Method for determining four main monoterpene glycoside compounds in radix paeoniae rubra based on HPLC (High Performance Liquid Chromatography)
Through the gradient elution procedure and specific chromatography conditions of high performance liquid chromatography, the problem of HPLC for too long determination of active ingredients in red peony root is solved, and the four monoterpene glycoside compounds in red peony root extract were achieved quickly. Accurate inspection ensures the reliability and precision of the test results.
Patent Information
- Application Number
- CN202411691256.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-01-10
AI Technical Summary
The existing HPLC method has been used to determine the active ingredients in red peony root for too long, making it difficult to efficiently test the content of four major monoterpene glycoside compounds in red peony root extract at the same time.
High performance liquid chromatography is used to prepare standard solution and sample solution to be tested, combining gradient elution procedure and specific chromatographic conditions, including mobile phase 0.2% formic acid water and acetonitrile, and elution procedure is 0-5min and 15%B ; 5-8min, 15-30%B; 8-15min, 30-50%B; 15-18min, 80%B; 18-20min, 80%B, using an Agilent Extend-C18 chromatography column, the detection wavelength is 245nm, The flow rate is 1 mL/min, the column temperature is 30°C, and the injection volume is 10 μL, so as to achieve the separation and determination of oxidized peonylin, peonylin, peonylin and benzoyl peonylin.
The rapid and accurate detection of four major monoterpene glycoside compounds in Paeonia lactobacillus was achieved, ensuring the reliability and precision of the detection results, and meeting the requirements of linearity, stability and repeatability.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of traditional Chinese medicine analysis, and in particular relates to a method for determining four main monoterpene glycoside compounds in red peony root based on HPLC. Background Art
[0002] As a traditional Chinese medicine with a long history, red peony root has been applied in a variety of industries, such as brewing and cosmetics, from medicine to winemaking and cosmetics, with the development of the times. It has broad development prospects. The main medicinal ingredients in red peony root are monoterpene glycosides, and the main ingredients of monoterpene glycosides are paeoniflorin, paeoniflorin lactone glycosides, oxidized paeoniflorin, and benzoylpeoniflorin. Paeoniflorin has good anti-inflammatory, immunomodulatory and anti-tumor effects. Paeoniflorin lactone glycosides have the effect of calming the liver and relieving depression. Oxidized paeoniflorin can prevent acute lung injury in mice caused by lipopolysaccharide. Benzoylpeoniflorin has anti-allergic effects and is a potential candidate drug for the treatment of allergic diseases.
[0003] High performance liquid chromatography is a common separation and analysis technology, which mainly uses two liquids of different polarities as mobile phases. When the sample enters the chromatographic column, the mobile phase elutes the mixture sample. Since the polarities of each substance in the mixture sample are different, the elution time is also different, thereby achieving the purpose of separating and testing the mixture sample.
[0004] The existing literature "HPLC method for simultaneous determination of the contents of 9 active ingredients in red peony root" established a method for simultaneous determination of 9 active ingredients (peonidin, oxidized paeonidin, paeoniflorin, benzoylpeonidin, benzoic acid, catechin, paeonol, gallic acid, 1,2,3,4,6-O-galloylglucose) in red peony root extract by high performance liquid chromatography (HPLC) ("HPLC method for simultaneous determination of the contents of 9 active ingredients in red peony root", Wang Hua et al., Medical and Health Science and Technology). However, this method has the problem of too long determination time.
[0005] Therefore, there is an urgent need for a method for simultaneously detecting four monoterpene glycosides, namely, paeoniflorin, paeoniflorin lactone glycosides, oxidized paeoniflorin, and benzoylpaeoniflorin, in red peony root extract using HPLC to solve the above problems. Summary of the invention
[0006] In order to solve the above technical problems, the present invention proposes a method for determining four main monoterpene glycosides in red peony root based on HPLC.
[0007] To achieve the above object, the present invention provides the following technical solutions:
[0008] The present invention provides a method for determining four main monoterpene glycoside compounds in red peony root based on HPLC, comprising the following steps:
[0009] Prepare standard solutions of four main monoterpene glycosides in red peony root; prepare sample solutions to be tested; perform high performance liquid chromatography on the standard solutions and the sample solutions to be tested; draw a standard curve using the test results of the standard solutions; calculate the contents of the four main monoterpene glycosides in the sample to be tested based on the test results of the sample solutions to be tested and the standard curve;
[0010] Wherein, the chromatographic conditions of the high performance liquid chromatography detection are:
[0011] Mobile phase: 0.2% formic acid water A and acetonitrile B;
[0012] Elution program: gradient elution, 0-5 min, 15% B; 5-8 min, 15-30% B; 8-15 min, 30-50% B; 15-18 min, 80% B; 18-20 min, 80% B.
