抗脑衰胶囊的质量控制方法研究
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摘要
抗脑衰胶囊由人参、制何首乌、党参、黄芪、熟地黄、山药、丹参、黄芩、枸杞子、白芍、远志、茯神、石菖蒲、葛根、酸枣仁、麦冬、龙骨(粉)、香附、菊花等19味药材组成。用于因肾精不足、肝气血亏所引起的精神疲惫、失眠多梦、头晕目眩、体乏无力、记忆力减退。药理实验表明本品对神经衰弱、记忆力减退、老年性痴呆症、血管障碍型痴呆症、脑萎缩、脑梗塞、脑血栓后遗症、高血压所致脑循环不全,以及脑外伤所致的记忆力障碍、体虚乏力、厌食、失眠及大脑发育不全等症有独特的疗效。
本研究采用反相高效液相色谱法建立了同时测定抗脑衰胶囊中七种有效成分的分析方法。建立了不同生产批次,不同生产厂家抗脑衰胶囊的HPLC指纹图谱,并对抗脑衰胶囊中主要色谱特征峰进行了分析,确定了主要色谱特征峰在抗脑衰胶囊组成药材中的归属,为阐明中药复方制剂的药效物质基础提供了有效的分析方法。方法专属性强,灵敏快速、结果准确可靠,精密度和稳定性良好。本研究对抗脑衰胶囊进行了综合的质量控制与评价,为保证中药复方制剂的质量提供了良好的质量控制与评价方法。
第一部分
抗脑衰胶囊中有效成分的多组分同时测定
目的:用反相高效液相色谱法建立中药复方制剂抗脑衰胶囊中有效成分的多组分同时测定方法,并将建立的方法用于不同批次,不同生产厂家的抗脑衰胶囊中有效成分的含量测定,并对含量测定结果加以比较,为科学有效的评价并控制抗脑衰胶囊的质量提供新的方法。
方法:(1)提取方法:比较了不同提取方式、提取溶剂及提取时间对抗脑衰胶囊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)高效液相色谱条件的优化:选用合适的色谱柱及检测方法,优化流动相的组成、配比、梯度洗脱程序、柱温等影响因素,确定最佳的色谱条件。(3)系统适用性试验:计算抗脑衰胶囊中有效成分黄芩苷的理论塔板数,及各有效成分与相邻峰的分离度。(4)标准曲线的制备:配制一系列浓度的混合对照品溶液,进样分析测定峰面积;以峰面积为纵坐标,浓度为横坐标,绘制标准曲线,经回归处理得各标准曲线方程。(5)定量限以及检测限试验:将混合对照品溶液逐步稀释并进行测定,以信噪比S/N≥10时确定为定量限,以信噪比S/N≥3时确定为检测限。(6)仪器精密度试验:取同一对照品溶液,重复进样6次,以所测有效成分的色谱保留时间以及色谱峰面积计算仪器精密度。(7)日内及日间精密度试验:取同一批次样品,在同一天精密称取6份,平行制备样品溶液,并对样品溶液进样进行分析,以所测有效成分的保留时间和峰面积计算日内精密度;取同一批次样品,在同一天精密称取2份,平行制备样品溶液,对样品溶液进样进行分析,连续测定3天,以所测有效成分的保留时间和峰面积计算日间精密度。(8)稳定性试验:取同一样品溶液置于室温,并分别于0、2、4、6、8、24、48h进样分析并进行有效成分的含量测定。(9)加样回收率试验:取同一批次并已知含量的样品,精密称定9份,分别精密加入高、中、低3种浓度的对照品混合物,按照供试品溶液制备方法进行制备,对样品溶液进样进行分析,并对有效成分的含量进行测定并计算加样回收率。(10)专属性试验:在上述色谱条件下,对阴性对照样品进样分析,将所得色谱图与样品色谱图进行比较,以排除抗脑衰胶囊中其它组成药材中化学物质的干扰。(11)抗脑衰胶囊中有效成分的含量测定:对不同批次,不同生产厂家的抗脑衰胶囊中有效成分的含量进行测定。
结果:抗脑衰胶囊中有效成分的多组分同时测定方法(1)提取方法:精密称取恒温干燥后的抗脑衰胶囊内容物约0.1g,用50%甲醇超声提取30 min,使各有效成分在提取方式相同的条件下同时达到最大的提取效率,且提取方法稳定。(2)HPLC色谱条件:采用Waters SunfireTM-C18色谱柱(150 mm×4.6 mm I.D., 5μm),以乙腈(A)-0.05%磷酸(B)为流动相进行梯度洗脱,梯度洗脱程序为:0~15 min,12%~20% A;15~30 min,20%~45% A;30~40 min,45%~75% A;40~50 min,75%~100% A;50~55 min,100%~12% A。采用Variable Wavelength Detector(VWD)紫外检测器以波长转换的方式进行检测,VWD检测波长时间变化程序为:0.00~6.10 min,320 nm;6.10~8.50 min,248 nm;8.50~15.00min,230 nm;15.00~25.00 min,320 nm;25.00~27.10 min,275 nm;27.10~32.50 min,286 nm;32.50~50.00 min,275 nm。(3)系统适用性试验:在此色谱条件下,以黄芩苷计算的理论塔板数大于6000,且其与其相邻色谱峰的分离度均大于1.5。(4)标准曲线的制备:对混合对照品溶液进样分析,以最小二乘法对峰面积和浓度进行线性回归得到各组分的标准曲线方程,其线性关系均符合要求。(5)定量限以及检测限试验:成分1~7的检测限分别为4.90、16.40、13.30、4.26、1.98、3.88、1.04 ng/ml;成分1~7的定量限分别为19.58、65.68、6.65、17.03、7.92、7.76、2.08 ng/ml。(6)仪器精密度试验:以有效成分的保留时间计算所得仪器精密度的RSD%均小于0.24;以有效成分的峰面积计算所得仪器精密度的RSD%均小于0.85。(7)日内及日间精密度试验:以有效成分的保留时间计算所得日间精密度的RSD%均小于0.37,以有效成分的峰面积计算所得日间精密度的RSD%均小于3.03;以有效成分的保留时间计算所得日内精密度的RSD%均小于0.46,以有效成分的峰面积计算所得日内精密度的RSD%均小于2.94(8)稳定性试验:以有效成分1~7的保留时间计算所得稳定性试验的RSD%均小于1.35,以有效成分1~7的峰面积计算所得稳定性试验的RSD%均小于1.11,样品在48h内稳定。(9)加样回收率试验:高、中、低3种浓度的加样回收率都在90.0%~108.3%之间,RSD%小于4.72。(10)专属性试验:在上述色谱条件下,对阴性对照样品进样分析,将所得色谱图与样品色谱图进行比较,阴性样品溶液在样品有效成分出峰位置没有干扰。(11)抗脑衰胶囊中有效成分的含量测定:依据所建立的色谱条件对不同批次,不同生产厂家的抗脑衰胶囊中有效成分的含量进行同时测定,由标准曲线计算得到各个样品中有效成分的含量。
结论:本研究采用反相高效液相色谱法建立了抗脑衰胶囊中有效成分的多组分同时测定方法。采用波长转换的方式以各待测组分的最大吸收波长作为相应组分的检测波长,最大程度的发挥了紫外检测器的检测效能,提高了检测灵敏度。方法专属性强,灵敏快速、结果准确可靠,精密度和稳定性良好。本研究建立的方法可以用于抗脑衰胶囊中多个有效成分含量的同时测定,为保证中药复方制剂的质量提供了良好的质量控制方法。
