GHTMDD - 347

Received: May 17, 1999Accepted: November 15, 1999

Bulletin of the Chemists and Technologists of Macedonia, V01.19, No. 1, pp. 27-33 (2000)ISSN 0350 - 0136

UDC: 632.952.028 : 544.3] : 635.64

Original scientific paper

DETERMINA TION OF PROPICONAZOLE RESIDUESIN TOMATOES BY GAS CHROMATOGRAPHY

Tatjana Kadifkova Panovska, Zoran Kavrakovski, Stevan Bauer

Faculty of Pharmacy, The "Sv. Kiril & Metodij" University,Vodnjanska 17, MK-1000 Skopje, Republic of Macedonia

A simple method for the determination of propiconazole residues in tomatoes, grown in greenhouses, has beendeveloped. Two procedures for extraction (acetone/cyc\ohexane; tartaric acid/ethanol) of the analyte from the samplematrix are suggested. Silica gel and Florisil column chromatography were used for purification of sample solution.The technique used for detection was Capillary Gas Chromatography equipped with flame ionization detector. GC-MSwasusedas a confirmatorymethod.The recoveriesrangedfrom97.5- 99.1 % for acetone/cyc\ohexaneextrac-tion and from 92.4 - 98.8 % for tartaric acid/ethanol extraction. Tomatoes treated with propiconazole using commer-

cial fungicide formulation - Tilt 250 EC were analyzed using both procedures. There are differences between the testresults obtained by the two procedures at 5% significance level. The acetone/cyc\ohexane extraction is recommendedfor use at determination of propiconazole in tomatoes.

Key words: fungicide; gas chromatography; tomato; pesticide

INTRODUCTION

Propiconazole (cis/trans-l-(2-12,4-dihlorfenil/-4-propil-I,3-dioksalan-2-il-metil)-lH-I,2,4-triazol)is a systemic fungicide applied to wheat, barleyand grape to control rust disease [1]. Besides thisapplication, in agricultural practice, this fungicideis often used for treating tomatoes, grown ingreenhouses. Its mode of action is to inhibited er-gosterol biosynthesis [2].

In modern agricultural food production, goodresults can be obtained only with the help of pesti-cides, but these materials contaminate the envi-ronment and some more or less persistent pesticideresidues remain on the surface of or inside the

products. Methods for pesticide residue determina-tions for the qualification of food products are un-der rapid development. The residue limits given bythe World Health Organization are becoming evenlower creating an ever-increasing demand for more

selective and sensitive methods. As the concentra-

tion of the pesticide residues in food material is inthe nanogram, picogram or sometimes femtogramper gram range, their evaluation can be carried outonly by extremely selective and sensitive detectionmethods [3].

The method for the determination of pesticideresidues in food materials consists of two steps:sample preparation and analysis. Chromatographycan be used as a preliminary concentrating proce-dure, which can be continued with column chroma-tography as a part of the clean up. Moreover,chromatography also plays a very important role inthe determination step [4].

We have established a method for single resi-due analysis of propiconazole in tomatoes, by cap-illary gas chromatography with flame ionizationdetector (FID).

Materials

EXPERIMENTAL

Pesticide standard. Pesticide standard refer-

ence material was purchased from Ciba-Geigy. The

purity of propiconazole was more than 95 % bygas liquid chromatography (GLC).

Pesticide working solutions. Pesticide stan-dard solutions (1000 Ilg/ml) were prepared by dis-

28 T. Kadifkova Panovska, Z. Kavrakovski, S. Eauer

solving the pesticide standard in distilled methanoland diluting to suitable concentrations with thesame solvent.

Organic solvents and reagents. Acetone,cyc1ohexane, n-hexane, ethanol, methanol were ofspecial grade for pesticide residue analysis.

Kieselgel 60 (70-230 mesh) and acidum tar-taric were obtained from Merck & Co. Inc. (Darm-stadt, Germany). Anhydrous sodium sulfate(Na2S04) was of analytical grade. These reagentswere used without prewashing. Florisil PR (60-100mesh) was obtained from Varian, U.S.A. FlorisilPR and anhydrous Na2S04 were heated overnightat 130°C and desiccated before use. Kieselgel 60was used without any treatment.

Samples. Tomatoes were obtained fromgreenhouses, treated with commercial fungicideformulation - Tilt 250 EC.

Apparatus

Gas chromatograph. Varian Star Model 3400CX equipped with a mass spectrometric (MS) de-tector was used for determination of propicona-zole. Fused-silica capillary columns DB-5 (30 m x0.25 mm i.d., film thickness 0.25 /lm) were usedfor pesticide content determination.

Chromatographic tube for column chromato-graphy. A glass column of 20 cm x 13 mm i.d. wasused in silica gel or Florisil column chromatogra-phy for purification of sample solution.

