【作者】 徐维盛；

【导师】 牛长群；

【作者基本信息】 河北医科大学 ， 药物分析学， 2008， 硕士

【摘要】 免疫抑制剂是一类具有免疫抑制作用的药物,可抑制机体异常的免疫反应,目前已广泛用于器官移植抗排斥反应和自身免疫性疾病的治疗。免疫抑制剂的不断更新及在临床的实际应用,使当今的器官移植取得了巨大的成功。脱氧精胍菌素(Deoxyspergualin,DSG)是一种全新的免疫抑制剂,不仅能有效地阻止移植器官排异,而且能逆转急性排异反应,由于DSG的免疫抑制作用强,毒性较低,特别是对肝、肾以及神经几乎没有毒副作用,因此,该新药为临床免疫抑制剂的应用提供了又一新的选择。为了研究开发脱氧精胍菌素,将该新药尽快推向临床,必须建立准确、可靠的分析方法,为合成工艺研究、制剂研究、质量研究和稳定性研究打下基础,本文建立了脱氧精胍菌素的反相离子对色谱分离分析方法,有效的解决了脱氧精胍菌素在反相色谱柱上不保留的问题,为脱氧精胍菌素的含量及有关物质测定提供了一个简便有效的分析手段。同时,本文还建立了脱氧精胍菌素的高效毛细管电泳分离分析方法,该法简便快速,灵敏度高,特异性好,可作为该药物高效液相分析方法的补充和完善,为该药物提供了一种新的分离分析手段。目的:建立脱氧精胍菌素及其有关物质的高效液相色谱分析方法和高效毛细管电泳分析方法,并对新方法进行方法学研究,分析测定实际样品中的有关物质和含量,为脱氧精胍菌素的研究开发提供可靠的分析方法。方法:1反相离子对色谱法。(1)通过对不同色谱柱,不同流动相的组成、配比、流速及柱温的优化选择,确定最佳的色谱分离条件,并对所建立的新方法进行方法学验证。(2)系统适用性试验:在已确定的反相液相色谱条件下,考察脱氧精胍菌素主峰的理论板数,并计算拖尾因子。(3)专属性试验:在合适的条件下,对脱氧精胍菌素样品进行热、碱、酸、氧化、光的破坏,考察加速破坏所产生的杂质与主峰的分离。(4)线性与范围:配制一系列浓度的脱氧精胍菌素对照品溶液,测定峰面积;以浓度为横坐标,以脱氧精胍菌素主峰面积为纵坐标,绘制标准曲线。(5)精密度试验:取一份对照品溶液,重复进样6次,记录峰面积,计算峰面积的RSD值。(6)稳定性试验:取脱氧精胍菌素供试品溶液适量,室温下放置,分别于0、2、4、6、8小时,取样注入液相色谱仪,记录色谱图,计算峰面积的RSD值。(7)回收率:取同一批脱氧精胍菌素供试品适量,按供试品溶液浓度的80%、100%、120%分别配制低、中、高3种浓度的溶液各3份,作为供试品溶液。分别取上述溶液各20μL注入液相色谱仪,记录色谱图及峰面积,计算回收率。(8)最低检测限:将脱氧精胍菌素对照品溶液逐步稀释,使峰高为噪音峰高的3倍,此时对应的脱氧精胍菌素的量即为最低检测限。(9)样品测定:对三批脱氧精胍菌素样品按照已确定的色谱条件进行测定。2.高效毛细管电泳法。(1)通过对不同缓冲液浓度、pH值、温度及电压的选择,确定最佳的高效毛细管电泳分离分析条件。(2)系统适用性试验:在已确定的高效毛细管电泳色谱条件下,以脱氧精胍菌素主峰面积考察系统理论板数,并计算拖尾因子。(3)专属性试验:在合适的条件下,对脱氧精胍菌素样品进行热、碱、酸、氧化、光的破坏,考察加速破坏所产生的杂质与主峰的分离情况。(4)线性与范围:配制一系列浓度的脱氧精胍菌素对照品溶液,测定峰面积;以浓度为横坐标,以脱氧精胍菌素主峰面积为纵坐标,绘制标准曲线。(5)精密度试验:取一份对照品溶液,重复进样6次,记录峰面积,计算峰面积的RSD值。