[0013] Preferably, the four main monoterpene glycosides are oxidized paeoniflorin, paeoniflorin, paeoniflorin and benzoylpaeoniflorin.
[0014] Preferably, the preparation of standard solutions of four main monoterpene glycosides in red peony root comprises the following steps: dissolving oxidized paeoniflorin standard, paeoniflorin lactone standard, paeoniflorin standard and benzoylpaeoniflorin standard with methanol to obtain 1 mg / mL oxidized paeoniflorin standard solution, 2 mg / mL paeoniflorin lactone standard solution, 3 mg / mL paeoniflorin standard solution and 1 mg / mL benzoylpaeoniflorin standard solution, respectively.
[0015] Preferably, the preparation of the test sample solution comprises the following steps: mixing red peony root powder and methanol solution, performing ultrasonic extraction and centrifugation to obtain a first supernatant; mixing the residue obtained by the centrifugation with the methanol solution, repeating the ultrasonic extraction and centrifugation to obtain a second supernatant; evaporating the first supernatant, and then adding the second supernatant to continue evaporation to obtain a test sample; redissolving the test sample in a methanol solution, performing ultrasonic extraction, centrifugation, re-centrifugation and filtering to obtain a test sample solution.
[0016] Preferably, the preparation method of the red peony root powder is: crushing and sieving the red peony root to obtain the red peony root powder.
[0017] Preferably, the dosage ratio of the red peony root powder and the methanol solution is 0.1 g:5 mL.
[0018] Preferably, the volume concentration of the methanol solution is 70%.
[0019] Preferably, the temperature of the ultrasonic extraction is 40°C and the time is 45 minutes.
[0020] Preferably, the pore size of the filter membrane used for filtration is 0.22 μm.
[0021] Preferably, the chromatographic column for HPLC detection is an Agilent Extend-C18 (4.6 mm×250 mm, 5 μm) chromatographic column, the detection wavelength is 245 nm, the flow rate is 1 mL / min, the column temperature is 30° C., and the injection volume is 10 μL.
[0022] Compared with the prior art, the present invention has the following advantages and technical effects:
[0023] The present invention adopts high performance liquid chromatography to determine four main monoterpene glycosides in red peony root. By controlling the chromatographic conditions of high performance liquid chromatography detection, the four main monoterpene glycosides, paeoniflorin, paeoniflorin, oxidized paeoniflorin, and benzoylpaeoniflorin, show a good linear relationship within their respective concentration ranges, and the precision, stability, repeatability, and spike recovery rate all meet the requirements. The method provided by the present invention is reliable and accurate, and can be used for the detection of four target compounds in red peony root. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:
[0025] Figure 1 The structural formulas of the four main monoterpenoid glycosides in red peony root are shown;
[0026] Figure 2 The chromatograms are of the standard solution (A) and the sample solution (B) obtained after detection using the method in Example 1;
[0027] Figure 3 The chromatograms are of the standard solution and the sample solution obtained after detection using the method in Comparative Example 1-2. DETAILED DESCRIPTION
[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.
[0030] The embodiment of the present invention provides a method for determining four main monoterpene glycosides in red peony root based on HPLC, comprising the following steps:
[0031] Prepare standard solutions of four main monoterpene glycosides in red peony root; prepare sample solutions to be tested; perform high performance liquid chromatography on the standard solutions and the sample solutions to be tested; draw a standard curve using the test results of the standard solutions; calculate the contents of the four main monoterpene glycosides in the sample to be tested based on the test results of the sample solutions to be tested and the standard curve;
[0032] Wherein, the chromatographic conditions of the high performance liquid chromatography detection are:
[0033] Mobile phase: 0.2% formic acid water A and acetonitrile B;
[0034] Elution program: gradient elution, 0-5 min, 15% B; 5-8 min, 15-30% B; 8-15 min, 30-50% B; 15-18 min, 80% B; 18-20 min, 80% B.
