A method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction

Abstract

The present invention provides a method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction, belonging to the technical field of traditional Chinese medicine component analysis, and comprising the following steps: after decocting the raw materials of Fangji Huangqi Decoction with water, ultrasonic extraction is carried out by using a methanol solution to obtain a test solution, and then the UHPLC-MS / MS method is adopted to precisely absorb the mixed reference solution and the test solution and inject them into an analytical instrument for determination. Compared with the traditional ultraviolet detector, the mass spectrometry can accurately give the mass number information of the precursor ion and the product ion of the analyte while accurately determining the component content, and different from the retention time, the mass number information is more stable, reliable and highly specific as the basis for qualitative identification.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis of traditional Chinese medicine components, and particularly relates to a method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction. Background Art

[0002] Fangji Huangqi Decoction is composed of four herbs: Stephania tetrandra, Astragalus membranaceus, Atractylodes macrocephala, and roasted Glycyrrhiza uralensis, and has the effects of replenishing qi and removing dampness, strengthening the spleen and promoting diuresis. It is a representative prescription for replenishing qi and promoting diuresis in traditional Chinese medicine clinical practice. Because of its significant curative effect and good clinical application safety, it is currently widely used in the treatment of nephrotic syndrome, various types of edema, rheumatoid arthritis, etc. In the prior art, the research on the chemical components of Fangji Huangqi Decoction is still very limited, and it is not comprehensive enough in terms of depth and breadth. The chemical composition of Fangji Huangqi Decoction is rich and complex, and the compounds belong to different basic structural categories. Traditional detection techniques such as HPLC-DAD, UV, HPLC-ELSD, etc. have disadvantages such as low sensitivity, long running time, and inability to simultaneously determine the contents of components with different chemical structures due to the limitations of the detectors, especially unable to accurately quantify some components with low abundance and small content in the prescription. A large number of basic studies have shown that the low abundance of traditional Chinese medicine components does not equal low efficacy, but due to the inability of detection means, the accurate analysis and determination of some active components with low content will undoubtedly directly affect the quality control of traditional Chinese medicine compound prescriptions and cannot guarantee the safety and effectiveness of clinical medication.

[0003] Four components, calycosin, medicarpin, formononetin, and atractylenolide II, have been reported in a large number of modern pharmacodynamic literature to have various activities and medicinal values. However, up to now, due to the technical limitation of the insufficient sensitivity of the existing analysis methods, it is still impossible to accurately determine the contents of the above 4 active components in Fangji Huangqi Decoction.

[0004] Therefore, how to provide a method for detecting the contents of calycosin, medicarpin, formononetin, and atractylenolide II in Fangji Huangqi Decoction is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes a method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction, comprising the following steps:

[0008] After decocting the raw materials of Fangji Huangqi Decoction with water, ultrasonic extraction is carried out with methanol solution to obtain the test solution, and then the mixed reference solution and the test solution are precisely pipetted into the analytical instrument for determination by UHPLC-MS / MS method;

[0009] In the UHPLC-MS / MS method, the chromatographic conditions are as follows:

[0010] C 18 Agilent BDS chromatographic column (2.1 mm×50 mm, 1.8 μm);

[0011] Flow rate: 0.3 mL / min;

[0012] Column temperature: 40 °C;

[0013] Injection volume: 3 μL;

[0014] Gradient elution conditions: The mobile phase is a mixture of an aqueous solution of formic acid and tetrahydrofuran and methanol. From 0 to 5 min, the volume fraction of methanol linearly increases from 10% to 85%, and the volume fraction of the aqueous solution of formic acid and tetrahydrofuran linearly decreases from 90% to 15%. From 5 to 6 min, the volume fraction of methanol remains at 85%, and the volume fraction of the aqueous solution of formic acid and tetrahydrofuran remains at 15%. From 6 to 7 min, the volume fraction of methanol linearly decreases to 10%, and the volume fraction of the aqueous solution of formic acid and tetrahydrofuran linearly increases to 90%. From 7 to 8 min, the volume fraction of methanol remains at 10%, and the volume fraction of the aqueous solution of formic acid and tetrahydrofuran remains at 90%.

