Method for determining content of five flavonoid components in hypericum perforatum medicinal material

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a method for determining the content of five flavonoid components in a hypericum perforatum medicinal material. The content determination method comprises the following steps: preparing rutin, hyperoside, isoquercitrin, quercitrin and quercetin into a mixed reference solution; extracting a hypericum perforatum medicinal material by using an extraction solvent to prepare a test solution; the mixed reference substance solutions and the test solution with different volumes are subjected to high performance liquid chromatography respectively, a mobile phase A is acetonitrile, a mobile phase B is a phosphoric acid aqueous solution with the volume concentration of 0.08-0.12%, and elution gradient is adopted. The content determination method has the advantages of high sensitivity, simplicity and convenience in operation, low cost and high detection speed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine analysis, in particular to a content determination method of five flavonoid components in Hypericum perforatum medicinal materials. BACKGROUND

[0002] Hypericum perforatum is the dry aboveground part of Hypericum perforatum L. of Guttiferae, which is harvested in the flowering season of summer and autumn, and is prepared by drying or low-temperature drying. Hypericum perforatum has the characteristics of light odor and slightly bitter and astringent taste, and is pungent and cold in nature, and belongs to the liver meridian. It has the effects of soothing the liver and relieving depression, clearing heat and reducing dampness, and reducing swelling and promoting lactation. It is used in traditional Chinese medicine to treat liver qi stagnation, emotional incoordination, chest depression, joint swelling and pain, and mastitis and less milk.

[0003] The chemical composition of Hypericum perforatum mainly includes naphthodianthrones, flavonoids and phloroglucinols. Pharmacological studies have shown that Hypericum perforatum has therapeutic effects on various mental and emotional disorders (such as post-traumatic stress disorder, obsessive-compulsive disorder, anxiety, depression, etc.), which are closely related to the rich flavonoids contained therein. The main flavonoids in Hypericum perforatum are rutin, hyperoside, isoquercitrin, quercitrin and quercetin. Simultaneous detection of these components is beneficial to better tracking of the quality and quantity transmission changes in the production process, and can comprehensively control the quality of Hypericum perforatum medicinal materials and preparations. SUMMARY

[0004] Therefore, one or more embodiments of the present application provide a content determination method which can simultaneously detect the contents of five flavonoid components (rutin, hyperoside, isoquercitrin, quercitrin and quercetin) in Hypericum perforatum medicinal materials, and has the advantages of high sensitivity and fast detection speed.

[0005] The content determination method of the five flavonoid components in Hypericum perforatum medicinal materials of the present application comprises the following steps:

[0006] Mix the standard samples of rutin, hyperoside, isoquercitrin, quercitrin and quercetin, dissolve them with a solvent to obtain a mother liquor; and the mother liquor is diluted step by step to prepare mixed control sample solutions with different concentrations;

[0007] Extracting Hypericum perforatum medicinal materials with an extraction solvent to prepare a test sample solution;

[0008] Performing high performance liquid chromatography analysis on the mixed control sample solutions with different concentrations to establish a concentration and peak area standard curve of rutin, hyperoside, isoquercitrin, quercitrin and quercetin;

[0009] The test product solution is subjected to high performance liquid chromatography analysis to determine the concentrations of rutin, hyperoside, isoquercitrin, quercitrin and quercetin in the test product solution;

[0010] The conditions of the high performance liquid chromatography analysis include gradient elution with acetonitrile as mobile phase A and 0.08-0.12% phosphoric acid aqueous solution by volume concentration as mobile phase B;

[0011] The program of the gradient elution includes:

[0012] 0-25 min, the volume percentage of the mobile phase A is maintained at 14%,

[0013] 25-45 min, the volume percentage of the mobile phase A is increased from 14% to 30%,

[0014] 45-46 min, the volume percentage of the mobile phase A is decreased from 30% to 14%,

[0015] 46-56 min, the volume percentage of the mobile phase A is maintained at 14%.

[0016] In some embodiments, the extraction is performed by ultrasonic extraction.

