Synthesis Of Fluorescent Derivative Reagents Of Coumarins And Carbazoles And It's Application In The Residue Determination Of Fipronil

Fipronil is a broad-spectrum insecticide.This insecticide can kill pests by binding to?-aminobutyric acid(GABA,neurotransmitter)receptor,which will severely affect the function of central nervous system.Fipronil is widely used because of its good insecticidal effect and non-toxic effect on crops.However,fipronil has a long half-life and is harmful to the environment.It can be enriched in animals and human bodies and therefore impairs their health.Consequently,it is necessary to detect and control fipronil residues.At present,there are several methods for fipronil residue determination,such as high performance liquid chromatography(HPLC),high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS),gas chromatography(GC)and gas chromatography-mass spectrometry(GC-MS).But the detection limits of HPLC and GC are too high to meet the needs of trace determination.Although HPLC-MS/MS and GC-MS have good sensitivity,their applications are still confined by the expensive sophisticated determination instruments and the high cost.The fluorescence detection method has extremely high sensitivity,good stability,strong selectivity and low interference,and therefore it is very suitable for micro-detection and trace detection.Because of its weak fluorescence,fipronil can not be detected by fluorescence detector(FLD)directly.In the present paper,two novel pre-column derivatization reagents of coumarin and carbazole with high fluorescence intensity were designed and synthesized,which were further used to develop HPLC-FLD method for fipronil determination by pre-column derivatization respectively,furthernore,this HPLC-FLD method was applied to the determination of residual fipronil in eggs.The main results and conclusions are as follows:1.Novel fluorescent derivative reagent were designed and synthesized using coumarin as precursor.The target coumarin fluorescent derivative reagent named as7-(diethylamino)-2-oxo-2H-chromone-3-carboxylic acid(L1)was synthesized using4-(diethylamino)salicylaldehyde as precursor,according to the following procedures:firstly,4-(diethylamino)salicylaldehyde and diethyl malonate was condensed into a ring compound under the catalysis of piperidine;secondly,ester hydrolysis was carried out to produce the target fluorescent derivative reagent(7-(diethylamino)-2-oxo-2H-chromone-3-carboxylic acid).The structures of the synthesized intermediate compounds and final products were all characterized and identified by ¹H NMR,¹³C NMR and HRMS analysis,which indicated that these compounds were the right target compounds.L1 can send out strong fluorescence under excitation.The excitation wavelength(?_ex)and emission wavelength(?_em)of L1 are 454 nm and 480 nm,respectively.2.A HPLC-FLD(high performance liquid chromatography-fluorescence detection method)method for fipronil determination was established using L1 as pre-column fluorescence derivative reahent.In the pre-column derivatization reaction(condensation reaction),EDC/DMAP was selected as catalytic agent,and dichloromethane was used as solvent as well.The reaction completed within 60 min in a water bath at 45?when the ratio of fipronil to L1 was 1:11.After derivative reaction,the product(N-(3-cyano-1-(2pyryl-6-dichloro-4-(trifluoromethyl)phenyl)-4-(trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)-7-(diethylamino)-2-oxo-2H-chromone-3-formamide,named as SF1)was isolated and indentified.SF1 can emit strong fluorescence when excited,which was further used to develop a HPLC-FLD method for fipronil determination.The main detection conditions of the established method were as followsing:C₁₈chromatographic column;isometric elution,mobile phase:acetonitrile/water(80:20),flow rate of mobile phase:1.0 mL/min;?_ex=454 nm,?_em=490 nm;injection volume:10?L,column temperature:40?.The HPLC-FLD detection completed within 20 min with a detection limit of 0.01?g/L and a quantitative limit of 0.036?g/L respectively.The RSD of retention time of precision,stability and reproducibility were all less than0.06%,meanwhile the RSD of peak area were all less than 2.5%.The above results showed that the established HPLC-FLD method for fipronil determination had a very low detection limit and fine precision,stability and reproducibility as well.3.Novel fluorescent derivative reagent were designed and synthesized using carbazole as precursor.The target carbazole fluorescent derivative reagent named as4-(2-(9-ethyl-9H-carbazol-3-yl)-1H-phenanthro[9,10-d]imidazol-1-yl)butanoic acid(L2)was synthesized using 9-ethyl-9H-carbazole as precursor,according to the following procedures:after the formylation of 9-ethyl-9H-carbazole,the product was cyclized with phenanthrenequinone by principle of substructure splicing;finaly,halogenation and hydrolysis was carried out to produce the target fluorescent derivative reagent(4-(2-(9-ethyl-9H-carbazol-3-yl)-1H-phenanthro[9,10-d]imidazol-1-yl)butanoic acid).The structures of the synthesized intermediate compounds and final products were all characterized and identified by ¹H NMR,¹³C NMR and HRMS analysis,which indicated that these compounds were the right target compounds.L2 can send out strong fluorescence under excitation.The excitation wavelength(?