第二部分
抗脑衰胶囊的高效液相色谱指纹图谱研究
目的:用反相高效液相色谱法建立中药复方制剂抗脑衰胶囊的HPLC指纹图谱,将所建立的方法用于建立不同批次,不同生产厂家的抗脑衰胶囊的HPLC指纹图谱,并对指纹图谱结果加以比较,为科学有效的评价并控制抗脑衰胶囊的质量提供新的方法。同时将建立的方法用于抗脑衰胶囊组成药材的HPLC指纹图谱的建立,并对抗脑衰胶囊中主要色谱特征峰在组成药材中的来源进行归属,为阐明抗脑衰胶囊的药效物质提供科学有效的方法。
方法:(1)提取方法:比较了不同提取方式、提取溶剂及提取时间对抗脑衰胶囊有效成分的提取效果,选择提取成分多,提取率高的提取条件。(2)HPLC色谱指纹图谱条件的优化:选用合适的色谱柱及紫外检测器检测波长,优化流动相的组成、配比、梯度洗脱程序,柱温等影响因素,确定最佳的HPLC指纹图谱条件。(3)系统适用性试验:计算抗脑衰胶囊中有效成分黄芩苷的理论塔板数,及其与相邻色谱峰的分离度。(4)仪器精密度试验:取同一供试品溶液,重复进样6次,以高效液相色谱图中主要成分的色谱保留时间以及色谱峰面积计算仪器精密度。(5)重复性试验:取同一批次样品,精密称取6份,平行制备样品溶液,对样品溶液进样分析,根据高效液相色谱图中主要成分的色谱保留时间以及色谱峰面积对重复性进行评价。(6)稳定性试验:取同一样品溶液置于室温,并分别于0、2、4、6、8、24、48h对样品溶液进行分析,根据高效液相色谱图中主要成分的色谱保留时间以及色谱峰面积对稳定性进行评价。(7)专属性试验:在上述色谱条件下对空白溶液、样品溶液进样分析并进行对比,以排除空白干扰。(8)抗脑衰胶囊指纹图谱分析:对不同批次,不同生产厂家的抗脑衰胶囊以及抗脑衰胶囊组成药材进行样品制备,并进行高效液相色谱指纹图谱分析。(9)指纹图谱数据分析:采用指纹图谱相似度计算软件对所得高效液相色谱指纹图谱数据进行分析和比较,使得数据结果可以有效的反映不同生产批次以及不同生产厂家的抗脑衰胶囊质量的差异。并对所得抗脑衰胶囊以及抗脑衰胶囊组成药材的高效液相色谱指纹图谱进行分析,对抗脑衰胶囊中主要色谱特征峰在组成药材中的来源进行归属。
结果:抗脑衰胶囊的HPLC指纹图谱研究(1)提取方法:精密称取恒温干燥后的抗脑衰胶囊样品,用50%甲醇超声提取30min,各主要成分在提取方式相同的条件下同时达到最大的提取效率,且提取方法稳定。(2)HPLC色谱指纹图谱条件:采用Waters SunfireTM-C18色谱柱(150 mm×4.6 mm I.D., 5μm),以乙腈(A)-0.05%磷酸(B)为流动相进行梯度洗脱,梯度洗脱程序为:0~30 min,3%~30% A;30~40 min,30%~65% A;40~50 min,65%~100% A;50~55 min,100%~3% A。采用Photodiode Array Detector(PDA)紫外检测器进行检测,检测波长为200 nm,波长扫描范围为:200~400 nm。(3)系统适用性试验:在此色谱条件下,以黄芩苷计算理论塔板数大于6000且与其相邻色谱峰的分离度均大于1.5。(4)仪器精密度试验:以主要成分的色谱保留时间计算所得仪器精密度的RSD%均小于0.07;以主要成分的色谱峰面积计算所得仪器精密度的RSD%均小于1.59。(5)重复性试验:以主要成分的色谱保留时间计算所得日间精密度的RSD%均小于1.46,以主要成分的色谱峰面积计算所得日间精密度的RSD%均小于3.96。(6)稳定性试验:以主要成分的色谱保留时间计算所得稳定性试验的RSD%均小于1.35,以主要成分的色谱峰面积计算所得稳定性试验的RSD%均小于1.11,样品在48h内稳定。(7)专属性试验:供试品溶液中各色谱峰分离较好,空白对照溶液无杂质干扰。(8)抗脑衰胶囊指纹图谱分析:对不同批次,不同生产厂家的抗脑衰胶囊以及抗脑衰胶囊组成药材进行样品制备,并依据所建立的HPLC色谱指纹图谱条件进行色谱指纹图谱分析。(9)指纹图谱数据分析:采用指纹图谱相似度软件对所得高效液相色谱指纹图谱数据进行分析和比较,所得数据结果可以有效的反映不同生产批次以及不同生产厂家的抗脑衰胶囊质量的差异。对所得抗脑衰胶囊以及抗脑衰胶囊组成药材的高效液相色谱指纹图谱进行分析并对抗脑衰胶囊中主要色谱特征峰在组成药材中的来源进行归属,确定了主要色谱特征峰在抗脑衰胶囊组成药材中的来源。
结论:本研究采用反相高效液相色谱法建立了不同生产批次,不同生产厂家抗脑衰胶囊的HPLC指纹图谱,并采用指纹图谱相似度计算软件对不同生产批次,不同生产厂家的抗脑衰胶囊的HPLC指纹图谱进行了分析和比较,对抗脑衰胶囊的质量进行了综合评价。同时建立了抗脑衰胶囊组成药材的HPLC指纹图谱,并对抗脑衰胶囊中主要色谱特征峰进行分析,确定了主要色谱特征峰在抗脑衰胶囊组成药材中的归属,为阐明中药复方制剂的药效物质基础提供了有效的方法。方法精密度和稳定性良好,所得数据结果可以有效的反映不同生产批次以及不同生产厂家的抗脑衰胶囊的质量差异,为保证中药复方制剂的质量提供了良好的质量控制方法。
Kangnaoshuai capsule (KNS) is an over-the-counter fufang preparation consisted of 19 herbs and the herbs are Radix Polygoni Multiflori, Radix rehmanniae preparata, Fructus lycii, Radix Ginseng, Radix Astragali, Rhizoma Dioscoreae, Radix Salviae Miltiorrhiae, Radix Paeoniae Alba, Rhizome Acori Talarinowii, radix polygalae, Semen ziziphi spinosae, Rhizoma Cyperi, Radix Scutellariae, Radix codonopsis, Indian bread with host wood, Radix ophiopogonis, Radix Puerariae, Flos Chrysanthemi. It has been used for the treatment of neurolysis, insomnia and dreamful sleep, neurastheria, memory deterioration. It has also been reported to be beneficial in the treatments for age-related dementia, encephalatrophy, cerebral infarction, cerebral hypoplasia, sequelae of cerebral thrombosis, brain injuries caused by stroke, cerebral trauma and encephalitis.