Rotary evaporator. Devarot equipped withwater bath and vacuum pump 2DS4 (Electromedi-cina, Ljubljana) were used to concentrate the or-ganic solvents. A water bath was set at 35-40 QC.

Methods

Extraction

1. Acetonelcyclohexane extraction. - Approxi-mately 1000 g of whole tomatoes were mechani-cally minced to provide a homogeneous tomatomix, from which sub-samples were taken. A sub-sample of 10 g was placed in a blender cup. Onehundred milliliters of an acetone/cyc1ohexanemixure (1: 1 VIV) was added. The homogenate wasfiltered through No. 5A filter paper, and the resi-due was rehomogenized with 100 ml of the samemixture and then filtered again. The filtrate wascombined, than dehydrated with 20 g of anhydrous

Na2S04, and evaporated to dryness using the rotaryevaporator. The residue was redissolved in metha-nol and made up to final volume of 3 ml (the sam-ple solution).

2. Tartaric acid/ethanol extraction. - A sub-

sample of 10 g of tomatoes was transferred to ablender cup, to which 60 ml of 80 % ethanol and 5g acidum tartaric had been added. The contentswere then blended for 30 minutes. The mixture

was filtered through No. 5A filter paper, and theresidue was rehomogenized with the same amountof ethanol and acidum tartaric and then filtered

again. The filtrate was combined, than dehydratedwith 20 g of anhydrous Na2S04, and evaporated todryness using the rotary evaporator. The residuewas redissolved in methanol and made up to finalvolume of 3 ml (the sample solution).

Column chromatography

(a) Silica Gel column chromatography. Tengrams of Kieselgel 60 suspended in adequateamounts of n-hexane was placed in a chroma-tographic tube plugged with cotton wool, and then10 g of anhidrous Na2S04 was added continuouslyand n-hexane drained.

Three milliliters of the sample solution wastransferred completely to a silica gel column alongwith 5 ml of the acetone/n-hexane mixture (3:7VIV). Propiconazole was eluted with 100 ml ofthis mixture. The eluate was concentrated using arotary evaporator, and the volume was adjusted to3 ml with methanol (sample solution).

(b) Florisil column chromatography. Tengrams of Florisil PR suspended in adequate amountsof n-hexane was placed in a chromatographic tubeplugged with cotton wool, and then 10 g of anhy-drous Na2S04 was added continuously and n-hexane drained.

Sample solution from Silica Gel columnchromatography was transferred completely to aFlorisil column along with 5 ml of the acetone/n-hexane mixture (3:7 VIV). Propiconazole waseluted with 100 ml of this mixture. The eluate was

concentrated using a rotary evaporator, and thevolume was adjusted to 2 ml with methanol (thetest solution).

Determination of pesticides. Test solutionprepared in (b) was subjected to GLC for propi-conazole content determination under the follow-

ing conditions: apparatus, Varian Star 3400 CX;column DB-5; column temperature, 160 to 260°C

Bull. Chem. Technol. Macedonia. 19, 1,27-33 (2000)

Determination ofpropiconazole residues in tomatoes by gas chromatography 29

at 8 QCmin-I and hold 4 min; injector temperature220 QC;detector (FID) temperature 300 QC;carrier

gas, He; flow rate, split ratio 100: 1; injection vol-ume, 1 Ill.

RESULTS AND DISCUSSION

. Basically, most procedures for the determina-tion of pesticide residues could be subdivided intothree steps, viz., extraction, and clean-up and in-strumental determination. With a view to improv-ing efficiency, improvements were made in thesethree aspects as to achieve a simple and rapid de-termination of propiconazole residues in tomatoes.

Extraction

Polar solvents, such as acetone or methanol,are most commonly used to extract pesticide resi-dues from food samples. As these solvents are mis-cible with water, they can penetrate the food matri-ces more effectively [5]. However, for subsequentconcentration, clean-up and determination, a sol-vent-partitioning step is often required to extractthe pesticides from this water-containing layer to awater-immisible organic layer [6-9]. In this study,an acetone/cyclohexane mixture (extraction 1) andtartaric acid/ethanol (extraction 2) was directlyemployed in the extraction of the sample. Thus, anadditional solvent partitioning step was not neces-sary. These solvents were chosen owing to theirproven applicability in the determination of or-ganonitrogenous pesticides [10, 11]. To enhancethe extraction efficiency, a sufficient amount ofanhydrous sodium sulfate was added to adsorb thewater originally present in the sample [12]. Afterfiltration, a measured volume of the extract wastaken out, concentrated and reconstituted for col-umn chromatographic clean up.

