Detection method of traditional Chinese medicine composition

Abstract

The invention relates to the technical field of traditional Chinese medicine composition detection, and discloses a traditional Chinese medicine composition detection method, which comprises: crushing a traditional Chinese medicine composition according to a ratio, mixing, adding a mixed extraction solvent, carrying out ultrasonic extraction, and purifying to obtain a purified extract; eluting the purified extracting solution by using methanol water, filtering, evaporating and concentrating, and washing to a constant volume to obtain a sample solution; performing UPLC-MS / MS detection on the sample solution to obtain quantitative ion peak areas of all target components; the method comprises the following steps: preparing a mixed standard substance solution, carrying out UPLC-MS / MS detection on the mixed standard substance solution, generating a standard curve regression equation of each component according to a detection result, and calculating the content of the corresponding target component according to the quantitative ion peak area of each target component and the standard curve regression equation. According to the method, the detection time is short, the characteristic effective components of the seven medicinal materials in the traditional Chinese medicine composition can be covered through one-time detection, all-component quality verification is achieved, and the defect that quality control is not comprehensive is overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of traditional Chinese medicine compositions, and in particular to a method for detecting traditional Chinese medicine compositions. Background Art

[0002] The existing methods for detecting Chinese medicine compositions have the following deficiencies: Limitations of single-component testing: Most tests focus on a single component (such as astragaloside IV and salvianolic acid B), and cannot simultaneously verify the active ingredients of multiple traditional Chinese medicines, resulting in incomplete quality control. The interference problem is prominent: Different Chinese herbal medicine ingredients have large differences in polarity (such as the coexistence of saponins, alkaloids, and phenolic acids). Traditional single solvent extraction is prone to introduce impurities, affecting detection accuracy. Low efficiency: The method mostly adopts step-by-step extraction and multiple detection modes, and a single test takes 4-6 hours, which cannot meet the quality screening needs of batch samples.

[0003] For traditional Chinese medicine compositions containing multiple drugs, existing methods cannot achieve efficient and accurate multi-index simultaneous detection. Therefore, a detection method for traditional Chinese medicine compositions is proposed, which has important practical significance. Summary of the Invention

[0004] In view of this, the present invention proposes a method for detecting a traditional Chinese medicine composition, aiming to solve at least one of the background technologies.

[0005] The present invention provides a method for detecting a Chinese medicine composition, comprising the following steps: 30 parts of Astragalus, 30 parts of dried Dendrobium, 30 parts of Pueraria, 30 parts of Salvia miltiorrhiza, 6 parts of Coptis chinensis, and 15 parts of Bambusa chinensis were ground and mixed to obtain a sample; Methanol and formic acid water are mixed in a volume ratio of 7:3 to obtain a mixed extraction solvent, the sample and the mixed extraction solvent are mixed and weighed, and then sealed under negative pressure, and then ultrasonic extraction is performed. After the end, the negative pressure is released, and the sample is weighed again, and the mixed extraction solvent is used to make up for the mass loss to obtain an extract, the extract is filtered, and the filtrate is collected. The filtrate is mixed with a composite adsorbent and then shaken to obtain a purified extract; activating a solid phase extraction column, injecting the purified extract into the activated solid phase extraction column, then adding methanol water for elution, discarding the eluate, then adding methanol water for elution, collecting the eluate, and performing a rotary evaporation on the eluate. After the evaporation, adding formic acid water for washing, then adding formic acid water again to adjust the volume, and filtering the adjusted solution to obtain a sample solution; The sample solution was injected into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for UPLC-MS / MS detection to obtain the quantitative ion peak area of ​​each target component; Astragaloside IV, dendrobium, puerarin, notoginsenoside R1, salvianolic acid B, berberine, rutin, and tanshinone IIA were prepared into a mixed standard solution, which was injected into an ultra-performance liquid chromatography-triple quadrupole mass spectrometer for detection. The concentration of each component was used as the independent variable and the quantitative ion peak area was used as the dependent variable. The weighted least squares method was used for linear regression to obtain the regression equation of the standard curve of each component. The quantitative ion peak area of ​​each target component is substituted into the standard curve regression equation to calculate the concentration of the corresponding target component, and the content of the corresponding target component is calculated based on the concentration of the corresponding target component.

[0006] Furthermore, the ultrasonic extraction process is specifically as follows: The extraction was carried out at a temperature of 35°C and an ultrasonic power of 300W for 10 minutes, then the temperature was raised to 40°C and the extraction was carried out for 15 minutes, and finally the temperature was lowered to 35°C and the ultrasonic power was reduced to 250W and the extraction was carried out for 5 minutes.

[0007] Furthermore, the activation treatment is specifically as follows: A C18 solid phase extraction column was taken, and 5 ml of methanol was added to the C18 solid phase extraction column at a flow rate of 0.5 mL / min. After the addition, 5 ml of ultrapure water was added at a flow rate of 0.5 mL / min.

[0008] Furthermore, the temperature of the rotary evaporation treatment was 45° C., the rotation speed was 60 r / min, and the vacuum degree was -0.08 MPa.

[0009] Furthermore, the washing process is specifically as follows: Add formic acid water to the eluate after rotary evaporation, shake for 1 minute, and then transfer to a volumetric flask. Then add formic acid water to the original container, shake for 1 minute, and then transfer to the volumetric flask. Finally, add formic acid water to the original container, shake for 1 minute, and then transfer to the volumetric flask.