A reversed phase high performance liquid chromatography method for simultaneous determination of the seven chemical components in the fufang preparation of kangnaoshuai capsule was developed. High performance chromatographic fingerprints of the constituent herbs and kangnaoshuai capsule at different production batches and different manufactures were constructed. The chemical characteristic of kangnaoshuai capsule was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components. The developed method was precise and accurate and the method can be effectively applied for the quanlity control and evaluation of kangnaoshuai capsule at different production batches and different manufactures. This chemical evidence based analysis can also provide valuable information for better understanding about the relationship between the chemical characteristic and the therapeutic efficacy of the kangnaoshuai capsule. Part one
Simultaneous determination of the major chemical components in kangnaoshuai capsule by HPLC
Objective: To establish a reversed phase high performance liquid chromatography method for simultaneous determination of the major chemical components in the fufang preparation of kangnaoshuai capsule. The established method can be applied for determination of the key chemical components in kangnaoshuai capsule of different production batches and different manufactures. The results of the quantitative analyses were compared for quality control and evaluation of Kangnaoshuai capsule.
Methods: (1) Optimization of the extraction method: different extraction methods, extraction solvents of different solvent composition and extration time were tested for optimization of the extraction efficacy of kangnaoshuai capsule (KNS). The most efficacy extration method was selected for the extraction of the major chemical components of KNS. (2) Optimization of the chromatographic conditions: chromatographic conditions such as analytical columns, detection method, mobile phase systems, gradient programs and column temperatures were tested for optimization of the chromatographic conditions. (3) System suitability test: Under the above conditions, the resolution of the major chemical components and the theoretical plate number the peak of baicalin was calculated. (4) A series of seven calibration solutions were prepared by diluting the stock standard solution to appropriate concentrations for construction of the regression equations. The calibration curve was constructed by plotting the peak area of each key chemical component against its concentration. (5) The test of LOD and LOQ: the limits of detection (LOD) and quantification (LOQ) were determined at a signal-to-noise ratio of about 3 and 10 by analyzing the diluted standard solution. (6) The test of injection precision: The injection precision was evaluated by analyzing the six repeated injection of the standard solution. The retention times and the peak areas of the seven chemical components were analyzed for evaluation of the precision. (7) The test of the intra-day and inter-day precision: intra-day precision was evaluated by analyzing six replications prepared from the KNS sample within a day. Duplicate copies of the KNS sample were prepared within a day for the consecutive three days, and inter-day precision was evaluated by analyzing the six sample solutions of three days. (8) The test of stability: stability was tested by determining the same stock standard solution stored at room temperature at different time of 0, 2, 4, 6, 8, 24, 48 h. (9) The test of recovery: Accurately weighted amounts of each reference compound at three levels were mixed with a fixed amount of the contents of KNS sample. Three replications at each level were extracted and analyzed to evaluate the accuracy of the developed method. (10) The test of selectivity: negative control samples were analyzed to evaluate the selectivity of the developed method. The results were compared to confirm that the chemical components were determined without the interference of other chemical components originated from the coexisting herbs. (11) The established method was applied for determination of the seven chemical components in kangnaoshuai capsule of different production batches and different manufactures.
Results: The simultaneous determination of the seven chemical components in kangnaoshuai capsule (1) The extraction method: An accurately weighted dried powder of the contents of KNS capsule was extracted with 50% methanol by a sonifier at room temperature for 30 min. The extraction method of the major chemical components of KNS was efficacy and stable. (2) The chromatographic condition: A Waters SunfireTM-C18 column (150 mm×4.6 mm I.D., 5μm) was used throughout. The mobile phase consisted of acetonitrile (A) and 0.1% aqueous phosphoric acid (v/v, B). The gradient program for quantitative analysis was: 0~15 min, 12%~20% A; 15~30 min, 20%~45% A; 30~40 min, 45%~75% A; 40~50 min, 75%~100% A, 50~55 min, 100%~12% A. The detection wavelength was programmed as: 0.00~6.10 min, 320 nm; 6.10~8.50 min, 248 nm; 8.50~15.00 min, 230 nm; 15.00~25.00 min, 320 nm; 25.00~27.10 min, 275 nm; 27.10~32.50 min, 286 nm; 32.50~50.00 min, 275 nm. (3) System suitability test: Under the above conditions, the peak of baicalin was separated well with the resolution of more than 1.2 and about 6000 of theoretical plate number. (4) The calibration curve was constructed by plotting the peak area of each key chemical component against its concentration for construction of the regression equations. All calibration curves showed good linearity within the test ranges. (5) The limits of LOD and LOQ: The limits of LOQ of the seven chemical components were 4.90、16.40、13.30、4.26、1.98、3.88、1.04 ng/ml;The limits of LOD of the seven chemical components were 19.58、65.68、6.65、17.03、7.92、7.76、2.08 ng/ml. (6) Injection precision: the RSD% of the retention times for the tests of injection precision was not more than 0.24; The RSD% of the peak areas for the tests of injection precision was not more than 0.85. (7) Intra-day and inter-day precision: the RSD% of the retention times for the tests of intra-day precision was not more than 0.37, the RSD% of the peak areas for the tests of intra-day precision was not more than 3.03;the RSD% of the retention times for the tests of inter-day precision was not more than 0.46, the RSD% of the peak areas for the tests of inter-day precision was not more than 2.94. (8) Stability: the RSD% of the retention times for the tests of injection precision was not more than 1.35; The RSD% of the peak areas for the tests of injection precision was not more than 1.11. The sample solution was stable within 48 hours. (9) Recovery test: the results of the recovery test at three levels were from 90.0%~108.3% and the RSD% were not more than 4.72. (10) Selectivity: 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 interference of other chemical components originated from the coexisting herbs. (11) The established method was applied for determination of the major chemical components in kangnaoshuai capsule of different production batches and different manufactures. The content of each chemical component was calculated according to the established calibration curves.
Conclusion: The reversed phase high performance liquid chromatography method for the simultaneous determination of the major chemical components in the fufang preparation of kangnaoshuai capsule was established. The UV detector was programmed to monitor at each component’s maximum absorption wavelength by varying the detection wavelength during a single elution process and the detection sensitivity was greatly improved by using this detection method. The developed methods was sensitive, precise, accurate and selective and can be applied for simultaneous determination of the major chemical components in kangnaoshuai capsule of different production batches and different manufactures. The results of the simultaneous determination could provide sufficient information for efficient quality control and evaluation of kangnaoshuai capsule.
Part two
Studies on the fingerprinting analysis of kangnaoshuai capsule
Objective: To establish a reversed phase high performance liquid chromatography method for the fingerprinting analysis of kangnaoshuai capsule. The established method can be applied for fingerprinting analysis of kangnaoshuai capsule at different production batches and different manufactures. The established method can also be applied for the construction of the HPLC fingerprints of the constituent herbs of kangnaoshuai capsule. The results of the fingerprinting analyses were compared for quality control and evaluation of Kangnaoshuai capsule. The chemical characteristic of kangnaoshuai capsule was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components.
