Quality detection method of traditional Chinese medicine compound fructus choerospondiatis three-ingredient decoction

Abstract

The invention discloses a quality detection method of a traditional Chinese medicine compound fructus choerospondiatis three-ingredient decoction. Chemical components in fructus choerospondiatis, radix sophorae flavescentis and fructus chebulae in the fructus choerospondiatis three-ingredient decoction are qualitatively analyzed by a UPLC-Q-Exactive Orbitrap-MS / MS (Ultra Performance Liquid Chromatography-Quality Exactive Orbitrap-Mass Spectrometry-Mass Spectrometry) analysis method; and determining the contents of sophocarpidine, sophoridine, gallic acid, vanillic acid and rutin in the fructus choerospondiatis three-ingredient decoction by using a high performance liquid chromatography method. The UPLC-Q-Exactive Orbitrap-MS / MS analysis method has the advantages of high resolution, good quality precision, strong qualitative ability and the like, and can be used for widely and accurately detecting the chemical components in the complex traditional Chinese medicine compound; high performance liquid chromatography can provide detection results with high accuracy, high sensitivity and good repeatability, operation is simple and easy to implement, methodology verification is passed, the linear relation is good, precision, stability, repeatability, the recovery rate and the like all meet regulations, and the combination of qualitative analysis and quantitative analysis lays a foundation for establishment of the quality standard of the fructus choerospondiatis three-ingredient decoction.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a quality detection method for a traditional Chinese medicine compound Guangzao Sanwei Decoction. Background Art

[0002] Guangzao Sanwei Decoction belongs to the category of Mongolian medicine. In the work carried out by the State Administration of Traditional Chinese Medicine to promote the inheritance and innovative development of traditional Chinese medicine, it was included in the "Catalogue of Ancient Classic Prescriptions (Second Batch)". The Mongolian medicine Guangzao Sanwei Decoction has a long history of treating coronary heart disease, which can be traced back to the 19th century. This classic prescription is composed of Guangzao, bitter ginseng and Terminalia chebula. It is used in Mongolian medicine to treat "bloody heart pain" and is a classic prescription for preventing and treating coronary heart disease.

[0003] As a traditional Chinese medicine, Guangzao Sanwei Decoction needs to be extracted with appropriate solvents to make liquid preparations for clinical use. Therefore, water is used as the solvent for extraction. The main active ingredients of Guangzao Sanwei Decoction come from three medicinal materials: Guangzao, Kushen and Terminalia chebula. In order to study the quality level of Guangzao Sanwei Decoction, an efficient detection method needs to be established for qualitative and quantitative research. Summary of the invention

[0004] The purpose of the invention is to design a quality detection method for a traditional Chinese medicine compound Guangzao Sanwei Decoction.

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

[0006] A quality detection method for Guangzao Sanwei Decoction is provided. The method comprises the following steps: firstly, a UPLC-Q-Exactive Orbitrap-MS / MS analysis method is used to perform qualitative analysis on three medicinal materials, namely, Guangzao, Kushen and Terminalia chebula, in Guangzao Sanwei Decoction; and then a high performance liquid chromatography method is used to determine the contents of matrine, sophoridine, gallic acid, vanillic acid and rutin in Guangzao Sanwei Decoction.

[0007] Compared with traditional liquid chromatography and thin layer chromatography qualitative methods, the UPLC-Q-Exactive Orbitrap-MS / MS analysis method of the present invention has a wider quality detection range, higher sensitivity and peak capacity, and can perform a large-scale qualitative analysis of complex traditional Chinese medicine compounds.

[0008] Compared with quantitative methods such as ultraviolet spectrophotometry and thin-layer chromatography, the present invention has higher accuracy, sensitivity, precision and a wider linear range, and utilizes the absorption of compounds at specific wavelengths to carry out quantitative analysis of the effective ingredients matrine, sophoridine, gallic acid, vanillic acid and rutin in the Guangzao Sanwei Decoction.