[0017] In some embodiments, the extraction solvent includes an aqueous solution of an alcohol by volume percentage of 50-70%, and optionally, the alcohol is an organic alcohol; and / or,

[0018] The weight to volume ratio of the Hypericum perforatum medicinal material and the extraction solvent is 0.6-1 g: 100 mL.

[0019] In some embodiments, the conditions of the high performance liquid chromatography analysis further include that the chromatographic column uses octadecylsilane-bonded silica gel as the filler.

[0020] In some embodiments, the column length of the chromatographic column is 240-260 mm, the inner diameter is 4.4-4.8 mm, and the particle size of the filler is 4-6 μm.

[0021] In some embodiments, the conditions of the high performance liquid chromatography analysis further include that the detection wavelength is 350-370 nm.

[0022] In some embodiments, the conditions of the high performance liquid chromatography analysis further include that the column temperature is 30-40℃.

[0023] In some embodiments, the conditions of the high performance liquid chromatography analysis further include that the flow rate is 0.6-1 mL / min.

[0024] In some embodiments, the conditions of the high performance liquid chromatography analysis further include that the injection volume of the test product solution is 5-20 μL.

[0025] In some embodiments, the mass concentrations of rutin, hyperoside, isoquercitrin, quercitrin and quercetin in the mixed control solution are independently 0.5-160 μg / mL.

[0026] The content determination method of the present application can simultaneously detect the contents of rutin, hyperoside, isoquercitrin, quercitrin and quercetin in Hypericum perforatum medicinal materials, without changing the detection wavelength or chromatographic conditions, and is simple, easy to operate and high in detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0028] Figure 1 The chromatogram of the control solution mixed solution of Example 1 of the present application (injection amount 10 μL).

[0029] Figure 2 The chromatogram of the blank solution of the negative solution of Example 1 of the present application.

[0030] Figure 3 The chromatogram of the sample solution of Example 1 of the present application.

[0031] Figure 4 The comparison results of the chromatograms detected under different mobile phase conditions of Comparative Example 1 of the present application.

[0032] Figure 5 The comparison results of the chromatograms detected under different elution program conditions of Comparative Example 2 of the present application (elution programs 1-4).

[0033] Figure 6 The comparison results of the chromatograms detected under different elution program conditions of Comparative Example 2 of the present application (elution programs 5-7).

[0034] Figure 7 The comparison results of the chromatograms detected under different flow rate conditions of Comparative Example 3 of the present application.

[0035] Figure 8 The comparison results of the chromatograms detected under different injection amount conditions of Comparative Example 4 of the present application.

[0036] Figure 9 The comparison results of the chromatograms detected under different column temperature conditions of Comparative Example 5 of the present application. DETAILED DESCRIPTION

[0037] The application will be further described below in connection with embodiments and examples. It should be understood that these examples are only used to illustrate the application and not intended to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content taught in the application, and these equivalent forms also fall within the protection scope of the claims of the application.

[0038] 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. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0039] The method for determining the content of five flavonoid components in Hypericum perforatum medicinal material of the application comprises the following steps:

[0040] Mixing standard samples of rutin, hyperoside, isoquercitrin, quercitrin and quercetin, dissolving with a solvent to obtain a mother liquor; diluting the mother liquor step by step to prepare mixed control sample solutions with different concentrations;

[0041] Extracting Hypericum perforatum medicinal material with an extraction solvent to prepare a test sample solution;

[0042] Performing high performance liquid chromatography analysis on the mixed control sample solutions with different concentrations to prepare concentration and peak area standard curves of rutin, hyperoside, isoquercitrin, quercitrin and quercetin;

[0043] Performing high performance liquid chromatography analysis on the test sample solution to determine the concentrations of rutin, hyperoside, isoquercitrin, quercitrin and quercetin in the test sample solution;

[0044] The conditions of the high performance liquid chromatography analysis include: gradient elution with acetonitrile as mobile phase A and 0.08-0.12% phosphoric acid aqueous solution by volume concentration as mobile phase B;

[0045] The program of the gradient elution includes:

[0046] 0-25min, the volume percentage of the mobile phase A is maintained at 14%,

[0047] 25-45min, the volume percentage of the mobile phase A is increased from 14% to 30%,

[0048] 45-46min, the volume percentage of the mobile phase A is decreased from 30% to 14%,

[0049] 46-56min, the volume percentage of the mobile phase A is maintained at 14%.

