WO2022252443A1 - Method for qualitatively analyzing phospholipid components in platycodon grandiflorum and performing c=c localization on phospholipid components in platycodon grandiflorum - Google Patents

Method for qualitatively analyzing phospholipid components in platycodon grandiflorum and performing c=c localization on phospholipid components in platycodon grandiflorum Download PDF

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WO2022252443A1
WO2022252443A1 PCT/CN2021/120010 CN2021120010W WO2022252443A1 WO 2022252443 A1 WO2022252443 A1 WO 2022252443A1 CN 2021120010 W CN2021120010 W CN 2021120010W WO 2022252443 A1 WO2022252443 A1 WO 2022252443A1
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platycodon grandiflorum
esi
phospholipid
phospholipid components
sample
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季申
胡青
兰岚
周恒�
黄卫珍
毛秀红
苗水
潘惠勤
李雯婷
袁佳佳
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上海市食品药品检验研究院
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    • G01N30/02—Column chromatography
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    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
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    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
    • G01N2030/484—

Definitions

  • Platycodon grandiflorum is the dry root of Platycodon grandiflorum (Jacq.) A.DC., a plant of Campanulaceae family, which has the functions of clearing the lung, relieving the throat, eliminating phlegm and draining pus.
  • Platycodon grandiflorum is not only a commonly used bulk medicinal material, but also a functional food for both medicine and food in many Asian countries. With the increase in market demand, the quality of platycodon grandiflorum from different sources is uneven, and the research on its quality control is particularly necessary.
  • Platycodon grandiflora mainly contains triterpenoid saponins, flavonoids, phenolic acids, polyacetylenes, polysaccharides and other components, among which saponins are considered as its main medicinal components.
  • a complete quality evaluation system for bellflower is still lacking.
  • the 2020 edition of "Chinese Pharmacopoeia” only has clear content control requirements for bellflower saponin D, and the monitoring target is single.
  • Simply relying on the current quality detection methods to detect the content changes of individual chemical components is not enough to indicate the real change in the quality of Platycodon grandiflorum. Therefore, improving the material basis and chemical characterization of platycodon grandiflora will help to improve the quality controllability of platycodon grandiflorum overall and systematically, and have guiding significance for the formulation of scientific standards.
  • the present invention will adopt the Matyash method to extract the phospholipid components in bellflower, and perform chromatographic separation on a HILIC column, and use ultra-high-efficiency liquid
  • PB Patern ⁇ -Büchi
  • ⁇ Analzer photochemical reactor
  • Step S1 using the Matyash method to extract the phospholipid components in Platycodon grandiflorum as an analysis sample;
  • Step S2 injecting the analyzed sample for the first time, and separating it into phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine and lysophosphatidylcholine after ultra-high performance liquid chromatography-high resolution mass spectrometry, and simultaneously obtain ESI + MS E and ESI - MS E data, deduce its molecular formula;
  • Step S2 according to the parent ion information obtained from the ESI - MSE data, inject the analysis sample for the second time, collect the ESI - MS/MS data, and determine the composition of the fatty acyl chain on the phospholipid;
  • the Matyash method is used to extract the phospholipids in Platycodon grandiflorum, and the specific steps are as follows:
  • platycodon root test sample powder Take 50 mg of platycodon root test sample powder, add 0.3 mL methanol and 1 mL MTBE, ultrasonically extract for 10 min, add 0.25 mL water to induce phase separation, centrifuge at 14000 rpm for 10 min, collect the upper organic layer, repeat the extraction of the remaining residue, and combine the organic phases to obtain The analyzed samples.
  • one or more of parent ion information, characteristic fragments of phospholipid molecules, neutral loss fragments and precise molecular weights of added ions are obtained from ESI + MSE data and ESI - MSE data.
  • the ultra-high performance liquid chromatography conditions are as follows:
  • Mobile phase A is: 10mmol/L ammonium acetate aqueous solution
  • Mobile phase B is: 50/50 acetone/acetonitrile (v/v);
  • the high-resolution mass spectrometry conditions are as follows:
  • Ion source Electrospray
  • Source temperature 120°C
  • Atomization gas temperature 450°C
  • Atomized gas flow rate 800L/h
  • step S2 the ESI + MS E mode is adopted, the low collision energy is 6eV, and the high collision energy ramp is 15-40eV.
  • step S3 the ESI - MS/MS mode is adopted, and the collision energy is respectively set as: phosphatidylethanolamine 35eV, lysophosphatidylethanolamine and lysophosphatidylcholine 30eV, phosphatidylcholine 38eV.
  • step S4 the ESI + MS/MS mode is adopted, and the collision energy is respectively set as: phosphatidylethanolamine 35eV, lysophosphatidylethanolamine and lysophosphatidylcholine 30eV, phosphatidylcholine 38eV.
  • the wavelength of the ultraviolet light in the photochemical microreactor is 190-260 nm; further, the wavelength is 200-260 nm; more preferably, the wavelength is 254 nm.
  • the present invention adopts the above technical scheme, and compared with the prior art, it has the following technical effects:
  • HILIC-PB-MS/MS ultra-high performance liquid chromatography-high resolution mass spectrometry system series connection online Patern ⁇ -Büchi (PB) photochemical reactor ( ⁇ Analzer)
  • Figure 1A-1C is the total ion current chromatogram ( Figure 1A) of Platycodon grandiflora sample and phospholipid standard under ESI + MS E mode, the secondary mass spectrum of PE 16:0_18:1 ( ⁇ 9) ( Figure 1B), PC 18 :0_18:1 ( ⁇ 9) of the secondary mass spectrum (Fig. 1C);
  • Fig. 2A is the secondary mass spectrogram of PE 16:0_18:1( ⁇ 9)ESI - mode
  • Fig. 2B is the secondary mass spectrogram under PC 18:0_18:1( ⁇ 9)ESI - mode;
  • Figure 2C is the secondary mass spectrum of PE 16:0_18:1( ⁇ 9)ESI + mode after PB reaction;
  • Figure 2D is the secondary mass spectrum in PC 18:0_18:1( ⁇ 9)ESI + mode after PB reaction;
  • Fig. 3A is the ESI - MS/MS spectrogram of PE 34:2 in the bellflower sample
  • Fig. 3B is the ESI + MS/MS spectrum of PE 34:2 in the platycodonoid sample after PB reaction;
  • Fig. 3 C is the ESI - MS/MS spectrogram of PC 36:3 in bellflower sample
  • Figure 3D is the ESI+MS/MS spectrum of PC 36:3 in Platycodon grandiflora samples after PB reaction.
