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

Abstract

The application discloses a kind of based on UPLC-Q-TOF-MS / MS technology detection chemical component in Yangwei Shu granules and application, including the preparation of test solution, chemical component detection analysis and chemical component identification etc.Steps.The method of the application is simple, and the loss of component is less, more accurate and sensitive detection, and time-consuming is short, 150 kinds of chemical components in Yangwei Shu granules can be effectively separated within 38min, solve the problem that prior art means and method are difficult to comprehensively, systematically characterize the chemical components in Yangwei Shu granules, realize the high-throughput detection and analysis identification of chemical components in Yangwei Shu granules, can quickly, comprehensively analyze the chemical components in Yangwei Shu granules, provide basis for further study Yangwei Shu granules efficacy material basis and comprehensive quality control.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting chemical components in Yangweishu granules based on UPLC-QTOF-MS / MS technology and application. BACKGROUND

[0002] Yangweishu granules are granule preparations composed of yam, radix codonopsis, fried atractylodes, fried south hawthorn, dried tangerine or orange peel, dried ginger, radix scrophulariae, radix adatodis, steamed rhizoma polygonati, semen cuscutae and fructus mume. Yangweishu granules have the effects of benefiting qi and consolidating the body, nourishing yin and stomach, regulating the middle warmer, promoting qi and relieving constipation, and are often used in the treatment of stomach burning and distending pain, chronic atrophic gastritis and chronic gastritis with the above symptoms.

[0003] Yangweishu granules are traditional Chinese medicine compound preparations. At present, the research on the chemical components of Yangweishu granules still mainly adopts liquid chromatography (HPLC), which can only qualitatively and quantitatively analyze a few chemical components, and it is difficult to comprehensively and systematically characterize the chemical components in Yangweishu granules. For example, only five components, namely harpagide, hesperidin, chlorogenic acid, cinnamic acid and vitexin rhamnoside, in Yangweishu granules are quantitatively analyzed in the method for simultaneously determining the contents of chlorogenic acid, vitexin rhamnoside and hesperidin in Yangweishu granules. However, the active components in Yangweishu granules are complex and diverse, and the determination of a few index components cannot fully reflect the quality of traditional Chinese medicine compound preparations. Therefore, it is necessary to study the high-throughput screening technology of the chemical components of Yangweishu granules. SUMMARY

[0004] In order to solve the above technical problems, the purpose of the present application is to provide a method for detecting chemical components in Yangweishu granules based on UPLC-QTOF-MS / MS technology. The method can quickly and comprehensively analyze the chemical components in Yangweishu granules, and solve the problem that the prior art methods cannot comprehensively and systematically characterize the chemical components in Yangweishu granules.

[0005] The purpose of the present application is achieved by 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 sample solution

[0008] Precisely weigh the Yangweishu granule sample, add the extraction solvent, mix uniformly, perform ultrasonic crushing treatment, centrifuge to obtain the supernatant, and obtain the test sample solution;

[0009] (2) Detection and analysis of chemical components

[0010] The chemical components in the Yangweishu granules are detected and analyzed by using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) technology.

[0011] The chromatographic conditions are as follows: a C18 chromatographic column, a mobile phase A of 0.1% formic acid aqueous solution, a mobile phase B of acetonitrile, a column temperature of 30 DEG C, and an elution gradient program of 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 are as follows: an electrospray ion source ESI, a capillary voltage of 3.0 KV and 2.0 KV in positive and negative ion modes respectively, an ion source temperature of 150 DEG C, a sample cone hole voltage of 30 V, a cone hole gas volume flow rate of 50 L / h, a nitrogen degassing temperature of 350 DEG C, a degassing volume flow rate of 800 L / h, and a scanning range of m / z 50-2000.

[0013] (3) Chemical component identification

[0014] The total ion chromatograms of the test sample solution collected in positive and negative ion modes are extracted by using an analysis software to obtain ion fragment information data, the molecular formula of a compound is determined according to a quasi-molecular ion peak, and the chemical components in the Yangweishu granules are identified by referring to secondary mass spectrometry fragment information in literature.