[0015] Preferably, the preparation method of the test solution specifically includes the following steps:

[0016] Precisely weigh 6 g of Stephania tetrandra, 7.5 g of Astragalus membranaceus, 4.5 g of Atractylodes macrocephala, and 3 g of roasted Glycyrrhiza uralensis and place them in a 500 mL round-bottom flask. First, add 210 mL of water, soak for 30 min, bring to a boil over high heat, and then keep it boiling gently over low heat for 30 min. Then filter out the medicinal liquid with four layers of gauze. Add 168 mL of water to the obtained medicinal residues, bring to a boil over high heat, and continue to keep it boiling gently over low heat for 30 min. Then filter out the medicinal liquid with four layers of gauze. Combine the two medicinal liquids, add water to make up to 500 mL, precisely transfer 2 mL of the medicinal liquid into a 50 mL volumetric flask, add 15 mL of 60% methanol solution, ultrasonicate for 15 min, cool, dilute to the mark with 60% methanol solution, centrifuge at 13000 r / min for 8 min, aspirate the supernatant and filter it through a 0.22 μm microporous filter membrane to obtain the test solution.

[0017] Preferably, the low-content active chemical components are calycosin, medicarpin, formononetin, and atractylenolide II.

[0018] Preferably, the concentration of the methanol solution is 60%.

[0019] Preferably, the mixed reference substance solution is obtained by dissolving calycosin, medicarpin, formononetin and atractylenolide II in a 60% methanol solution;

[0020] Among them, the concentration of calycosin is 112.200 ng / mL, the concentration of medicarpin is 41.650 ng / mL, the concentration of formononetin is 41.400 ng / mL, and the concentration of atractylenolide II is 15.675 ng / mL.

[0021] Preferably, in the aqueous solution of formic acid and tetrahydrofuran, the volume fraction of formic acid is 0.1% and the volume fraction of tetrahydrofuran is 1%.

[0022] Preferably, in the UHPLC-MS / MS method, the scanning mode of the mass spectrometry is the multiple reaction monitoring mode, and mass spectrometry analysis is carried out under two modes of ESI+ and ESI- respectively.

[0023] Preferably, the conditions of the mass spectrometry are as follows:

[0024] Ion source temperature: 500 °C;

[0025] Q1 / Q3 (m / z): 285.1 / 269.9; 269.1 / 254.1; 267.0 / 252.1; 233.2 / 159.1;

[0026] Declustering potential (DP): 7.41; -135.07; -122.47; 136.43 V;

[0027] Collision energy (CE): 30.92; -22.98; -27.12; 28.60 eV;

[0028] Nebulizing gas: 60 psi; Auxiliary heating gas: 60 psi; Curtain gas: 40 psi;

[0029] Nitrogen is used as the spray assist gas.

[0030] Compared with the prior art, the present invention has the following advantages and technical effects:

[0031] Using the detection method provided by the present invention, 4 low-content chemical components in the Fangji Huangqi Decoction can be effectively separated within 8 minutes under the gradient elution conditions of a methanol-(0.1% formic acid + 1% tetrahydrofuran) water system, enabling accurate and precise analysis and determination of the content of each component. The method has good accuracy and extremely high sensitivity. The screening and determination of the specific stationary phase and mobile phase composition in the present invention are the key factors for the successful simultaneous quantitative analysis of 4 different types of low-content active compounds in the Fangji Huangqi Decoction. Compared with the traditional ultraviolet detector, the method provided by the present invention can accurately determine the component content while precisely providing the mass numbers of the precursor ion and product ion of the analyte. Different from the retention time, the mass number information is more stable, reliable, and highly specific as the basis for qualitative identification. Description of the Drawings

[0032] The drawings constituting a part of this application are used to provide a further understanding of this application. The schematic embodiments and descriptions thereof of this application are used to explain this application and do not constitute an improper limitation to this application. In the drawings:

[0033] Figure 1 is the extracted ion current mass chromatogram of 4 components in the mixed reference solution of the present invention;

[0034] Among them, A. Calycosin (3.29 min); B. Medicarpin (4.18 min); C. Formononetin (3.88 min); D. Atractylenolide II (5.17 min);

[0035] Figure 2 is the chemical structure diagram of 4 analytes in the test solution of the present invention. Detailed Embodiments

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the 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 protection scope of the present invention.

