CN113295799A - 一种桔梗中磷脂类成分定性分析及其c=c定位的方法 - Google Patents
一种桔梗中磷脂类成分定性分析及其c=c定位的方法 Download PDFInfo
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Abstract
本发明公开了一种桔梗中磷脂类成分定性分析及其C=C定位的方法,采用Matyash方法提取桔梗中的磷脂类成分,HILIC柱进行色谱分离,并以超高效液相色谱‑高分辨质谱系统串联在线PB光化学反应器组成磷脂定性及C=C定位系统,通过三步实验对桔梗样品中磷脂类成分进行精准分析。本发明在桔梗样品中共分析鉴定出包括C=C异体在内的180个磷脂分子,可为桔梗的物质基础研究及全面质量控制拓宽思路并提供技术支撑。
Description
技术领域
本发明属于中药分析检测领域,具体涉及桔梗中磷脂类成分定性分析及其C=C定位的方法。
背景技术
桔梗为桔梗科植物桔梗Platycodon grandiflorum(Jacq.)A.DC.的干燥根,具有宣肺、利咽、祛痰、排脓等功效。桔梗既是常用的大宗药材,又是许多亚洲国家药食两用的功能性食品,伴随着市场需求量的增大,不同来源的桔梗品质参差不齐,对其质量控制的研究尤为必要。
桔梗主要含有三萜皂苷类、黄酮类、酚酸类、聚炔类、多糖类等成分,其中皂苷类化合物被认为是其主要药效成分。目前完善的桔梗质量评价体系仍然缺失,例如2020年版《中国药典》仅对桔梗皂苷D有明确的含量控制要求,监测目标单一。单纯依赖通行的质量检测方法检测个别化学成分的含量变化不足以表明桔梗药材质量的真正变化。因此,完善桔梗的物质基础和化学表征研究将有助于整体和系统地提高桔梗质量的可控性,并对科学标准的制定有着指导意义。
目前已有文献对桔梗的物质基础进行了研究,但是,对于桔梗中化学成分的表征还不够全面,例如,一些微量成分可能未完全暴露,且质谱图中的一些高丰度成分仍未被鉴定等。我们通过UPLC/IM-QTOF-MS技术对桔梗进行全成分分析过程中,首次发现了磷脂类化合物,且其在质谱图中具有非常高的丰度。研究表明,磷脂作为生物膜的主要成分具有广泛的生物活性。由于头部基团、骨架和脂肪酰基链的不同组合,以及C=C和sn位置不同,磷脂在生物样本中具有复杂的分子结构,这使得磷脂的分离和鉴定仍面临诸多挑战。结构决定功能,特别是C=C的引入对磷脂的结构和功能产生显著影响。而在以往研究中,针对于中药或食品中磷脂类化合物的鉴定多局限于头部基团和脂肪酸链的鉴定,而关于其不饱和脂肪酸链上的C=C双键位置鉴定的研究较少。
因此,有必要对桔梗中磷脂类化合物进行研究,进一步阐明其精细结构,为全面提高桔梗的质量控制及药用和营养价值评价提供技术支撑。
发明内容
为了鉴定桔梗中的磷脂类成分,并对其不饱和脂肪酸链上的C=C位置进行定位,本发明将采用Matyash方法提取桔梗中的磷脂类成分,HILIC柱进行色谱分离,并以超高效液相色谱-高分辨质谱系统串联在线Paternò-Büchi(PB)光化学反应器(ΩAnalzer)组成磷脂定性及C=C定位系统,通过三步实验对桔梗样品中磷脂类成分进行精准分析。
本发明提供一种桔梗中磷脂类成分定性分析及其C=C定位的方法,包括以下步骤:
步骤S1、采用Matyash方法提取桔梗中的磷脂类成分作为分析样品;
步骤S2、第一次进样所述分析样品,经超高效液相色谱-高分辨质谱后分离为磷脂酰乙醇胺、溶血磷脂酰乙醇胺、磷脂酰胆碱和溶血磷脂酰胆碱,同时得到ESI+MSE及ESI-MSE数据,推测出其分子式;
