A method for determining the content of ginsenoside Rb1 in Shenmeng Shuangxin granules

Abstract

The application discloses a method for determining the content of ginsenoside Rb1 in Shenlou Shuangxin granules. The application provides a detection method for ginsenoside Rb1, which is characterized by comprising the following steps: detecting a sample to be measured by using liquid chromatography; the preparation of the sample to be measured comprises the following steps: extracting Shenlou Shuangxin granules containing ginsenoside Rb1 by using a solvent, separating by using a solid-phase extraction column, and then detecting; the detection condition of the liquid chromatography comprises that a C18 reversed-phase chromatographic column is used as the chromatographic column, acetonitrile is used as mobile phase A, and water is used as mobile phase B; and the elution mode of the liquid chromatography is linear gradient elution. The detection method provided by the application has high accuracy, high sensitivity, good repeatability and reliable results, and provides a basis for quality control and evaluation of Shenlou Shuangxin granules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the content of ginsenoside Rb1 in Shenlou Shuangxin Granules. Background Technology

[0002] Shenlou Shuangxin Granules are derived from the traditional Chinese medicine clinical experience formula Huoxue Anxin Fang, which is composed of seven Chinese herbs: Panax notoginseng, Salvia miltiorrhiza, Astragalus membranaceus, Ziziphus jujuba var. spinosa, Trichosanthes kirilowii, Allium macrostemon, and Ginkgo biloba. It has the effects of invigorating qi and restoring pulse, promoting blood circulation and removing blood stasis, and calming the mind and soothing the nerves. Clinically, it is intended to be used for chest pain, shortness of breath, dark purple tongue, and white greasy tongue coating caused by phlegm and blood stasis; and for angina pectoris in coronary heart disease with the above symptoms. Panax notoginseng is an important component of this formula, and ginsenoside Rb1 is an effective component of Panax notoginseng. Existing research shows (References: [1] Chen Meiqing, Li Weihua, Shi Guixiu. Research progress on the pharmacological effects of ginsenoside Rb1 on the cardiovascular system [J]. Medical Review, 2015, 21(03): 506-509. [2] Yang Yanping. Overview of research on the pharmacological effects of ginsenoside Rb1 [J]. Chinese Pharmacist, 2010, 13(02): 280-281.) that ginsenoside Rb1 can play an antiarrhythmic role by affecting multiple ion channels such as calcium and potassium; it can protect myocardial cells from damage caused by ischemia / reperfusion or other chemical substances, inhibit ventricular remodeling after acute myocardial infarction, inhibit myocardial hypertrophy and ventricular hypertrophy, and protect cardiac contractile function; it can lower blood pressure by stimulating endothelial cell-dependent vasodilation; and it can also play a role in the prevention and treatment of various cardiovascular diseases (such as hypertension and coronary heart disease) by improving the function of vascular endothelial cells. In addition, it has a wide range of effects on the central nervous system, immune system, and anti-tumor activity.

[0003] Ginseng and Sanlou Shuangxin Granules are a new traditional Chinese medicine. Literature review revealed a lack of research on quality standards. To effectively control the quality of this product and ensure its safety and efficacy in clinical use, solid-phase extraction-high performance liquid chromatography (SPE-HPLC) was used to determine the content of ginsenoside Rb1. The content determination method established in this invention has high accuracy, high sensitivity, good repeatability, and reliable results, providing a basis for the quality control and evaluation of Ginseng and Sanlou Shuangxin Granules. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for determining the content of ginsenoside Rb1 in Shenlou Shuangxin Granules. This method has high accuracy, high sensitivity, good repeatability, and reliable results, providing a basis for the quality control and evaluation of Shenlou Shuangxin Granules.

[0005] The present invention solves the above-mentioned technical problems through the following solutions.

[0006] This invention provides a method for detecting ginsenoside Rb1, which includes the following steps:

[0007] The sample to be tested was detected using liquid chromatography.

