HPLC Detection Method of Paeoniflorin and Baicalin in Kunyuean

Abstract

The present invention relates to the field of liquid chromatography analysis and detection, and provides an efficient liquid phase detection method for paeoniflorin and baicalin in Kunyuean. The chromatographic conditions of the efficient liquid phase detection method are: methanol as mobile phase A, 0.08% phosphoric acid solution as mobile phase B, gradient elution; wherein the gradient elution program is as follows: 0-50min, the volume ratio occupied by mobile phase A is: 20%→80%, and the volume ratio occupied by mobile phase B is 80%→20%. The present invention realizes the simultaneous and accurate determination of the content of white peony root and scutellaria in Kunyuean granules, and the present invention provides a high-performance liquid phase method for simultaneously determining the content of paeoniflorin and baicalin in Kunyuean granules. The detection method of the present invention has good precision, stability and repeatability, improves the accuracy and efficiency of detection, and can better control product quality.

Description

Technical Field

[0001] The invention relates to the field of liquid chromatography analysis and detection, and in particular to a high-performance liquid phase detection method for paeoniflorin and baicalin in Kunyue'an. Background Art

[0002] Kunyuean Granule is an exclusive product produced by Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd. It is composed of 15 medicinal materials, including white peony root, spiny jujube seed (fried), mistletoe, Dipsacus asper, Ophiopogon japonicus, Dendrobium officinale, chrysanthemum, Tribulus terrestris, Scutellaria baicalensis (fried), Gardenia jasminoides (fried), Gentiana scabra (fried), Citrus aurantium, Curcuma aromatica, Albizzia julibrissin peel, and Luffa sponge. It has the effects of nourishing yin and blood, soothing the liver and relieving depression. It is used for menstrual dizziness, headache, breast swelling, body pain, irritability and premenstrual tension syndrome caused by blood deficiency and liver depression, yin deficiency and liver hyperactivity.

[0003] Due to the complex composition of Chinese medicine compositions, it is difficult to use the same method to measure different Chinese medicine compositions. Different Chinese medicine compositions need to establish their own unique detection methods based on the characteristics of their compositions, and strict methodological research is required before they can be used to control the content determination of the Chinese medicine compositions. The content determination of two or more components is even more difficult.

[0004] At present, the detection method of the prior art is to extract the Kunyuean granules sample by ethanol reflux, purify it by column chromatography silica gel D101 macroporous resin, and then determine the content of paeoniflorin by C18 column, methanol: 0.08% phosphoric acid (30:70) as mobile phase, and detection wavelength 230nm. The pretreatment process of this determination method is not high for the extraction rate of baicalin, the active ingredient of the prescription medicinal material Scutellaria baicalensis, and the active components determined are limited. The present invention provides a high-performance liquid phase method for simultaneously determining the content of paeoniflorin and baicalin in Kunyuean granules, improves the extraction rate of baicalin, the active ingredient of the prescription medicinal material Scutellaria baicalensis in the content determination project of Kunyuean granules, improves the accuracy and efficiency of detection, and can better control product quality. Summary of the invention

[0005] The purpose of the present invention is to provide a high-performance liquid phase detection method for paeoniflorin and baicalin in Kunyue'an, and the test solution and the reference solution are respectively injected into a high-performance liquid chromatograph for determination, characterized in that the chromatographic conditions of the high-performance liquid phase detection method are: methanol is used as mobile phase A, 0.08% phosphoric acid solution is used as mobile phase B, and the volume ratio of mobile phase A: mobile phase B is 20-80:80-20, and linear gradient elution is performed.

[0006] The chromatographic conditions also include: the detector used is a diode array detector, dual wavelength detection is adopted, the detection wavelength of paeoniflorin is 230nm, and the detection wavelength of baicalin is 280nm.

[0007] The chromatographic conditions also include: using an octadecylsilyl bonded silica gel chromatographic column, controlling the flow rate to 1 ml / min, the column temperature to 25° C., the injection volume to 10 μl, and the theoretical plate number calculated based on the paeoniflorin and baicalin peaks to be no less than 5000.