步骤S2、根据ESI-MSE数据得到的母离子信息,第二次进样所述分析样品,采集ESI-MS/MS数据,判断磷脂上的脂肪酰基链组成;
步骤S3、开启串联在所述超高效液相色谱和高分辨质谱之间的PB光化学微型反应器,根据ESI+MSE数据获得的母离子信息,第三次进样所述分析样品,所述分析样品与所述光化学微型反应器中的流动相中的丙酮进行PB反应,采集[M+58]+的ESI+MS/MS数据,脂肪酰基链上的每个C=C会对应生成质量差26Da的诊断离子对,从而可判断C=C的数量和位置。
优选地,采用Matyash方法提取桔梗中的磷脂类成分,具体操作步骤如下:
取桔梗供试品粉末50mg,加入0.3mL甲醇和1mL MTBE,超声提取10min,加入0.25mL水诱导相分离,在14000rpm下离心10min后,收集上部有机层,剩余残渣重复提取一遍,合并有机相得到所述分析样品。
优选地,从ESI+MSE数据及ESI-MSE数据获得母离子信息、磷脂分子的特征碎片、中性丢失片段和加和离子的精确分子量中的一种或几种。
优选地,超高效液相色谱条件如下:
流动相A为:10mmol/L乙酸铵水溶液;
流动相B为:50/50丙酮/乙腈(v/v);
柱温:30℃;
流速:0.4ml/min;
进样量:2μL;
洗脱方式:梯度洗脱。
进一步地,所述梯度洗脱程序如下:
时间(min) | 流动相A(%) | 流动相B(%) | 曲线 |
0 | 10 | 90 | 初始 |
1.5 | 15 | 85 | 6 |
2.5 | 15 | 85 | 6 |
5 | 16 | 84 | 6 |
6 | 10 | 90 | 1 |
优选地,高分辨质谱条件如下:
离子源:电喷雾;
毛细管电压:2.0kV;
锥孔电压:40V;
源温度:120℃;
雾化气温度:450℃;
雾化气流速:800L/h;
锥孔气流速:50L/h。
优选地,步骤S2中,采用ESI+MSE模式,低碰撞能量为6eV,高碰撞能量ramp为15-40eV。
优选地,步骤S3中,采用ESI-MS/MS模式,碰撞能分别设定为:磷脂酰乙醇胺35eV,溶血磷脂酰乙醇胺和溶血磷脂酰胆碱30eV,磷脂酰胆碱38eV。
优选地,步骤S4中,采用ESI+MS/MS模式,碰撞能分别设定为:磷脂酰乙醇胺35eV,溶血磷脂酰乙醇胺和溶血磷脂酰胆碱30eV,磷脂酰胆碱38eV。
优选地,所述光化学微型反应器中紫外光的波长为190~260nm;进一步地,波长为200~260nm;更优选地,波长为254nm。
本发明采用以上技术方案,与现有技术相比,具有如下技术效果:
(1)本发明在桔梗样品中共分析鉴定出包括C=C异体在内的180个磷脂分子,可为桔梗的物质基础研究及全面质量控制拓宽思路并提供技术支撑;
(2)本发明以超高效液相色谱-高分辨质谱系统串联在线Paternò-Büchi(PB)光化学反应器(ΩAnalzer)组成磷脂定性及C=C定位系统(HILIC-PB-MS/MS),可以及时快速地得到精确的检测结果,避免了人为操作带来的污染和误差。
(3)采用Matyash方法提取桔梗中的磷脂类成分,有机相位于上层,分离时得到更纯净的有机相,提高提取的纯度。
附图说明
图1A-1C为ESI+MSE模式下,桔梗样品与磷脂标准品的总离子流色谱图(图1A),PE16:0_18:1(△9)的二级质谱图(图1B),PC 18:0_18:1(△9)的二级质谱图(图1C);
图2A为PE 16:0_18:1(△9)ESI-模式下的二级质谱图;