  • Step S1 using the Matyash method to extract the phospholipid components in Platycodon grandiflorum as an analysis sample, the specific operations are as follows:
  • Step S2 the analysis sample is injected for the first time, separated into phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine and lysophosphatidylethanolamine after ultra-high performance liquid chromatography-high resolution mass spectrometry, and ESI is collected at the same time + MS E and ESI - MS E data, obtain the information of phospholipids such as parent ion information, characteristic fragments of phospholipid molecules, neutral loss fragments and the precise molecular weight of additive ions, etc., and infer its molecular formula;
  • Step S3 according to the parent ion information obtained from the ESI - MSE data, inject the analysis sample for the second time, collect the ESI - MS/MS data, and determine the composition of the fatty acyl chain on the phospholipid;
  • Step S4 turn on the PB (Patern ⁇ -Büchi) photochemical microreactor ( ⁇ Analzer) connected in series between the ultra-high performance liquid chromatography and the high-resolution mass spectrometer, and the sample reacts with acetone in the mobile phase with PB to generate M+58Da
  • PB Pattern ⁇ -Büchi
  • ⁇ Analzer photochemical microreactor
  • Mobile phase A is: 10mmol/L ammonium acetate aqueous solution
  • Mobile phase B is: 50/50 acetone/acetonitrile (v/v);
  • ESI electrospray
  • the ⁇ Analyzer (Qingpu Analytical Instrument Co., Ltd.) photochemical microreactor is connected in series between the chromatographic column and the mass spectrometer.
  • the mass spectrometry parameters were set as follows:
  • Source temperature 120°C
  • Atomization gas temperature 450°C
  • Atomized gas flow rate 800L/h
  • This example provides an analysis method for the molecular formula of phospholipids.
  • the phospholipid components in the analysis sample PG can be separated according to the category on the HILIC chromatographic column.
  • the elution order is shown in Figure 1A. Compared with the standard, it is determined that the order is phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC).
  • PE phosphatidylethanolamine
  • LPE lysophosphatidylethanolamine
  • PC phosphatidylcholine
  • LPC lysophosphatidylcholine
  • phospholipid class information such as characteristic fragments or neutral loss of phospholipid molecules can be obtained through ESI + MS E data.
  • PE phospholipid molecules tend to neutrally lose a fragment of 141.02Da (C 2 H 8 O 4 NP); as shown in Figure C, the PC-like phospholipid molecule was able to generate a characteristic fragment m/z 184.07 representing its polar head group ([C 5 H 15 NO 4 P] + ).
  • This example provides an analysis method for the composition of fatty acyl chains on phospholipids of phospholipids.
  • the molecular weight information of the precursor ions was obtained from the ESI - MS E data and used for subsequent ESI - MS/MS analysis.
  • the additive ion form of PE and LPE is [MH] -
  • the additive ion form of PC and LPC is [M+CH 3 COO] - .
  • Fragment information representing fatty acyl chains of phospholipid molecules can be obtained from the MS/MS mass spectrum, and the results are shown in Table 3 below.
  • PE 16:0_18:1( ⁇ 9) and PC 18:0_18:1( ⁇ 9) are taken as examples for illustration.
  • PE 16:0_18:1( ⁇ 9) produces two main fragment ions of m/z 255 and 281 under the collision energy of 35eV, representing two fatty acyl groups of 16:0 and 18:1 respectively chain.
  • Acetone is added to the mobile phase of ⁇ Analzer as a PB reaction reagent, and the ⁇ Analzer is turned on.
  • the mobile phase carries the sample through the ⁇ Analzer.
  • Step S1 take the collected platycodon root sample powder obtained from the Anhui medicinal material base, and use the Matyash method to extract the phospholipid components in the platycodon root as an analysis sample: take 50 mg of the platycodon root test sample powder, add 0.3 mL methanol and 1 mL MTBE, ultrasonically extract for 10 min, add 0.25 mL of water induces phase separation, after centrifugation at 14000 rpm for 10 min, the upper organic layer is collected, the remaining residue is extracted again, and the organic phase is combined to obtain the analysis sample.
  • Step S2 injecting the analyzed sample for the first time, and separating it into phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine and lysophosphatidylcholine after ultra-high performance liquid chromatography-high resolution mass spectrometry, and simultaneously obtain ESI + Based on MS E and ESI - MS E data, the molecular formulas of the two compounds are deduced to be PE 34:2 ([MH]-, m/z 714.51) and PC 36:3 ([M+CH3COO]-, m/z 842.57);
  • Step S3 according to the parent ion information obtained from the ESI - MSE data, inject the analysis sample for the second time, collect the ESI - MS/MS data, and determine the fatty acyl chain composition on the phospholipid:
  • PE 34:2 produces two fragments m/z 255 (16:0) and m/z 279 (18:2), which can be inferred to be PE 16:0_18 :2.
  • PC 36:3 [M+CH 3 COO] - , m/z 842.57)
  • PC 36:3 produces four fats with m/z 277, 279, 281 and 283 Fragmentation of the acyl chain, it can be speculated that the ion exists as isomers PC 18:3_18:0 and PC 18:2_18:1.
  • PC 36:3 includes PC 18:2( ⁇ 9,12)_18:1( ⁇ 9), PC 18:2( ⁇ 9,12)_18:1( ⁇ 11) and PC 18:3( ⁇ 9,12, 15) Three isomers of _18:0.