[0008] Preferably, the chromatographic conditions are: methanol as mobile phase A, 0.08% phosphoric acid solution as mobile phase B, and the linear gradient elution conditions adopted by the mobile phase are: 0-50min, the volume ratio of mobile phase A is 20%→80%, and the volume ratio of mobile phase B is 80%→20%, and linear gradient elution is performed.

[0009] The method for preparing the test solution is as follows: taking a sample to be tested, accurately weighing it, accurately adding an ethanol aqueous solution with a volume fraction of 50-70% to extract it, and weighing the weight; heating and refluxing, weighing the weight again, supplementing the lost weight with ethanol with a volume fraction of 50-70%, filtering it with a microporous filter membrane, taking the filtrate, and obtaining the test solution.

[0010] Preferably, the method for preparing the test solution is: take 5.0g of Kunyuean granules, accurately weigh the sample weight, accurately add 50ml of 70% by volume ethanol, weigh the weight, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with 70% by volume ethanol, shake well, filter with a microporous filter membrane, discard the initial filtrate, and take the subsequent filtrate as the test solution.

[0011] The pore size of the microporous filter membrane is 0.45 μm.

[0012] The reference solution preparation method is as follows: paeoniflorin and baicalin reference substances are taken respectively, dissolved in 50-70% ethanol by volume, and reference solutions of 0.128 mg / ml paeoniflorin and 0.4 mg / ml baicalin are prepared respectively.

[0013] Preferably, the reference solution preparation method is: take paeoniflorin and baicalin reference substances respectively, add 70% by volume ethanol to dissolve, and prepare reference solutions of 0.128 mg / ml paeoniflorin and 0.4 mg / ml baicalin respectively.

[0014] Preferably, the detection method comprises the following steps:

[0015] (1) Preparation of the test solution: Take 5.0 g of Kunyuean granules, accurately weigh the sample, accurately add 50 ml of 70% ethanol by volume, weigh the sample, heat and reflux for 30 min, cool, weigh again, make up the lost weight with 70% ethanol by volume, shake well, filter with a microporous filter membrane with a pore size of 0.45 μm, discard the primary filtrate, and take the subsequent filtrate as the test solution;

[0016] (2) Preparation of reference solution: Paeoniflorin and baicalin reference substances were taken separately and dissolved in 70% ethanol to prepare reference solutions of 0.128 mg / ml paeoniflorin and 0.4 mg / ml baicalin respectively;

[0017] (3) Chromatographic conditions: an octadecylsilyl bonded silica gel column was used, methanol was used as mobile phase A, 0.08% phosphoric acid was used as mobile phase B, gradient elution, flow rate was 1 ml / min, column temperature was 25°C, diode detector, detection wavelength for paeoniflorin was 230 nm, detection wavelength for baicalin was 280 nm, the number of theoretical plates calculated based on the peaks of paeoniflorin and baicalin should be not less than 5000, wherein the gradient elution program was as follows: wherein the gradient elution program was as follows: 0-50 min, the volume ratio of mobile phase A was 20%→80%, the volume ratio of mobile phase B was 80%→20%;

[0018] (4) Chromatographic peak determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, calculate the peak area at the same retention time, substitute it into the standard curve equation, and then calculate the content of paeoniflorin and baicalin.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention can simultaneously and accurately determine the contents of white peony root and scutellaria baicalensis in the preparation, improve the accuracy and efficiency of detection, and better control the product quality.

[0021] (2) The precision (RSD% < 0.7%), stability (RSD% < 0.4%), and repeatability (RSD% < 0.6%) of the HPLC method established in the present invention show that the detection method has the advantages of accurate determination, simple operation, and good stability.