图2B为PC 18:0_18:1(△9)ESI-模式下的二级质谱图;
图2C为经PB反应后,PE 16:0_18:1(△9)ESI+模式下的二级质谱图;
图2D为经PB反应后,PC 18:0_18:1(Δ9)ESI+模式下的二级质谱图;
图3A为桔梗样品中PE 34:2的ESI-MS/MS谱图;
图3B为经PB反应后,桔梗样品中PE 34:2的ESI+MS/MS谱图;
图3C为桔梗样品中PC 36:3的ESI-MS/MS谱图;
图3D为经PB反应后,桔梗样品中PC 36:3的ESI+MS/MS谱图。
具体实施方式
为了鉴定桔梗中的磷脂类成分,并对其不饱和脂肪酸链上的C=C位置进行定位,本发明将采用Matyash方法提取桔梗中的磷脂类成分,而后采用HILIC-PB-MS/MS方法,依照三步实验的顺序对桔梗中的磷脂类成分的亚类信息、脂肪酰基链组成以及C=C位置进行鉴定。
具体地,本发明提供的一种桔梗中磷脂类成分定性分析及其C=C定位的方法,包括以下步骤S1-S4:
步骤S1、采用Matyash方法提取桔梗中的磷脂类成分作为分析样品,具体操作如下:
取桔梗供试品粉末50 mg,加入0.3mL甲醇和1mL MTBE,超声提取10min,加入0.25mL水诱导相分离,在14000 rpm下离心10min后,收集上部有机层,剩余残重复提取一次,合并有机相并作为分析样品(PG)。
步骤S2、第一次进样所述分析样品,经超高效液相色谱-高分辨质谱后分离为磷脂酰乙醇胺、溶血磷脂酰乙醇胺、磷脂酰胆碱和溶血磷脂酰胆碱,同时采集得到ESI+MSE及ESI-MSE数据,获得母离子信息、磷脂分子的特征碎片、中性丢失片段和加和离子的精确分子量等磷脂类别信息,推测出其分子式;
步骤S3、根据ESI-MSE数据得到的母离子信息,第二次进样所述分析样品,采集ESI-MS/MS数据,判断磷脂上的脂肪酰基链组成;
步骤S4、开启串联在所述超高效液相色谱和高分辨质谱之间的PB(Paternò-Büchi)光化学微型反应器(ΩAnalzer),样品与流动相中的丙酮进行PB反应,会产生M+58Da的化合物,根据ESI+MSE获得的母离子信息,采集[M+58]+的ESI+MS/MS数据,脂肪酰基链上的每个C=C会对应生成质量差26Da的诊断离子对,从而可判断C=C的数量和位置。
本发明在桔梗样品中共分析鉴定出包括C=C异体在内的180个磷脂分子,如下表4中所示,可为桔梗的物质基础研究及全面质量控制拓宽思路并提供技术支撑。
下面通过具体实施例对本发明进行详细和具体的介绍,以使更好的理解本发明,但是下述实施例并不限制本发明范围。
样品制备
采用Matyash方法提取桔梗中的磷脂类成分,具体操作步骤如下:
取桔梗供试品粉末50mg,加入0.3mL甲醇和1mL MTBE,超声提取10min,加入0.25mL水诱导相分离,在14000rpm下离心10min后,收集上部有机层,重复提取一次,合并有机相并作为分析样品(PG)。
色谱与质谱条件
1、超高效液相色谱条件
采用美国Waters公司Acquity超高效液相色谱仪;
流动相A为:10mmol/L乙酸铵水溶液;
流动相B为:50/50丙酮/乙腈(v/v);
柱温:30℃;
流速:0.4ml/min;
进样量:2μL;
梯度洗脱程序见表1。
表1梯度洗脱程序
时间(min) | 流动相A(%) | 流动相B(%) | 曲线 |
0 | 10 | 90 | 初始 |
1.5 | 15 | 85 | 6 |
2.5 | 15 | 85 | 6 |
5 | 16 | 84 | 6 |
