Method for determining chemical components in caraway

Abstract

The invention provides a method for determining chemical components in caraway, which adopts ultra-high performance liquid chromatography-quadrupole rod-electrostatic field orbitrap high-resolution mass spectrometry to determine the chemical components in caraway, quickly identifies main chemical components in caraway extract, totally identifies 33 compounds, and has the advantages of high accuracy, high sensitivity, high accuracy and high sensitivity. The raw materials mainly comprise flavonoids, alkaloids and saccharides, and further comprise chemical components such as proanthocyanidins and amino acids. The method has the characteristics of rapidness, simplicity, convenience, feasibility, small sample dosage, high-throughput analysis and the like, realizes separation and identification of the effective components of the caraway extract, lays a good material foundation for pharmacological research, quantitative detection analysis and quality control of the effective components in the caraway extract, and has wide application prospects. Meanwhile, an important research basis is provided for pharmacodynamic association of effective components in the extract. And an important research basis is provided for pharmacological research, quantitative detection analysis and quality standard establishment of effective components in the caraway extract.

Description

Technical Field

[0001] The invention relates to the technical field of chemical component analysis, and in particular to a method for determining chemical components in a caraway extract by using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry. Background Art

[0002] Carum carvi L. is the fruit of the dicotyledonous plant Carum of the Umbelliferae family. In traditional Chinese medicine, it has the medicinal value of dispelling wind and cold, warming the middle and strengthening the spleen. Its fruit and root can be used as medicine. It is warm in nature, slightly spicy, and aromatic. It is used to treat stomachache caused by cold stagnation, hiccups caused by stomach cold, back pain, abdominal pain caused by cold stagnation, hernia, etc. It has rich pharmacological activities, such as antioxidant, antibacterial, anti-inflammatory, antiviral, anti-stress, anticonvulsant, antidepressant, anti-tumor, anti-obesity and other active effects.

[0003] At present, there are few studies on the quality control methods of Tibetan fennel extracts. There is no record in the Chinese Pharmacopoeia, and there are only basic identifications such as source and identification in the ministerial standards. The research on its components and pharmacological effects is still incomplete. CN202310792717.3 discloses a Tibetan fennel identification method and its application, which is to compare Tibetan fennel extracted by methanol reflux with a reference substance by TLC method to achieve the purpose of identification. It cannot characterize the specific components contained in Tibetan fennel, cannot further study the efficacy of its components, and cannot comprehensively control the product quality. Therefore, there is an urgent need for a method that can effectively determine and identify multiple chemical components in Tibetan fennel extracts. Summary of the invention

[0004] The purpose of the present invention is to provide a method for determining chemical components in carambola in view of the deficiencies of the prior art. The method uses ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry to determine the chemical components in carambola, realizes the separation and identification of multiple effective components in carambola extract, and provides an important research basis for the pharmacological study of effective components in carambola extract, quantitative detection analysis and establishment of quality standards.

[0005] The method for determining the chemical components in caraway described in the present invention adopts ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry to determine the chemical components in caraway, and the specific operation is carried out according to the following steps:

[0006] Sample pretreatment:

[0007] a. Mix the caraway powder and the solvent, 70-75% ethanol or methanol, at a material-liquid ratio of 1:5-20, perform ultrasonic treatment for 15-40 min, cool, weigh the mass, make up the lost mass with the solvent ethanol, extract with petroleum ether, remove the aqueous phase, evaporate the solvent, add methanol to the scale, shake well, filter with a 0.22 μm organic microporous filter membrane, and take the filtrate to obtain the caraway extract test sample;

[0008] b. Using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry to perform liquid phase separation and mass spectrometry detection on the test sample obtained in step a.

[0009] Chromatographic conditions: chromatographic column Waters BEH C1 8, 2.1×1 50 mm, 1.7 μm; mobile phase 0.05-0.15% formic acid in water (A)-methanol (B) for gradient elution, 0-5 min, 5% B-5% B; 5-10 min, 5% B-13% B; 10-14 min, 13% B-20% B; 14-25 min, 20% B-27% B; 25-31 min, 27% B-32% B; 31-37 min, 32% B-39% B; 37-67 min, 39% B-46% B; 67-70min, 46% B-52% B; 70-72min, 52% B-55% B; 72-80min, 55% B-95% B; 80-90min, 95% B-95% B; flow rate 0.18-0.22mL / min; column temperature 20-40℃, injection volume 2-20μL, detection wavelength 254nm, obtain the peaks of each chemical component in caraway, and conduct qualitative analysis;