[0022] (3) The present invention omits the step of passing through the column in the original standard, simplifies the sample extraction method, and reduces the loss of the components to be tested due to passing through the column during the extraction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Paeoniflorin standard curve

[0024] Figure 2 Baicalin standard curve

[0025] Figure 3 HPLC spectrum of the test solution of Example 1

[0026] Figure 4 HPLC spectrum of the mixed control solution of paeoniflorin and baicalin in Example 1

[0027] Figure 5HPLC spectrum of the negative control solution of Kunyuean granules (lacking white peony root) in Example 1

[0028] Figure 6 This is the HPLC spectrum of the negative control solution of Kunyuean Granules (lacking Scutellaria baicalensis) in Example 1

[0029] Figure 7 HPLC spectrum of Example 2

[0030] Figure 8 HPLC spectrum of Example 3

[0031] Fig. 9 HPLC spectrum of Comparative Example 1

[0032] Fig.10 HPLC spectrum of Comparative Example 2

[0033] Fig.11 HPLC spectrum of Comparative Example 3

[0034] Fig.12 HPLC spectrum of Comparative Example 4 DETAILED DESCRIPTION

[0035] The following examples are used for further explanation, but the present invention is not limited to these examples. In the examples and comparative examples, unless otherwise specified, the ethanol concentrations are expressed in volume percentages.

[0036] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0037] The instruments and reagents involved in the following embodiments include:

[0038] Instruments: American Waters 515 pump, Waters 717 automatic sample injector, Waters 2487 detector;

[0039] Reagents: Paeoniflorin reference substance (provided by the National Institute for the Control of Pharmaceutical and Biological Products, batch number: 110736-200423);

[0040] Baicalin reference substance (provided by the National Institute for the Control of Pharmaceutical and Biological Products, batch number: 110715-200212);

[0041] Reagents: Acetonitrile (chromatographic grade, Fisher, USA).

[0042] Example 1

[0043] (1) Preparation of standard curves for paeoniflorin and baicalin content: Accurately pipette 0.5, 2.0, 5.0, and 8.0 ml of the reference solution into 10 ml volumetric flasks, add 70% ethanol to the mark, and shake well to obtain mixed reference solutions of each series of concentrations. Pipet 10 μl of the mixed reference solution of each series of concentrations, inject into the liquid chromatograph, and measure. Draw the standard curve with the reference sample volume (μg) as the horizontal axis and the peak area integral value as the vertical axis. The results are shown in Table 1. Figure 1 , Figure 2 The regression equation of paeoniflorin was calculated as: y = 147133x-5822; the regression equation of baicalin was calculated as: y = 3592294x + 25665. The results showed that the paeoniflorin injection amount had a good linear relationship within the range of 1.024 μg; the baicalin injection amount had a good linear relationship within the range of 3.2 μg. The results of the standard curve test are shown in Table 1.

[0044] Table 1 Standard curve test results

[0045]

[0046] (2) Preparation of test solution: Grind Kunyuean granules, accurately weigh 5.0 g, place in a 250 ml conical flask, accurately add 50 ml of 70% ethanol, weigh the solution, heat and reflux in a water bath for 30 minutes, cool, weigh the solution, make up the lost weight with 70% ethanol, shake well, filter through a microporous membrane with a pore size of 0.45 μm, discard the primary filtrate, and take the subsequent filtrate as the test solution;

[0047] (3) Preparation of reference solution: dissolve the reference peony inoside and baicalin in 70% ethanol to prepare a solution containing 0.128 mg / ml of paeoniflorin and 0.4 mg / ml of baicalin.

[0048] (4) Preparation of negative control solution: According to the prescription composition, take the remaining medicinal ingredients except white peony root and scutellaria baicalensis, prepare a preparation without white peony root and scutellaria baicalensis according to the preparation process requirements, and prepare a negative control solution according to the method of step (2);

[0049] (5) Chromatographic conditions: an octadecylsilyl bonded silica gel column was used, methanol was used as mobile phase A, 0.08% phosphoric acid was used as mobile phase B, gradient elution, flow rate was 1 ml / min, column temperature was 25°C, diode detector, detection wavelength for paeoniflorin was 230 nm, detection wavelength for baicalin was 280 nm, the number of theoretical plates calculated based on the peaks of paeoniflorin and baicalin should be not less than 5000, the gradient elution program was as follows: 0-50 min, the volume ratio of mobile phase A was 20%→80%, the volume ratio of mobile phase B was 80%→20%;

[0050] (6) The test solution, reference solution and negative control solution obtained in steps (2) to (4) were measured according to the chromatographic conditions described in step (5). The measurement results are shown in Figure 3-6 The contents of paeoniflorin and baicalin in the test solution are shown in Table 2.

