阅读说明：本技术 一种快速识别分析洋甘菊中化学成分的方法 (Method for rapidly identifying and analyzing chemical components in chamomile ) 是由 江洁怡 肖观林 毕晓黎 胥爱丽 陈伟韬 李养学 李素梅 陈昭 张靖年 于 2021-09-17 设计创作，主要内容包括：本发明公开了一种快速识别分析洋甘菊中化学成分的方法,其包括以下步骤：制备供试品溶液；建立超高效液相色谱-质谱检测程序,并获得色谱质谱总离子流原始数据；根据所述色谱质谱总离子流原始数据匹配目的组份,结合比对色谱保留时间、准确相对分子质量以及碎片离子碎片信息进行快速的结构鉴定,以实现对洋甘菊中化学成分定性。本发明中采用UPLC-Q-TOF-MS/MS法对洋甘菊所含化学成分进行分析测定,为洋甘菊中化学成分鉴定提供了一种快速、准确和高效的鉴定方法,为研究洋甘菊药材的药效物质基础、质量变化规律及质量标准提升提供理论依据和技术参考。(The invention discloses a method for rapidly identifying and analyzing chemical components in chamomile, which comprises the following steps: preparing a test solution; establishing an ultra-high performance liquid chromatography-mass spectrometry detection program, and obtaining total ion flow original data of a chromatography-mass spectrometry; matching target components according to the original data of the total ion flow of the chromatographic mass spectrum, and carrying out rapid structural identification by combining and comparing chromatographic retention time, accurate relative molecular mass and fragment ion fragment information so as to realize the qualitative determination of chemical components in chamomile. According to the invention, the UPLC-Q-TOF-MS/MS method is adopted to analyze and measure the chemical components contained in the chamomile, so that a rapid, accurate and efficient identification method is provided for identifying the chemical components in the chamomile, and theoretical basis and technical reference are provided for researching the pharmacodynamic substance basis, the mass change rule and the quality standard improvement of the chamomile medicinal material.)

1. A method for rapidly identifying and analyzing chemical components in chamomile is characterized by comprising the following steps:

step 1, preparing a test solution;

step 2, establishing an ultra-high performance liquid chromatography-mass spectrometry detection program, and obtaining original data of total ion current of a chromatography-mass spectrometry;

and 3, matching target components according to the original data of the total ion flow of the chromatographic mass spectrum, and performing rapid structural identification by combining and comparing chromatographic retention time, accurate relative molecular mass and fragment ion fragment information so as to realize the qualitative determination of chemical components in the chamomile.

2. The method for rapidly identifying and analyzing chemical components in chamomile according to claim 1, wherein in the step 1, 0.25g of chamomile powder is taken, sieved by a No. 3 sieve, placed in a 50mL conical flask with a plug, 25mL of methanol is precisely added, weighed and treated by ultrasonic treatment for 30min at a power of 300W and a frequency of 40KHz, and is cooled and shaken to 13000 r-min^-1Centrifuging at the rotation speed of (1) for 10min, collecting supernatant, filtering with 0.22 μm microporous membrane, and collecting filtrate to obtain the sample solution.

3. The method for rapidly identifying and analyzing chemical components in chamomile according to claim 1, wherein the chromatographic conditions in the step 2 are as follows: the chromatographic column is Waters ACQUITY UPLC BEH C18, the specification of the chromatographic column is 150mm multiplied by 2.1mm, 1.7 μm, and the mobile phase: methanol (A) -0.1% formic acid water solution (B), gradient elution, volume flow 0.2mL min^-1The column temperature was 40 ℃ and the amount of sample was 1 mL.

4. The method for rapid identification and analysis of chemical components in chamomile according to claim 3, wherein the gradient elution procedure is: 0-4 min, 5% -25% A; 4-9 min, 25% -65% A; 9-15 min, 65% -87% A; 15-19 min, 87% -98% A; 19-22 min, 98% A.

5. The method for rapidly identifying and analyzing chemical components in chamomile according to claim 4, wherein the mass spectrum conditions in the step 2 are as follows: negative ion mode scan measurement, electrospray ionization source ESI, instrument parameters were as follows: the scanning range m/z is 50-1500, the atomization gas is 55psi, the auxiliary gas is 55psi, the gas curtain gas is 35psi, the atomization temperature is 500 ℃, the negative ions are subjected to full-scanning ESI, the capillary voltage is 4500V, the cracking voltage is-80V, and the collision energy is 35 eV.

