determination of procymidone in vegetables by a commercial competitive inhibition enzyme immunoassay

ANALYTICA CHIMICA ACM

ELSEVIER Analytica Chimica Acta 311(1995) 371-376

Determination of Procymidone in vegetables by a commercial competitive inhibition enzyme immunoassay

Amadeo R. Fernandez-Alba a,* , Antonio Valverde a, Ana Agiiera b, Mariano Contreras b, Dolores Rodriguez ’

a Pesticide Residues Research Group, Facultad de Ciencias de Abner& 04071 Abner&z, Spain b Laboratorio de An&is Agricola de COEXPhWL (Cosecheros-Exportaa’ores de Hortalizas de Almeria), El Viso, 04070 Almeria, Spain

’ Centro National de Biotecnologia C.S.I.C., Campus UniversidadAut&oma, Cantoblanco, 28049 Madrid, Spain

Received 20 September 1994, revised 8 February 1995; accepted 13 February 1995

Abstract

An ELISA commercial kit was used to quantitate residues of Procymidone at very low levels (< 20 pg/kg) in pepper samples. Samples were extracted with ethyl acetate-sodium sulphate and an aliquot is evaporated to dryness and reconstituted in 10 ml of light petroleum. Sample clean up is accomplished by aspirating 2 ml of the light petroleum extract through a silica gel solid phase disposable cartridge. Following aspiration, the sample was eluted with 2 ml of ethyl ether-petroleum ether (1:l). The eluted fraction was evaporated and dissolved with sonication in 2 ml of water before immunoassay analysis. The limit of Procymidone detection was 0.6 pg/kg. The assay logarithmic response was linear from 5 to 80 pg/kg of Procymidone residue. Irma-assay percentage coefficients of variation (%C.V.) ranged from 3.5 to 18.0 and inter-assays %C.V.s varied from 8.0 to 16.0. Comparison studies between gas chromatography with electron capture detection and ELBA analyses showed an acceptable coincidence at levels of 8 pg/kg. However, lower levels of ca. 2 pg/kg were detectable by ELISA only. No cross-reactivity problems were found in the ELBA test associated with the presence of other dichloroaniline derivatives and other pesticides studied.

Keywords: Immunoassay; Enzymatic methods; Biosensors; Procymidone; Pesticides; Environmental analysis; ELISA, Vegetables

1. Introduction

Procymidone (N-(3,5-dichlorophenyl)-1,2-di- methylcyclopropane-1,2-dicarboximide) is a system- atic fungicide that is widespread used in mediter- ranean agricultural areas as a preharvest treatment on fruits and vegetables to prevent Botrytis [l]. This pesticide is of special concern due to its chlorinated

character and its persistence in the environment [2-41. It is therefore an important objective in monitoring food analysis. Tolerances in different countries/commodities for residues of Procymidone in vegetables are very variable and range from around 2.0 mg/kg in the EEC [5] to “nil” tolerance in the USA [6] and Switzerland [7], which is understood to be less than the limit of detection (L.O.D.) of 0.02 mg/kg bl.

Currently the * Corresponding author. in vegetables is

0003-2670/95/$09.50 8 1995 Elsevier Science B.V. All rights reserved

SSDI 0003-2670(95)00121-2

presence of Procymidone residues detected by applying well-known

372 A.R. Fernandez-Alba et al. /Analytica Chimica Acta 311 (1995) 371-376

multi-residue methods (MRMs) [8-131 which gener- ally involve extraction, evaporation and clean-up steps followed by gas chromatography (GC) with electron capture detection (ECD). The detection lim- its in routine GC methods are around 0.02 mg/kg [14,15]. In such a situation the possibility of obtaining false positives or negatives near the L.O.D. with complex vegetable matrices such as pepper is sub- stantial. Although confirmation analyses by GC-MS [16-181 can easily be performed by skilled analysts in well-equipped laboratories, these analyses are la- borious, expensive and time consuming, especially at very low levels [19]. Enzyme-linked immunosorbent assay (ELISA) is a simple and fast confirmatory and quantitative technique that can fulfil the needs of sensitivity [20,21].

In previous works [22,23] we developed a simple protocol to analyze organochlorine and organophos- phorus pesticides in vegetal matrices. The present study deals with the application of ELISA for the confirmation and quantitation of low levels ( < 0.02 mg/kg) of Procymidone residues in pepper samples.

