Study On Azithromycin Residues And Its Elimination In Tissues Of Chickens With Method Of HPLC-FLD

Azithromyin(AZM) is a kind of the second filial generation macrolides antibiotics which has been attracted lots of farmers to use it in practical works because of its various good qualities,even thoμgh it does not belong to veterinary clinical medication, what will also bring us the hidden danger of animal food.In order to enhance monitoring of AZM residue in animal foodstuffs, we have established a precolumn derivation high performance liquid chromatography with fluorescence detection(HPLC-FLD) method to determine azithromycin residues in chicken tissues.The samples(plasma,muscle,liver) has been alkalified by NaOH, then extracted by N-hexane,after that, quantity sufficient organic layer was transferred and evaporated at 45℃under a flow of nitrogen.The residue was dissolved in derivative reagent: 9-fluorenylmethyloxycarbonyl chloride(FMOC-Cl)and borate buffer in water.The reaction vials was incubated in a waterbath of 50℃for 40 min.Finally,add some glycine act as stop buffer in the reaction liquid which will be analyzed by HPLC. The separation was performed on an Agilent C18 column(4.6mm×250mm);using the mobile phase composed of sodium phosphate buffer containing 0.2﹪tritthylamine and acetonitrile,which volume ration was 29:71,and pH value was 5.8.Excitationwavelength is 265 nm and emission wavelength of 315 nm, Flow rate is 1.2mL/min,column temperature is 35℃and 20μL of the solution was injected onto the chromatographic system. The limit of detection of AZM in blood plasma is 0.02μg/mL. The limit of detection of AZM in muscles and livers is 0.03μg/mL.The limit of quantitaton 0.04μg/mL. The calibration curve was linear in the range of 0.04～5μg/mL. At 0.1,1,3μg/mL (μg/g) concentration,the mean CV﹪within day was 0.82﹪～1.04﹪for plasma,0.91﹪～1.15﹪for mucle , 1.03﹪～1.81﹪for liver ; The mean CV﹪between day was 1.03﹪～1.27﹪,1.16﹪～1.31﹪,1.20﹪～2.12﹪for plasma,muscle,liver respectively.The recoveries of AZM added to the tissues at levels of 0.1,1,3μg/mL (μg/g) were 95.2±0.20﹪,76.3±0.16﹪,77.7±0.93﹪for plasma,muscle,liver respectively. From the datas in the experiment we can see that the HPLC-FLD method is simple and sensitive, which can monitor the safe of animal food safe and accorded with the requirement for determining residues of azithromycin in broilers.In the course of animality experiment,chickens were administrated by single-dose (20mg/kg)AZM,then collected the plasma,muscle and liver at 2,4,6,8,10,12, 24,36,48,60,72h after administeration.The AZM was not found at 48h by the HPLC-FLD method,while the elimination time of AZM is longer in muscle and liver tissues ,which were not found at 72h.The elimination characteristics of AZM in these tissues can be described by the equations as follows:C_plasma=7.7224e^-0.1362t ,C_muscle=21.668e^-0.0732t,C_liver=21.052e^-0.0567t.The elimination half-life time of these tissues respectly is t_1/2plasma=1.91 (h), t_1/2muscle=10.56(h),t_{1/2 liver}=13.12(h). It can be discerned that the speed of elimination of AZM in plasma is quick, while the speed of elimination of AZM is very slow and residue time is very long in these chicken muscle and liver tissues.