Environmental Immunochemical Methods - American Chemical Society

Chapter 15

Validation of an Immunoassay for Screening Chlorpyrifos-methyl Residues on Grain 1,4

1,4

1

Brian A. Skoczenski , Titan S. Fan , Jonathan J. Matt , J. Terry Pitts , and J. Larry Zettler Downloaded by STANFORD UNIV GREEN LIBR on October 8, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch015

2

3

1

Millipore Corporation, 80 Ashby Road, Bedford, MA 01730 Gustafson, Inc., 1400 Preston Road, Suite 400, Plano, TX 75093 Agricultural Research Service, U.S. Department of Agriculture, 2021 South Peach Avenue, Fresno, CA 93727

2

3

A competitive, enzyme labeled, heterogeneous immunoassay (EnviroGardChlorpyrifos-methylScreening Kit) was validated for the rapid detection of residues of the post-harvest insecticide chlorpyrifos-methyl(CPM). Grain samples were treated with C P M in a manner simulating commercial field applications and were analyzed by the immunoassay and simultaneously by aPAMII instrumental method. Analysis was repeated at t = 30 days after storage of the grain under field conditions. The initial phase was performed in the developer's lab (ImmunoSystems, Inc.). This was duplicated at the United States Department of Agriculture Stored-Product Insects Research and Development Laboratory, Savannah, GA. Field trials were performed by individuals selected to represent the typical user. Recently, the U.S. EPA, Office of Prevention, Pesticides and Toxic Substances, Analytical Chemistry Branch independently validated the method. Chlorpyrifos-methyl (0,0-dimethyl 0-3,5,6-trichloro-2-pyridyl phosphorothionate) is a broad range insecticide. Post-harvest application prior to storage is indicated for pest protection. Because of the need to limit grain application to a single treatment and the numerous times that grain may change hands between harvest and ultimate use, there exists a need to analyze grain samples to determine if treatment is appropriate. We have developed an immunoassay which can be used at remote locations without dedicated instruments for screening grain samples for the presence of C P M residues. Results can be interpreted visually and the assay can reliably detect residues at 0.25 mg/kg (ppm). 4

Current address: Beacon Analytical Systems, 4 Washington Avenue, Scarborough, ME 04074

0097-6156/96/0646-0161$15.00/0 © 1996 American Chemical Society In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

162

ENVIRONMENTAL IMMUNOCHEMICAL METHODS

Grain samples were treated at 0, 0.1, 0.25, 1.0 and 6.0 ppm active ingredient.

Downloaded by STANFORD UNIV GREEN LIBR on October 8, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch015

Treatment levels were verified by analysis of three to five replicates of each sample using the P A M II instrumental method (acetone extraction and gas chromatography/electron capture detector). Five replicates of each treatment level were extracted and analyzed using the immunoassay method. Samples were then stored under ambient conditions for a period of thirty days, at which time both the GC and immunoassay analyses were repeated. The study was designed and performed in accordance with Pesticide Assessment Guidelines promulgated by the US E P A in compliance with Good Laboratory Practices Standard (1). Immunoassay Development Antisera were raised in rabbits against a derivitized chlorpyrifos compound attached to a protein carrier. These antibodies were purified and immobilized on 12x75 mm polystyrene test tubes. A similar derivitized chlorpyrifos compound was covalently attached to the enzyme horseradish peroxidase. The design criteria for the assay was a rapid (< 30 minute) protocol which could be utilized at remote locations with a minimum of dedicated equipment or specialized training. The assay needed to be able to identify grain samples which had been previously treated with CPM; defined as containing ^ 0.25 ppm C P M residues. A method of sample extraction was developed which involves volumetric measurement of the grain sample, addition of rubbing alcohol (70% isopropyl alcohol) and shaking for two minutes. Studies performed by gas chromatographic analysis of extracts demonstrated that this extraction technique was approximately 50% efficient in the removal of C P M residues. The actual C P M content of the calibrator was adjusted to allow for an easily visible distinction between the calibrator and samples which contain ^ 0.25 ppm CPM. Because of this necessity, the assay will also yield a high percentage of positive results for grain samples containing ^ 0.1 ppm CPM. Kits are packaged to contain all materials required for the test, except the rubbing alcohol required for the grain extraction. The immunoassay protocol can be summarized as follows: 1. Add grain sample to 15 mL mark on extraction vial (« 10 grams). 2. Add rubbing alcohol to the 45 mL mark, cap and shake for 2 minutes. 3. Add 5 drops of assay diluent to the antibody-coated tubes.

In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

15. SKOCZENSKI ET AL.

Screening CPM Residues on Grain

163

4. Add 3 drops of calibrator or sample extract to the appropriate tubes. 5. Add 5 drops of enzyme conjugate to the tubes and allow to incubate for 10 minutes.


6. Wash the tubes, add 10 drops of substrate and allow to incubate for 10 minutes. 7. Interpret results by comparing color of sample tubes to color of calibrator tube: If a sample tube contains more color than the calibrator tube, then the sample is negative for C P M . If a sample tube contains less color than the calibrator tube, the sample is positive for C P M .