2. Experimental

2.1. Chemicals

EnviroGard’” EIA kits were obtained from Milli- pore (Bedford, MA), which consist of polystyrene test tubes coated with Procymidone antibodies and an enzyme conjugate (horseradish peroxidase bound to ‘Procymidone). Hydrogen peroxide is used as substrate and tetramethylbenzidine as chromogen.

Pesticide-grade ethyl acetate, petroleum ether, methanol, anhydrous sodium sulfate (12-60 mesh) and reagent grade water were obtained from Merck (Darmstadt). Silica gel solid phase extraction disposable columns of 6 ml and 500 mg were obtained from Varian (Harbor City, CA). The Procymidone standard was obtained from Riedel-de HHen (Seelze). 100.0 mg/l standard stock solutions were prepared by dissolving 10.0 mg of purity certified pesticide in 100 ml of petroleum ether. Working standard solutions were prepared by transferring 0.2 ml of standard stock solution to a 100 ml volumetric flask and made up to volume with petroleum ether. This gives a 0.2 mg/l working standard solution. Pesticide

standard solutions for immunoassay were prepared by adding 30,50,100,200,300,800,1000 and 2000 ~1 aliquots of the working standard solution to 2 ml of ethyl ether-petroleum ether (1:l) extract of un- treated and not fortified pepper samples (see Section 2.3) in 10 ml vials. After evaporating to dryness by nitrogen stream the extract was redissolved with 2 ml of water. This procedure gives a concentration range of S-200 pg/l for the immunoassay standard solution. Pesticide standard solutions for GC were prepared in the same way but using 50% ethyl ether in petroleum ether instead of water to redissolve the extract.

2.2. Apparatus

A Perkin Elmer 8600 (Beaconsfield) equipped with a

g$s chromatograph Ni electron capture

detector and an HP1 fused silica capillary column (30 m X 0.53 mm i.d., 2.65 pm particle size) coated with methylsilicone (Hewlett-Packard, Palo Alto, CA) was used for GC analysis.

A Shimadzu (Kyoto) UV-160 spectrophotometer was used for spectrophotometric determinations.

2.3. Sample preparation

Fresh pepper samples were collected from different greenhouses in the vicinity of Almerfa (Spain), in which Procymidone was not applied. A number of pepper samples were fortified at l-10 pg/kg with Procymidone as previously described [22,23]. Sam- ples were extracted in our laboratory according to the following procedure. Pepper samples (50 g) chopped in a high-speed blender were thoroughly mixed with 40 g of anhydrous sodium sulfate and then 100 ml of ethyl acetate were added and the mixture was blended for 5 min more. The liquid supernatant was filtered by suction through a filter paper and a layer of 20 g of anhydrous sodium sulfate. The filter was rinsed with 50 ml of ethyl acetate and the combined extracts were evaporated on a vacuum rotary evaporator using a 40-60°C water-bath. The residue was redissolved in 10 ml of petroleum ether. In order to achieve a cleaner extract an optimized clean up step was added [23] by pass- ing 2 ml of this vegetable extract through to a SPE silica gel (500 mg) disposable cartridge previously

A.R. Fermdez-Alba et al./Analytica Chimica Acta 311 (1995) 371-376 373

equilibrated with 5 ml of petroleum ether. The SPE minicolumn was eluted with 2 ml of 50% ethyl ether in petroleum ether at a flow of l-2 ml/mm The eluted fraction was evaporated carefully to dryness and the residue dissolved in 2 ml of petroleum ether for GC-ECD analysis or 2 ml of water with sonication for immunoassay analysis.

2.4. Immunoassay of Procymidone

Immunoassay standard solutions and extracts from fortified samples were analyzed according to the following procedure: 160 ~1 of either standard solution or sample extract were added to coated tubes of the immunoassay kit followed by 160 ~1 enzyme conjugate (as many as 8 tubes can be prepared simultaneously). Tubes were incubated for 15 min at room temperature. The tubes were rinsed 5 times with water to remove unreacted sample and enzyme conjugate. The reaction was developed by addition of 160 ~1 of substrate and 160 ~1 of chromogen. Tubes were incubated at room temperature for 10 min before adding 1 drop 1.25 M sulfuric acid to stop the reaction. Finally 500 ~1 of purified water were added. The concentration of Procymidone in each sample was determined by measuring the absorbance at 450 nm. A control tube (zero standard) tube was included with each set of tubes to calculate %B/B, values of standard and samples (absorbance at 450 nm of standard or sample/absorbance at 450 nm of control X 100). The observed sample results were compared to a linear regression line (between the log of the Procymidone concentration and %B/B,) calculated after the analysis of 5, 10, 30 and 80 pg/l standard solutions of Procymidone.

