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1、金银花复方制剂的质量控制方法研究 金银花复方制剂的质量控制方法研究 Research of Quality Control Method for Honeysuckle Compound Preparations【中文摘要】 金银花为常用中药,现代药理研究表明,此中药具有抑菌、抗病毒等作用。据文献报道,已应用于临床的金银花复方药物制剂达数十种,金银花的主要有效成分为绿原酸。药品的质量可控性是药品的基本属性,是药品的安全性和有效性的基础。质量标准是监控药品质量的重要手段,是质量可控性的具体体现。经查阅大量文献,多种金银花复方药物制剂的质量控制方法已有报道,但其中仍有一些金银花复方制剂未见质量控制
2、方法的报道或只有以其中一种或两种有效成分作为质量控制指标的报道,不能全面评价药品的内在质量,还有一些质量控制方法需要进一步改进和完善。本课题选定三种金银花复方药物制剂银翘解毒片、热毒宁注射液和鼻炎滴剂,以药物中主要有效成分作为质量控制指标,采用反相高效液相色谱法,对药物的质量控制进行了较为系统的方法学研究。建立了同时测定银翘解毒片中绿原酸、连翘苷和甘草酸的反相高效液相色谱法;热毒宁注射液中绿原酸和栀子苷的反相高效液相色谱法;鼻炎滴剂中黄芩苷和盐酸麻黄碱、绿原酸和黄芩苷及单一成分绿原酸的反相高效液相色谱法。所建立的方法专属性强,灵敏度高、精密度和稳定性良好,结果准确可靠,可做为上述金银花复方制剂
3、的质量控制方法。第一部分三波长检测梯度洗脱RP-HPLC同时测定银翘解毒片中绿原酸、连翘苷和甘草酸的含量目的:建立反相高效液相色谱法同时分离测定银翘解毒片中绿原酸、连翘苷和甘草酸的含量。为银翘解毒片提供一种以3种有效成分作为质量控制指标的分析方法。方法:(1)提取方法:比较不同提取溶剂及提取时间对银翘解毒片有效成分的提取效率,选择提取成分多,提取效率高的提取方法。(2)反相高效液相色谱条件的优化:选择合适的固定相,调整流动相的组成、配比、流速,设计梯度洗脱程序及检测方法等色谱条件,以使绿原酸、连翘苷和甘草酸色谱峰与其它峰完全分离。(3)系统适用性实验:在已确定的色谱条件下,考察3种待测成分色谱
4、峰的分离度、理论塔板数、对称因子是否符合要求。(4)制备标准曲线;进行最低检测限实验、精密度实验、稳定性实验和重复性实验。(5)加样回收率实验。(6)样品含量测定。结果:(1)提取方法:精密称取银翘解毒片样品,用50%甲醇超声提取30min,使各有效成分在此提取条件下达到最大的提取效率。(2)反相高效液相色谱条件:采用DiamonsilTMC18色谱柱(250 mm4.6 mm, 5m),以乙腈(A)-0.1%磷酸水溶液(B)为流动相进行梯度洗脱,梯度洗脱程序为:05 min,17 % A;510 min,17 %30 % A;1015 min,30 % A;1516 min,30 %43.5
5、 % A;1628 min,43.5 % A;2829 min,43.5 %17 % A(A+B=100 %)。流速为1.0 ml/min ;柱温为30;三波长检测,绿原酸检测波长1为326 nm,连翘苷检测波长2为228 nm,甘草酸检测波长3为252 nm。(3)系统适用性实验:在此色谱条件下,3种指标成分色谱峰的理论塔板数均大于5000,与相邻色谱峰的分离度均大于2。(4)专属性实验:在上述色谱条件下进样,阴性对照样品的色谱图与样品色谱图进行比较,阴性对照样品无干扰。(5)标准曲线的制备:绿原酸、连翘苷和甘草酸进样量分别在0.3962.77g、0.5934.15g和0.5583.91g范
6、围内呈良好的线性关系,相关系数分别为0.9995、0.9999和0.9999。(6)最低检测限实验:绿原酸、连翘苷和甘草酸的最低检测限分别为0.39、0.65、0.61g/ml。(7)精密度实验:三种有效成分峰面积的RSD分别为0.51 %、0.24 %、0.33 %。(8)稳定性实验:三种有效成分峰面积的RSD分别为1.4%、1.6%、1.8%,样品在24h内稳定。(9)重现性实验:以三种有效成分的峰面积计算所得重现性实验的RSD分别为1.8%、1.2%、1.5%(10)加样回收率实验:绿原酸、连翘苷和甘草酸的平均加样回收率分别为98.2%、97.9 %和96.2 %,RSD分别为1.4 %
7、、1.5 %和1.2 %。(11)含量测定:在所建立的色谱条件下,对不同批次、不同生产厂家的银翘解毒片中有效成分的含量进行同时测定,由外标法计算样品中有效成分的含量。结论:采用三波长检测梯度洗脱RP-HPLC同时测定银翘解毒片中绿原酸、连翘苷和甘草酸的含量。该法具有分析时间短、线性范围宽、柱效高、专属性强,精密度和准确度良好等优点,可用于银翘解毒片的质量控制。第二部分RP-HPLC测定热毒宁注射液中绿原酸和栀子苷的含量目的:研究建立反相高效液相色谱法同时测定热毒宁注射液中绿原酸和栀子苷的含量。方法:采用DiamonsilTM C18色谱柱(250 mm4.6 mm,5m),流动相:乙腈-0.4
8、%磷酸水溶液(10:90),流速1.0 ml/min,柱温30,检测波长240 nm。结果:在上述色谱条件下,绿原酸和栀子苷的色谱峰与相邻峰的分离度均大于2,理论塔板数以绿原酸计为9280,以栀子苷计为10455。绿原酸和栀子苷在0.1941.36g和0.1621.14g范围内线性关系良好,相关系数分别为0.9998和1.000。绿原酸和栀子苷的平均回收率分别为99.0%和101.5 %,RSD分别为1.6 %和1.5 %。结论:采用RP-HPLC法可同时测定热毒宁注射液中绿原酸和栀子苷的含量,该方法具有分析时间短,线性范围宽,柱效高,制剂辅料无干扰,专属性好等优点,可用于热毒宁注射液的质量控
9、制。第三部分鼻炎滴剂的质量控制方法研究(一)双波长检测梯度洗脱RP-HPLC同时测定鼻炎滴剂中黄芩苷和盐酸麻黄碱的含量目的:建立反相高效液相色谱法同时测定鼻炎滴剂中黄芩苷和盐酸麻黄碱的含量。方法:采用DiamonsilTMC18色谱柱(250mm4.6mm, 5m ),以乙腈(A)-1.0%磷酸溶液(含0.4%三乙胺)(B)为流动相进行梯度洗脱,梯度洗脱程序为:03 min,12% A;312 min,12%27% A;1225 min,27% A;2526 min,27%12% A(A+B=100 %);梯度洗脱周期为35分钟,流速1.0 ml/min,检测波长:07 min,210 nm;
10、735 min,278 nm。柱温30。结果:在此色谱条件下,黄芩苷和盐酸麻黄碱的理论塔板数均大于5000,与相邻色谱峰的分离度均大于2.0。黄芩苷和盐酸麻黄碱在0.635 3.02g和0.203 0.966g范围内线性关系良好,相关系数分别为0.9999和0.9997。黄芩苷和盐酸麻黄碱的平均回收率分别为101.5%和101.0%,RSD < 1%。结论:采用反相高效液相色谱法可同时测定鼻炎滴剂中黄芩苷和盐酸麻黄碱的含量。方法专属性好,灵敏度高,结果准确可靠,可用于鼻炎滴剂的质量控制。(二)RP-HPLC测定鼻炎滴剂中绿原酸和黄芩苷的含量目的:建立反相高效液相色谱法同时测定鼻炎滴剂中绿
