Method for detecting chemical components in Weiweishu granules based on UPLC-Q-TOF-MS / MS technology and application

Abstract

The invention discloses a method for detecting chemical components in nourishing stomach soothing granules based on a UPLC-Q-TOF-MS / MS (Ultra Performance Liquid Chromatography-Quality-Time of Flight-Mass Spectrometry-Mass Spectrometry) technology and application. The method comprises the steps of preparing a test solution, detecting and analyzing the chemical components, identifying the chemical components and the like. The method disclosed by the invention is simple in pretreatment, less in component loss, more accurate and sensitive in detection and short in consumed time, 150 chemical components in the stomach nourishing granules can be effectively separated within 38min, the problem that the chemical components in the stomach nourishing granules are difficult to comprehensively and systematically represent by means and methods in the prior art is solved, and the method is suitable for popularization and application. High-throughput detection and analysis identification of the chemical components in the Weiweishu granules are realized, the chemical components in the Weiweishu granules can be rapidly and comprehensively analyzed, and a basis is provided for further research on the pharmacodynamic material basis and comprehensive quality control of the Weiweishu granules.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and specifically relates to a method and application of detecting chemical components in Yangweishu granules based on UPLC-QTOF-MS / MS technology. Background Art

[0002] Yangweishu Granules are a granule preparation composed of Chinese yam, Codonopsis pilosula, Atractylodes macrocephala (fried), Crataegus pinnatifida (fried), Tangerine peel, Dried ginger, Scrophularia ningpoensis, Adenophora sibiricum, Polygonatum sibiricum (steamed), Cuscuta australis and Prunus mume. It has the effects of invigorating Qi and strengthening the body, nourishing Yin and nourishing the stomach, regulating the middle jiao, and promoting Qi and digestion. It is commonly used in the clinic to treat epigastric burning pain and distension, chronic atrophic gastritis, and chronic gastritis with the above symptoms.

[0003] Yangweishu Granules are a compound preparation of traditional Chinese medicine. At present, the research on the chemical components of Yangweishu Granules still mostly uses liquid chromatography (HPLC), which can only perform qualitative and quantitative analysis on several chemical components, and it is difficult to comprehensively and systematically characterize the chemical components in Yangweishu Granules. For example, in the "HPLC method for simultaneous determination of the content of chlorogenic acid, rhamnoside of vitexin and hesperidin in Yangweishu Granules", only five components of harpagoside, hesperidin, chlorogenic acid, cinnamic acid, and rhamnoside of vitexin in Yangweishu Granules were quantitatively analyzed. However, the active ingredients in Yangweishu Granules are complex and diverse, and the content determination of a few index components can no longer fully reflect the quality of traditional Chinese medicine compound preparations. Therefore, it is very necessary to study the high-throughput screening technology of the chemical components of Yangweishu Granules. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a method for detecting the chemical components in Yangweishu granules based on UPLC-QTOF-MS / MS technology. This method can quickly and comprehensively analyze the chemical components in Yangweishu granules, solving the problem that the existing technical means and methods are difficult to comprehensively and systematically characterize the chemical components in Yangweishu granules.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology, comprising the following steps:

[0007] (1) Preparation of test solution

[0008] Accurately weigh the Yangweishu granules sample, add the extraction solvent and mix well, ultrasonically crush it, and then centrifuge to obtain the supernatant to obtain the test solution;

[0009] (2) Chemical composition detection and analysis

[0010] Ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) technology was used to detect and analyze the chemical components in the Yangweishu granules test solution;

[0011] The chromatographic conditions are as follows: C18 chromatographic column; mobile phase A is a formic acid aqueous solution with a volume concentration of 0.1%, and mobile phase B is acetonitrile; column temperature is 30°C; elution gradient program is as follows: 0-6 min, 95-90% A; 6-10 min, 90-86% A; 10-23 min, 86-77% A; 23-24 min, 77% A; 24-25 min, 77-70% A; 25-29 min, 70-60% A; 29-31 min, 60-50% A; 31-32 min, 50-5% A; 32-34 min, 5% A; 34-35 min, 5-95% A; 35-38 min, 95% A;