[0051] Example 2

[0052] (1) Preparation of test solution: Grind Kunyuean granules, accurately weigh 5.0 g, place in a 250 ml conical flask, accurately add 50 ml of 50% ethanol, weigh the solution, heat and reflux in a water bath for 30 minutes, cool, weigh the solution, make up the lost weight with 50% ethanol, shake well, filter through a microporous membrane with a pore size of 0.45 μm, discard the primary filtrate, and take the subsequent filtrate as the test solution;

[0053] (2) Preparation of reference solution: dissolve the reference substance of paeoniflorin and baicalin in 50% ethanol to prepare a solution containing 0.128 mg / ml of paeoniflorin and 0.4 mg / ml of baicalin.

[0054] (3) Preparation of negative control solution: According to the prescription composition, take the remaining medicinal ingredients except white peony root and scutellaria baicalensis, prepare a preparation without white peony root and scutellaria baicalensis according to the preparation process requirements, and prepare a negative control solution according to the method of step (1);

[0055] (4) The remaining chromatographic conditions were the same as those in Example 1;

[0056] (5) The test solution obtained in step (1) was measured according to the chromatographic conditions described in step (4). The results are shown in Table 2 and Figure 7 .

[0057] Example 3

[0058] Preparation of test solution: Grind Kunyuean granules, accurately weigh 5.0g, place in a 250ml conical flask, accurately add 50ml of 60% ethanol, weigh, heat and reflux in a water bath for 30 minutes, cool, weigh, make up the lost weight with 60% ethanol, shake well, filter with a microporous membrane with a pore size of 0.45μm, discard the initial filtrate, and take the subsequent filtrate as the test solution;

[0059] (2) Preparation of reference solution: dissolve the reference substance of paeoniflorin and baicalin in 60% ethanol to prepare a solution containing 0.128 mg / ml of paeoniflorin and 0.4 mg / ml of baicalin;

[0060] (3) Preparation of negative control solution: According to the prescription composition, take the remaining medicinal ingredients except white peony root and scutellaria baicalensis, prepare a preparation without white peony root and scutellaria baicalensis according to the preparation process requirements, and prepare a negative control solution according to the method of step (1);

[0061] (4) The remaining chromatographic conditions were the same as those in Example 1;

[0062] (5) The test solution obtained in step (1) was measured according to the chromatographic conditions described in step (4). The results are shown in Table 2 and Figure 8 .

[0063] Comparative Example 1

[0064] Preparation of test solution: Grind Kunyuean granules, accurately weigh about 5g, place in a 250ml conical flask, accurately add 50ml of 95% ethanol, weigh the weight, heat and reflux in a water bath for 30 minutes, cool, weigh the weight, make up the lost weight with 95% ethanol, filter, accurately measure 25ml of the filtrate, mix with 0.3ml of 1.0mol / L sodium hydroxide solution, place on a 3g silica gel H column (inner diameter 15mm, dry column, vacuum tighten), flow rate 1-4ml per minute, use 95% ethanol (1.0mol / L Sodium hydroxide solution (pH adjusted to 9) 20ml, 70% ethanol solution 30ml sequentially eluted, the eluents were collected separately, the ethanol eluent was evaporated to dryness, the residue was dissolved with 30ml water for each time, placed in a 20ml treated D101 macroporous resin column (inner diameter 15mm), eluted with water and 50% ethanol 100ml each in sequence, the 50% ethanol eluent was collected, placed in a 100ml volumetric flask, diluted to the scale with ethanol, shaken well, filtered with a microporous filter membrane (pore size 0.45μm), discarded the initial filtrate, and the subsequent filtrate was used as the test solution;

[0065] The remaining steps of preparing the reference solution are the same as those in Example 1;

[0066] Chromatographic conditions: octadecylsilane bonded silica gel as filler, methanol:0.08% phosphoric acid (30:70) as mobile phase, UV detector, detection wavelength of 230nm, theoretical plate number calculated based on paeoniflorin peak, should be no less than 1500;

[0067] The test results of the test products are shown in Table 2 and Fig. 9 .