11、原酸和黄芩苷的含量。方法:采用DiamonsilTMC18色谱柱(250mm4.6mm ,5m ),以甲醇-水-磷酸(52:48:0.1)为流动相,检测波长320 nm,流速1.0 ml/min,柱温30。结果:绿原酸和黄芩苷的浓度分别在4.3034.4g/ml和15.5109g/ml范围内线性关系良好,相关系数分别为1.000和0.9998。绿原酸和黄芩苷的平均回收率分别为102.0%和100.7%,RSD < 2%。结论:采用RP-HPLC法可同时测定鼻炎滴剂中绿原酸和黄芩苷的含量。此法专属性好,灵敏度高,操作简便,对鼻炎滴剂的质量控制有一定意义。(三)RP-HPLC测定鼻炎滴剂中绿
12、原酸的含量目的:建立反相高效液相色谱法测定鼻炎滴剂中绿原酸的含量。为鼻炎滴剂的质量控制提供一种快速分析方法。方法:采用DiamonsilTMC18色谱柱(250mm4.6mm,5m ),以甲醇-水-冰醋酸(25:75:1)为流动相,流速为1.0 ml/min,检测波长328nm。结果:在上述色谱条件下,绿原酸的色谱峰与相邻峰的分离度大于1.7,理论塔板数n >5000,绿原酸在4.3430.4g/mL范围内线性关系良好,相关系数0.9997。绿原酸的平均回收率100.5%,RSD=0.86%。结论:采用反相高效液相色谱法,鼻炎滴剂中绿原酸的峰形对称,柱效高,分析时间短,方法灵敏,操作简便
13、,结果准确可靠,可用于鼻炎滴剂中绿原酸的快速测定。【英文摘要】 Honeysuckle is a common Chinese herb. The modern pharmacology research shows that honeysuckle has the functions of anti-bacterial, anti-viral, ect. It has been reported that tens of honeysuckle compound pharmaceutical preparations have been applied to clinic,and the c
14、hlorogenic acid is the main active component of honeysuckle.Quality control for drug is an essential attribute and the basis for security and validity of medicine. Quality standard is a main means to monitor drug quality and also a detailed reflection of quality control. Through looking up large num
15、ber of literatures, it can be found that quality control methods of many honeysuckle compound pharmaceutical preparations have been reported yet. However, the quality control of some honeysuckle compound pharmaceutical preparations has not been reported, or only one or two active components for qual
16、ity control has been reported. It can not totally evaluate the quality of these drugs. Furthermore, some quality control methods reported before need to be improved too.This research selected three kinds of honeysuckle compound pharmaceutical preparations to study, such as Yinqiao Jiedu tablet, Redu
17、ning injection and coryza dripping solution. The main active components were used as the quality control indices. And systematical methodology research of drug quality control based on RP-HPLC method was studied. The author established the assay of RP-HPLC to determine the chlorogenic acid,forsythin
18、 and glycyrrhetic acid in Yinqiao Jiedu tablet,the assay of RP-HPLC to determine the chlorogenic acid and gardenin in Reduning injection,and the assay of RP-HPLC to determine the baicalin and ephedrine hydrochloride, the chlorogenic acid and baicalin, and the only chlorogenic acid in coryza dripping
19、 solution. The method built in this research has several advantages, such as strong specificity, high sensitivity, good accuracy and stability. Therefore, it can be used for the quality control of the above mentioned honeysuckle compound pharmaceutical preparations.Part OneSimultaneous Determination
20、 of Chlorogenic Acid, Forsythin and Glycyrrhetic Acid in Yinqiao Jiedu Tablet by RP-HPLC with Three-Wavelength Detection and Gradient ElutionObjective:To establish a RP-HPLC method for separation and determination of chlorogenic acid, forsythin and glycyr- rhetic acid in Yinqiao Jiedu tablet. A anal
