| Sparfloxacin(SPFX) and enrofloxacin(ENR) were both antibacterial drugs that belong to the fluroroquinolone class, and they have been developed for the treatment of a variety of microbial infections in both animal and human medicine. However, based on published results and data on file with the manufacture of them, some adverse effects would occur in some patients. Therefore, in order to further effectively utilize SPFX and ENR and enhance the clinical safety, a simple, rapid and efficient method is needed for determination of them in clinical monitor. Recently, modern analytical techniques are the main methods for biopharmaceutical analysis, however, these methods have shown some disadvantages for the analysis of drugs, such as low specificity, high background, interference with the other components and requirement of sample pre-treatment. Taking all these factors into consideration, it is necessary to establish a more simple and reliable analytical method for determination of SPFX and ENR in biological samples and foods. In this study, the antisera against SPFX and ENR were obtained by immunizing rabbits with the artificial immunogen. Based on these antisera, the ELISA methods for the determination of SPFX and ENR in pharmaceuticals and rat plasma and tissues have been developed.The coating antigen (SPFX-OVA) and the immunogen (SPFX-BSA) was obtained by mixed anhydride method with sparfloxacin(SPFX) conjugated directly with bovine serum (BSA)and ovalbumin(OVA). Two kinds of artificial antigens were identified by ultraviolet spectrum and infrared spectrophotometry, and through the ultraviolet spectrum method to calculate SPFX in various protein on the coupling number. Two female Japan white rabbits were subcutaneously immunized with the immunogen SPFX-BSA at multiple sites. First immunization,2 mg immunogen dissolved in 2 mL of physiological saline solution and emulsified with the same volume of CFA. Every 10 days to booster immunizations.5 days after the last booster, blood was collected from carotid artery of each rabbit. After phalanx titration, ELISA proved that the titres of antiserum for rabbit was 1/102400. The ideal coating antigen was 1μg/mL and the available antiserum dilution was 1/25600. After the procedure was optimized, The atandard curve and correlation corfficient of SPFX were y=-0.1636x+0.8881 and 0.9942, respectively, the practical measuring rang were from 5 ng/ml to 2μg/mL. The antiserum specificity was evaluated by cross-reactivity assays with various similar compounds, and all compounds tested showed no cross reactivity. The result indicated the antiserum has a high specificity for SPFX. To validate the precision, satisfactory analytical recovery were obtained for SPFX in milk (93.7%-110.3%), in pork(98.3%-104.9%), in rat plasma(105.6%-113.3%) and in rat urine (90.2%-98.1%), the relative standard deviation for milk, pork, rat plasma and rat urine were 6.6%to 10.6%,7.4% to 19.5%,4.8% to 8.2% and 3.5% to 9.6%, respectively. The ELISA method and an HPLC method were compared for measuring 9 samples containing known amounts of atandard of SPFX, and obtained the regression equation and the correlation corfficient were y=1.0235x+0.1347 and R2=0.9918, respectively. In addition, the determination of SPFX in tablet and capsule were also performed, and the results were similar. The result showed that there was a good correlation between two methods. In order to further application ELISA method, a preliminary pharmacokinetic study of SPFX in rat was performed, and AUCo-∞, Tmax and Cmax were 2.169 mg h L-1,3 h and 346.06 ng mL-1, respectively. Moreover, the preliminary tissue distribution of SPFX in rats was performed.The coating antigen (ENR-OVA) and the immunogen (ENR-BSA) was obtained by mixed anhydride method with sparfloxacin(ENR) conjugated directly with bovine serum (BSA)and ovalbumin(OVA). Two kinds of artificial antigens were identified by ultraviolet spectrum, infrared spectrophotometry and fluorescence spectrum, and through the ultraviolet spectrum method to calculate ENR in various protein on the coupling number. Through tha same immune dose and immune method, preparation of serum resistance. After phalanx titration, ELISA proved that the titres of antiserum for rabbit was 1/102400. The ideal coating antigen was 500 ng/mL and the available antiserum dilution was 1/25600. After the procedure was optimized, The atandard curve and correlation corfficient of SPFX were y=-0.2288x+1.0147 and 0.9852, respectively, the practical measuring rang were from 5 ng/ml to 2μg/mL. The antiserum specificity was evaluated by cross-reactivity assays with various similar compounds, and all compounds tested showed no cross reactivity. The result indicated the antiserum has a high specificity for ENR. To validate the precision, satisfactory analytical recovery were obtained for ENR in milk (98.4%-105.8%), in pork(94.2%-102.4%), in rat plasma(91.7%-101.2%) and in rat urine (97.0%-110.3%), the relative standard deviation for milk, pork, rat plasma and rat urine were 5.6% to 10.6%,5.7% to 17.9%,4.8% to 7.2% and 4.5% to 9.6%, respectively. The ELISA method and an HPLC method were compared for measuring 9 samples containing known amounts of atandard of ENR, and obtained the regression equation and the correlation corfficient were y=0.9866x+0.5906 and R2=0.9892, respectively. In addition, the determination of SPFX in injection was also performed, and the results were similar. The result showed that there was a good correlation between two methods. In order to further application ELISA method, a preliminary pharmacokinetic study of SPFX in rat was performed, and AUCo-∞, Tmax and Cmax were 33.533 mg h L-1,0.75 h and 3.011mg L-1, respectively. Moreover, the preliminary tissue distribution of ENR in rats was performed. |
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