[0037] To make the above objects, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0038] Stephania tetrandra in the embodiments of the present invention was purchased from Anhui Jiuhetang Chinese Medicine Co., Ltd. (Jiangxi, China); Atractylodes macrocephala was purchased from Jiangsu Donglian Pharmaceutical Co., Ltd. (Zhejiang, China); Astragalus membranaceus and Radix Glycyrrhizae Preparata were purchased from Bozhou Yonggang Decoction Pieces Factory Co., Ltd. (Gansu, China); all were identified as genuine products by Professor Chen Jianwei of the School of Pharmacy, Nanjing University of Chinese Medicine.

[0039] The instruments used in the embodiments of the present invention include:

[0040] Shimadzu CBM20Alite high performance liquid chromatography system (Shimadzu LC20ADXR quaternary pump, SPD 30AUV detector, SIL 30A CXR autosampler, CT030AC column oven);

[0041] UHPLC-TQ-MS mass spectrometry system: Triple Quadrupole 5500 mass spectrometer (AB SCIEX, USA), ESI ion source;

[0042] Mass spectrometry workstation: Analyst TF 1.6; Ultrasonic cleaner (KQ-250E, Kunshan Ultrasonic Instruments Co., Ltd.);

[0043] QUINTIX35-1CN electronic balance (Sartorius Scientific Instruments Co., Ltd., model: SQP, d = 0.01mg);

[0044] Pure water instrument (Merck Millipore, model: IX70XX);

[0045] High-speed centrifuge (Gene Co., Ltd., model: X1).

[0046] The test drugs in the embodiments of the present invention include:

[0047] Calycosin (104038-221001) (purity > 99%), Medicarpin (HB0003) (purity > 98.5%), Formononetin (102832) (purity > 98.5%), Atractylenolide II (100653) (purity > 98%) were all purchased from Jiangsu Yongjian Medical Science and Technology Co., Ltd.

[0048] Methanol (batch number: CAEQ-4-000306-4000), formic acid (batch number: 4.000308.4000) were of mass spectrometry purity (Shanghai Anpu Scientific Instruments Co., Ltd.), tetrahydrofuran was of chromatographic purity (Anaqua Chemicals, batch number: FA-3116-0500), ultrapure water (Millipore), and the rest of the reagents were of analytical purity.

[0049] In the (0.1% formic acid + 1% tetrahydrofuran) aqueous solution in the embodiments of the present invention, the volume fraction of formic acid in the solution is 0.1%, and the volume fraction of tetrahydrofuran is 1%.

[0050] Example 1

[0051] A method for simultaneously determining 4 low-content active chemical components in Stephania Tetrandra and Astragalus Decoction, comprising the following steps:

[0052] 1. Preparation of Mixed Reference Substance Solution

[0053] Accurately weigh appropriate amounts of calycosin, medicarpin, formononetin, and atractylenolide II reference substances respectively, dissolve and dilute them with 60% methanol, shake well to obtain a mixed reference substance solution with the concentrations of calycosin, medicarpin, formononetin, and atractylenolide II being 112.200 ng / mL, 41.650 ng / mL, 41.400 ng / mL, and 15.675 ng / mL respectively;

[0054] 2. Preparation of Test Solution

[0055] According to the prescription ratio of Fangji Huangqi Decoction, accurately weigh 6 g of Stephania tetrandra, 7.5 g of Astragalus membranaceus, 4.5 g of Atractylodes macrocephala, and 3 g of roasted Glycyrrhiza uralensis in a 500 mL round-bottom flask. First, add 210 mL of water, soak for 30 min, bring to a boil over high heat, then keep it boiling gently over low heat for 30 min, filter out the medicinal liquid with four layers of gauze. Add 168 mL of water to the medicinal residues, bring to a boil over high heat, then keep it boiling gently over low heat for 30 min, filter out the medicinal liquid with four layers of gauze. Combine the two decoctions, add an appropriate amount of water to make the volume up to 500 mL. Accurately transfer 2 mL of the medicinal liquid into a 50 mL volumetric flask, add 15 mL of 60% methanol solution, sonicate for 15 min, let it cool, dilute to the mark with 60% methanol solution, centrifuge at 13000 r / min for 8 min, aspirate the supernatant and filter it through a 0.22 μm microporous filter membrane to obtain the test solution;