[0010] Furthermore, the chromatographic column used in the UPLC-MS / MS detection was Waters ACQUITY UPLC C18, which was flushed with 0.1% by volume formic acid water at a flow rate of 0.3 mL / min for 30 minutes before use.

[0011] Furthermore, the mobile phase A used in the UPLC-MS / MS detection was 0.1% by volume formic acid water, and the mobile phase B was acetonitrile.

[0012] Furthermore, the elution program in the UPLC-MS / MS detection was set as follows: flow rate 0.3 mL / min, column temperature 35°C, injection volume 2 μL; The elution process is as follows: during 0-3 min, the mobile phase A is 95%, the mobile phase B is 5%, and the ion source adopts positive ion scanning mode; during 3-8 min, the mobile phase A linearly decreases from 95% to 75%, the mobile phase B linearly increases from 5% to 25%, and the ion source is switched from positive ion scanning to negative ion scanning; during 8-15 min, the mobile phase A linearly decreases from 75% to 50%, the mobile phase B linearly increases from 25% to 50%, and the ion source is switched from negative ion scanning to positive ion scanning; during 15-18 min, the mobile phase A linearly decreases from 50% to 40%, the mobile phase B linearly increases from 50% to 60%, and the ion source remains positive ion scanning; during 18-20 min, the mobile phase A linearly decreases from 40% to 5%, the mobile phase B linearly increases from 60% to 95%, and the ion source is switched from positive ion scanning to negative ion scanning; during 20-22 min, the mobile phase A remains 5%, the mobile phase B remains 95%, and the ion source remains negative ion scanning; during 22-25 min, the mobile phase A linearly increases from 5% to 95%, the mobile phase B linearly decreases from 95% to 5%, and the ion source is switched from negative ion scanning to positive ion scanning.

[0013] Further, the sample liquid is injected into the UPLC-MS / MS, and the sample liquid is used to rinse the injection needle 3 times, 5 μL each time.

[0014] Further, the method for configuring the mixed standard solution is as follows: Take 10 mg of astragaloside, dendrobium alkaloid, puerarin, panax notoginseng saponin R1, salvianolic acid B, berberine, rutin and tanshinone IIA respectively, and put them into 8 10 mL volumetric flasks, dissolve and constant volume to the calibration line with methanol to prepare single component stock solutions with a concentration of 1 mg / mL; Take 8 kinds of the single component stock solutions, dilute them with the mobile phase A to prepare 8 concentration gradient mixed standard solutions with concentrations of 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL and 50 μg / mL.

[0015] Compared with the prior art, the present application has the following beneficial effects: (1) The present application simultaneously detects 8 kinds of core index components, i.e. astragaloside, dendrobium alkaloid, puerarin, panax notoginseng saponin R1, salvianolic acid B, berberine, rutin and tanshinone IIA by adopting methanol-formic acid water mixed solvent synergistic extraction + UPLC-MS / MS positive and negative ion synchronous scanning technology, so that the characteristic effective components of 7 kinds of medicinal materials in the traditional Chinese medicine composition can be covered by one detection, without step-by-step detection of single components, full component quality verification is realized, and the defect of incomplete quality control is solved.

[0016] (2) The application extracts by first using a mixed solvent suitable for dissolving saponins, alkaloids and phenolic acids to perform ultrasonic extraction, and then purifying through a solid-phase extraction column and precisely locking target ions in a multiple reaction monitoring (MRM) mode, so that the impurities dissolved in the extraction process are reduced, the interfering substances are effectively removed, only the target components are responded during detection, the impurity interference rate is greatly reduced, and the detection accuracy is significantly improved.

[0017] (3) The application realizes that the single detection time is shortened to 1.5 hours through UPLC-MS / MS detection, the efficiency is improved by 3-4 times compared with the traditional single-component HPLC method (4-6 hours), the quality screening demand of industrialized batch samples can be met, and the problem of low detection efficiency is solved. DETAILED DESCRIPTION

[0018] The various illustrative embodiments of the application will now be described in detail in connection with the following figures. This Description is not to be considered as limited to the particular details shown herein, but is intended to be broadly within the scope of the claims. There are many other variations to which the application can be practiced. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0019] Further, for numerical ranges that are expressly recited herein, it is specifically intended that each and every intermediate value and sub-range within the recited ranges be explicitly enumerated. In any statement of a range, any intervening value or intervening sub-range, are specifically contemplated and should be considered as included within the range. The upper and lower limits of these intervening ranges can independently be included or excluded in the range.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0021] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations warrant the patentable subject matter under the provisions of 35 U.S.C. § 101 and the like. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0022] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to permit but not limit the inclusion of elements or the number of elements, and that do not exclude additional elements or additional elements that are not recited.

