CN110146611B - 一种快速识别驴胶补血颗粒中化学成分的方法 - Google Patents
一种快速识别驴胶补血颗粒中化学成分的方法 Download PDFInfo
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Abstract
一种快速识别驴胶补血颗粒化学成分的分析方法,属于中药制剂分析领域,旨在为驴胶补血颗粒的化学成分分析提供方法,包括如下步骤:(1)取驴胶补血颗粒样品经研细、提取、除杂,制得供试品溶液;(2)将供试品溶液注入UHPLC‑Q Exactive轨道肼高分辨质谱联用仪进行测定;(3)建立系列成分的UHPLC‑Q Exactive轨道肼高分辨质谱分析方法,结合色谱保留时间、紫外吸收光谱特征、分子量及多级裂解离子碎片信息等进行结构鉴定。本发明可以避免样品前处理方法,最大程度地保留样品中的原有成分;提高原位色谱分离效果,使微量成分的浓度得到富集而被检测,具有高分离能力、高灵敏度和专属性强的特点。
Description
技术领域
本发明属于中药制剂分析技术领域,具体涉及一种采用UHPLC-Q Exactive轨道肼高分辨质谱技术快速识别驴胶补血颗粒中主要化学成分的方法。
背景技术
驴胶补血颗粒是九芝堂股份有限公司生产的现代中药制剂,具有滋阴补血、健脾益气的功效,对于久病体虚、血亏气虚、疲乏无力、手术后失血过多、及肿瘤病人化疗后白细胞减少等症有显著疗效。该制剂驴胶为君药,配以白术、党参、黄芪等健脾益气之品,再辅以当归养血,从而达到气血双补气血足、强身健体精力旺的功效。然而,驴胶补血颗粒中的主要化学成分以及功效物质基础研究报道较少,多为单纯使用高效液相色谱进行分析,具有进样量大、分析时间长、需大量标准品进行指认且无法获得全部化学成分信息等缺点。因此,全面快速表征驴胶补血颗粒化学成分对于进一步阐明该药物药效物质基础以及作用机制具有重要意义和价值。
当前,单纯依靠传统的提取、分离、结构鉴定三部曲的天然产物研究方法以及以活性追踪为核心的天然产物研究方法,虽然能比较系统、深入地研究天然药物中的化学成分或活性成分,但是耗时长、成本高、盲目性强,且无法获得天然药物中所有成分的结构信息,具有很多的局限性。因此,复杂物质体系分析方法的研究已成为药物分析领域的前沿课题之一。
液质联用技术具有高分离能力、高灵敏度、专属性强和耗时短的特点,在中药及天然产物的定性鉴别中发挥着重要的作用。UHPLC-Q Exactive轨道高分辨质谱将四级杆母离子选择性与高分辨率和准确质量数(HRAM)Orbitrap检测相结合,具有高灵敏度,在一次扫描中同时检测痕量级和高丰度化合物,可用于中药复杂体系的快速分析和成分识别。本发明采用UHPLC-Q Exactive轨道肼高分辨质谱技术快速鉴定驴胶补血颗粒中的主要化学成分,基于中成药中复杂化学成分的结构鉴定策略,旨在通过提高原位色谱分离效果,使微量成分的浓度得到富集而被检测,以期为驴胶补血颗粒复杂的化学成分分析提供方法。
发明内容
本发明采用UHPLC-Q Exactive轨道肼高分辨质谱对驴胶补血颗粒的化学成分进行分析鉴定,得到所含成分的准分子离子峰及蕴含丰富结构信息的碎片离子峰,从而对其结构进行准确、快速的鉴定,为驴胶补血颗粒质量控制和药效物质基础研究奠定依据。
本发明采用如下技术方案:
一种对驴胶补血颗粒化学成分的定性检测方法,包括以下步骤:
(1)供试品溶液的制备,取驴胶补血颗粒样品经研细、提取、除杂,制得供试品溶液;
(2)将供试品溶液注入UHPLC-Q Exactive轨道肼高分辨质谱联用仪进行测定;
(3)建立系列成分的UHPLC-Q Exactive轨道肼高分辨质谱分析方法,并结合色谱保留时间、紫外吸收光谱特征、分子量以及多级裂解离子碎片信息等进行快速的结构鉴定。
