Quality detection method and application of Shuidi decoction

Abstract

The invention provides a quality detection method and application of Shuidi decoction, and belongs to the technical field of medicine detection.The quality detection method comprises the steps that the Shuidi decoction is used for preparing a test solution, acetonitrile (A)-0.08-0.12 wt% phosphoric acid aqueous solution (B) serves as a mobile phase, and the quality of the Shuidi decoction is detected through ultra-high performance liquid chromatography-tandem mass spectrometry; the obtained detection result can be used for the quality evaluation or control of the whole process of research / development / production / clinical application of the Shuidi decoction. According to the present invention, the UPLC-Q-TOF-MS method is adopted to perform qualitative analysis on the substance-based chemical components of the Shuandii decoction, such that the scientific basis is provided for the substance-based substance fundamental research of the Shuandii decoction, and the main chemical components of the Shuandii decoction are clarified;

Description

Technical Field

[0001] The present invention relates to drug detection technology, and in particular to a quality detection method and application of Liangdi Decoction. Background Art

[0002] Liangdi Decoction comes from the book "Fu Qingzhu's Gynecology" written by Fu Shan (styled Qingzhu), a medical doctor in the late Ming and early Qing dynasties. Its prescription composition includes prepared rehmannia root, scrophularia root, prepared white peony root, ophiopogon root, cortex lycii radicis, and donkey-hide gelatin. It is widely used clinically and is an effective prescription commonly used for regulating menstruation in gynecology. The main effects of Liangdi Decoction are nourishing yin and clearing heat, cooling blood and regulating menstruation, and it is used to treat symptoms such as early menstruation, scanty menstrual flow, prolonged menstrual period, and functional uterine bleeding caused by yin deficiency and internal heat.

[0003] Liangdi Decoction is made from 6 traditional Chinese medicines. The compound components of traditional Chinese medicines are complex, containing various active substances such as alkaloids, flavonoids, and saponins. The comprehensive analysis of its effective substance basis has always been a difficult point in the modern research of traditional Chinese medicines. Traditional analysis methods such as high-performance liquid chromatography (HPLC) are limited in separation efficiency and difficult to achieve synchronous and accurate identification of multiple components, which restricts the research on quality control and action mechanisms. Ultra-high performance liquid chromatography tandem mass spectrometry technology has high analysis efficiency and speed, can obtain accurate molecular structure information, and can obtain the primary and multi-stage mass spectrometry information of each compound, providing a good technical means for the multi-index component analysis of complex traditional Chinese medicine systems. Therefore, it is necessary to develop a method for analyzing Liangdi Decoction using ultra-high performance liquid chromatography tandem mass spectrometry technology to more comprehensively evaluate the quality of Liangdi Decoction. Summary of the Invention

[0004] In view of the above problems, the present invention provides a quality detection method and application of Liangdi Decoction.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A quality detection method of Liangdi Decoction, the quality detection method includes the following steps:

[0007] S1. Take Liangdi Decoction to prepare a test solution;

[0008] S2. Take the test solution and use ultra-high performance liquid chromatography tandem mass spectrometry to detect the quality of Liangdi Decoction;

[0009] The mobile phase A for the ultra-high performance liquid chromatography detection is acetonitrile, and the mobile phase B is an aqueous phosphoric acid solution with a concentration of 0.08 - 0.12 wt%;

[0010] The elution mode for the ultra-high performance liquid chromatography detection is gradient elution;

[0011] The elution conditions for the gradient elution are as follows:

[0012] 0 - 1 min, 1% → 5% mobile phase A, 99% → 95% mobile phase B;

[0013] 1 - 4.5 min, mobile phase A from 5% to 15%, mobile phase B from 95% to 85%;

[0014] 4.5 - 8.5 min, mobile phase A from 15% to 17%, mobile phase B from 85% to 83%;

[0015] 8.5 - 10.5 min, mobile phase A from 17% to 26%, mobile phase B from 83% to 74%;

[0016] 10.5 - 18 min, mobile phase A from 26% to 51%, mobile phase B from 74% to 49%;

[0017] 18 - 21 min, mobile phase A from 51% to 80%, mobile phase B from 49% to 20%;

[0018] 21 - 22 min, mobile phase A from 80% to 1%, mobile phase B from 20% to 99%.

