Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2008
Publication Date: 7/11/2008
Citation: Shelver, W.L., Shappell, N.W., Franek, M., Rubio, F.R. 2008. ELISA for sulfonamides and its application for screening in water contamination. Journal of Agricultural and Food Chemistry 56:6609-6615.
Interpretive Summary: Medicines used in agriculture practice can find their way into the environment and re-cycle through or alter the food web. Similarly, chemicals used as human medicines are excreted and find their way into the sewer system. If the particular medicine is not destroyed in sewage and not removed by the wastewater treatment process, that agent will persist in the environment. Since medicines by definition demonstrate the capability to alter biological processes, persistent agents pose a potential food safety and environmental problem. The class of antibacterials known as sulfonamides have been found in modest concentrations in wastewater treatment plants and waterways in Switzerland, Canada, Korea, and the United States. Two particular compounds of interest are sulfamethazine (used in veterinary medicine) and sulfamethoxazole (used in human medicine). To monitor these two chemicals and follow their environmental fate we tested two ELISA systems, one to measure each compound, and verified the results with instrumental analysis (LC-MS/MS). The ELISA systems are simple to use, portable so they can be used at the site being monitored, and capable of analyzing very large numbers of samples each day. They are also specific and sensitive so they are useful to accurately determine the target molecule in many different samples and in identifying samples containing higher than normal amounts. The LC-MS/MS method is expensive to use and cannot economically analyze large number of samples in a short period of time. However, this method is excellent at confirming the presence of a suspected contaminant. We demonstrated the sulfamethazine ELISA worked well in analysis of the wastewater from swine raising facilities. Similarly, the sulfamethoxazole ELISA worked well to determine the amount of sulfamethoxazole in two different wastewater treatment plants. The results were confirmed by separate analysis using LC-MS/MS. The test kits could be used to assist in the monitoring of wastewater processing systems so as not to pollute our environment and thus ensure food safety.
Technical Abstract: Two sulfonamide enzyme linked immunosorbent assays (ELISAs) were tested for their suitability in analyte measurement in wastewater from various stages in wastewater treatment plants (WWTPs), the river into which the wastewater is discharged, and two swine rearing facilities. The sulfamethoxazole ELISA cross-reacts with several compounds achieving detection limits of < 0.04 microgram/L for sulfamethoxazole (SMX), sulfamethoxypyridine, sulfachloropyridine, and sulfamethoxine; while the sulfamethazine (SMZ) ELISA is more compound specific, with detection limit of < 0.03 microgram/L. Samples from various stages of wastewater purifications gave 0.6 to 3.1 microgram/L by SMX-ELISA, while river samples were ~10 fold lower, ranging from below detection to 0.09 microgram/L. Swine wastewater samples analyzed by the SMX-ELISA, were either at or near detectable limits from one facility, while the other facility had concentrations ~ 0.5 microgram/L, although LC-MS/MS did not confirm the presence of SMX. Sulfamethazine ELISA detected no SMZ in either WWTP or river samples. In contrast, wastewater samples from swine facilities analyzed by SMZ-ELISA were found to contain 33.2 microgram/L (50-100 lb piglet wastewater) and 7.2 microgram/L (market weight hog wastewater). Sulfamethazine ELISA analyses of wastewater from another swine facility found concentrations to be near or below detection limits. A solid phase extraction method was used to isolate and concentrate sulfonamides from water samples prior to LC-MS/MS multi-residue confirmatory analysis. The recoveries at 1 microgram/L fortification ranged from 42.3 +/- 4.1 % for SMZ to 87.5 +/- 3.8 % for SMX (n = 6). The ELISA results in the WWTPs were confirmed by LC-MS/MS, as sulfonamide multi-residue confirmatory analysis identified SMX, sulfapyridine, and sulfasalazine to be present in the wastewater. Sulfamethazine presence at one swine rearing facility was also confirmed by LC-MS/MS, demonstrating the usefulness of the ELISA technique as rapid and high-throughput screening method.