Immunoassay Performance One consideration in the design and use of immunoassays is the limited dynamic range compared to many instrumental methods. The dynamic range is the range of analyte concentrations over which the assay will respond with a corresponding change in signal generated. This range is determined by a number of factors including antibody affinity, antibody concentration, enzyme conjugate concentration and sample size. Since all of these factors are static for the normal immunoassay user, the situation would be somewhat comparable to an instrumental method where the detector gain can not be adjusted. Figure 1 shows the response of the assay for οΜοφντί&8-πιβυιν1. Generally, the dynamic range is considered to be limited to 20 to 80% of the signal generated at 0 concentration. For the current assay, this would correspond to approximately 0.5 to 10 ppb. While the standard grain treatment level is 6 ppm, the design criteria required the ability to easily distinguish 0.25 ppm concentrations on grain as positive while also easily distinguishing untreated grain as negative. Visual inteφretation requires greater differences in color between calibrators and samples for unambiguous results as compared to that required for inteφretation with a photometer. Based on the design criteria, the dynamic range of the assay and the sample extraction; the calibrator was set at 5 ppb C P M . Figure 2 shows the location of the calibrator and the theoretical content of extracts from 0.1 ppm and 0.25 ppm grain samples. It is obvious from the response curve that the assay is not be able to distinguish, for instance 6 ppm from 1 ppm, but for this application, the important factor is that all of these can visually be distinguished as "positive" for ΰηΙοφνΓΪίοβ-ηιβίηνΙ. A result of this approach is that the assay will potentially yield positive results for grain samples containing less than 0.25 ppm. Theoretically, the assay would be able to identify grain samples with concentrations as low as 0.03 ppm as positive for C P M .

Grain Treatment. The test commodity was corn grain, variety Funk's Blend, which had been harvested and stored without the application of post-harvest insecticides. The test substance was Reldan 4E and was GLP certified to contain 43.2% active ingredient.



164


0.01

0.1

1

10

100

1000

ppb Chlorpyrifos-methyl Figure 1. Dynamic Range for Chlorpyrifos-methyl


10000

15. SKOCZENSKI ET AL.

Screening CPM Residues on Grain

165


The analytical standard was GLP certified to be 99.8% pure. Ten pound samples of the test commodity were treated in a manner to simulate commercial field application. In addition to an untreated blank, samples were treated at 0.10, 0.25, 1.0 and 6.0 ppm (6.0 ppm is the standard application rate). The samples were split into two 5 pound aliquots and one was immediately frozen for shipment to the USDA facility. These frozen aliquots were shipped on dry ice to the USDA facility and were reportedly receivedfrozenand in good condition. In addition to the treated grain samples, a vial of the analytical standard was shipped in a separate container. Gas Chromatographic Analysis Grain samples were analyzed for chlorpyrifos-methyl according to a modification of the method of Kuper, 1979 entitled "Determination of Residues of Chlorpyrifosmethyl in Grains" (DowElanco; unpublished). Briefly, 10 g samples of grain were weighed and ground with 40 mL of acetone using a high-speed blender. The blended grain/acetone was then homogenized for 1 minute and mixed overnight on an orbital mixer at 200 R P M . The extracts were then centrifuged prior to injection. Gas Chromatograph Conditions: Column: Detector: Carrier Gas: Make Up Gas. Oven Temperature: Injector Temperature: Detector Temperature. Injection Volume: Scale:

15m χ 0.53mm methyl phenyl cyano silica ECD Helium @ 7 mL/min. Argon/Methane (95:5) @ 50 mL/min. 140-260°C@8°C/min. 250°C 350°C 1 μΐ 1 Volt

Fortified samples were analyzed prior to analysis of treated grain samples to demonstrate the functionality of the analytical method. In the initial phase, fortifications were at five levels and four replicates were analyzed at day 0 and again at day 30. Recoveries ranged from 92.5% to 103% and averaged 97.8%. In the validation phase, fortifications were at three levels and three replicates were analyzed at day 0 and day 30. Recoveries ranged from 95.6% to 109% and averaged 104%. This data are summarized in Table I. In the initial phase, five replicates of each of the treatment levels were analyzed. In the validation phase, three replicates of duplicate samples were analyzed. Analysis was performed at Day 0 and repeated at Day 30. For the initial phase, recoveries averaged 88% at day 0 and 76% at day 30.


166


Table I - Recovery of Chlorpyrifos-methyl from Fortified Samples


I) Initial Phase (Average of four replicates) Recovery at Day 0 ppm % 0.0074 92.5 0.041 102 0.206 103 0.979 97.9 1.874 93.7

ppm Added 0.008 0.04 0.2 1.0 2.0

Recovery at Day 30 ppm % 0.0082 102 0.0038 95.0 0.196 98.0 0.975 97.5 1.924 96.2

II) Validation Phase (Average of three replicates of duplicate samples) Recovery at Day 0 ppm % 0.131 95.6 1.45 106 6.73 103

ppm Added 0.137 1.37 6.56

Recovery at Day 30 ppm % 0.150 109 1.44 105 6.78 103

Table II - Parts per Million of Chlorpyrifos-methyl on Treated Grain Samples

I) Initial Phase Sample #(ppm> 921025-14 921025-15 921025-16 921025-17 921025-18

Target Rate (ppm) 0 0.1 0.25 1.0 6.0

1

Day 0 (ppm)

Environmental Immunochemical Methods - American Chemical Society

Recommend Documents