Development of a New Ultra High Performance Liquid Chromatographic Method with Fluorescence and Dad Detection for Efficient Separation and Analysis of Antiparasitic (Antibiotics, Pesticides, Antioxidants) Agents in Fish

Abstract

The use of antiparasitic agents in aquaculture is of a major concern due to its adverse effect on human health. This research developed and validated a new rapid Ultra High Performance Liquid Chromatography (UHPLC) method using fluorescence and Diode Array (DAD) detection for identification and quantification of antiparasitic agents (antibiotics, pesticides antioxidants) in fish samples. This was done in accordance with the European Commission 2000 guidelines. Separation and identification of antiparasitic agents was achieved on a Syvchronis C18 analytical column (100 mm ×2.1 mm I.D., particle size 1.7 μm) with mobile phase acetonitrile/ acetic acid, magnesium nitrate at a flow rate of 0.6 mL min−1 and at 35°C. The detection wavelength of the detector was set at excitation wavelength (λexc) 350nm and emission wavelength (λem) 451nm for sarafloxacin and ethoxyquin, 251nm absorption wavelength (ℷabs) for diflubenzuron and teflubenzuron and finally, absorption wavelength (λabs) 596 nm for crystal violet and malachite green. Under the optimum chromatographic conditions, standard calibration curves were measured with good linearity (r 2 = 0.99991) for all the analytes (n = 10). The method detected lower concentrations of these antiparasitic agents studied in this research with the detection limit of 0.056, 0.0122, 5.080, 1.575, 0.2654, 0.3870 μ/kg for Sarafloxacin, Ethoxyquin, Diflubenzuron, Teflubenzuron, Crystal Violet and Malachite Green respectively. These detection limits were lower than the Maximum Residual Limit set by the European Union. Recoveries obtained for all the various type of antiparasitic agents in fish were between 80%-99.73%.