[0012] The mass spectrometry conditions were as follows: electrospray ionization source ESI, capillary voltages of 3.0 KV and 2.0 KV in positive and negative ion modes, respectively; ion source temperature of 150 °C, sample cone voltage of 30 V, cone gas volume flow rate of 50 L / h, nitrogen degassing temperature of 350 °C, degassing volume flow rate of 800 L / h; scanning range of m / z 50-2000;

[0013] (3) Chemical composition identification

[0014] The total ion current of the test solution collected in the positive and negative ion modes was extracted using analysis software to obtain ion fragment information data. The molecular formula of the compound was determined based on the quasi-molecular ion peak, and the chemical components in Yangweishu Granules were identified by referring to the secondary mass spectrometry fragment information in the literature.

[0015] The Yangweishu granules of the invention are granule preparations composed of yam, codonopsis pilosula, stir-fried atractylodes macrocephala, stir-fried southern hawthorn, tangerine peel, dried ginger, scrophularia ningpoensis, northern adenophora, steamed polygonatum, dodder seed and ebony.

[0016] Preferably, in step (1), the extraction solvent is selected from pure water or methanol, preferably pure water.

[0017] Preferably, in step (1), the mass volume ratio of the Yangweishu granule sample to the extraction solvent is (0.5-5.0 g): (5-60 mL), preferably 1.0 g: (5-20 mL).

[0018] Preferably, in step (1), the ultrasonic crushing time is 30 to 60 minutes; the ultrasonic power is 220 to 250 kW; and the ultrasonic frequency is 30 to 50 kHz.

[0019] Preferably, in step (1), the centrifugal speed is 6000-10000 rpm, and the time is 10-20 min.

[0020] Preferably, in step (2), the chromatographic column is ACQUITY HSS T3, specifications are 2.1×100mm, 1.8μm.

[0021] Preferably, in step (3), a total of 150 chemical components are identified in the Yangweishu granules, including flavonoids, organic acids, terpenoids and nitrogen-containing compounds.

[0022] The present invention also provides application of the above method in quality control of Yangweishu granules, and quality control or identification of Yangweishu granules is performed by detecting the chemical components of the Yangweishu granules.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology. The method has simple pretreatment, less component loss, more accurate and sensitive detection, and short time consumption. 150 chemical components in Yangweishu granules can be effectively separated within 38 minutes, which solves the problem that the existing technical means and methods are difficult to comprehensively and systematically characterize the chemical components in Yangweishu granules, realizes high-throughput detection and analytical identification of the chemical components in Yangweishu granules, can quickly and comprehensively analyze the chemical components in Yangweishu granules, and provides a basis for further studying the material basis of the efficacy of Yangweishu granules and comprehensive quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 1 is a TIC mass spectrum obtained in the negative ion mode of Comparative Example 1 of the present invention, wherein (A) is a TIC mass spectrum of elution gradient 1; (B) is a TIC mass spectrum of elution gradient 2; (C) is a TIC mass spectrum of elution gradient 3;

[0026] Figure 2 It is the TIC mass spectra obtained in the negative ion mode of Comparative Example 2 of the present invention, wherein (A) is the TIC mass spectra of the methanol solution of Yangweishu granules in the negative ion mode; (B) is the TIC mass spectra of the aqueous solution of Yangweishu granules in the negative ion mode;

[0027] Figure 3It is the TIC mass spectra obtained in the negative ion mode in Comparative Example 3 of the present invention, wherein (A) is the TIC mass spectra of Yangweishu granules: pure water (1:5) in the negative ion mode; (B) is the TIC mass spectra of Yangweishu granules: pure water (1:10) in the negative ion mode; (C) is the TIC mass spectra of Yangweishu granules: pure water (1:20) in the negative ion mode;

[0028] Figure 4 It is the total ion current chromatogram (TIC) of Yangweishu granules in positive and negative ion modes in Example 1 of the present invention, wherein (A) is the TIC mass spectrum of Yangweishu granules in positive ion mode; (B) is the TIC mass spectrum of Yangweishu granules in negative ion mode;

[0029] Figure 5 This is the cracking method of some chemical components in Yangweishu Granules. DETAILED DESCRIPTION

[0030] In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. However, the implementation methods of the present invention are not limited to the following embodiments.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0032] Example 1: Method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology 1. Preparation of Yangweishu granule test solution

[0033] Take Yangweishu granules, accurately weigh 1.0 g, put it in a stoppered conical flask, add 10 mL of pure water respectively, stopper it, weigh it, treat it with ultrasonic wave (power 250 W, frequency 40 kHz) for 60 min, let it cool, weigh it again, make up the lost weight with pure water, shake it well, centrifuge it at 8000 rpm for 10 min several times until the solution is clear, and the Yangweishu test solution is obtained.