6 | 10 | 90 | 1 |
2、质谱条件
采用美国Watres公司Vion IMS-QTOF高分辨质谱仪,离子源:电喷雾(ESI)。ΩAnalyzer(清谱分析仪器有限公司)光化学微型反应器串联在色谱柱和质谱仪之间。
质谱参数设置如下:
毛细管电压:2.0kV;
锥孔电压:40 V;
源温度:120℃;
雾化气温度:450℃;
雾化气流速:800L/h;
锥孔气流速:50L/h。
分析目的和对应模式:
(1)磷脂类别分析:采用ESI+MSE模式,低碰撞能量为6eV,高碰撞能量ramp为15-40eV,并采集ESI-MSE数据,为后续脂肪酰基链的分析提供母离子信息。
(2)磷脂连接的脂肪酰基链鉴定:采用ESI-MS/MS模式,碰撞能分别设定为:35eV(PE),30eV(LPE、LPC),38eV(PC)。
(3)脂肪酰基链上的C=C鉴定:采用ESI+MS/MS模式,碰撞能分别设定为:35eV(PE),30eV(LPE、LPC),38eV(PC)。
【实施例1】
本实施例提供磷脂类化合物分子式的分析方法。
分析样品PG中的磷脂成分能够在HILIC色谱柱上按照类别进行分离,其洗脱顺序如图1A所示,通过标准品进行了比对,确定依次为磷脂酰乙醇胺(PE),溶血磷脂酰乙醇胺(LPE),磷脂酰胆碱(PC),溶血磷脂酰胆碱(LPC)。根据ESI+MSE及ESI-MSE模式下加和离子的精确分子量可推测出其分子式,结果如下表2中所示。
同时,通过ESI+MSE数据可获得磷脂分子的特征碎片或中性丢失等磷脂类别信息,如图1B中,PE类磷脂分子倾向于中性丢失一个141.02Da的片段(C2H8O4NP);如图C中所示,PC类磷脂分子能够产生代表其极性头部基团([C5H15NO4P]+)的特征碎片m/z 184.07。
LPE和LPC的质谱裂解行为分别与PE和PC相似。
【实施例2】
本实施例提供磷脂类化合物的磷脂上的脂肪酰基链组成的分析方法。
从ESI-MSE数据中获得的母离子的分子量信息,并用于后续的ESI-MS/MS分析。其中PE和LPE加和离子形式为[M-H]-,PC和LPC加和离子形式为[M+CH3COO]-。从MS/MS质谱图上可获得代表磷脂分子脂肪酰基链的碎片信息,结果见下表3。
本实施例中以PE 16:0_18:1(△9)和PC 18:0_18:1(△9)为例进行说明。
参见图2A中,二级质谱中,PE 16:0_18:1(△9)在35eV碰撞能下产生m/z 255和281两个主要碎片离子,分别代表16:0和18:1两条脂肪酰基链。
参见图2B中,PC 18:0_18:1(△9)生成代表其脂肪酰基链的碎片m/z 281(18:1)和283(18:0)。同时,生成丰度较大的[M-CH3]-(m/z 772.59)碎片,这是由于其头部基团易丢失一个甲基。
对于LPE与LPC则只产生一个脂肪酰基链碎片。
【实施例3】
本实施例提供磷脂类化合物脂肪酰基链上的C=C数量和位置分析。
丙酮作为PB反应试剂被加到ΩAnalzer的流动相中,开启ΩAnalzer,流动相带着样品流经ΩAnalzer,在254nm紫外光的照射下,样品中磷脂分子不饱和脂肪酰基链上的C=C与丙酮进行PB反应,会产生一对M+58Da的化合物。
根据ESI+MSE获得的母离子信息,采集[M+58]+的ESI+MS/MS数据,这对产物将碎裂成质量差26Da的诊断离子对,每一对△26Da的离子对对应一个C=C,从而可判断C=C的数量和位置。
