ELISA M ETHODS FOR P ESTICIDES ELISA is a common format that has been reported in the literature for determining

4.2.3 ELISA M ETHODS FOR P ESTICIDES ELISA is a common format that has been reported in the literature for determining

pesticides and their metabolites in foods, as well as environmental and biological sample matrices [2,5,23,28,30 –49]. These pesticides include organochlorine (OC) and organophosphorus (OP) compounds, carbamates, sulfonylurea pyrethroids, and many herbicides. Depending on the specificity of the antibody and the design of the hapten, ELISA methods can be very selective for a specific target pesticide and used for quantitative measurements. Other methods employing less selective anti- bodies, having a high cross-reactivity for structurally similar pesticides, can be used as qualitative monitoring tools or to develop exposure equivalency indices. Tables 4.1 and 4.2 summarize some of the ELISA methods developed for foods as well as environmental and biological samples.

Assay performance must be demonstrated before applying the ELISA method to field or study samples. For laboratory-based ELISA methods, immunoreagents such as antibodies and coating antigens may only be available from the source laboratories while enzyme conjugates and substrates are commercially available. Generally, the protocols provided by the source laboratories should be used as

starting points for determining optimal concentrations of immunoreagents for the particular analysis. Checkerboard titrations can be performed to determine the optimal concentrations of the antibodies and coating antigens. Whenever new lots of immunoreagents are used, they should be examined for their performance with previously used reagents. Protocols provided with commercial testing kits should be followed in the specified manner and reagents used within the expiration date. Most ELISA methods can offer comparable or better analytical precision (e.g., within

instrument methods for analyzing pesticides. Calibration curves based on standard solutions must reflect the composition of the sample extract. Standards should be prepared in the same buffer=solvent solution as the samples. Ideally, the standards should also include the same amount of matrix as the samples. This is particularly important when sample dilution is used as the cleanup. For example, if a food extract contains 20% orange juice the standards should also contain 20% orange juice (analyte-free before spiking). When assay performance is extremely well- documented as to the extent of the matrix effect, the matrix may be omitted and

a conversion factor applied to the buffer standard curve to account for the matrix in the sample. Recently, a laboratory-based ELISA method was adapted to determine 3-phenoxy benzoic acid (3-PBA) in human urine samples collected in subsets from two obser- vational field studies. 3-PBA is a common urinary metabolite for several pyrethroid pesticides (cypermethrin, cyfluthrin, deltamethrin, esfenvalerate, permethrin) that contain the phenoxybenzyl group. The anti-PBA antibody had negligible cross- reactivity toward the parent pyrethroids but also recognized and reacted with 4-fluoro- 3-PBA (FPBA). The cross-reactivity to the structurally similar FPBA was 72%

Immunoassays

TABLE 4.1 Examples of ELISA Methods for Determining Pesticides and Metabolites in Foods

and Analyte

References Biosensors 2,4-D

Food Matrix

Assay Format

LOD

[34] Acephate

Apple, grape, potato, orange, peach

Magnetic particle, DC ELISA

5 ppb

Analyte-fortified tap water,

IC ELISA

2 ng=mL

mulberry leaves, lettuce

Acetamiprid

[46] Alachlor, carbofuran,

Fruits, vegetables

DC ELISA

0.053 ng=g

[33] atrazine, benomyl, 2,4-D

Beef liver, beef

Magnetic particle DC ELISA

1 –14 ppb

(per each analyte)

Atrazine

[50] Azoxystrobin

Extra virgin olive oil

Plate DC and DC sensor ELISA

0.7 ng=mL

Grape extract

ELISA, FPIA, TR-FIA

3 pg=mL (ELISA)

36 pg=mL (PFIA) 28 pg=mL (TR-FIA)

Carbaryl (1-naphthyl

[15] methyl carbamate)

Apple, Chinese cabbage,

Test tube, ELISA

0.7 ng=g

rice, barley

Carbaryl, endosulfan

Rice, oat, carrot, green pepper

Flow-through and lateral-flow,

10 –100 ng=mL

membrane-based gold particles

Chlorpyrifos

[45] Chlorpyrifos

Fruits and vegetables

DC ELISA

0.32 ng=mL

[42] DDT and metabolites

Olive oil

Microtiter plate IC ELISA

0.3 ng=mL

Drinking water, various foods

ELISA-CL

0.06 ng=mL (DDT)

[37] 0.04 ng=mL (metabolites)

(continued ) 101

TABLE 4.1 (continued) Examples of ELISA Methods for Determining Pesticides and Metabolites in Foods Analyte

References Difenzoquat

Food Matrix

Assay Format

LOD

Beer, cereal, bread

IC ELISA

0.8 ng=mL (beer)

[35] Analysis

16.0 ng=g (cereals)