[0035] The present invention adopts high performance liquid chromatography to determine four main monoterpene glycoside compounds in red peony root, and establishes a reliable and accurate method for detecting four target compounds in red peony root by controlling the chromatographic conditions of high performance liquid chromatography detection. The present invention adopts the above-mentioned mobile phase and elution procedure, and the four target chemicals can be eluted out at different times, so as to achieve the purpose of separating and detecting the four main monoterpene glycoside compounds in red peony root, and at the same time avoids interference between the four target compounds, thereby improving the reliability and accuracy of the detection result.
[0036] In a preferred embodiment, the four main monoterpene glycosides are oxidized paeoniflorin, paeoniflorin, paeoniflorin and benzoylpaeoniflorin.
[0037] In a preferred embodiment, the preparation of standard solutions of four main monoterpene glycosides in red peony root comprises the following steps: dissolving oxidized paeoniflorin standard, paeoniflorin lactone standard, paeoniflorin standard and benzoylpeoniflorin standard with methanol to obtain 1 mg / mL oxidized paeoniflorin standard solution, 2 mg / mL paeoniflorin lactone standard solution, 3 mg / mL paeoniflorin standard solution and 1 mg / mL benzoylpeoniflorin standard solution, respectively.
[0038] In a preferred embodiment, the preparation of the test sample solution comprises the following steps: mixing red peony root powder and methanol solution, performing ultrasonic extraction and centrifugation to obtain a first supernatant; mixing the residue obtained by the centrifugation with methanol solution, repeating the ultrasonic extraction and centrifugation to obtain a second supernatant; evaporating the first supernatant, then adding the second supernatant to continue evaporating to obtain a test sample; redissolving the test sample in a methanol solution, ultrasonically, centrifuging, re-centrifuging and filtering to obtain a test sample solution. The present invention prepares a test sample solution by adopting the above method, which has the advantages of fully dissolving the compound in a solvent and having few impurities, and is conducive to improving the accuracy of the test results.
[0039] In a preferred embodiment, the dosage ratio of the red peony root powder to the methanol solution is 0.1 g:5 mL; and the volume concentration of the methanol solution is 70%.
[0040] In a preferred embodiment, the preparation method of the red peony root powder is: crushing and sieving the red peony root to obtain the red peony root powder; the red peony root comes from the Institute of Natural Ecology of Heilongjiang Academy of Sciences of China; the crushing equipment is a grinder; the mesh size of the sieve used for sieving is 60 mesh.
[0041] In a preferred embodiment, the temperature of the ultrasonic extraction is 40° C. and the time is 45 min. The ultrasonic extraction of the present invention under the above conditions can achieve sufficient extraction of the target compound in the sample.
[0042] In a preferred embodiment, the pore size of the filter membrane used for filtration is 0.22 μm. The present invention uses the filter membrane with the above pore size for filtration, thereby avoiding the interference of impurities on the detection results and reducing the loss of samples.
[0043] In a preferred embodiment, the chromatographic column for the HPLC detection is an Agilent Extend-C18 (4.6 mm×250 mm, 5 μm) chromatographic column, the detection wavelength is 245 nm, the flow rate is 1 mL / min, the column temperature is 30° C., and the injection volume is 10 μL. The HPLC detection of the present invention under the above conditions is conducive to the smooth elution of the four target chemicals, ensuring the reliability and accuracy of the detection results.
[0044] The room temperature in the embodiments of the present invention refers to "25±2°C".
[0045] In the following examples, oxidized paeoniflorin standard (purity ≥ 98%), paeoniflorin standard (purity ≥ 98%), paeoniflorin standard (purity ≥ 98%), and benzoylpeoniflorin standard (purity ≥ 98%) were all purchased from Chengdu Munster Biotechnology Co., Ltd. Acetonitrile, methanol, and formic acid (analytical grade) were all purchased from Beijing Bailingwei Company.
