thiabendazole in potatoes according to Italian legislation and Ministerial regulation of 6/6/1985. Active principle
Condition of the sample to be analysed
Maximum permissible residue (mg/kg)
Suspension period (days)
Chlorpropham Propham Thiabendazole
Peeled potatoes Peeled potatoes Washed potatoes
0-5 a 0-5 a 4-0
30 30 30
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a
As the sum of propham and chlorpropham.
Saxton 1987, Tonagai etal. 1987, Voznakova et al. 1988); the number of TLC procedures is much smaller (Ambrus et al. 1981, Korsos and Lantos 1984, Lawerenz et al. 1986, Osselton and Snelling 1986, Wachalz et al. 1988). Experimental
Apparatus The plates used were Merck C8 HPTLC, 10 x 10 cm, 0-2 mm thick (Merck, Darmstadt, Germany). Spotting was performed using Hirschmann Laborgerate (Germany) Minicaps calibrated capillaries. Densitometric scanning of the Chromatographie plates was performed with a CAMAG Scanner connected to a Perkin-Elmer R 100 A recorder. For the HPLC investigation a Perkin-Elmer series 410 Chromatograph with variable wavelength LC 85B spectrophotometric detector and R 100 A recorder were used. The column was a Supelco Supelcosil LCis (particle size 5 pm, internal diameter 4-6 mm, length 15 cm). For the preliminary Chromatographie separation, Merck Si6o TLC plates (20x20 cm, 0*25 mm thick) were used. Spotting was made with a Desaga 80 Microdoser with a graduated syringe. Materials Merck Lichrosolv solvents were used for chromatography and Merck RP dichloromethane to extract the residues from the potatoes. The analytical standards were purchased from Labo Service (Bologna, Italy). Extraction of residues from potatoes Potato (100 g) was carefully washed or peeled as stipulated by Italian law in relation to analysis for residues (table 1) and minced in a meat-mincer to give a homogeneous mixture. An exact weight of around 25 g was placed in a separating funnel, 20 ml of a saturated aqueous solution of NaCl added and the mixture extracted with three 50 ml portions of dichloromethane. The pooled organic phases were dried over Na2SC>4 and evaporated to dryness on the rotavapor. The residue was redissolved in 1 ml dichloromethane and 400 p.\ of this solution was spotted on Silica 60 TLC plates with an automatic doser under the conditions described below. Chromatographie conditions Initial TLC clean up of the samples extracts. Four 400 ^1 portions of potato
HPTLC in the control of pesticide residues
609
Table 2. Preparation of the samples for analysis. Elution of single compounds (cm)
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1st plate elution 2nd plate elution Band removed for analysis
Mobile phase
CIPC
IPC
TBZ
Total plate elution (cm) Total plate elution (cm)
CHC13 :triethylamine 1:0-05 CHC13:CH3OH 1:0-05
15-3
15-3
9-3
19-0
—
—
11-8
14-0
14-5-16-0
14-5-16 •0
11-1-12-2
extract were spotted on to a plate along with a fifth, similar spot containing only the standards. The spots were spaced at a distance of 0-7 cm along a 3-cm line. Deposition was performed so as to have a margin of 1 cm from the edges of the plate and 2 cm from the side immersed in the mobile phase. After development using the two eluents shown in table 2, the layers corresponding to the compounds CIPC and IPC and that corresponding to the compound TBZ were removed by scraping with a metal spatula. The compounds were eluted from the silica by shaking with 400 /x\ methanol and then separated from the solid phase by centrifuging. The solution was spotted directly on HPTLC plates for quantitative analysis. For HPLC the residual solution was diluted to 800 /tl for compounds CIPC and IPC and to 2-5 ml for compound TBZ; the solutions were filtered through a 0-45 fiva membrane before injection. Quantitative analysis on HPTLC plates. On the RP8 HPTLC plates the spots were placed 0*9 cm apart and 1-5 cm from the upper edge of the plate. Development was carried to within 0-5 cm of the upper edge of the plate. The Rt values were 0-35 for CIPC, 0-48 for IPC and 0-33 for TBZ. The plates were read at 248 nm and the spots were of 5 /tl of the methanol fraction containing the CIPC and IPC and 2 fA of the fraction containing TBZ, with sensitivity at 10, span 8, plate speed 30 mm/min and baseline 200 mV. In order to optimize elution performance, the mobile phase consisting of a methanol/water mixture (1:0-3) was placed in the Chromatographie chamber 12 h before elution. The position of the plate was kept constant and the internal temperature of the chamber (20°C ±1-5) was kept constant by a thermostat. HPLC analysis. The analysis was carried out with detection at 248 nm with a flow of 2 ml/min, volume of injection 6/J, mobile phase methanol/0-1 M phosphate buffer pH 6-6 (3:2). Calibration curve for the three compounds Different volumes of a methanol standard solution containing chlorpropham (1 mg/ml), propham (1 mg/ml) and thiabendazole (4 mg/ml) were added to 25 g of homogenate of washed or peeled potatoes (as specified by the legislation). The range of calibration is reported in table 3.
