CN106053700A - 一种首乌益智胶囊的指纹图谱测定方法 - Google Patents
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Abstract
本发明涉及首乌益智胶囊的质量控制方法,具体涉及一种首乌益智胶囊的指纹图谱测定方法,属于药物分析技术领域,该测定方法是通过以下步骤实现的:(1)对照品溶液的制备;(2)内标溶液的制备;(3)供试品溶液的制备;(4)利用高效液相色谱进行分析测定;本发明创新性的在中药制剂复杂的指纹图谱中定量加入一个内标峰,以此对制剂中的色谱峰进行标定,使建立的方法准确简便、灵敏度高、重复性好。本发明能够更全面地监控半成品、成品质量,监控生产工艺的稳定性。
Description
技术领域
本发明涉及一种首乌益智胶囊的质量控制方法,具体涉及一种首乌益智胶囊的指纹图谱测定方法,属于药物分析技术领域。
背景技术
首乌益智胶囊是由制何首乌、益智、黄芪等10种中药及提取物配伍,经特定加工提取工艺制成的中药胶囊剂。首乌益智胶囊全方通补结合,补而不滞,诸药配合,滋补温养精血,通行血脉, 具有补肾填精、化瘀通窍的功效,临床主要用于治疗肾精亏虚,瘀血阻络型血管性痴呆,能明显提高患者的认知能力、记忆能力和日常生活能力,有效改善中医临床症状,提高患者的生活质量。由于口服何首乌及其制剂有可能引起肝损伤的风险,本品所用为制何首乌,虽然制何首乌的风险低于生品,但不断提高其质量控制水平是保证产品质量均一、稳定的必要手段。
由于全球气候变暖、环境污染加剧、农药残留超标、品种退化及混杂等原因,中药原材料的安全稳定性已成为亟待解决的关键问题。中药指纹图谱能全面反映中药内在化学成分的品种与数量,进而反映重要的质量,现阶段中药的有效成分绝大多数没有明确,采用中药指纹图谱的方式,将有效的表征中药质量,指纹图谱已经被国际社会所认可,有利于中药进入国际市场。指纹图谱为获得中药化学成分的谱图,具有特征性强、整体性和模糊性等特点,可以用于中药及中药制剂真伪、一致性和稳定性的评价。
中国专利CN103487516A提供了一种含有何首乌中药制剂的指纹图谱测定方法,该方法选用制剂中含有的成分做参照峰,并对方中的9个成分进行了指认,选用自身含有的成分做对照峰不能客观反应制剂中各共有峰成分的相对含量,虽然指认了9个成分,分别来源于4种药物,占方中药物比例低(全方共18种药物),不能全面反应和控制制剂质量;文献《何首乌配方颗粒的UPLC指纹图谱及化学成分归属》,该方法以何首乌配方颗粒为研究对象,进行了UPLC指纹图谱研究,指认了5个共有峰,在指纹图谱分析中采用自身含有的成分做对照,但该方法不能进行定量测定。
发明内容
本发明就是针对现有技术不能全面、客观、科学的评价首乌益智胶囊的质量的问题,提出的一种HPLC指纹图谱测定方法。该方法在成分复杂的样品溶液中定量加入一个内标,使建立的方法准确简便、灵敏度高、重复性好。
本发明是按照以下技术方案实现的:
本发明提供了一种首乌益智胶囊的指纹图谱测定方法,包括以下步骤:
(1)对照品溶液的制备:分别称取大黄素、大黄素甲醚、2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、丹酚酸B、丹参酮ⅡA,丹酚酸B加75%甲醇溶解,其余对照品加甲醇溶解,分别制成对照品溶液;
(2)内标溶液的制备:称取汉黄芩苷,加入甲醇制成浓度为70µg/mL的内标溶液;
(3)供试品溶液制备:取首乌益智胶囊内容物粉末0.5g,加入75%甲醇25mL,回流提取,放凉,用75%甲醇补足减失的重量,摇匀,滤过,取续滤液适量,加入浓度为75µg/mL的内标溶液,混匀,过0.45µm微孔滤膜,即得;
(4)利用高效液相色谱法进行分析测定。
进一步的,步骤(1)中,所述大黄素对照品溶液的浓度为10.26µg/mL、大黄素甲醚对照品溶液的浓度为9.68µg/mL、2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷对照品溶液的浓度为64.08µg/mL、丹酚酸B对照品溶液的浓度为0.15mg/mL、丹参酮ⅡA对照品溶液的浓度为20µg/mL。
上述2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、丹参酮ⅡA的对照品溶液需避光保存。
本发明回流提取的时间为30min,沸水浴条件下进行回流提取。
进一步的,步骤(3)中,所述续滤液同内标溶液的体积比为1∶1。
本发明所述的高效液相色谱分离使用Thermo Syncronis C18色谱柱:250 mm×4.6 mm,5 μm,在柱温30 ℃,流速1.0 mL/min;检测波长280 nm;进样量10 μL条件下,以体积分数为0.1%甲酸水溶液为流动相A,乙腈为流动相C,进行梯度洗脱。