[0019] [[ID=2,0]]Further, the test solution is prepared by adding methanol to Liangdi Decoction for dissolution, followed by filtration.

[0020] Further, the weight - volume ratio of Liangdi Decoction to the prepared test solution is 0.9 - 1.1 g: 10 mL. [[ID=2,4]]

[0021] Further, the detection wavelength for the ultra - high performance liquid chromatography detection is 190 - 215 nm.

[0022] Further, the flow rate for the ultra - high performance liquid chromatography detection is 0.35 - 0.45 mL / min and the column temperature is 33 - 37 °C.

[0023] Further, the chromatographic column for the ultra - high performance liquid chromatography detection is packed with octadecylsilyl silica gel.

[0024] Further, the mass spectrometry detection is carried out in positive ion mode and negative ion mode respectively. [[ID=,37]]

[0025] Further, the conditions for the mass spectrometry detection are: drying gas temperature 345 - 355 °C, drying gas flow rate 8 - <12 L / min, nebulizing gas pressure 34 - 36 psi, sheath gas temperature 345 - 355 °C, sheath gas flow rate 11 - 13 L / min, capillary voltage in positive ion mode 3400 - 3600 V, capillary voltage in negative ion mode 3400 - 3600 V.

[0026] Further, during the mass spectrometry detection, the first - stage mass spectrometry uses the MS mode, the mass scanning range is 100 - 1200 m / z, and the second - stage mass spectrometry uses the Auto MSMS mode.

[0027] Application of the test results obtained by the above-mentioned quality inspection method in the quality evaluation or control of the whole process of research / development / production / clinical application of Liangditan;

[0028] Among them, the test results can be the liquid chromatography-mass spectrometry fingerprint of Liangditan (including but not limited to UPLC-UV chromatogram and / or UPLC-MS total ion current chromatogram), can also be the primary analysis results of Liangditan (such as Table 1 of the examples), and can also be the secondary mass spectrometry analysis results of Liangditan (such as Table 1 of the examples), such as peak number and retention time.

[0029] The beneficial effects of the quality inspection method and application of Liangditan of the present invention are as follows:

[0030] The present invention uses the UPLC-Q-TOF-MS method to qualitatively analyze the chemical components of the reference substance of Liangditan, provides a scientific basis for the study of the material basis of the reference substance of Liangditan, and simultaneously clarifies its main chemical composition;

[0031] The present invention uses the UPLC-Q-TOF-MS method to qualitatively analyze the chemical components of the reference substance of Liangditan. It has high analysis efficiency and speed, can obtain accurate molecular structure information in Liangditan, and can obtain the primary and multi-stage mass spectrometry information of each compound, providing a good technical means for the multi-index component analysis of identifying the complex traditional Chinese medicine system of Liangditan;

[0032] The present invention uses the UPLC-Q-TOF-MS method to qualitatively analyze the chemical components of the reference substance of Liangditan, provides a scientific basis for the study of the material basis of Liangditan, and simultaneously clarifies its main chemical composition;

[0033] The quality inspection method of the present invention can jointly detect all the medicinal material components in the whole prescription of Liangditan;

[0034] The present invention utilizes the characteristics that the physicochemical properties of different active components in Liangditan are different, changes the chromatographic conditions, so as to achieve the purpose of well separating different active components in Liangditan;

[0035] By selecting specific chromatographic conditions and mass spectrometry conditions, the present invention can obtain a better chromatogram of Liangditan;

[0036] The quality inspection method of the present invention also has good feasibility, stability and reproducibility;