[0050] In some embodiments, the extraction method comprises ultrasonic extraction.

[0051] In some embodiments, the extraction solvent comprises an aqueous solution of an alcohol at a volume percentage of 50%-70%, and the alcohol is optionally an organic alcohol; and / or,

[0052] The weight-to-volume ratio of the Hypericum perforatum medicinal material to the extraction solvent is 0.6-1 g: 100 mL.

[0053] In some embodiments, the conditions of the high-performance liquid chromatography analysis further comprise that the chromatographic column uses octadecylsilane-bonded silica gel as the filler.

[0054] In some embodiments, the column length of the chromatographic column is 240-260 mm, the inner diameter is 4.4-4.80 mm, and the particle size of the filler is 4-6 μm.

[0055] In some embodiments, the conditions of the high-performance liquid chromatography analysis further comprise that the detection wavelength is 350-370 nm.

[0056] In some embodiments, the conditions of the high-performance liquid chromatography analysis further comprise that the column temperature is 30-40℃.

[0057] In some embodiments, the conditions of the high-performance liquid chromatography analysis further comprise that the flow rate is 0.6-1 mL / min.

[0058] In some embodiments, the conditions of the high-performance liquid chromatography analysis further comprise that the injection volume of the test sample solution is 5-20 μL.

[0059] In some embodiments, the mass concentrations of rutin, hyperoside, isoquercitrin, quercitrin, and quercetin in the mixed control solution are independently 0.5-160 μg / mL.

[0060] The following are some specific embodiments.

[0061] In the following specific embodiments, the experimental parameters not specified can be preferably referred to the guidance given in the present application file, and can also be referred to the experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturers.

[0062] In the following specific embodiments, the medicinal materials, samples, and reagents involved can be commercially available or can be prepared by those skilled in the art according to known means.

[0063] Embodiment 1

[0064] 1. Medicinal materials, samples, and instruments

[0065] Hypericum perforatum was purchased from Hebei Jiaheng Lengbei Pharmaceutical Co., Ltd.; rutin reference substance (batch number 100080-202012), hyperoside reference substance (batch number 111521-202310), isoquercetin reference substance (batch number 111809-202205), quercetin reference substance (batch number 111538-202308), and quercetin reference substance (batch number 100081-201610) were all purchased from the China Food and Drug Inspection Institutes; an analytical balance MSU205DU was used; a high-performance liquid chromatograph (Vanquish, Thermo Fisher Scientific) was used, including a diode array detector, a quaternary gradient pump, an online degasser, and an autosampler; and an As series ultrasonic cleaner was used.

[0066] 2. Sample preparation

[0067] 2.1 Preparation of mixed reference solution

[0068] Accurately weigh 10 mg each of rutin reference substance, hyperoside reference substance, isoquercetin reference substance, quercetin reference substance, and quercetin reference substance, place them in 10 mL volumetric flasks, add methanol to dissolve, and dilute to the scale to make solutions containing 1 mg per mL, which are used as stock solutions ①②③④⑤ respectively.

[0069] Accurately measure 1 mL each of stock solutions ①②③④⑤, add 5 mL of methanol solution, and prepare a mixed solution of 100 μg / mL each of rutin reference substance, hyperoside reference substance, isoquercetin reference substance, quercetin reference substance, and quercetin reference substance as the mixed reference solution.

[0070] 2.2 Preparation of test solution

[0071] Weigh about 0.4 g of Hypericum perforatum powder (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 60% ethanol, stopper it tightly, weigh it, perform ultrasonic extraction for 30 minutes, weigh it, make up the lost weight with 60% ethanol, shake well, filter, and take the filtrate to obtain the product.

[0072] 2.3 Negative solution and blank solution

[0073] Accurately add 50 mL of 60% ethanol to a stoppered conical flask, seal it tightly, weigh it, perform ultrasonic extraction for 30 minutes, weigh it again, make up the lost weight with 60% ethanol, shake well, filter, and take the filtrate to obtain the product.