上述梯度洗脱的条件为:0~30min,95~75%流动相A,5~25%流动相C;30~50min,75~73%流动相A,25~27%流动相C;50~60min,73~70%流动相A,27~30%流动相C;60~80min,70~30%流动相A,30~70%流动相C;80~95min,30~25%流动相A,70~75%流动相C;95~105min,25~0%流动相A,75~100%流动项C;所述百分比均为体积百分比。
本发明首次在首乌益智胶囊的指纹图谱测试中将供试品溶液中定量加入一个内标溶液,在图谱分析中,采用共有峰与内标峰的相对峰面积比值作为该指纹图谱的数学表达,这样比通常选用中药自身成分做参照计算各共有峰的相对峰面积,更能体现各共有峰含量的绝对变化,同时,避免系统性的误差。
本发明获得了如下有益效果。
(1)本发明建立的首乌益智胶囊HPLC指纹图谱测定方法符合指纹图谱方法学研究的要求,进一步阐释了其物质基础;
(2)本发明选用内标法,对含有内标物质的供试品溶液进行指纹图谱测定,采用共有峰与内标峰的相对峰面积比值作为该指纹图谱的数学表达,更能体现各共有峰含量的绝对变化;
(3)本发明针对药物所含有效成分的理化性质和结构特点,对供试品溶液的配制方法、流动相组成、洗脱程序等条件进行了筛选和优化,所选用的方法重复性好、稳定性佳;
(4)本发明有效的解决了现有技术不能全面、客观、科学的评价首乌益智胶囊的质量的问题,有效的保证了首乌益智胶囊的产品质量,可用于半成品、成品的质量控制与评价,监控生产工艺的稳定性。
附图说明
图1为实施例1得到的首乌益智胶囊指纹图谱。
图2为本发明对10批首乌益智胶囊进行了指纹图谱测定,采用中位数法生成对照指纹图谱。
具体实施方式
下面通过实施例对本发明进行进一步的阐述,下述说明仅为了解释本发明,并不对其内容进行限定。
1.1仪器
高效液相色谱仪(美国Waters公司:Waters e2695 ), BP211D型电子分析天平(瑞士梅特勒-托利多公司),Simplicity纯水仪(美国密理博公司)。
1.2试药
大黄素(110756-200110)、大黄素甲醚(110758-201415)、2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷(110844-200404)、丹酚酸B(111562-200504)、丹参酮ⅡA(110766-200416)均购自中国药品生物制品检定所,汉黄芩苷(13120401)购自成都普瑞法科技开发有限公司。
液相色谱用甲醇为色谱纯,其他试剂均为分析纯。
试验用首乌益智胶囊由山东仙河药业有限公司提供。
实施例1
一种首乌益智胶囊的指纹图谱测定方法,包括以下步骤:
(1)对照品溶液的制备:分别称取大黄素、大黄素甲醚、2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、丹酚酸B、丹参酮ⅡA对照品适量,丹酚酸B加75%甲醇溶解,其余对照品加甲醇溶解,分别制成对照品溶液,其中大黄素对照品溶液浓度为10.26µg/mL、大黄素甲醚对照品溶液浓度为9.68µg/mL、2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷对照品溶液浓度为64.08µg/mL、丹酚酸B对照品溶液浓度为0.15mg/mL、丹参酮ⅡA对照品溶液浓度为20µg/mL,2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、丹参酮ⅡA对照品溶液避光保存;
(2)内标溶液的制备:称取汉黄芩苷对照品,加入甲醇适量,制成浓度为70µg•mL-1的内标溶液;
(3)供试品溶液制备方法:取首乌益智胶囊内容物粉末0.5g,精密称定,加入75%甲醇25mL,回流提取30分钟,放凉,用75%甲醇补足减失的重量,摇匀,滤过,取续滤液适量,精密加入等体积浓度为75µg•mL-1的内标溶液,混匀,过0.45µm微孔滤膜,即得;
(4)利用高效液相色谱法进行分析测定:
色谱条件 :Thermo Syncronis C18色谱柱(250mm×4.6mm,5µm),在柱温30 ℃,流速1.0 mL/min;检测波长280 nm;进样量10 μL条件下,以体积分数为0.1%甲酸水溶液为流动相A,乙腈为流动相C,进行高效液相色谱检测;
所述梯度洗脱的条件为,具体见流动相梯度洗脱表1。
表1 流动相梯度洗脱表
本发明对内标物的选择:内标物的选择难度大,要求高。经过大量的理论研究发现:汉黄芩苷的保留时间为52.5 min左右,与样品中成分无干扰,与相邻峰达到基线分离,相邻峰的分离度均>1.5,且与溶于供试液,稳定性好,适宜作为该样品供试液的内标物质。
(5)按照上述建立的指纹图谱测定方法,得到色谱图,如图1所示:
对首乌益智胶囊进行指纹图谱分析,以汉黄芩苷为参照峰(S),标定了24个共有峰,共有峰面积占总面积的99.3%,通过对照品保留时间和紫外吸收光谱的比对,确定了共有峰1、7、18、19、23来源于制何首乌,共有峰1、2、4、6、8、9、10、11来源于银杏叶提取物,共有峰2、5来源于天麻,共有峰12、13、15、21、22、24来源于丹参,共有峰14、20来源于川芎,共有峰17来源于远志,其中7、15、19、23、24号峰分别为2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、丹酚酸B、大黄素、大黄素甲醚和丹参酮ⅡA,生成的对照指纹图谱的共有峰保留时间见表2。
