CN114200069A - 一种白术优质药材指纹图谱及指标性成分含量测定的方法 - Google Patents
一种白术优质药材指纹图谱及指标性成分含量测定的方法 Download PDFInfo
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Abstract
本发明涉及一种白术优质药材指纹图谱及指标性成分含量测定的方法,采用包括如下步骤:步骤1:采用高效液相全波长可见紫外检测法,建立白术优质药材的指纹图谱;步骤2:利用液质联用技术对步骤1中指纹图谱的色谱峰进行指认,确定紫外检测共有的指纹峰及非共有峰;步骤3:采用高效液相蒸发光散射检测法对2个结构明确且易测的指标性成分进行含量测定。
Description
技术领域
本发明属于中药分析及中药材含量测定领域,具体涉及一种白术优质药材指纹图谱及指标性成分含量测定的方法。
背景技术
中药白术为菊科苍术属植物白术(Atractylodes macrocephala Koidz.)的干燥根茎,在我国安徽、浙江、江西、湖南、四川等大部分地区都有栽培。临床主要用于补脾气,性温,味甘、微辛带苦,功能健脾益气、燥湿利水、止汗安胎,用于脾虚食少、腹胀泄泻、痰饮、眩悸、水肿、自汗、胎动不安等症。
中药材药效的强弱与药材质量的优劣密切相关,而药材质量的优劣与药材中所含主要化学成分含量的高低紧密相关。中药材中多种已知或未知的化学成分,通过多条途径作用于多靶点发挥其药理作用。目前关于白术药材的定性分析,主要为指纹图谱分析,但之前建立的方法耗时较长,损耗试剂较多,不利于高通量分析,对于复杂成分的指认也不充分;对白术药材中化学成分的定量分析,目前未有统一标准,难以准确、全面地体现中药材的内在质量。中药多指标质量控制和评价模式需要有各种成分的对照品,但部分中药对照品分离难度大、单体不稳定、难以供应或供应价格高等问题的存在,使其不能满足中药质量监控和新药研发等方面的需求,目前白术药材中已测定含量的化合物数量较少。故针对上述复杂成分指认不充分以及定量化学成分较少等缺陷,本发明提供一种白术药材指纹图谱及指标性成分含量测定的方法,该方法能够较全面地对远志药材中化合物进行定性及定量分析。
发明内容
本发明的目的在于提供一种白术优质药材指纹图谱及指标性成分含量测定的方法,该方法能够较全面地对白术药材中化合物进行定性及定量分析。
为实现上述目的,本发明提供的技术方案为:
—种远志药材指纹图谱及指标性成分的含量测定方法,步骤如下:
步骤1:采用高效液相全波长可见紫外检测法,建立白术优质药材的指纹图谱;
混合对照品溶液的制备:取苍术酮对照品适童,精密称定,加甲醇制成每l ml各含0.2mg的溶液,即得。并置于4℃冰箱中备用;
供试品溶液的制备:取白术药材粉末(过四号筛)1.0g,精密称定,置具塞锥形瓶中,精密加入甲醇20ml,称重,超声提取(功率 250W,频率40kHz)30min,放冷后再次称重,用甲醇补足失重,摇匀,取续滤液,即得。
指纹图谱的测定:分别精密吸取参照物溶液和供试品溶液各10μl,注入液相色谱仪,测定,记录色谱图,即得。
其中,色谱条件如下:
以十八烷基硅烷键合硅胶为填充剂(柱长为25cm,内径为4.6 mm,粒径为5μm);以乙腈为流动相A,以水为流动相B,按下表中的规定进行梯度洗脱;流速每分钟为1.0ml;检测波长为220nm;柱温为35℃。理论板数按苍术酮峰计算应不低于20 000。
步骤2:利用液质联用技术对步骤1中指纹图谱的色谱峰进行指认,确定紫外检测共有的指纹峰及非共有峰,其中液相色谱条件同步骤1,质谱条件如下:
离子源:电喷雾离子源(ESI);扫描方式:正负离子同时扫描;喷雾电压:3.5kV;鞘气流速:35psi(lpsi~6.9kPa);辅助气流速:10psi;毛细管温度:320℃;探头加温器温度:300℃;最大喷雾电流:100A;S-Lens分辨率:55;扫描范围:m/z 100-2000;质量分辨率:70000;
步骤3:考虑到指标性成分结构确定且易测,筛选出2个指标性成分(蔗果三糖、蔗果四糖),采用高效液相蒸发光散射检测法对上述 2个指标性成分进行含量测定;
测定方法为:
混合对照品溶液的制备:取蔗果三糖和蔗果四糖对照品适量,精密称定,加水制成每1ml含蔗果三糖70.45μg、蔗果四糖98.72μg的溶液,即得混合对照品溶液。并置于4℃冰箱中备用;