[0008] The preparation of the sample to be tested includes the following steps: extracting the Ginseng and Trichosanthes dual-core granules containing ginsenoside Rb1 with a solvent, separating them by solid-phase extraction column, and then detecting them;

[0009] The detection conditions for the liquid chromatography method include:

[0010] The chromatographic column is a C18 reversed-phase column;

[0011] Mobile phase A is acetonitrile;

[0012] Mobile phase B is water;

[0013] The elution method of the liquid chromatography is linear gradient elution, and the elution procedure is as follows:

[0014]

[0015] For example, "0→20 minutes, the volume of mobile phase A is 20%" means that within the range of 0 to 20 minutes, the volume of mobile phase A (the percentage of mobile phase A in the total volume of the mobile phase) is fixed at 20%; "20→33 minutes, the volume of mobile phase A is 20%→24%" means that within the range of 20 to 33 minutes, the volume of mobile phase A (the percentage of mobile phase A in the total volume of the mobile phase) changes linearly from 20% to 24%.

[0016] In some embodiments of the present invention, in the preparation of the sample to be tested, the solvent may be an alcohol solvent or a mixture of an alcohol solvent and water, such as a 25% methanol aqueous solution, a 50% methanol aqueous solution, a 70% methanol aqueous solution, or methanol, preferably methanol; wherein “%” refers to the percentage of methanol content (by volume) in the solvent.

[0017] In some embodiments of the present invention, in the preparation of the sample to be tested, the extraction can be performed by ultrasonic extraction, reflux extraction or soaking extraction, preferably reflux extraction;

[0018] Preferably, the reflux extraction is performed by heating in a water bath to reflux; the reflux extraction time is 30-90 min, preferably 60 min.

[0019] In some embodiments of the present invention, in the preparation of the sample to be tested, the ratio (g / mL) of the ginseng and halomena dual-core granules to the solvent is 1:(20-80), for example 1:80, 1:40, 1:20, preferably 1:80.

[0020] In some embodiments of the present invention, in the preparation of the sample to be tested, the solid-phase extraction column may be a C18 solid-phase extraction column, for example... HLB SPE column C18SPE column or SelectCore HR-C18, preferably C18SPE column, 500mg / 6ml.

[0021] In some embodiments of the present invention, the separation process in the preparation of the sample to be tested includes the following steps: activation, sample loading, rinsing, and elution;

[0022] The activation is preferably performed using methanol and water; the volume ratio of methanol to water is preferably 1:1.

[0023] The rinsing agent can be water, a 20% methanol aqueous solution, a 30% methanol aqueous solution, or a 40% methanol aqueous solution, preferably water;

[0024] The eluent used for elution can be an 80% aqueous methanol solution or methanol, preferably an 80% aqueous methanol solution.

[0025] In some embodiments of the present invention, the preparation of the sample to be tested includes the following steps: mixing the ginseng and halomena dual-core granule powder with methanol solution, reflux extraction, filtration, evaporating the filtrate to dryness, dissolving it in water, and separating the sample to be tested by solid-phase extraction column.

[0026] In some embodiments of the present invention, the particle size of the chromatographic column packing is 5 μm.

[0027] In some embodiments of the present invention, the length of the chromatographic column is 250 mm.

[0028] In some embodiments of the present invention, the inner diameter of the chromatographic column is 4.6 mm.

[0029] In some embodiments of the present invention, the chromatographic column is Waters Xselect CSH. TM Alternatively, a NanoChrom C18 or GL Sciences AQ-C18 column may be used; preferably, the column is a Waters Xselect CSH column. TM 4.6mm×250mm, 5μm or NanoChrom C18 250×4.6mm, 5μm.

[0030] In some embodiments of the present invention, the column temperature of the chromatographic column may be 25–40°C, for example, 30°C.

[0031] In some embodiments of the present invention, the detector of the detection method may be an ultraviolet detector; the detection wavelength of the detector is preferably 203 nm.

[0032] In some embodiments of the present invention, the flow rate of the mobile phase may refer to the conventional flow rate used in chromatographic detection in the art, for example, 1.0 mL / min.

[0033] In some embodiments of the present invention, the injection volume of the liquid chromatography method may refer to the conventional injection volume of chromatographic detection and analysis in the art, such as 10 μL.

[0034] In some embodiments of the present invention, the detection method further includes using a reference solution, the preparation of which includes the following steps: mixing ginsenoside Rb1 with an organic solvent.

