Synthesis of Fluorescent Coumarin Derivatization Reagent and Development of a Pre-column Derivatization-High Performance Liquid Chromatography with Fluoresence Detection Method for Fipronil Determination

A novel fluorescent coumarin derivatization reagent, 7-(diethylamino)-2-oxo-2H-chromone-3-carboxylic acid (named as L1), was synthesized by condensation-cyclization and hydrolysis reactions using 4-(diethylamino) salicylaldehyde as the raw material. The derivatization reaction system of L1 and fipronil was established and optimized. The optimal reaction conditions were obtained as follows: condensation catalyst, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/4-dimethylaminopyridine; reaction solvent, dichloromethane; ratio of L1 to fipronil, 11:1; reaction time, 60 min; and derivatization temperature, 45 ℃. The derivative of fipronil, N-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-(trifluoromethyl) sulfonyl)-1H-pyrazole-5-yl)-7-(diethylamino)-2-oxo-2H-chromone-3-formamide (named as SF1), was isolated and identified. On this basis, a high performance liquid chromatography-fluorescence detection method (HPLC-FLD) based on pre-column derivatization was established for fipronil determination, with a detection limit of 0.01 μg/L and quantitative limit of 0.036 μg/L. In summary, the HPLC-FLD method presented good sensitivity, accuracy and reliability, and therefore could be applied to the detection of fipronil residue in agricultural products.