供试品溶液的制备:取白术粗粉约1g,精密称定,精密量取水25ml,称重,超声(500w,40kHZ)30min,再称定重量,加水补足减失的重量;精密量取溶液2mL于10mL容量瓶中,加甲醇稀释至刻度,摇匀,1200r/min离心10min,滤过,取续滤液过0.45μm膜,即得供试品溶液。
色谱条件:
色谱柱选用Waters XBridge Amid(3.5μm,4.6×250nm);流动相:乙腈(A)、水(B),梯度洗脱(0-30min,85%-55%A;30-50min, 55%-45%A;50-55min,45%-85%A;55-60min,85%A);流速1.0ml/min;柱温:40℃;进样量:10μl;蒸发光散射检测器,漂移管温度:105℃;氮气流速:2.6L/min。
相比于现有技术,本发明通过HPLC“指纹图谱定性+指标性成分定量”的方式,从整体和关键组分两方面共同实现质控标准的提升, 不仅全面而特定定量的确定了产品的质量可控性,而且使得白术药材指纹图谱的复杂性表征得以实现。
附图说明
图1为本发明提供的45批白术药材指纹图谱及标准指纹图谱;其中样品编号见表1,R为对照指纹图谱;
图2为各批次白术对照图谱;
图3为白术化学成分二级质谱图
图4为蔗果三糖、蔗果四糖混合对照品溶液的HPLC色谱图; 14min为蔗果三糖,16min为蔗果四糖;
图5为8批白术药材供试品溶液的HPLC色谱图。
具体实施方式
以下实施例用于说明本方法,但不用来限制本发明的范围。
为了更好地理解本发明内容,下面结合附图和具体实施例对本发明作进一步说明。
步骤1:采用高效液相全波长可见紫外检测法,建立白术优质药材的指纹图谱
1.试验材料、仪器与试剂
试验材料:所用白术药材样本是从安徽省不同产地收集到的8批白术药材全根,粉碎,过药典四号筛,备用。试验材料具体信息见表 1。
表1白术样品信息表
仪器:Waters e2695高效液相色谱仪(包括2998PDA二极管阵列检测器,美国Waters公司);KH-250B型超声清洗器(昆山市超声仪器有限公司);AL210型万分之一电子分析天平(梅特勒-托利多仪器有限公司);MS105十万分之一电子分析天平(梅特勒-托利多仪器有限公司);TGL-16B高速离心机(上海安亭科学仪器厂)。中药色谱指纹图谱相似度评价系统2012版。
试剂:乙腈(色谱纯,Fisher公司),甲酸(色谱纯,Fisher公司),超纯水(自制),甲醇(国产分析纯)。
2.混合对照品溶液的制备
取苍术酮对照品适量,精密称定,加甲醇制成每l ml各含0.2 mg的溶液,即得。并置于4℃冰箱中备用;
3.供试品溶液的制备
取白术药材粉末(过四号筛)1.0g,精密称定,置具塞锥形瓶中,精密加入甲醇20ml,称重,超声提取(功率250W,频率40kHz) 30min,放冷后再次称重,用甲醇补足失重,摇匀,取续滤液,即得。
4.指纹图谱的测定
分别精密吸取参照物溶液和供试品溶液各10μl,注入液相色谱仪,测定,记录色谱图,即得。
其中,色谱条件如下:
以十八烷基硅烷键合硅胶为填充剂(柱长为25cm,内径为4.6 mm,粒径为5μm);以乙腈为流动相A,以水为流动相B,按下表中的规定进行梯度洗脱;流速每分钟为1.0ml;检测波长为220nm;柱温为35℃。理论板数按苍术酮峰计算应不低于20 000。
采用“中药色谱指纹图谱相似度评价系统2012版”软件对8批白术药材HPLC指纹图谱进行数据处理,确定共有峰12个(如图1所示)。从图中可以直观看出,相似度<0.900的8个批次白术其生成的对照图谱,峰9(苍术酮)明显低于其余批次白术生成的对照图谱,峰2(白术内酯III)及峰5(白术内酯I)高于其余批次白术生成的对照图谱。
以该参照峰计算各共有峰相对保留时间和相对峰面积,进行指纹图谱方法学考察,测定方法与步骤1中相同。结果显示,同一样品溶液连续进样6次,各共有峰相对保留时间RSDV0.15%,相对峰面积RSDV0.98%,精密度良好;稳定性考察各共有峰相对保留时间RSDV0.42%,相对峰面积RSDV1.83%;重复性考察各共有峰相对保留时间RSDV 0.35%,相对峰面积RSDV2.10%。
8批白术样品的HPLC指纹图谱见图1,各产地远志药材指纹图谱与对照指纹图谱比相似度介于0.863-0.986,结果见表2。