21、ysis method can be provided for the quality control of Yinqiao Jiedu tablet by using three kinds of active ingredients as indicators. Methods: (1) Optimization of the extraction method: different extraction solvents and extration time were tested for optimization of the extraction efficacy of Yinqia
22、o Jiedu tablet. The most efficient extration method was selected for the extraction of the major chemical components of Yinqiao Jiedu tablet. (2) Optimization of the chromatographic conditions: chromatographic conditions such as stationary phase, mobile phase systems, velocity, gradient programs and
23、 detection method were tested for optimization of the chromatographic conditions,so that the chromatographic peaks of chlorogenic acid,forsythin and glycyrrhetic acid were completely separated with other peaks. (3) System suitability test: under the above conditions, the resolution, theoretical plat
24、e number and symmetry factor of the three chemical components were studied. (4) Constructing the calibration curve; testing the limits of detection (LOD), precision, stability and reproducibility. (5) Testing the recovery. (6) Determinating the samples.Results: (1) The extraction method: an accurate
25、ly weighted powder of Yinqiao Jiedu tablet was extracted with 50% methanol by a sonifier at room temperature for 30 min. The extraction method for the major chemical components of Yinqiao Jiedu tablet was efficient. (2) The chromatographic condition: a DiamonsilTM C18 column(250 mm4.6 mm , 5m) was u
26、sed throughout. The mobile phase consisted of acetonitrile (A) and 0.1% phosphoric acid (v/v, B). The gradient program for quantitative analysis was: 05 min, 17 % A; 510 min, 17 %30 % A;1015 min,30 % A;1516 min,30 %43.5 % A;1628 min,43.5 % A;2829 min,43.5 %17 % A(A+B=100 %).The flow rate was 1.0 ml/
27、min and the column temperature was 30. The detection wavelength was programmed as: chlorogenic acid at 326 nm, forsythin at 228 nm and glycyrrhetic acid at 252 nm. (3) System suitability test:under the above conditions,the peaks of chlorogenic acid,forsythin and glycyrrhetic acid were separated well
28、 with the resolution of more than 2, and the theoretical plate number of each was larger than 5000. (4) Specialization: negative control samples were analyzed and the results were compared with that of the sample preparation.The results showed that the chemical components were determined without the
29、 interference of other chemical components originated from the coexisting herbs. (5) The calibration curve:the linearities of chlorogenic acid, forsythin and glycyrrhetic acid were respectively good in the range of 0.3962.77g(r=0.9995), 0.5934.15g(r=0.9999)and 0.558 3.91g (r=0.9999). (6) The limits
30、of detection (LOD): the LOD of chlorogenic acid,forsythin and glycyrrhetic acid were 0.39, 0.65 and 0.61g/ml. (7) Precision: the RSD% of the peak areas were 0.51%, 0.24%, 0.33%, respectively. (8) Stability:the RSD% of the peak areas were 1.4%, 1.6%, 1.8%, respectively. The sample solution was stable