[0023] The present invention provides a method for detecting a Chinese medicine composition, comprising the following steps: 30 parts of Astragalus, 30 parts of dried Dendrobium, 30 parts of Pueraria, 30 parts of Salvia miltiorrhiza, 6 parts of Coptis chinensis, and 15 parts of Bambusa chinensis were ground and mixed to obtain a sample; Methanol and formic acid water are mixed in a volume ratio of 7:3 to obtain a mixed extraction solvent, the sample and the mixed extraction solvent are mixed and weighed, and then sealed under negative pressure, and then ultrasonic extraction is performed. After the end, the negative pressure is released, and the sample is weighed again, and the mixed extraction solvent is used to make up for the mass loss to obtain an extract, the extract is filtered, and the filtrate is collected. The filtrate is mixed with a composite adsorbent and then shaken to obtain a purified extract; activating a solid phase extraction column, injecting the purified extract into the activated solid phase extraction column, then adding methanol water for elution, discarding the eluate, then adding methanol water for elution, collecting the eluate, and performing a rotary evaporation on the eluate. After the evaporation, adding formic acid water for washing, then adding formic acid water again to adjust the volume, and filtering the adjusted solution to obtain a sample solution; The sample solution was injected into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for UPLC-MS / MS detection to obtain the quantitative ion peak area of ​​each target component; Astragaloside IV, dendrobium, puerarin, notoginsenoside R1, salvianolic acid B, berberine, rutin, and tanshinone IIA were prepared into a mixed standard solution, which was injected into an ultra-performance liquid chromatography-triple quadrupole mass spectrometer for detection. The concentration of each component was used as the independent variable and the quantitative ion peak area was used as the dependent variable. The weighted least squares method was used for linear regression to obtain the regression equation of the standard curve of each component. The quantitative ion peak area of ​​each target component is substituted into the standard curve regression equation to calculate the concentration of the corresponding target component, and the content of the corresponding target component is calculated based on the concentration of the corresponding target component.

[0024] Specifically, 30 parts of Astragalus, 30 parts of dried Dendrobium, 30 parts of Pueraria, 30 parts of Salvia miltiorrhiza, 6 parts of Coptis chinensis slices, and 15 parts of Lophatherum gracile prepared according to the ratio were taken, and the above 6 medicinal materials were placed in a cyclone mill (model FW100) respectively, the speed was set to 2800r / min, the single crushing amount was controlled to ≤10g, and after crushing, they were sieved through 80 mesh respectively.

[0025] Specifically, the formic acid water is 0.1% by mass formic acid water, which is prepared by dissolving analytical pure formic acid in ultrapure water at a volume ratio of 0.1:99.9.

[0026] Specifically, the negative pressure value of the negative pressure seal is -0.03 MPa.

[0027] Specifically, the composite adsorbent is mixed by neutral alumina and diatomite at a mass ratio of 1:2, and the particle size is 100-200 mesh. When used, it is activated at 120°C for 4h.

[0028] Specifically, the methanol water is prepared by mixing methanol and ultrapure water at a volume ratio of 5:95.

[0029] Specifically, when the sample solution is obtained by filtering the constant volume solution, a 0.22μm nylon organic filter membrane is used to filter the constant volume solution. Before filtering, the filter membrane is rinsed with 5mL of mobile phase A, and the rinse solution is discarded.

[0030] Specifically, in the UPLC-MS / MS detection, the mass spectrometry conditions are set as follows: the collision chamber pressure is 3.0×10 -3 mbar, multi-reaction monitoring (MRM) is used, and the quantitative ions and qualitative ions are set for the eight target components, as follows: Calycosin (positive ion mode): parent ion m / z 829.5, quantitative daughter ion m / z 787.4 (cone voltage 35V, collision energy 25eV, residence time 100ms), qualitative daughter ion m / z 645.3 (cone voltage 35V, collision energy 30eV, residence time 100ms); Shihubase (positive ion mode): parent ion m / z 206.2, quantitative daughter ion m / z 174.1 (cone voltage 20V, collision energy 18eV, residence time 100ms), qualitative daughter ion m / z 146.1 (cone voltage 20V, collision energy 22eV, residence time 100ms); Puerarin (negative ion mode): parent ion m / z 416.9, quantitative daughter ion m / z 267.0 (cone voltage 25V, collision energy 20eV, residence time 100ms), qualitative daughter ion m / z 152.9 (cone voltage 25V, collision energy 25eV, residence time 100ms); Panax notoginseng saponin R1 (positive ion mode): parent ion m / z 987.6, quantitative daughter ion m / z 945.5 (cone voltage 40V, collision energy 28eV, residence time 100ms), qualitative daughter ion m / z 783.4 (cone voltage 40V, collision energy 32eV, residence time 100ms); Danshensuan B (negative ion mode): parent ion m / z 717.1, quantitative daughter ion m / z 519.0 (cone voltage 30V, collision energy 22eV, residence time 100ms), qualitative daughter ion m / z 321.0 (cone voltage 30V, collision energy 28eV, residence time 100ms); Berberine (positive ion mode): parent ion m / z 336.1, quantitative daughter ion m / z 321.0 (cone voltage 22 V, collision energy 15 eV, residence time 100 ms), qualitative daughter ion m / z 293.0 (cone voltage 22 V, collision energy 20 eV, residence time 100 ms); Rutin (negative ion mode): parent ion m / z 610.9, quantitative daughter ion m / z 300.8 (cone voltage 28 V, collision energy 24 eV, residence time 100 ms), qualitative daughter ion m / z 270.8 (cone voltage 28 V, collision energy 29 eV, residence time 100 ms); Tanshinone ⅡA (positive ion mode): parent ion m / z 297.2, quantitative daughter ion m / z 279.1 (cone voltage 25 V, collision energy 21 eV, residence time 100 ms), qualitative daughter ion m / z 251.0 (cone voltage 25 V, collision energy 26 eV, residence time 100 ms); Specifically, the content of the corresponding target component is calculated according to the concentration of the corresponding target component, and the formula is: Content (mg / g) = (c x V x D) / m In the formula, c is the concentration of the target component in the sample solution (μg / mL), V is the constant volume (10 mL), D is the dilution multiple, and m is the sample mass.