Clean-up

Vegetables contain a wide variety of com-pounds that are extractable and may give rise tointerferences or other problems. More insidiouseffects arise when coextractives cause adsorptionor decomposition of pesticides. Lipids, chloro-phyll,or other high molecular materials accumulat-ing in the injection region of gas chromatographsareparticularly problematic. Vegetables are gener-allylow in lipids [13], but can have relatively highlevelsof pigments and phenols. Thus, clean up byFlorisil or silica gel column chromatography was

f]Jac. xeM. TeXHOJI. MaKel10Hlfja, 19, 1,27-33 (2000)

necessary in the quantitation of organonitrogenpesticides.

Gas chromatographic determination

Gas chromatography remains the basis for de-termination of residues in most single or multiresi-due methods. This is largely because GC detectorshave the high sensitivity and selectivity requiredfor residue work. The advent of fused silica opentubular (FSOT) columns with bonded polymericliquids phased has aided residue determination byprovided stable, reproducible, highly inert columnwith much higher resolving power than previouspacked column technology. Gas chromatogra-phy/mass spectrometry (GC/MS) has become thepreeminent technique for identification and deter-mination of pesticide residues [14, 15]. Accordingto the Pesticide Analytical Manual [16], there arelarge differences in the retention times of propi-conazole under isothermal conditions. In order toachieve an effective separation of these fungicideswithin a shorter period of time, temperature pro-gramming was thus attempted. According to theapplied temperature program, the retention time ofpropiconazole was 13.675 minutes. Figure 1 showsthe gas chromatogram of the standard solution ofpropiconazole (0.5 Ilg ml-3) using the DB-5 col-umn with an optimized temperature program.

Under this condition, propiconazole gave riseto doublet peaks in the chromatogram due to theseparation of diastereomers. Figure 2 shows thegas chromatogram of the test solution of tomatoafter extraction and clean-up.

Table 1 summarizes the slope and correlationcoefficients for the calibration curves of the propi-conazole. As indicated by the high correlation co-efficient, responses for the fungicide is linearwithin the concentration ranges studied.

The identity of the peak was confirmed byGC-MS (Fig. 3). The electron ionization massspectrum (70 eV) of isolated propiconazole wascompared with its mass spectrum from the NISTLibrary (Fig. 4).

30 T. Kadifkova Panovska, Z. Kavrakovski, S. Bauer

mVolts

13.675

2.0

1.0

0.0 10.0 20.0 mm

Fig. 1. Capillary gas chromatogram of the standard solution of propiconazole (0.5 Ilg mr') in methanol.Gas chromatographic conditions are described in the text

mVolts

13.6752.0

1.0

0.0 10.0 20.0 mm

Fig. 2. Capillary gas chromatogram of the test solution of tomato in methanol after extraction with acetone/cyclohexane 1:1.Gas chromatographic conditions are described in the text

Bull. Chem. Technol. Macedonia, 19, 1,27-33 (2000)

Detennination ofpropiconazole residues in tomatoes by gas chromatography 31

Table I

Linear regression output for the calibration curves of propiconazole

(%) 100 259

8069

60

17340

14555

20 109191

20786

050 100 150 200 250 300 Mass

Fig 3. GC-MS electron ionization mass spectrum (70 eV) of isolated propiconazole from tomato

(%) 100 69173

80 259

60

40 128145

55 86 109

751 96 1

19120

0

50 100 150 200 250 300 Mass

Fig 4. GC-MS electron ionization mass spectrum (70 eV) of propiconazole from the NIST Library

fJIac. xeM. TeXHOJI. MaKe110HIIja, 19, 1, 27-33 (2000)

Fungicide Concentration range Correlation coefficient Slope Intercept

(/_.tgmr1) (r) (/lg-mrl per unit area) (unit area)

Propiconazole 0.05 - 0.5 0.9996 2194.1 11.281

32 T. Kadifkova Panovska, Z. Kavrakovski, S. Bauer

For the estimation of detection limits, sampleextracts were spiked with the fungicides at lowconcentration levels so that they gave rise to mar-ginally identifiable peaks in the chromatogram.Gas chromatographic determination was then re-peated 10 times. The standard deviation (s) of thedetermination was calculated. The detection limit

was defined as 3 s after taking into account theconcentration factor achieved in the sample prepa-ration steps. The detection limit for the propicona-zole achieved by the present method is 0.005 !J.8"g-1.This value is well below the maximum residue lev-

els published by Codex Alimentarius Commission(0.05 !J.8"g-J)[17].