Methods: (1) Optimization of the extraction method: different extraction method, extraction solvents of different solvent composition and extration time were tested for optimization of the extraction efficacy of kangnaoshuai capsule (KNS). The most efficacy extration method was selected for the extraction of the major chemical components of KNS. (2) Optimization of the chromatographic conditions: chromatographic conditions such as analytical columns, detection method, mobile phase systems, gradient programs and column temperatures were tested for optimization of the chromatographic conditions. (3) System suitability test: Under the above conditions, the resolution and the theoretical plate number the peak of baicalin was calculated. (4) The test of injection precision: The injection precision was evaluated by analyzing the six repeated injection of the same sample solution. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the precision. (5) The test of reproducibility: reproducibility was evaluated by analyzing six replications prepared from the KNS sample within a day. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the reproducibility. (6) The test of stability: the test of stability was tested by determining the same sample solution stored at room temperature at different time of 0, 2, 4, 6, 8, 24, 48 h. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the stability. (7) The established method was applied for construction of the chromatographic figerprints of kangnaoshuai capsule at different production batches and different manufactures, and the constituent herbs. (8) Similarity analysis of the fingerprinting data and analysis of the correlation: similarities of the HPLC fingerprints of KNS were calculated by the professional software recommended by State Food and Drug Administration of China. The analysis results can be applied for qualtity evaluation of kangnaoshuai capsule at different production batches and different manufactures. Analysis was carried out for the chromatographic figerprints of kangnaoshuai capsule, the constituent herbs of kangnaoshuai capsule. The HPLC fingerprints of kangnaoshuai capsule were compared with that of the constituent herbs to identify the sub-chemical characteristic of the constituent herbs.
Results: The fingerprinting analysis of kangnaoshuai capsule (1) The extraction method: An accurately weighted powder of the contents of KNS capsule and the constituent herbs were extracted with 50% methanol by a sonifier at room temperature for 30 min. The extraction method for the major chemical components of KNS was efficacy and stable. (2) The chromatographic condition: A Waters SunfireTM-C18 column (150 mm×4.6 mm I.D., 5μm) was used throughout. The mobile phase consisted of acetonitrile (A) and 0.1% aqueous phosphoric acid (v/v, B). The gradient program for fingerprinting analysis was: 0~30 min, 3%~30% A; 30~40 min, 30%~65% A; 40~50 min, 65%~100% A; 50~55 min, 100%~3% A. The detection wavelength was set at 200 nm. (3) System suitability test: Under the above conditions, the peak of baicalin was separated well with the resolution of more than 1.2 and about 6000 of theoretical plate number. (4) Injection precision: the RSD% of the retention times for the tests of injection precision was not more than 0.07; The RSD% of the peak areas for the tests of injection precision was not more than 1.59. (5) Reproducibility: the RSD% of the retention times for the tests of injection precision was not more than 1.46; The RSD% of the peak areas for the tests of injection precision was not more than 3.96. (6) Stability: the RSD% of the retention times for the tests of injection precision was not more than 1.35; The RSD% of the peak areas for the tests of injection precision was not more than 1.11. The sample solution was stable within 48 hours. (7) The established method was applied for the construction of the HPLC fingerprints of kangnaoshuai capsule at different production batches and different manufactures, and the constituent herbs. The results were analyzed for quality evaluation. The chromatographic fingerprint of kangnaoshuai capsule was compared with that of the constituent herbs and the sub-chemical characteristic of the constituent herbs were identified. (8) Similarity analysis of the fingerprinting data and analysis of the correlation: similarities of the HPLC fingerprints of KNS were calculated by the professional software recommended by State Food and Drug Administration of China. The analysis results can effectively show the difference of the quality of kangnaoshuai capsule at different production batches and different manufactures. Analysis was carried out on the chromatographic figerprints of kangnaoshuai capsule, and the constituent herbs. The chromatographic fingerprint of kangnaoshuai capsule was compared with that of the constituent herbs and the sub-chemical characteristic of the constituent herbs were identified.