[0031] It can be understood that the present application simultaneously detects eight core index components of astragaloside, smithosine, puerarin, panax notoginseng saponin R1, salvianolic acid B, berberine, rutin and tanshinone ⅡA by adopting methanol-formic acid water mixed solvent synergistic extraction + UPLC-MS / MS positive and negative ion synchronous scanning technology, so that one detection can cover the characteristic effective components of 7 medicinal materials in the traditional Chinese medicine composition, without step-by-step detection of single components, realizing full-component quality verification, and solving the defect of incomplete quality control.

[0032] It can be understood that the present application first extracts by ultrasonic extraction with a mixed solvent suitable for dissolving saponins, alkaloids and phenolic acids, and then purifies by a solid phase extraction column + multi-response monitoring (MRM) mode to precisely lock target ions, reduces the dissolution of impurities in the extraction process, effectively removes interfering substances, and only responds to target components during detection, greatly reduces the impurity interference rate, and significantly improves the detection accuracy.

[0033] It can be understood that the present application shortens the single detection time to 1.5 hours through UPLC-MS / MS detection, which is 3-4 times more efficient than the traditional single-component HPLC method (4-6 hours), can meet the quality screening needs of industrial batch samples, and solves the problem of low detection efficiency.

[0034] In some embodiments of the present application, the ultrasonic extraction process is specifically: At a temperature of 35℃, extract for 10 minutes at an ultrasonic power of 300W, then increase the temperature to 40℃, extract for 15 minutes, and finally reduce the temperature to 35℃, reduce the ultrasonic power to 250W, and extract for 5 minutes.

[0035] It can be understood that the first stage is a low-temperature flexible extraction at 35℃, which accurately avoids the problem that the degradation of dendrobium alkaloids is easy at high temperature in the initial stage of extraction, and ensures the detection accuracy of the heat-sensitive component; the second stage increases the temperature to 40℃ and maintains a power of 300W, which uses moderate temperature rise to improve the solubility of saponins (such as astragaloside A and panax notoginseng saponin R1) and phenolic acids (such as salvianolic acid B) to the solvent, promotes the complete dissolution of the target components, and avoids the low content detection caused by incomplete extraction; finally, the third stage falls back to 35℃ and reduces the power to 250W, which is targeted to protect salvianolic acid B from heat damage, solves the defect of high degradation rate of salvianolic acid B in traditional constant temperature ultrasonic extraction, and at the same time, the design of water bath water level being 1cm higher than the liquid level in the bottle ensures the uniform and stable temperature in the extraction process, further guarantees the accurate landing of each stage parameter, and finally realizes the effect of "less degradation of heat-sensitive components, high dissolution of target components, and stable temperature control in the extraction process", which provides high-quality extract for subsequent purification and detection.

[0036] In some embodiments of the present application, the activation process is specifically: Take a C18 solid phase extraction column, add 5ml of methanol to the C18 solid phase extraction column at a flow rate of 0.5mL / min, and then add 5ml of ultrapure water at a flow rate of 0.5mL / min after the end.

[0037] It can be understood that first adding 5mL of methanol and controlling the flow rate of 0.5mL / min can fully soak the C18 filler and make the hydrophobic group fully stretch, avoid "cracks" in the column bed due to dryness, ensure uniform penetration of the sample solution during subsequent sample loading, and prevent fluctuations in purification effect caused by uneven local flow rate; secondly, after washing with methanol, 5mL of ultrapure water is used for activation at the same flow rate, which can gradually replace the residual methanol in the column bed with an aqueous environment, so that the C18 filler is in a "hydrated equilibrium state", which is polar and matches the "aqueous filtrate" (filtrate containing a large amount of water phase treated by composite adsorbent) for subsequent sample loading, which can reduce the non-specific adsorption of target components (such as astragaloside A and panax notoginseng saponin R1) in the initial stage of sample loading; in addition, the stable flow rate control of 0.5mL / min can avoid column bed compaction caused by too fast flow rate or low activation efficiency caused by too slow flow rate, ensure that the activation process is repeatable and easy to control, and finally provide an efficient and stable purification carrier for the subsequent filtrate treated by the composite adsorbent, ensure complete impurity removal and stable target component recovery in subsequent detection, and further improve the accuracy and reliability of the overall detection method.

[0038] In some embodiments of the present application, the temperature of the rotary evaporation treatment is 45℃, the rotation speed is 60r / min, and the vacuum degree is -0.08MPa.

[0039] In some embodiments of the present application, after the eluent after the rotary evaporation treatment is added with formic acid water and shaken for 1min, it is transferred to a volumetric flask, then the original container is added with formic acid water and shaken for 1min, and the eluent is transferred to the volumetric flask, and finally the original container is added with formic acid water and shaken for 1min, and the eluent is transferred to the volumetric flask.

[0040] It can be understood that multiple washing of the original container with formic acid water can ensure complete dissolution and transfer of the residues in the original container.

[0041] In some embodiments of the present application, the chromatographic column used in the UPLC-MS / MS detection is Waters ACQUITY UPLC C18, which is flushed with 0.1% formic acid water at a flow rate of 0.3mL / min for 30min before use.

[0042] Specifically, the specification of Waters ACQUITY UPLC C18 is 100mm×2.1mm, 1.7μm.

[0043] It can be understood that flushing with 0.1% formic acid water at a flow rate of 0.3mL / min for 30min before use can ensure stable column efficiency.