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Abstract

A method for qualitatively analyzing phospholipid components in Platycodon grandiflorum and performing C=C localization on phospholipid components in Platycodon grandiflorum. Phospholipid components in Platycodon grandiflorum are extracted by using a Matyash method, chromatographic separation is performed by means of an HILIC column, an ultra-high performance liquid chromatography-high-resolution mass spectrometry system is connected in series to an online PB photochemical reactor to form a phospholipid qualitative and C=C localization system, and phospholipid components in a Platycodon grandiflorum sample are analyzed by means of a three-step experiment. By means of the method for qualitatively analyzing phospholipid components in Platycodon grandiflorum and performing C=C localization on phospholipid components in Platycodon grandiflorum, a total of 180 phospholipid molecules comprising C=C allosome are analyzed and identified from a Platycodon grandiflorum sample.

Description

一种桔梗中磷脂类成分定性分析及其C＝C定位的方法A method for qualitative analysis of phospholipid components in bellflower and its C=C positioning 技术领域technical field
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。The invention belongs to the field of analysis and detection of traditional Chinese medicines, and in particular relates to a qualitative analysis of phospholipid components in bellflower and a method for C=C positioning thereof.
背景技术Background technique
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。Platycodon grandiflorum is the dry root of Platycodon grandiflorum (Jacq.) A.DC., a plant of Campanulaceae family, which has the functions of clearing the lung, relieving the throat, eliminating phlegm and draining pus. Platycodon grandiflorum is not only a commonly used bulk medicinal material, but also a functional food for both medicine and food in many Asian countries. With the increase in market demand, the quality of platycodon grandiflorum from different sources is uneven, and the research on its quality control is particularly necessary.
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***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。Platycodon grandiflora mainly contains triterpenoid saponins, flavonoids, phenolic acids, polyacetylenes, polysaccharides and other components, among which saponins are considered as its main medicinal components. At present, a complete quality evaluation system for bellflower is still lacking. For example, the 2020 edition of "Chinese Pharmacopoeia" only has clear content control requirements for bellflower saponin D, and the monitoring target is single. Simply relying on the current quality detection methods to detect the content changes of individual chemical components is not enough to indicate the real change in the quality of Platycodon grandiflorum. Therefore, improving the material basis and chemical characterization of platycodon grandiflora will help to improve the quality controllability of platycodon grandiflorum overall and systematically, and have guiding significance for the formulation of scientific standards.
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。At present, there have been studies on the material basis of bellflower, but the characterization of the chemical components in bellflower is not comprehensive enough. For example, some trace components may not be fully exposed, and some high-abundance components in the mass spectrum have not yet been identified. Wait. We found phospholipids for the first time in the process of analyzing the total components of Platycodon grandiflorum by UPLC/IM-QTOF-MS technology, and it has a very high abundance in the mass spectrum. Studies have shown that phospholipids, as the main components of biological membranes, have a wide range of biological activities. Phospholipids have complex molecular structures in biological samples due to different combinations of head groups, backbones and fatty acyl chains, as well as C=C and sn positions, which make the separation and identification of phospholipids still face many challenges. Structure determines function, especially the introduction of C=C has a significant impact on the structure and function of phospholipids. In previous studies, the identification of phospholipids in traditional Chinese medicine or food was mostly limited to the identification of head groups and fatty acid chains, and there were few studies on the identification of C=C double bond positions on unsaturated fatty acid chains. .
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。Therefore, it is necessary to study the phospholipids in Platycodon grandiflorum, further clarify its fine structure, and provide technical support for comprehensively improving the quality control and evaluation of medicinal and nutritional value of Platycodon grandiflorum.
发明内容Contents of the invention
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***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***，通过三步实验对桔梗样品中磷脂类成分进行精准分析。In order to identify the phospholipid components in bellflower and locate the C=C position on its unsaturated fatty acid chain, the present invention will adopt the Matyash method to extract the phospholipid components in bellflower, and perform chromatographic separation on a HILIC column, and use ultra-high-efficiency liquid The phase chromatography-high-resolution mass spectrometry system is connected in series with the online Paternò-Büchi (PB) photochemical reactor (ΩAnalzer) to form a phospholipid qualitative and C=C positioning system. Through three-step experiments, the phospholipid components in the bellflower sample are accurately analyzed.
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：The invention provides a method for qualitative analysis of phospholipid components in Platycodon grandiflora and its C=C positioning, comprising the following steps:
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；Step S1, using the Matyash method to extract the phospholipid components in Platycodon grandiflorum as an analysis sample;
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 +MS E及ESI -MS E数据，推测出其分子式； Step S2, injecting the analyzed sample for the first time, and separating it into phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine and lysophosphatidylcholine after ultra-high performance liquid chromatography-high resolution mass spectrometry, and simultaneously obtain ESI + MS E and ESI - MS E data, deduce its molecular formula;
步骤S2、根据ESI -MS E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； Step S2, according to the parent ion information obtained from the ESI - MSE data, inject the analysis sample for the second time, collect the ESI - MS/MS data, and determine the composition of the fatty acyl chain on the phospholipid;
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 +MS E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。 Step S3, turn on the PB photochemical microreactor connected in series between the ultra-high performance liquid chromatography and the high-resolution mass spectrometer, and inject the analysis sample for the third time according to the precursor ion information obtained from the ESI + MS E data, the The analysis sample is reacted with acetone in the mobile phase in the photochemical microreactor for PB reaction, and the ESI + MS/MS data of [M+58] + is collected, and each C=C on the fatty acyl chain will generate a corresponding mass difference 26Da diagnostic ion pair, so that the quantity and position of C=C can be judged.
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：Preferably, the Matyash method is used to extract the phospholipids in Platycodon grandiflorum, and the specific steps are as follows:
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。Take 50 mg of platycodon root test sample powder, add 0.3 mL methanol and 1 mL MTBE, ultrasonically extract for 10 min, add 0.25 mL water to induce phase separation, centrifuge at 14000 rpm for 10 min, collect the upper organic layer, repeat the extraction of the remaining residue, and combine the organic phases to obtain The analyzed samples.