[0046] Example 1
[0047] A method for determining four main monoterpene glycosides in red peony root based on HPLC:
[0048] (1) Weigh 10 mg of oxidized paeoniflorin standard, 20 mg of paeoniflorin standard, 30 mg of paeoniflorin standard and 10 mg of benzoylpaeoniflorin standard, place them in a 10 mL volumetric flask, and then add methanol to dissolve them to obtain 1 mg / mL oxidized paeoniflorin standard solution, 2 mg / mL paeoniflorin standard solution, 3 mg / mL paeoniflorin standard solution and 1 mg / mL benzoylpaeoniflorin standard solution, respectively;
[0049] (2) The root of red peony root was crushed by a grinder, and then sieved with a 60-mesh sieve to obtain red peony powder; 0.1000 g of red peony powder was placed in a 10 mL centrifuge tube, 5 mL of a methanol solution with a volume concentration of 70% was added, and then ultrasonic extraction was performed at 40° C. for 45 minutes, and then centrifuged for 10 minutes to obtain a first supernatant; the residue obtained by centrifugation was mixed with 5 mL of a methanol solution with a volume concentration of 70%, and ultrasonic extraction was performed again at 40° C. for 45 minutes, and centrifuged for 15 minutes to obtain a second supernatant; the first supernatant was evaporated by a vacuum centrifugal concentrator, and then the second supernatant was added and evaporated to obtain a sample to be tested; the sample to be tested was redissolved in 1 mL of a methanol solution with a volume concentration of 70%, and then ultrasonicated, and then centrifuged for 15 minutes to obtain a third supernatant, the third supernatant was centrifuged again for 10 minutes, and then the supernatant obtained by the second centrifugation was filtered with a 0.22 μm filter membrane to obtain a sample solution to be tested;
[0050] (3) performing high performance liquid chromatography on the standard solution obtained in step (1) and the sample solution obtained in step (2);
[0051] The chromatographic conditions for HPLC detection were:
[0052] Mobile phase: 0.2% formic acid water A and acetonitrile B; chromatographic column: Agilent Extend-C18 (4.6mm×250mm, 5μm) chromatographic column; detection wavelength: 245nm; flow rate: 1mL / min; column temperature: 30℃; injection volume: 10μL; elution program is shown in Table 1.
[0053] Table 1 Elution procedure of Example 1
[0054] Time (min) Mobile phase A (%) Mobile phase B (%) 0 85 15 5 85 15 8 70 30 15 50 50 18 20 80 20 20 80
[0055] Figure 1 The following are the structural formulas of the four main monoterpenoid glycosides in red peony root. A is oxidized paeoniflorin, B is paeoniflorin lactone, C is paeoniflorin, and D is benzoylpaeoniflorin.
[0056] Test results:
[0057] 1. Chromatography of sample solution and standard solution
[0058] The chromatograms of the standard solution (A) and the sample solution (B) are shown in Figure 2 As shown. Figure 2 It can be seen that the method of Example 1 of the present invention can simultaneously determine the four target compounds of oxidized paeoniflorin, paeoniflorin, paeoniflorin, and benzoylpeoniflorin in the sample solution to be tested and the standard solution, and they do not interfere with each other during the detection process. The chromatographic peak times of the four target compounds in the sample solution to be tested and the standard solution also correspond one to one, indicating that the liquid chromatography conditions in the present invention can be used to detect the four target compounds in the red peony root extract.
[0059] 2. The content of four target compounds in the sample solution to be tested
[0060] The test results of the standard solution are used to draw a standard curve; the contents of the four main monoterpene glycoside compounds in the sample to be tested are calculated according to the test results of the sample solution to be tested and the standard curve.
[0061] The calculation formula for the target compound content is:
[0062]
[0063] Where: Q is the target compound content, mg / g; K is the dilution factor; C is the mass concentration of the sample solution to be tested, mg / L; V is the volume of the extract, L; M is the mass of the sample to be tested, g.
[0064] The contents of the four target compounds in the sample solution were measured to be: 0.86 mg of oxidized paeoniflorin, 9.95 mg of paeoniflorin lactone glycoside, 33.00 mg of paeoniflorin, and 0.48 mg of oxidized paeoniflorin.
[0065] 3. Linear Relationship
[0066] The four standard solutions in step (1) of Example 1 were diluted in half with methanol solution to obtain standard solutions with concentrations of 3 mg / mL, 2 mg / mL, 1.5 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL. The standard solutions of different concentrations were measured according to the chromatographic conditions in step (3) of Example 1, and then the peak area was used as the ordinate (Y) and the mass concentration of the standard was used as the abscissa (X) to obtain the linear regression equations of the four target compounds of oxidized paeoniflorin, paeoniflorin, paeoniflorin, and benzoylpaeoniflorin, as shown in Table 2.