[0015] The Yangweishu granules disclosed by the application are granule preparations composed of yam, radix codonopsis, fried atractylodes, fried south hawthorn, dried tangerine or orange peel, dried ginger, radix scrophulariae, north radix scrophulariae, steamed yellow ginseng, dodder seed and Chinese plum.

[0016] Preferably, in step (1), the extraction solvent is selected from pure water or methanol, and preferably is 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), and preferably is 1.0 g:(5-20 mL).

[0018] Preferably, in step (1), the ultrasonic crushing time is 30-60 min, the ultrasonic power is 220-250 kW, and the ultrasonic frequency is 30-50 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, specification 2.1*100mm, 1.8um.

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

[0022] The application also provides application of the above method in quality control of Yangweishu granules, which controls or identifies the quality of Yangweishu granules by detecting the chemical components of Yangweishu granules.

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

[0024] The application provides a method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology, which has the advantages of simple pretreatment, less component loss, more accurate and sensitive detection, short time consumption, and 150 chemical components in Yangweishu granules can be effectively separated within 38 min, solves the problem that the prior art method cannot comprehensively and systematically characterize the chemical components in Yangweishu granules, realizes high-throughput detection and analysis identification of the chemical components in Yangweishu granules, and can quickly and comprehensively analyze the chemical components in Yangweishu granules, thereby providing a basis for further research on the pharmacodynamic material basis and comprehensive quality control of Yangweishu granules. BRIEF DESCRIPTION OF DRAWINGS

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

[0026] Figure 2 is a TIC mass spectrum obtained by the application in the negative ion mode, wherein (A) is a TIC mass spectrum of Yangweishu granules methanol solution in the negative ion mode, and (B) is a TIC mass spectrum of Yangweishu granules water solution in the negative ion mode;

[0027] Figure 3is the TIC mass spectrum obtained in negative ion mode in the present application comparative example 3, wherein (A) is the TIC mass spectrum of Yangweishu granules: pure water (1:5) in negative ion mode; (B) is the TIC mass spectrum of Yangweishu granules: pure water (1:10) in negative ion mode; (C) is the TIC mass spectrum of Yangweishu granules: pure water (1:20) in negative ion mode;

[0028] Figure 4 is the total ion chromatogram (TIC) of Yangweishu granules in positive and negative ion modes in the present application example 1, 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 is the fragmentation mode of part of the chemical components in Yangweishu granules. DETAILED DESCRIPTION

[0030] In order for those skilled in the art to better understand the technical solutions of the present application, the technical solutions described in the present application will be further described in detail below in combination with the drawings and specific embodiments. However, the embodiments of the present application are not limited by the following examples.

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

[0032] Example 1: Method 1 for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology

[0033] Take Yangweishu granules, accurately weigh 1.0 g, put it in a conical flask with a plug, add pure water 10 mL, tightly plug, weigh, ultrasonic crushing treatment (power 250 W, frequency 40 kHz) for 60 min, cool, weigh again, add pure water to make up the weight loss, shake well, centrifuge at 8000 rpm for 10 min multiple times until the solution is clear, and obtain the Yangweishu test solution.

[0034] 2. Chemical component detection and analysis

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

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

[0037] The mobile phase: the mobile phase A is 0.1% formic acid aqueous solution by volume concentration, and the mobile phase B is acetonitrile;

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

[0039] Table 1

[0040]

[0041] Mass spectrometry conditions: an electrospray ion source (ESI) was used; the capillary voltage was 3.0 kV in positive ion mode and 2.0 kV in negative ion mode. The ion source temperature was 150 DEG C, the sample cone hole voltage was 30 V, the cone hole gas volume flow rate was 50 L / h, the nitrogen degassing temperature was 350 DEG C, the degassing volume flow rate was 800 L / h, and the scan range was m / z 50-2000. 3. Chemical component identification in Yangweishu granules

[0042] The total ion current chromatograms (TIC graphs, see Figure 4 ) of the sample solutions collected in positive and negative ion modes were extracted using Masslynx 4.2 software, ion fragment information data were obtained, the compound molecular formula was determined according to the quasi-molecular ion peak, and the chemical components in the Yangweishu granules were identified by referring to the literature secondary mass spectrometry fragment information.