[0074] 3. Chromatographic analysis

[0075] Accurately pipette the control sample stock solution into the same 10 mL volumetric flask, dilute to the mark with methanol, shake well, and dilute with methanol to prepare a mixed control sample solution containing rutin, hyperoside, quercetin, isoquercitrin, and astragalin at concentrations of 2.5-160 μg / mL, 1.25-80 μg / mL, 0.5-32 μg / mL, 1.25-8 μg / mL, and 2.5-160 μg / mL, respectively.

[0076] Table 1 Linear concentration content of 5 components in Hypericum perforatum

[0077]

[0078] According to the above chromatographic conditions, sample analysis was performed, and the chromatogram was recorded. The detection results are shown in Table 1. Figure 1 Figure 1 Peak 1-rutin, 2-hyperoside, 3-isoquercitrin, 4-quercitrin, and 5-quercetin. The standard curve was plotted with the control sample solution concentration (X, μg / mL) as the abscissa and the peak area (Y) as the ordinate. The regression equation and the correlation coefficient (r) were calculated to investigate the linear relationship of the 5 active ingredients within a certain concentration range. The results are shown in Table 6.

[0079] Table 2 Investigation results of linear relationship of 5 components in Hypericum perforatum

[0080]

[0081] Accurately pipette 10 μL of the negative solution blank solution into the liquid chromatograph for high performance liquid chromatography analysis. The chromatogram is shown in Table 1. Figure 2

[0082] Accurately pipette 10 μL of the test sample solution into the liquid chromatograph for high performance liquid chromatography analysis. The chromatogram is shown in Table 1. Figure 3 Figure 3 and Figure 1 As can be seen from the above, the peak 1-rutin, 2-hyperoside, 3-isoquercitrin, 4-quercitrin, and 5-quercetin in the test sample solution were obtained. The peak area of rutin, hyperoside, isoquercitrin, quercitrin, and quercetin in the test sample solution was measured to determine the concentration.

[0083] ​​​The conditions of high performance liquid chromatography analysis are as follows: octadecylsilane-bonded silica gel is used as filler (250 mm x 4.6 mm), the particle size of the filler is 5 μm, and the type is Hypersil Gold C18 chromatographic column; the column temperature is 35 °C; the flow rate is 0.7 mL / min, the injection amount is 10 μL, and the detection wavelength is 360 nm; acetonitrile is used as mobile phase A, and 0.1% phosphoric acid aqueous solution is used as mobile phase B for gradient elution; the gradient elution procedure is as follows: 0-25 min, the volume percentage of mobile phase A is maintained at 14%, 25-45 min, the volume percentage of mobile phase A is increased from 14% to 30%, 45-46 min, the volume percentage of mobile phase A is decreased from 30% to 14%, and 46-56 min, the volume percentage of mobile phase A is maintained at 14%.

[0084] 4. Methodology

[0085] 4.1. Specificity

[0086] Take 10 μL of the test solution, mixed control solution, negative sample solution and blank excipient solution respectively into the HPLC chromatograph, and confirm whether there is interference.

[0087] 4.2. Precision

[0088] Take the test solution, and continuously inject 6 times under the chromatographic conditions in item 3, record the peak area and calculate the relative standard deviation (RSD) of each peak area.

[0089] Table 3. Precision test results

[0090]

[0091] 4.3. Reproducibility

[0092] Take the same batch of samples, and prepare 6 test solution samples in parallel according to the method in item 2.2, and determine and record the peak area under the chromatographic conditions in item 3.

[0093] Table 4. Reproducibility test results

[0094]

[0095] 4.4. Stability

[0096] Take the same test solution, and inject 10 μL under the chromatographic conditions in item 3 at 0, 4, 8, 12, 18, 24 and 36 h respectively to determine and record the peak area of the five components, and calculate the relative standard deviation (RSD) of the peak area of each component within 36 h.

[0097] Table 5. Stability test results

[0098]

[0099] 4.5 Sample recovery rate investigation

[0100] Take 5 portions of the known sample, take about 0.4 g of each portion, accurately weigh, and place them in conical flasks respectively. Accurately add appropriate amount of reference stock solution of rutin, hyperoside, isoquercetin, quercetin, and quercetin. Prepare the test solution as described above, and inject 10 μL of each sample for determination under the chromatographic conditions of the three items, and calculate the recovery rate.