In the recovery studies, methanolic solutionsof fungicide standard were spiked to comminuted

Table 2

tomatoes, which were previously tested to be freeof these fungicides. The spiked concentrations for

propiconazole were 0.1 !J.8"g-l,0.2 !J.8"g-l,and 0.3!J.gg-l. The spiked samples were processed accord-ing to the two newly developed procedures and theresults are summarized in Table 2. Recoveries of

propiconazole were 97.5 - 99.1 % (with ace-tone/cyclohexane) and 92.4 - 98.8 % (with tartaricacid/ethanol). No significant differences in the re-covery of propiconazole were evident regardless oforganic solvents used at the extraction step. Be-cause the acetone/cyclohexane extraction gave thebetter recoveries of propiconazole then the tartaricacid/ethanol extraction, it is recommended for useat determination of propiconazole in tomatoes.

No.

Recovery of propiconazole from spiked tomatoes

Added

(/lgg-l)

Calculated

(/lgg-l)

Determined

(/lgg-l)

R(%)

Acetone/cyclohexane extraction

2

3

0.1

0.2

4 0.3

0.243

0.343

0.143

0.237 97.5

99.10.340

0.4390.443 99.1

Tartaric acidlethanol extraction

2

3

0.1

0.2

4 0.3

These two procedures of extraction were alsoverified by the analysis of tomatoes, grown ingreenhouses, treated with propiconazole usingcommercial fungicide formulation - Tilt 250 EC.These treated tomatoes were analyzed using both

Table 3

0.292

0.392

0.192

0.270 92.4

98.50.386

0.4860.492 98.8

procedures. The results obtained are summarized inTable 3. A Hest was performed [18]. There aredifferences between the test results obtained by thetwo procedures at 5 % significance level.

Results of analysis of tomatoes treated with Tilt 250 EC

Concentration :t Sl/lg-g-lFungicide

Extraction 1 Extraction 2

0.192:t 0.0025 40.12Propiconazole 0.143:t 0.0011

Extraction 1 - acetone!cyclohexane extractionExtraction 2 - tartaric acidlethanol extractionn=5

Bull. Chem. Technol. Macedonia, 19, 1,27-33 (2000)

Determination ofpropiconazole residues in tomatoes by gas chromatography 33

CONCLUSION

A simple and rapid analytical procedure forthe determination of propiconazole in tomatoeswasdeveloped. The speed, accuracy and sensitivityof this method are its advantages, which couldwarrantits introduction for routine residue analysis

of propiconazole in tomatoes. With minor adapta-tions to the extraction procedure, propiconazolecould also be determinate in a wide range of otherproduce.

REFERENCES

[1]Pesticide Manual, Crop Protection Publications, 855(1995).

[2]J. M. Gou-Ot, Phytoma, 474, 12 (1995).

[3]K. Fodor-Csorba, Journal of Chromatography, 624, 353(1992).

[4]W. Horowitz, Official Methods of Analysis of the Asso-ciation of Official Analytical Chemists, AOAC, 14, 29(1985).

[5]G. Becker, P. Schug, Dtsch. Lebensm-Rundsch. 86, 239(1990);Chem. Abstr. 229799s, 113 (1990).

[6]A. Di Muccio, A. Ausili, R. Dommarco, D. AttardBarbini, A. Santi1io, F. Vergoli, G. De Merulius,L.Sernicola, J. Chromatogr., 552, 241 (1991).

[7]W. F. Beckert, V. Lopez Avila, Environ. Sci. Res., 42,113 (1992).

[8]D. W. Beyers, C. A. Carison, J. D. Tessari, Environ.Toxico/. Chem., 10, 1425 (1991).

[9]H. J. Borburgh, W. E. Hammers, Toxicol. Environ.Chem.,35, 79 (1992).

[10] Association of Official Analytical Chemists, OfficialMethods of Analysis, 1, (1990).

[11] L. G. M. T. Tuinstra, A. H. Roos, A. M. Matser, W. A.Traag, J. A. Van Rhijn, Fresenius' J. Anal. Chem., 339,384 (1991).

[12] G. A. Va1verde, E. Gonza1es Pradas, J. Martinez Vidal,A. Aguera Lopez, J. Agric. Food Chem., 39, 2188 (1991).

[13] M. A. Luke, H. T. Masumoto, Academic Press, 15, 161(1986).

[14] T. Cairns, E. G. Siegmund, J. J. Stamp, Mass Spectrom.Rev., 8, 93 (1989).

[15] P. T. Holland, Pure and Appl. Chem., 62, 317 (1990).

[16] Pesticide Analytical Manual, U. S. Department of Healthand Human Services. Food and Drug Administration,Washington, DC, 1 (1994).

[17] Codex Alimentarius Commission, Codex Alimentarius,Pesticide Residues in Food; Maximum Limits for Pesti-cide Residues, World Health Organization, Rome, 2(1993).

[18] S. C. Miller, J. N. Miller, Statistics for Analytical Chem-istry, Ellis Horwood, Chichester, 2nd edn. (1988).

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