优选地，从ESI +MS E数据及ESI -MS E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。 Preferably, one or more of parent ion information, characteristic fragments of phospholipid molecules, neutral loss fragments and precise molecular weights of added ions are obtained from ESI + MSE data and ESI - MSE data.
优选地，超高效液相色谱条件如下：Preferably, the ultra-high performance liquid chromatography conditions are as follows:
色谱柱：ACQUITY
Figure PCTCN2021120010-appb-000001
BEH HILIC column(2.1mm×100mm,1.7μm)； Column: ACQUITY
Figure PCTCN2021120010-appb-000001
BEH HILIC column (2.1mm×100mm, 1.7μm);
流动相A为：10mmol/L乙酸铵水溶液；Mobile phase A is: 10mmol/L ammonium acetate aqueous solution;
流动相B为：50/50丙酮/乙腈(v/v)；Mobile phase B is: 50/50 acetone/acetonitrile (v/v);
柱温：30℃；Column temperature: 30°C;
流速：0.4ml/min；Flow rate: 0.4ml/min;
进样量：2μL；Injection volume: 2μL;
洗脱方式：梯度洗脱。Elution method: Gradient elution.
进一步地，所述梯度洗脱程序如下：Further, the gradient elution procedure is as follows:
时间(min)time (min) 流动相A(％)Mobile phase A(%) 流动相B(％)Mobile phase B(%) 曲线 curve
00 1010 9090 初始initial
1.51.5 1515 8585 66
2.52.5 1515 8585 66
55 1616 8484 66
66 1010 9090 11
优选地，高分辨质谱条件如下：Preferably, the high-resolution mass spectrometry conditions are as follows:
离子源：电喷雾；Ion source: Electrospray;
毛细管电压：2.0kV；Capillary voltage: 2.0kV;
锥孔电压：40V；Cone voltage: 40V;
源温度：120℃；Source temperature: 120°C;
雾化气温度：450℃；Atomization gas temperature: 450°C;
雾化气流速：800L/h；Atomized gas flow rate: 800L/h;
锥孔气流速：50L/h。Cone gas flow rate: 50L/h.
优选地，步骤S2中，采用ESI +MS E模式，低碰撞能量为6eV，高碰撞能量ramp为15-40eV。 Preferably, in step S2, the ESI + MS E mode is adopted, the low collision energy is 6eV, and the high collision energy ramp is 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。 Preferably, in step S3, the ESI - MS/MS mode is adopted, and the collision energy is respectively set as: phosphatidylethanolamine 35eV, lysophosphatidylethanolamine and lysophosphatidylcholine 30eV, phosphatidylcholine 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。 Preferably, in step S4, the ESI + MS/MS mode is adopted, and the collision energy is respectively set as: phosphatidylethanolamine 35eV, lysophosphatidylethanolamine and lysophosphatidylcholine 30eV, phosphatidylcholine 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。Preferably, the wavelength of the ultraviolet light in the photochemical microreactor is 190-260 nm; further, the wavelength is 200-260 nm; more preferably, the wavelength is 254 nm.
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：The present invention adopts the above technical scheme, and compared with the prior art, it has the following technical effects:
(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；(1) The present invention analyzes and identifies 180 phospholipid molecules including C=C variants in the bellflower sample, which can broaden the thinking and provide technical support for material basis research and overall quality control of bellflower;
(2)本发明以超高效液相色谱-高分辨质谱***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***(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。(2) The present invention forms phospholipid qualitative and C=C localization system (HILIC-PB-MS/MS) with ultra-high performance liquid chromatography-high resolution mass spectrometry system series connection online Paternò-Büchi (PB) photochemical reactor (ΩAnalzer), can Accurate detection results can be obtained in time and quickly, and pollution and errors caused by human operation can be avoided.
(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。(3) The phospholipids in Platycodon grandiflorum are extracted by the Matyash method, the organic phase is located in the upper layer, and a purer organic phase is obtained during separation to improve the purity of the extraction.
附图说明Description of drawings
图1A-1C为ESI +MS E模式下，桔梗样品与磷脂标准品的总离子流色谱图(图1A)，PE 16:0_18:1(Δ9)的二级质谱图(图1B)，PC 18:0_18:1(Δ9)的二级质谱图(图1C)； Figure 1A-1C is the total ion current chromatogram (Figure 1A) of Platycodon grandiflora sample and phospholipid standard under ESI + MS E mode, the secondary mass spectrum of PE 16:0_18:1 (Δ9) (Figure 1B), PC 18 :0_18:1 (Δ9) of the secondary mass spectrum (Fig. 1C);
图2A为PE 16:0_18:1(Δ9)ESI -模式下的二级质谱图； Fig. 2A is the secondary mass spectrogram of PE 16:0_18:1(Δ9)ESI - mode;
图2B为PC 18:0_18:1(Δ9)ESI -模式下的二级质谱图； Fig. 2B is the secondary mass spectrogram under PC 18:0_18:1(Δ9)ESI - mode;
图2C为经PB反应后，PE 16:0_18:1(Δ9)ESI +模式下的二级质谱图； Figure 2C is the secondary mass spectrum of PE 16:0_18:1(Δ9)ESI + mode after PB reaction;
图2D为经PB反应后，PC 18:0_18:1(Δ9)ESI +模式下的二级质谱图； Figure 2D is the secondary mass spectrum in PC 18:0_18:1(Δ9)ESI + mode after PB reaction;
图3A为桔梗样品中PE 34:2的ESI -MS/MS谱图； Fig. 3A is the ESI - MS/MS spectrogram of PE 34:2 in the bellflower sample;
图3B为经PB反应后，桔梗样品中PE 34:2的ESI +MS/MS谱图； Fig. 3B is the ESI + MS/MS spectrum of PE 34:2 in the platycodonoid sample after PB reaction;
图3C为桔梗样品中PC 36:3的ESI -MS/MS谱图； Fig. 3 C is the ESI - MS/MS spectrogram of PC 36:3 in bellflower sample;
图3D为经PB反应后，桔梗样品中PC 36:3的ESI+MS/MS谱图。Figure 3D is the ESI+MS/MS spectrum of PC 36:3 in Platycodon grandiflora samples after PB reaction.
