【作者】 张伟；

【导师】 黄金林； 周伟；

【作者基本信息】 扬州大学 ， 制药工程， 2019， 硕士

【摘要】 庆大霉素是多组分混合物,在采用管碟法测定其含量时,常常存在标准品与样品之间不同质问题。因此,需要建立新的庆大霉素含量测定的方法并与管碟法进行比较。经过查阅文献,发现近年来庆大霉素的含量测定方法有微生物检定法、旋光法、光谱法、色谱法等。与管碟法比较,旋光法与光谱法也可以测定庆大霉素全部组分的含量,但旋光法的灵敏度太低。通过对光谱法的比较分析,发现紫外可见分光光度法具有灵敏度高、检测快速等优点,这可以作为建立庆大霉素含量测定方法的研究方向。根据庆大霉素的化学性质,本文寻找到化学性质稳定的水杨醛试剂。经过试验发现,水杨醛与庆大霉素在常温下发生衍生化学反应,且衍生产物在400nm波长处有最大紫外可见吸收。采用单因素多水平设计方法,优化反应条件。结果显示水杨醛与庆大霉素在pH5.5的磷酸盐缓冲液中,经过50℃水浴20分钟,衍生产物的吸收度值最大,实验条件最佳。采用最佳实验条件,依据《中国药典》规定的指导原则,进行紫外可见分光光度法测定庆大霉素含量的方法学验证。结果显示本方法的特异性好;在280～519 U/ml的浓度范围内,衍生产物的吸光度值(A)与庆大霉素浓度值(C)之间具有良好的线性关系,标准曲线A=0.0007C+0.0186,R 值达到 0.999;平均回收率为 100.09±0.8%,RSD 为 0.7%(n=7);衍生产物在0～10分钟内稳定。同时,采用管碟法测定庆大霉素的含量。按照菌悬液制备、培养基制备、灭菌、供试液制备、双碟制备、滴加供试液、培养、抑菌圈测量的流程进行试验,并将测量的抑菌圈直径按照生物检定统计法进行可靠性检测、可信限率及效价计算。独立平行实验4次。结果显示,1次实验结果的可靠性检测参数试品间(F1)不符合判定要求;1次实验结果的可信限率为7.96%,不符合硫酸庆大霉素注射液标准规定(不超过7%)的要求;含量结果平均值为 101.10%,RSD 为 1.7%(n=4)。最后,将紫外可见分光光度法与管碟法进行比较研究。首先分别用紫外可见分光光度法与管碟法测定硫酸庆大霉素注射液中庆大霉素的含量,并将各自检测结果进行比较:然后将紫外可见分光光度法与管碟法的方法学验证结果、硫酸庆大霉素注射液中庆大霉素含量测定结果的显著差异性进行比较。结果显示紫外可见分光光度法测定庆大霉素含量平均值为100.43%,RSD为1.2%(n=104);管碟法测定庆大霉素含量的平均值为101.10%,RSD为1.7%(n=4);紫外可见分光光度法的线性与范围为280～519 U/ml、线性关系R值为0.999、回收率为100.09%±0.8%、RSD为0.7%;管碟法的线性与范围为2～12 U/ml、回收率为100.80%±1.5%/98.7%±1.5%、RSD为1.7%;根据t分布检验法,在P<0.05的置信区间内,t=0.88,小于 1.98[t(0.05,106)]。本文建立紫外可见分光光度法测定庆大霉素含量的方法,具有检测快速,操作方便的特点。同时方法的特异性好、灵敏度与准确度高、重现性好,但是衍生产物的稳定性欠佳。与管碟法比较,两个方法的结果无显著性差异,但紫外可见分光光度法的准确性与重现性更优。因此,紫外可见分光光度法满足庆大霉素含量测定的常量分析要求,能为控制其质量提供新的参考依据,进而保障庆大霉素使用的安全性与有效性。更多还原

