CN107607654B - A kind of analysis method of flavonoids in walnut flower - Google Patents

Abstract

The invention discloses a method for analyzing the chemical components of flavonoids in walnut flowers, which comprises the following steps: extracting a walnut flower medicinal material by adopting an ethanol heating reflux method, concentrating, redissolving, carrying out ultrasonic treatment, filtering to obtain a test solution, detecting the test solution by adopting an ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry method to obtain second-level mass spectrum information of a compound, and analyzing chemical component information in a detection result. The method can quickly and efficiently analyze the flavonoid compounds in the walnut flower, provides a basis for the research and the quick and accurate identification of the drug effect substances of the walnut flower, and is beneficial to the development and the utilization of the walnut flower.

Description

A kind of analysis method of flavonoids in walnut flower

technical field

The invention relates to a method for analyzing chemical components, in particular to a method for analyzing chemical components of flavonoids in walnut flowers based on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry technology.

Background technique

Walnut flower is the style of walnut, also known as walnut new, longevity vegetable, dragon mustard vegetable. Folks eat it as a cold dish, and clinically it is made into a tincture to remove warts. Its main components are glycerol linoleate, protein, carbohydrates and vitamins, etc., which have the functions of promoting human hematopoietic function, preventing arteriosclerosis, calming asthma and moistening the intestines. At present, the research on the components of walnut flowers is mainly based on flavonoids, and there is no report on the analysis of flavonoids in walnut flowers by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a method for analyzing the chemical components of flavonoids in walnut flowers. Through the combined technique of ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, it provides a method for analyzing the chemical components of flavonoids in walnut flowers. A fast and efficient analysis method was developed.

A method for detecting flavonoid chemical components in walnut flowers, comprising the following steps:

The walnut flower medicinal material was extracted by ethanol heating and refluxing, concentrated, reconstituted, sonicated, and filtered to obtain a test solution, which was detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry.

The method for detecting flavonoids in walnut blossoms according to the present invention, wherein, using Analyst TF1.7.1 software to set the chromatographic conditions in the ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry method, parameter The settings are as follows:

The chromatographic column was ACQUITY BEH C ₁₈ , 100mm×2.1mm, 1.7μm; 0.1% formic acid-water was used as mobile phase A, methanol was used as mobile phase B, linear gradient elution, 0～1min, 20%～35%B; 1min～5min, 35%～80%B; 5min～15min, 80%～100%B; 15min～17min, 100%B; 17.1min～20min, 20%B, the volume flow is 0.4mL/min; the column temperature is 40°C; injection volume 10 μL.

The method for detecting flavonoids in walnut blossoms according to the present invention, wherein, using Analyst TF1.7.1 software to set the mass spectrometry conditions in the ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry method, parameter The settings are as follows:

UPLC-Triple-TOF 5600+ time-of-flight LC/MS: positive ion scanning mode; scanning range: m/z100～1200; ion source temperature: 150℃; ion source voltage: 4500V; desolvation gas N ₂ : 110psi; Desolvation gas temperature: 600°C; curtain gas N ₂ : 35psi; primary scan: declustering voltage DP: 80V; secondary scan: use TOF MS~Product Ion~IDA mode to collect mass spectrometry data, CID energy is 20~50eV.

The method for detecting flavonoids in walnut flower of the present invention, wherein, the preparation of the test solution specifically includes the following steps: taking 1.0 g of walnut flower medicinal material to a 50 mL flask, heating and refluxing 50 mL of 50% ethanol to extract twice, The first extraction was 30 minutes, the second extraction was 1 hour, filtered, the two filtrates were combined, concentrated to dryness under reduced pressure, reconstituted in 5 mL of methanol, sonicated for 30 minutes, filled with methanol to make up the original total weight, filtered with a 0.22 μm microporous membrane, And stored in a 4 ℃ refrigerator as a test solution.