[0037] The quality inspection method of the present invention can detect and clarify the source attribution of the medicinal flavors of 36 peak substances and the qualitative analysis of the main chromatographic peaks; among them, a total of 25 monoterpenoids and their glycosides were identified, namely catalpol, isomucronulatol glycoside, acteoside, harpagide, harpagoside, paeoniflorin, rehmannioside D, leonuride, digitalis leaf glycoside C, jiao rehmannioside A1 / A2, rehmannia ionoside A / B, 8-O-coumaroyl harpagoside, jiao rehmannioside B1 / B2, moutan cortex glycoside I, angoroside C, rehmannioside / iso-rehmannioside, harpagoside isomer, 6-O-α-D-galactosyl harpagoside, benzoylpaeoniflorin, isobenzoylpaeoniflorin, adenosine, β-gentiobiosylpaeoniflorin, galloylpaeoniflorin, zhanlongjian glycoside, albiflorin; a total of 2 alkaloid components were identified, namely physalin B / physalin A, betaine. Among them, physalin A / physalin B is the exclusive component of cortex lycii radicis; a total of 1 flavonoid component was identified, which is catechin; a total of 1 tannin component was identified, and gallic acid is the exclusive component of processed white peony root; a total of 3 organic acid components were identified, namely citric acid, 8-epistrychnine acid, cinnamic acid;

[0038] Using the quality inspection method of the present invention, the presence, absence and characteristics of the common characteristic peaks in the chromatogram of Liangditan can comprehensively monitor the quality of the raw materials, semi-finished products and finished products of Liangditan. By comparing the similarity degree of chromatographic characteristics, the advantages and disadvantages of Liangditan can be evaluated, the stability and consistency can be investigated, making up for the deficiencies of the current quality control methods. At the same time, the stability of the production process of Liangditan can also be monitored to ensure the stability, uniformity and controllability of its quality;

[0039] Using the quality inspection method of the present invention, the chromatogram of Liangditan obtained improves the quality control standards for the finished products and semi-finished products of Liangditan, effectively preventing the occurrence of product forgery events and ensuring the normal production and circulation order of Liangditan; on the basis of the present invention, the correlation study between the chromatogram information detected by the UPLC-Q-TOF-MS method and the pharmacodynamic activity information can also be carried out, so as to deeply clarify the correlation between the internal chemical components of Liangditan and the efficacy of the preparation.

[0040] The quality inspection method described in the present invention can be used for the quality evaluation or control of the whole process of research / development / production / clinical application of Liangditan. Description of the Drawings

[0041] Figure 1 It is the UPLC-UV chromatogram and the UPLC-MS total ion current chromatogram of Liangditan in Example 1 of the present invention, where from top to bottom are the UPLC-MS total ion current chromatogram (positive ion mode TIC diagram), the UPLC-MS total ion current chromatogram (negative ion mode TIC diagram) and the UPLC-UV chromatogram of Liangditan (chromatogram under the condition of ultraviolet 310nm). Detailed implementation manners

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0043] Embodiment 1 Quality detection method and application of Liangdi Decoction

[0044] In this embodiment, the reference substance of Liangdi Decoction is used for the quality detection of Liangdi Decoction. The specific preparation method of the reference substance of Liangdi Decoction is as follows:

[0045] Take 30 grams of stir-fried Rehmanniae Radix with wine, 30 grams of Scrophulariae Radix, 15 grams of Paeoniae Radix Alba with wine, 15 grams of Ophiopogonis Radix, 9 grams of Cortex Lycii Radicis, and 9 grams of Colla Corii Asini, place them in a casserole, decoct twice with 10 times the amount of water each time, filter, combine the filtrates, concentrate under reduced pressure, and freeze-dry to obtain a dry extract, that is, the reference substance of Liangdi Decoction (i.e., dry extract powder).

[0046] This embodiment is a quality detection method and application of Liangdi Decoction, which specifically includes the following steps:

[0047] S1. Solution preparation

[0048] S11. Preparation of test solution

[0049] Take about 1 g of the reference substance of Liangdi Decoction, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol to dissolve, tightly stopper, ultrasonically treat (power 480 W, frequency 40 kHz) for 30 minutes, let it cool, dilute to the mark with methanol, shake well, filter, and obtain the test solution.