【Abstract】 Gentamicin is a multi-component mixture.When the content of Gentamicin is determined by tube-plate method,there is often a problem of different quality between the standard and the sample.Therefore,it is necessary to establish a new method for the determination of gentamicin and compare it with the tube-plate method.After consulting the literature,it was found that the determination methods of gentamicin in recent years include microbiological assay,polarimetry,spectroscopy,chromatography and so on.Compared with tube-plate method,polarimetry and spectroscopy could also determine all the content of gentamicin,but the sensitivity of polarimetry was too low.Through comparative analysis of spectrophotometry,it was found that ultraviolet-visible spectrophotometry had the advantages of high sensitivity and fast detection,which could be used as the research direction of establishing the determination method of gentamicin content.According to the chemical properties of gentamicin,salicylaldehyde reagents with stable chemical properties were found in this paper.It was found by experiment that the derivatization of Salicylaldehyde and gentamicin took place at room temperature,and the derivatives had the maximum ultraviolet-visible absorption at 400 nm wavelength.The single factor and multi-level design method was used to optimize the reaction conditions.The results showed that salicylaldehyde and gentamicin had the highest absorptivity in phosphate buffer at pH 5.5 after 20 minutes of water bath at 50℃,and the experimental conditions were the best.With the best experimental conditions and according to the guiding principles stipulated in the Chinese Pharmacopoeia,the methodological verification of the determination of gentamicin by ultraviolet-visible spectrophotometry was carried out.The results showed that the specificity of the method was good.In the concentration range of 280-519 U/ml,there was a good linear relationship between the absorbance value(A)of derivatives and the concentration value of gentamicin(C).The standard curve A was 0.0007C+0.0186,and the R value was 0.999.The average recovery was 100.09±0.8%,RSD was 0.7%(n=7),and the derivatives were stable in 0-10 minutesMeanwhile,the content of gentamicin was determined by tube-plate method.According to the process of suspension preparation,culture medium preparation,sterilization,test solution preparation,double dish preparation,dripping test solution,culture and measurement of bacteriostasis circle.The measured diameter of bacteriostasis circle was tested by reliability test,reliability limit efficiency and titer calculation according to the statistical method of biological verification.Four independent parallel experiments were conducted.The results showed that the reliability test parameters(F1)of the one of four experiment did not meet the judgment requirement;the reliability limit rate of the one of four experiment was 7.96%,which did not meet the requirements of the standard of gentamicin sulfate injection(no more than 7%);the average content of the results was 101.10%,and RSD was 1.7%(n=4).Finally,the ultraviolet-visible spectrophotometry and tube-plate method were compared.Firstly,the content of gentamicin in gentamicin sulfate injection was determined by ultraviolet-visible spectrophotometry and tube-plate method respectively,and the results were compared with each other.Then,the methodological verification results of ultraviolet-visible spectrophotometry and tube-plate method and the significant differences of the determination results of gentamicin sulfate injection were compared.The results showed that the average content of gentamicin was 100.43%and RSD was 1.2%(n=104)by ultraviolet-visible spectrophotometry.The average content of gentamicin was 101.10%and RSD was 1.7%(n=4)by tube-plate method.The linearity and range of ultraviolet-visible spectrophotometry were 280-519 U/ml,R value was 0.999,recovery is 100.09%±0.8%,RSD was 0.7%.The linearity and range of tube-plate method were 2-12 U/ml,recovery was 100.80%±1.5%/98.7%±1.5%,RSD was 1.7%.According to the t-distribution test,in the confidence interval of P<0.05,t=0.88,less than 1.98[t(0.05,106)].A method for the determination of gentamicin by ultraviolet-visible spectrophotometry was established.The method was rapid and easy to operate.At the same time,the method had good specificity,sensitivity,accuracy and reproducibility,but the stability of the derivatives was not good.Compared with the tube-plate method,the results of the two methods had no significant difference,but the accuracy and reproducibility of ultraviolet-visible spectrophotometry were better.Therefore,ultraviolet-visible spectrophotometry could meet the requirement of constant analysis for the determination of gentamicin content,and provide a new reference basis for controlling its quality,and ensure the safety and effectiveness of gentamicin use.更多还原