A method for analyzing flavonoid chemical components in walnut flowers, comprising the steps of: using the method of the present invention to detect the flavonoid chemical components in walnut flowers, and obtaining a total ion current map, primary mass spectrometry information and secondary mass spectrometry information of the compound , and use the PeakView 2.2 software to analyze the chemical composition information in the test results. The PeakView 2.2 includes the MasterView 1.1 plug-in:

(1) Open the collected walnut flower sample information in MasterView 1.1, and set the analysis parameters as follows: Intensity (response value): >1000counts; S:N (signal-to-noise ratio): >10; Default XIC Width (extracted ion width): 0.02Da; Default Retention Time Width: 2min; Default Threshold (threshold): 100cps; Minimum Retention Time: 0.3min; Mass Error (molecular weight error): <10ppm;

(2) According to the existing database containing 948 kinds of flavonoids in traditional Chinese medicine and the flavonoids found in Juglans, the molecular formula was imported into MasterView 1.1, and the molecular ion of each compound was set in MasterView 1.1. The sum of the form is [M+H] ⁺ or [M+Na] ⁺ , according to the molecular formula and molecular weight and walnut flower data to match using MasterView 1.1, get all imported compound information and experimental data matching results list; The information of compounds that meet the parameters set in step (1) will be displayed in green, if not, it will not be displayed in green. The MasterView 1.1 software is used to screen out the compounds that meet the parameter settings, that is, the compounds that are displayed in green, and the compounds that meet the parameter settings will be listed. The information in the derivation includes Formula (molecular formula), Mass (theoretical molecular weight), Adduct (actual molecular weight), Extraction (summation form), Error (error), Intensity (response value) and Found At RT (retention time) );

At the same time, the information of each compound will be clicked, and the primary mass spectrum and secondary mass spectrum corresponding to the clicked compound displayed in MasterView 1.1 will be exported and saved one by one for further analysis;

(3) In order to prevent the effective information of the compounds reported in the database and literature from being filtered due to errors during the software matching process, in the parameters set in step (1), Mass Error (molecular weight error): <10ppm, this range is relatively large, Therefore, further analysis and screening are required one by one;

Apply Analyst TF 1.7.1 "Calculators" Isotopic Distribution to calculate the exact mass of the molecular ion of each compound molecular formula, and the compound first-level mass spectrometry information recorded in step (2), according to the formula:

Error (ppm)=(actual molecular weight-theoretical molecular weight)/theoretical molecular weight×10 ⁶

Eliminate the compounds with an error greater than 5ppm in the calculation results;

(4) In order to improve the reliability of the identification, the results after removing some compounds in step (3) were analyzed by secondary mass spectrometry: the cracking pathways of the compounds were analyzed according to the literature, mzCloud mass spectrometry database and the cracking rules of flavonoids. If there are two fragment ions in the secondary mass spectrometry information obtained in , which conform to the fragmentation law of the compound, and the error range between the actual molecular weight of the fragment ion and the theoretical molecular weight is less than 15 ppm, it is considered that the compound exists in the experimental sample. The error calculation formula is the same as that in step (3) in the same.

The difference between the analytical method of the flavonoid chemical composition in the walnut flower of the present invention and the prior art is:

The method for analyzing flavonoids in walnut flowers of the present invention is the first to analyze and measure the flavonoids in walnut flowers by UHPLC-Triple-TOF-MS, which provides a fast and efficient identification method for the identification of chemical components in walnut flowers. Provide technical reference for basic research and quality control of pharmacodynamics of chemical components.

The method for analyzing the flavonoid chemical components in the walnut flower of the present invention will be further described below in conjunction with the accompanying drawings.

Description of drawings

Fig. 1 is the ion chromatogram of 36 compounds extracted in the embodiment of the present invention.

Detailed ways

1. Instruments and Materials

The walnut flower sample was identified by Professor Lei Haimin of Beijing University of Traditional Chinese Medicine as the walnut flower column of the genus Jugaceae; methanol and formic acid were chromatographically pure (PA company in the United States), water was Milli-Q ultrapure water, and the rest of the reagents were analytically pure ( Beijing Chemical Plant Company).