参见图2C中,可观察到m/z 467/493离子对,对应PE 16:0_18:1(△9)中18:1脂肪酰基链△9位的C=C,同时可看到其中性丢失141Da后的碎片离子m/z 635.5。
参见图2D中,m/z 678/704为PC 18:0_18:1(△9)上18:1脂肪酰基链△9位的C=C的诊断离子对,m/z 184.07为PC及LPC类磷脂分子头部基团的特征碎片。
【实施例4】
本实施例以PE 34:2([M-H]-,m/z 714.51)和PC 36:3([M+CH3COO]-,m/z842.57)为例,进行桔梗中磷脂类成分定性分析及其C=C定位的方法的说明,具体包括以下步骤:
步骤S1、取收集的安徽药材基地所得桔梗药材样品粉末,采用Matyash方法提取桔梗中的磷脂类成分作为分析样品:取桔梗供试品粉末50 mg,加入0.3mL甲醇和1mL MTBE,超声提取10min,加入0.25mL水诱导相分离,在14000rpm下离心10min后,收集上部有机层,剩余残渣重复提取一遍,合并有机相并得到所述分析样品。
步骤S2、第一次进样所述分析样品,经超高效液相色谱-高分辨质谱后分离为磷脂酰乙醇胺、溶血磷脂酰乙醇胺、磷脂酰胆碱和溶血磷脂酰胆碱,同时得到ESI+MSE及ESI-MSE数据,推测出两化合物的分子式为PE 34:2([M-H]-,m/z714.51)和PC 36:3([M+CH3COO]-,m/z 842.57);
步骤S3、根据ESI-MSE数据得到的母离子信息,第二次进样所述分析样品,采集ESI-MS/MS数据,判断磷脂上的脂肪酰基链组成:
参见图3A中,在ESI-MS/MS图谱中,PE 34:2产生m/z 255(16:0)与m/z 279(18:2)两个碎片,可推断其为PE 16:0_18:2。
参见图3C中,对于PC 36:3([M+CH3COO]-,m/z 842.57),其ESI-MS/MS图谱中,PC36:3产生m/z 277,279,281和283共四个脂肪酰基链的碎片,可推测该离子存在同分异构体PC 18:3_18:0和PC 18:2_18:1。
步骤S4、开启串联在所述超高效液相色谱和高分辨质谱之间的PB光化学微型反应器,根据ESI+MSE数据获得的母离子信息,第三次进样所述分析样品,所述分析样品与所述光化学微型反应器中的流动相中的丙酮进行PB反应,采集[M+58]+的ESI+MS/MS数据,脂肪酰基链上的每个C=C会对应生成质量差26Da的诊断离子对,从而判断C=C的数量和位置:
参见图3B中,经PB反应后,m/z 774.52([M+H+58]+)在MS/MS条件下产生m/z 467/493和m/z 507/533两对诊断离子对,可推断其18:2脂肪酰基链上的两个C=C分别位于△9和△12,故可推断m/z 714.51([M-H]-)其为PE 16:0_18:2(Δ9,12)。
参见图3D中,经PB反应后,m/z 842.57([M+H+58]+)在MS/MS条件下产生7对质量差△26Da的诊断离子对,说明其存在C=C位置异构体。根据对应的脂肪酰基链碎片丰度以及诊断离子对丰度,可推断m/z 676/702和m/z 716/742代表C18:2上C=C的诊断离子对,m/z674/700和m/z 702/728分别表示C=C位于Δ9和Δ11,而m/z 678/704,m/z 718/744和m/z758/784则代表C18:3上C=C的诊断离子对。因此,可推断PC 36:3包括PC 18:2(Δ9,12)_18:1(Δ9),PC 18:2(Δ9,12)_18:1(Δ11)和PC 18:3(Δ9,12,15)_18:0三个同分异构体。
