| Cefquinome Sulfate, the fourth-generation animal-special antibiotic produced by the German Hoechst (Hoechst AG) in the early1980s, is firstly approved for sale in1993. Owing to such features as broad widely antibacterial spectrum, strong antibacterial activity, fine pharmacokinetics characteristics and low toxicity, it has been used for the clinical prevention and treatment on bacterial infections of pigs and cattle in Europe and South america. Based on the provision of suspension-type injection in "People’s Republic of Veterinary Pharmacopoeia ", this research develops Cefquinome Sulfate suspension injection, and makes an investigation on quality control, stability, bioequivalence,clinical application, residue depletion, etc., hoping a kind of injection with equal biologic effects as the imported will be gained, which could be popularly applied in clinical veterinary in china.1. The development of Cefquinome Sulfate injectionAccording to the physicochemical properties of Cefquinome Sulfate, the research is carried on the formula optimization and preparation manufacturing technique of Cefquinome Sulfate injection (2.5%), where we make the raw material superfine comminute, choose injection grade soybean oil as solvent, establish the optimized technics in aseptic condition of filling-in nitrogen. Through testing the influence of temperature and light on the suspension injection stability, we establish the aseptic preparation techniques and the storage conditions in which the injection must be stored under20℃avoid light.2. Quality inspection and Stability and the preliminary irritant of injected intramuscularly test Through the characters monitoring, illumination test, distributed test and sedimentation test, we conclude that the injection of Cefquinome sulfate is oil suspension, which will be layered if long placed, and becomes almost white or light yellow after being shaken. According to Ultraviolet-visible spectrophotometry, it is confirmed that Cefquinome Sulfate has maximum absorption at wavelength of265nm±2nm. Based on these facts that the hydroximic acid. generated by chromogenic reaction of cephalosporin and hydroxylamine in alkaline condition, can react with high iron ion in dilute acids condition; permanent white precipitation will appear after Sulfate mixing with barium chloride test solution; the peak retention time (tR) of test sample together with the peak retention time (tR) of reference substance can be shown by liquid chromatography, it is confirmed that the color-display method, precipitation reaction and liquid chromatographic method can be used to identify Cefquinome Sulfate.A HPLC method is established for the determination of Cefquinome sulfate in Cefquinome sulfate injection, in which the assay is performed on C18column (250mm×4.6mm,5μm), a mixture of0.025mol/L perchlorate solution-phosphoric acid-acetonitrile (500:6:57, pH3.5±0.1), chosen as mobile phase, is delivered at a flow rate of1mL/min, and the detection wavelength is set at269nm while sample size at20μL. The results indicate that the Cefquinome sulfate within the range from11.78μg/mL to235.6μg/mL is linearly correlated(r=0.9998), the average recovery is99.70%(n=9), and the RSD is0.435%.The three batches of Cefquinome sulfate injection respectively, accelerated and long-term stability test, by measuring the appearance of the characters, decomposition products, and content indicators, compared with0. Results show that, the colour and lustre of the injection deepen when it is put in the condition of40±2℃for6months, and the total impurities are already more than4%by liquid-phase determination. However, when it is put in the condition of30±2℃for6months, the colour and lustre somewhat deepen, the peaks of2,3-cyclohexyl pyridine, other single biggest peak and total impurity peak are neither overproof, and the content is also in compliance range. In addition, related materials increase gradually, but still in the prescribed standards range when in cool and dark place for18months.Four healthy domestic rabbits are selected, and divided into two groups to carry on the preliminary irritant experiment. The first group of two rabbits is injected in the quadriceps with the Cefquinome sulfate injection suspension, whose appetite and desire to drink slightly decreased within48hours after injection, and four limbs do not obviously change. It is found that thighbone quadriceps is mild bleeding, where there are scattered bleeding points, but no degenerative lesions in muscles after anatomy. Neither the appetite and drink desire of the second group, during7days after injected, significantly drop, nor can obvious changes be observed from the appearance of four limbs. The