具体实施方式Detailed ways
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。In order to identify the phospholipid components in Platycodon grandiflorum and locate the C=C position on its unsaturated fatty acid chain, the present invention will adopt the Matyash method to extract the phospholipid components in Platycodon grandiflorum, and then adopt the HILIC-PB-MS/MS method, According to the order of the three-step experiment, the subclass information, fatty acyl chain composition and C=C position of the phospholipid components in Platycodon grandiflora were identified.
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：Specifically, the present invention provides a method for qualitative analysis of phospholipid components in Platycodon grandiflora and its C=C positioning, comprising the following steps 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：Step S1, using the Matyash method to extract the phospholipid components in Platycodon grandiflorum as an analysis sample, the specific operations are as follows:
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，剩余残重复提取一次，合并有机相并作为分析样品(PG)。Take Platycodon grandiflorum test sample powder 50mg, add 0.3mL methanol and 1mL MTBE, ultrasonically extract for 10min, add 0.25mL water to induce phase separation, centrifuge at 14000rpm for 10min, collect the upper organic layer, repeat the extraction of the remaining residue once, combine the organic phases and As an analytical sample (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 +MS E及ESI -MS E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； Step S2, the analysis sample is injected for the first time, separated into phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine and lysophosphatidylethanolamine after ultra-high performance liquid chromatography-high resolution mass spectrometry, and ESI is collected at the same time + MS E and ESI - MS E data, obtain the information of phospholipids such as parent ion information, characteristic fragments of phospholipid molecules, neutral loss fragments and the precise molecular weight of additive ions, etc., and infer its molecular formula;
步骤S3、根据ESI -MS E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； Step S3, according to the parent ion information obtained from the ESI - MSE data, inject the analysis sample for the second time, collect the ESI - MS/MS data, and determine the composition of the fatty acyl chain on the phospholipid;
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 +MS E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。 Step S4, turn on the PB (Paternò-Büchi) photochemical microreactor (ΩAnalzer) connected in series between the ultra-high performance liquid chromatography and the high-resolution mass spectrometer, and the sample reacts with acetone in the mobile phase with PB to generate M+58Da According to the precursor ion information obtained by ESI + MS E , the ESI + MS/MS data of [M+58] + is collected, and each C=C on the fatty acyl chain will generate a diagnostic ion pair with a mass difference of 26Da. Thus the quantity and position of C=C can be judged.
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。The present invention analyzed and identified 180 phospholipid molecules including C=C variants in the bellflower sample, as shown in the following table 4, which can broaden the thinking and provide technical support for the material basis research and overall quality control of bellflower.
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。In the following, the present invention will be described in detail and specifically through specific examples, so as to better understand the present invention, but the following examples do not limit the scope of the present invention.
样品制备Sample Preparation
采用Matyash方法提取桔梗中的磷脂类成分，具体操作步骤如下：Adopt the Matyash method to extract the phospholipids component in Platycodon grandiflorum, concrete operation steps are as follows:
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)。Take 50 mg of Platycodon grandiflorum test sample powder, add 0.3 mL methanol and 1 mL MTBE, ultrasonically extract for 10 min, add 0.25 mL of water to induce phase separation, centrifuge at 14000 rpm for 10 min, collect the upper organic layer, repeat the extraction once, combine the organic phase and use it as an analysis Sample (PG).
色谱与质谱条件Chromatography and Mass Spectrometry Conditions
1、超高效液相色谱条件1. Ultra-high performance liquid chromatography conditions
采用美国Waters公司Acquity超高效液相色谱仪；Acquity ultra-high performance liquid chromatograph from Waters Company of the United States is adopted;
色谱柱：ACQUITY
Figure PCTCN2021120010-appb-000002
BEH HILIC column(2.1mm×100mm,1.7μm)； Column: ACQUITY
Figure PCTCN2021120010-appb-000002
BEH HILIC column (2.1mm×100mm, 1.7μm);
流动相A为：10mmol/L乙酸铵水溶液；Mobile phase A is: 10mmol/L ammonium acetate aqueous solution;
流动相B为：50/50丙酮/乙腈(v/v)；Mobile phase B is: 50/50 acetone/acetonitrile (v/v);
柱温：30℃；Column temperature: 30°C;
流速：0.4ml/min；Flow rate: 0.4ml/min;
进样量：2μL；Injection volume: 2μL;
梯度洗脱程序见表1。The gradient elution program is shown in Table 1.
表1梯度洗脱程序Table 1 Gradient elution program
时间(min)time (min) 流动相A(％)Mobile phase A(%) 流动相B(％)Mobile phase B(%) 曲线 curve
00 1010 9090 初始initial
1.51.5 1515 8585 66
2.52.5 1515 8585 66
55 1616 8484 66
66 1010 9090 11
2、质谱条件2. Mass Spectrometry Conditions
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。Vion IMS-QTOF high-resolution mass spectrometer from Watres, USA, ion source: electrospray (ESI). The ΩAnalyzer (Qingpu Analytical Instrument Co., Ltd.) photochemical microreactor is connected in series between the chromatographic column and the mass spectrometer.
质谱参数设置如下：The mass spectrometry parameters were set as follows:
毛细管电压：2.0kV；Capillary voltage: 2.0kV;
锥孔电压：40V；Cone voltage: 40V;
源温度：120℃；Source temperature: 120°C;
雾化气温度：450℃；Atomization gas temperature: 450°C;
雾化气流速：800L/h；Atomized gas flow rate: 800L/h;
锥孔气流速：50L/h。Cone gas flow rate: 50L/h.