Fenazaquin Apple and pear

[40] Fenitrothion

IC ELISA

8 ng=mL

[47] of Fenthion

Apple and peach

DC ELISA microtiter plate

20.0 ng=g

Vegetable samples

Microtiter plate DC ELISA

0.1 ng=mL (plate)

[53] Pesticides

and dipstick ELISA

0.5 ng=mL (dipstick)

Imidacloprid Fortified water samples

[54] Imidacloprid

Microtiter plate IC ELISA

0.5 ng=mL

[49] Iprodione

Fruit juices

Microtiter DC ELISA

5 –20 ng=mL

[48] in Isofenphos

Apple, cucumber, eggplant

Microtiter plate DC ELISA

0.3 ng=g

[55] Food Methyl parathion

Fortified rice and lettuce

IC ELISA

5.8 ng=mL

0.05 ng=mL (methyl parathion), [56] and parathion

Water and several food matrices

DC ELISA

and Methyl parathion

0.5 ng=mL (parathion)

Vegetable, fruit

IC and DC ELISA; FPIA

IC: 0.08 ng=mL; DC: 0.5 ng=mL; [41]

Environmental Pirimiphos-methyl

FPIA: 15 ng=mL

[57] Tebufenozide

Spiked grains

IC ELISA

0.07 ng=mL

[58] CL, Chemiluminescence; DC, direct competitive; IC, indirect competitive; PFIA, fluorescence polarization immunoassay; TR-FIA, time-resolved fluorescence immunoassay;

Red and white wine

DC ELISA

10 ng=mL

ELISA, enzyme-linked immunosorbent assay. Samples

Immunoassays

TABLE 4.2 and

Examples of ELISA Methods for Determining Pesticides and Metabolites in Biological and Environmental Samples Biosensors Analyte

References 2,4-D

Sample Matrix

Assay Format

Microtiter plate IC ELISA

30 ng=mL in urine

[38] 3,5,6-TCP

Urine

Microtiter plate IC ELISA

1 ng=mL in urine

0.25 ng=mL in assay buffer [38] 4-Nitrophenol parathion

Dust, soil

Magnetic particle DC ELISA

[25] Atrazine mercapturic acid

Soil

Microtiter plate IC ELISA

0.2 –1 ng=mL buffer

[22,28] DDE

Urine

Microtiter plate IC ELISA

0.05 –0.3 ng=mL in urine

[59] Glycine conjugate of cis=trans-DCCA

Soil

Microtiter plate IC ELISA

IC 50 ¼ 20 ng=mL

[27] Glyphosate, atrazine,

Urine

Microtiter plate IC ELISA

1 ng=mL in urine

[60] metolachlor mercapturate

Water, urine

Multiplexed fluorescence

0.03 –0.11 ng=mL

microbead immunoassay

Methyl parathion

Soil

Microtiter plate IC and

0.08 ng=mL (IC)

DC ELISA and FPIA

0.5 ng=mL (DC) 15 ng=mL (FPIA)

Triazine herbicides

Surface water,

Test tube DC ELISA

0.2 –2 ng=mL in water

groundwater

FPIA, Fluorescence polarization immunoassay; IC, indirect competitive; DC, direct competitive; ELISA, enzyme-linked immunosorbent assay.

104 Analysis of Pesticides in Food and Environmental Samples as reported by the source laboratory [61]. FPBA is the metabolite for cyfluthrin

(a pyrethroid pesticide containing a fluorophenoxybenzyl group). This high cross- reactivity is advantageous as this 3-PBA ELISA can be used as a monitoring tool for determining a broad exposure to pyrethroids. For assay development, the anti-PBA antibody, coating antigen, and initial assay protocol were provided by the source laboratory. Checkerboard titration experiments were performed to determine the optimal concentrations of anti-PBA antibody, coating antigen, and a commercial enzyme-conjugated secondary antibody. The optimal conditions established for the 3-PBA ELISA were 0.5 ng=mL of coating antigen, a 1:4000 dilution of anti- PBA antibody, and a dilution of 1:6000 of the commercial enzyme-labeled secondary antibody conjugate (goat anti-rabbit labeled with horseradish peroxidase). The assay procedures were modified by preparing the standard solutions in a 10% metha- nol extract of 10% hydrolyzed drug-free urine in PBS. Calibration curves (Figure 4.3) for 3-PBA were generated based on 10 concentration levels ranging from 0.00256 to 500 ng=mL (1:5 dilution series). The percent relative standard deviation (%RSD) values of the triplicate analyses were <20% for the standard solutions. Day-to-day variation for the quality control (QC) standard solution (1.0 ng=mL) was

detection limit was 0.2 ng=mL. Quantitative recoveries of 3-PBA were achieved urine samples were prepared and analyzed by the ELISA method. Different aliquots

of the urine samples were also analyzed by gas chromatography=mass spectrometry (GC=MS). The GC=MS results indicated that 3-PBA was detected in 95% of the samples, whereas FPBA was only detected in 8.4% (10 out of 119 samples) of the samples. Similar results suggesting that FPBA was detected at much lower rate than 3-PBA in human urine samples collected from residential settings was also