Conclusion: The reversed phase high performance liquid chromatography method for construction of the chromatographic fingerprints of kangnaoshuai capsule and the constituent herbs was established. Analysis was carried out for the chromatographic figerprints of kangnaoshuai capsule and the constituent herbs. Analysis of the chromatographic fingerprints of kangnaoshuai capsule at different production batches and different manufactures was carried out. The results of the fingerprinting analyses were analyzed and compared by the professional software recommended by State Food and Drug Administration of China for the quality evaluation of Kangnaoshuai capsule. The chemical characteristic of kangnaoshuai capsule was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components. The developed method was precise and accurate and the analysis result can effectively show the difference of the quality of kangnaoshuai capsule at different production batches and different manufactures. This chemical evidence based analysis can also provide valuable information for better understanding about the relationship between the chemical characteristic and the therapeutic efficacy of the kangnaoshuai capsule.
本研究采用反相高效液相色谱法建立了同时测定抗脑衰胶囊中七种有效成分的分析方法。建立了不同生产批次,不同生产厂家抗脑衰胶囊的HPLC指纹图谱,并对抗脑衰胶囊中主要色谱特征峰进行了分析,确定了主要色谱特征峰在抗脑衰胶囊组成药材中的归属,为阐明中药复方制剂的药效物质基础提供了有效的分析方法。方法专属性强,灵敏快速、结果准确可靠,精密度和稳定性良好。本研究对抗脑衰胶囊进行了综合的质量控制与评价,为保证中药复方制剂的质量提供了良好的质量控制与评价方法。
第一部分
抗脑衰胶囊中有效成分的多组分同时测定
目的:用反相高效液相色谱法建立中药复方制剂抗脑衰胶囊中有效成分的多组分同时测定方法,并将建立的方法用于不同批次,不同生产厂家的抗脑衰胶囊中有效成分的含量测定,并对含量测定结果加以比较,为科学有效的评价并控制抗脑衰胶囊的质量提供新的方法。
方法:(1)提取方法:比较了不同提取方式、提取溶剂及提取时间对抗脑衰胶囊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)高效液相色谱条件的优化:选用合适的色谱柱及检测方法,优化流动相的组成、配比、梯度洗脱程序、柱温等影响因素,确定最佳的色谱条件。(3)系统适用性试验:计算抗脑衰胶囊中有效成分黄芩苷的理论塔板数,及各有效成分与相邻峰的分离度。(4)标准曲线的制备:配制一系列浓度的混合对照品溶液,进样分析测定峰面积;以峰面积为纵坐标,浓度为横坐标,绘制标准曲线,经回归处理得各标准曲线方程。(5)定量限以及检测限试验:将混合对照品溶液逐步稀释并进行测定,以信噪比S/N≥10时确定为定量限,以信噪比S/N≥3时确定为检测限。(6)仪器精密度试验:取同一对照品溶液,重复进样6次,以所测有效成分的色谱保留时间以及色谱峰面积计算仪器精密度。(7)日内及日间精密度试验:取同一批次样品,在同一天精密称取6份,平行制备样品溶液,并对样品溶液进样进行分析,以所测有效成分的保留时间和峰面积计算日内精密度;取同一批次样品,在同一天精密称取2份,平行制备样品溶液,对样品溶液进样进行分析,连续测定3天,以所测有效成分的保留时间和峰面积计算日间精密度。(8)稳定性试验:取同一样品溶液置于室温,并分别于0、2、4、6、8、24、48h进样分析并进行有效成分的含量测定。(9)加样回收率试验:取同一批次并已知含量的样品,精密称定9份,分别精密加入高、中、低3种浓度的对照品混合物,按照供试品溶液制备方法进行制备,对样品溶液进样进行分析,并对有效成分的含量进行测定并计算加样回收率。(10)专属性试验:在上述色谱条件下,对阴性对照样品进样分析,将所得色谱图与样品色谱图进行比较,以排除抗脑衰胶囊中其它组成药材中化学物质的干扰。(11)抗脑衰胶囊中有效成分的含量测定:对不同批次,不同生产厂家的抗脑衰胶囊中有效成分的含量进行测定。
结果:抗脑衰胶囊中有效成分的多组分同时测定方法(1)提取方法:精密称取恒温干燥后的抗脑衰胶囊内容物约0.1g,用50%甲醇超声提取30 min,使各有效成分在提取方式相同的条件下同时达到最大的提取效率,且提取方法稳定。(2)HPLC色谱条件:采用Waters SunfireTM-C18色谱柱(150 mm×4.6 mm I.D., 5μm),以乙腈(A)-0.05%磷酸(B)为流动相进行梯度洗脱,梯度洗脱程序为:0~15 min,12%~20% A;15~30 min,20%~45% A;30~40 min,45%~75% A;40~50 min,75%~100% A;50~55 min,100%~12% A。采用Variable Wavelength Detector(VWD)紫外检测器以波长转换的方式进行检测,VWD检测波长时间变化程序为:0.00~6.10 min,320 nm;6.10~8.50 min,248 nm;8.50~15.00min,230 nm;15.00~25.00 min,320 nm;25.00~27.10 min,275 nm;27.10~32.50 min,286 nm;32.50~50.00 min,275 nm。(3)系统适用性试验:在此色谱条件下,以黄芩苷计算的理论塔板数大于6000,且其与其相邻色谱峰的分离度均大于1.5。(4)标准曲线的制备:对混合对照品溶液进样分析,以最小二乘法对峰面积和浓度进行线性回归得到各组分的标准曲线方程,其线性关系均符合要求。(5)定量限以及检测限试验:成分1~7的检测限分别为4.90、16.40、13.30、4.26、1.98、3.88、1.04 ng/ml;成分1~7的定量限分别为19.58、65.68、6.65、17.03、7.92、7.76、2.08 ng/ml。(6)仪器精密度试验:以有效成分的保留时间计算所得仪器精密度的RSD%均小于0.24;以有效成分的峰面积计算所得仪器精密度的RSD%均小于0.85。(7)日内及日间精密度试验:以有效成分的保留时间计算所得日间精密度的RSD%均小于0.37,以有效成分的峰面积计算所得日间精密度的RSD%均小于3.03;以有效成分的保留时间计算所得日内精密度的RSD%均小于0.46,以有效成分的峰面积计算所得日内精密度的RSD%均小于2.94(8)稳定性试验:以有效成分1~7的保留时间计算所得稳定性试验的RSD%均小于1.35,以有效成分1~7的峰面积计算所得稳定性试验的RSD%均小于1.11,样品在48h内稳定。(9)加样回收率试验:高、中、低3种浓度的加样回收率都在90.0%~108.3%之间,RSD%小于4.72。(10)专属性试验:在上述色谱条件下,对阴性对照样品进样分析,将所得色谱图与样品色谱图进行比较,阴性样品溶液在样品有效成分出峰位置没有干扰。(11)抗脑衰胶囊中有效成分的含量测定:依据所建立的色谱条件对不同批次,不同生产厂家的抗脑衰胶囊中有效成分的含量进行同时测定,由标准曲线计算得到各个样品中有效成分的含量。
结论:本研究采用反相高效液相色谱法建立了抗脑衰胶囊中有效成分的多组分同时测定方法。采用波长转换的方式以各待测组分的最大吸收波长作为相应组分的检测波长,最大程度的发挥了紫外检测器的检测效能,提高了检测灵敏度。方法专属性强,灵敏快速、结果准确可靠,精密度和稳定性良好。本研究建立的方法可以用于抗脑衰胶囊中多个有效成分含量的同时测定,为保证中药复方制剂的质量提供了良好的质量控制方法。
第二部分
抗脑衰胶囊的高效液相色谱指纹图谱研究
目的:用反相高效液相色谱法建立中药复方制剂抗脑衰胶囊的HPLC指纹图谱,将所建立的方法用于建立不同批次,不同生产厂家的抗脑衰胶囊的HPLC指纹图谱,并对指纹图谱结果加以比较,为科学有效的评价并控制抗脑衰胶囊的质量提供新的方法。同时将建立的方法用于抗脑衰胶囊组成药材的HPLC指纹图谱的建立,并对抗脑衰胶囊中主要色谱特征峰在组成药材中的来源进行归属,为阐明抗脑衰胶囊的药效物质提供科学有效的方法。
方法:(1)提取方法:比较了不同提取方式、提取溶剂及提取时间对抗脑衰胶囊有效成分的提取效果,选择提取成分多,提取率高的提取条件。(2)HPLC色谱指纹图谱条件的优化:选用合适的色谱柱及紫外检测器检测波长,优化流动相的组成、配比、梯度洗脱程序,柱温等影响因素,确定最佳的HPLC指纹图谱条件。(3)系统适用性试验:计算抗脑衰胶囊中有效成分黄芩苷的理论塔板数,及其与相邻色谱峰的分离度。(4)仪器精密度试验:取同一供试品溶液,重复进样6次,以高效液相色谱图中主要成分的色谱保留时间以及色谱峰面积计算仪器精密度。(5)重复性试验:取同一批次样品,精密称取6份,平行制备样品溶液,对样品溶液进样分析,根据高效液相色谱图中主要成分的色谱保留时间以及色谱峰面积对重复性进行评价。(6)稳定性试验:取同一样品溶液置于室温,并分别于0、2、4、6、8、24、48h对样品溶液进行分析,根据高效液相色谱图中主要成分的色谱保留时间以及色谱峰面积对稳定性进行评价。(7)专属性试验:在上述色谱条件下对空白溶液、样品溶液进样分析并进行对比,以排除空白干扰。(8)抗脑衰胶囊指纹图谱分析:对不同批次,不同生产厂家的抗脑衰胶囊以及抗脑衰胶囊组成药材进行样品制备,并进行高效液相色谱指纹图谱分析。(9)指纹图谱数据分析:采用指纹图谱相似度计算软件对所得高效液相色谱指纹图谱数据进行分析和比较,使得数据结果可以有效的反映不同生产批次以及不同生产厂家的抗脑衰胶囊质量的差异。并对所得抗脑衰胶囊以及抗脑衰胶囊组成药材的高效液相色谱指纹图谱进行分析,对抗脑衰胶囊中主要色谱特征峰在组成药材中的来源进行归属。
结果:抗脑衰胶囊的HPLC指纹图谱研究(1)提取方法:精密称取恒温干燥后的抗脑衰胶囊样品,用50%甲醇超声提取30min,各主要成分在提取方式相同的条件下同时达到最大的提取效率,且提取方法稳定。(2)HPLC色谱指纹图谱条件:采用Waters SunfireTM-C18色谱柱(150 mm×4.6 mm I.D., 5μm),以乙腈(A)-0.05%磷酸(B)为流动相进行梯度洗脱,梯度洗脱程序为:0~30 min,3%~30% A;30~40 min,30%~65% A;40~50 min,65%~100% A;50~55 min,100%~3% A。采用Photodiode Array Detector(PDA)紫外检测器进行检测,检测波长为200 nm,波长扫描范围为:200~400 nm。(3)系统适用性试验:在此色谱条件下,以黄芩苷计算理论塔板数大于6000且与其相邻色谱峰的分离度均大于1.5。(4)仪器精密度试验:以主要成分的色谱保留时间计算所得仪器精密度的RSD%均小于0.07;以主要成分的色谱峰面积计算所得仪器精密度的RSD%均小于1.59。(5)重复性试验:以主要成分的色谱保留时间计算所得日间精密度的RSD%均小于1.46,以主要成分的色谱峰面积计算所得日间精密度的RSD%均小于3.96。(6)稳定性试验:以主要成分的色谱保留时间计算所得稳定性试验的RSD%均小于1.35,以主要成分的色谱峰面积计算所得稳定性试验的RSD%均小于1.11,样品在48h内稳定。(7)专属性试验:供试品溶液中各色谱峰分离较好,空白对照溶液无杂质干扰。(8)抗脑衰胶囊指纹图谱分析:对不同批次,不同生产厂家的抗脑衰胶囊以及抗脑衰胶囊组成药材进行样品制备,并依据所建立的HPLC色谱指纹图谱条件进行色谱指纹图谱分析。(9)指纹图谱数据分析:采用指纹图谱相似度软件对所得高效液相色谱指纹图谱数据进行分析和比较,所得数据结果可以有效的反映不同生产批次以及不同生产厂家的抗脑衰胶囊质量的差异。对所得抗脑衰胶囊以及抗脑衰胶囊组成药材的高效液相色谱指纹图谱进行分析并对抗脑衰胶囊中主要色谱特征峰在组成药材中的来源进行归属,确定了主要色谱特征峰在抗脑衰胶囊组成药材中的来源。