[0056] 3. Chromatographic Conditions and Mass Spectrometry Parameter Conditions

[0057] Chromatographic conditions: C 18 Agilent BDS chromatographic column (2.1 mm × 50 mm, 1.8 μm); Flow rate: 0.3 mL / min; Column temperature: 40 °C; Injection volume: 3 μL; Gradient elution conditions: The mobile phase is a mixture of (0.1% formic acid + 1% tetrahydrofuran) aqueous solution and methanol. From 0 to 5 min, the volume fraction of methanol linearly increases from 10% to 85%, and the volume fraction of (0.1% formic acid + 1% tetrahydrofuran) aqueous solution linearly decreases from 90% to 15%. From 5 to 6 min, the volume fraction of methanol remains at 85%, and the volume fraction of (0.1% formic acid + 1% tetrahydrofuran) aqueous solution remains at 15%. From 6 to 7 min, the volume fraction of methanol linearly decreases to 10%, and the volume fraction of (0.1% formic acid + 1% tetrahydrofuran) aqueous solution linearly increases to 90%. From 7 to 8 min, the volume fraction of methanol remains at 10%, and the volume fraction of (0.1% formic acid + 1% tetrahydrofuran) aqueous solution remains at 90%.

[0058] Mass spectrometry parameter conditions:

[0059] The mass spectrometry scanning mode is the multiple reaction monitoring mode (MRM), and the following parameters are used:

[0060] Ion source temperature: 500 °C;

[0061] Q1 / Q3 (m / z): 285.1 / 269.9; 269.1 / 254.1; 267.0 / 252.1; 233.2 / 159.1;

[0062] Declustering potential (DP): 7.41; -135.07; -122.47; 136.43 V;

[0063] Collision energy (CE): 30.92; -22.98; -27.12; 28.60 eV;

[0064] Nebulizing gas: 60 psi; Auxiliary heating gas: 60 psi; Curtain gas: 40 psi;

[0065] Nitrogen is used as the spray assist gas.

[0066] 4. Investigation of linear relationship

[0067] Precisely pipette the mixed reference solution, and serially dilute it to obtain a series of standard solutions with dilutions of 1, 2, 4, 8, 16, and 32 times and measure them. Using the peak area (Y) as the ordinate and the reference substance concentration (X) as the abscissa, perform linear regression to obtain the regression equation and correlation coefficient of each compound. The results are shown in Table 1. The results indicate that the four chemical components have a good linear relationship within the set range.

[0068] Table 1 Linear equations, correlation coefficients, and linear ranges of four chemical components in Decoction for Invigorating Qi and Supplementing Astragalus

[0069] Reference substance Linear equation R <![CDATA[Linear range ng·mL -1 > Calycosin <![CDATA[y = 1.26819e 5 x + 3.63138e 5 > 0.9996 3.506～112.200 Medicarpin y = 901.24004x - 148.54782 0.9999 1.302～41.650 Formononetin <![CDATA[y = 5.67090e 4 x + 13709.52865]]> 0.9999 1.294～41.400 Atractylenolide II y = 11728.13899x + 8589.30427 0.9998 0.490～15.675

[0070] 5. Precision experiment

[0071] Precisely pipette the same mixed reference solution: a mixed solution with concentrations of calycosin, medicarpin, formononetin, and atractylenolide II being 28.050, 5.206, 5.175, and 1.959 ng / mL respectively, inject samples continuously for 6 times, and record the peak areas of each. The results show that the RSD (n = 6) of the peak areas of calycosin, medicarpin, formononetin, and atractylenolide II are 1.7%, 1.6%, 1.9%, and 1.6% respectively, indicating good instrument precision.