[0035] In some embodiments of the present invention, the organic solvent used in the preparation of the reference solution is an alcohol solvent, preferably methanol.

[0036] In some embodiments of the present invention, the concentration of the reference solution is 5 to 95 μg / mL, preferably 23 to 27 μg / mL, for example 23 μg / mL.

[0037] This invention also provides a method for determining the content of ginsenoside Rb1, which includes the following steps:

[0038] Step 1: Detect the sample to be tested using the detection method described in any of the foregoing schemes of this invention;

[0039] Step 2: Calculate the content of ginsenoside Rb1 in the sample using the external standard method.

[0040] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0041] The reagents and raw materials used in this invention are all commercially available.

[0042] The positive and progressive effects of this invention are as follows:

[0043] (1) This invention establishes a method for determining the content of Ginseng and Trichosanthes dual-core granules for the first time by pretreatment of samples using a C18 solid-phase extraction column.

[0044] (2) The present invention uses ginsenoside Rb1 as a quality marker for content determination. The method for determining the content of ginsenoside Rb1 in Shenlou Shuangxin Granules is highly accurate, sensitive, repeatable, and reliable. It provides a basis for quality control of Shenlou Shuangxin Granules and ensures that the product is safer and more effective. Attached Figure Description

[0045] Figure 1 The HPLC chromatogram is the result of the specificity investigation.

[0046] Figure 2 This is a linear relationship diagram. Detailed Implementation

[0047] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0048] 1. Instruments and reagents

[0049] 1.1 Instruments: Thermo Scientific Vanquish high-performance liquid chromatograph (Thermo Fisher Scientific, USA); UV detector; HH-8 digital display constant temperature water bath (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); MS204DU electronic balance (Mettler-Toledo Instruments Co., Ltd.); AL204 electronic balance (Mettler-Toledo Instruments Co., Ltd.); STX1202ZH electronic balance [Ohaus Instruments (Changzhou) Co., Ltd.]; C18SPE column (500mg / 6ml); Waters XselectCSH TM Chromatographic column (4.6 mm × 250 mm, 5 μm).

[0050] 1.2 Reagents: Methanol (Tenet, Inc., chromatographic grade; Sinopharm Chemical Reagent Co., Ltd., analytical grade); Acetonitrile (Tenet, Inc., chromatographic grade); Ethanol (Tianjin Fuyu Fine Chemical Co., Ltd., analytical grade); Formic acid (Tianjin Kemio Chemical Reagent Co., Ltd., analytical grade); Phosphoric acid (Tianjin Kemio Chemical Reagent Co., Ltd., analytical grade); Triethylamine (Tianjin Kemio Chemical Reagent Co., Ltd., analytical grade); Double-distilled water; Ginsenoside Rb1 (Chengdu Mansite Biotechnology Co., Ltd., MUST-17071711, 99.02%).

[0051] 1.3 Shenlou Shuangxin Granules: Xinjiang Uygur Pharmaceutical Co., Ltd.; Que Sanqi Granules: Xinjiang Uygur Pharmaceutical Co., Ltd.

[0052] 2. Sample testing

[0053] 2.1 Chromatographic conditions

[0054] Column: Waters Xselect CSH TM C18 column (4.6 mm × 250 mm, 5 μm);

[0055] Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is water;

[0056] Detection wavelength: 203nm;

[0057] Column temperature: 30℃;

[0058] Flow rate: 1 mL·min -1 ;

[0059] Injection volume: 10 μL;

[0060] Linear gradient elution procedure:

[0061]

[0062] 2.2 Solution Preparation

[0063] (1) Preparation of reference solution: Accurately weigh ginsenoside Rb1 reference standard, place it in a volumetric flask, and add methanol solution to prepare a reference solution of 23 μg / mL.