[0101] Table 6 Sample recovery

[0102]

[0103]

[0104] 4.6 Limit of Quantitation Investigation

[0105] Collect a blank baseline using the chromatographic conditions in Item 3 and measure the baseline noise. Separately, obtain the reference solution in Item 2.4 and measure the peak heights of each peak using the chromatographic conditions in Item 2.4. Record the peak heights of the five components in the reference solution when they are approximately 10 times the baseline noise.

[0106] Table 7 Limit of quantification and detection limit

[0107]

[0108] Comparative Example 1

[0109] The test solution was tested according to the chromatographic conditions under item 3, and the content was determined by using methanol-water, acetonitrile-water, and methanol-0.1% phosphoric acid water as the mobile phase. The test results were as follows: Figure 4 ,according to Figure 4 It can be seen that only by using the mobile phase under item 3 can all chromatographic peaks be detected, while some chromatographic peaks cannot be detected when other mobile phases are used.

[0110] Comparative Example 2

[0111] The test solution was tested according to the chromatographic conditions under item 3, and different elution procedures (elution procedures 1-7) were used for content determination. The test results were as follows: Figures 5-6 ,according to Figures 5-6 It can be seen that only by adopting the 3-item elution procedure can all chromatographic peaks be detected with good separation, while using other elution procedures may result in some chromatographic peaks being unable to be detected or having very poor separation, resulting in very low accuracy.

[0112] Table 8 Elution procedure 1

[0113]

[0114] Table 9 Elution procedure 2

[0115]

[0116] Table 10 Elution procedure 3

[0117]

[0118] Table 11 Elution procedure 4

[0119]

[0120] Table 12 Elution procedure 5

[0121]

[0122] Table 13 Elution procedure 6

[0123]

[0124] Table 14 Elution procedure 7

[0125]

[0126] Comparative Example 3

[0127] The test solution was detected basically according to the chromatographic conditions in the three items, and different flow rates of 0.5 mL / min, 0.6 mL / min, 0.8 mL / min, 0.9 mL / min and 1.0 mL / min were used for content determination, and the detection results were as follows Figure 7 According to the chromatographic conditions in the three items, it can be seen that the flow rates of 0.5 mL / min, 0.6 mL / min, 0.8 mL / min, 0.9 mL / min and 1.0 mL / min can detect all the chromatographic peaks, and the separation degree is good, while using other flow rates, some chromatographic peaks cannot be detected, or there is a situation of very poor separation degree, resulting in very low accuracy. Figure 7

[0128] Comparative Example 4

[0129] The test solution was detected basically according to the chromatographic conditions in the three items, and different sample injection amounts of 5 μL, 15 μL and 20 μL were used for content determination, and the detection results were as follows Figure 8 According to the chromatographic conditions in the three items, it can be seen that the sample injection amounts of 5 μL, 15 μL and 20 μL can obtain chromatographic peaks with good peak shape, and the separation degree between peaks is good, while using other sample injection amounts, some chromatographic peaks have poor peak shape, or there is a situation of very poor separation degree, resulting in very low accuracy. Figure 8

[0130] Comparative Example 5

[0131] The test solution was detected basically according to the chromatographic conditions in the three items, and the column temperature was set to 25℃, 30℃ and 40℃ respectively for content determination, and the detection results were as follows Figure 9 According to the chromatographic conditions in the three items, it can be seen that the column temperatures of 25℃, 30℃ and 40℃ can detect all the chromatographic peaks, and the separation degree is good, while using other column temperatures, some chromatographic peaks cannot be detected, or there is a situation of very poor separation degree, resulting in very low accuracy. Figure 9 ​​It can be seen that the column temperature has less influence on the chromatographic peak than other factors, but when the column temperature is 3, the chromatographic peak has the best peak shape and separation degree, and the baseline is more stable.