[0010] Mass spectrometry conditions:

[0011] Electrospray ion source, positive and negative ion scanning mode, sheath gas flow rate 40L / min, auxiliary gas flow rate 10L / min, spray voltage 3kV, capillary ion transfer tube temperature 300℃ positive ion mode or positive and negative ion mode, 350℃ negative ion mode, maximum spray current 100μA, auxiliary gas temperature 350℃, mass scanning range m / z100-1500Da, MSE mode was used to collect bar graphs at each time point, scanning interval 0.2s, collision energy 6eV was transmitted during low energy scanning, and collision energy 25-50eV was transmitted during high energy scanning; leucine enkephalin with a mass concentration of 1.0ng / L was selected for real-time mass correction, flow rate 10μL / min, and data acquisition was controlled by MassLynx 4.1 software.

[0012] The solvent in step a is 55-80% ethanol; the solid-liquid ratio is 1:5-15; the ultrasonic treatment time is 20-35 minutes;

[0013] In step b, the column temperature is 25-30° C.; the injection volume is 3-10 μL; the flow rate is 0.2 mL / min; and the volume concentration of the aqueous solution of formic acid is 0.1%.

[0014] The method for determining the chemical components in caraway described in the present invention has the following beneficial effects compared with the prior art:

[0015] The present invention uses ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-Q-Orbitrap-HRMS Plus) technology to quickly identify the main chemical components in the caraway extract, and a total of 33 compounds are identified, mainly flavonoids, alkaloids, sugars, and also contain chemical components such as proanthocyanidins and amino acids. The method is fast, simple, easy, has the characteristics of small sample usage and high-throughput analysis, and realizes the separation and identification of the effective components of the caraway extract, laying a good material foundation for the pharmacological research, quantitative detection and analysis, and quality control of the effective components in the caraway extract, and at the same time provides an important research basis for the drug-effect correlation of the effective components in the extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Total ion chromatogram (TIC) of mass spectrometry of carveri extract in negative ion mode measured by the determination method of Example 1;

[0017] Figure 2 The peak number corresponding to the mass spectrum of the caraway extract in the negative ion mode under the determination method of Example 1;

[0018] Figure 3 Total ion chromatogram (TIC) of mass spectrometry of carveri extract in positive ion mode measured by the determination method of Example 1;

[0019] Figure 4 The peak numbers are corresponding to the mass spectrogram of the Tibetan fennel extract in the positive ion mode according to the determination method of Example 1. DETAILED DESCRIPTION

[0020] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in the field or the product instructions of the relevant products are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0021] Example 1

[0022] Sample pretreatment:

[0023] Take 20.0g of fennel powder, accurately weigh it, put it in a 250mL beaker, accurately add 200mL of 70% ethanol, weigh the mass, power 250W, frequency 35kHz, ultrasonic treatment for 15min, cool, weigh the mass again, use 70% ethanol to make up the lost mass, shake well, filter under reduced pressure, rotary evaporate the filtrate until there is no alcohol taste, add 10ml of distilled water to dissolve, extract with petroleum ether, remove the aqueous phase, evaporate the solvent, put it in a 10mL volumetric flask, add methanol to the scale, shake well, filter with 0.22μm organic microporous membrane, take the filtrate, and obtain the fennel extract test sample;

[0024] The components of the extract of Caraway were detected by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry (UPLC-Q-Orbitrap-HRMSPlus):

[0025] Liquid phase conditions:

[0026] Chromatographic column: Waters BEH C1 8 (2.1×1 50 mm, 1.7 μm);

[0027] Column temperature: 30°C;

[0028] Mobile phase: A-0.1% formic acid in water; B-methanol;

[0029] Gradient elution conditions:

[0030] Time <![CDATA[A% [H2O (0.01% formic acid)]]]> B%(MeoH) 0.000 95.0 5.0 5.000 95.0 5.0 10.000 87.0 13.0 14.000 80.0 20.0 25.000 73.0 27.0 31.000 68.0 32.0 37.000 61.0 39.0 67.000 54.0 46.0 70.000 48.0 52.0 72.000 45.0 55.0 80.000 5.0 95.0 90.000 5.0 95.0

[0031] Mass spectrometry conditions:

[0032] Electrospray ion source (HESI), positive and negative ion scanning mode, sheath gas flow rate 40L / min, auxiliary gas flow rate 10L / min, spray voltage 3kV, capillary ion transfer tube temperature 300℃ positive ion mode or positive and negative ion mode, temperature 350℃ negative ion mode, maximum spray current 100μA, auxiliary gas temperature 350℃, mass scanning range m / z100-1500 Da, MSE mode was used to collect bar graphs at each time point, scanning interval 0.2s, transmission collision energy 6eV during low energy scanning, transmission collision energy 25-50eV during high energy scanning; leucine enkephalin with a mass concentration of 1.0ng / L was selected for real-time mass calibration, flow rate 10μL / min, data acquisition was controlled by MassLynx 4.1 software;

[0033] The sample pre-treated caraway extract was injected into an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer (UPLC-Q-Orbitrap-HRMS Plus) for determination;

[0034] Test results:

[0035] Test results such as Figure 1 , 3 As shown, the components of the peaks of the caraway extract in the mass spectrum were analyzed, and Table 1 is as follows:

[0036] Table 1 List of ingredients of caraway extract

[0037]

[0038]

[0039] Example 2

[0040] Sample pretreatment:

[0041] Take 20.0g of fennel powder, accurately weigh it, put it in a 250mL beaker, accurately add 200mL of 75% ethanol, weigh the mass, ultrasonically treat it at a power of 250W and a frequency of 35kHz for 40min, cool it, weigh it again, make up the lost mass with 75% ethanol, shake it well, filter it under reduced pressure, rotary evaporate the filtrate until there is no alcohol taste, add 10ml of distilled water to dissolve it, extract it with petroleum ether, remove the aqueous phase, evaporate the solvent, put it in a 10mL volumetric flask, add methanol to the scale, shake it well, filter it with a 0.22μm organic microporous filter membrane, and take the filtrate to obtain the fennel extract test sample;

[0042] The components of the extract of Caraway were detected by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry (UPLC-Q-Orbitrap-HRMSPlus):

[0043] Liquid phase conditions:

[0044] Chromatographic column: Waters BEH C1 8 (2.1×1 50 mm, 1.7 μm);

[0045] Column temperature: 20°C;

[0046] Mobile phase: A-0.1% formic acid in water; B-methanol;

[0047] Gradient elution conditions:

[0048] Time <![CDATA[A% [H2O (0.01% formic acid)]]]> B%(MeoH) 0.000 95.0 5.0 5.000 95.0 5.0 10.000 87.0 13.0 14.000 80.0 20.0 25.000 73.0 27.0 31.000 68.0 32.0 37.000 61.0 39.0 67.000 54.0 46.0 70.000 48.0 52.0 72.000 45.0 55.0 80.000 5.0 95.0 90.000 5.0 95.0

[0049] Mass spectrometry conditions:

[0050] Electrospray ion source (HESI), positive and negative ion scanning mode, sheath gas flow rate 40L / min, auxiliary gas flow rate 10L / min, spray voltage 3kV, capillary ion transfer tube temperature 300℃ positive ion mode or positive and negative ion mode, temperature 350℃ negative ion mode, maximum spray current 100μA, auxiliary gas temperature 350℃, mass scanning range m / z100-1500 Da, MSE mode was used to collect bar graphs at each time point, scanning interval 0.2s, transmission collision energy 6eV during low energy scanning, transmission collision energy 25-50eV during high energy scanning; leucine enkephalin with a mass concentration of 1.0ng / L was selected for real-time mass calibration, flow rate 10μL / min, data acquisition was controlled by MassLynx 4.1 software;

[0051] The sample pre-treated caraway extract was injected into an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer (UPLC-Q-Orbitrap-HRMS Plus) for determination;

[0052] Test results:

[0053] Compound 1-33 can be identified based on the spectrum information, and its structural formula is:

[0054]

[0055]

[0056] Example 3

[0057] Sample pretreatment:

[0058] Take 20.0g of fennel powder, accurately weigh it, put it in a 250mL beaker, accurately add 200mL of 70% methanol, weigh the mass, and ultrasonically treat it at a power of 250W and a frequency of 35kHz for 30min. Cool it, weigh it again, make up the lost mass with 70% methanol, shake it well, filter it under reduced pressure, rotary evaporate the filtrate until there is no alcohol taste, add 10ml of distilled water to dissolve it, extract it with petroleum ether, remove the aqueous phase, evaporate the solvent, put it in a 10mL volumetric flask, add methanol to the scale, shake it well, filter it with a 0.22μm organic microporous filter membrane, and take the filtrate to obtain the fennel extract test sample;

[0059] The components of the extract of Caraway were detected by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry (UPLC-Q-Orbitrap-HRMSPlus):

[0060] Liquid phase conditions:

[0061] Chromatographic column: Waters BEH C1 8 (2.1×1 50 mm, 1.7 μm);