表2 8批白术药材间的相似度计算结果
步骤2:利用液质联用技术对步骤1中指纹图谱的色谱峰进行指认, 确定紫外检测共有的指纹峰
1.试验材料、仪器与试剂
试验材料及试剂:同步骤1。
仪器:Thermo Scientific Q Exactive LC-MS,美国Thermo;其他仪器同步骤1。
2.质谱条件如下:
离子源:电喷雾离子源(ESI);扫描方式:正负离子同时扫描;喷雾电压:3.5kV;鞘气流速:35psi(lpsi~6.9kPa);辅助气流速:10psi;毛细管温度:320℃;探头加温器温度:300℃;最大喷雾电流:100A; S-Lens分辨率:55;扫描范围:m/z 100-2000;质量分辨率:70000;用步骤1中色谱条件对8批远志药材进行HPLC指纹图谱分析。对各样品的12个共有峰紫外光谱图进行对比分析,所得紫外图谱如图1 所示。
采用国家药典委员会推荐的《中药色谱指纹图谱相似度评价系统》生成白术药材标准指纹图谱,其中有12个共有峰,峰号依次记为1-12 号。在指纹图谱中,以2号峰为例,正离子模式下扫描显示以下碎片结果:m/z249[M+H]+,m/z231[M+H-H20]+,m/z213[M+H-2H20]+,m/z185[M+H-2H20-CO]+,m/z105[M+H-H2O-CO-CH2CO-C4H8]+。依据文献中白术内酯III裂解数据并以白术内酯III对照品对照,鉴定 2号峰为白术内酯III。其余成分按该方法,依照相关文献数据[1及对照品比对,结合一级与二级离子碎片信息进行鉴定。
表3白术指纹图谱中成分鉴定
步骤3:考虑到指标性成分结构确定且易测,筛选出2个指标性成分(蔗果三糖、蔗果四糖),采用高效液相蒸发光散射检测法对上述2 个指标性成分进行含量测定
1.试验材料、仪器与试剂同步骤1。
2.混合对照品溶液的制备
取蔗果三糖和蔗果四糖对照品适量,精密称定,加水制成每 1ml含蔗果三糖70.45μg、蔗果四糖98.72μg的溶液,即得混合对照品溶液。并置于4℃冰箱中备用;
3.供试品溶液的制备
取白术粗粉约1g,精密称定,精密量取水25ml,称重,超声(500w, 40kHZ)30min,再称定重量,加水补足减失的重量;精密量取溶液 2mL于10mL容量瓶中,加甲醇稀释至刻度,摇匀,1200r/min离心 10min,滤过,取续滤液过0.45μm膜,即得供试品溶液。
4.色谱条件:
色谱柱选用Waters XBridge Amid(3.5μm,4.6×250nm);流动相:乙腈(A)、水(B),梯度洗脱(0-30min,85%-55%A;30-50min, 55%-45%A;50-55min,45%-85%A;55-60min,85%A);流速 1.0ml/min;柱温:40℃;进样量:10μl;蒸发光散射检测器,漂移管温度:105℃;氮气流速:2.6L/min。
5.指标性成分的含量测定
分别精密吸取上述混合对照品溶液和供试品溶液各10μl注入高效液相色谱仪,确定各待测成分的色谱峰,并将混合对照品溶液逐级稀释,建立2个指标性成分的线性回归方程,分别计算供试品溶液中蔗果三糖、蔗果四糖2个指标性成分的含量,其中,2个指标性成分的线性回归方程见表4、表5。
表4蔗果三糖线性关系考察结果
表5蔗果四糖线性关系考察结果
6.精密度考察
精密度试验精密量取蔗果三糖、蔗果四糖混合对照品溶液10μl, 按步骤3中的色谱条件进行测定,连续进样6次,记录上述2个化合物的色谱峰面积,并计算计算目标成分的浓度(mg·ml-1)并计算目标成分浓度的相对标准偏差RSD值分别为1.56%、1.64%表明仪器精密度良好。具体数据见表6-7。
表6对照品精密度试验结果(峰面积)
7.稳定性试验
取同一批样品,按步骤3中备样方法平行制备6份,室温下放置, 按步骤1中色谱条件分别在0、2、4、8、12、18、24h时进样分析,并计算蔗果三糖、蔗果四糖峰面积的相对标准偏差RSD。结果蔗果三糖、蔗果四糖稳定性的RSD值均小于3.00%,表明该含测方法重复性好,见表8。
表8样品稳定性试验结果(峰面积)
8.重复性
按照步骤3条件制备供试品,平行6份,各进2针,改变混合对照品溶液的进样量作为随行对照,计算目标成分的浓度(mg·ml-1) 并计算目标成分浓度的相对标准偏差。结果表明目标成分重复性的 RSD值均小于2.00%,表明该含测方法重复性好,见表9。