6. The method for rapidly identifying and analyzing chemical components in chamomile according to claim 1, wherein the step 3 comprises the following specific steps: and matching the collected total ion flow original data of the chromatographic mass spectrum with component information prestored in a Natural Products HR-MS/MS database by using MasterView data software, and deriving the name, molecular formula, theoretical molecular weight, actual molecular weight, addition form, error, response value and retention time of chemical components.

7. The method for rapidly identifying and analyzing chemical components in chamomile according to claim 6, wherein the main parameter settings are as follows: response value>1000counts, signal-to-noise ratio of 3, extracted ion width of 0.02Da, retention time width of 2min, threshold of 100cps, and molecular weight error<5ppm, according to the formula: error ppm ═ actual molecular weight-theoretical molecular weight)/theoretical molecular weight × 10⁶And removing compounds with the error of more than 5ppm in the calculation result.

8. The method for rapidly identifying and analyzing chemical components in chamomile according to claim 1, wherein a control solution is prepared before the step 2, and the method comprises the following specific steps: precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, palmitic acid, linoleic acid and oleic acid, respectively, adding appropriate amount of methanol, ultrasonic dissolving, standing at room temperature for clarification, collecting appropriate amount of supernatant, filtering with 0.22 μm microporous membrane, and placing in a sample bottle as mixed reference solution.

Technical Field

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for rapidly identifying and analyzing chemical components in chamomile.

Background

Chamomile (Matricaria chamomilla L.) is an annual herb of Matricaria (Matricaria) in the family of Compositae (Compositae), also known as chamomile or chamomile in Germany, Uygur medicine is also called babu's and Bamuo, is bitter in taste and sweet in flavor, is an important medicinal plant and spice resource, has the functions of generating dry heat, softening hardness and reducing swelling, warming stomach and stimulating appetite, dissipating qi and diminishing inflammation, dispelling wind and relieving pain, improving eyesight and the like, is mainly used for treating wet-cold or viscous liquid diseases, such as wet-cold various hard lump inflammation, joint swelling, dyspepsia, tendon and muscle relaxation, amenorrhea, anuresis and the like, and is collected in national drug standard Uygur medicine handbook, Chinese department of health medicine standard and the like. At present, chamomile is widely applied abroad, and dried chamomile flowers and essential oil are collected in United states Pharmacopeia, European Pharmacopeia and British Pharmacopeia.

The chemical components of chamomile are various, the chemical components of chamomile mainly comprise volatile oil, flavonoid, coumarin, organic acid and the like in previous researches, the main study on the components of the chamomile is the volatile oil, and the main study on the quality mainly comprises microscopic identification and content measurement. The quality evaluation of the traditional Chinese medicine is usually to evaluate the quality of the medicinal materials by one or more components in the medicinal materials, but for the traditional Chinese medicine, because the chemical components are complex, compatibility relationship exists among the components, certain limitation exists in the quality control research of the preparation and the medicinal materials only by adopting the traditional HPLC-UV fingerprint spectrum and multi-index content measurement, and common peaks, different components, compounds without obvious chromophore, and the like existing in the fingerprint spectrum cannot be accurately analyzed and identified. At present, the basic research on the drug effect substances of chamomile is not deep enough, comprehensive qualitative analysis on chemical components in the chamomile is lacked, and no report on the analysis of the chemical components in the chamomile by adopting an ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry technology exists. Therefore, the research on the drug effect substance basis of the chamomile has important practical significance for improving the quality standard of the chamomile medicinal material and the preparation thereof.

The ultra-high performance liquid phase combined quadrupole-tandem time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology becomes an important tool for researching chemical components and qualitative analysis in a traditional Chinese medicine complex system, has the advantages of strong chemical identification and analysis such as high scanning speed, high sensitivity, high resolution, accurate mass-to-charge ratio of a measured compound, wide ion scanning range and the like, and can perform structure prediction and analysis on the components in the absence of a reference substance. According to the information of accurate mass number, fragment ions, cracking rules and the like, qualitative analysis is carried out on the detected chemical components by combining a database, literature data, a reference substance, a mass spectrum database and the like, and the method is an effective means for systematically clarifying the basis of the chemical substances of the traditional Chinese medicine.