分析目的和对应模式：Analysis purpose and corresponding mode:
(1)磷脂类别分析：采用ESI +MS E模式，低碰撞能量为6eV，高碰撞能量ramp为15-40eV，并采集ESI -MS E数据，为后续脂肪酰基链的分析提供母离子信息。 (1) Analysis of phospholipids: ESI + MS E mode is used, the low collision energy is 6eV, and the high collision energy ramp is 15-40eV, and ESI - MS E data are collected to provide parent ion information for the subsequent analysis of fatty acyl chains.
(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)。 (2) Identification of phospholipid-linked fatty acyl chains: ESI - MS/MS mode was used, and the collision energies were set as: 35eV (PE), 30eV (LPE, LPC), and 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)。 (3) Identification of C=C on the fatty acyl chain: ESI + MS/MS mode is adopted, and the collision energies are respectively set as: 35eV (PE), 30eV (LPE, LPC), and 38eV (PC).
【实施例1】【Example 1】
本实施例提供磷脂类化合物分子式的分析方法。This example provides an analysis method for the molecular formula of phospholipids.
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 +MS E及ESI -MS E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。 The phospholipid components in the analysis sample PG can be separated according to the category on the HILIC chromatographic column. The elution order is shown in Figure 1A. Compared with the standard, it is determined that the order is phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC). According to the precise molecular weight of the added ion in ESI + MS E and ESI - MS E modes, its molecular formula can be deduced, and the results are shown in Table 2 below.
同时，通过ESI +MS E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(C 2H 8O 4NP)；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([C 5H 15NO 4P] +)的特征碎片m/z 184.07。 At the same time, phospholipid class information such as characteristic fragments or neutral loss of phospholipid molecules can be obtained through ESI + MS E data. As shown in Figure 1B, PE phospholipid molecules tend to neutrally lose a fragment of 141.02Da (C 2 H 8 O 4 NP); as shown in Figure C, the PC-like phospholipid molecule was able to generate a characteristic fragment m/z 184.07 representing its polar head group ([C 5 H 15 NO 4 P] + ).
LPE和LPC的质谱裂解行为分别与PE和PC相似。The MS fragmentation behaviors of LPE and LPC are similar to PE and PC, respectively.
【实施例2】[Example 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。This example provides an analysis method for the composition of fatty acyl chains on phospholipids of phospholipids.
从ESI -MS E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+CH 3COO] -。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。 The molecular weight information of the precursor ions was obtained from the ESI - MS E data and used for subsequent ESI - MS/MS analysis. The additive ion form of PE and LPE is [MH] - , and the additive ion form of PC and LPC is [M+CH 3 COO] - . Fragment information representing fatty acyl chains of phospholipid molecules can be obtained from the MS/MS mass spectrum, and the results are shown in Table 3 below.
本实施例中以PE 16:0_18:1(Δ9)和PC 18:0_18:1(Δ9)为例进行说明。In this embodiment, PE 16:0_18:1(Δ9) and PC 18:0_18:1(Δ9) are taken as examples for illustration.
参见图2A中，二级质谱中，PE 16:0_18:1(Δ9)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。See Figure 2A, in the secondary mass spectrometry, PE 16:0_18:1(Δ9) produces two main fragment ions of m/ z 255 and 281 under the collision energy of 35eV, representing two fatty acyl groups of 16:0 and 18:1 respectively chain.
参见图2B中，PC 18:0_18:1(Δ9)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-CH 3] -(m/z 772.59)碎片，这是由于其头部基团易丢失一个甲基。 See Figure 2B, PC 18:0_18:1 (Δ9) generates fragments m/z 281 (18:1) and 283 (18:0) representing its fatty acyl chain. At the same time, a [M-CH 3 ] - (m/z 772.59) fragment with relatively large abundance is generated, because its head group easily loses a methyl group.
对于LPE与LPC则只产生一个脂肪酰基链碎片。For LPE and LPC, only one fatty acyl chain fragment is produced.
【实施例3】[Example 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。This example provides analysis of the number and position of C=C on the fatty acyl chain of phospholipid compounds.
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。Acetone is added to the mobile phase of ΩAnalzer as a PB reaction reagent, and the ΩAnalzer is turned on. The mobile phase carries the sample through the ΩAnalzer. Under the irradiation of 254nm ultraviolet light, the C=C on the unsaturated fatty acyl chain of the phospholipid molecule in the sample and the acetone A PB reaction will produce a pair of compounds with M+58Da.
根据ESI +MS E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Δ26Da的离子对对应一个C＝C，从而可判断C＝C的数量和位置。 According to the precursor ion information obtained by ESI + MS E , collect [M+58] + ESI + MS/MS data, this pair of products will be fragmented into diagnostic ion pairs with a mass difference of 26Da, and each pair of Δ26Da ion pairs corresponds to one C=C, so the quantity and position of C=C can be judged.
参见图2C中，可观察到m/z 467/493离子对，对应PE 16:0_18:1(Δ9)中18:1脂肪酰基链Δ9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。Referring to Figure 2C, the m/z 467/493 ion pair can be observed, corresponding to the C=C of the Δ9 position of the 18:1 fatty acyl chain in PE 16:0_18:1(Δ9), and it can be seen that after the neutral loss of 141Da The fragment ion m/z 635.5.
参见图2D中，m/z 678/704为PC 18:0_18:1(Δ9)上18:1脂肪酰基链Δ9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。See Figure 2D, m/z 678/704 is the diagnostic ion pair of C=C at the Δ9 position of the 18:1 fatty acyl chain on PC 18:0_18:1(Δ9), m/z 184.07 is PC and LPC phospholipid molecules Characteristic fragments of the head group.
【实施例4】【Example 4】
本实施例以PE 34:2([M-H]-,m/z 714.51)和PC 36:3([M+CH3COO]-,m/z 842.57)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：In this embodiment, taking PE 34:2 ([M-H]-, m/z 714.51) and PC 36:3 ([M+CH3COO]-, m/z 842.57) as examples, the qualitative analysis of phospholipids in Bellflower and its The description of the method for C=C positioning specifically includes the following steps:
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，合并有机相并得到所述分析样品。Step S1, take the collected platycodon root sample powder obtained from the Anhui medicinal material base, and use the Matyash method to extract the phospholipid components in the platycodon root as an analysis sample: take 50 mg of the platycodon root test sample powder, add 0.3 mL methanol and 1 mL MTBE, ultrasonically extract for 10 min, add 0.25 mL of water induces phase separation, after centrifugation at 14000 rpm for 10 min, the upper organic layer is collected, the remaining residue is extracted again, and the organic phase is combined to obtain the analysis sample.