[0072] 6. Repeatability experiment

[0073] Six samples of the test solution of Fangji Huangqi Decoction were prepared in parallel from the same batch of Chinese herbal pieces and injected for determination, and the concentration of each compound was calculated. The average concentrations of calycosin, medicarpin, formononetin, and atractylenolide II were 39.25, 17.86, 19.52, and 6.678 ng / mL, respectively; the RSDs were 1.4%, 2.1%, 2.4%, and 2.4%, respectively, indicating good repeatability of the method.

[0074] 7. Stability experiment

[0075] An aliquot of the test solution of Fangji Huangqi Decoction was accurately pipetted and allowed to stand at room temperature. Samples were injected for analysis at 0, 2, 4, 8, 12, and 24 h, and the peak areas were recorded. The results showed that the RSDs of calycosin, medicarpin, formononetin, and atractylenolide II were 2.3%, 1.9%, 1.9%, and 1.4%, respectively, indicating good stability of the test solution within 24 h at room temperature.

[0076] 8. Recovery experiment

[0077] An aliquot of 1.0 mL of the Fangji Huangqi Decoction sample with known content was accurately pipetted in six parallels and placed in a 50-mL volumetric flask. An aliquot of 1.0 mL of a mixed reference solution with a certain concentration (the concentrations of calycosin, medicarpin, formononetin, and atractylenolide II were 40.80, 17.85, 18.63, and 6.688 ng / mL, respectively) was accurately added. 14 mL of 60% methanol solution was added, and the mixture was sonicated for 15 min, cooled, diluted to the mark with 60% methanol solution, centrifuged at 13000 r / min for 8 min, the supernatant was taken, filtered through a 0.22-μm microporous membrane, and then injected for analysis. The peak areas were recorded and the spike recoveries were calculated. The results are shown in Table 2. It can be seen that the average recoveries for the determination of the four components were 98.9% - 104.4%, and the RSDs < 3.5%, indicating that the method is accurate and reliable.

[0078] Table 2 Recoveries of four chemical components (n = 6)

[0079]

[0080] 9. Determination of sample content

[0081] Three batches of decoction samples were prepared from the Chinese herbal pieces in the prescription of Fangji Huangqi Decoction, and the test solutions were prepared and injected for analysis. The peak areas were recorded and the compound contents were calculated. The determination results are shown in Table 3.

[0082] Table 3 Determination results of the contents of four chemical components in Fangji Huangqi Decoction (ng / mL, n = 3)

[0083]

[0084]

[0085] The present invention uses the UHPLC-MS / MS method to analyze four chemical components in the Fangji Huangqi Decoction. According to the properties of each compound, the multiple reaction monitoring mode is adopted, and mass spectrometry analysis needs to be carried out under two modes of ESI+ and ESI- respectively to obtain satisfactory mass spectrometry responses: Calycosin and Atractylenolide II have good responses in the positive ion mode, while Medicarpin and Formononetin have better responses in the negative ion mode.

[0086] The conditions of the stationary phase and the mobile phase in the present invention are the key factors for the successful separation and final accurate quantification of the four compounds. The BDS bonded phase has a full peak tailing technology, and can give a more symmetrical normal distribution chromatographic peak compared with the ODS bonded phase commonly used in the field. This advantage is particularly important when determining low-content components. The mobile phase system is screened and optimized many times. Methanol-water, acetonitrile-water, acetonitrile-0.1% formic acid water, methanol-0.1% formic acid water and the mixture of (0.1% formic acid + 1% tetrahydrofuran) aqueous solution and methanol are investigated and compared successively (Table 4). It is found that when the latter is used as the mobile phase system for gradient elution, the chromatographic peak resolution and peak shape are better, and the retention time is short, and the experimental efficiency is high. Therefore, it is finally determined as the mixture of (0.1% formic acid + 1% tetrahydrofuran) aqueous solution and methanol, combined with the gradient elution method. The addition of tetrahydrofuran is a necessary factor, making the chromatographic peak resolution and peak shape of each compound good, meeting the conditions for accurate quantitative analysis and determination.