[0050] S12. Preparation of single-herb solution

[0051] Weigh 2 g of each medicinal material slice in the formula, place them in a round-bottom flask respectively, add 50 mL of water, decoct for 30 minutes, filter, take the subsequent filtrate, evaporate to dryness in a water bath, add an appropriate amount of methanol to dissolve, tightly stopper, ultrasonically treat (power 480 W, frequency 40 kHz) for 30 minutes, let it cool, dilute to the mark with methanol, shake well, filter, and obtain the single-herb solution corresponding to each medicinal material.

[0052] S13. Preparation of reference solution

[0053] Take appropriate amounts of gallic acid, harpagide, lycium barbarum L. ethyl acetate, paeoniflorin, cinnamic acid, and harpagoside, accurately weigh them, and prepare solutions containing 0.3 mg per 1 mL with methanol respectively to obtain the corresponding reference solutions.

[0054] S2. Quality detection

[0055] Take the test solution for UPLC-Q-TOF-MS detection to obtain the UPLC-UV chromatogram and the total ion current chromatogram (TIC chromatograms in positive and negative ion modes) of Liangdi Decoction, which are the chromatograms of Liangdi Decoction. See specifically Figure 1 . Figure 1 The substances of chromatographic peaks numbered 1 to 36 marked in

[0056] Among them, the instrument and equipment for UPLC-Q-TOF-MS detection are as follows:

[0057] Agilent 1290 Infinity II ultra-high performance liquid chromatograph (UPLC), connected to a G6530C quadrupole-time of flight tandem mass spectrometer (Q-TOF-MS). Equipped with an independent quaternary pump, an auto-sampler, a column oven, a diode array detector (DAD), and an electrospray ionization source (ESI).

[0058] The chromatographic conditions for UPLC-Q-TOF-MS detection are as follows:

[0059] Chromatographic separation uses a Waters HSST3 C18 UPLC column (2.1×100 mm, 1.8 μm), equipped with an online filter;

[0060] The column temperature is 35 °C;

[0061] The flow rate is 0.4 mL / min;

[0062] The detection wavelength is 210 nm;

[0063] The injection volume is 3 μL;

[0064] Use acetonitrile as mobile phase A and 0.1 wt% phosphoric acid aqueous solution as mobile phase B;

[0065] The elution method is gradient elution, and the elution conditions are as follows:

[0066] 0 - 1 min, 1% → 5% mobile phase A, 99% → 95% mobile phase B;

[0067] 1 - 4.5 min, 5% → 15% mobile phase A, 95% → 85% mobile phase B;

[0068] 4.5 - 8.5 min, 15% → 17% mobile phase A, 85% → 83% mobile phase B;

[0069] 8.5 - 10.5 min, 17% → 26% mobile phase A, 83% → 74% mobile phase B;

[0070] 10.5 - 18 min, mobile phase A from 26% to 51%, mobile phase B from 74% to 49%;

[0071] 18 - 21 min, mobile phase A from 51% to 80%, mobile phase B from 49% to 20%;

[0072] 21 - 22 min, mobile phase A from 80% to 1%, mobile phase B from 20% to 99%.

[0073] The mass spectrometry conditions for UPLC - Q - TOF - MS detection are as follows:

[0074] Mass spectrometry detection is carried out in positive and negative ion modes respectively. The drying gas temperature is 350 °C, the drying gas flow rate is 10 L / min, the nebulizing gas pressure is 35 psi, the sheath gas temperature is 350 °C, the sheath gas flow rate is 12 L / min, and the capillary voltage is 3500 V (positive mode) and 3500 V (negative mode). The first - stage mass spectrometry selects the MS mode, and the mass scanning range is 100 - 1200 m / z. The second - stage mass spectrometry selects the Auto MSMS mode, and the collision voltage is adjusted appropriately according to different compounds. The obtained LC - MS data are collected by Agilent MassHunter (B.08.00) software. Data processing is carried out using Agilent software Qualitative Navigator (B.08.00) and QualitativeWorkflows (B.08.00).