UPLC-Triple-TOF/MS system: ExionLC ^TM AD type high performance liquid chromatograph (AB SCIEX, USA), Triple TOF 5600+ mass spectrometer, equipped with ^ESI ion source (AB SCIEX, USA)

Second, the detection method of flavonoids in walnut flowers:

1. Chromatography and mass spectrometry conditions

Chromatographic conditions: ACQUITY BEH C ₁₈ (100mm×2.1mm, 1.7μm); 0.1% formic acid-water as mobile phase A, methanol as mobile phase B, linear gradient elution, 0～1min, 20%～35 %B; 1min～5min, 35%～80%B; 5min～15min, 80%～100%B; 15min～17min, 100%B; 17.1min～20min, 20%B, the volume flow is 0.4mL/min; The column temperature was 40°C; the injection volume was 10 μL.

Mass spectrometry conditions UPLC-Triple-TOF 5600 ⁺ time-of-flight LC/MS: positive ion scan mode; scan range: m/z 100～1200; ion source temperature: 150°C; ion source voltage: 4500V; desolvation gas ( N ₂ ): 110 psi; desolvation temperature: 600°C; curtain gas (N ₂ ): 35 psi; primary scan: declustering voltage (DP): 80 V; secondary scan: acquisition using TOF MS~Product Ion~IDA mode Mass spectrometry data, CID energy 20-50 eV; data analysis software: Analyst TF1.7.1, PeakView 2.2 and MasterView 1.1.

2. Preparation of the test solution

Take 1.0 g of walnut flower medicinal materials into a 50 mL flask, extract 2 times with 50 mL of 50% ethanol under reflux, the first extraction is 30 min, the second extraction is 1 h, filtered, the two filtrates are combined, concentrated to dryness under reduced pressure, reconstituted in 5 mL of methanol, Ultrasonic for 30min, make up to the original total weight with methanol, filter with 0.22μm microporous membrane, and store in a 4°C refrigerator as a test solution.

3. Analysis method of flavonoids in walnut flowers

The method of the present invention is used to detect the flavonoid chemical components in the walnut flowers, to obtain the total ion current map, primary mass spectrometry information and secondary mass spectrometry information of the compound, and the PeakView 2.2 software is used to analyze the chemical composition information in the detection result, The PeakView 2.2 includes the MasterView 1.1 plugin:

(1) Open the collected walnut flower sample information in MasterView 1.1, and set the analysis parameters as follows: Intensity: >1000counts; S:N: >10; Default XIC Width: 0.02Da; Default Retention TimeWidth: 2min; Default Threshold: 100cps; Minimum Retention Time: 0.3min; Mass Error: <10ppm;

(2) According to the existing database containing 948 kinds of flavonoids in traditional Chinese medicine and the flavonoids found in Juglans, the molecular formula was imported into MasterView 1.1, and the molecular ion of each compound was set in MasterView 1.1. The additive form (Adduct) is [M+H] ⁺ or [M+Na] ⁺ , according to the molecular formula and molecular weight and the walnut flower data to match using MasterView 1.1, get all imported compound information and experimental data matching result list ; The information of the compounds that meet the parameters set in (1) in the result will be displayed in green, if not, it will not be displayed in green. The MasterView 1.1 software is used to screen out the compounds in the results that match the parameter settings, that is, the compounds that match the parameter settings will be displayed in green. Export of information in the compound list including Formula, Mass, Adduct, Extraction, Error, Intensity and Found At RT;

At the same time, the information of each compound will be clicked, and the primary mass spectrum and secondary mass spectrum corresponding to the clicked compound displayed in MasterView 1.1 will be exported and saved one by one for further analysis;

(3) In order to prevent the effective information of the compounds reported in the database and literature from being filtered due to errors in the software matching process, in the parameters set in (1), Mass Error: <10ppm, this range is large, so it is necessary to further carry out one by one. analysis and screening;

Apply Analyst TF 1.7.1"Calculators"Isotopic Distribution to calculate the exact mass of the molecular ion of each compound molecular formula, and the compound first-level mass spectrometry information recorded in (2), according to the formula:

Error (ppm)=(actual molecular weight-theoretical molecular weight)/theoretical molecular weight×10 ⁶

Eliminate the compounds with an error greater than 5ppm in the calculation results;

(4) In order to improve the reliability of identification, the results of (3) after removing some compounds were subjected to secondary mass spectrometry analysis: the cracking pathways of the compounds were analyzed according to the literature, mzCloud mass spectrometry database and the cracking rules of flavonoids. The obtained secondary mass spectrometry information has two fragment ions (flavonoid glycosides have at least one fragment ion, because it is easy to lose the sugar group to obtain an aglycone fragment ion), which is in line with the fragmentation law of the compound, and the actual molecular weight of the fragment ion and the theoretical molecular weight. If the error range is less than 15ppm (the error calculation formula is the same as (3)), it is considered that the compound exists in the experimental sample.

According to the above analysis method, a total of 36 flavonoids in walnut flowers were identified, of which 25 compounds were presumed to have not been reported in the previous literature. The sum of the extracted ion chromatograms is shown in Figure 1, and the relevant information of the 36 compounds is shown in Table 1.

Fourth, the experimental method optimization

1. Extraction method, time and solvent selection

Comparing the two extraction methods of ultrasonic and heating and refluxing, the extraction was carried out within 20-60 minutes, and 50% methanol, 50% ethanol, 80% methanol and 80% ethanol were used to extract them respectively. extraction efficiency. The results showed that the extraction efficiency of 50% ethanol under reflux for 30min was higher.

2. Column selection

Under the same experimental conditions, three chromatograms of ACQUITY BEH C ₁₈ (100mm×2.1mm, 1.7μm), ACQUITY BEHC ₁₈ (150mm×2.1mm, 1.7μm) and ACQUITY HSS C ₁₈ (100mm×2.1mm, 1.8μm) were compared Separation efficiency of the column. BEH-type chromatographic columns can provide better separation efficiency and peak shape for most compounds, and are universal. ACQUITYHSS C ₁₈ (100mm×2.1mm, 1.8μm) has lower separation efficiency than ACQUITY BEH C ₁₈ (100mm×2.1mm, 1.7μm), while ACQUITY BEH C ₁₈ (150mm×2.1mm, 1.7μm) results in a higher column pressure.

V. Conclusion

In this experiment, UHPLC-Triple-TOF-MS was used for rapid analysis of flavonoids in walnut flower samples in positive ion mode, and a total of 36 flavonoids were inferred, 25 of which had not been reported in the previous literature. It provides some reference for further chemical composition, pharmacological research and quantitative research. The method can quickly and efficiently analyze flavonoids in walnut flowers, provides a basis for the research and rapid and accurate identification of medicinal substances in walnut flowers, and is beneficial to the development and utilization of walnut flowers.

The above-mentioned embodiments merely describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. Variations and improvements should fall within the protection scope determined by the claims of the present invention.

Claims (4)