Disclosure of Invention

In order to overcome the technical problem, the invention discloses a method for rapidly identifying and analyzing chemical components in chamomile.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a method for rapidly identifying and analyzing chemical components in chamomile comprises the following steps:

step 1, preparing a test solution;

step 2, establishing an ultra-high performance liquid chromatography-mass spectrometry detection program, and obtaining original data of total ion current of a chromatography-mass spectrometry;

and 3, matching target components according to the original data of the total ion flow of the chromatographic mass spectrum, and performing rapid structural identification by combining and comparing chromatographic retention time, accurate relative molecular mass and fragment ion fragment information so as to realize the qualitative determination of chemical components in the chamomile.

In the step 1, 0.25g of chamomile powder is taken, sieved by a No. 3 sieve, placed in a 50mL conical flask with a plug, precisely added with 25mL of methanol, weighed to obtain a certain mass, subjected to ultrasonic treatment for 30min, the power is 300W, the frequency is 40KHz, and the mixture is cooled, shaken uniformly and subjected to 13000 r.min^-1Centrifuging at the rotation speed of (1) for 10min, collecting supernatant, filtering with 0.22 μm microporous membrane, and collecting filtrate to obtain the sample solution.

The method for rapidly identifying and analyzing chemical components in chamomile, wherein the chromatographic conditions in the step 2 are as follows: the chromatographic column is Waters ACQUITY UPLC BEH C18, the specification of the chromatographic column is 150mm multiplied by 2.1mm, 1.7 μm, and the mobile phase: methanol (A) -0.1% formic acid water solution (B), gradient elution, volume flow 0.2mL min-1, column temperature 40 ℃, sample volume 1 mL.

4. The method for rapid identification and analysis of chemical components in chamomile according to claim 3, wherein the gradient elution procedure is: 0-4 min, 5% -25% A; 4-9 min, 25% -65% A; 9-15 min, 65% -87% A; 15-19 min, 87% -98% A; 19-22 min, 98% A.

The method for rapidly identifying and analyzing chemical components in chamomile, wherein in the step 2, the mass spectrum conditions are as follows: negative ion mode scan measurement, electrospray ionization source ESI, instrument parameters were as follows: the scanning range is m/z 50-1500, the atomization gas is 55psi, the auxiliary gas is 55psi, the gas curtain gas is 35psi, the atomization temperature is 500 ℃, the negative ions are subjected to full-scanning ESI, the capillary voltage is 4500V, the cracking voltage is-80V, and the collision energy is 35 eV.

The method for rapidly identifying and analyzing the chemical components in the chamomile comprises the following specific steps in the step 3: and matching the collected total ion flow original data of the chromatographic mass spectrum with component information prestored in a Natural Products HR-MS/MS database by using MasterView data software, and deriving the name, molecular formula, theoretical molecular weight, actual molecular weight, addition form, error, response value and retention time of chemical components.

The method for rapidly identifying and analyzing the chemical components in the chamomile comprises the following main parameter settings: response value>1000counts, signal-to-noise ratio of 3, extracted ion width of 0.02Da, retention time width of 2min, threshold of 100cps, and molecular weight error<5ppm, according to the formula: error ppm ═ actual molecular weight-theoretical molecular weight)/theoretical molecular weight × 10⁶And removing compounds with the error of more than 5ppm in the calculation result.

In the above method for rapidly identifying and analyzing chemical components in chamomile, before performing step 2, a reference solution is prepared, and the specific steps are as follows: precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, palmitic acid, linoleic acid and oleic acid, respectively, adding appropriate amount of methanol, ultrasonic dissolving, standing at room temperature for clarification, collecting appropriate amount of supernatant, filtering with 0.22 μm microporous membrane, and placing in a sample bottle as mixed reference solution.

The invention has the beneficial effects that:

(1) depending on the ultra-high efficiency separation capability of UPLC and the high sensitivity and accurate qualitative and quantitative capability of Q-TOF-MS/MS, the ultra-high efficiency liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) combined technology can simultaneously detect trace-level and high-abundance compounds in one scanning, can be used for rapid analysis and component identification of a chamomile traditional Chinese medicine complex system, has the advantages of high resolution, high sensitivity, high accuracy, good reproducibility, short analysis time, wide scanning range and the like, can avoid complicated and complicated sample pretreatment, and can identify chemical components in natural chamomile medicines by combining related literature data according to the accurate molecular mass, fragment ion peak, chromatographic retention time and reference substance information of the compounds obtained by the high resolution mass spectrometry;

(2) on one hand, the method can avoid a complicated sample pretreatment method, and furthest retain the original chemical components in the sample, on the other hand, the analysis method of the chemical components in the chamomile firstly uses UPLC-Q-TOF-MS/MS to carry out analysis and determination on the chemical components in the chamomile, thereby providing a quick, accurate and efficient identification method for identifying the chemical components in the chamomile, and providing theoretical basis and technical reference for researching the pharmacodynamic material basis, the quality change rule and the quality standard improvement of the chamomile medicinal material.

