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DEVELOPMENT AND APPLICATION OF IMMUNOAFFINITY COLUMN CHROMATOGRAPHY AS A CLEANUP METHOD FOR THE DETERMINATION OF ATRAZINE IN COMPLEX ENVIRONMENTAL SAMPLE MEDIA
CHUANG, J. C., J. M. VAN EMON, R. JONES, J. DURNFORD, AND B. LORDO. DEVELOPMENT AND APPLICATION OF IMMUNOAFFINITY COLUMN CHROMATOGRAPHY AS A CLEANUP METHOD FOR THE DETERMINATION OF ATRAZINE IN COMPLEX ENVIRONMENTAL SAMPLE MEDIA. ANALYTICA CHIMICA ACTA. Elsevier Science Ltd, New York, NY, 583(1):32-39, (2007).
Effective on-site environmental monitoring often requires methods that are rapid, inexpensive and easy to perform. In many instances, immunoassay methods can offer these advantages. Several immunochemical methods have demonstrated appropriate applications for environmental field work under the Superfund Innovative Technology Evaluation (SITE) program. Field screening immunoassay methods have directed site activities in real-time, minimizing both time and cost. Immunoassay methods also provide for a high sample throughput which is important for the timely analysis of a large number of samples. Laboratory-based quantitative immunoassays can support in-depth site characterization and exposure assessment studies. Immunoassays for parent compounds, environmental degradation products, and biomarkers of exposure can be configured for appropriate environmental and biological matrices to support an integrated multimedia approach to environment monitoring and risk assessment. In situ immunochemical detection methods such as electrochemical immunosensors may be feasible for down-hole monitoring, or the dynamic monitoring of waste streams or other effluents. Methods capable of continuous, in situ remote operations could be useful for the detection of episodic releases of hazardous materials into rivers or aquifers and to monitor ground water. This task will supply other techniques such as immunoaffinity chromatography for streamlining sample preparations for instrumental detection methods such as mass spectrometry. Instrumental sample preparations will also be coupled with immunoassays to leverage the advantages of both technologies. Each completed method will be evaluated based on real-world samples, or if more appropriate, field tested at a hazardous site. A user guide and SOPs for individual methods have been written. Results are communicated to the Program Office and EPA Regions through presentation, publications and direct communications. Yearly research meetings have also been conducted to discuss results and to stay current with state-of-the-art bioanalytical technologies. These research meetings enable effective communications with clients and other researchers.
A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine effectively bound to the IA columns with loading solvents of either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffer. Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating the compound did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96±2% (2.1%). Recoveries of the 500, 50, and 5 ng mL-1 atrazine standard solutions from the four IA columns were 107±7%(6.5%), 122±14% (12%), and 114±9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (~0.16 micro g of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and be reused more than 50 times with no change in performance. The IA columns were challenged with real-world soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) and ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.