结论:本研究采用反相高效液相色谱法建立了不同生产批次,不同生产厂家抗脑衰胶囊的HPLC指纹图谱,并采用指纹图谱相似度计算软件对不同生产批次,不同生产厂家的抗脑衰胶囊的HPLC指纹图谱进行了分析和比较,对抗脑衰胶囊的质量进行了综合评价。同时建立了抗脑衰胶囊组成药材的HPLC指纹图谱,并对抗脑衰胶囊中主要色谱特征峰进行分析,确定了主要色谱特征峰在抗脑衰胶囊组成药材中的归属,为阐明中药复方制剂的药效物质基础提供了有效的方法。方法精密度和稳定性良好,所得数据结果可以有效的反映不同生产批次以及不同生产厂家的抗脑衰胶囊的质量差异,为保证中药复方制剂的质量提供了良好的质量控制方法。
Kangnaoshuai capsule (KNS) is an over-the-counter fufang preparation consisted of 19 herbs and the herbs are Radix Polygoni Multiflori, Radix rehmanniae preparata, Fructus lycii, Radix Ginseng, Radix Astragali, Rhizoma Dioscoreae, Radix Salviae Miltiorrhiae, Radix Paeoniae Alba, Rhizome Acori Talarinowii, radix polygalae, Semen ziziphi spinosae, Rhizoma Cyperi, Radix Scutellariae, Radix codonopsis, Indian bread with host wood, Radix ophiopogonis, Radix Puerariae, Flos Chrysanthemi. It has been used for the treatment of neurolysis, insomnia and dreamful sleep, neurastheria, memory deterioration. It has also been reported to be beneficial in the treatments for age-related dementia, encephalatrophy, cerebral infarction, cerebral hypoplasia, sequelae of cerebral thrombosis, brain injuries caused by stroke, cerebral trauma and encephalitis.
A reversed phase high performance liquid chromatography method for simultaneous determination of the seven chemical components in the fufang preparation of kangnaoshuai capsule was developed. High performance chromatographic fingerprints of the constituent herbs and kangnaoshuai capsule at different production batches and different manufactures were constructed. The chemical characteristic of kangnaoshuai capsule was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components. The developed method was precise and accurate and the method can be effectively applied for the quanlity control and evaluation of kangnaoshuai capsule at different production batches and different manufactures. This chemical evidence based analysis can also provide valuable information for better understanding about the relationship between the chemical characteristic and the therapeutic efficacy of the kangnaoshuai capsule. Part one
Simultaneous determination of the major chemical components in kangnaoshuai capsule by HPLC
Objective: To establish a reversed phase high performance liquid chromatography method for simultaneous determination of the major chemical components in the fufang preparation of kangnaoshuai capsule. The established method can be applied for determination of the key chemical components in kangnaoshuai capsule of different production batches and different manufactures. The results of the quantitative analyses were compared for quality control and evaluation of Kangnaoshuai capsule.
Methods: (1) Optimization of the extraction method: different extraction methods, extraction solvents of different solvent composition and extration time were tested for optimization of the extraction efficacy of kangnaoshuai capsule (KNS). The most efficacy extration method was selected for the extraction of the major chemical components of KNS. (2) Optimization of the chromatographic conditions: chromatographic conditions such as analytical columns, detection method, mobile phase systems, gradient programs and column temperatures were tested for optimization of the chromatographic conditions. (3) System suitability test: Under the above conditions, the resolution of the major chemical components and the theoretical plate number the peak of baicalin was calculated. (4) A series of seven calibration solutions were prepared by diluting the stock standard solution to appropriate concentrations for construction of the regression equations. The calibration curve was constructed by plotting the peak area of each key chemical component against its concentration. (5) The test of LOD and LOQ: the limits of detection (LOD) and quantification (LOQ) were determined at a signal-to-noise ratio of about 3 and 10 by analyzing the diluted standard solution. (6) The test of injection precision: The injection precision was evaluated by analyzing the six repeated injection of the standard solution. The retention times and the peak areas of the seven chemical components were analyzed for evaluation of the precision. (7) The test of the intra-day and inter-day precision: intra-day precision was evaluated by analyzing six replications prepared from the KNS sample within a day. Duplicate copies of the KNS sample were prepared within a day for the consecutive three days, and inter-day precision was evaluated by analyzing the six sample solutions of three days. (8) The test of stability: stability was tested by determining the same stock standard solution stored at room temperature at different time of 0, 2, 4, 6, 8, 24, 48 h. (9) The test of recovery: Accurately weighted amounts of each reference compound at three levels were mixed with a fixed amount of the contents of KNS sample. Three replications at each level were extracted and analyzed to evaluate the accuracy of the developed method. (10) The test of selectivity: negative control samples were analyzed to evaluate the selectivity of the developed method. The results were compared to confirm that the chemical components were determined without the interference of other chemical components originated from the coexisting herbs. (11) The established method was applied for determination of the seven chemical components in kangnaoshuai capsule of different production batches and different manufactures.