[0009] As a preferred embodiment of the quality detection method of the Guangzao Sanwei Decoction of the present invention, the UPLC-Q-Exactive Orbitrap-MS / MS analysis method is used to analyze the three medicinal materials of Guangzao Sanwei Decoction, Radix Achyranthis Bidentatae, and Fructus Terminaliae, and the chromatographic column is ACQUITY UPLC BEH C 18 (2.1mm×100mm, 1.7μm), with acetonitrile containing 0.1% formic acid (mobile phase B)-water containing 0.1% formic acid (mobile phase A) as the mobile phase, flow rate of 0.3mL / min, column temperature of 30℃, injection volume of 10μL, gradient elution. UPLC-Q-Exactive-Orbitrap MS / MS high-resolution mass spectrometry was used for mass spectrometry data acquisition, and the detection mode was Full MS-dd-MS 2 , positive and negative ion modes were scanned simultaneously, the scanning range (m / z) was 100-1500, the capillary temperature was 320°C; the evaporation temperature was 350°C; the spray voltage was 3500 / 3200V (+ / -); the auxiliary gas flow rate was 10Arb; the sheath gas flow rate was 35Arb; the collision energy was 20, 40, 60, 80%; the full scan resolution was 120000.

[0010] As a preferred scheme of the quality detection method of Guangzao Sanwei Decoction described in the present invention, wherein: the gradient elution is specifically, at 0-5 minutes, the mobile phase B is 5%; at 5-20 minutes, the mobile phase B is increased from 5% to 95%; at 20-30 minutes, the mobile phase B is 95%; at 30-35 minutes, the mobile phase B is reduced from 95% to 5%; at 35-40 minutes, the mobile phase B is 5%.

[0011] As a preferred embodiment of the quality detection method of Guangzao Sanwei Decoction described in the present invention, wherein: the content of matrine, sophoridine, gallic acid, vanillic acid and rutin is detected by high performance liquid chromatography. The chromatographic column is InertsilODS-3 (4.6mm×250mm, 5μm), with water (mobile phase A)-acetonitrile (mobile phase B) containing 0.1% phosphoric acid as the mobile phase, the wavelength is 210nm, the flow rate is 0.6mL / min, the column temperature is 30°C, the injection volume is 10μL, and gradient elution is performed. At 0.01-6min, the mobile phase B is 5%; at 6-60min, the mobile phase B is increased from 5% to 17%; at 60-80min, the mobile phase B is 17%; at 80-80.01min, the mobile phase B is reduced from 17% to 5%; at 80.01-90min, the mobile phase B is 5%.

[0012] The following beneficial effects can be obtained through the above technical solution:

[0013] The present invention first uses UPLC-Q-Exactive Orbitrap-MS / MS analysis method to qualitatively analyze the chemical components of three medicinal materials, namely, jujube, bitter ginseng and terminalia chebula in Guangzao Sanwei Decoction; and then uses high performance liquid chromatography to determine the contents of matrine, sophoridine, gallic acid, vanillic acid and rutin in Guangzao Decoction. The UPLC-Q-Exactive Orbitrap-MS / MS analysis method has the advantages of high resolution, good mass accuracy, strong qualitative ability, etc., and can perform extensive and accurate detection of chemical components in complex traditional Chinese medicine compounds; high performance liquid chromatography can provide detection results with high accuracy, high sensitivity and good repeatability, and the operation is simple and easy, and has passed the methodological verification, and has a good linear relationship, and the precision, stability, repeatability and recovery rate are all in compliance with the regulations. The combination of qualitative and quantitative analysis has laid a foundation for the establishment of the quality standard of Guangzao Sanwei Decoction. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the total ion current chromatogram of Guangzao Sanwei Decoction in positive (A) and negative (B) ion modes;

[0015] Figure 2 It is a comparative chromatogram of blank solution, reference solution and test solution;

[0016] Figure 3 It is the standard curve of matrine, sophoridine, gallic acid, vanillic acid and rutin reference substances. DETAILED DESCRIPTION

[0017] The following is attached Figure 1-3 The present invention is further described in conjunction with the embodiments:

[0018] Embodiment 1:

[0019] A quality detection method for Guangzao Sanwei Decoction is disclosed. The method comprises the following steps: firstly using UPLC-Q-Exactive Orbitrap-MS / MS analysis method to perform qualitative analysis on three medicinal materials, namely, Guangzao, Kushen and Terminalia chebula in Guangzao Sanwei Decoction; and then using high performance liquid chromatography to determine the contents of matrine, sophoridine, gallic acid, vanillic acid and rutin in Guangzao Decoction.