[0064] (2) Preparation of the test solution: Take an appropriate amount of Ginseng and Trichosanthes Double Heart Granules (batch number: SLYF231104), grind them into a fine powder, pass them through an 80-mesh sieve, accurately weigh 0.25g of the powder and place it in an Erlenmeyer flask, add 20mL of methanol, weigh it, and reflux extract (the material-liquid ratio is 1:80, and the extraction is carried out by reflux in a water bath for 60min). Cool it down, make up the weight loss with methanol, filter it, take 10mL of the filtrate, evaporate it to dryness, dissolve the residue in 5mL of water and add it to a solid-phase extraction column ( C18 SPE column, 500 mg / 6 mL (pre-activated with 5 mL methanol and 5 mL water), discard the eluent, rinse with 10 mL water, discard the eluent, elute with 4.5 mL 80% methanol into a 5 mL volumetric flask, dilute to the mark, filter through a 0.45 μm filter membrane to obtain the test solution.

[0065] (3) Preparation of negative solution: Take an appropriate amount of Panax notoginseng granules (batch number: SLYX231102), grind them into a fine powder, pass them through an 80-mesh sieve, accurately weigh about 0.25g of the powder and place it in an Erlenmeyer flask, add 20mL of methanol, weigh it, and reflux extract (water bath heating and reflux extraction for 60min). Cool it, replenish the lost weight with methanol, filter it, take 10mL of the filtrate, evaporate it to dryness, dissolve the residue in 5mL of water and add it to a solid phase extraction column. C18 SPE column (500 mg / 6 mL, pre-activated with 5 mL methanol and 5 mL water), discard the eluent, rinse with 10 mL water, discard the eluent, elute with 4.5 mL 80% methanol into a 5 mL volumetric flask, dilute to the mark, filter through a 0.45 μm filter membrane to obtain the negative solution.

[0066] 2.3 Specificity Examination

[0067] Accurately pipette 10 μL each of the reference solution, test solution, and negative control solution, and determine the concentration according to the prescribed chromatographic conditions. The results show that the retention time and UV spectrum of the target peak in the chromatogram of the test solution are similar to those of the ginsenoside Rb1 reference standard. Figure 1 The results show a resolution greater than 1.5 and peak purity above 98%, indicating that the established method has strong specificity. Figure 1 As shown.

[0068] 2.4 Examination of the linear range

[0069] Take 1 mL of a reference solution containing 115.26144 μg of ginsenoside Rb1 and dilute it serially. Inject the solutions sequentially into the liquid chromatograph under the chromatographic conditions described in section "2.1" and record the peak areas. Plot a standard curve with peak area (Y) on the ordinate and concentration (X) on the abscissa, and perform linear regression to obtain the regression equation for ginsenoside Rb1: Y = 0.0465X + 0.0221(r 2 =0.9998). The results showed that ginsenoside Rb1 exhibited good linearity in the concentration range of 5.244–92.209 μg / mL. (See Table 1). Figure 2 As shown.

[0070] Table 1. Results of the linear range investigation (n=6)

[0071]

[0072] 2.5 Precision Test

[0073] A precise amount of ginsenoside Rb1 reference solution (23 μg / mL) was injected six times consecutively under the prescribed chromatographic conditions, 10 μL each time. The peak area of ​​the ginsenoside Rb1 chromatographic peak was measured, and the calculated RSD value of the peak area was 0.72%, which meets the technical requirements for the validation of the analytical method of the quality standard (RSD within 2.0%), indicating that the instrument precision is good. The results are shown in Table 2.

[0074] Table 2. Precision test results (n=6)

[0075]

[0076] 2.6 Repeatability Test

[0077] Six test solutions were prepared from the same batch of test granules according to the prescribed method. Each solution was injected into the solution under the prescribed chromatographic conditions, with a volume of 10 μL per solution. The average content of ginsenoside Rb1 was 950.1975 μg / g, and the calculated RSD was 2.90%, which meets the requirements for validation of the analytical method (RSD within 3.0%), indicating good repeatability of the method. The results are shown in Table 3.

[0078] Table 3. Results of repeatability tests (n=6)

[0079]

[0080] 2.7 Stability Test

[0081] One sample solution was prepared from the same batch of medicinal material as used in the repeatability test, using the prescribed method. Under the prescribed chromatographic conditions, 10 μL of the solution was injected at 0 h, 1 h, 3 h, 5 h, 7 h, 14 h, 28 h, and 48 h, respectively, and the peak area of ​​ginsenoside Rb1 was determined. The RSD of the peak area was calculated to be 1.56%, indicating that the sample solution was stable within 48 h. The results are shown in Table 4.