进一步地,所述UHPLC-Q Exactive轨道肼高分辨质谱分析方法中样品溶液的制备包括如下步骤:
(1)制剂供试品溶液的制备:取3批驴胶补血颗粒混匀,称取2g研细,置于50mL锥形瓶中,加50%甲醇25mL,室温超声处理30min,静置冷却至室温,用定性滤纸过滤,过0.22μm微孔滤膜,取续滤液,即得;
(2)对照品溶液的制备:分别取各对照品适量,精密称定,用甲醇制备含绿原酸、新绿原酸、隐绿原酸、阿魏酸,咖啡酸、黄芪甲苷Ⅳ、芒柄花苷、黄芪皂苷Ⅰ、白术内酯Ⅰ、白术内酯Ⅱ对照品各1.0mg/mL的对照品储备液,各取200μL到5mL量瓶,用50%甲醇溶液定容,制备成混合对照品溶液。
进一步地,所述UHPLC-Q Exactive轨道肼高分辨质谱分析方法UHPLC条件如下:
以色谱柱为Waters Acquity UPLC BEH C18液相色谱柱(2.1mm×100mm,1.7μm),流动相为A:0.1%甲酸水溶液;B:乙腈。梯度洗脱,0~3min,1%B;3~8min,1%~3%B;8~10min,3%~20%B;10~15min,20%B;15~18min,20%~35%B;18~20min,35%B;20~23min,35%~45%B;23~28min,45%~55%B;28~30min,55%~90%B,30~32min,90%B;32~32.5min,90%~1%B;32.5~35min,1%B。检测波长为290nm,柱温为45℃,流速为0.2mL/min,进样量为1μL。
进一步地,所述UHPLC-Q Exactive轨道肼高分辨质谱分析方法质谱条件如下:
Full MS/DD MS2(TOPN);离子源:电喷雾离子源(HESI);扫描方式:正负离子同时扫描;毛细管温度:320℃;鞘气体积流量为35psi(1psi≈6.9kPa),辅助气体积流量为10psi;喷雾电压:正离子模式为3.5kV,负离子模式为2.5kV,透镜电压55kPa;探头加温器温度:300℃;最大喷雾电流:100V;NEC:10,30,50;质量扫描范围均为m/z 100~1500,质量分辨率70000。
进一步地,所述UHPLC-Q Exactive轨道肼高分辨质谱分析方法的数据处理流程如下:
将采集到的原始质谱数据运用Xcalibur 3.2数据软件进行峰提取、峰匹配分析处理。将采集到的原始质谱数据导入Compound Discover 2.0软件,与数据库进行计算匹配,导出样品名称、保留时间、分子式、质荷比、以及对应的离子强度组成的数据集,主要参数设置如下:保留时间范围0~25min;质荷比范围为100~1500Da;偏差为5ppm:信噪比为3;保留时间漂移值为0.1min。
相对于现有技术,本发明具有以下优势:
中药成分复杂,分离困难,薄层色谱(TLC)或高效液相色谱(HPLC)有时很难对中药成分做出分析检测。而UHPLC-Q Exactive轨道肼高分辨质谱分析方法可以避免繁琐和复杂的样品前处理,同时能得到化合物保留时间、分子量以及特征结构碎片等丰富的信息。此外,UHPLC-Q Exactive轨道肼高分辨质谱分析方法相对于其他方法,具有快速、高效、灵敏等优点。
附图说明
图1为快速识别驴胶补血颗粒化学成分的分析方法研究策略图;
图2为驴胶补血颗粒正离子模式下的UHPLC-Q Exactive轨道肼高分辨质谱总离子流图;
图3为驴胶补血颗粒负离子模式下的UHPLC-Q Exactive轨道肼高分辨质谱总离子流图。
具体实施方式
一种快速识别驴胶补血颗粒化学成分的分析方法,包括以下步骤:
(1)材料与试剂