[0020] The UPLC-Q-Exactive Orbitrap-MS / MS analysis method was used to conduct qualitative analysis on the three medicinal materials of Guangzao Sanwei Decoction, namely, Guangzao, Kushen and Terminalia chebula. Compared with the traditional HPLC and thin layer chromatography qualitative methods, the UPLC-Q-Exactive Orbitrap-MS / MS analysis method has a wider quality detection range, higher sensitivity and peak capacity, and can conduct a large-scale qualitative analysis of complex Chinese medicine compound prescriptions; including the following contents:

[0021] (1) Chromatographic and mass spectrometry conditions

[0022] The chromatographic column is ACQUITY UPLC BEH C 18 (2.1mm×100mm, 1.7μm), acetonitrile containing 0.1% formic acid (mobile phase B)-water containing 0.1% formic acid (mobile phase A) as the mobile phase, flow rate of 0.3mL / min, column temperature of 30℃, injection volume of 10μL, gradient elution, time program as shown in Table 1. UPLC-Q-Exactive-Orbitrap MS / MS high-resolution mass spectrometry was used for mass spectrometry data acquisition, detection mode was Full MS-dd-MS2, positive ion and negative ion mode were scanned simultaneously, the scanning range (m / z) was 100-1500, capillary temperature was 320℃; evaporation temperature was 350℃; spray voltage was 3500 / 3200V (+ / -); auxiliary gas flow rate was 10Arb; sheath gas flow rate was 35Arb; collision energy was 20, 40, 60, 80%; full scan resolution was 120000.

[0023] Table 1 Gradient elution table

[0024]

[0025] (2) Preparation of mixed reference solution:

[0026] Take appropriate amount of adenosine, citric acid, ellagic acid, quinic acid, gallic acid, isorhamnetin, matrine, nicotinamide, oxysophocarpine, sophoracarpine, sophoridine, caffeic acid, vanillic acid, hyperoside, rutin, chlorogenic acid and quercetin reference substances respectively, place them in a brown volumetric flask, dissolve and dilute with methanol, and make up to 25 mL to prepare a 200 μg / mL reference substance stock solution, take appropriate amount of each reference substance stock solution, and dilute it into a mixed reference substance solution of about 5 μg / mL.

[0027] (3) Preparation of test solution

[0028] According to the prescription, weigh 40g of the three medicinal materials of Guangzao Sanwei Decoction, add 12 times the amount of water, extract for 120min, extract twice, combine the filtrate, and concentrate under reduced pressure (60℃) on a rotary evaporator to 250mL. Accurately transfer 1mL of the concentrate to a 50mL volumetric flask, dilute with distilled water and adjust to the scale, shake well, and obtain the Guangzao Sanwei Decoction test solution (3.2mg / mL).

[0029] In this study, UPLC-Q-Exactive Orbitrap-MS / MS was used to collect mass spectrometric information of the water extract of the three-flavor decoction of Atractylodes lancea in positive and negative ion modes. The total ion current chromatogram is shown in Figure 1According to the self-built "Guangzao Sanwei Decoction Compounds" database and Xcalibur qualitative software, combined with the molecular ions and secondary fragment ions of the compounds, a total of 76 compounds in the traditional Chinese medicine compound Guangzao Sanwei Decoction were compared, including 23 flavonoid compounds, 8 alkaloid compounds, 6 phenolic acid compounds, 6 coumarin compounds, 4 organic acid compounds, 4 phenylpropanoid compounds, 3 amino acid compounds, 3 tannin compounds, 2 sesquiterpene lactone compounds, 1 iridoid compound, 1 phenylpeptide compound, 1 polyphenol compound, 1 nucleoside compound, 1 anthraquinone compound, 1 triterpenoid saponin compound, 1 fatty acid compound and 10 other organic compounds. See Table 2. Among them, 17 compounds were accurately identified based on the chromatographic peaks and mass spectrometry data of the reference substances, including: 4 alkaloid compounds (matrine, sophoridine, sophorocarpine and oxysophocarpine), 4 flavonoid compounds (quercetin, isorhamnetin, rutin and hyperoside), 3 phenolic acid compounds (gallic acid, vanillic acid and caffeic acid), 3 organic acid compounds (quinic acid, citric acid and ellagic acid), 1 phenylpropanoid compound (chlorogenic acid), 1 nucleoside compound (adenosine) and 1 organic compound (nicotinamide). The other 59 compounds were inferred by comparing the molecular ions and secondary fragment ions with the natural compound database.