表2桔梗药材中磷脂类成分的亚类信息
表3桔梗药材中磷脂类成分上脂肪酰基链组成信息
表4桔梗药材中磷脂类成分脂肪酰基链上的C=C位置信息
(/:无对应的C=C诊断离子对)
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
Claims (10)
1.一种桔梗中磷脂类成分定性分析及其C=C定位的方法,其特征在于,包括以下步骤:
步骤S1、采用Matyash方法提取桔梗中的磷脂类成分作为分析样品;
步骤S2、第一次进样所述分析样品,经超高效液相色谱-高分辨质谱后分离为磷脂酰乙醇胺、溶血磷脂酰乙醇胺、磷脂酰胆碱和溶血磷脂酰胆碱,同时得到ESI+MSE及ESI-MSE数据,推测出其分子式;
步骤S3、根据ESI-MSE数据得到的母离子信息,第二次进样所述分析样品,采集ESI-MS/MS数据,判断磷脂上的脂肪酰基链组成;
步骤S4、开启串联在所述超高效液相色谱和高分辨质谱之间的PB光化学微型反应器,根据ESI+MSE数据获得的母离子信息,第三次进样所述分析样品,所述分析样品与所述光化学微型反应器中的流动相中的丙酮进行PB反应,采集[M+58]+的ESI+MS/MS数据,脂肪酰基链上的每个C=C会对应生成质量差26Da的诊断离子对,从而判断C=C的数量和位置。
2.根据权利要求1中所述的桔梗中磷脂类成分定性分析及其C=C定位的方法,其特征在于,采用Matyash方法提取桔梗中的磷脂类成分,具体操作步骤如下:取桔梗供试品粉末50mg,加入0.3mL甲醇和1mL MTBE,超声提取10min,加入0.25mL水诱导相分离,在14000rpm下离心10min后,收集上部有机层,剩余残渣重复提取一遍,合并有机相得到所述分析样品。
3.根据权利要求1中所述的桔梗中磷脂类成分定性分析及其C=C定位的方法,其特征在于,从ESI+MSE数据及ESI-MSE数据获得母离子信息、磷脂分子的特征碎片、中性丢失片段和加和离子的精确分子量中的一种或几种。
5.根据权利要求4中所述的桔梗中磷脂类成分定性分析及其C=C定位的方法,其特征在于,所述梯度洗脱程序如下:
6.根据权利要求1中所述的桔梗中磷脂类成分定性分析及其C=C定位的方法,其特征在于,高分辨质谱条件如下:
离子源:电喷雾;
毛细管电压:2.0kV;
锥孔电压:40V;
源温度:120℃;
雾化气温度:450℃;
雾化气流速:800L/h;
锥孔气流速:50L/h。
7.根据权利要求1中所述的桔梗中磷脂类成分定性分析及其C=C定位的方法,其特征在于,步骤S2中,采用ESI+MSE模式,低碰撞能量为6eV,高碰撞能量ramp为15-40eV。
8.根据权利要求1中所述的桔梗中磷脂类成分定性分析及其C=C定位的方法,其特征在于,步骤S3中,采用ESI-MS/MS模式,碰撞能分别设定为:磷脂酰乙醇胺35eV,溶血磷脂酰乙醇胺和溶血磷脂酰胆碱30eV,磷脂酰胆碱38eV。
9.根据权利要求1中所述的桔梗中磷脂类成分定性分析及其C=C定位的方法,其特征在于,步骤S4中,采用ESI+MS/MS模式,碰撞能分别设定为:磷脂酰乙醇胺35eV,溶血磷脂酰乙醇胺和溶血磷脂酰胆碱30eV,磷脂酰胆碱38eV。
10.根据权利要求1中所述的桔梗中磷脂类成分定性分析及其C=C定位的方法,其特征在于,所述PB光化学微型反应器中紫外光的波长为190~260nm。
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