驴胶补血颗粒由九芝堂股份有限公司提供。绿原酸、新绿原酸、隐绿原酸、阿魏酸,咖啡酸、黄芪甲苷Ⅳ、芒柄花苷、黄芪皂苷Ⅰ、白术内酯Ⅰ、白术内酯Ⅱ等对照品购自购自四川省维克奇生物科技有限公司和自江苏永健医药有限公司,其结构经1H-NMR、13C-NMR等手段得以确定,纯度经HPLC归一化法检测,均大于95%,可用于定性研究。
超纯水由Milli-Q Integral Water Purification System(美国Millipore公司)制备,甲酸、乙腈为色谱纯(美国Thermo公司),甲醇(分析纯)购于天津市大茂化学试剂厂。
(2)仪器
Thermo fisher U3000超高效液相色谱仪,配置在线脱气机、四元梯度泵、柱温箱、自动进样器(美国Thermo Fisher Scientific公司),Thermo Scientific Q Exactive组合型四极杆Orbitrap质谱仪;十万分之一天平(梅特勒-托利多仪器有限公司)。
(3)样品溶液的制备
制剂供试品溶液的制备:取3批驴胶补血颗粒混匀,称取2g研细,置于50mL锥形瓶中,加50%甲醇25mL,室温超声处理30min,静置冷却至室温,用定性滤纸过滤,过0.22μm微孔滤膜,取续滤液,即得;
对照品溶液的制备:分别取各对照品适量,精密称定,用甲醇制备含绿原酸、新绿原酸、隐绿原酸、阿魏酸,咖啡酸、黄芪甲苷Ⅳ、芒柄花苷、黄芪皂苷Ⅰ、白术内酯Ⅰ、白术内酯Ⅱ对照品各1.0mg/mL的对照品储备液,各取200μL到5mL量瓶,用50%甲醇溶液定容,制备成混合对照品溶液。
(4)UHPLC-Q Exactive轨道肼高分辨质谱分析方法
UHPLC条件:以色谱柱为Waters Acquity UPLC BEH C18液相色谱柱(2.1mm×100mm,1.7μm),流动相为A:0.1%甲酸水溶液;B:乙腈。梯度洗脱,0~3min,1%B;3~8min,1%~3%B;8~10min,3%~20%B;10~15min,20%B;15~18min,20%~35%B;18~20min,35%B;20~23min,35%~45%B;23~28min,45%~55%B;28~30min,55%~90%B,30~32min,90%B;32~32.5min,90%~1%B;32.5~35min,1%B。检测波长为290nm,柱温为45℃,流速为0.2mL/min,进样量为1μL。
质谱条件:Full MS/DD MS2(TOPN);离子源:电喷雾离子源(HESI);扫描方式:正负离子同时扫描;毛细管温度:320℃;鞘气体积流量为35psi(1psi≈6.9kPa),辅助气体积流量为10psi;喷雾电压:正离子模式为3.5kV,负离子模式为2.5kV,透镜电压55kPa;探头加温器温度:300℃;最大喷雾电流:100V;NEC:10,30,50;质量扫描范围均为m/z 100~1500,质量分辨率70000。
(5)数据处理
将采集到的原始质谱数据运用Xcalibur 3.2数据软件进行峰提取、峰匹配分析处理。将采集到的原始质谱数据导入Compound Discover 2.0软件,与数据库进行计算匹配,导出样品名称、保留时间、分子式、质荷比、以及对应的离子强度组成的数据集,主要参数设置如下:保留时间范围0~25min;质荷比范围为100~1500Da;偏差为5ppm:信噪比为3;保留时间漂移值为0.1min。
(6)分析结果
根据文献报道、TCMSP数据库和标准品,结合色谱保留行为、分子量信息以及质谱裂解规律,对其中的化学成分进行鉴定,结果见表1和表2。