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 +MS E及ESI -MS E数据，推测出两化合物的分子式为PE 34:2([M-H]-,m/z 714.51)和PC 36:3([M+CH3COO]-,m/z 842.57)； Step S2, injecting the analyzed sample for the first time, and separating it into phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine and lysophosphatidylcholine after ultra-high performance liquid chromatography-high resolution mass spectrometry, and simultaneously obtain ESI + Based on MS E and ESI - MS E data, the molecular formulas of the two compounds are deduced to be PE 34:2 ([MH]-, m/z 714.51) and PC 36:3 ([M+CH3COO]-, m/z 842.57);
步骤S3、根据ESI -MS E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： Step S3, according to the parent ion information obtained from the ESI - MSE data, inject the analysis sample for the second time, collect the ESI - MS/MS data, and determine the fatty acyl chain composition on the phospholipid:
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。See Figure 3A, in the ESI-MS/MS spectrum, PE 34:2 produces two fragments m/z 255 (16:0) and m/z 279 (18:2), which can be inferred to be PE 16:0_18 :2.
参见图3C中，对于PC 36:3([M+CH 3COO] -,m/z 842.57)，其ESI-MS/MS图谱中，PC 36:3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。 See Figure 3C, for PC 36:3 ([M+CH 3 COO] - , m/z 842.57), in its ESI-MS/MS spectrum, PC 36:3 produces four fats with m/ z 277, 279, 281 and 283 Fragmentation of the acyl chain, it can be speculated that the ion exists as isomers PC 18:3_18:0 and 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 +MS E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： Step S4, turn on the PB photochemical microreactor connected in series between the ultra-high performance liquid chromatography and the high-resolution mass spectrometer, and inject the analysis sample for the third time according to the precursor ion information obtained from the ESI + MS E data, the The analysis sample is reacted with acetone in the mobile phase in the photochemical microreactor for PB reaction, and the ESI + MS/MS data of [M+58] + is collected, and each C=C on the fatty acyl chain will generate a corresponding mass difference 26Da diagnostic ion pair, so as to determine the number and position of C=C:
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Δ9和Δ12，故可推断m/z 714.51([M-H] -)其为PE 16:0_18:2(Δ9,12)。 See Figure 3B, after the PB reaction, m/z 774.52 ([M+H+58] + ) generates two diagnostic ion pairs of m/z 467/493 and m/z 507/533 under MS/MS conditions, It can be inferred that the two C=Cs on its 18:2 fatty acyl chain are located at Δ9 and Δ12, so it can be inferred that m/z 714.51 ([MH] - ) is 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Δ26Da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C 18:2上C＝C的诊断离子对，m/z 674/700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/z 758/784则代表C 18:3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。 See Figure 3D, after the PB reaction, m/z 842.57 ([M+H+58] + ) produced 7 pairs of diagnostic ion pairs with a mass difference of Δ26Da under MS/MS conditions, indicating the presence of C=C positional isomerism body. According to the abundance of the corresponding fatty acyl chain fragments and the abundance of the diagnostic transition, it can be inferred that m/z 676/702 and m/z 716/742 represent the diagnostic transition of C=C on C 18:2, m/z 674/ 700 and m/z 702/728 represent C=C at Δ9 and Δ11, respectively, while m/z 678/704, m/z 718/744 and m/z 758/784 represent C=C on C 18:3 Diagnostic transitions. Therefore, it can be deduced that PC 36:3 includes PC 18:2(Δ9,12)_18:1(Δ9), PC 18:2(Δ9,12)_18:1(Δ11) and PC 18:3(Δ9,12, 15) Three isomers of _18:0.
表2桔梗药材中磷脂类成分的亚类信息Table 2 Subclass information of phospholipid components in Platycodon grandiflora
Figure PCTCN2021120010-appb-000003
Figure PCTCN2021120010-appb-000003
Figure PCTCN2021120010-appb-000004
Figure PCTCN2021120010-appb-000004
Figure PCTCN2021120010-appb-000005
Figure PCTCN2021120010-appb-000005
Figure PCTCN2021120010-appb-000006
Figure PCTCN2021120010-appb-000006
Figure PCTCN2021120010-appb-000007
Figure PCTCN2021120010-appb-000007
表3桔梗药材中磷脂类成分上脂肪酰基链组成信息Table 3 Composition information of fatty acyl chains on phospholipids in Platycodon grandiflora
Figure PCTCN2021120010-appb-000008
Figure PCTCN2021120010-appb-000008
Figure PCTCN2021120010-appb-000009
Figure PCTCN2021120010-appb-000009
Figure PCTCN2021120010-appb-000010
Figure PCTCN2021120010-appb-000010
Figure PCTCN2021120010-appb-000011
Figure PCTCN2021120010-appb-000011
Figure PCTCN2021120010-appb-000012
Figure PCTCN2021120010-appb-000012
Figure PCTCN2021120010-appb-000013
Figure PCTCN2021120010-appb-000013
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Table 4 C=C position information on the fatty acyl chain of the phospholipid components in Platycodon grandiflorum
Figure PCTCN2021120010-appb-000014
Figure PCTCN2021120010-appb-000014
Figure PCTCN2021120010-appb-000015
Figure PCTCN2021120010-appb-000015
Figure PCTCN2021120010-appb-000016
Figure PCTCN2021120010-appb-000016
Figure PCTCN2021120010-appb-000017
Figure PCTCN2021120010-appb-000017
Figure PCTCN2021120010-appb-000018
Figure PCTCN2021120010-appb-000018
Figure PCTCN2021120010-appb-000019
Figure PCTCN2021120010-appb-000019
Figure PCTCN2021120010-appb-000020
Figure PCTCN2021120010-appb-000020
Figure PCTCN2021120010-appb-000021
Figure PCTCN2021120010-appb-000021
(/：无对应的C＝C诊断离子对)(/: no corresponding C=C diagnostic transition)
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。The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

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：A method for qualitative analysis of phospholipid components in bellflower and its C=C positioning, characterized in that it comprises the following steps:
    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；Step S1, using the Matyash method to extract the phospholipid components in Platycodon grandiflorum as an analysis sample;
    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 +MS E及ESI -MS E数据，推测出其分子式； Step S2, injecting the analyzed sample for the first time, and separating it into phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine and lysophosphatidylcholine after ultra-high performance liquid chromatography-high resolution mass spectrometry, and simultaneously obtain ESI + MS E and ESI - MS E data, deduce its molecular formula;
    步骤S3、根据ESI -MS E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； Step S3, according to the parent ion information obtained from the ESI - MSE data, inject the analysis sample for the second time, collect the ESI - MS/MS data, and determine the composition of the fatty acyl chain on the phospholipid;
    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 +MS E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。 Step S4, turn on the PB photochemical microreactor connected in series between the ultra-high performance liquid chromatography and the high-resolution mass spectrometer, and inject the analysis sample for the third time according to the precursor ion information obtained from the ESI + MS E data, the The analysis sample is reacted with acetone in the mobile phase in the photochemical microreactor for PB reaction, and the ESI + MS/MS data of [M+58] + is collected, and each C=C on the fatty acyl chain will generate a corresponding mass difference 26Da diagnostic ion pair, so as to determine the number and position of C=C.