2.5. Chromatographic analysis

Following the silica clean up step the extracts were analyzed by GC. Helium was the carrier gas flowing at 8 ml/min. The temperatures of the injec- tor and detector were maintained at 240°C and 300°C respectively. The oven temperature program was 150°C (1 min hold) to 215°C (6 min hold) at 30”C/min then to 260°C at lS”C/min, the injection volume was 1 ~1. External standard calibration graphs were obtained by using the GC standard

solutions of Procymidone described above. The detection limit of Procymidone was 8 pg/kg [23].

3. Results and discussion

3.1. Linear range studies

The immunoassay standard response curve, using the standard solutions described in the Experimental section, showed a linear relationship (R = 0.999) from 5 to 80 pg/l of Procymidone. This is equiva- lent to Procymidone values of 1-16 pg/kg in fresh pepper sample. Fig. 1 illustrates the mean dose response curve for four standard solutions selected to calibrate the Procymidone-ELlSA test. For samples with concentrations > 80 pg/l a simple dilution must be performed on the sample extract. The dis- placement at 10 pg/l (it represents 2 pg/kg in fresh pepper sample) is significant, 73% B/B,. The assay sensitivity was estimated to be 3 pg/l, 0.6 pg/kg in sample, using the 90% B/B, displace- ment [24]. This L.O.D. is much lower than those obtained by standard GC methods and fulfils the‘ needs of sensitivity.

3.2. Matrix effects

Some tests were carried out in order to assess the need of the clean up step with silica previous to the

-I 01 IO0

I

IO' 102

c- of Racymidone (&L)

Fig. 1. Dose response curve for Procymidone. Each data point

represents the mean of five determinations. Vertical bars indicate

f 2S.D. of the mean.


Table 1

Percentage of inhibition and coefficient of variation (C.V.) of

Procymidone antibodies for different types of pepper sample

blanks

Pepper type %Inhibition

Without C.V. With C.V.

clean up (o/o) a clean up (o/o) a

Yellow 28 20 12 6

Green 25 18 15 8

Red 31 21 11 9

a Percent coefficients of variation based on 4 determinations in 1

day.

Procymidone-ELBA assay for pepper samples. Table 1 shows the values of Procymidone antibody inhibition for different pepper blank extracts obtained by the described sample preparation procedure, with or without silica clean-up step. This inhibition percentage is referred to purified water ELISA values. However, while inhibition values > 25% were obtained when the clean-up is omitted, values < 15% were obtained when the clean-up was performed. In addition, the high variability in the percentage of inhibition obtained with the same type of pepper (C.V. r 18%) and with different pepper types (C.V. = 21%) when the clean-up step was not performed, indicates the need for an adequate clean-up previous to the Procymidone ELBA analysis for pepper samples.

Table 3

Cross-reactivity of Procymidone antibody towards a selection of

fungicides

Compound ( cLg/I) Cross reaction (o/o)

Procymidone

Vinclozolin

Iprodione

Carbendazime

Clozolinate

Benomyl

Thiabendazole

30

30

3000

30

3000

30

3000

30

3000

30

3000

30

3000

100

< 0.01

< 0.01

< 0.01

< 0.01

< 0.01

< 0.01

< 0.01

< 0.01

< 0.01

< 0.01

< 0.01

< 0.01

3.3. Precision of the assay

Results for the Procymidone-ELlSAs in cleaned- up extracts from different pepper samples and fortified at 2, 6 and 10 pg/kg are presented in Table 2. High reproducibility was achieved when different analyses were performed either intra-assay and inter-assay obtaining coefficients of variation rang- ing from 3.5% to 18.0%. Procymidone levels determined by ELlSAs in fortified pepper samples referred to in Table 3 ranged between 1.4-2.3 pug/kg, 4.8-7.3 pg/kg and 7.3-10.8 pug/kg for samples fortified at 2, 6 and 10 pg/kg, respectively. The