[0034] 2. Chemical composition detection and analysis

[0035] Chromatographic conditions: The chromatographic column is ACQUITY HSS T3 (2.1×100mm, 1.8μm);

[0036] The flow rate was 0.3 mL / min; the column temperature was 30 °C; the injection volume was 4 μL;

[0037] Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile;

[0038] The gradient elution program is shown in Table 1:

[0039] Table 1

[0040]

[0041] Mass spectrometry conditions: Electrospray ionization source (ESI) was used; capillary voltage, positive ion mode was 3.0 k V, negative ion mode was 2.0 k V. Ion source temperature was 150 ° C, sample cone voltage was 30 V, cone gas volume flow rate was 50 L / h, nitrogen degassing temperature was 350 ° C, degassing volume flow rate was 800 L / h, and the scanning range was m / z 50-2000. 3. Identification of chemical components in Yangweishu granules

[0042] Masslynx 4.2 software was used to extract the total ion current (TIC) of the sample solution collected in the positive and negative ion modes. Figure 4 ), obtain ion fragment information data, determine the molecular formula of the compound according to the quasi-molecular ion peak, and identify the chemical components in Yangweishu granules by referring to the secondary mass spectrometry fragment information in the literature.

[0043] 4. Experimental Results

[0044] The present invention is a fast, efficient and comprehensive method for analyzing the chemical components of Yangweishu granules based on UPLC-Q-TOF-MS / MS technology. It can accurately identify compounds and generate their molecular formulas by setting conditions such as full scan of positive and negative ion modes, molecular ion peak pairing, accurate molecular weight mass error, and isotope peak abundance. The identification of compounds is completed based on a self-built compound database, and a total of 150 compounds are identified, including flavonoid compounds, organic acids, terpenoid compounds, nitrogen-containing compounds, etc. The analysis results of the chemical components in Yangweishu granules are shown in Table 2.

[0045] Table 2

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] The main ingredients in Yangweishu Granules include flavonoids, organic acids, terpenoids, nitrogen-containing compounds, etc. Flavonoids are prone to Diels-Alder reaction (RDA) cleavage, demethylation, decarbonylation, glycosidic bond cleavage, etc. Taking compound 135 as an example, the parent ion of compound 135 is m / z 403.1387[M+H]+, and its main fragment ion in the secondary mass spectrum is m / z 388.1128[M+H-CH3] + 、m / z 373.0870[M+H-2CH3] + 、m / z 355.0767[M+H-2CH3-H2O] + , combined with the database and literature, it is inferred that the compound is nobiletin, and its specific cleavage process is shown in Figure 5 As shown in A.

[0056] Organic acid compounds are a common natural product. The main fragmentation pathway of this type of compound is the easy loss of branched groups such as CH3 and CO, and the easy breakage at the carbonyl group to form fragment ions. Taking compound 61 as an example, in the negative ion mode, the quasi-molecular ion peak m / z 193.0507 [MH] - It is easy to remove a methyl group at the methoxy group of the aromatic ring to form a fragment ion m / z 178.0275 [MH-CH3] - , and it is easy to remove a formate group at the carbonyl group to form a fragment ion m / z 149.0602 [MH-COO] - In addition, the quasi-molecular ion peak can also simultaneously remove one formic acid group and one methyl group to form a fragment ion m / z134.0373[MH-CH3-COO] - Combined with mass spectrometry information and related literature reports, it can be determined that the compound is an organic acid compound, ferulic acid. Its possible fragmentation pathways are shown in Figure 5 As shown in B.