[0044] In some embodiments of the present application, the mobile phase A used in the UPLC-MS / MS detection is 0.1% formic acid water, and the mobile phase B is acetonitrile.

[0045] In some embodiments of the present application, the elution program in the UPLC-MS / MS detection is set as follows: flow rate 0.3mL / min, column temperature 35℃, injection volume 2μL; The elution process was as follows: during the period of 0-3 min, the mobile phase A was 95%, the mobile phase B was 5%, and the ion source was in positive ion scanning mode; during the period of 3-8 min, the mobile phase A was linearly decreased from 95% to 75%, the mobile phase B was linearly increased from 5% to 25%, and the ion source was switched from positive ion scanning to negative ion scanning; during the period of 8-15 min, the mobile phase A was linearly decreased from 75% to 50%, the mobile phase B was linearly increased from 25% to 50%, and the ion source was switched from negative ion scanning to positive ion scanning; during the period of 15-18 min, the mobile phase A was linearly decreased from 50% to 40% %, mobile phase B linearly increased from 50% to 60%, and the ion source maintained positive ion scanning; during 18-20 min, mobile phase A linearly decreased from 40% to 5%, mobile phase B linearly increased from 60% to 95%, and the ion source switched from positive ion scanning to negative ion scanning; during 20-22 min, mobile phase A was maintained at 5%, mobile phase B was maintained at 95%, and the ion source maintained negative ion scanning; during 22-25 min, mobile phase A linearly increased from 5% to 95%, mobile phase B linearly decreased from 95% to 5%, and the ion source switched from negative ion scanning to positive ion scanning.

[0046] Specifically, the present invention adopts a dynamic gradient elution program: the flow rate is set to 0.3 mL / min, the column temperature is 35°C, the injection volume is 2 μL, and the gradient elution process is synchronized with the ion source scanning mode, as follows: 0-3min: Mobile phase A was 95%, mobile phase B was 5%, and the ion source was in positive ion scanning mode to specifically detect astragaloside IV and dendrobine (strong polar components); 3-8 min: Mobile phase A was linearly decreased from 95% to 75%, and mobile phase B was linearly increased from 5% to 25%. The ion source was switched from positive ion scanning to negative ion scanning to specifically detect puerarin and rutin (phenolic acid components); 8-15 min: Mobile phase A was linearly decreased from 75% to 50%, and mobile phase B was linearly increased from 25% to 50%. The ion source was switched from negative ion scanning to positive ion scanning to specifically detect salvianolic acid B and berberine (medium polarity components). 15-18 min: Mobile phase A was linearly decreased from 50% to 40%, and mobile phase B was linearly increased from 50% to 60%. The ion source maintained positive ion scanning to specifically detect notoginsenoside R1 (saponin component). 18-20 min: Mobile phase A was linearly decreased from 40% to 5%, and mobile phase B was linearly increased from 60% to 95%. The ion source was switched from positive ion scanning to negative ion scanning to specifically detect tanshinone IIA (a weakly polar component). 20-22 min: Mobile phase A is maintained at 5%, mobile phase B is maintained at 95%, and the ion source is maintained at negative ion scanning to flush out residual components in the column; 22-25min: Mobile phase A was linearly increased from 5% to 95%, and mobile phase B was linearly decreased from 95% to 5%. The ion source was switched from negative ion scanning to positive ion scanning, and the initial conditions were restored to prepare for the next injection.

[0047] In some embodiments of the present invention, before the sample solution is injected into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer, the injection needle is rinsed three times with 5 μL each time.

[0048] It is understood that rinsing the injection needle with the sample solution three times can avoid cross contamination.

[0049] In some embodiments of the present invention, the method for preparing the mixed standard solution is: 10 mg each of astragaloside IV, dendrobine, puerarin, notoginsenoside R1, salvianolic acid B, berberine, rutin, and tanshinone IIA were placed in eight 10 mL volumetric flasks, dissolved in methanol, and diluted to the mark to prepare a single-component stock solution with a concentration of 1 mg / mL. Eight single-component stock solutions were taken and diluted with the mobile phase A to prepare eight concentration gradient mixed standard solutions with concentrations of 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL.

[0050] Example 1 S1. Take 30 parts of Astragalus, 30 parts of Dried Dendrobium, 30 parts of Pueraria, 30 parts of Salvia miltiorrhiza, 6 parts of Coptis chinensis, and 15 parts of Lophatherum gracile (1 g each) prepared according to the ratio, and place the above 6 medicinal materials into a cyclone mill (model FW100) at a speed of 2800 r / min. The single crushing amount is controlled to ≤10 g (to avoid uneven particle size caused by overloading). After crushing, pass the crushed materials through an 80-mesh sieve to obtain samples; S2. Take 14 mL of methanol and 6 mL of 0.1% formic acid water, pour them into a 25 mL volumetric flask, cover the flask tightly with the stopper and shake it upside down 5 times to make a mixed extraction solvent of methanol-0.1% formic acid water. Weigh 0.5 g of the evenly mixed sample and put the weighed sample into a 50 mL stoppered conical flask. Add 20 mL of the above mixed extraction solvent to the 50 mL stoppered conical flask containing the sample, cover the sealing cap (with a negative pressure interface), connect the interface to the negative pressure device, adjust the negative pressure value to -0.03 MPa, and stabilize it for 5 minutes.