_ex)and emission wavelength(?_em)of L2 are 310 nm and 398 nm,respectively.4.A HPLC-FLD(high performance liquid chromatography-fluorescence detection method)method for fipronil determination was established using L2 as pre-column fluorescence derivative reahent.In the pre-column derivatization reaction(condensation reaction),DCC/DMAP was selected as catalytic agent,and dichloromethane was used as solvent as well.The reaction completed within 60 min in a water bath at 55?when the ratio of fipronil to L2 was 1:11.After derivative reaction,the product(N-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl)-4-(2-(9-ethyl-9H-carbazol-3-yl)-1H-phenanthro[9,10-d]imidazol-1-yl)buta namide,named as SF2)was isolated and indentified.SF2 can emit strong fluorescence when excited,which was further used to develop a HPLC-FLD method for fipronil determination.The main detection conditions of the established method were as followsing:C₁₈ chromatographic column;isometric elution,mobile phase:acetonitrile/water(85:15),flow rate of mobile phase:1.0 mL/min;?_ex=310 nm,?_em=398 nm;injection volume:10?L,column temperature:40?.The HPLC-FLD detection completed within 20 min with a detection limit of 0.007?g/L and a quantitative limit of0.025?g/L respectively.The RSD of retention time of precision,stability and reproducibility were all less than 0.1%,meanwhile the RSD of peak area were all less than 1.5%.The above results showed that the established HPLC-FLD method for fipronil determination had a very low detection limit and fine precision,stability and reproducibility as well.5.A HPLC-FLD(high performance liquid chromatography-fluorescence detection method)method for fipronil sulfide determination was established using L2 as pre-column fluorescence derivative reahent.In the pre-column derivatization reaction(condensation reaction),EDC/DMAP was selected as catalytic agent,and dichloromethane was used as solvent as well.The reaction completed within 75 min in a water bath at 45?when the ratio of fipronil sulfide to L2 was 1:8.After derivative reaction,theproduct(N-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)thio)-1H-pyr azol-5-yl)-4-(2-(9-ethyl-9H-carbazol-3-yl)-1H-phenanthro[9,10-d]imidazol-1-yl)butanam ide,named as DX1)was isolated and indentified.DX1 can emit strong fluorescence when excited,which was further used to develop a HPLC-FLD method for fipronil sulfide determination.The main detection conditions of the established method were as followsing:C₁₈ chromatographic column;isometric elution,mobile phase:acetonitrile/water(80:20),flow rate of mobile phase:1.0 mL/min;?_ex=310 nm,?_em=404 nm;injection volume:20?L,column temperature:40?.The HPLC-FLD detection completed within 20 min with a detection limit of 0.033?g/L and a quantitative limit of0.092?g/L respectively.The RSD of retention time of precision,stability and reproducibility were all less than 0.1%,meanwhile the RSD of peak area were all less than 2.1%.The above results showed that the established HPLC-FLD method for fipronil sulfide determination had a very low detection limit and fine precision,stability and reproducibility as well.6.A HPLC-FLD method for the determination of residual fipronil and fipronil sulfide in eggs was established.Acetonitrile was used as solvent to extract residual fipronil and fipronil sulfide from eggs with a ratio of 20 mL/5 g(acetonitrile/egg).The extraction completed within 20 min at room temperature(about 20?)with the assistance of oscillation.The synthesized carbazole fluorescence derivative reagent was selected for the fluorescence derivatization detection of residual fipronil and fipronil sulfide in eggs.The recovery rate of the established HPLC-FLD detection method was more than 84%,at the mean time the relative standard deviation(RSD)ranged from 0.95 to 5.09%(n=3).It should be noted that the established method showed an very low detection limit and quantitative limit,the detection limit of fipronil and fipronil sulfide were 0.025?g/kg?0.052?g/kg respectively and the quantitative limit of fipronil and fipronil sulfide were0.082?g/kg?0.132?g/kg respectively.The above results indicated that the established HPLC-FLD method for the detection of residual fipronil and its metabolite-fipronil sulfide in eggs using L2 as pre-column fluorescence derivative reagents showed a high sensitivity and a low detection limit,and therefore had a good prospect in application.In conclusion,two novel pre-column derivatization reagents of coumarin and carbazole with high fluorescence intensity were designed and synthesized,and two HPLC-FLD methods for fipronil determination were developed with high sensitivity accordingly;the method using L2 as fluorescent derivative reagent showed a relatively lower detection limit and a relatively high sensitivity,and therefore was applied in the determination of residual fipronil and its metabolites-fipronil sulfide in eggs with a detection limit of 0.025?g/kg?0.052?g/kg respectively,which was obviously better than that of HPLC-MS/MS and GC-MS method(0.1?g/kg and 3?g/kg respectively).Consequently,the developed HPLC-FLD method had a promising application prospect in the determination of residual fipronil and its metabolites in eggs.