综上,采用UHPLC-Q Exactive轨道肼高分辨质谱对驴胶补血颗粒中的各类成分进行色谱分离、质谱数据采集以及固有成分的结构鉴定,最终从采用UHPLC-Q Exactive轨道肼高分辨质谱中鉴定氨基酸、有机酸、环烯醚萜苷、皂苷、内酯、黄酮等各类成分共65个,为进一步研究其药效物质基础提供了依据和前提。
表1驴胶补血颗粒负离子模式下化学成分质谱信息
表2驴胶补血颗粒正离子模式下化学成分质谱信息
Claims (1)
1.一种快速识别驴胶补血颗粒化学成分的分析方法,其特征在于:包括如下步骤:
步骤一,供试品溶液的制备,取驴胶补血颗粒样品经研细、提取、除杂,制得供试品溶液;
步骤二,将样品溶液注入UHPLC-Q Exactive 轨道肼高分辨质谱联用仪进行测定;
步骤三,建立系列成分的UHPLC-Q Exactive 轨道肼高分辨质谱分析方法,并结合色谱保留时间、紫外吸收光谱特征、分子量以及多级裂解离子碎片信息进行快速的结构鉴定;
所述供试品溶液的制备,包括如下步骤:
取3批驴胶补血颗粒混匀,称取2g研细,置于50mL锥形瓶中,加50%甲醇25mL,室温超声处理30min,静置冷却至室温,用定性滤纸过滤,过0.22μm微孔滤膜,取续滤液,即得;
对照品溶液的制备:分别取各对照品适量,精密称定,用甲醇制备含绿原酸、新绿原酸、隐绿原酸、阿魏酸,咖啡酸、黄芪甲苷Ⅳ、芒柄花苷、黄芪皂苷Ⅰ、白术内酯Ⅰ、白术内酯Ⅱ对照品各1.0 mg/mL的对照品储备液,各取200 µL到5 mL 量瓶,用50%甲醇溶液定容,制备成混合对照品溶液;
所述UHPLC-Q Exactive 轨道肼高分辨质谱分析方法的UHPLC条件如下:
以色谱柱为 Waters Acquity UPLC BEH C18 液相色谱柱,所述色谱柱2.1 mm×100mm,1.7 µm,流动相为 A: 0.1%甲酸水溶液; B: 乙腈;梯度洗脱,0 ~ 3 min,1% B;3 ~ 8min,1% ~ 3% B;8 ~ 10 min,3% ~ 20% B;10 ~ 15 min,20% B;15 ~ 18 min,20% ~ 35%B;18 ~ 20 min,35 % B;20 ~ 23 min,35% ~ 45% B;23 ~ 28 min,45% ~ 55% B;28 ~ 30min,55% ~ 90% B,30 ~ 32 min,90% B;32 ~ 32.5 min,90% ~ 1% B;32.5 ~ 35 min,1%B;检测波长为 290 nm,柱温为 45℃,流速为 0.2 mL/min,进样量为1 μL;
所述UHPLC-Q Exactive 轨道肼高分辨质谱分析方法的质谱条件如下:
Full MS/DD MS2;离子源:电喷雾离子源;扫描方式:正负离子同时扫描;毛细管温度:320 ℃;鞘气体积流量为 35 psi,辅助气体积流量为10 psi;喷雾电压:正离子模式为3.5kV,负离子模式为2.5 kV,透镜电压55 kPa;探头加温器温度:300℃;最大喷雾电流:100 V;NEC:10,30,50;质量扫描范围均为m/z 100~1500,质量分辨率70000;
所述UHPLC-Q Exactive 轨道肼高分辨质谱分析方法的数据处理流程如下:
将采集到的原始质谱数据运用Xcalibur 3.2 数据软件进行峰提取、峰匹配分析处理;将采集到的原始质谱数据导入 Compound Discover 2.0软件,与数据库进行计算匹配,导出样品名称、保留时间、分子式、质荷比、以及对应的离子强度组成的数据集,主要参数设置如下:保留时间范围0~25 min;质荷比范围为 100 ~ 1500 Da;偏差为 5 ppm:信噪比为3;保留时间漂移值为0.1 min。
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