[0068] Comparative Example 2

[0069] Chromatographic conditions: octadecylsilane bonded silica gel as filler, methanol:0.08% phosphoric acid (30:70) as mobile phase, UV detector, detection wavelength of 230nm, theoretical plate number calculated based on paeoniflorin peak, should be no less than 1500;

[0070] The remaining steps of preparing the test solution and the reference solution are the same as those in Example 1;

[0071] The test results of the test products are shown in Table 2 and Fig.10 .

[0072] Comparative Example 3

[0073] Preparation of test solution: Grind Kunyuean granules, accurately weigh about 5g, place in a 250ml conical flask, accurately add 50ml of 95% ethanol, weigh the weight, heat and reflux in a water bath for 30 minutes, cool, weigh the weight, make up the lost weight with 95% ethanol, filter, accurately measure 25ml of the filtrate, mix with 0.3ml of 1.0mol / L sodium hydroxide solution, place on a 3g silica gel H column (inner diameter 15mm, dry column, vacuum tighten), flow rate 1-4ml per minute, use 95% ethanol (1.0mol / L Sodium hydroxide solution (pH adjusted to 9) 20ml, 70% ethanol solution 30ml sequentially eluted, the eluents were collected separately, the ethanol eluent was evaporated to dryness, the residue was dissolved with 30ml water for each time, placed in a 20ml treated D101 macroporous resin column (inner diameter 15mm), eluted with water and 50% ethanol 100ml each in sequence, the 50% ethanol eluent was collected, placed in a 100ml volumetric flask, diluted to the scale with ethanol, shaken well, filtered with a microporous filter membrane (pore size 0.45μm), discarded the initial filtrate, and the subsequent filtrate was used as the test solution;

[0074] The remaining chromatographic conditions, reference solution preparation and other steps are the same as those in Example 1;

[0075] The test results of the test products are shown in Table 2 and Fig.11 .

[0076] Comparative Example 4

[0077] Chromatographic conditions: using an octadecylsilyl bonded silica gel column, methanol as mobile phase A, 0.08% phosphoric acid as mobile phase B, gradient elution, flow rate 1 ml / min, column temperature: 25°C, diode detector, peony inoside detection wavelength of 230 nm, baicalin detection wavelength of 280 nm, theoretical plate number calculated based on peony inoside and baicalin peaks should not be less than 5000, wherein the gradient elution program is as follows: wherein the gradient elution program is as follows: 0-50min, the volume ratio of mobile phase A is: 28%→65%, the volume ratio of mobile phase B is 72%→35%;

[0078] The remaining steps of preparing the test solution and the reference solution are the same as those in Example 1;

[0079] The test results of the test products are shown in Table 2 and Fig.12 .

[0080] The test results of the test solutions of Examples 1-3 and Comparative Examples 1-4 are shown in Table 2.

[0081] Table 2 Determination results of the test solution of the embodiment and the comparative example

[0082] serial number Paeoniflorin content (mg / g) Baicalin content (mg / g) Example 1 1.74 4.70 Example 2 1.67 4.61 Example 3 1.68 4.61 Comparative Example 1 1.42 / Comparative Example 2 1.38 / Comparative Example 3 1.40 1.6 Comparative Example 4 / 4.58

[0083] Conclusion: The detection method of Examples 1-3 improves the preparation method of the test sample, omits the original standard column passing step, simplifies the sample extraction method, reduces the loss of the test components caused by column passing during the extraction process, and adopts a gradient elution method and a diode detector, so the contents of paeoniflorin and baicalin can be accurately detected at the same time.

[0084] The methods of Comparative Examples 1 and 2 were unable to measure the baicalin content because they used an ultraviolet detector with a detection wavelength of 230 nm.

[0085] In the method of Comparative Example 3, the test solution was passed through a column, which resulted in the loss of the components to be tested. Therefore, the contents of paeoniflorin and baicalin could not be accurately detected.

[0086] In Comparative Example 4, the elution gradient was unreasonable and the peaks could not be separated, so the paeoniflorin content could not be measured.