[0051] S3. Place the conical flask in the water bath of an ultrasonic extractor (model KQ-300DE), ensuring the water level is 1 cm above the liquid level in the flask. Set the ultrasonic parameters and perform extraction in three stages: The first stage (0-10min): the water temperature is controlled at 35℃ (temperature difference ±1℃), the ultrasonic power is 300W, and low-temperature flexible extraction is carried out to avoid degradation of dendrobium alkaloids in the initial stage; The second stage (10-25 minutes): the water temperature is raised to 40°C (temperature difference ± 1°C), and the ultrasonic power is maintained at 300W to promote the dissolution of saponins and phenolic acids; The third stage (25-30 min): the water temperature was lowered to 35°C (temperature difference ± 1°C), and the ultrasonic power was reduced to 250W to protect salvianolic acid B from thermal degradation; S4. After the extraction is completed, first turn off the negative pressure device, then open the sealing lid, use an analytical balance to weigh the total mass of the conical flask (accurate to 0.001g), and record the mass loss value; use the mixed extraction solvent to make up for the lost mass (for example, if the loss is 0.8g, add 0.8mL of solvent), close the stopper and shake it upside down for 30s, then slowly pour the extract in the conical flask into a Büchner funnel covered with double-layer qualitative filter paper (pore size 1-2μm), connect the suction filtration device, adjust the negative pressure to -0.02MPa and filter; discard the initial 5mL of filtrate, and collect the subsequent filtrate into a clean beaker; S5. Take 5 mL of the above filtrate and pour it into a 10 mL centrifuge tube. Add 0.2 g of the composite adsorbent. Cover the centrifuge tube tightly and place it in a vortex oscillator at a speed of 3000 r / min. Oscillate for 2 min and then let it stand for 5 min to obtain the purified extract. S6. Take a C18 solid phase extraction column (specification 500 mg / 6 mL), install it on a solid phase extraction device, first add 5 mL of methanol), open the device valve to control the flow rate to 0.5 mL / min, and after the methanol has completely flowed into the column bed, add 5 mL of ultrapure water, also controlling the flow rate to 0.5 mL / min to activate the column bed, slowly inject 4 mL of the purified extract into the column, controlling the flow rate to 0.3 mL / min, then add 5 mL of 5% methanol water, controlling the flow rate to 0.5 mL / min, elute the residual polysaccharides that have not been adsorbed by the composite adsorbent, discard the eluate, add 10 mL of 80% methanol water, controlling the flow rate to 0.4 mL / min, collect the eluate into a rotary evaporation flask, and after the elution is completed, rinse the solid phase extraction column outlet with a small amount of 80% methanol water (about 1 mL), and merge the rinse into the rotary evaporation flask; S7. Install the rotary evaporation bottle containing the eluent on the rotary evaporator, set the temperature to 45°C, the speed to 60r / min, and the vacuum degree to -0.08MPa, start the instrument for reduced pressure evaporation, and stop evaporating when the remaining liquid in the bottle is ≤0.5mL. Remove the rotary evaporation bottle, add 2ml of 0.1% formic acid water, put it into a vortex oscillator and oscillate for 1min, transfer the solution to a 10mL volumetric flask, then add 2ml of 0.1% formic acid water to the rotary evaporation bottle, put it into a vortex oscillator and oscillate for 1min, transfer the solution to the 10mL volumetric flask, and finally add 2ml of 0.1% formic acid water to the rotary evaporation bottle, put it into a vortex oscillator and oscillate for 1min, transfer the solution to the 10mL volumetric flask.

