Establishment and Application of HPLC-FLD Method for Fipronil Sulfoxide Detection by Pre-column Fluorescence Derivatization

A method of precolumn fluorescence derivatization of fipronil sulfoxide (a metabolite of fipronil) was established and applied in the residue detection of chicken eggs. The main optimized parameters of precolumn fluorescence derivatization were as follows: catalyzer for condensation: EDC/DMAP; reaction solvent: dichloromethane; ratio of fipronil sulfoxide to fluorescence derivatization reagent: 1/8; reaction temperature: 45 ℃ (water bath); reaction time: 75 min. The target product of the fluorescence derivatization reaction was (N-(3-cyano-1-(2, 6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)thio)-1H-pyrazol-5-yl)-4-(2-(9-ethyl-9H-carbazol-3-yl)-1H-phenanthro9, 10-dimidazol-1-yl)butanamide (DX1), with high fluorescence intensity when excited. The main conditions for HPLC-FLD of DX1 were as following: C₁₈ chromatographic column; isocratic elution; mobile phase: acetonitrile/water (80/20, v/v), flow rate: 1.0 mL/min, wavelength of the exciting light: 310 nm, emission wavelength: 404 nm, injection volume: 20 μL, column temperature: 40 ℃. Under the above-mentioned conditions, the limit of detection (LOD) and limit of quantitation (LOQ) were 0.033 and 0.092 μg/L respectively. Meanwhile, the extraction and determination method for the residual fipronil sulfoxide in chicken eggs were further developed with a LOD of 0.052 µg/kg and a LOQ of 0.132 µg/kg, whose RSDs of the retention time and peak area of precision, stability and reproducibility were respectively less than 0.04% and 2.7%. What's more, the operation of the HPLC-FLD detection could be completed in 20 min. The method had good sensitivity, accuracy and reliability, and could be used to the determination of residual fipronil sulfoxide in eggs.