610
P. Corti et al.
Table 3. Calibration curve parameters for HPTLC and HPLC analysis of chlorpropham, propham and thiabendazole added to the biological matrix. Intercept ± p 1CT2
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Product
Slope ± P
Coefficient of correlation
Detection limits (*4g/g)
Quantification limits (fg/g)
Chlorpropham
a b
0-020 ±0-210 1-056 ±0-012 0-081 ±0-530 1-660 ±0-091
0-9982 0-9998
0-023 0-002
0-075 0-007
Propham
a b
0-024 ±0-180 1-204 ±0-014 0-072 ±0-470 1-351 ±0-080
0-9994 0-9987
0-019 0-002
0-065 0-008
Thiabendazole
a -0-011 ±0-100 0-572 ±0-070 b 0-112 ±0-720 2-140 ±0-130
0-9988 0-9991
0-100 0-0005
0-350 0-0017
The parameters were calculated from peak height (mm) and concentration (ng/dep or ng/injection). a and b refer to the curves obtained with the HPTLC and HPLC respectively. 1st confidence interval p calculated on five calibration curves (five points in the range for each) was below that determined for p = 0-05 in all cases. The calibration range was 5-300 ng/deposit or ng/injection in all cases.
Results Preparation of the sample Table 2 summarizes the conditions for the preparation of samples for analysis. Elution of the plate with a mobile phase consisting of chloroform/ triethylamine (1:0-05) enabled the fraction corresponding to compounds CIPC and IPC to be isolated in a relatively pure state. The second elution with chloroform/methanol (1:0-05) eliminated certain components from the thiabendazole fraction, which interfere in the successive HPTLC and HPLC analysis from the first elution solvent with the above mobile phases. The widths of the two bands of silica to be removed were the maximum widths obtained by repeating many spottings on many plates, as described below. The groups of untreated potatoes of the varieties Kennebec and Majestic grown in different parts of the Province of Siena, were checked for significant differences in endogenous components in the fractions of extract to be analysed. The repeatability of the widths of the silica layer to be removed was also examined. Out of ten samples from each group it was found that there were no variations in the type of components in the two silica bands removed for analysis of the three compounds. No Chromatographie signals interfering with compounds CIPC, IPC or TBZ were found by TLC or HPLC. The results of the analysis of potatoes which had either been washed or peeled, gave similar results. The reproducibility of the width of the band of silica to remove for the three standards was also satisfactory. In the ten plates analysed, also with a reference spot containing a mixture of the three standards (a total of five spots per plate), a coefficient of variation of 5-4% was obtained for thiabendazole and 3-4% for the other two components. In order to understand in detail the behaviour of the TLC plates used in this preliminary phase, spots (400 jtl distributed over 3 cm) of a solution containing CIPC (12-5 jtg/ml), IPC (12-5/tg/ml) and TBZ (100^g/ml) were also made to determine the reproducibility of the spots in quantitative terms. Deposits were made on six plates from three different lots of production and the single fractions were analysed by HPTLC.