[0067] Table 2 Linear regression equations of target compounds
[0068] Element Linear regression equation <![CDATA[R 2 ]]> <![CDATA[Linear range (mg·mL -1 )]]> Oxidized paeoniflorin Y=8025.7X+108.38 0.9991 0.015625～1 Paeoniflorin Y=8260.2X+158.31 0.9992 0.015625～2 Paeoniflorin Y=4596.2X+100.12 0.9994 0.015625～3 Benzoylpaeoniflorin Y=11440X+236.54 0.9991 0.015625～1
[0069] 4. Precision experiment
[0070] Take 10 μL of the standard solution in step (1) of Example 1, and perform the measurement according to the chromatographic conditions in step (3) of Example 1. Repeat the injection 6 times to obtain the peak areas and elution times of the four target compounds in the standard solution, and calculate the relative standard deviation (RSD). The results are shown in Table 3.
[0071] The calculation formula of RSD is:
[0072]
[0073] Table 3 Precision test results
[0074]
[0075]
[0076] Note: RT is the peak time; PA is the peak area; AVG is the average value.
[0077] It can be seen from Table 3 that the RSDs of the four target compounds are all less than 2%, indicating that the detection method provided by the present invention has good precision performance.
[0078] 5. Stability test
[0079] The sample solution to be tested in step (2) of Example 1 was tested at 0 h, 2 h, 4 h, 8 h, 16 h, and 24 h after preparation according to the chromatographic conditions in step (3) of Example 1. The peak areas and elution times of the four target compounds in the sample solution to be tested at different times were measured, and the RSD was calculated. The results are shown in Table 4.
[0080] Table 4 Stability test results
[0081]
[0082]
[0083] Note: RT is the peak time; PA is the peak area; AVG is the average value.
[0084] As can be seen from Table 4, the RSDs of the four target compounds in the sample solutions to be tested were all less than 2%, indicating that the red peony root sample solution was relatively stable within 24 hours.
[0085] 6. Repeatability Experiment
[0086] Take the same batch of red peony root powder as in Example 1, and prepare 6 identical sample solutions to be tested by the method in step (2) of Example 1, then test the samples according to the chromatographic conditions in step (3) of Example 1, measure the peak areas and elution times of the four target compounds in the sample solutions to be tested, and calculate the RSD. The results are shown in Table 5.
[0087] Table 5 Repeatability test results
[0088]
[0089] Note: RT is the peak time; PA is the peak area; AVG is the average value.
[0090] It can be seen from Table 5 that the RSDs of the four target compounds in the six sample solutions to be tested are all less than 2%, indicating that the detection method provided by the present invention has good repeatability in measuring the red peony root sample solution and meets the requirements for measuring the sample solution.
[0091] 8. Spike recovery experiment
[0092] A standard solution of known concentration was added to the sample solution with known component content according to the addition amount in Table 6, and then the test sample solution was prepared according to the method of step (2) of Example 1. Then, the sample was tested according to the chromatographic conditions in step (3) of Example 1, and the spiked recoveries of the four target compounds in the test sample solution were determined, and the RSD was calculated. The results are shown in Table 6.
[0093] Table 6 Spiked recovery experimental results
[0094]
[0095] It can be seen from Table 6 that the RSDs of the four target compounds in the test sample solutions are all less than 2%, indicating that the detection results of the detection method provided by the present invention for the four target compounds are very accurate and reliable, and meet the requirements for the determination of the sample solution.
[0096] Comparative Example 1
[0097] The difference from Example 1 is that the mobile phase in step (3) is: 0.1% phosphoric acid aqueous solution A and acetonitrile B.
[0098] Comparative Example 2
[0099] The difference from Example 1 is that in step (3), the chromatographic conditions of the HPLC detection are as follows: the chromatographic column is an Agilent Extend-C18 (4.6 mm×250 mm, 5 μm) chromatographic column, the detection wavelength is 254 nm, the flow rate is 1 mL / min, the column temperature is 30° C., and the injection volume is 10 μL. Mobile phase: 0.2% formic acid water A and acetonitrile B; elution program: gradient elution, 0-10 min, 10% B; 10-25 min, 10-30% B; 25-40 min, 30-50% B; 40-50 min, 50%-80% B; 50-60 min, 80% B.