anatomy, made7days later, shows that thighbone quadriceps is almost not bleeding, and muscles show no degenerative lesions, either. The results indicated that the local irritation test is weak in animal muscle.3. The pharmacokinetics bioequivalence test and the residue depletion test in porcineThe pharmacokinetics bioequivalence testing of cefquinome Sulfate injection (2.5%) was taken in porcine in this research, and the pharmacokinetic process expressed as a two-compartment open model. The experimental group absorbed more slowly, peak time is longer, eliminated slower when compared with the control group, The AUC90%confidence interval of experimental group was104.1%-120.0%, which was in range of80%-120%control group average; Cmax90%confidence interval was99.6%-116.7%, which was in range of70%-130%control group average. No differences between the test and the reference were observed in individuals, cycles and dosage forms. Both of them meet the statistics request of bioequivalence, and are biological equivalent preparations.A solid phase extraction and high performance liquid chromatography (SPE-HPLC) method was developed for the determination of cefquinome residues in porcine tissues. Porcine tissues were treated with phosphate buffer and acetonitrile, centrifuged, evaporated and then passed through preconditioned C18solid phase extraction (SPE) cartridge. The SPE column was washed with phosphate buffer and eluted with acetonitrile solution. The elutant was collected, evaporated dry and redissolved. The final solution was analyzed by HPLC at265nm.The limit of detection (LOD) and the limit of quantitation (LOQ) of cefquinome in porcine tissues were5～30ng/g and20～100ng/g, respectively. Mean recoveries of cefquinome in all porcine tissues at a concentration range of0.02~2.0μg/g were55.7～79.4%with coefficient of variation below6.7%. The technical parameters, established in the cephalosporins residue determination in pig tissue, can meet the requirements of determination.Healthy pigs were administered cefquinome intramuscularly at a dosage of2mg/kg once daily for five days. Five pigs were slaughtered at days0.5,1,2,3,5,7, and9after the last administration. Tissues including muscle, fat, liver and kidney were collected for the determination of cefquinome residues. The residue levels of cefquinome from high to low were kidney, liver, fat and muscle, indicating that kidney and liver were the target organs for cefquinome. Cefquinome residues in all examined organs were below the accepted maximum residue limits (MRLs) recommended by The European Medicines Agency (EMEA) at3third withdrawal day. The results showed that the depletion of cefquinome in swine is rapid with a short residue time. It was proposed that withdrawal time is3days.4. Treatment of cow mastitis caused by E. coli treated and the residue depletion test in cow milk24clinical sick cows infected with Eschrichia coli in breast were randomly divided into cefquinome-treated group and ampicillin-treated group. Both cefquinome and ampicillin had therapeutic efficacy on cow mammitis infected by E. coli. The effect of cefquinome on controlling course of disease and production of milk was obviously better than ampicillin. Sick cows were administered cefquinome intramuscularly at a dosage of lmg/kg once daily for two days. The clinical cure rate was more than90%, and the bacteriology cure rate was more than80%.The test method of cefquinom residual were established with HLCP. The extracted solution was evaporated to dry and added flowing phase. The flowing phase was acetate buffer (pH3.6)-mathanol (83:17); detection wavelength was270nm. Under these chromatography conditions, cefquinome concentration in milk had a good liner coefficient relation(R2≥0.9997) in the range of0.02～2μag/mL. The average recoveries at0.02μg/mL、1μg/mL、2μg/mL levels in milk were85.28%～98.56%. The within-day CV and between-day CV were1.55%～4.36%and0.40%～2.01%respectively. The lowest detectable limit was0.01μg/mL, and the detection quantity was 0.02μg/ml.Residural depletion of a cefquinome sulfate injection in milk.Four healthy adult Holstein cows which were not used cefquinome before, were used in residural depletion studies. The cows were intramuscularly injected cefquinome sulfate suspention:once a day, for two days. The milk samples were collected at a given time point after administration. Cefquinome concentrations in milk were determinted by an established method above. The results indicated that the CEF concentration reached its peak at24h, fell below0.02μg/mL at84h, all below the detection limit0.01μg/mL at96h. So the milk withdral time of this drug can be adviced as4days. |
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