CN103926330B - 一种酒炙豨莶草的检测方法 - Google Patents
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Abstract
本发明涉及一种酒炙豨莶草的检测方法,该方法采用UPLC/Q-TOF-MS进行,本发明提供的检测方法操作简单、耗时短、区分明显。
Description
技术领域
本发明涉及鉴别方法,具体涉及一种酒炙豨莶草的检测方法。
背景技术
豨莶草为菊科豨莶属植物豨莶(Siegesbeckia.orientalisL)、腺梗豨莶(SiegesbeckiaPuescensMakina)及毛梗豨莶(Siegesbeckia.glabrescensMakino)的干燥地上部分。性味寒、苦、微辛。具有祛风湿,利关节,解毒的功效[1]。其主要成分有萜类、内酯类、苷类等。豨莶草生品及炮制品广泛用于临床,经典方剂豨莶丸、首乌丸均以酒炙豨莶草入药。传统认为豨莶草酒炙后气味香美,药性转寒为温,活血祛风之性未改,而温养之力更加益元气,祛风逐湿之中有补益肝肾之功。
豨莶草的鉴别方法与含量方法详见《中国药典》2010年版,具体为:色谱条件与系统适用性试验:以十八烷基硅烷键合硅胶为填充剂;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;检测波长为215nm。理论板数按奇壬醇峰计算应不低于5000。
时间(分钟) | 流动相A(%) | 流动相B(%) |
0~5 | 5→24 | 95→76 |
5~30 | 24 | 76 |
对照品溶液的制备:取奇壬醇对照品适量,精密称定,加甲醇制成每1ml含20μg的溶液,即得。
供试品溶液的制备:取本品粉末(过三号筛)约1g,精密称定,置具塞锥形瓶中,精密加入甲醇50ml,称定重量,加热回流5小时,放冷,再称定重量,用甲醇补足减失的重量,摇匀,滤过,取续滤液,即得。
测定法:分别精密吸取对照品溶液与供试品溶液各20μl,注入液相色谱仪,测定,即得。
虽然上述鉴别或含量方法可以用于炮制或未炮制的豨莶草,但是不利于对炮制后的豨莶草,尤其是酒炙豨莶草的鉴别。
UPLC/Q-TOF-MS分析方法是超高效液相色谱-四极杆联合飞行时间串联质谱分析方法,通过高效液相的联用,可以方便得到每个样品的主要结构信息,并针对每个组分进行准确的定性分析。四级杆质谱是在交变电场的作用下没事某些符合要求的离子通过四级杆到达检测器,飞行时间质谱(TOF)是应用不同的m/z离子的飞行速度不同,离子飞行通过相同的路径到达检测器的时间不同而获得质量分离。四级杆串联飞行时间质谱不仅用于一级质谱的分析而且可以实现二级质谱分析,一级质谱可得出化合物的分子量信息,二级质谱则可以得到化合物的碎片离子等结构信息。
目前,UPLC/Q-TOF-MS可以广泛运用中药化学成分的快速鉴定定性分析,它可以使样品的分离和定性及定量成为一个连续的过程,费时费力而且对环境不好,UPLC/Q-TOF-MS的快速分析鉴别,能够在短时有效成分分离的基础上,获得大量的化合物结构信息,在中药化学成分分析中发挥了重要的作用。
目前尚未见到UPLC-Q-TOF/MS在用于酒炙豨莶草检测方面的报道。
发明内容
本发明的目的是提供一种酒炙豨莶草的检测方法。
本发明提供的一种酒炙豨莶草的检测方法,该方法采用UPLC/Q-TOF-MS分析方法进行。
具体的,所述检测方法包括以下步骤:
色谱分析条件为:WatersACQUITYUPLCTMBEHC18Column(50mm×2.1mm.id.,1.7μm)色谱柱;二元梯度洗脱,A相为水,B相为乙腈,流速0.25mL/min;进样量5μL。
Q-TOF/MS条件:采用电离离子源,正/负离子模式检测:质量扫描范围为50-1200。
优选地,所述检测方法包括以下步骤:
色谱条件:色谱柱:采用WatersACQUITYUPLCTMBEHC18Column(50mm×2.1mm。id.,1.7μm)色谱柱;柱温为30℃,流速为0.25mL/min,进样量为5μL;流动相A为水;流动相B为乙腈;如下表的梯度洗脱:
时间(分钟) | 流动相A(%) | 流动相B(%) |
0-4 | 95%→76% | 5→24 |
4-14 | 76%→70% | 24→30% |
14→17 | 70%→50% | 30%→50% |
17→20 | 50%→38% | 50%→62% |
20→23 | 38%→20% | 62%→80% |
23→27 | 20%→95% | 80%→5% |
