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

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

Post on 15-Jun-2016

212 views

Category:

Documents

0 download

TRANSCRIPT

<ul><li><p>ANALYTICA CHIMICA ACM </p><p>ELSEVIER Analytica Chimica Acta 311(1995) 371-376 </p><p>Determination of Procymidone in vegetables by a commercial competitive inhibition enzyme immunoassay </p><p>Amadeo R. Fernandez-Alba a,* , Antonio Valverde a, Ana Agiiera b, Mariano Contreras b, Dolores Rodriguez </p><p>a Pesticide Residues Research Group, Facultad de Ciencias de Abner&amp; 04071 Abner&amp;z, Spain b Laboratorio de An&amp;is Agricola de COEXPhWL (Cosecheros-Exportaaores de Hortalizas de Almeria), El Viso, 04070 Almeria, Spain </p><p> Centro National de Biotecnologia C.S.I.C., Campus UniversidadAut&amp;oma, Cantoblanco, 28049 Madrid, Spain </p><p>Received 20 September 1994, revised 8 February 1995; accepted 13 February 1995 </p><p>Abstract </p><p>An ELISA commercial kit was used to quantitate residues of Procymidone at very low levels (&lt; 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. </p><p>Keywords: Immunoassay; Enzymatic methods; Biosensors; Procymidone; Pesticides; Environmental analysis; ELISA, Vegetables </p><p>1. Introduction </p><p>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 </p><p>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. </p><p>Currently the * Corresponding author. in vegetables is </p><p>0003-2670/95/$09.50 8 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(95)00121-2 </p><p>presence of Procymidone residues detected by applying well-known </p></li><li><p>372 A.R. Fernandez-Alba et al. /Analytica Chimica Acta 311 (1995) 371-376 </p><p>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 obtain- ing 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]. </p><p>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 ( &lt; 0.02 mg/kg) of Procymidone residues in pepper samples. </p><p>2. Experimental </p><p>2.1. Chemicals </p><p>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 sub- strate and tetramethylbenzidine as chromogen. </p><p>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 dispos- able 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 solu- tions were prepared by transferring 0.2 ml of stan- dard 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 </p><p>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. </p><p>2.2. Apparatus </p><p>A Perkin Elmer 8600 (Beaconsfield) equipped with a </p><p>g$s chromatograph Ni electron capture </p><p>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. </p><p>A Shimadzu (Kyoto) UV-160 spectrophotometer was used for spectrophotometric determinations. </p><p>2.3. Sample preparation </p><p>Fresh pepper samples were collected from differ- ent 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 40-60C 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 </p></li><li><p>A.R. Fermdez-Alba et al./Analytica Chimica Acta 311 (1995) 371-376 373 </p><p>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 sonica- tion for immunoassay analysis. </p><p>2.4. Immunoassay of Procymidone </p><p>Immunoassay standard solutions and extracts from fortified samples were analyzed according to the following procedure: 160 ~1 of either standard solu- tion 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 ab- sorbance 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. </p><p>2.5. Chromatographic analysis </p><p>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 240C and 300C respectively. The oven temperature program was 150C (1 min hold) to 215C (6 min hold) at 30C/min then to 260C at lSC/min, the injection volume was 1 ~1. External standard calibration graphs were obtained by using the GC standard </p><p>solutions of Procymidone described above. The de- tection limit of Procymidone was 8 pg/kg [23]. </p><p>3. Results and discussion </p><p>3.1. Linear range studies </p><p>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 re- sponse curve for four standard solutions selected to calibrate the Procymidone-ELlSA test. For samples with concentrations &gt; 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. </p><p>3.2. Matrix effects </p><p>Some tests were carried out in order to assess the need of the clean up step with silica previous to the </p><p>-I 01 IO0 </p><p>I </p><p>IO' 102 </p><p>c- of Racymidone (&amp;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. </p></li><li><p>374 A.R. Fernandez-Alba et al. /Analytica Chimica Acta 311 (1995) 371-376 </p><p>Table 1 </p><p>Percentage of inhibition and coefficient of variation (C.V.) of Procymidone antibodies for different types of pepper sample blanks </p><p>Pepper type %Inhibition </p><p>Without C.V. With C.V. clean up (o/o) a clean up (o/o) a </p><p>Yellow 28 20 12 6 Green 25 18 15 8 Red 31 21 11 9 </p><p>a Percent coefficients of variation based on 4 determinations in 1 day. </p><p>Procymidone-ELBA assay for pepper samples. Table 1 shows the values of Procymidone antibody inhibi- tion for different pepper blank extracts obtained by the described sample preparation procedure, with or without silica clean-up step. This inhibition percent- age is referred to purified water ELISA values. However, while inhibition values &gt; 25% were ob- tained when the clean-up is omitted, values &lt; 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 per- formed, indicates the need for an adequate clean-up previous to the Procymidone ELBA analysis for pepper samples. </p><p>Table 3 Cross-reactivity of Procymidone antibody towards a selection of fungicides </p><p>Compound ( cLg/I) Cross reaction (o/o) </p><p>Procymidone Vinclozolin </p><p>Iprodione </p><p>Carbendazime </p><p>Clozolinate </p><p>Benomyl </p><p>Thiabendazole </p><p>30 30 </p><p>3000 30 </p><p>3000 30 </p><p>3000 30 </p><p>3000 30 </p><p>3000 30 </p><p>3000 </p><p>100 &lt; 0.01 &lt; 0.01 &lt; 0.01 &lt; 0.01 &lt; 0.01 &lt; 0.01 &lt; 0.01 &lt; 0.01 &lt; 0.01 &lt; 0.01 &lt; 0.01 &lt; 0.01 </p><p>3.3. Precision of the assay </p><p>Results for the Procymidone-ELlSAs in cleaned- up extracts from different pepper samples and forti- fied 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 deter- mined by ELlSAs in fortified pepper samples re- ferred 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 </p><p>Table 2 Recovery and reproducibility of the Procymidone tube immunoassay for fortified pepper samples </p><p>Pepper Fortification Recovery C.V. (%) </p><p>sample level ( pg/kg) (%I (intra-assay) a </p><p>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 </p><p>C.V. (o/o) (inter-assay) b </p><p>16.0 15.0 11.0 13.0 15.0 9.3 </p><p>14.0 8.0 </p><p>16.0 </p><p>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. </p></li><li><p>A.R. Fernundez-Alba et al. /Analytica Chimica Acta 311 (1995) 371-376 375 </p><p>Table 4 Mean levels ( pg/kg) and standard deviations (n = 5) of Procymidone in fortified peppper samples determined by ELISA and GC-ECD </p><p>Technique Batch A Batch B </p><p>Fortification level ( pg/kg) Fortification level ( pg/kg) </p><p>2 8 2 8 </p><p>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 </p><p> Not detected. </p><p>inter-assay study was conducted over three months with several batches of tube immunoassay kits. </p><p>3.4. Antibody specificity </p><p>Cross-reactivity studies were conducted with 30 pg/l Procymidone standard solutions containing </p><p>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- </p><p>A B </p><p>I 0 5 10 Timohin) b 5 10 liwhin) </p><p>Fig. 2. GC-ECD analysis of fortified pepper samples (see Experimental). Concentration of Procymidone: (A) 2 pg/kg, (B) 8 pg/kg. </p></li><li><p>376 A.R. Fernandez-Alba et al. /Analytica Chimica Acta 311 (1995) 371-376 </p><p>strated a remarkable high specificity towards Pro- cymidone. </p><p>3.5. Results obtained with pepper samples </p><p>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 chroma...</p></li></ul>

Recommended

View more >