| Immunosuppressant is a kind of drug that has an immunosuppressive effect, which has suppressive activities on abnormal immune responses.At present ,it has been widely used in treating the transplant rejection and the autoimmune disease. The great success of the organ transplantation was owning to the continuous renewal and the clinical application of the immunosuppressant.Deoxyspergualin is a new immunosuppressant,which not only can prevent the transplant rejection effectively, but also can reverse the outbreak of acute rejection. Because of its high immunosuppressive activity and low side-effect, deoxyspergualin provides a new alternative of immunosuppressant for clinical application.In order to the investigation and exploitation of deoxyspergualin, an accurate and reliable method required to be established, it is also the foundation of the quality control and stability test. An simple but effective method has been established for analysis of deoxyspergualin by reversed-phase ion-pair high performance liquid chromatography. At the same time, another precise and fast method, that is, high-performance capillary electrophoresis, has also been established. This new method proved to be alternative to RP-HPLC for analysis of deoxyspergualin.Objective: To qualitative and quantitative analysis of deoxyspergualin ,two new methodes were established ,i.e. the RP-HPLC method and the HPCE method.Method: 1. RP-HPLC method. (1) Optimization chromatographic condition: the best separation condition was chosen by optimizing different columns, adjusting solvent proportion of mobile phase and column temperature. (2) System suitability test: On the optimized chromatographic condition, the theoretical plate of deoxyspergualin and the tailing factor were determinated. (3) Specificity test: After treated with heating, base, acid, hydrogen peroxide (H2O2) and strong light, the sample of deoxyspergualin were analyzed. (4) Linearity and range of calibration curve: Prepared a series of the reference solutions and determined peaks areas, then calibration curve was obtained by the contents of deoxyspergualin and the peaks areas. (5) Precision test: The sample solution was analyzed for six times; the peak area of deoxyspergualin was determinaed, and relative standard deviation was calculated. (6) Stability test: By determinaed sample solutions at different time on the room temperature, the stability of the sample solution was determined. (7) Recovery test: The sample solutions were prepared at three level of concentration, i.e. low, middle and high, three portions for each level., The peaks areas were determined and recovery were calculated.(8) Limit of detection test: Dilute the reference solution until the ratio of signal and noise ( S/N )was not less than 3.The limit of detection was determinated. (9) Sample analysis: Determination the related substances the content of three batchs of deoxyspergualin. 2. HPCE method. (1) By optimizing factors which affect the separation, such as concentration of buffers, the pH value , the supplied voltage and temperature, the optimum conditions for separation were selected. (2) System suitablity test: On the optimized analysis condition, the theoretical plate together with the tailing factor of deoxyspergualin were determinated. (3) Specificity test: After treated with heating, base, acid, hydrogen peroxide (H2O2) and strong light, the sample of deoxyspergualin were analyzed. (4) Linearity and range of calibration curve: Prepared a series of the reference solutions and determined peaks areas, then calibration curve was obtained by the contents of deoxyspergualin and the peaks areas. (5) Precision test: The sample solution was analyzed for six times; the peaks areas of deoxyspergualin were determined, and relative standard deviation was calculated. (6) Stability test: By determinated sample solutions at different time on the room temperature, the stability of the sample solution was determined. (7) Limit of detection test: Dilute the reference solution until the ratio of signal and noise ( S/N )was not less than 3.The limit of detection was determinaed. (8) Sample analysis:Determination the related substances and the content of three batchs of deoxyspergualin.Results: 1. RP-HPLC method. (1)The RP-HPLC separation was performed on a Gemini-C18 analytical column(250×4.60mm,5μm), with a mobile phase consisting of acetonitrile, 5mmol K2HPO4 and 5mmol sodium pentanesulfonate. The pH value was adjusted to 3.6±0.3 by phosphoric acid. The flow rate was 1.0 ml/min. The detection wavelength was 210 nm. The column temperature was set at 30℃. Injection volume was 20μl. (2) System suitablitily test: On the optimized chromatographic condition, the theoretical plate of deoxyspergualin was about 8000, and the tailing factor was 1.05. (3) Specificity test: By analyzed accelerate samples, the specificity of the system was proved. (4) Linearity and range of calibration curve: The linear range for deoxyspergualin was 0.05-2.0mg/ml. The calibration curve was Y=5.7×106X+27336, (r2=1.000). (5) Precision test: The Precision of main peak at six times was good and the RSD of the peaks areas of deoxyspergualin was 0.26%. (6) Stability test: The RSD of the peak area of deoxyspergualin was 0.40%. The test solution was stable in 8 hours. (7) Recovery test: The average recovery at three level of concentration were 98.5%,99.8% and 98.2% respectively. (8) Limit of detection test: The detection limit of deoxyspergualin was 0.5μg/mL. (9) Sample analysis: The related substances of deoxyspergualin was 1.3%, 1.4% and 1.3% respectively; and the content of deoxyspergualin was 98.8%,98.5% and 98.9% respectively. 2. HPCE method. (1) The HPCE separation was performed on a fused silica capillary column. The runing buffer was Na2HPO4 solution adjusted to pH 2.5 by 5% phosphoric acid. (2) System suitablitily test: On the optimized separation condition, the theoretical plate of deoxyspergualin were about 200000, and the tailing factor was 1.01. (3) Specificity test: By analyzed accelerate samples,the specificity was proved. (4) Linearity and range of calibration curve: The linear range for deoxyspergualin was 0.1-2.0mg/ml. The calibration curve was Y=0.8116X+0.07141, (r=0.9996). (5) Precision test: The Precision of main peak at six times was good and the RSD of the peaks areas of deoxyspergualin was 0.47%. (6) Stability test: The RSD of the peaks areas of deoxyspergualin was 0.45%.The test solution was stable in 8 hours. (7) Limit of detection (LOD) test: The limit of detection of deoxyspergualin was 0.19%.(8) Sample analysis: The related substances of deoxyspergualin was 1.3%,1.4% and 1.3% respectively; and the content of deoxyspergualin for three batchs were 98.7%,98.6% and 98.8% .Conclusion: Two new methodes were established, i.e. the RP-HPLC method and the HPCE method. By validation, the two new methods were proved to be specific, accurate and sensitive. They may be used to qualitative and quantitative analysis of deoxyspergualin in the new drug development for stability study and quality control.
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