[0087] Experimental Example 1 Precision Test

[0088] This experimental example involves the determination of the precision of the method of the present invention, comprising the following steps:

[0089] The reference solution (peonidin 0.128 mg / ml, baicalin 0.4 mg / ml) was accurately aspirated and measured six times continuously according to the chromatographic conditions of Example 1. The results are shown in Table 3.

[0090] Table 3 Precision test results

[0091] Serial number Paeoniflorin peak area Baicalin peak area 1 185353 1451442 2 184416 1452781 3 186006 1458368 4 183606 1449578 5 186603 1460874 6 186645 1462612 Mean 185353 1451442 RSD(%) 0.7 0.4

[0092] According to the above results, the relative standard deviation of the peak area integral value of paeoniflorin is 0.7%, and the relative standard deviation of the peak area integral value of baicalin is 0.4%, indicating that the precision of this method is good.

[0093] Experimental Example 2 Stability Test

[0094] The test solution was taken and measured according to the method at 0, 2, 4, 8, 12, 18, and 24 hours respectively. The results showed that paeoniflorin and baicalin were basically stable within 24 hours of the test. The determination method was the same as in Example 1, and the results are shown in Table 4.

[0095] Table 4 Stability test results

[0096] Time (hours) Relative retention time of paeoniflorin Relative retention time of baicalin Paeoniflorin peak area Baicalin peak area 0 16.28 31.06 1895865 14516193 2 16.30 31.08 1894923 14550922 4 16.31 31.08 1905813 14542308 8 16.32 31.11 1909750 14531700 12 16.32 31.13 1892975 14602919 18 16.37 31.20 1910769 14611096 24 16.43 31.26 1910907 14588541 RSD(%) 0.3 0.2 0.4 0.3

[0097] The experimental results show that when the test solution is measured within 24 hours, the relative retention time RSD of the characteristic peaks of paeoniflorin and baicalin is less than 0.3%, and the relative peak area RSD is less than 0.4%, indicating that the results remain stable when the test solution is measured within 24 hours.

[0098] Experimental Example 3 Repeatability Test

[0099] Accurately weigh 6 portions of Kunyuean granules of the same batch number, prepare test solutions according to the law, and measure the contents of paeoniflorin and baicalin respectively. The determination method is according to Example 1. The results are shown in Table 5.

[0100] Table 5 Reproducibility test results

[0101] Serial number Paeoniflorin content (mg / g) Baicalin content (mg / g) 1 1.73 4.70 2 1.74 4.69 3 1.73 4.67 4 1.74 4.69 5 1.73 4.67 6 1.72 4.62 Mean 1.73 4.67 RSD(%) 0.4 0.6

[0102] Results: The average content of paeoniflorin in the test samples was 1.73 (mg / g), and its relative standard deviation was 0.4%; the average content of baicalin was 4.67 (mg / g), and its relative standard deviation was 0.6%, indicating that the reproducibility of this method was good.

[0103] Experimental Example 4 Spiked recovery test

[0104] The spiked recovery method was adopted. A preparation sample with a known content was taken, and paeoniflorin and baicalin reference substances were added respectively. The samples were determined according to the method and the recovery rate was calculated. The determination method was the same as that in Example 1. The results are shown in Table 6.

[0105] Table 6 Recovery test results

[0106]

[0107] Results: The average recovery rate of paeoniflorin was 98.1%; the average recovery rate of baicalin was 101.2%. The results showed that this method has a good recovery rate.

