Apparatus and glassware: A Microplate reader equipped with a 450nm filter (ELx800TM , BioTek, USA), Waterbath (281 ANALOG, Cole Parmer), 4 decimal points analytical balance (AND, Germany), Centrifuge (5810, Eppendorf, Germany), Vortex (Barnsteed, Germany), Micropipettes (Eppendorf, Germany), Nitrogen evaporator (Organomation Associates Int.), Polypropylenecentrifuge tubes (Griner, B 532), glass test tube (Pyrex) and Volumetricflasks (Schoot Duran) were used to prepare samples and perform an extraction. Reagents, chemicals and standards: All chemicals and solvents used were of analytical reagent grade except Ethyl acetate that was HPLC grade. Deionized water was obtained from Barnstead smart2 pure system (Thermo Scientific, Germany). Ethyl acetate, n-Hexane, HCl, NaOH, K2HPO4, and N, N–dimethylformamide were obtained from Sigma Aldrich, Germany. The nitrofuran metabolites ELISA kits (NF3461-3463, NF3465) produced by Randox Laboratories Ltd, UK were used. Nitrofuran Metabolites (AOZ, AMOZ, AHD, SEM) Standards were obtained from Sigma Aldrich, Germany. Stock standard solutions of the four compounds (100ng/ml) were prepared by dissolving each one in N, N–dimethylformamide and store at -20?C. Working mixed standard solutions (10ng/ml) of the compounds were freshly prepared from the stock standard solutions. Sample preparation: After brought to the laboratory, known blackfish and shrimp samples were washed with tap water. Head, shard of shrimps and scales of fish were removed from the body. Flesh was taken out from the bone of fish. Around 200g homogenized paste was prepared from the samples using a blender and stored at -20oC. The extraction procedure: 1g of homogenized muscle was taken into a 50 ml polypropylene tube and 4 ml double-distilled H2O, 0.5ml 1M HCl, 75μl 10mM 4-Nitrobenzaldehyde (in DMSO) were added and vortexed for 1 min. The mixture was incubated for 2 hrs at 50oC in a water bath and then kept at room temperature for 10 min. 5ml 0.1M K2HPO4, 0.4ml 1M NaOH and 6ml ethyl acetate were added to the sample and vortexed for 1 min. After centrifugation (10 min at 4000rpm at 25oC) 3ml of the upper ethyl acetate layer was evaporated at 60oC under N2 stream and re-dissolved in 1ml hexane and 1ml dilution buffer provided with the ELISA kit. After 10 min centrifugation at 4000rpm at 25oC, 50μl of lower phase was used for the analysis. Validation study: According to 2002/675/EC for validation of qualitative screening method, determination of detection capability CCβ (the smallest content of the analyte that may be detected, identified and/or quantified in a sample with an error probability of β), specificity/selectivity and applicability/ruggedness/stability are mandatory. Validation of screening methods (whether qualitative or semi-quantitative) also requires identification of a Cut-Off Level at, or above which the sample is categorized as 'screen positive' and liable to physicochemical confirmation (CRL 2010). Detection capability CCβ and cutoff level: The CCβ and cut-off level were established by analysing 20 blank samples and 20 spiked samples for each investigated matrix. Matrix blank samples were spiked with nitrofuran metabolites at the screening target concentration. In case of SEM, 40 blank and 40 spiked samples for each investigated matrix were tested. Minimum Required Performance Limit (MRPL) for each of the nitrofuran metabolites is 1μg/kg set by European Union (EC Decision 181, 2003), so screening target concentration was set at 0.5μg/kg for AOZ, AMOZ and AHD and 0.75μg/kg for SEM. Selectivity/specificity and stability: The selectivity/specificity was tested by analyzing 20 representative blank samples for each matrix under examination. The stability of the analyte in matrix extracted was tested by analyzing spiked sample at screening target concentration. Matrix Extracted solution was divided into 3 aliquots. one aliquot was analyzed immediately. The remaining aliquots were stored at -200C and analyzed them in two other occasions.The calculation of the concentration of the analyte in each aliquot carried out by using the formula: Solution of the analyte freshly prepared at the time of analysis as 100 %. alyte Remaining (%) = Ci × 100/Cfresh Ci= concentration at time point Cfresh= concentration of fresh solution