Table 2

Recovery and reproducibility of the Procymidone tube immunoassay for fortified pepper samples

Pepper Fortification Recovery C.V. (%)

sample level ( pg/kg) (%I (intra-assay) a

Yellow 1 2 70.2 8.5

Yellow 2 6 99.0 7. 1 Yellow 3 10 73.4 12.0

Green 1 2 75.8 3.5

Green 2 6 120.9 18.0

Green 3 10 90.2 8.5

Red 1 2 119.4 12.0

Red 2 6 80.5 9.4

Red 3 10 108.7 15.0

C.V. (o/o)

(inter-assay) b

16.0

15.0 11.0

13.0

15.0

9.3

14.0

8.0

16.0

a Percent coefficients of variation based on 3 determinations in 1 day.

b Percent coefficients of variation based on 3 determinations performed in 3 different days.

A.R. Fernundez-Alba et al. /Analytica Chimica Acta 311 (1995) 371-376 375

Table 4

Mean levels ( pg/kg) and standard deviations (n = 5) of Procymidone in fortified peppper samples determined by ELISA and GC-ECD

Technique Batch A Batch B

Fortification level ( pg/kg) Fortification level ( pg/kg)

2 8 2 8

ELISA 1.6 f 0.2 10.1 f 2.0 1.5 f 0.3 6.7 f 1.3

GC-ECD _a 8.8 f 0.4 _a 9.2 f 0.5

’ Not detected.

inter-assay study was conducted over three months with several batches of tube immunoassay kits.

3.4. Antibody specificity

Cross-reactivity studies were conducted with 30 pg/l Procymidone standard solutions containing

3000 pg/l of different 3,Sdichloroaniline derivative fungicides (Vinclozolin, Clozolinate or Iprodione) or other pesticides with similar structures such as Car- bendazime, Benomyl and Thiabendazole. No cross- reactivity was found for these fungicides at this level. Although a limited number of pesticides have been tested for cross-reactivity the antibody demon-

A B

I 0 5 10 Timohin) b ’ 5 10 liwhin)

Fig. 2. GC-ECD analysis of fortified pepper samples (see Experimental). Concentration of Procymidone: (A) 2 pg/kg, (B) 8 pg/kg.


strated a remarkable high specificity towards Pro- cymidone.

3.5. Results obtained with pepper samples

Table 4 shows the different Procymidone residue values obtained when two pepper batches (A and B) spiked at 2 and 8 pg/kg were analyzed by the GC and ELISA techniques. Fig. 2 shows an example of the chromatograms obtained by GC-ECD analysis of two pepper samples spiked with 2 and 8 kg/kg of Procymidone. Quantitative results obtained by ELISA at spike levels of 8 pg/kg are in acceptable coincidence with those obtained by GC-ECD although the %C.V. is less when the chromatographic method is applied. As can be observed in Fig. 2, the most remarkable aspect is the good detection of Procymi- done residues by ELISA at very low levels (2 pg/kg), while at this concentration no detection was achieved by GC-ECD.

On the other hand, ELISA analysis makes it possible that the entire analysis can be completed within 45 min, and that up to 8 samples can be analyzed simultaneously. It represents a significant increase in the number of samples that can be analyzed compared to a gas chromatographic method.

4. Conclusions

In the present report the applicability of an ELISA commercial kit for detection and quantification of residues of Procymidone at very low levels ( < 20 pg/kg) in pepper samples was described. Pepper samples require extraction and clean up steps previous to ELISA assay. The performance of the assays evaluated by spike and recovery studies showed good recoveries and acceptable accuracy and precision. These assays do not detect similar pesticides other than Vinclozolin, Iprodione, Clozolinate, Car- bendazime, Benomyl and Thiabendazole.

This Procymidone method was less precise than GC methods but has the advantages of higher sensitivity and throughput, and lower expenses. Due to its high sensitivity this ELISA test can be used for segregation of vegetables for “Procymidone-free” markets.

Acknowledgements

This study was supported by the F.I.A.P.A. Pro- ject 22/3/93.

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determination of procymidone in vegetables by a commercial competitive inhibition enzyme immunoassay

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