Claims

1. The high performance liquid phase detection method of paeoniflorin and baicalin in Kunyuean is to inject the test solution and the reference solution into a high performance liquid chromatograph for determination, characterized in that: The chromatographic conditions of the high performance liquid phase detection method are as follows: methanol is used as mobile phase A, 0.08% phosphoric acid solution is used as mobile phase B, the volume ratio of mobile phase A: mobile phase B is 20-80:80-20, and linear gradient elution is performed; an octadecylsilyl bonded silica gel chromatographic column is used, the flow rate is controlled to be 1 ml / min, the column temperature is 25°C, the injection volume is 10 μl, and the theoretical plate number should be not less than 5000 based on the peaks of paeoniflorin and baicalin; the chromatographic conditions are as follows: methanol is used as mobile phase A, 0.08% phosphoric acid solution is used as mobile phase B, and the volume ratio of mobile phase A: mobile phase B is 20-80:80-20, and linear gradient elution is performed; an octadecylsilane bonded silica gel chromatographic column is used, the flow rate is controlled to be 1 ml / min, the column temperature is 25°C, the injection volume is 10 μl, and the theoretical plate number is calculated based on the peaks of paeoniflorin and baicalin and should be not less than 5000; The linear gradient elution condition adopted by the mobile phase is: 0-50min, the volume ratio of mobile phase A is 20%→80%, the volume ratio of mobile phase B is 80%→20%, and linear gradient elution is performed; the preparation method of the test solution is: taking the sample to be tested, accurately weighing, accurately adding 50-70% ethanol aqueous solution by volume fraction for extraction, and weighing the weight; heating and reflux, weighing again, supplementing the lost weight with 50-70% ethanol by volume fraction, filtering with a microporous filter membrane, taking the filtrate, and obtaining the test solution.

2. The high performance liquid phase detection method according to claim 1, characterized in that: The chromatographic conditions also include: the detector used is a diode array detector, dual wavelength detection is adopted, the detection wavelength of paeoniflorin is 230nm, and the detection wavelength of baicalin is 280nm.

3. The high performance liquid phase detection method according to claim 1, characterized in that: The method for preparing the test solution is as follows: take 5.0g of Kunyuean granules, accurately weigh the sample weight, accurately add 50ml of 70% by volume ethanol, weigh the weight, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with 70% by volume ethanol, shake well, filter with a microporous filter membrane, discard the initial filtrate, and take the subsequent filtrate as the test solution.

4. The high performance liquid phase detection method according to claim 3, characterized in that: The pore size of the microporous filter membrane is 0.45 μm.

5. The high performance liquid phase detection method according to any one of claims 1 to 2, characterized in that: The reference solution preparation method is as follows: paeoniflorin and baicalin reference substances are taken respectively, dissolved in 50-70% ethanol by volume, and reference solutions of 0.128 mg / ml paeoniflorin and 0.4 mg / ml baicalin are prepared respectively.

6. The high performance liquid phase detection method according to claim 5, characterized in that: The reference solution preparation method is as follows: paeoniflorin and baicalin reference substances are taken respectively, dissolved in 70% ethanol by volume, and reference solutions of 0.128 mg / ml paeoniflorin and 0.4 mg / ml baicalin are prepared respectively.

7. The high performance liquid phase detection method according to claim 1, characterized in that: The detection method comprises the following steps: (1) Preparation of the test solution: Take 5.0 g of Kunyuean granules, accurately weigh the sample, accurately add 50 ml of 70% ethanol by volume, weigh the sample, heat and reflux for 30 min, cool, weigh again, make up the lost weight with 70% ethanol by volume, shake well, filter with a microporous filter membrane with a pore size of 0.45 μm, discard the primary filtrate, and take the subsequent filtrate as the test solution; (2) Preparation of reference solution: Paeoniflorin and baicalin reference substances were taken separately and dissolved in 70% ethanol to prepare reference solutions of 0.128 mg / ml paeoniflorin and 0.4 mg / ml baicalin respectively; (3) Chromatographic conditions: an octadecylsilyl bonded silica gel column was used, methanol was used as mobile phase A, 0.08% phosphoric acid was used as mobile phase B, gradient elution, flow rate was 1 ml / min, column temperature was 25°C, diode detector, detection wavelength for paeoniflorin was 230 nm, detection wavelength for baicalin was 280 nm, the number of theoretical plates calculated based on the peaks of paeoniflorin and baicalin should be not less than 5000, wherein the gradient elution program was as follows: 0-50 min, the volume ratio of mobile phase A was 20%→80%, the volume ratio of mobile phase B was 80%→20%; (4) Chromatographic peak determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, calculate the peak area at the same retention time, substitute it into the standard curve equation, and then calculate the content of paeoniflorin and baicalin.

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