(6)稳定性试验:取脱氧精胍菌素供试品溶液适量,室温下放置,分别于0、2、4、6、8小时,取样注入液相色谱仪,记录色谱图,计算峰面积的RSD值。(7)检测限的确定:将脱氧精胍菌素对照品溶液逐步稀释,测量最高噪音峰的峰高,使样品峰高为噪音峰高的3倍,此时对应的脱氧精胍菌素的量即为最低检测限。(8)样品测定:对三批脱氧精胍菌素样品按照已确定的色谱条件进行测定。结果:1反相离子对色谱法。(1)色谱条件:色谱柱:Gemini C18色谱柱(250×4.60mm,5μm Phenomenex公司);流动相: 5mmol/L磷酸氢二钾水溶液(内含5mmol/L戊烷磺酸钠,用磷酸调pH3.6±0.3)-乙腈(90:10);检测波长:210nm;柱温:30℃;进样量:20μl;流速:1.0ml/min。(2)系统适用性试验:以脱氧精胍菌素峰计算的理论板数为8000,拖尾因子为1.05。(3)专属性试验:脱氧精胍菌素样品进行热、碱、酸、氧化、光破坏,降解产物可与主成分峰达基线分离,表明该方法专属性良好。(4)线性与范围:脱氧精胍菌素浓度在0.05-2mg/ml范围内与峰面积呈良好的线性关系,线性方程为Y=5.7×106X+27336,相关系数r2为1.000(n=5)。(5)精密度:按上述条件平行测定6次,脱氧精胍菌素峰面积的RSD为0.26%,表明方法精密度良好。(6)溶液稳定性:供试品溶液室温下放置0、2、4、6、8小时,脱氧精胍菌素峰面积的RSD为0.40%,表明样品溶液在8 h内稳定。(7)回收率:低、中、高三个浓度的平均回收率分别为98.5%、99.8%和98.2%,RSD%值分别为0.32%、0.35%和0.29%。试验结果表明,该方法回收率高。(8)最低检测限:当S/N=3时,测得本方法的最低检测浓度为0.5μg/mL,计算最低检测量为10ng。(9)样品测定:三批供试品含量分别为98.8%、98.5%和98.9%;有关物质分别为1.3%、1.4%和1.3%。2.高效毛细管电泳法。(1)色谱条件:未涂层石英毛细管67cm×50μm(有效长度60cm);缓冲液为75mmol/L磷酸氢二钠缓冲液(用5%磷酸调节pH 2.5);检测波长为214nm;电压为25kV;温度为30℃;压力进样5s。(2)系统适用性:理论板数按脱氧精胍菌素峰计算为200000,拖尾因子为1.01。(3)线性与范围:浓度在0.1-2.0mg/mL范围内脱氧精胍菌素峰面积与浓度值之间呈良好的线性关系,回归方程为:Y=0.8116X+0.07141,相关系数r为0.9996(n=5)。(4)精密度:按上述条件平行测定6次,脱氧精胍菌素峰面积的RSD为0.47%,表明方法精密度良好。(5)溶液稳定性:供试品溶液室温下放置0、2、4、6、8小时,脱氧精胍菌素峰面积的RSD为0.45%,表明样品溶液在8 h内稳定。(6)最低检测限:当S/N=3时,测得本方法的最低检测浓度为3μg/mL,计算最低检测限为0.19%。(7)样品测定:对三批脱氧精胍菌素在已确定的色谱条件下进行了测定,测得脱氧精胍菌素含量分别98.7%、98.6%和98.8%;有关物质分别为1.3%、1.4%和1.3%。结论:本文建立了脱氧精胍菌素的反相离子对色谱和高效毛细管电泳测定方法,并进行了方法学研究,对脱氧精胍菌素进行了含量和有关物质测定。为该药的稳定性研究、质量控制研究及质量标准的建立提供了可靠、有效的分析手段。更多还原

【Abstract】 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.更多还原