[0087] Table 4 Optimization and screening results of the mobile phase system

[0088]

[0089]

[0090] Note: "-" represents not involved

[0091] The selection of the extraction solvent has been optimized many times. The extraction efficiencies of methanol, 40% methanol, 60% methanol and 60% ethanol solutions for the components are investigated and compared successively. It is found that the 60% methanol solution can most efficiently extract four components, namely Calycosin, Medicarpin, Formononetin and Atractylenolide II, in the Fangji Huangqi Decoction, ensuring the accuracy and reliability of the subsequent analysis and determination results (Table 5).

[0092] Table 5 Effects of different extraction solvents on the extraction recoveries (%) of four components to be measured

[0093]

[0094] From the above experimental data, it can be seen that the present invention has established a method for simultaneously determining 4 low-content active chemical components in Fangji Huangqi Decoction by using UHPLC-MS / MS technology. Under the conditions selected in the present invention, the separation effect is good and a certain response is achieved. The determination and analysis results of the contents of each component are accurate and have good stability. It is a reliable method for determining low-content active chemical components of various structural types in Fangji Huangqi Decoction, and can provide an objective and scientific experimental basis for the improvement of the quality evaluation method of Fangji Huangqi Decoction and the formation of a comprehensive quality evaluation system.

[0095] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction, characterized in that, It includes the following steps: After decocting the raw materials of Fangji Huangqi Decoction with water, ultrasonically extract with methanol solution to obtain the test solution, and then precisely pipette the mixed reference solution and the test solution into the analytical instrument for determination by UHPLC-MS / MS method; The chromatographic conditions in the UHPLC-MS / MS method are as follows: C 18 Agilent BDS chromatographic column, with a specification of 2.1 mm × 50 mm, 1.8 μm; Flow rate: 0.3 mL / min; Column temperature: 40 °C; Injection volume: 3 μL; Gradient elution conditions: The mobile phase is a mixture of an aqueous solution of formic acid and tetrahydrofuran and methanol. From 0 to 5 min, the volume fraction of methanol linearly increases from 10% to 85%, and the volume fraction of the aqueous solution of formic acid and tetrahydrofuran linearly decreases from 90% to 15%. From 5 to 6 min, the volume fraction of methanol remains at 85%, and the volume fraction of the aqueous solution of formic acid and tetrahydrofuran remains at 15%. From 6 to 7 min, the volume fraction of methanol linearly decreases to 10%, and the volume fraction of the aqueous solution of formic acid and tetrahydrofuran linearly increases to 90%. From 7 to 8 min, the volume fraction of methanol remains at 10%, and the volume fraction of the aqueous solution of formic acid and tetrahydrofuran remains at 90%; The low-content active chemical components are calycosin, medicarpin, formononetin, and atractylenolide II.

2. The method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction according to claim 1, wherein, The concentration of the methanol solution is 60%.

3. A method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction according to claim 1, characterized in that, The mixed reference solution is obtained by dissolving calycosin, medicarpin, formononetin, and atractylenolide II in a 60% methanol solution; Among them, the concentration of calycosin is 112.200 ng / mL, the concentration of medicarpin is 41.650 ng / mL, the concentration of formononetin is 41.400 ng / mL, and the concentration of atractylenolide II is 15.675 ng / mL.

4. A method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction according to claim 1, characterized in that, In the aqueous solution of formic acid and tetrahydrofuran, the volume fraction of formic acid is 0.1%, and the volume fraction of tetrahydrofuran is 1%.

5. A method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction according to claim 1, characterized in that, In the UHPLC-MS / MS method, the scanning mode of the mass spectrometry is the multiple reaction monitoring mode, and mass spectrometry analysis is carried out in two modes of ESI+ and ESI- respectively.

6. A method for simultaneously determining low-content active chemical components in Fangji Huangqi Decoction according to claim 5, characterized in that, The conditions of the mass spectrometry are as follows: Ion source temperature: 500 °C; Q1 / Q3 (m / z): 285.1 / 269.9；269.1 / 254.1；267.0 / 252.1；233.2 / 159.1； Declustering voltage: 7.41; -135.07; -122.47; 136.43 V; Collision energy: 30.92; -22.98; -27.12; 28.60 eV; Nebulizing gas: 60 psi; Auxiliary heating gas: 60 psi; Curtain gas: 40 psi; Nitrogen is used as the spray assist gas.

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