[0030] Table 2 Chemical composition information of Guangzao Sanwei Decoction

[0031]

[0032]

[0033]

[0034] Embodiment 2 A method for determining the content of Guangzao Sanwei Decoction comprises the following contents:

[0035] High performance liquid chromatography was used to detect the contents of matrine, sophoridine, gallic acid, vanillic acid and rutin in Guangzao Sanwei Decoction. Compared with the traditional ultraviolet spectrophotometry, this step has high accuracy, high sensitivity and good repeatability. It is simple and easy to operate and can be used to determine the contents of multiple components in complex traditional Chinese medicines. The related research on content determination is as follows:

[0036] (1) Chromatographic conditions

[0037] The chromatographic column was Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm), with water (mobile phase A)-acetonitrile (mobile phase B) containing 0.1% phosphoric acid as the mobile phase, a wavelength of 210 nm, a flow rate of 0.6 mL / min, a column temperature of 30°C, an injection volume of 10 μL, and gradient elution. The time program is shown in Table 3.

[0038] Table 3 Gradient elution table

[0039]

[0040] (2) Preparation of test solution

[0041] According to the prescription, weigh 40g of the three medicinal materials of Guangzao Sanwei Decoction, add 12 times the amount of water, extract for 120min, extract twice, combine the filtrate, and concentrate under reduced pressure (60℃) on a rotary evaporator to 250mL. Accurately transfer 1mL of the concentrate to a 50mL volumetric flask, dilute with distilled water and adjust to the scale, shake well, and obtain the Guangzao Sanwei Decoction test solution (3.2mg / mL).

[0042] (3) Preparation of reference substance stock solution

[0043] Take appropriate amounts of matrine, sophoridine, gallic acid, vanillic acid and rutin reference substances, place them in brown volumetric flasks, dissolve and dilute them with methanol, and make up to 20 mL to prepare 500 μg / mL stock solutions of each reference substance.

[0044] (4) Preparation of mixed reference solution

[0045] Take an appropriate amount of each reference substance stock solution and dilute it into a mixed photo solution containing about 15 μg / mL of matrine, about 50 μg / mL of sophoridine, about 10 μg / mL of gallic acid, about 2.5 μg / mL of vanillic acid and about 5 μg / mL of rutin.

[0046] Example 3 Investigation of the linear range

[0047] A series of mixed reference solutions of matrine, sophoridine, gallic acid, vanillic acid and rutin were prepared respectively, and the samples were injected and measured according to the liquid phase method of "Claim 3". The peak area was used as the ordinate (Y) and the solution concentration (μg / mL) was used as the abscissa (X) to perform linear plotting, and the linear equations of each reference solution were obtained. The results are shown in Tables 4 and Figure 3 , indicating that the linear relationship of each reference solution is good.

[0048] Table 4 Linearity and range

[0049]

[0050] Example 4 Precision Experiment

[0051] The mixed reference solution was sampled six times continuously according to the liquid phase method of "Claim 3", and the peak area RSDs of matrine, sophoridine, gallic acid, vanillic acid and rutin were calculated to be 4.3%, 4.4%, 1.5%, 2.8% and 1.9%, respectively, indicating that the method has good instrument precision.

[0052] Example 5 Stability Test

[0053] After the test solution was placed at room temperature for 0, 4, 8, 12, 24 and 48 hours, the peak areas of matrine, sophoridine, gallic acid, vanillic acid and rutin were calculated to be 3.9%, 3.7%, 1.2%, 2.7% and 3.9%, respectively, indicating that the test solution was stable within 48 hours.