[0057] Terpenoids are one of the main components of Atractylodes macrocephala, and their main molecular structure contains one or more isoprene structures. After high-energy bombardment, sesquiterpenoids lose one molecule of -H2O and then one molecule of -CO when there is hydroxyl substitution; when there is no hydroxyl substitution, one molecule of -HCOOH is lost, followed by one molecule of -C2H4 or -C3H6. Taking compound 134 as an example, the quasi-molecular ion peak in positive ion mode is m / z 249.1466 [M+H] + The molecular formula is estimated to be C 15 H 20 O3. In its secondary spectrum, the main fragment ion peak is located at 231.1384 [M+H-H2O] + 、203.1432[M+H-H2O-CO] +Combined with the accurate relative molecular mass and literature comparison, this compound was confirmed to be Atractylodes lactone III. The specific cleavage method is shown in Figure 5 As shown in C.

[0058] The nitrogen-containing compounds in the Yangweishu granules identified by the present invention mainly include amino acids and alkaloids. The cleavage pattern of such compounds is generally centered on the N atom, and the substituents are gradually lost along the side chain toward the benzene ring, and the amino group is further removed. Sometimes the benzene ring can also be seen to crack. Taking compound 26 as an example, the quasi-molecular ion peak scanned in the positive ion mode is 182.0812 [M+H] + , and the fragment ion m / z165.0540[M+H-NH3] was obtained in the secondary mass spectrometry. + 、m / z 136.0723[M+H-HCOOH] + , m / z 119.0489[M+H-HCOOH-NH3] + Combined with mass spectrometry information and related literature reports, it is speculated that compound 26 is tyrosine, and its specific cleavage process is shown in Figure 5 As shown in D.

[0059] Comparative Example 1: Screening and Optimization of Liquid Chromatography Detection Conditions

[0060] The present invention has found through exploration that the most influential factor for the UPLC-Q-TOF-MS / MS analysis of the Yangweishu granules is the gradient elution condition. Therefore, after preliminarily determining the optimization conditions such as the chromatographic column and injection volume, the gradient elution condition is re-studied.

[0061] For comparison, the present invention lists the chromatographic conditions used in the optimization process and the optimization process of the gradient elution program, as shown below. Except for the different chromatographic conditions, the rest of the treatment is the same as in Example 1.

[0062] Using ACQUITY HSS T3 (2.1×100mm, 1.8μm) chromatographic column; column temperature 30℃; flow rate 0.3mL / min; injection volume 4μL; mobile phase A in the elution system is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile, gradient elution is performed according to the provisions in Tables 3 to 5, and the liquid chromatogram detected is recorded. Figure 1 .

[0063] Table 3: Investigation of elution gradient 1

[0064]

[0065]

[0066] Table 4: Investigation of elution gradient 2

[0067]

[0068] Table 5: Investigation of elution gradient 3

[0069]

[0070]

[0071] The results showed that the chromatographic peaks obtained by gradient elution 1 and gradient elution 2 had uneven baselines, and the chromatographic peaks obtained had poor peak shapes and fewer peaks, and the separation was poor. The mass spectrum required by the present invention should display as many chromatographic peaks and chemical components in Yangweishu Granules as possible. The chromatographic peaks obtained by gradient elution 3 had rich information and could comprehensively display the chemical components of Yangweishu Granules. Therefore, gradient elution 3 was finally selected as the gradient elution condition.

[0072] Comparative Example 2: Determination of the Extraction Solvent of the Test Solution of Yangweishu Granules

[0073] Take 1.0 g of Yangweishu granules and accurately weigh them, put them in a stoppered conical flask, add 10 mL of methanol and pure water respectively, stopper it, weigh it, and treat it with ultrasound (power 250 W, frequency 40 kHz) for 60 min. Let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, and centrifuge it at 8000 rpm for 10 min several times until the solution is clear to obtain the Yangweishu test solution.

[0074] According to the chromatographic conditions of Example 1, record the detected liquid chromatogram Figure 2 .

[0075] The results showed that using pure water as the solvent resulted in better chromatographic peak shape, more chromatographic peak information, lower preparation cost of the test solution, and a simpler method. Therefore, pure water was selected as the extraction solvent for Yangweishu Granules.

