氟虫腈亚砜荧光衍生化、HPLC-FLD检测方法的建立及应用

建立了氟虫腈代谢物-氟虫腈亚砜的柱前荧光衍生化方法，并将其应用于鸡蛋中的残留检测。优化后的柱前荧光衍生化反应主要条件如下:催化缩合剂为EDC/DMAP；反应溶剂为二氯甲烷；氟虫腈亚砜与荧光衍生试剂的用量比为1:8；45 ℃水浴中反应75 min。衍生产物为N-（3-氰基-1-（2, 6-二氯-4-（三氟甲基）苯基)-4-（（三氟甲基）硫代）-1H-吡唑-5-基）-4-（2-（9-乙基-9H-咔唑-3-基）-1H-菲并9, 10-d]咪唑-1-基）丁酰胺（DX1），具有很高的荧光强度。衍生化产物DX1的高效液相色谱-荧光检测（HPLC-FLD）的主要条件如下:C₁₈色谱柱；等度洗脱；流动相:乙腈/水（80/20，v/v）；流速:1.0 mL/min；λex=310 nm，λem=404 nm；进样量20 μL；柱温40 ℃。在上述检测条件下，氟虫腈亚砜HPLC-FLD检测方法的检测限为0.033 μg/L，定量限为0.092 μg/L。在此基础上，建立了鸡蛋残留氟虫腈亚砜提取和检测的HPLC-FLD方法，检测限和定量限分别达到了0.052和0.132 μg/kg，精密度、稳定性和重现性在保留时间和峰面积上的RSD分别小于0.04%和2.7%，上机检测可在20 min内完成。该方法具有较好的灵敏度、精确性和可靠性，可应用于鸡蛋中残留氟虫腈亚砜的检测。

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-d]imidazol-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.