[0054] Example 6 Repeatability

[0055] Six test solutions were prepared in parallel and sampled and measured according to the liquid phase method of "Claim 3". The RSDs of matrine, sophoridine, gallic acid, vanillic acid and rutin were calculated to be 4.1%, 1.3%, 1.0%, 0.5% and 3.1%, respectively, indicating that the method had good repeatability.

[0056] Example 7 Sample recovery test

[0057] Accurately pipette the test solution with known matrine, sophoridine, gallic acid, vanillic acid and rutin content, and add a certain amount of mixed reference solution. Prepare 6 solutions in parallel and inject and measure according to the liquid phase method of "Claim 3". The results are shown in Table 5. The average sample recovery rates of matrine, sophoridine, gallic acid, vanillic acid and rutin are 96.68%, 100.57%, 105.38%, 101.29% and 98.54%, respectively, indicating that the accuracy of this method is good.

[0058] Table 5. Sample recovery test results

[0059]

[0060]

[0061] Example 8 Content Determination

[0062] Six test sample solutions were prepared in parallel and tested by the liquid phase method of "Claim 3". The results are shown in Table 6, and the average contents of matrine, sophoridine, gallic acid, vanillic acid and rutin were calculated to be 4.66 mg / g, 13.04 mg / g, 3.43 mg / g, 1.20 mg / g and 1.55 mg / g, respectively.

[0063] Table 6 Content determination results

[0064]

[0065] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various modifications to the present invention in equivalent forms all belong to the protection scope of the claims attached to this application. The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various modifications to the present invention in equivalent forms all belong to the protection scope of the claims attached to this application.

Claims

1. A quality detection method for a Chinese medicine compound Guangzao Sanwei Decoction, characterized in that: The traditional Chinese medicine compound Guangzao Sanwei Decoction is composed of three medicinal materials: Guangzao, Kushen and Terminalia chebula. The specific detection method is as follows: S1: Qualitative analysis of Fructus Achyranthis Bidentatae, Radix Sophorae Flavescentis and Fructus Terminaliae were performed using UPLC-Q-Exactive Orbitrap-MS / MS analysis method; S2: HPLC was used to determine the contents of matrine, sophoridine, gallic acid, vanillic acid and rutin in Guangzao Sanwei Decoction; The content determination method is as follows: Step 1: Chromatographic conditions The chromatographic column was Inertsil ODS-3, with water-acetonitrile containing 0.1% phosphoric acid as the mobile phase, the wavelength was 210nm, the flow rate was 0.6mL / min, the column temperature was 30℃, the injection volume was 10μL, and gradient elution was performed. Step 2: Preparation of test solution According to the prescription, weigh 40g of the three medicinal materials of Guangzao Sanwei Decoction, add 12 times the amount of water, extract for 120min, extract twice, combine the filtrate, and concentrate to 250mL under reduced pressure on a rotary evaporator; accurately transfer 1mL of the concentrate, place it in a 50mL volumetric flask, dilute it with distilled water and make up to the scale, shake well, and the Guangzao Sanwei Decoction test solution is obtained; Step 3: Preparation of reference stock solution Take appropriate amounts of matrine, sophoridine, gallic acid, vanillic acid and rutin reference substances, place them in brown volumetric flasks, dissolve and dilute them with methanol, and make up to 20 mL to prepare 500 μg / mL stock solutions of each reference substance; Step 4: Preparation of mixed reference solution Take an appropriate amount of each reference substance stock solution and dilute it into a mixed photo solution containing about 15 μg / mL of matrine, about 50 μg / mL of sophoridine, about 10 μg / mL of gallic acid, about 2.5 μg / mL of vanillic acid and about 5 μg / mL of rutin.