  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。According to the qualitative analysis of phospholipid components in Platycodon grandiflorum described in claim 1 and the method for C=C location thereof, it is characterized in that, adopt Matyash method to extract the phospholipid component in Platycodon grandiflorum, concrete operation steps are as follows: Get the test sample powder of Platycodon grandiflorum 50mg, add 0.3mL methanol and 1mL MTBE, ultrasonically extract for 10min, add 0.25mL water to induce phase separation, centrifuge at 14000rpm for 10min, collect the upper organic layer, repeat the extraction of the remaining residue, and combine the organic phases to obtain the analysis sample.
  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 +MS E数据及ESI -MS E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。 According to the method for the qualitative analysis of phospholipid composition in Platycodon grandiflorum described in claim 1 and its C=C location, it is characterized in that, from ESI + MS E data and ESI - MS E data obtain the characteristic fragment of parent ion information, phospholipid molecule One or more of the exact molecular weights of , neutral loss fragments, and adducted ions.
  4. 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，超高效液相色谱条件如下：According to the method for the qualitative analysis of phospholipid components in Platycodon grandiflorum described in claim 1 and its C=C location, it is characterized in that, the ultra-high performance liquid chromatography condition is as follows:
    色谱柱：
    Figure PCTCN2021120010-appb-100001
    BEH HILIC column(2.1mm×100mm,1.7μm)；     Column:
    Figure PCTCN2021120010-appb-100001
    BEH HILIC column (2.1mm×100mm, 1.7μm);
    流动相A为：10mmol/L乙酸铵水溶液；Mobile phase A is: 10mmol/L ammonium acetate aqueous solution;
    流动相B为：50/50丙酮/乙腈(v/v)；Mobile phase B is: 50/50 acetone/acetonitrile (v/v);
    柱温：30℃；Column temperature: 30°C;
    流速：0.4ml/min；Flow rate: 0.4ml/min;
    进样量：2μL；Injection volume: 2μL;
    洗脱方式：梯度洗脱。Elution method: Gradient elution.
  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：According to the method for the qualitative analysis of phospholipid components in Platycodon grandiflorum described in claim 4 and C=C localization thereof, it is characterized in that, described gradient elution procedure is as follows:
    时间(min)time (min) 流动相A(％)Mobile phase A(%) 流动相B(％)Mobile phase B(%) 曲线curve 00 1010 9090 初始initial 1.51.5 1515 8585 66 2.52.5 1515 8585 66 55 1616 8484 66 66 1010 9090 11
  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：According to the method for the qualitative analysis of phospholipid components in Platycodon grandiflorum described in claim 1 and C=C localization thereof, it is characterized in that, high-resolution mass spectrometry condition is as follows:
    离子源：电喷雾；Ion source: Electrospray;
    毛细管电压：2.0kV；Capillary voltage: 2.0kV;
    锥孔电压：40V；Cone voltage: 40V;
    源温度：120℃；Source temperature: 120°C;
    雾化气温度：450℃；Atomization gas temperature: 450°C;
    雾化气流速：800L/h；Atomized gas flow rate: 800L/h;
    锥孔气流速：50L/h。Cone gas flow rate: 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 +MS E模式，低碰撞能量为6eV，高碰撞能量ramp为15-40eV。 According to the method for the qualitative analysis of phospholipid composition in Platycodon grandiflorum described in claim 1 and its C=C location, it is characterized in that, in step S2, adopt ESI + MS E mode, low collision energy is 6eV, and high collision energy ramp is 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。 According to the method for qualitative analysis of phospholipid components in Platycodon grandiflorum described in claim 1 and its C=C location, it is characterized in that, in step S3, adopt ESI - MS/MS mode, collision energy is respectively set as: phosphatidylethanolamine 35eV, lysophosphatidylethanolamine and lysophosphatidylcholine 30eV, phosphatidylcholine 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。 According to the method for qualitative analysis of phospholipid components in Platycodon grandiflorum described in claim 1 and its C=C location, it is characterized in that, in step S4, adopt ESI + MS/MS mode, collision energy is respectively set as: phosphatidylethanolamine 35eV, lysophosphatidylethanolamine and lysophosphatidylcholine 30eV, phosphatidylcholine 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。The method for qualitative analysis of phospholipid components in Platycodon grandiflorum and its C=C positioning according to claim 1, characterized in that the wavelength of ultraviolet light in the PB photochemical microreactor is 190-260nm.
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