3-PBA standard curve

Mean OD (450 nm) 0.39 0.29 0.19

Concentration (ng/mL)

y = ((A ⫺ D )/(1 + (x /C ) B )) + D: A B C D R 2 Std PBA Curve (Standards: Conc. (ng/mL) vs. Mean OD) 0.961

Immunoassays and Biosensors 105 reported in the CDC third National Report on Human Exposure to Environmental

Chemicals [62]. The ELISA-derived 3-PBA concentrations correlated well with the GC=MS results. The Pearson correlation coefficient between the 3-PBA concen- trations of the two methods was 0.952, which was statistically significant ( p < 0.0001). A nonsignificance outcome (p ¼ 0.756) was also observed from the paired t-test indicating that there was no significant difference in measurements between the two analytical methods (ELISA vs. GC=MS) for a given sample. This study demonstrated that the ELISA method could be used as a monitoring tool for the urinary biomarker, 3-PBA in human urine samples, for assessing human exposure to pyrethroids.

As most fruit and vegetable baby food preparations generally contain a signifi- cant amount (>80%) of water, ELISA methods have the advantage over instrumental methods in determining pesticides in this aqueous sample matrix. We investigated various sample preparation methods for determining pesticides in baby foods using either GC=MS or ELISA methods [26]. A streamlined direct ELISA method con- sisting of dilution, filtration, and ELISA was evaluated on spiked baby foods at 1, 2,

5, 10, or 20 ppb. Quantitative recoveries (90% –140%) were achieved for atrazine in the nonfat baby foods (i.e., pear, apple sauce, carrot, banana=tapioca, green bean). The performance of other ELISA testing kits was not as good as the atrazine-ELISA testing kit. Over-recoveries were observed for carbofuran and metolachlor testing kits in banana=tapioca and green bean. This was probably due to a sample matrix interference that was not completely removed by dilution. An off-line coupling of enhanced solvent extraction (ESE) with ELISA was developed to determine atrazine in a more complex sample matrix of fatty baby foods. The results indicated that the extraction temperature was an important factor to recover atrazine. The ESE-ELISA method consisted of extracting the food at 1508C and 2000 psi with water and performing ELISA on the aqueous extract.

In an on-going study, different sample preparation procedures are being inves- tigated for a magnetic particle ELISA analysis for permethrin. Quantitative recover- ies (>90%) were obtained when the fortified soil samples were extracted with sonication using DCM, methyl-t-butyl ether (MTBE) or 10% ethyl ether (EE) in hexane. Recoveries were <50% from the fortified soil samples when the shaking method was employed (shaking with methanol for 1 h). A longer shaking time (16 h, overnight) was evaluated, using methanol, yielding recoveries of over 200% by ELISA. The longer shaking time extracted substances that interfered with the ELISA detection. This interference was also detected in the GC=MS analysis and

persisted even after the SPE cleanup. Satisfactory recovery data (>90%) for post- spiked dust samples and a spiked dust sample were obtained. DCM was selected as the extraction solvent, as it was easily evaporated, facilitating the solvent-exchange step. The collected field samples were extracted with DCM using sonication. The DCM extract was concentrated and solvent exchanged into methanol. The methanol extract was diluted with reagent water (1:1) before ELISA.

Interferences caused by sample matrix components are a concern for both conventional instrument methods and ELISA methods. In immunoassays, sample matrix effects may result from nonspecific binding of the analyte to the matrix as well as the matrix to the antibody or enzyme or denaturation of the antibody or

106 Analysis of Pesticides in Food and Environmental Samples

a practical detection limit can still be achieved [23]. Alternatively, cleanup methods for instrumental methods (e.g., SPE or column chromatographic separation) can also be performed before ELISA detection. Another effective cleanup method is immunoaffinity column chromatography that can be applied for the purification of sample extracts for either instrumental or ELISA detection [2,63].

In a recent study [64], an effective bioanalytical method for atrazine in complex sample media (soil, sediment, and duplicate-diet food samples) was developed. The method consisted of an ASE procedure with DCM, followed by immunoaffinity column cleanup with detection by a magnetic particle ELISA. Quantitative recover-

data for these samples (the Pearson correlation coefficient was 0.994 for soil and sediment and 0.948 for food). However, the ELISA values were slightly higher than those obtained by GC=MS. This was probably the result of the solvent-exchange step required for the GC=MS but not the ELISA. This bioanalytical approach is more streamlined than the GC=MS analysis and could be applied to future large-scale environmental monitoring and human exposure studies.