[0076] Comparative Example 3: Determination of the material ratio of the test solution of Yangweishu granules

[0077] Take 1.0 g of Yangweishu granules and accurately weigh them. Place them in a stoppered conical flask, add 5 mL, 10 mL, and 20 mL of pure water respectively, stopper them, weigh them, and perform ultrasonic treatment (power 250 W, frequency 40 kHz) for 60 min. Let cool, weigh them again, make up the lost weight with pure water, shake well, and centrifuge at 8000 rpm for 10 min several times until the solution is clear to obtain the Yangweishu test solution.

[0078] According to the chromatographic conditions of Example 1, record the detected liquid chromatogram Figure 3 .

[0079] The results showed that when pure water was used as the solvent and 10 mL of extraction solvent was added to 1.0 g of Yangweishu extract, the obtained peak response values, peak shapes and separation were better, which could more comprehensively display the chemical components in Yangweishu granules. Therefore, the sample preparation method for the test solution of Yangweishu granules in the present invention was to add 10 mL of pure water to 1.0 g of Yangweishu granules.

[0080] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology, characterized in that: The following steps are involved: (1) Preparation of test solution Accurately weigh the Yangweishu granules sample, add the extraction solvent and mix well, ultrasonically crush it, centrifuge and take the supernatant to obtain the test solution; (2) Chemical composition detection and analysis Ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) was used to detect and analyze the chemical components in the Yangweishu granules test solution. The chromatographic conditions are as follows: C18 column; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile; column temperature is 30°C; elution gradient program is: 0-6min, 95-90%A; 6-10min, 90-86%A; 10-23min, 86-77%A; 23~24min, 77%A, 24~25min, 77~70%A; 25~29min, 70~60%A; 29~31min, 60~50%A; 31~32min, 50~5%A, 32~34min, 5%A; 34~35min, 5~95%A; 35~38min, 95%A; The mass spectrometry conditions were as follows: electrospray ionization source ESI, capillary voltages of 3.0 KV and 2.0 KV in positive and negative ion modes, respectively; ion source temperature of 150 °C, sample cone voltage of 30 V, cone gas volume flow rate of 50 L / h, nitrogen degassing temperature of 350 °C, degassing volume flow rate of 800 L / h; scanning range of m / z 50-2000; (3) Chemical composition identification The total ion current of the test solution collected in the positive and negative ion modes was extracted using analysis software to obtain ion fragment information data. The molecular formula of the compound was determined based on the quasi-molecular ion peak, and the chemical components in Yangweishu Granules were identified by referring to the secondary mass spectrometry fragment information in the literature.

2. A method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology according to claim 1, characterized in that: The Yangweishu granules are a granule preparation composed of yam, codonopsis pilosula, stir-fried atractylodes macrocephala, stir-fried southern hawthorn, tangerine peel, dried ginger, scrophularia ningpoensis, northern adenophora, steamed polygonatum, dodder seed and ebony.

3. The method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology according to claim 1, characterized in that: In step (1), the extraction solvent is selected from pure water or methanol, preferably pure water.

4. The method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology according to claim 1, characterized in that: In step (1), the mass volume ratio of the Yangweishu granule sample to the extraction solvent is (0.5-5.0 g): (5-60 mL), preferably 1.0 g: (5-20 mL).

5. The method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology according to claim 1, characterized in that: In step (1), the ultrasonic fragmentation time is 30-60 min; the ultrasonic power is 220-250 kW; and the ultrasonic frequency is 30-50 kHz.

6. The method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology according to claim 1, characterized in that: In step (1), the centrifugal speed is 6000-10000 rpm and the time is 10-20 min.

7. The method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology according to claim 1, characterized in that: In step (2), the chromatographic column is ACQUITY UPLC ® HSS T3, specifications are 2.1 ×100 mm, 1.8 μm.

8. The method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology according to claim 1, characterized in that: In step (3), a total of 150 chemical components were identified in Yangweishu Granules, including flavonoids, organic acids, terpenoids and nitrogen-containing compounds.

9. Use of the method according to any one of claims 1 to 8 in quality control of Yangweishu granules.

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