determination of procymidone in vegetables by a commercial competitive inhibition enzyme immunoassay
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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-Exportaaores 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
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
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  to nil tolerance in the USA  and Switzerland , 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 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 . 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.
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.
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
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.
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 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  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 sonica- tion 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 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.
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 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
solutions of Procymidone described above. The de- tection limit of Procymidone was 8 pg/kg .
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 line