1. a detection method of flavonoid chemical composition in walnut flower, is characterized in that: comprise the steps: Using 50% ethanol heating and refluxing to extract the medicinal materials of walnut flower, concentrating, reconstituting, sonicating, and filtering to obtain the test solution, which was subjected to ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. detection; Use Analyst TF 1.7.1 software to set the chromatographic conditions in the ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry method, and the parameters are set as follows: The chromatographic column was ACQUITY BEH C ₁₈ , 100mm×2.1mm, 1.7μm; 0.1% formic acid-water was used as mobile phase A, methanol was used as mobile phase B, linear gradient elution, 0～1min, 20%～35%B; 1min～5min, 35%～80%B; 5min～15min, 80%～100%B; 15min～17min, 100%B; 17.1min～20min, 20%B, the volume flow is 0.4mL/min; the column temperature is 40°C; injection volume 10 μL. 2. the detection method of flavonoids chemical composition in the walnut flower according to claim 1, is characterized in that: application Analyst TF 1.7.1 software to described ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry method. The mass spectrometry conditions are set, and the parameters are set as follows: UPLC-Triple-TOF 5600+ time-of-flight LC/MS: positive ion scan mode; scan range: m/z 100～1200; ion source temperature: 150℃; ion source voltage: 4500V; desolvation gas N ₂ : 110psi ; Desolvation temperature: 600°C; Curtain gas N ₂ : 35psi; Primary scan: declustering voltage DP: 80V; Secondary scan: use TOF MS～Product Ion～IDA mode to collect mass spectrometry data, CID energy is 20～50eV . 3. the detection method of flavonoid chemical composition in walnut flower according to claim 2, it is characterized in that: described need testing solution preparation specifically comprises the steps: get walnut flower medicinal material 1.0g to 50mL flask, 50mL 50% ethanol Heated and refluxed for 2 extractions, the first extraction was 30 min, the second extraction was 1 h, filtered, the two filtrates were combined, concentrated to dryness under reduced pressure, redissolved in 5 mL of methanol, sonicated for 30 min, and made up to the original total weight with methanol, 0.22 μm Filtered through a microporous membrane and stored in a 4°C refrigerator as a test solution. 4. an analytical method of flavonoid chemical composition in walnut flower, it is characterized in that: comprise the steps: adopt the method of claim 2 to detect flavonoid chemical composition in walnut flower, obtain the total ion flow diagram of compound, one-level Mass spectrometry information and secondary mass spectrometry information, and use PeakView 2.2 software to analyze the chemical composition information in the detection results. The PeakView 2.2 includes the MasterView 1.1 plug-in: (1) Open the collected walnut flower sample information in MasterView 1.1, and set the analysis parameters as follows: response value>1000counts; signal-to-noise ratio:>10; extracted ion width: 0.02Da; retention time width: 2min; threshold: 100cps; minimum Retention time: 0.3min; molecular weight error: <10ppm; (2) According to the existing database containing 948 kinds of flavonoids in traditional Chinese medicine and the flavonoids found in Juglans, the molecular formula was imported into MasterView 1.1, and the molecular ion of each compound was set in MasterView 1.1. The sum of the form is [M+H] ⁺ or [M+Na] ⁺ , according to the molecular formula and molecular weight and walnut flower data to match using MasterView 1.1, get all imported compound information and experimental data matching results list; The information of compounds that meet the parameters set in step (1) will be displayed in green, if not, it will not be displayed in green. The MasterView 1.1 software is used to screen out the compounds that meet the parameter settings, that is, the compounds that are displayed in green, and the compounds that meet the parameter settings will be listed. The information in is derived, the information includes molecular formula, theoretical molecular weight, actual molecular weight, addition form, error, response value, retention time; At the same time, the information of each compound will be clicked, and the primary mass spectrum and secondary mass spectrum corresponding to the clicked compound displayed in MasterView 1.1 will be exported and saved one by one for further analysis; (3) In order to prevent the effective information of the compounds reported in the database and literature from being filtered due to errors in the software matching process, the molecular weight error in the parameters set in step (1): <10ppm, this range is large, so it is necessary to further carry out Analyze and filter one by one; Apply Analyst TF 1.7.1>>Calculators>>Isotopic Distribution software to calculate the exact mass of the molecular ion of each compound molecular formula, and the compound first-level mass spectrometry information recorded in step (2), according to the formula: Error ppm=(actual molecular weight-theoretical molecular weight)/theoretical molecular weight×10 ⁶ Eliminate the compounds with an error greater than 5ppm in the calculation results; (4) In order to improve the reliability of the identification, the results after removing some compounds in step (3) were analyzed by secondary mass spectrometry: the cracking pathways of the compounds were analyzed according to the literature, mzCloud mass spectrometry database and the cracking rules of flavonoids. If there are two fragment ions in the secondary mass spectrometry information obtained in , which conform to the fragmentation law of the compound, and the error range between the actual molecular weight of the fragment ion and the theoretical molecular weight is less than 15 ppm, it is considered that the compound exists in the experimental sample. The error calculation formula is the same as that in step (3) in the same.

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