表2 首乌益智胶囊对照指纹图谱共有峰及其保留时间数据表
1.1精密度试验
取首乌益智胶囊供试品溶液,连续进样6次,按照实施例1中提供的方法进行测定,以汉黄芩苷为参照,计算24个共有峰的相对峰面积和相对保留时间。结果显示24个主要峰的相对峰面积RSD均<2.90%,相对保留时间RSD均<0.51%,表明仪器精密度良好。
重复性试验
取同一批号首乌益智胶囊,按照实施例1提供的方法制备供试品溶液,分别进样6次,记录液相色谱图,以汉黄芩苷为参照,计算24个共有峰的相对峰面积和相对保留时间。结果显示24个共有峰的相对峰面积RSD均<2.96%,相对保留时间RSD均<0.22%,表明方法的重复性良好。
稳定性试验
取首乌益智胶囊供试品溶液,按照实施例1提供的方法,于供试品溶液制备0小时开始,在630min(10.5h)内连续进样6次进行测定,以汉黄芩苷色谱峰为参照峰,计算24个共有峰的相对峰面积和相对保留时间。结果显示24个主要峰的相对峰面积RSD均<2.90%,相对保留时间RSD均<0.51%,表明供试品溶液在630min(10.5h)内稳定性良好。
按照实施例1提供的方法,对10批首乌益智胶囊进行了指纹图谱测定,采用《中药色谱指纹图谱相似度评价系统(2004A版)》,采用中位数法生成对照指纹图谱,见图2。各批样品相似度均大于0.812,各批样品与生成的对照图谱相似度均大于0.925,具体结果见表3。
表3 10批首乌益智胶囊指纹谱图相似度计算结果
由表3可知,各批样品与生成的对照图谱比较,相似度均大于0.925,各批样品间相似度均大于0.812,说明首乌益智胶囊工艺相对稳定,产品均一性较好,但由于各批次样品投料用药材的不同,导致了各批样品间有一定差异,用该方法对首乌益智胶囊进行质量控制较可靠。
Claims (7)
1.一种首乌益智胶囊的指纹图谱测定方法,其特征在于,包括以下步骤:
(1)对照品溶液的制备:分别称取大黄素、大黄素甲醚、2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、丹酚酸B、丹参酮ⅡA,丹酚酸B加75%甲醇溶解,其余对照品加甲醇溶解,分别制成对照品溶液;
(2)内标溶液的制备:称取汉黄芩苷,加入甲醇制成浓度为70µg/mL的内标溶液;
(3)供试品溶液制备:取首乌益智胶囊内容物粉末0.5g,加入75%甲醇25mL,回流提取,放凉,用75%甲醇补足减失的重量,摇匀,滤过,取滤液适量,加入浓度为75µg/mL的内标溶液,混匀,过0.45µm微孔滤膜,即得;
(4)利用高效液相色谱法进行分析测定。
2.根据权利要求1所述的指纹图谱测定方法,其特征在于,步骤(1)中,所述大黄素对照品溶液的浓度为10.26µg/mL、大黄素甲醚对照品溶液的浓度为9.68µg/mL、2 , 3 , 5 ,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷对照品溶液的浓度为64.08µg/mL、丹酚酸B对照品溶液的浓度为0.15mg/mL、丹参酮ⅡA对照品溶液的浓度为20µg/mL。
3.根据权利要求2所述的指纹图谱测定方法,其特征在于,所述2, 3, 5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、丹参酮ⅡA的对照品溶液需避光保存。
4.根据权利要求1所述的指纹图谱测定方法,其特征在于,所述回流提取的时间为30min。
5.根据权利要求1所述的指纹图谱测定方法,其特征在于,步骤(3)中,所述滤液同内标溶液的体积比为1:1。
6.根据权利要求1-5任一项所述的指纹图谱测定方法,其特征在于,所述高效液相色谱分离使用Thermo Syncronis C18色谱柱:250 mm×4.6 mm,5μm,在柱温30 ℃,流速1.0 mL/min;检测波长280 nm;进样量10 μL条件下,以体积分数为0.1%甲酸水溶液为流动相A,乙腈为流动相C,进行梯度洗脱。
7.根据权利要求6所述的指纹图谱测定方法,其特征在于,所述梯度洗脱的条件为:0~30min,95~75%流动相A,5~25%流动相C;30~50min,75~73%流动相A,25~27%流动相C;50~60min,73~70%流动相A,27~30%流动相C;60~80min,70~30%流动相A,30~70%流动相C;80~95min,30~25%流动相A,70~75%流动相C;95~105min,25~0%流动相A,75~100%流动项C;所述百分比均为体积百分比。
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