The Studies On Determination Of Residues Of Three Fluoroquinolones In Grass Carp (Ctenopharyngodon Idellus)

Fluoroquinolones(FQNS)is the third generation of quinolones antibacterial drugs,which is highly against Gram-positive bacteria,Gram-Negative bacillus,coccobacteria and gonococci and which also has powerful inhibiting effect to some of the chlamydia,sprirochete,mycoplasma and anaerobic bacterium,so it has been widely used to treat various bacterial infectious diseases of livestock,poultry and aquatic animal.In generally,the residues of FQNS in aquatic products have become a hot concern.The residues of FQNS in aquatic products not only have bad effect on human beings and enviroment,but also impact the export of China's aquatic products,market competitiveness,social effects,etc.Therefore,it is imperative to detect and monitor the residues of fluoroquinolones in fishery product.There are three parts.In the first part,extraction,concentration,purification and some other component elements were sieved and the mobile phase,detector,wavelength of detection and some other chromatographic conditions were selected and optimized.Danofloxain Mesylate was used as the internal standard substance.On the Basis of national standard method,the high performance liquid chromatographic method-fluorescence detector(HPLC-FLD)was established for the multi-residues determination of Norfloxacin,Enrofloxacin,Ciprofloxacin in Grass carp muscle tissue.The result demonstrated that acetonitrile mixed with phosphoric acid used as the extracting solution,the method of vacuum rotary evaporation used at 55℃for concentration,the method of liquid-liquid extraction and n-hexane used for defatting and purifying samples,fluorescence detector used at excitation wavelength of 280nm and emission wavelength of 450nm.The calibration graphs for three drugs were linear from 0.01μg/g to 10μg/g.Extraction recovery rate was between 79.89% and 99.45%and method recovery rate was between 97.60%～102.34%.The intra-coefficients of variation were less than 4%and the inter-coefficients of variation were less than 7%.The limits of detection for three drugs were 0.006μg/g.The establishment of this method laid the foundation for the studies on the residues determination and residue elimination law of above-mentioned drugs in Grass carp.In the second part,the residues of Norfloxacin,Enrofloxacin and Ciprofloxacin in aquatic product in parts of the markets of aquatic product in Chongqing were investigated by the multi-residues determination method.It was discovered that there were no residues in Grass carp muscle tissues in the Meishiba fry breeding ground in Rongchang Chongqing,but there were residues of three drugs in Xiema Market in Beibei and the aquatic market in Changshou,Tongnan, Wanzhou and Dianjiang.The positive rate was less than 8%,and the quantities of residues were below the MRLs stipulated by the nation.It proved that the use of three drugs in the cultivation of Grass carp was comparatively standardized in the above-mentioned regions.In the third part,the residue elimination law of Enrofloxacin and its metabolite in Grass carp muscle tissues was studied by the multi-residues determination method.Actual aquacultural conditions were simulated in this study.Grass carp were administrated enrofloxacin by eating bait (in the morning and afternoon,respectively)at a dose of 40 mg/kg(body weight)for five consecutive days.The samples were got when 3h after the last time of administration every day and 3h,6h,12h,24h,48h,96h,144h,192h,240h,288h,384h,528h,720h,960h, 1104 h,1176 h after drug withdrawal.Every time five samples were collected.The squama of fish were wiped off and the muscle including skin were fetched fllow the back of fish.After preparation and determination of the samples,the results were as follow.The drug concentration-time data of Enrofloxacin and its metabolite Ciprofloxacin were best described by two-compartmental model. The pharmacokinetic parameters of Enrofloxacin and Ciprofloxacin in Grass carp muscle tissues were respectively as follows:T_1/2(half-time of absorption)were 7.085h and 183.606h,Tmax(time to peak time)were 5.635h and 4.498h,Cmax(peak concentration)were 7.045μg/g and 0.188μg/g, AUC(area under concentration-time curve)were 366.045μg.h/g and 30.927μg.h/g,V_B(apparent volume)were 68.000L/kg and 0.918L/kg,T_2/2(half-life of elimination)were 86.265h and 3.281h, CL_B(total body clearance)were 0.546g/(kg.h)and 0.194g/(kg.h).The results showed that the elimination of Enrofloxacin in Grass carp muscle tissues was slow.So,it was suggested that if the drug was used in aquiculture,the withdrawal time of enrofloxacin should be postponed or the other durgs were used as the substitutes.At this experiment conditions,the predicted withdrawal time of enrofloxacin was 36d for Grass carp.For the practical cultivation,considering the temperature of water,the quality of water,the density of fish and the uniformity of the bait,the predicted withdrawal time of enrofloxacin was 59d for Grass carp.