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Evaluation of the coefficient of variation for individual plates gave a maximum CV of 2-4% for TBZ; comparison of plates gave CV of 1-0% for CIPC, 0-9% for IPC and 1*3% for TBZ. Comparison with direct analysis of the solutions of the three active principles gave mean per cent recoveries in this phase of 99 • 7 for CIPC, 101-2 for IPC and 99-3 for TBZ. Overall recovery and repeatability were evaluated adding appropriate quantities of standards (0-25, 0-50, 1 -00 /ig/g for CIPC and IPC and 1-00, 2-00, 4-00 jig/g for TBZ) to homogenate of untreated potatoes. The mean recoveries and coefficient of variation were respectively 92-9% and 5-1 for CIPC, 90-7% and 4* 1 for IPC, 94-6% and 5-1 for TBZ. Assay by HPTLC The best analytical conditions for the assay of the three compounds were obtained with HPTLC RP8 plates and methanol/water (1:0-3) mobile phase. For other types of layers (silica, silica derivatized with propylamine or propionitrile) the separation of the three compounds from the coextracted matrix was not satisfactory. Figure 1 and table 3 give the Chromatographie resolutions and a summary of the calibration curve parameters for the standards extracted from the biological matrix. The limits of detection and quantification were well below those stipulated by the legislation, making the method suitable for surveillance and enforcement purposes. On this basis we decided to check certain parameters in order to validate the method. First we found that the performance of the plates was the same for all points of deposition. It is known that the lateral parts of the plate may suffer variations in the elution process as a result of a possible slight non-linearity of the solvent front. When this happens, one may have different retention distance (Rf value) and spot distribution for a given compound at a given concentration, with
TBZ
CIPC IPC
min
min
Figure 1. HPTLC resolution of the blank fractions of potato extract with the addition of propham, chlorpropham and thiabendazole. The contents of CIPC and IPC in the biological matrix were 0-12 jig/g and 0-15 jig/g respectively. The content of thiabendazole in the biological matrix was l-0pg/g.
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a consequent difference in Chromatographie peak height between depositions which are central and lateral on the plate. On three plates from three different lots, five deposits of a mixture of CIPC and IPC and five of TBZ were made. The deposits for CIPC and IPC were started on one half of the plate, and those for TBZ on the other. The solutions run had a content of active principle equal to that of the highest point used for the calibrations. For the elution evaluated on the maximum Chromatographie peak, no significant differences were found between the central and lateral spots on individual plates; nor were statistically significant differences found (/? = ()• 05) when this parameter was compared between plates. As far as the quantitative evaluation is concerned (the parameter used was Chromatographie peak height), the results were completely satisfactory. Between individual plates, the percentage differences in peak height between lateral and corresponding central spots was always less than 1-95%, which was the value obtained for a deposit of propham. Comparison between plates did not give statistically significant differences (p = 0-05). On the basis of these results it was decided to perform ten deposits per plate so as to utilize the whole available width. Later we determined the variability of the method in the calibration range used for the three compounds in relation to the plates and to the system of manual deposition used. Diluting the standard solutions of CIPC, IPC and TBZ appropriately, two sets of calibration curves were deposited
IPC TBZ
J 10 mJn
min
Figure 2. HPLC resolution of the blank fractions of potato extract with the addition of propham, chlorpropham and thiabendazole. The contents of CIPC and IPC in the biological matrix were 0 • 40 pg/g and 0 • 20 /jg/g respectively. The content of TBZ in the biological matrix was 0 • 68 jig/g.
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on six plates from three different lots for the mixture of propham and chlorpropham and for thiabendazole. A similar test was performed using different operators. The slopes and intercepts of the different calibrations were not statistically different (p = 0 • 05) on individual plates, between different plates or for deposits performed by one or two operators. Assay by HPLC The Chromatographie resolutions of the fractions for the assay of the three compounds and the parameters of the regressive curve are summarized in figure 2 and table 3. The mobile phase chosen was able to satisfactorily separate the mixture of the three compounds. Again the precision of the method was tested. For each active principle, five points in the calibration range were injected five times in succession. The band of regression lines obtained, in each case, lay within the confidence interval calculated for p = 0-05. A similar result was obtained injecting five points for each active principle on successive days (a set of calibrations over the period of a week). Conclusions At the end of the study, the results obtained by the two methods were compared for the three compounds, adding the latter randomly but within the calibration range, to the biological matrix. The distribution of the results is summarized in figures 3-5 among with the coefficients of correlation (r). The coefficients of correlation between corresponding points shows good linearity and consequently similarity between the HPTLC and HPLC results. Clearly, the sensitivities are different, but although HPTLC is more limited than column chromatography, it is still quite sensitive enough for detecting residues of IPC, CIPC and TBZ in potatoes down to approximately 1/1 Oth of the Maximum
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