[0052] S8. Add 0.1% formic acid to a 10 mL volumetric flask to the mark, cap the flask tightly, and shake it upside down 10 times. Take 3 mL of the diluted solution and filter it through a 0.22 μm nylon organic filter membrane. Collect the filtrate into a clean injection bottle to obtain the sample solution. S9. Rinse the injection needle three times with 5 μL of sample solution each time, then place the needle in the autosampler of an ultra-performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-MS / MS, model Waters Xevo TQ-XS) for injection. Start the detection program, perform three parallel injections of each sample solution, and record the quantitative ion peak area of ​​each target component for each injection. The chromatographic conditions are set as follows: A Waters ACQUITY UPLC C18 column (100 mm × 2.1 mm, 1.7 μm, column lot number 20240115) was used and flushed with mobile phase A for 30 min (flow rate 0.3 mL / min) before use. Mobile phase A was 0.1% formic acid in water (ultrasonic degassed for 15 min after preparation), and mobile phase B was acetonitrile (chromatographic grade, ultrasonic degassed for 15 min); Dynamic gradient elution program: Set the flow rate to 0.3 mL / min, column temperature to 35°C, injection volume to 2 μL, and synchronize the gradient elution process with the ion source scan mode as follows: 0-3min, mobile phase A 95%, mobile phase B 5%, positive ion scan mode; 3-8min, mobile phase A linearly decreased from 95% to 75%, mobile phase B linearly increased from 5% to 25%, ion source switched from positive ion scan to negative ion scan; 8-15min, mobile phase A linearly decreased from 75% to 50%, mobile phase B linearly increased from 25% to 50%, ion source switched from negative ion scan to positive ion scan; 15-18min, mobile phase A linearly decreased from 50% to 40%, mobile phase B linearly increased from 50% to 60%, ion source remained positive ion scan; 18-20min, mobile phase A linearly decreased from 40% to 5%, mobile phase B linearly increased from 60% to 95%, ion source switched from positive ion scan to negative ion scan; 20-22min, mobile phase A remained 5%, mobile phase B remained 95%, ion source remained negative ion scan; 22-25min, mobile phase A linearly increased from 5% to 95%, mobile phase B linearly decreased from 95% to 5%, ion source switched from negative ion scan to positive ion scan; Mass spectrometry conditions were set as follows: collision chamber pressure 3.0x10 -3 mbar; multiple reaction monitoring (MRM) was used, and parameters were as follows: quantitative ions and qualitative ions were set for 8 target components, and specific parameters were as follows: astragaloside IV (positive ion mode): parent ion m / z 829.5, quantitative daughter ion m / z 787.4 (cone voltage 35V, collision energy 25eV, residence time 100ms), qualitative daughter ion m / z 645.3 (cone voltage 35V, collision energy 30eV, residence time 100ms); dendrobium alkaloid (positive ion mode): parent ion m / z 206.2, quantitative daughter ion m / z 174.1 (cone voltage 20V, collision energy 18eV, residence time 100ms), qualitative daughter ion m / z 146.1 (cone voltage 20V, collision energy 22eV, residence time 100ms); puerarin (negative ion mode): parent ion m / z 416.9, quantitative daughter ion m / z 267.0 (cone voltage 25V, collision energy 20eV, residence time 100ms), qualitative daughter ion m / z 152.9 (cone voltage 25V, collision energy 25eV, residence time 100ms); notoginsenoside R1 (positive ion mode): parent ion m / z 987.6, quantitative daughter ion m / z 945.5 (cone voltage 40V, collision energy 28eV, residence time 100ms), qualitative daughter ion m / z 783.4 (cone voltage 40V, collision energy 32eV, residence time 100ms); Salvianolic acid B (negative ion mode): parent ion m / z 717.1, quantitative product ion m / z 519.0 (cone voltage 30 V, collision energy 22 eV, dwell time 100 ms), qualitative product ion m / z 321.0 (cone voltage 30 V, collision energy 28 eV, dwell time 100 ms); Berberine (positive ion mode): parent ion m / z 336.1, quantitative product ion m / z 321.0 (cone voltage 22 V, collision energy 15 eV, dwell time 100 ms), qualitative product ion m / z 293.0 (cone voltage 22 V, collision energy 20 eV, dwell time 100 ms); Rutin (negative ion mode): parent ion m / z 610.9, quantitative product ion m / z 300.8 (cone voltage 28 V, collision energy 24 eV, dwell time 100 ms), qualitative product ion m / z 270.8 (cone voltage 28 V, collision energy 29 eV, dwell time 100 ms); Tanshinone IIA (positive ion mode): parent ion m / z 297.2, quantitative product ion m / z 279.1 (cone voltage 25 V, collision energy 21 eV, dwell time 100 ms), qualitative product ion m / z 251.0 (cone voltage 25 V, collision energy 26 eV, dwell time 100 ms); S10. Weigh 10 mg (accurate to 0.0001 g) each of astragaloside IV, dendrobium, puerarin, notoginsenoside R1, salvianolic acid B, berberine, rutin, and tanshinone IIA reference substances, place them separately into eight 10 mL volumetric flasks, dissolve them in methanol, and dilute to the mark to prepare a single-component stock solution with a concentration of 1 mg / mL. Take the above eight single-component stock solutions and dilute them with mobile phase A to prepare eight concentration gradient mixed standard solutions with concentrations of 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL; prepare three replicates for each concentration gradient. S11, inject 8 mixed standard solutions with concentration gradients according to the detection conditions of step S9, use the concentration of each component (abscissa x, unit μg / mL) as the independent variable, and the quantitative ion peak area (ordinate y) as the dependent variable, and use the weighted least squares method (weight 1 / x 2 ) to perform linear regression to obtain the standard curve regression equation of each component; S12. Statistically analyze the peak areas of three parallel injections of each sample solution and calculate the average value. Substitute the average peak area into the standard curve regression equation of the corresponding component to calculate the concentration of the component in the sample solution (unit: μg / mL). Then calculate the content of the component in the sample (unit: mg / g) according to the following formula: Content (mg / g) = (c×V×D) / m Where: c is the concentration of the target component in the sample solution (μg / mL), V is the fixed volume (10 mL), D is the dilution factor (no additional dilution in this protocol, D = 1), and m is the sample mass (0.5 g). Note the unit conversion (1 mg = 1000 μg) when calculating. Test results: The peak areas of three parallel injections of each sample solution in Example 1 are shown in Table 1: Table 1 Peak area of ​​three parallel injections of each sample solution

[0053] The standard curve regression equations of the components in Example 1 are shown in Table 2: Table 2 Standard curve regression equations for each component

[0054] In the table, y=ax+b (y is the quantitative ion peak area, AU×s; x is the component concentration, μg / mL; a is the slope, and b is the intercept) The contents of the various components in the sample in Example 1 are shown in Table 3: Table 3 Content of each component in the sample

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered by the scope of protection of the claims of the present invention.