2. The quality detection method of the Chinese medicine compound Guangzao Sanwei Decoction according to claim 1 is characterized in that: S1 conducts qualitative analysis on the three medicinal materials of Guangzao Sanwei Decoction, i.e., 20g Guangzao, 10g Sophora flavescens, and 10g Terminalia chebula, including the following steps: Step 1: Chromatographic and mass spectrometric conditions; Step 2: Preparation of mixed reference solution: Take appropriate amount of adenosine, citric acid, ellagic acid, quinic acid, gallic acid, isorhamnetin, matrine, nicotinamide, oxysophocarpine, sophoracarpine, sophoridine, caffeic acid, vanillic acid, hyperoside, rutin, chlorogenic acid and quercetin reference substances respectively, place them in a brown volumetric flask, dissolve and dilute with methanol, and make up to 25mL to prepare a 200μg / mL reference substance stock solution, take appropriate amount of each reference substance stock solution, dilute to a mixed reference substance solution of about 5μg / mL. Step 3: Preparation of test solution: According to the prescription, weigh 40g of the three medicinal materials of Guangzao Sanwei Decoction, add 12 times the amount of water, extract for 120min, extract twice, combine the filtrate, and concentrate to 250mL under reduced pressure on a rotary evaporator. Accurately transfer 1mL of the concentrate, place it in a 50mL volumetric flask, dilute it with distilled water and make up to the scale, shake well, and you will get the Guangzao Sanwei Decoction test solution; After determining the chromatography-mass spectrometry conditions, take an appropriate amount of the mixed reference solution and the test solution, pass them through a 0.22 μm microporous filter membrane, and introduce them into the UPLC-Q-Exactive Orbitrap-MS / MS chromatography-mass spectrometry instrument for detection. The compounds in Guangzao Sanwei Decoction can be accurately identified based on the chromatographic peaks and mass spectrometry data of the reference, and other compounds can be inferred by comparing with the molecular ions and secondary fragment ions of the natural compound database.

3. The quality detection method of the Chinese medicine compound Guangzao Sanwei Decoction according to claim 1 is characterized in that: The content was measured by high performance liquid chromatography, with water-acetonitrile containing 0.1% phosphoric acid as the mobile phase, water as mobile phase A, acetonitrile as mobile phase B, the flow rate was 0.6 mL / min, the column temperature was 30°C, the injection volume was 10 μL, and gradient elution was performed. From 0.01 to 6 min, the mobile phase B was 5%; from 6 to 60 min, the mobile phase B increased from 5% to 17%; from 60 to 80 min, the mobile phase B was 17%; from 80 to 80.01 min, the mobile phase B decreased from 17% to 5%; from 80.01 to 90 min, the mobile phase B was 5%.

4. The quality detection method of the Chinese medicine compound Guangzao Sanwei Decoction according to claim 2 is characterized in that: Chromatography-mass spectrometry method, the chromatographic column is ACQUITY UPLC BEH C 18 , 2.1mm×100mm, 1.7μm, with mobile phase B containing 0.1% formic acid-mobile phase A containing 0.1% formic acid as the mobile phase, flow rate of 0.3mL / min, column temperature of 30℃, injection volume of 10μL, gradient elution; UPLC-Q-Exactive-Orbitrap MS / MS high-resolution mass spectrometry was used for mass spectrometry data acquisition, and the detection mode was Full MS-dd-MS 2 , positive and negative ion modes were scanned simultaneously, the scanning range m / z was 100~1500, the capillary temperature was 320℃; the evaporation temperature was 350℃; the spray voltage was 3500 / 3200V (+ / -); the auxiliary gas flow rate was 10Arb; the sheath gas flow rate was 35Arb; the collision energy was 20, 40, 60, 80%; the full scan resolution was 120000.

5. The quality detection method of the Chinese medicine compound Guangzao Sanwei Decoction according to claim 4 is characterized in that: The gradient elution is specifically as follows: at 0-5 min, the mobile phase B is 5%; at 5-20 min, the mobile phase B is increased from 5% to 95%; at 20-30 min, the mobile phase B is 95%; at 30-35 min, the mobile phase B is reduced from 95% to 5%; at 35-40 min, the mobile phase B is 5%.

6. The quality detection method of the Chinese medicine compound Guangzao Sanwei Decoction according to claim 1 is characterized in that: The contents of matrine, sophoridine, gallic acid, vanillic acid and rutin in Guangzao Sanwei Decoction are 4.66 mg / g, 13.04 mg / g, 3.43 mg / g, 1.20 mg / g and 1.55 mg / g, respectively.

Images