Results: The simultaneous determination of the seven chemical components in kangnaoshuai capsule (1) The extraction method: An accurately weighted dried powder of the contents of KNS capsule was extracted with 50% methanol by a sonifier at room temperature for 30 min. The extraction method of the major chemical components of KNS was efficacy and stable. (2) The chromatographic condition: A Waters SunfireTM-C18 column (150 mm×4.6 mm I.D., 5μm) was used throughout. The mobile phase consisted of acetonitrile (A) and 0.1% aqueous phosphoric acid (v/v, B). The gradient program for quantitative analysis was: 0~15 min, 12%~20% A; 15~30 min, 20%~45% A; 30~40 min, 45%~75% A; 40~50 min, 75%~100% A, 50~55 min, 100%~12% A. The detection wavelength was programmed as: 0.00~6.10 min, 320 nm; 6.10~8.50 min, 248 nm; 8.50~15.00 min, 230 nm; 15.00~25.00 min, 320 nm; 25.00~27.10 min, 275 nm; 27.10~32.50 min, 286 nm; 32.50~50.00 min, 275 nm. (3) System suitability test: Under the above conditions, the peak of baicalin was separated well with the resolution of more than 1.2 and about 6000 of theoretical plate number. (4) The calibration curve was constructed by plotting the peak area of each key chemical component against its concentration for construction of the regression equations. All calibration curves showed good linearity within the test ranges. (5) The limits of LOD and LOQ: The limits of LOQ of the seven chemical components were 4.90、16.40、13.30、4.26、1.98、3.88、1.04 ng/ml;The limits of LOD of the seven chemical components were 19.58、65.68、6.65、17.03、7.92、7.76、2.08 ng/ml. (6) Injection precision: the RSD% of the retention times for the tests of injection precision was not more than 0.24; The RSD% of the peak areas for the tests of injection precision was not more than 0.85. (7) Intra-day and inter-day precision: the RSD% of the retention times for the tests of intra-day precision was not more than 0.37, the RSD% of the peak areas for the tests of intra-day precision was not more than 3.03;the RSD% of the retention times for the tests of inter-day precision was not more than 0.46, the RSD% of the peak areas for the tests of inter-day precision was not more than 2.94. (8) Stability: the RSD% of the retention times for the tests of injection precision was not more than 1.35; The RSD% of the peak areas for the tests of injection precision was not more than 1.11. The sample solution was stable within 48 hours. (9) Recovery test: the results of the recovery test at three levels were from 90.0%~108.3% and the RSD% were not more than 4.72. (10) Selectivity: 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 interference of other chemical components originated from the coexisting herbs. (11) The established method was applied for determination of the major chemical components in kangnaoshuai capsule of different production batches and different manufactures. The content of each chemical component was calculated according to the established calibration curves.
Conclusion: The reversed phase high performance liquid chromatography method for the simultaneous determination of the major chemical components in the fufang preparation of kangnaoshuai capsule was established. The UV detector was programmed to monitor at each component’s maximum absorption wavelength by varying the detection wavelength during a single elution process and the detection sensitivity was greatly improved by using this detection method. The developed methods was sensitive, precise, accurate and selective and can be applied for simultaneous determination of the major chemical components in kangnaoshuai capsule of different production batches and different manufactures. The results of the simultaneous determination could provide sufficient information for efficient quality control and evaluation of kangnaoshuai capsule.
Part two
Studies on the fingerprinting analysis of kangnaoshuai capsule
Objective: To establish a reversed phase high performance liquid chromatography method for the fingerprinting analysis of kangnaoshuai capsule. The established method can be applied for fingerprinting analysis of kangnaoshuai capsule at different production batches and different manufactures. The established method can also be applied for the construction of the HPLC fingerprints of the constituent herbs of kangnaoshuai capsule. The results of the fingerprinting analyses were compared for quality control and evaluation of Kangnaoshuai capsule. The chemical characteristic of kangnaoshuai capsule was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components.
Methods: (1) Optimization of the extraction method: different extraction method, extraction solvents of different solvent composition and extration time were tested for optimization of the extraction efficacy of kangnaoshuai capsule (KNS). The most efficacy extration method was selected for the extraction of the major chemical components of KNS. (2) Optimization of the chromatographic conditions: chromatographic conditions such as analytical columns, detection method, mobile phase systems, gradient programs and column temperatures were tested for optimization of the chromatographic conditions. (3) System suitability test: Under the above conditions, the resolution and the theoretical plate number the peak of baicalin was calculated. (4) The test of injection precision: The injection precision was evaluated by analyzing the six repeated injection of the same sample solution. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the precision. (5) The test of reproducibility: reproducibility was evaluated by analyzing six replications prepared from the KNS sample within a day. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the reproducibility. (6) The test of stability: the test of stability was tested by determining the same sample solution stored at room temperature at different time of 0, 2, 4, 6, 8, 24, 48 h. The retention times and the peak areas of the major chemical components were analyzed for evaluation of the stability. (7) The established method was applied for construction of the chromatographic figerprints of kangnaoshuai capsule at different production batches and different manufactures, and the constituent herbs. (8) Similarity analysis of the fingerprinting data and analysis of the correlation: similarities of the HPLC fingerprints of KNS were calculated by the professional software recommended by State Food and Drug Administration of China. The analysis results can be applied for qualtity evaluation of kangnaoshuai capsule at different production batches and different manufactures. Analysis was carried out for the chromatographic figerprints of kangnaoshuai capsule, the constituent herbs of kangnaoshuai capsule. The HPLC fingerprints of kangnaoshuai capsule were compared with that of the constituent herbs to identify the sub-chemical characteristic of the constituent herbs.
Results: The fingerprinting analysis of kangnaoshuai capsule (1) The extraction method: An accurately weighted powder of the contents of KNS capsule and the constituent herbs were extracted with 50% methanol by a sonifier at room temperature for 30 min. The extraction method for the major chemical components of KNS was efficacy and stable. (2) The chromatographic condition: A Waters SunfireTM-C18 column (150 mm×4.6 mm I.D., 5μm) was used throughout. The mobile phase consisted of acetonitrile (A) and 0.1% aqueous phosphoric acid (v/v, B). The gradient program for fingerprinting analysis was: 0~30 min, 3%~30% A; 30~40 min, 30%~65% A; 40~50 min, 65%~100% A; 50~55 min, 100%~3% A. The detection wavelength was set at 200 nm. (3) System suitability test: Under the above conditions, the peak of baicalin was separated well with the resolution of more than 1.2 and about 6000 of theoretical plate number. (4) Injection precision: the RSD% of the retention times for the tests of injection precision was not more than 0.07; The RSD% of the peak areas for the tests of injection precision was not more than 1.59. (5) Reproducibility: the RSD% of the retention times for the tests of injection precision was not more than 1.46; The RSD% of the peak areas for the tests of injection precision was not more than 3.96. (6) Stability: the RSD% of the retention times for the tests of injection precision was not more than 1.35; The RSD% of the peak areas for the tests of injection precision was not more than 1.11. The sample solution was stable within 48 hours. (7) The established method was applied for the construction of the HPLC fingerprints of kangnaoshuai capsule at different production batches and different manufactures, and the constituent herbs. The results were analyzed for quality evaluation. The chromatographic fingerprint of kangnaoshuai capsule was compared with that of the constituent herbs and the sub-chemical characteristic of the constituent herbs were identified. (8) Similarity analysis of the fingerprinting data and analysis of the correlation: similarities of the HPLC fingerprints of KNS were calculated by the professional software recommended by State Food and Drug Administration of China. The analysis results can effectively show the difference of the quality of kangnaoshuai capsule at different production batches and different manufactures. Analysis was carried out on the chromatographic figerprints of kangnaoshuai capsule, and the constituent herbs. The chromatographic fingerprint of kangnaoshuai capsule was compared with that of the constituent herbs and the sub-chemical characteristic of the constituent herbs were identified.