[0043] 4. Experimental results

[0044] The method for analyzing the chemical components of the Yangweishu granules is fast, efficient and comprehensive, and is based on the UPLC-Q-TOF-MS / MS technology. The compounds can be accurately identified, the molecular formula can be generated, the compound identification can be completed based on the self-built compound database by setting the conditions of full scanning in positive and negative ion modes, molecular ion peak pairing, accurate molecular weight mass error and isotope peak abundance. A total of 150 compounds, such as flavonoids, organic acids, terpenes and nitrogen-containing compounds, were identified. The analysis results of the chemical components in the Yangweishu granules are shown in Table 2.

[0045] Table 2

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

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

[0056] Organic acid compounds are a common natural product. The main cleavage pathway of this type of compound is to easily lose branched groups such as CH3 and CO, and to easily break at the carbonyl group to form fragment ions. For example, in the negative ion mode, the quasi-molecular ion peak of compound 61 is m / z 193.0507 [M-H] - , which is prone to lose one methyl group at the aromatic ring substituted methoxy group to form a fragment ion m / z 178.0275 [M-H-CH3] - , and prone to lose one formic acid group at the carbonyl group to form a fragment ion m / z 149.0602 [M-H-COO] - . In addition, the quasi-molecular ion peak can also simultaneously lose one formic acid group and one methyl group to form a fragment ion m / z 134.0373 [M-H-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, and its possible fragment cleavage pathway is shown in Figure 5 B.

[0057] Terpenoids are one of the main component types of Baizhu, and contain one or more isoprene structures in their main molecular structure. After high-energy bombardment, sesquiterpenes with hydroxyl substitution lose one molecule of-H2O, and then lose one molecule of-CO; without hydroxyl substitution, they lose one molecule of-HCOOH, and then lose one molecule of-C2H4 or-C3H6. For example, the quasi-molecular ion peak of compound 134 in the positive ion mode is m / z 249.1466 [M+H] + , and the molecular formula is C 15 H 20 O3. In its secondary spectrum, the main fragment ion peaks are at 231.1384 [M+H-H2O] + , 203.1432 [M+H-H2O-CO] +, combined with accurate relative molecular mass and literature comparison, it is confirmed that the compound is atractylodinol III, and the specific cleavage mode is shown in Figure 5 C.

[0058] The nitrogen-containing compounds in the Yangweishu granules identified by the present application mainly include amino acids and alkaloids. The cleavage rules of such compounds are generally that the N atom is taken as the center, and the substituents are gradually lost along the side chain towards the benzene ring direction, and further, the amino group is removed, and sometimes, the benzene ring is also cracked. Taking compound 26 as an example, the quasi-molecular ion peak is 182.0812[M+H] + , and the fragment ions m / z 165.0540[M+H-NH3] + , m / z 136.0723[M+H-HCOOH] + , m / z 119.0489[M+H-HCOOH-NH3] + , combined with mass spectrum information and related literature reports, it is speculated that compound 26 is tyrosine, and the specific cleavage process is shown in Figure 1 D.

[0059] Comparative Example 1: Screening and optimization of liquid chromatography detection conditions

[0060] The present application finds that, for the UPLC-Q-TOF-MS / MS analysis of the Yangweishu granules, the most influential factor is the gradient elution condition. Therefore, after the optimization conditions such as the chromatographic column and the sample amount are preliminarily determined, the gradient elution condition is further researched.

[0061] As a comparison, the present application 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 processing is the same as Example 1.

[0062] ACQUITY HSS T3(2.1×100mm,1.8μm) chromatographic column; the column temperature is 30℃; the flow rate is 0.3mL / min; the sample amount is 4μL; the mobile phase A in the elution system is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and gradient elution is carried out according to the provisions in Tables 3-5, respectively, and the detected liquid chromatography Figure 2 is recorded.