[0100] The chromatograms of the sample solution and the standard solution obtained after detection by the method in Comparative Example 1-2 are shown in Figure 3 , wherein Figure A is the chromatogram of the four target compounds in the standard solution of Comparative Example 1, Figure B is the chromatogram of the four target compounds in the standard solution of Comparative Example 2, and Figure C is the chromatogram of the four target compounds in the sample solution of Comparative Example 2. Figure 3 It can be seen that the use of 0.1% phosphoric acid aqueous solution as the mobile phase in Comparative Example 1 will cause the four target compounds to elute quickly, affecting the accuracy and reliability of the detection results. The use of a different elution process in Comparative Example 2 will result in an elution time that is too long. At the same time, there are certain differences in the peak areas detected by the four target compounds at different wavelengths.
[0101] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.
Claims
1. A method for determining four main monoterpene glycosides in red peony root based on HPLC, characterized in that: The following steps are involved: Prepare standard solutions of four main monoterpenoid glycosides in red peony root; prepare sample solutions to be tested; Performing high performance liquid chromatography on the standard solution and the sample solution to be tested; drawing a standard curve using the test results of the standard solution; The contents of four main monoterpene glycosides in the sample to be tested are calculated based on the test results of the sample solution to be tested and the standard curve; Wherein, the chromatographic conditions of the high performance liquid chromatography detection are: Mobile phase: 0.2% formic acid water A and acetonitrile B; Elution program: gradient elution, 0-5 min, 15% B; 5-8 min, 15-30% B; 8-15 min, 30-50% B; 15-18 min, 80% B; 18-20 min, 80% B.
2. The method for determining four main monoterpene glycosides in red peony root based on HPLC according to claim 1, characterized in that: The four main monoterpene glycosides are oxidized paeoniflorin, paeoniflorin, paeoniflorin and benzoylpaeoniflorin.
3. The method for determining four main monoterpene glycosides in red peony root based on HPLC according to claim 2, characterized in that: The preparation method comprises the following steps: dissolving an oxidized paeoniflorin standard, a paeoniflorin lactone glycoside standard, a paeoniflorin standard and a benzoylpaeoniflorin standard in methanol to obtain a 1 mg / mL oxidized paeoniflorin standard solution, a 2 mg / mL paeoniflorin lactone glycoside standard solution, a 3 mg / mL paeoniflorin standard solution and a 1 mg / mL benzoylpaeoniflorin standard solution, respectively.
4. The method for determining four main monoterpene glycosides in red peony root based on HPLC according to claim 1, characterized in that: The preparation of the sample solution to be tested comprises the following steps: mixing red peony root powder and a methanol solution, performing ultrasonic extraction and centrifugation to obtain a first supernatant; mixing the residue obtained by the centrifugation with a methanol solution, repeating the ultrasonic extraction and centrifugation to obtain a second supernatant; evaporating the first supernatant, then adding the second supernatant and continuing to evaporate to obtain a sample to be tested; and redissolving the sample to be tested in a methanol solution, performing ultrasonic extraction, centrifugation, re-centrifugation and filtering to obtain a sample solution to be tested.
5. The method for determining four main monoterpene glycosides in red peony root based on HPLC according to claim 4, characterized in that: The preparation method of the red peony root powder is as follows: crushing and sieving the red peony root to obtain the red peony root powder.
6. The method for determining four main monoterpene glycosides in red peony root based on HPLC according to claim 4, characterized in that: The dosage ratio of the red peony root powder and the methanol solution is 0.1g:5mL.
7. The method for determining four main monoterpene glycosides in red peony root based on HPLC according to claim 4 or 6, characterized in that: The volume concentration of the methanol solution is 70%.
8. The method for determining four main monoterpene glycosides in red peony root based on HPLC according to claim 4, characterized in that: The temperature of the ultrasonic extraction is 40° C. and the time is 45 min.
9. The method for determining four main monoterpene glycosides in red peony root based on HPLC according to claim 4, characterized in that: The pore size of the filter membrane used for the filtration is 0.22 μm.
10. The method for determining four main monoterpene glycosides in red peony root based on HPLC according to claim 1, characterized in that: The chromatographic column for the HPLC detection was an Agilent Extend-C18 (4.6 mm×250 mm, 5 μm) chromatographic column, the detection wavelength was 245 nm, the flow rate was 1 mL / min, the column temperature was 30° C., and the injection volume was 10 μL.