Conclusion: The reversed phase high performance liquid chromatography method for construction of the chromatographic fingerprints of kangnaoshuai capsule and the constituent herbs was established. Analysis was carried out for the chromatographic figerprints of kangnaoshuai capsule and the constituent herbs. Analysis of the chromatographic fingerprints of kangnaoshuai capsule at different production batches and different manufactures was carried out. The results of the fingerprinting analyses were analyzed and compared by the professional software recommended by State Food and Drug Administration of China for the quality evaluation of Kangnaoshuai capsule. The chemical characteristic of kangnaoshuai capsule was analyzed to identify the sub-chemical characteristic of each constituent herb so as to clarify the complex composition of the chemical components. The developed method was precise and accurate and the analysis result can effectively show the difference of the quality of kangnaoshuai capsule at different production batches and different manufactures. This chemical evidence based analysis can also provide valuable information for better understanding about the relationship between the chemical characteristic and the therapeutic efficacy of the kangnaoshuai capsule.
引文
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21 邓六勤,钟鸣,陈怡禄等. 复方毛冬青注射液挥发性成分的研究.中药材,2006,29(3):303-305
22 袁子民,王静,吕佳等. 肉豆蔻饮片炮制前后挥发油成分的GC-MS分析. 中国中药杂志,2006,31(9):737-739
23 邱琴,张国英,丁玉萍等. 超临界CO2流体萃取法与水蒸气蒸馏法提取干姜片挥发油化学成分的比较. 上海中医药杂志,2005,39(3):55-57
24 胡浩斌,郑旭东. GC-MS测定超临界流体CO2萃取竹节草挥发油的化学成分. 中国中药杂志,2006,31(7):607-608
25 罗晓健,韩飞,柏健等. 香砂养胃浓缩丸挥发油化学成分在水蒸气蒸馏提取过程中的变化研究.中草药,2004,35(8):859-861
2 程瑞艳. 抗脑衰胶囊在脑挫裂伤后综合征中的应用. 浙江中西医结合杂志 2002,12(9):547-548
3 王传森,袁芳,孙绍江. 抗脑衰胶囊治疗 38 例脑梗塞临床及血液流变学分析. 云南医药,1998,19(3):201
4 谈跃,朱榆红,殷梅等. 抗脑衰胶囊治疗 46 例血管性痴呆的疗效观察. 云南医药,1998,19(3):200
5 张玉岭,吴海深. 抗脑衰胶囊治疗慢性心力衰竭 58 例疗效观察. 河北中医,2006,28(6):410
6 黄远光. 抗脑衰胶囊治疗失眠症 100 例临床分析. 实用医学杂志,2003,19(5):554-555
7 俞淑文,李维莲. 抗脑衰胶囊治疗血管性痴呆疗效观察. 山东医药,2001,41(19):47-48
8 程瑞艳. 石门牌抗脑衰胶囊治疗脑挫裂伤后综合症的疗效观察. 医学理论与实践,2002,15(5):542-543
1 中药注射剂指纹图谱研究的技术要求. 2003
2 中华人民共和国药典一部. 2005
1 金晓玲,何新霞,杜红岩等. 超临界流体萃取佛手挥发油的气相色谱/质谱分析. 浙江师范大学学报(自然科学版),2005,28(1):61-65
2 赵晨曦,梁逸曾,李晓宁等. 杜鹃嫩枝叶挥发油化学成分研究. 药学学报,2005,40(9):854-860
3 F.A. Oladimeji,O.O. Orafidiya,T.A.B. Ogunniyi et al. Pediculocidal and scabicidal properties of Lippia multiflora essential oil. Journal of Ethnopharmacology,2000,72(3):305-311
4 Jianqing Yu,Jiachuan Lei,Huaidong Yu et al. Chemical composition and antimicrobial activity of the essential oil of Scutellaria barbata. Phytochemistry,2004,65(12):881-884
5 Mozaina Kobaisy, Mario R. Tellez, Franck E. Dayan,et al. Phytotoxicity and volatile constituents from leaves of Callicarpa japonica Thunb. Phytochemistry,2002,61(5): 37-40
6 董岩,王新芳,魏兴国. 山东艾蒿挥发油化学成分的GC-MS研究. 中成药,2005,27(3):326-328
7 陈振德,许重远,谢 立. 超临界流体CO2萃取酸枣仁脂肪油化学成分的研究. 中草药,2001,32(11):976-977
8 吴志平,李祥,陈建伟. 明党参果实脂肪油成分GC/MSD分析. 南京中医药大学学报(自然科学版),2002,18(5):293-294
9 李澎灏,陈振德. 牵牛子脂肪油超临界CO2萃取及气相色谱-质谱测定. 中国药房,2003,14(7):431-432
10 李澎灏,陈振德. 沙苑子脂肪油超临界CO2萃取及GC-MS分析.中药材,2003,26(9):640-641
11 Guohua Hu , Yanhua Lu , Dongzhi Wei. Fatty acid composition of the seed oil of Allium tuberosum. Bioresource Technology,2005,96(11):1630-1632
12 俞作仁,吕娟涛,臧庶声. 西北蔷薇果挥发油及种籽脂肪油化学成分研究. 中草药,2001,32(50):403,416
13 李勇,孙秀燕,林翠英等. 3个品种莪术挥发油化学成分的比较. 中草药,2005,36(120:1785-1787
14 唐登峰,杨中林,程启厚等. 地蚕不同药用部位挥发油的气质联析. 中成药,2002,24(3):205-208
15 郭兰萍,刘俊英,吉力等. 茅苍术道地药材的挥发油组成特征分析. 中国中药杂志,2002,27(11):814-819
16 Christine Robles,Suzanne Garzino. Infraspecific variability in the essential oil composition of Cistus monspeliensis leaves. Phytochemistry,2000,53(6):71-75
17 朱顺英,杨扬,侯洁等. 青木香挥发油的化学成分分析及抗菌活性. 武汉大学学报,2005,51(6):757-761
18 姚煜,王英锋,王欣月等. 线纹香茶菜挥发油化学成分的GC-MS分析. 中国中药杂志,2006,31(8):695-696
19 张典瑞,任天池,夏方亮等. 苍耳子散挥发性成分的GC-MS 分析. 中国中药杂志,2003,28(11):1083-1086
20 魏刚,符红,王淑英等. GC-MS法建立广藿香挥发油指纹特征图谱研究. 中成药,2002,24(6):407-410
21 邓六勤,钟鸣,陈怡禄等. 复方毛冬青注射液挥发性成分的研究.中药材,2006,29(3):303-305
22 袁子民,王静,吕佳等. 肉豆蔻饮片炮制前后挥发油成分的GC-MS分析. 中国中药杂志,2006,31(9):737-739
23 邱琴,张国英,丁玉萍等. 超临界CO2流体萃取法与水蒸气蒸馏法提取干姜片挥发油化学成分的比较. 上海中医药杂志,2005,39(3):55-57
24 胡浩斌,郑旭东. GC-MS测定超临界流体CO2萃取竹节草挥发油的化学成分. 中国中药杂志,2006,31(7):607-608
25 罗晓健,韩飞,柏健等. 香砂养胃浓缩丸挥发油化学成分在水蒸气蒸馏提取过程中的变化研究.中草药,2004,35(8):859-861