31、 in 24 hours. (9) Reproducibility: the RSD% of the peak areas were 1.8%, 1.2%, 1.5%, respectively. (10) Recovery test: the average recovery rates of chlorogenic acid, forsythin and glycyrrhetic acid were 98.2%, 97.9%, 96.3% and the RSD% were 1.4%, 1.5%, 1.2%, respectively. (11) The established metho
32、d was applied for determination of the major chemical components in Yinqiao Jiedu tablet of different production batches and different manufactures. The content of each chemical component was calculated according to external standard method.Conclusions: Chlorogenic acid,forsythin and glycyrrhetic ac
33、id can be determined simultaneously by a RP-HPLC with three-wavelength detection and gradient elution. This method has several advantages, such as short analysis time, large linear range, high column efficiency, good specialization, good precision and accuracy. Therefore, it can be used for the qual
34、ity control of Yinqiao Jiedu tablet.Part Two Determination of Chlorogenic Acid and Gardenin in Reduning Injection by RP-HPLCObjective: To establish a RP-HPLC method for deter- mination of chlorogenic acid and gardenin in Reduning injection.Methods: A DiamonsilTM C18 column (2504.6mm,5m) was used thr
35、oughout.The mobile phase consisted of acetonitrile and 0.4% phosphoric acid (10:90); the flow rate was 1.0 ml/min; the column temperature was 30; the detection wavelength was 240nm.Results: Under the above conditions, the peaks of chlorogenic acid and gardenin were separated well with the resolution
36、 of more than 2. To the chlorogenic acid , theoretical plate number was 9280, and to the gardenin, theoretical plate number was 10455. The linearity of chlorogenic acid was good in the range of 0.1941.36g (r=0.9998) with average recovery rate reaching 99.0% (RSD=1.6%). The linearity of gardenin was
37、good in the range of 0.1621.14g (r=1.000) with the average recovery rate reaching 101.5 % (RSD=1.5 %).Conclusions: Chlorogenic acid and gardenin can be determined simultaneously by a RP-HPLC method. This method has several advantages, such as short analysis time, large linear range, high column effi
38、ciency, good specialization and no interference of the excipient in the Reduning injection. Therefore, it can be used for the quality control of Reduning injection. Part Three(1) Determination of Baicalin and Ephedrine Hydrochloride in Coryza Dripping Solution by RP-HPLC with double-wavelength detec
39、tion and Gradient elutionObjective: To establish a RP-HPLC method for determi -nation of baicalin and ephedrine hydrochloride in Coryza Dripping solution .Methods: A DiamonsilTM C18 column(2504.6mm,5m) was used throughout. The mobile phase consisted of acetonitrile (A) and 1.0% phosphoric acid (cont
40、ented 0.4% triethylamine ) (B). The gradient program for quantitative analysis was: 03 min,12% A;312min, 12%27% A;1225min,27% A;2526 min,27%12% A(A+B=100 %). The time of gradient elution was 35min and the flow rate was 1.0 ml/min. The detection wavelength was programmed as: 07min, 210nm; 735min, 278