Claims

1. A method for detecting a Chinese medicine composition, characterized in that: The following steps are involved: 30 parts of Astragalus, 30 parts of dried Dendrobium, 30 parts of Pueraria, 30 parts of Salvia miltiorrhiza, 6 parts of Coptis chinensis, and 15 parts of Bambusa chinensis were ground and mixed to obtain a sample; Methanol and formic acid water are mixed in a volume ratio of 7:3 to obtain a mixed extraction solvent, the sample and the mixed extraction solvent are mixed and weighed, and then sealed under negative pressure, and then ultrasonic extraction is performed. After the end, the negative pressure is released, and the sample is weighed again, and the mixed extraction solvent is used to make up for the mass loss to obtain an extract, the extract is filtered, and the filtrate is collected. The filtrate is mixed with a composite adsorbent and then shaken to obtain a purified extract; activating a solid phase extraction column, injecting the purified extract into the activated solid phase extraction column, then adding methanol water for elution, discarding the eluate, then adding methanol water for elution, collecting the eluate, and performing a rotary evaporation on the eluate. After the evaporation, adding formic acid water for washing, then adding formic acid water again to adjust the volume, and filtering the adjusted solution to obtain a sample solution; The sample solution was injected into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for UPLC-MS / MS detection to obtain the quantitative ion peak area of ​​each target component; Astragaloside IV, dendrobium, puerarin, notoginsenoside R1, salvianolic acid B, berberine, rutin, and tanshinone IIA were prepared into a mixed standard solution, which was injected into an ultra-performance liquid chromatography-triple quadrupole mass spectrometer for detection. The concentration of each component was used as the independent variable and the quantitative ion peak area was used as the dependent variable. The weighted least squares method was used for linear regression to obtain the regression equation of the standard curve of each component. The quantitative ion peak area of ​​each target component is substituted into the standard curve regression equation to calculate the concentration of the corresponding target component, and the content of the corresponding target component is calculated based on the concentration of the corresponding target component.

2. The method for detecting the Chinese medicine composition according to claim 1, wherein The ultrasonic extraction process is specifically as follows: The extraction was carried out at a temperature of 35°C and an ultrasonic power of 300W for 10 minutes, then the temperature was raised to 40°C and the extraction was carried out for 15 minutes, and finally the temperature was lowered to 35°C and the ultrasonic power was reduced to 250W and the extraction was carried out for 5 minutes.

3. The method for detecting the Chinese medicine composition according to claim 2, wherein The activation treatment is specifically as follows: A C18 solid phase extraction column was taken, and 5 ml of methanol was added to the C18 solid phase extraction column at a flow rate of 0.5 mL / min. After the addition, 5 ml of ultrapure water was added at a flow rate of 0.5 mL / min.

4. The method for detecting the Chinese medicine composition according to claim 3, wherein The temperature of the rotary evaporation treatment was 45° C., the rotation speed was 60 r / min, and the vacuum degree was -0.08 MPa.

5. The method for detecting the Chinese medicine composition according to claim 4, wherein The washing process is specifically as follows: Add formic acid water to the eluate after rotary evaporation, shake for 1 minute, and then transfer to a volumetric flask. Then add formic acid water to the original container, shake for 1 minute, and then transfer to the volumetric flask. Finally, add formic acid water to the original container, shake for 1 minute, and then transfer to the volumetric flask.

6. The method for detecting the Chinese medicine composition according to claim 5, wherein: The chromatographic column used in the UPLC-MS / MS detection was Waters ACQUITY UPLC C18, which was flushed with 0.1% by volume formic acid water at a flow rate of 0.3 mL / min for 30 minutes before use.

7. The method for detecting the Chinese medicine composition according to claim 6, wherein: The mobile phase A used in the UPLC-MS / MS detection was 0.1% by volume formic acid water, and the mobile phase B was acetonitrile.

8. The method for detecting the Chinese medicine composition according to claim 7, wherein: The elution program in the UPLC-MS / MS detection was set as follows: flow rate 0.3 mL / min, column temperature 35°C, injection volume 2 μL; The elution process was as follows: during the period of 0-3 min, the mobile phase A was 95%, the mobile phase B was 5%, and the ion source was in positive ion scanning mode; during the period of 3-8 min, the mobile phase A was linearly decreased from 95% to 75%, the mobile phase B was linearly increased from 5% to 25%, and the ion source was switched from positive ion scanning to negative ion scanning; during the period of 8-15 min, the mobile phase A was linearly decreased from 75% to 50%, the mobile phase B was linearly increased from 25% to 50%, and the ion source was switched from negative ion scanning to positive ion scanning; during the period of 15-18 min, the mobile phase A was linearly decreased from 50% to 40% %, mobile phase B linearly increased from 50% to 60%, and the ion source maintained positive ion scanning; during 18-20 min, mobile phase A linearly decreased from 40% to 5%, mobile phase B linearly increased from 60% to 95%, and the ion source switched from positive ion scanning to negative ion scanning; during 20-22 min, mobile phase A was maintained at 5%, mobile phase B was maintained at 95%, and the ion source maintained negative ion scanning; during 22-25 min, mobile phase A linearly increased from 5% to 95%, mobile phase B linearly decreased from 95% to 5%, and the ion source switched from negative ion scanning to positive ion scanning.

9. The method for detecting the Chinese medicine composition according to claim 8, wherein Before injecting the sample solution into ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, the injection needle was rinsed with the sample solution three times, 5 μL each time.

10. The method for detecting the Chinese medicine composition according to claim 8, characterized in that: The method for preparing the mixed standard solution is: 10 mg each of astragaloside IV, dendrobine, puerarin, notoginsenoside R1, salvianolic acid B, berberine, rutin, and tanshinone IIA were placed in eight 10 mL volumetric flasks, dissolved in methanol, and diluted to the mark to prepare a single-component stock solution with a concentration of 1 mg / mL. Eight single-component stock solutions were taken and diluted with the mobile phase A to prepare eight concentration gradient mixed standard solutions with concentrations of 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL.

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