[0075] At the same time, UPLC - Q - TOF - MS detection is carried out on each single - herb solution and each reference substance solution, and a comparative analysis is made with the detection results of the test solution, clarifying the medicinal flavor source attribution of 36 peak substances and the qualitative analysis of the main chromatographic peaks. For the six medicinal flavors of stir - fried Rehmannia glutinosa, Scrophularia ningpoensis, stir - fried Paeonia lactiflora, Cortex Lycii Radicis, Ophiopogon japonicus, and Colla Corii Asini in the formula, the peak attribution in the mass spectrometric fingerprint of the substance reference solution can be obtained.

[0076] Through the accurate mass number determination of high - resolution TOF - MS, first - stage mass spectrometry analysis is carried out, and Figure 1 the compounds in peaks 1 - 36 are analyzed. A total of 32 compounds are identified, and the results are shown in Table 1. It can be seen that in the positive mode, these compounds can generate adduct ions such as molecular ion peaks [M + H] + , and [M + Na] + , [M + K] + ; while in the negative mode, they can generate molecular ion peaks [M - H] - and [M + Cl] - , [M + HCOO] -Equivalent addition ions. Based on this addition ion information, the exact molecular weight and molecular formula of the compound can be accurately inferred (the mass determination error is ≤5 ppm, indicating that the matching result is correct), which helps with its subsequent structure identification. Among the primary mass spectrometry information of 36 compound peaks, most compounds respond in both positive and negative ion modes, and some compounds can only be detected in one mode.

[0077] Furthermore, the test solution was subjected to Auto MS / MS analysis (mainly in the negative mode), that is, second-order mass spectrometry analysis, under the same chromatographic conditions to confirm the structures of some compounds in Table 1. The results are shown in Table 1. Through second-order mass spectrometry analysis, the primary mass spectrometry identification results in Table 1 were further confirmed to a certain extent.

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] The test solutions of the material reference of Liangditan were analyzed by UPLC-Q-TOF-MS respectively, and a total of 36 components were detected. Through comparison with literature data, comparison of retention times with reference substances, and analysis of the accurate molecular weights given by UPLC-Q-TOF-MS, 32 components were identified in total. And the source medicinal flavors of the compounds were attributed.

[0087] a. Monoterpenoids and their glycosides

[0088] A total of 25 monoterpenoids and their glycoside components were identified, namely catalpol, isoiridoglucoside, acteoside, harpagide, harpagoside, paeoniflorin, rehmannioside D, leonuride, digitalis leaf glycoside C, jiaodihuangoside A1 / A2, rehmanniaviolanoside A / B, 8-O-coumaroyl harpagoside, jiaodihuangoside B1 / B2, moutanpioside I, angoroside C, rehmannioside / isodehydrorehmannioside, harpagoside isomer, 6-O-α-D-galactosyl harpagoside, benzoylpaeoniflorin, isobenzoylpaeoniflorin, adenosine, β-gentiobiosylpaeoniflorin, galloylpaeoniflorin, zhanlongjianside, albiflorin.

[0089] Among them, the three compounds, harpagoside, harpaoside and paeoniflorin, were compared with standard substances and their accurate chemical structures were confirmed.

[0090] b. Alkaloids

[0091] Two alkaloid components were identified, namely lycopodione / lycopodione and betaine. Among them, lycopodione / lycopodione is the exclusive component of Lycium bark.

[0092] c. Flavonoids

[0093] A total of one flavonoid component was identified, which was catechin.

[0094] d. Tannins

[0095] A total of one tannin component was identified, among which gallic acid is a unique component of white peony root.

[0096] e. Organic acids

[0097] Three organic acids were identified, namely citric acid, 8-epi-strychnine acid and cinnamic acid.

[0098] The quality detection method of Liangdi Decoction in this embodiment can be used for quality evaluation or control of the entire process of research / development / production / clinical application of Liangdi Decoction.

[0099] The liquid-mass fingerprint (including but not limited to UPLC-UV chromatogram and / or UPLC-MS total ion current diagram) obtained by the quality detection method in this embodiment, the primary analysis results (such as Table 1 of the embodiment), and the secondary mass spectrometry analysis results (such as Table 1 of the embodiment) can also be used for quality evaluation or control of the entire process of research / development / production / clinical application of Liangdi Decoction. For example, the peak number and retention time can be directly used for quality evaluation or control of the entire process of research / development / production / clinical application of Liangdi Decoction.