Q-TOF/MS条件,质谱采用电喷雾电离源(ESI),TOF离子飞行方式采用Ⅴ模式;四级杆质量扫描m/z范围50-1200,一次扫描时间为0.1s,离子源温度120℃,脱溶剂氮气流速800L/h,正离子电离模式,毛细管电离电压3kV,取样椎孔电压40V,碰撞能量(CE):30V;负离子电离模式,毛细管电离电压3kV,取样椎孔电压40V,碰撞能量(CE):30V。
另外,正离子电离模式质量校正质核比为m/z556.2771;负离子电离模式,质量校正质核比为m/z554.2617。
数据处理,LC-MC的原始数据文件通过WatersMicromassMassV4.1数据处理系统获得,对于每个离子的鉴定通过正离子和负离子模式下的二级质谱进一步确认。
本发明提供的酒炙豨莶草的质量控制方法具有以下优点:
1、本发明提供的检测方法操作简单、耗时短、区分明显。
2、本发明提供的UPLC/Q-TOF-MS分析方法,限定了梯度洗脱,色谱条件分离度较好,基线较平稳,适合豨莶草中成分的测定。
3、研究证实,豨莶草酒炙前后成分差异显著,其成分的化学变化是酒炙后药性变化和增效的基础,因而可通过合理的炮制应用于临床治疗。对于豨莶草的生品与酒炙品能很好的检测和区分,同时也为豨莶草药效物质基础的阐明提供了重要依据。
附图说明
图1-1:正离子模式下豨莶草酒炙品及生品UPLC-Q-TOF/MS的分析总离子流色谱图;
图1-2:负离子模式下豨莶草酒炙品及生品UPLC-Q-TOF/MS的分析总离子流色谱图;
图2-1、图2-2、图2-3、图2-4:均为豨莶草生品与酒炙豨莶草的主要成分离子变化趋势图,其中图2-1为豨莶酮的变化趋势图;图2-2为槲皮素的变化趋势图;图2-3为奇壬醇的变化趋势图;图2-4为豨莶酸的变化趋势图,图2-1、图2-2、图2-3、图2-4中,所有的横坐标123为生品中含量,456为酒炙品中含量。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例:检测方法
1、实验试剂
甲醇为色谱纯,美国Fisher公司;
超纯水,实验室ELGAPURELABClassic-UVF纯水机制备,该纯水机购自英国;
其他试剂均为市售国产分析纯。
2、实验样品
酒炙豨莶草:按照药典方法,即《中国药典》2010版收载了酒炙豨莶草,规定每100kg豨莶草,用黄酒20kg,照酒蒸法(附录ⅡD)蒸透,具体方法为:取净药材,加酒拌匀,置锅内,用文火炒至规定的程度时,取出,放凉。
豨莶草生品。
3、实验条件
3.1色谱条件为:色谱柱:采用WatersACQUITYUPLCTMBEHC18Column(50mm×2.1mm。id.,1.7μm)色谱柱;柱温为30℃,流速为0.25mL/min,进样量为5μL;流动相A为水;流动相B为乙腈;其梯度洗脱见表1:
表1:梯度洗脱
时间(分钟) | 流动相A(%) | 流动相B(%) |
0-4 | 95%→76% | 5→24 |
4-14 | 76%→70% | 24→30% |
14→17 | 70%→50% | 30%→50% |
17→20 | 50%→38% | 50%→62% |
20→23 | 38%→20% | 62%→80% |
23→27 | 20%→95% | 80%→5% |
3.2Q-TOF/MS条件,质谱采用电喷雾电离源,TOF离子飞行方式采用Ⅴ模式;四级杆质量扫描m/z范围50-1200,一次扫描时间为0.1s,离子源温度120℃,脱溶剂氮气流速800L/h,正离子电离模式,毛细管电离电压3kV,取样椎孔电压40V,碰撞能量:30V;负离子电离模式,毛细管电离电压3kV,取样椎孔电压40V,碰撞能量:30V。
3.3所述正离子电离模式质量校正质核比为m/z556.2771;负离子电离模式,质量校正质核比为m/z554.2617。
4、数据处理,LC-MC的原始数据文件通过WatersMicromassMassV4.1数据处理系统获得,对于每个离子的鉴定通过正离子和负离子模式下的二级质谱进一步确认。
5、实验结果:
5.1酒炙豨莶草及生品的总离子流色谱峰组分指纹信息:见图1-1、图1-2和表2
图1-1为正离子模式下酒炙豨莶草与豨莶草总离子流图;图1-2为负离子模式下酒炙豨莶草与豨莶草总离子流图。
对图1-1、图1-2的总离子流图组分的指纹信息进行分析,得到表2:
表2:酒炙豨莶草及生品的总离子流色谱峰组分指纹信息
表2结果显示:
正离子模式下(见图1-1),与生品比较,酒炙品中离子峰1﹑2﹑3﹑6即豨莶苷(darutoside)﹑3,,4,-去二磺酸基苍术苷(3′,4′-dedisulphatedatractyloside)﹑豆甾醇-3-O-β-D-吡喃葡萄糖苷(stig-masterol-3-O-β-D-glucopyranosid)﹑对映-16α,17-二羟基-19-羧酸(ent-16α,17-dihy-droxy-19-kauranoicacid)含量明显升高,;离子峰4﹑5﹑7﹑8即对映-18-乙酰氧基-16α,17-二羟基-贝壳杉烷-19-羧酸(ent-18-acetoxy-16α,17-dihydroxykauran-19-oicacid)﹑16-O-Acetyldarutoside﹑木犀草素(Luteolin)﹑对映-18-乙酰氧基-17-二羟基16βH-贝壳杉烷-19-羧酸(ent-18-acetoxy-17-hydroxy-16βH-kauran-19-oicacid)含量明显降低,。
负离子模式下(图1-2),与生品比较,酒炙品中离子峰1﹑2﹑9﹑10﹑13即豨莶苷(darutoside)﹑3,,4,-去二磺酸基苍术苷(3′,4′-dedisulphatedatractyloside)﹑2-氨基-3(3,-羟基-2,-甲氧苯基)1-丙醇(2-amino-3(3’-hydroxy-2’-methoxy-lphenyl)1-propanol)﹑hythiemosideB﹑γ-十二烷基-α羟基γ内酯(γ–dodecylalpha-α-hydroxy-γlactone)含量明显升高;离子峰11﹑12﹑14﹑15即矢车菊黄素(centaureidin)﹑4-羰基-α-卡拉布烷-11(13)-烯-12,8β-内酯(4-carbonylgroup-α-caraclothhydride-11(13)-olefinic-12,8β-lactone)﹑单棕榈酸甘油酯(glyeeroylmonopalnlitate)﹑D-甘露醇(D-mannito)含量明显下降。
5.2主要成分离子变化趋势:见图2-1、图2-2、图2-3、图2-4
图2-1、图2-2、图2-3、图2-4均为豨莶草生品与酒炙豨莶草的主要成分离子变化趋势图,其中图2-1为豨莶酮的变化趋势图;图2-2为槲皮素的变化趋势图;图2-3为奇壬醇的变化趋势图;图2-4为豨莶酸的变化趋势图,上述图中,所有的横坐标123为生品中含量,456为酒炙品中含量;中药炮制原理的核心是中药饮片炮制后其药性发生了改变,而根源还是炮制后其内在物质基础化学成分的改变。酒炙豨莶草成分的变化可能为酒炙后药性转寒为温的物质基础。酒炙豨莶草后,其主要活性成分奇壬醇(kirenol)、豨莶酸(siegesbeckicacid)、豨莶酮(siegesbeckicketone)、槲皮素(quercetin)含量均升高,而奇壬醇(kirenol)是其祛风除湿的主要活性成分,具有抗血栓、抗炎、抗菌等广泛的药理作用。是其酒炙后增效的物质基础。
研究证实豨莶草酒炙前后成分差异显著,其成分的化学变化是酒炙后药性变化和增效的基础,因而可通过合理的炮制应用于临床治疗。对于豨莶草的生品与酒炙品能很好的检测和区分,同时也为豨莶草药效物质基础的阐明提供了重要依据。
虽然,上文中已经用一般性说明、具体实施方式及试验,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)
1.一种酒炙豨莶草的检测方法,其特征在于,该方法采用UPLC/Q-TOF-MS进行,包括以下步骤:
色谱条件:色谱柱:采用WatersACQUITYUPLCTMBEHC18Column,50mm×2.1mm,id.,1.7μm,色谱柱;柱温为30℃,流速为0.25mL/min,进样量为5μL;流动相A为水;流动相B为乙腈;梯度洗脱程序如下:0-4分钟,流动相A95%→76%,流动相B5→24,4-14分钟,流动相A76%→70%,流动相B24→30%;14→17分钟,流动相A70%→50%,流动相B30%→50%;17→20分钟,流动相A50%→38%,流动相B50%→62%;20→23分钟,流动相A38%→20%,流动相B62%→80%;23→27分钟,流动相A20%→95%,流动相B80%→5%;
Q-TOF/MS条件,质谱采用电喷雾电离源,TOF离子飞行方式采用Ⅴ模式;四级杆质量扫描m/z范围50-1200,一次扫描时间为0.1s,离子源温度120℃,脱溶剂氮气流速800L/h,正离子电离模式,毛细管电离电压3kV,取样椎孔电压40V,碰撞能量:30V;负离子电离模式,毛细管电离电压3kV,取样锥孔电压40V,碰撞能量:30V。
2.根据权利要求1所述的方法,其特征在于,正离子电离模式质量校正质核比为m/z556.2771;负离子电离模式,质量校正质核比为m/z554.2617。
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