Drawings

The invention is further illustrated with reference to the following figures and examples.

FIG. 1 is a total ion flow diagram of a UPLC-Q-TOF-MS/MS high-resolution mass spectrum of chamomile in a negative ion mode.

Detailed Description

The present invention is further illustrated by the following specific examples, which are intended to facilitate the understanding and appreciation of the technical solutions of the present invention, rather than to limit the invention thereto.

Example 1:

1. materials and reagents

Chamomile was purchased from Guangdong Kangmei pharmaceutical Co., Ltd, and a chamomile sample was identified as chamomile (Matricaria chamomilla L.) of a Matricaria (Compositae) plant of the genus Matricaria by the second institute of traditional Chinese medicine and engineering, Guangdong province (institute of traditional Chinese medicine and engineering, Guangdong province) Liujin researchers; chlorogenic acid as a reference (batch No. 110753-201415 with the purity of 96.2%), caffeic acid (batch No. 110885-200102 with the purity of 98.0%), oleic acid (batch No. 111621-201205 with the mass fraction of 99.6%) were purchased from China food and drug testing institute, neochlorogenic acid (batch No. X-014-180410 with the purity of more than 98%), isochlorogenic acid B (batch No. Y-069-150624 with the purity of more than 98%), and Dougeisi Biotechnology, Inc; palmitic acid (batch number PRF9061144, purity 95%) was obtained from Dougeny scientific development, Inc., while cryptochlorogenic acid (17061401, purity 98%) was obtained from Dougeny biotechnology, Inc.; isochlorogenic acid C (batch number: AF20121801, purity > 98%), linoleic acid (batch number AF7060202, mass fraction 98%) were purchased from Chengdu method Biotech, and were used for qualitative research. Methanol, formic acid, acetonitrile (ms spec pure, Fisher, usa), distilled water (guangzhou drochen food & beverage limited), methanol (analytically pure) purchased from guangzhou chemical reagent plant.

2. Instrument for measuring the position of a moving object

UPLC-Q-TOF-MS/MS system: an ExionLC AC-type high performance liquid chromatograph (AB SCIEX, USA), an XTOF 500R mass spectrometer, equipped with an ESI ion source (AB SCIEX, USA), a Mettler TOLEDO XS 205-type electronic analytical balance (Mettler-Torledo instruments, Inc.), a Sorvall Legend Micro 17R microcentrifuge (radius 8.6cm) (Thermo Scientific, USA), and a KQ-700 DE-type numerically controlled ultrasonic cleaner (ultrasonic power 700W, operating frequency 40KHz) (ultrasound, Inc., Kunshan).

3. Preparation of sample solution

Preparation of a test solution: taking chamomile powder (0.25 g passing through a No. 3 sieve), placing in a 50mL conical flask with a plug, precisely adding 25mL of methanol, weighing for determining mass, performing ultrasonic treatment (power 300W and frequency 40KHz), treating for 30min, cooling, shaking uniformly, and centrifuging (13000 r.min)^-1) For 10min, collecting supernatant, filtering with 0.22 μm microporous membrane, and collecting filtrate.

Preparation of control solutions: precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, palmitic acid, linoleic acid and oleic acid, respectively, adding appropriate amount of methanol, ultrasonic dissolving, standing at room temperature for clarification, collecting appropriate amount of supernatant, filtering with 0.22 μm microporous membrane, and placing in a sample bottle as mixed reference solution.

UPLC-Q-TOF-MS/MS high-resolution mass spectrometry analysis method

UPLC conditions: the column was Waters ACQUITY UPLC BEH C18(150 mm. times.2.1 mm, 1.7 μm); mobile phase: methanol (a) -0.1% aqueous formic acid (B), gradient elution: 0-4 min, 5% -25% A; 4-9 min, 25% -65% A; 9-15 min, 65% -87% A; 15-19 min, 87% -98% A; 19-22 min, 98% A; volume flow rate 0.2 mL/min^-1(ii) a The column temperature is 40 ℃; the sample volume is 1 mL.

