POCIS sampling in combination with ELISA: screening of sulfonamide residues in surface and waste waters.

Sulfonamide antibiotics coming from both human and veterinary medicine are among the most common emerging pollutants in freshwater. The present paper shows the successful application of passive sampling using POCIS in combination with an immunochemical ELISA technique and HPLC/MS/MS analysis to study the distribution of sulfonamides in streams around small towns in the Czech Republic, as well as around a major agglomeration of the city of Brno, including its waste water treatment plant (WWTP). Results indicated the presence of sulfonamides at most studied sites with concentrations ranging from <20 up to 736 ng of sulfamethoxazole equivalents per POCIS. Very high levels were detected in both the influent and effluent of the Brno WWTP with maxima > 8000 ng SMX per POCIS. All samplers collected down-stream of the studied towns and WWTPs clearly showed an increase in sulfonamide drug residues. Higher concentrations were determined in rivers at the city of Brno agglomeration. In agreement with other available studies, these findings indicate low efficiency of conventional WWTPs to eliminate polar pharmaceuticals such as sulfonamides. Good performance and correlation with the LC/MS results, as well as ease of use, indicate good potential for the immunochemical ELISA technique to become the screening tool for sulfonamide determination in surface waters including passive samplers.

Determination of atrazine in surface waters by combination of POCIS passive sampling and ELISA detection.

Polar organic compound integrative samplers (POCIS) in combination with instrumental techniques such as LC-MS-MS were previously used to monitor environmental pollutants but the performance of alternative immunochemical methods such as ELISA (enzyme-linked immunosorbent assay) has been explored less. In the present study, POCIS technology was applied to surface water sampling in the Czech Republic, and ELISA was used as a detection technique for the herbicide atrazine. In the first study, 28 samples from streams around small municipal waste water treatment plants (WWTPs) were collected using two different devices (POCISpest and POCISpharm) over the course of 21 days. Elevated atrazine concentrations (up to 25 ng per POCIS) were found in samples down-stream of WWTPs. This observation was also confirmed in another two year study (4 sampling periods) investigating 7 river sites around a major city of Brno as well as the inlet and outlet of the city's WWTP. High atrazine levels were systematically determined at the outlet from the WWTPs (120-605 ng per POCIS). A decreasing trend in the atrazine concentrations in rivers around the city of Brno has been observed, with the highest levels observed within the first sampling period in spring 2007 (100-600 ng per POCIS, with an extreme value of 2760 ng per POCIS). Results of the atrazine ELISA were closely correlated with LC-MS/MS, which confirmed good applicability of ELISA as a cost-effective screening tool.

Analytical evaluation of a high-throughput enzyme-linked immunosorbent assay for acrylamide determination in fried foods.

The analytical performance and evaluation of a kit-based ELISA for the determination of acrylamide in fried potato and corn chip samples are described. The sample homogenate is subjected to clean-up using SPE, followed by analyte derivatization and ELISA detection. Accuracy, precision and linearity of the ELISA procedure have been validated using spiked samples. Analytical recovery ranged from 91.8% to 96.0% with coefficients of variation below 15%. Good linearity over a wide range of dilution and minimal assay drift was observed within a microtiter plate. IC50 value of the calibration curve was 110 ng/mL, with the limit of detection about 5 ng/mL and dynamic range from 10 to 1000 ng/mL. The high specificity of the ELISA was demonstrated by cross-reactivity study using 11 potential cross-reactants. A good correlation between the results obtained from the ELISA and GC-MS within the concentration range 120-1500 µg/kg was found in the chip samples (r=0.992, n=120). The data demonstrate that the evaluated and validated ELISA has a potential utility in a quick, simple and reliable acrylamide screening analysis for the medium- and large-sized food companies, as well as for residue laboratories and the food industry dealing with improving the chemical safety of foods available to the consumer.

In-house validation of an ELISA method for screening of semicarbazide in eggs.

An enzyme-linked immunosorbent assay (ELISA) method is described for the semi-quantitative determination of semicarbazide (SEM), the marker residue for the banned nitrofuran drug, nitrofurazone, in chicken eggs. The sample homogenate is subjected to acid hydrolysis and derivatisation with o-nitrobenzaldehyde, followed by ethyl acetate/hexane extraction and detection by ELISA. The ELISA procedure has been validated using 0.3, 1.0 and 3 microg kg(-1) of SEM in fortified samples. Detection capability (CC(ss)) was based on the acceptance of 5% false compliant results for a given concentration level according to Commission Decision 2002/657/EC and was determined to be 0.3 microg kg(-1) with a respective limit of detection of 0.13 microg kg(-1). A validated LC-MS/MS method was used for the analysis of incurred egg samples and the results compared with ELISA. A good correlation between the results obtained from ELISA and LC-MS/MS within the concentration range 0.12-20.3 microg kg(-1) was observed in samples collected from chickens fed with a medicated ration of nitrofurazone (r = 0.992, n = 14). Validated ELISA enabled reliable monitoring of SEM levels in eggs collected from incurred chickens over a 90-day period.

Broad-specificity immunoassays for sulfonamide detection: immunochemical strategy for generic antibodies and competitors.

Development of antibodies with broad specificity recognition for sulfonamide drugs was found to be surprisingly difficult when conventional immunochemical strategies were applied to hapten design. To improve the cross-reactivity pattern of antibodies for the family of sulfonamide drugs, a novel strategy based on the single-ring (fragment-derived) hapten moieties with different spacer substituent lengths was employed for the preparation of immunogens, coating conjugates, and enzyme competitors. The rabbit antibodies raised against a common (one-ring) p-aminobenzenesulfonamide hapten moiety (attached to a carrier protein through the N-1 position) in combination with a homologous hapten-peroxidase tracer allowed the detection of 15 sulfonamide species at the maximum residue limit level using direct ELISA. The two-ring 6-(4-aminobenzensulfonylamino)hexanoic hapten mimics, previously reported in the literature as a weak generic antigen, generated surprisingly superior immune responses in rabbits. The antibodies raised against this two-ring hapten were capable of detecting at least 19 and 17 sulfonamides in a direct ELISA system at the regulatory level with sensitivities corresponding to 20 and 50% binding inhibition, respectively. A negligible cross-reaction with N4 metabolites makes it possible to measure responses of parent sulfonamides in the presence of their metabolized forms. In skimmed milk, the highest limit of detection (LOD) for sulfacetamide defined as 20% inhibition was 65.2 microg x L(-1) (IC20 value), whereas the additional 18 sulfonamides tested exhibited LODs in the range of 0.2-36.8 microg x L(-1). This sensitivity allows simple multisulfonamide tests to be established for use in the laboratory or on site.