Fast screening immunoassay of sulfonamides in commercial fish samples.
An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed in plate to detect three sulfonamide residues (sulfamerazine (SMR), sulfadimetoxine (SDM), and sulfadiazine (SDZ)) in gilthead sea bream (Sparus aurata) samples. Different extraction methodologies--using methanol/water 1:1 (v/v) + ethylene diamine tetraacetic acid (EDTA) 0.5% (m/v), acetonitrile, phosphate-buffered saline (PBS) 10 mmol L(-1) pH 7 and acetate buffer 100 mmol L(-1) pH 5--and cleanup steps, based on solid-phase extraction (C(18), SCX, Si) or liquid extraction with hexane, were assayed. As optimum, a fast and simple method using acetonitrile was selected to extract the sulfonamide residues from the edible muscle of fish. Due to matrix effects, a standard addition calibration curve in fish extract is necessary for quantification purposes. Sulfonamide-free samples were spiked at different concentration levels (between 30 and 90 ng g(-1), 5-15 ng mL(-1) in plate) and average recoveries (n = 8), ranging from 71% to 95%, 65% to 79%, and 72% to 95%, were obtained for SMR, SDM, and SDZ, respectively. The assay detection limits for these antibiotics were lower than 100 microg kg(-1) (maximum residue level established by the European Union). The accuracy was evaluated by spiking blank fish extracts at different concentrations (10-40 ng mL(-1), 5-20 ng mL(-1) in plate), and the relative errors ranged between +/-20%. Finally, in order to confirm the utility of the developed ELISA as a screening methodology, fish samples from different supermarkets were analyzed, and results were compared with those obtained by a validated high-performance liquid chromatography (HPLC) method. The correlation between the results obtained by both ELISA and HPLC methods is satisfactory.
Cháfer-Pericás
Consuelo
C
Instituto de Reconocimiento Molecular y Desarrollo Tecnológico, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
Maquieira
Angel
A
Puchades
Rosa
R
Miralles
Javier
J
Moreno
Amelia
A
D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis*; D004350:Drug Residues / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D057230:Limit of Detection; D008460:Meat / Q000032:analysis*; D009132:Muscles / Q000737:chemistry; D021541:Sea Bream; D013449:Sulfonamides / Q000032:analysis*
D000900:Anti-Bacterial Agents; D013449:Sulfonamides