表9样品重复性试验结果
9.加样回收率试验
取蔗果三糖和蔗果四糖对照品适量,精密称定,加水制成每1ml 含蔗果三糖173.2μg、蔗果四糖257.6μg的溶液,即得加样混合对照品溶液。
按照步骤3的方法制备水煎液,按照低、中、高浓度原则各平行 3份。分别精密移取0.5g白术粉末至锥形瓶中,再分别加入1.6、2.0、 2.4ml的加样混合对照品溶液于锥形瓶中,精密量取水25ml,称重,超声(500w,40kHZ)30min,再称定重量,加水补足减失的重量;精密量取溶液2mL于10mL容量瓶中,加甲醇稀释至刻度,摇匀, 1200r/min离心10min,滤过,取续滤液过0.45μm膜,即得供试品溶液。
按照以下公式计算加样回收率,并计算相对标准偏差RSD:
加样回收率(%)=(mmea-msam)/madd×100%
其中mmea为加标样品测得的含量(mg),msam为样品理论的含量,madd为加标量。结果表明目标成分加样回收率在合理范围内,重复性良好,该检测方法具有一定的准确性,见表10-11。
表10蔗果三糖加样回收率结果
表11蔗果四糖加样回收率结果
10.样品含量测定
按步骤3中备样方法制备8批供试品溶液,分别测定8批白术样品的蔗果三糖、蔗果四糖含量。8批白术药材2个指标性成分含量测定结果见表12,混合对照品的色谱图如图4所示,白术样品的色谱图如图5所示。
表12 8批白术蔗果三糖、蔗果四糖含量测定
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (2)
1.本发明涉及一种白术优质药材指纹图谱及指标性成分含量测定的方法,包括如下步骤:
步骤1:采用高效液相全波长可见紫外检测法,建立白术优质药材的指纹图谱;
步骤2:利用液质联用技术对步骤1中指纹图谱的色谱峰进行指认,确定紫外检测共有的指纹峰及非共有峰;
步骤3:考虑到指标性成分结构确定且易测,筛选出2个指标性成分(蔗果三糖、蔗果四糖),采用高效液相蒸发光散射检测法对上述2个指标性成分进行含量测定。
2.一种白术优质药材指纹图谱及指标性成分含量测定的方法,其特征在于,步骤如下:
步骤1:采用高效液相全波长可见紫外检测法,建立白术优质药材的指纹图谱;
混合对照品溶液的制备:取苍术酮对照品适童,精密称定,加甲醇制成每lml各含0.2mg的溶液,即得。并置于4℃冰箱中备用;
供试品溶液的制备:取白术药材粉末(过四号筛)1.0g,精密称定,置具塞锥形瓶中,精密加入甲醇20ml,称重,超声提取(功率250W,频率40kHz)30min,放冷后再次称重,用甲醇补足失重,摇匀,取续滤液,即得。
指纹图谱的测定:分别精密吸取参照物溶液和供试品溶液各10μl,注入液相色谱仪,测定,记录色谱图,即得。
其中,色谱条件如下:
以十八烷基硅烷键合硅胶为填充剂(柱长为25cm,内径为4.6mm,粒径为5μm);以乙腈为流动相A,以水为流动相B,按下表中的规定进行梯度洗脱;流速每分钟为1.0ml;检测波长为220nm;柱温为35℃。理论板数按苍术酮峰计算应不低于20 000。
步骤2:利用液质联用技术对步骤1中指纹图谱的色谱峰进行指认,确定紫外检测共有的指纹峰及非共有峰,其中液相色谱条件同步骤1,质谱条件如下:
离子源:电喷雾离子源(ESI);扫描方式:正负离子同时扫描;喷雾电压:3.5kV;鞘气流速:35psi(lpsi~6.9kPa);辅助气流速:10psi;毛细管温度:320℃;探头加温器温度:300℃;最大喷雾电流:100A;S-Lens分辨率:55;扫描范围:m/z100-2000;质量分辨率:70000;
步骤3:考虑到指标性成分结构确定且易测,筛选出2个指标性成分(蔗果三糖、蔗果四糖),采用高效液相蒸发光散射检测法对上述2个指标性成分进行含量测定;
测定方法为:
混合对照品溶液的制备:取蔗果三糖和蔗果四糖对照品适量,精密称定,加水制成每1ml含蔗果三糖70.45μg、蔗果四糖98.72μg的溶液,即得混合对照品溶液。并置于4℃冰箱中备用;
供试品溶液的制备:取白术粗粉约1g,精密称定,精密量取水25ml,称重,超声(500w,40kHZ)30min,再称定重量,加水补足减失的重量;精密量取溶液2mL于10mL容量瓶中,加甲醇稀释至刻度,摇匀,1200r/min离心10min,滤过,取续滤液过0.45μm膜,即得供试品溶液。