[0132] According to the above examples and comparative examples, it can be seen that the content determination method of the present application can be used to simultaneously determine rutin, hyperoside, isoquercitrin, quercitrin and quercetin in Hypericum perforatum L. medicinal materials, and each component has high separation degree, good peak shape and stable baseline. The content determination method has good methodological verification effect, is simple to operate, has low cost and short detection time.

[0133] The technical features of the above-described embodiments and examples can be combined in any suitable manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, but it should be understood that any combination of the technical features that does not cause contradiction is within the scope of the present disclosure.

[0134] The above-described embodiments only express several embodiments of the present application, but should not be construed as limiting the scope of the patent application. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. In addition, it should be understood that after reading the above description of the present application, those skilled in the art can make various modifications or improvements to the present application, and the equivalent forms are also within the scope of the present application. It should also be understood that those skilled in the art can obtain technical solutions based on the technical solutions provided by the present application through logical analysis, reasoning or limited experiments, which are within the scope of the appended claims of the present application. Therefore, the scope of the patent of the present application should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A method for determining the contents of five flavonoid components in Hypericum perforatum medicinal material, characterized in that: The five flavonoid components in the Hypericum perforatum medicinal material are rutin, hyperoside, isoquercetin, quercetin and quercetin; The content determination method comprises the following steps: Mixing the standard products of rutin, hyperoside, isoquercetin, quercetin and quercetin, adding a solvent to dissolve them, and obtaining a mother solution; diluting the mother solution stepwise to prepare mixed reference solutions of different concentrations; The extracting solvent is used to extract the medicinal material Hypericum perforatum to prepare a test solution; The mixed reference solution of different concentrations was subjected to high performance liquid chromatography analysis to establish standard curves of concentration and peak area of ​​rutin, hyperoside, isoquercetin, quercetin and quercetin; The test solution is subjected to high performance liquid chromatography analysis to determine the concentrations of rutin, hyperoside, isoquercetin, quercetin and quercetin in the test solution; The conditions for the high performance liquid chromatography analysis include: using acetonitrile as mobile phase A and a 0.08-0.12% volume concentration of phosphoric acid aqueous solution as mobile phase B for gradient elution; The gradient elution procedure includes: From 0 to 25 min, the volume percentage of the mobile phase A was maintained at 14%. 25-45min, the volume percentage of the mobile phase A increased from 14% to 30%, 45-46 min, the volume percentage of the mobile phase A decreased from 30% to 14%, From 46 to 56 minutes, the volume percentage of the mobile phase A was maintained at 14%.

2. The method for determining the content of the compound according to claim 1, wherein: Extraction methods include ultrasound.

3. The content determination method according to claim 1, characterized in that The extraction solvent comprises an aqueous solution of 50% to 70% alcohol by volume, and optionally, the alcohol is an organic alcohol; and / or, The weight-to-volume ratio of the Hypericum perforatum medicinal material and the extraction solvent is 0.6-1 g:100 mL.

4. The content determination method according to claim 1, characterized in that The conditions for the high performance liquid chromatography analysis also include that the chromatographic column uses octadecylsilane bonded silica gel as a filler.

5. The content determination method according to claim 4, characterized in that The column length of the chromatographic column is 240-260 mm, the inner diameter is 4.4-4.8 mm, and the particle size of the filler is 4-6 μm.

6. The method for determining the content of a compound according to any one of claims 1 to 5, wherein: The conditions for the high performance liquid chromatography analysis also include a detection wavelength of 350-370 nm.

7. The method for determining the content of a compound according to any one of claims 1 to 5, wherein: The conditions for the HPLC analysis also include a column temperature of 30-40°C.

8. The method for determining the content of a compound according to any one of claims 1 to 5, wherein: The conditions for the high performance liquid chromatography analysis also include a flow rate of 0.6-1 mL / min.

9. The method for determining the content of a compound according to any one of claims 1 to 5, wherein: The conditions for the high performance liquid chromatography analysis also include an injection volume of the test solution of 5-20 μL.

10. The method for determining the content of a compound according to any one of claims 1 to 5, wherein: The mass concentrations of rutin, hyperoside, isoquercetin, quercetin and quercetin in the mixed reference solution are independently 0.5-160 μg / mL.

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