Linking SPE and ELISA to measure chlorpyrifos in fish tissue
P. WAN1, C. R. SANTERRE1, and D. C. Deardorff2. (1) Dept. of Foods and Nutrition, Purdue University, 1264 stone hall, west Lafayette, IN 47907-1264, (2) Strategic Diagnostic Inc., King of Prussia, PA 19406
JUSTIFICATION: A recent report indicated that 10.5% of farm-raised catfish contained detectable chlorpyrifos (Dursban®) residues. Data from the EPA’s National Contaminate Biomonitoring Program showed that chlorpyrifos was found in wild fish samples with average concentrations as high as 23 ppb. A rapid and low cost method for measuring chlorpyrifos in fish is needed due to heavy agriculture use of this insecticide and the potential for the contamination of fish. In a previous study, we demonstrated the capability of quantitatively analyzing chlorpyrifos in fish tissue by ELISA. However, the extensive amount of time required for the extraction and cleanup procedures reduced the benefits of an immunoassay. A more efficient column extraction and solid phase extraction (SPE) cleanup procedure was developed prior to an ELISA to measure chlorpyrifos in fish tissue.
OBJECTIVES: To develop an efficient extraction and cleanup procedure for chlorpyrifos analysis in fish tissue using a commercial ELISA kit.
METHODS: Ground, spiked fish tissue (3-5 g) was mixed with anhydrous KH2PO4, anhydrous Na2HPO4 and anhydrous Na2SO4. This mixture was then transferred to a prepacked column containing silica gel and anhydrous Na2SO4. Chlorpyrifos was extracted with 5% diethyl ether in hexane. Extracts were evaporated and diluted with 10% (v/v) acetone in water, passed through C18 or C8 SPE cartridges and eluted with methanol prior to analysis by ELISA or GC/ECD.
RESULTS: Recoveries of greater than 60% were achieved with a LOD below 0.05 ppm. The standard curve was linear from 0.05 to 2.0 ppm. The relative standard deviation of this method was under 10%. This ELISA method (6-8 hrs) offers a more time efficient procedure than traditional GC method (2-3 days). Solvent waste for this method was less than 200 ml per sample comparing to the GC method which produces 4 liters per sample.
SIGNIFICANCE: An efficient column extraction, which combines extraction, filtration, and cleanup in one step is reported. ELISA compared favorably with GC/ECD analysis for measurement of chlorpyrifos in fish tissue. Coupling of column extraction, SPE and ELISA technique provide a rapid means for analyzing a large number of fish samples.