[0062] Column temperature: 35°C;

[0063] Mobile phase: A-0.1% formic acid in water; B-methanol;

[0064] Gradient elution conditions:

[0065] Time <![CDATA[A% [H2O (0.01% formic acid)]]]> B%(MeoH) 0.000 95.0 5.0 5.000 95.0 5.0 10.000 87.0 13.0 14.000 80.0 20.0 25.000 73.0 27.0 31.000 68.0 32.0 37.000 61.0 39.0 67.000 54.0 46.0 70.000 48.0 52.0 72.000 45.0 55.0 80.000 5.0 95.0 90.000 5.0 95.0

[0066] Mass spectrometry conditions:

[0067] Electrospray ion source (HESI), positive and negative ion scanning mode, sheath gas flow rate 40L / min, auxiliary gas flow rate 10L / min, spray voltage 3kV, capillary ion transfer tube temperature 300℃ positive ion mode or positive and negative ion mode, 350℃ negative ion mode, maximum spray current 100μA, auxiliary gas temperature 350℃, mass scanning range m / z100-1500 Da, MSE mode was used to collect bar graphs at each time point, scanning interval 0.2s, transmission collision energy 6eV during low energy scanning, transmission collision energy 25-50eV during high energy scanning; leucine enkephalin with a mass concentration of 1.0ng / L was selected for real-time mass calibration, flow rate 10μL / min, data acquisition was controlled by MassLynx 4.1 software;

[0068] The sample pre-treated caraway extract was injected into an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer (UPLC-Q-Orbitrap-HRMS Plus) for determination;

[0069] Test results:

[0070] Compounds 1-33 can be identified based on the spectral information.

Claims

1. A method for determining the chemical components in caraway, characterized in that: Ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry was used to determine the chemical components in caraway. The specific operation was carried out according to the following steps: Sample pretreatment: a. Mix the fennel powder and the solvent, which is 70-75% ethanol or methanol, at a material-liquid ratio of 1:5-20, perform ultrasonic treatment for 15-40 min, cool, weigh the mass, make up the lost mass with the solvent ethanol, extract with petroleum ether, remove the aqueous phase, evaporate the solvent, add methanol to the scale, shake well, filter with a 0.22 μm organic microporous filter membrane, and take the filtrate to obtain the fennel extract test sample; b. Using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry to perform liquid phase separation and mass spectrometry detection on the test sample obtained in step a. Chromatographic conditions: chromatographic column Waters BEH C1 8, 2.1×1 50mm, 1.7μm; mobile phase 0.05-0.15% formic acid in water (A) - methanol (B) for gradient elution, 0-5 min, 5%B-5%B; 5-10 min, 5%B-13%B; 10-14 min, 13%B-20%B; 14-25 min, 20%B-27%B; 25-31 min, 27%B-32%B; 31-37 min, 32%B-39%B; 37-67 min, 39%B-46%B; 67-70 min, 46%B-52%B; 70-72 min, 52%B-55%B; 72-80 min, 55%B-95%B; 80-90 min, 95%B-95%B; flow rate 0.18-0.22 mL / min; column temperature 20-40℃, injection volume 2-20μL, detection wavelength 254nm, obtain the peaks of each chemical component in caraway and conduct qualitative analysis; Mass spectrometry conditions: Electrospray ion source, positive and negative ion scanning mode, sheath gas flow rate 40 L / min, auxiliary gas flow rate 10 L / min, spray voltage 3 kV, capillary ion transfer tube temperature 300℃ positive ion mode or positive and negative ion mode, 350℃ negative ion mode, maximum spray current 100μA, auxiliary gas temperature 350℃, mass scanning range m / z100-1500Da, bar graphs at each time point were collected, scanning interval 0.2 s, collision energy 6eV was transmitted during low energy scanning, and collision energy 25-50eV was transmitted during high energy scanning; leucine enkephalin with a mass concentration of 1.0 ng / L was selected for real-time mass calibration, flow rate 10 μL / min, and data acquisition was controlled by MassLynx 4.1 software.

2. The method for determining the chemical components in Caraway according to claim 1, characterized in that: The solvent in step a is 55-80% ethanol; the solid-liquid ratio is 1:5-15; and the ultrasonic treatment time is 20-35 min.

3. The method for determining the chemical components in caraway according to claim 1, characterized in that: In step b, the column temperature is 25-30° C.; the injection volume is 3-10 μL; the flow rate is 0.2 mL / min; and the volume concentration of the aqueous solution of formic acid is 0.1%.

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