色谱条件:
色谱柱选用Waters XBridge Amid(3.5μm,4.6×250nm);流动相:乙腈(A)、水(B),梯度洗脱(0-30min,85%-55%A;30-50min,55%-45%A;50-55min,45%-85%A;55-60min,85%A);流速1.0ml/min;柱温:40℃;进样量:10μl;蒸发光散射检测器,漂移管温度:105℃;氮气流速:2.6L/min。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102680631A (zh) * | 2012-05-24 | 2012-09-19 | 成都中医药大学 | 一种白术药材的检测方法 |
KR101831436B1 (ko) * | 2017-05-31 | 2018-02-22 | 서울대학교산학협력단 | 크로마토그래프지문의 유사성 비교를 통한 원료의 품질 평가 방법 |
CN109580857A (zh) * | 2018-12-29 | 2019-04-05 | 北京中研同仁堂医药研发有限公司 | 白术特征图谱构建方法和质量检测方法 |
CN109884219A (zh) * | 2019-04-15 | 2019-06-14 | 国药集团广东环球制药有限公司 | 白术药材uplc特征图谱的构建方法和检测方法 |
-
2021
- 2021-09-17 CN CN202111092172.2A patent/CN114200069A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102680631A (zh) * | 2012-05-24 | 2012-09-19 | 成都中医药大学 | 一种白术药材的检测方法 |
KR101831436B1 (ko) * | 2017-05-31 | 2018-02-22 | 서울대학교산학협력단 | 크로마토그래프지문의 유사성 비교를 통한 원료의 품질 평가 방법 |
CN109580857A (zh) * | 2018-12-29 | 2019-04-05 | 北京中研同仁堂医药研发有限公司 | 白术特征图谱构建方法和质量检测方法 |
CN109884219A (zh) * | 2019-04-15 | 2019-06-14 | 国药集团广东环球制药有限公司 | 白术药材uplc特征图谱的构建方法和检测方法 |
Non-Patent Citations (6)
Title |
---|
DAN ZHUANG ET AL.,: "Oligosaccharide-based quality evaluation of Atractylodis rhizome and a strategy for simplifying its quality control", BMC CHEMISTRY, 12 July 2019 (2019-07-12), pages 2 - 5 * |
ZHE LIN ET AL.: "Characterisation of oligosaccharides from Baizhu by HILIC-MS", NATURAL PRODUCT RESEARCH, pages 2 - 5 * |
刘国厂: "运动饮料中糖分的检测", 食品研究与开发, vol. 38, no. 11, pages 154 - 155 * |
孙学;文红梅;崔小兵;陆兔林;李伟;单晨啸;: "HPLC-PDA指纹图谱结合UFLC-Q-TOF/MS定性鉴别评价不同产地白术药材质量", 中草药, no. 19, 12 October 2016 (2016-10-12), pages 3495 - 3497 * |
孙学;文红梅;崔小兵;陆兔林;李伟;单晨啸;: "HPLC-PDA指纹图谱结合UFLC-Q-TOF/MS定性鉴别评价不同产地白术药材质量", 中草药, no. 19, pages 3495 - 3497 * |
陈向东;张光大;兰小勇;刘法锦;魏梅;: "HPLC指纹图谱对白术药材的质量评价研究", 中药材, no. 02 * |
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