[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, poor peak shapes, and fewer peaks, resulting in poor resolution. The mass spectrum required by this invention should display as many chromatographic peaks and chemical components as possible in Yangweishu granules. The chromatographic peaks obtained by gradient elution 3 are rich in information and can 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 Extraction Solvent for Yangweishu Granules Test Sample Solution

[0073] Accurately weigh 1.0g of Yangweishu granules and place them in a stoppered conical flask. Add 10mL of methanol and 10mL of pure water respectively, seal tightly, weigh, and sonicate (power 250W, frequency 40kHz) for 60min. Let cool, weigh again, and replenish the lost weight with the corresponding solvent. Shake well and centrifuge at 8000rpm for 10min several times until the solution is clear to obtain the Yangweishu test solution.

[0074] Record the detected liquid chromatograms according to the chromatographic conditions of Example 1. Figure 3 .

[0075] The results showed that using pure water as a solvent resulted in better peak shapes, more chromatographic information, lower sample solution preparation costs, and a simpler method. Therefore, pure water was chosen as the extraction solvent for Yangweishu granules.

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

[0077] Accurately weigh 1.0g of Yangweishu granules and place them in a stoppered conical flask. Add 5mL, 10mL, and 20mL of pure water respectively, seal tightly, weigh, and sonicate (power 250W, frequency 40kHz) for 60min. Let cool, weigh again, replenish the lost weight with pure water, shake well, and centrifuge at 8000rpm for 10min several times until the solution is clear. This gives you the Yangweishu test solution.

[0078] Record the detected liquid chromatograms according to the chromatographic conditions of Example 1. Figure 4 .

[0079] The results show that when 1.0 g Yangweishu extract is added with 10 mL extraction solvent, the response value of each peak is better, the peak type is better, and the separation degree is better, so that the chemical components in the Yangweishu granules can be more comprehensively displayed, and therefore the sample preparation method of the Yangweishu granules test solution in the present application is that 1.0 g Yangweishu granules is added with 10 mL pure water.

[0080] The above has specifically described the preferred embodiments of the present application, but the present application is not limited to the described embodiments, and those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the present application, and these equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. A method for detecting chemical components in Yangweishu granules based on UPLC-Q-TOF-MS / MS technology, wherein the Yangweishu granules are a granular preparation consisting of yam, radix codonopsis, fried atractylodes, fried south hawthorn, dried tangerine or orange peel, dried ginger, radix scrophulariae, radix notoginseng, steamed yellow ginseng, dodder and dark plum, characterized by, Comprising the following steps: ​ (1) Preparation of test solution Accurately weigh the Yangweishu granules sample, add pure water, mix well, centrifuge after ultrasonic crushing treatment, and take the supernatant to obtain the test solution; (2) Chemical component detection analysis The UPLC-Q-TOF-MS / MS technology is used to detect and analyze the chemical components in the test solution of Yangweishu granules; Among them, the chromatographic conditions are: C18 column; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile; column temperature is 30 ℃; 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 are: electrospray ion source ESI, the capillary voltage is 3.0K V and 2.0KV in positive and negative ion modes respectively; ion source temperature is 150 ℃, sample cone hole voltage is 30 V, cone hole gas volume flow rate is 50 L / h, nitrogen degassing temperature is 350 ℃, and degassing volume flow rate is 800 L / h; scanning range is m / z 50~2000; The chromatographic column is ACQUITY UPLC ® HSS T3, 2.1 x 100 mm, 1.8 μm; (3) Chemical component identification The total ion chromatogram of the test solution collected in positive and negative ion modes is extracted respectively using analysis software to obtain ion fragment information data, the molecular formula of the compound is determined according to the quasi-molecular ion peak, and the chemical components in Yangweishu granules are identified by referring to the literature secondary mass spectrometry fragment information. 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). 2.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 1.0 g: (5~20 mL).

3. The method according to claim 2, wherein the method is characterized in that, In step (1), the ultrasonic crushing time is 30~60 min; the ultrasonic power is 220~250 kW; and the ultrasonic frequency is 30~50 kHz.

4. The method according to claim 1, wherein the method is characterized in that, In step (1), the centrifugal speed is 6000~10000 rpm, and the time is 10~20 min.

5. The method according to claim 1, wherein the method is characterized in that, In step (3), 150 chemical components are identified in Yangweishu granules, including flavonoids, organic acids, terpenoids and nitrogen-containing compounds. 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, 7. The method according to any one of claims 1 to 6 for use in the quality control of Yangweishu granules. ​

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