41、nm. The column temperature was 30.Results: Under the above conditions, the peaks of baicalin and ephedrine hydrochloride were separated well with the resolution of more than 2.0, and the theoretical plate number of each was larger than 5000. The linearity of baicalin was good in the range of 0.6353.
42、02g (r=0.9999) with average recovery rate reaching 101.5%. The linearity of ephedrine hydrochloride was good in the range of 0.2030.966g (r=0.9997) with average recovery rate reaching 101.0%. The RSD% of the average recovery rate for baicalin and ephedrine hydrochloride were not more than 1.Conclusi
43、ons: Baicalin and ephedrine hydrochloride can be determined simultaneously by a RP-HPLC method. The method is special, sensitive and accurate. It can be used for the quality control of Coryza Dripping solution.(2) Determination of Chlorogenic Acid and Baicalin in Coryza Dripping Solution by RP-HPLCO
44、bjective: To establish a RP-HPLC method for determination of chlorogenic acid and baicalin in Coryza Dripping solution.Methods: A DiamonsilTM C18 column(2504.6mm,5m) was used throughout; the mobile phase consisted of methanol-water -phosphoric acid (52:48:0.1); the flow rate was 1.0 ml/min; the dete
45、ction wavelength was 320nm; the column temperature was 30.Results: The linearity of chlorogenic acid was good in the range of 4.3034.4g/ml (r=1.000) with average recovery rate reaching 102.0%. The linearity of baicalin was good in the range of 15.5108.6g/ml (r=0.9998) with average recovery rate reac
46、hing 100.7%. The RSD% of the average recovery rate for chlorogenic acid and baicalin were not more than 2.Conclusions: Chlorogenic acid and baicalin can be determined simultaneously by a RP-HPLC method. The method is special, sensitive and simple. It is with a certain significance for the quality co
47、ntrol of Coryza Dripping solution.(3) Determination of Chlorogenic Acid in Coryza Dripping Solution by RP-HPLCObjective: To establish a RP-HPLC method for determination of chlorogenic acid in Coryza Dripping solution. A rapid analysis method can be provided for the quality control of Coryza Dripping
48、 solution.Methods: A DiamonsilTM C18 column(2504.6mm,5m) was used throughout; the mobile phase consisted of methanolwater -acetic acid (25:75:1); the flow rate was 1.0 ml/min; the detection wavelength was 328nm.Results: Under the above conditions, the peak of chlorogenic acid was separated well with
49、 the resolution of more than 1.7,and the theoretical plate number was larger than 5000.The linearity of chlorogenic acid was good in the range of 4.3430.4g/ml (r=0.9995), and the average recovery rate was 100.5% (RSD=0.86 % ). Conclusions: The symmetrical peak shape, high column efficiency and short analysis time can be maked by a RP-HPLC method for the determination of chlorogenic acid in coryza dripping solution. The method is sensitive, simple and accurate. It can be used for the rapid determ