Mass spectrum conditions: sciex X500R QTOF Mass spectrometer: scanning and measuring in a negative ion mode; electrospray ion source (ESI); the instrument parameters were as follows: the scanning range m/z is 50-1500, the atomization gas is 55psi, the auxiliary gas is 55psi, the gas curtain gas is 35psi, the atomization temperature is 500 ℃, the negative ions are subjected to full scanning (ESI), the capillary voltage is 4500V, the cracking voltage is-80V, and the collision energy is 35 eV.

5. Data processing

The general ion flow diagram of the UPLC-Q-TOF-MS/MS high-resolution mass spectrum of chamomile in a negative ion mode is shown in figure 1.

Matching the collected total ion current original data of the chromatographic mass spectrum with prestored component information in a database (Natural Products HR-MS/MS, Version 1.0, AB Sciex, Forster City, USA) by using MasterView data software, and deriving a sample name, a molecular formula, a theoretical molecular weight, an actual molecular weight, an addition form, an error, a response value and retention time; the main parameters are set as follows: response value>1000 counts; signal-to-noise ratio: 3; and (3) extracting ion width: 0.02 Da; retention time width: 2 min; threshold value: 100 cps; error in molecular weight:<5ppm, according to the formula: error ppm ═ actual molecular weight-theoretical molecular weight)/theoretical molecular weight × 10⁶Removing compounds with error of more than 5ppm of calculation result;

6. optimization of experimental methods

(1) Extraction method, time and solvent selection

Comparing two extraction methods of ultrasonic extraction and heating reflux extraction, extracting within 20-40 min, respectively extracting with 50% methanol, 50% ethanol, methanol and water, and comparing the extraction efficiency of chamomile extract under different conditions. The result shows that the methanol ultrasonic extraction efficiency is higher in 30 min.

(2) Mobile phase, chromatographic column and positive and negative ion scanning mode selection

Under the same experimental conditions, the separation efficiency of two columns, ACQUITY BEH C18(100 mm. times.2.1 mm, 1.7 μm) and ACQUITY UPLC C18(100 mm. times.2.1 mm, 1.7 μm), were compared. The BEH type chromatographic column can provide better separation efficiency and peak shape for most compounds and has universality.

When the mobile phase is inspected, 2 solvent systems of methanol-water and acetonitrile-water are compared, and the result shows that the elution effect of the methanol-water is better than that of the acetonitrile-water, and the mass spectrum response of the negative ion mode can be more effectively improved by adding 0.1% formic acid, so that 0.1% formic acid water solution-methanol is adopted as the mobile phase.

Meanwhile, considering that the response modes of compounds contained in the chamomile medicinal material are different, in order to better estimate the compounds in the sample, the experiment adopts positive and negative 2 ion modes for scanning detection, and the result shows that most compounds can be identified in the negative ion mode, so that the negative ion mode is selected for detection.

7. Analysis results

According to comparison of reference substances, literature reports, database query of Scifinder, Chemspider, Pubchem, TCMSP, MassBank and the like, the chemical components are identified by combining chromatographic retention behavior, molecular weight information and mass spectrum cracking rules, and the results are shown in Table 1.

TABLE 1 analysis results of chemical components in Chamomilla recutita anion mode

In conclusion, in the experiment, under the negative ion mode, the UPLC-Q-TOF-MS/MS high-resolution mass spectrum is adopted to carry out chromatographic separation, mass spectrum data acquisition and structural identification of inherent components on various components in the chamomile, and 34 compounds are analyzed and identified in total, and mainly contain organic acid and flavonoid compounds. The method can quickly and efficiently analyze the compounds in the chamomile, provides a basis for research and quick and accurate identification of the pharmacodynamic substances of the chamomile, and is beneficial to subsequent development and utilization of the chamomile.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Those skilled in the art can make many possible variations and modifications to the invention using the above disclosed technical means and teachings, or can modify equivalent embodiments with equivalent variations, without departing from the scope of the invention. Therefore, all equivalent changes made according to the shape, structure and principle of the present invention should be covered by the protection scope of the present invention without departing from the contents of the technical scheme of the present invention.