[0100] Example 2-5 Quality detection method and application of Liangdi soup

[0101] Examples 2 to 5 are respectively a method for detecting the quality of two-place soup and its application. The steps thereof are basically the same as those of Example 1, and the only difference is the different process parameters. See Table 2 for details:

[0102] Table 2 List of process parameters in Examples 2 to 5

[0103]

[0104] The process steps and parameters of other parts of Examples 2 to 5 are the same as those of Example 1, and the obtained test results are also similar to those of Example 1, which will not be repeated here.

[0105] Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A quality inspection method for Liangdi Decoction, characterized in that The quality inspection method includes the following steps: S1. Prepare a test solution from Liangdì Decoction; S2. Use ultra-high performance liquid chromatography tandem mass spectrometry to detect the quality of Liangdì Decoction with the prepared test solution; In the ultra-high performance liquid chromatography detection, mobile phase A is acetonitrile, and mobile phase B is an aqueous phosphoric acid solution with a concentration of 0.08 - 0.12 wt%; The elution method for the ultra-high performance liquid chromatography detection is gradient elution; The elution conditions for the gradient elution are as follows: 0 - 1 min, 1% → 5% mobile phase A, 99% → 95% mobile phase B; 1 - 4.5 min, 5% → 15% mobile phase A, 95% → 85% mobile phase B; 4.5 - 8.5 min, 15% → 17% mobile phase A, 85% → 83% mobile phase B; 8.5 - 10.5 min, 17% → 26% mobile phase A, 83% → 74% mobile phase B; 10.5 - 18 min, 26% → 51% mobile phase A, 74% → 49% mobile phase B; 18 - 21 min, 51% → 80% mobile phase A, 49% → 20% mobile phase B; 21 - 22 min, 80% → 1% mobile phase A, 20% → 99% mobile phase B.

2. The quality inspection method of Liangdi Decoction according to claim 1, characterized in that, The test solution is prepared by adding Liangdì Decoction to methanol for dissolution and then filtration.

3. The quality inspection method of Liangdi Decoction according to claim 2, characterized in that, The weight - volume ratio of Liangdì Decoction to the prepared test solution is 0.9 - 1.1 g:10 mL.

4. The quality inspection method of Liangdi Decoction according to any one of claims 1-3, characterized in that The detection wavelength for the ultra-high performance liquid chromatography detection is 190 - 215 nm.

5. The quality inspection method of Liangdi Decoction according to any one of claims 1-3, characterized in that, The flow rate for the ultra-high performance liquid chromatography detection is 0.35 - 0.45 mL / min, and the column temperature is 33 - 37 °C.

6. The quality inspection method of Liangdi Decoction according to any one of claims 1-3, characterized in that The chromatographic column for the ultra-high performance liquid chromatography detection is packed with octadecylsilane chemically bonded silica gel.

7. The quality inspection method of Liangdi Decoction according to any one of claims 1-3, characterized in that, The mass spectrometry detection is carried out in both positive ion mode and negative ion mode.

8. The quality inspection method of Liangdi Decoction according to any one of claims 1-3, characterized in that, The conditions for the mass spectrometry detection are: drying gas temperature 345 - 355 °C, drying gas flow rate 8 - 12 L / min, nebulizing gas pressure 34 - 36 psi, sheath gas temperature 345 - 355 °C, sheath gas flow rate 11 - 13 L / min, capillary voltage in positive ion mode is 3400 - 3600 V, and capillary voltage in negative ion mode is 3400 - 3600 V.

9. The quality inspection method of Liangdi Decoction according to any one of claims 1-3, characterized in that, During the mass spectrometry detection process, the first - stage mass spectrometry uses the MS mode with a mass scanning range of 100 - 1200 m / z, and the second - stage mass spectrometry uses the Auto MSMS mode.

10. Application of the detection result obtained by the quality inspection method according to any one of claims 1 - 9 in the quality evaluation or control of the whole process of research / development / production / clinical application of Liangdì Decoction.

Images