[0082] Table 4. Stability test results (n=8)

[0083]

[0084] 2.8 Recovery Test

[0085] Six test particles, each approximately 0.125 g, from the same batch as those used in the repeatability test were accurately weighed and added to ginsenoside Rb1 reference standard at a 1:1 ratio. The test solutions were prepared according to the prescribed method. The solutions were injected and analyzed under the prescribed chromatographic conditions, and the peak area of ​​ginsenoside Rb1 was recorded. The average recovery rate of ginsenoside Rb1 was calculated to be 95.71%, with an RSD of 2.82%, meeting the requirements for validation of the analytical method in the quality standard (recovery limit of 90–108%), indicating high accuracy of the method. The results are shown in Table 5.

[0086] Table 5 Results of the recovery test (n=6)

[0087]

[0088] Note: Original amount = Sample weight * Repeatability content; Recovery rate = (Measured amount - Original amount) / Added amount * 100%.

[0089] 2.9 Sample Determination

[0090] Fifteen batches of Ginseng and Ginseng Double-Heart Granules, each approximately 0.25g, with two parallel samples per batch, were accurately weighed. Test solutions were prepared according to the prescribed method, and the solutions were injected and analyzed under the prescribed chromatographic conditions. Each sample was measured twice, and the peak area of ​​ginsenoside Rb1 was recorded. The content of ginsenoside Rb1 in the granules was calculated using the external standard method (external standard method). The results are shown in Table 6.

[0091] Table 6. Results of determination of 15 batches of Ginseng and Trichosanthes dual-core granules

[0092]

[0093] 3. Using the above sample detection method, change one of the conditions to conduct the following experiment.

[0094] 3.1 Chromatographic conditions

[0095] (1) In this experiment, the detection wavelength was screened using the 3D spectrum of the UV detector. The results showed that ginsenoside Rb1 is terminally absorbed. Based on the pharmacopoeia, 203nm was selected as the detection wavelength.

[0096] (2) Analysis was performed using NanoChrom C18 (250×4.6mm, 5μm) and GL Sciences AQ-C18 (250×4.6mm, 5μm) columns, respectively. The results are shown in Table 7:

[0097] Table 7 Results of investigation using different chromatographic columns

[0098]

[0099] (3) The prepared solutions were analyzed by setting the detection column temperature to 25℃ and 40℃ respectively. The results are shown in Table 8:

[0100] Table 8 Results of investigation at different column temperatures

[0101]

[0102] 3.2 Extraction conditions

[0103] (1) Using 25% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, and methanol solution as extraction solvents respectively, the results showed that methanol solution had the highest extraction efficiency (content) (results are shown in Table 9):

[0104] Table 9 Results of the investigation of different extraction solvents

[0105]

[0106] (2) Ultrasonic extraction, reflux extraction, and soaking extraction were used respectively. The results showed that reflux extraction had the highest extraction efficiency (content) (results are shown in Table 10):

[0107] Table 10 Results of different extraction methods

[0108]

[0109] (3) Extraction times were set to 30 min, 60 min, and 90 min respectively. The results showed that the extraction efficiency (content) of ginsenoside Rb1 was higher when the extraction time was 60 min (results are shown in Table 11).

[0110] Table 11 Results of the investigation at different extraction times

[0111]

[0112] (4) The material-to-liquid ratios were set to 1:80, 1:40, and 1:20, respectively. The results showed that the extraction efficiency (content) of ginsenoside Rb1 was relatively high at a material-to-liquid ratio of 1:80 (results are shown in Table 12).

[0113] Table 12 Results of the investigation under different sample sizes and liquid-to-solid ratios

[0114]

[0115] (5) Investigate different types of solid phase extraction columns respectively C18, SelectCore HR-C18 and The extraction efficiency of the HLB SPE column was demonstrated by the results. C18 showed the best extraction efficiency (content) (results are shown in Table 13):

[0116] Table 13 Results of investigation using different solid phase extraction columns (SPE columns)

[0117]

[0118] (6) The extraction efficiency (content) was higher when water was used as the eluent, according to four different eluents: water, 20% methanol aqueous solution, 30% methanol aqueous solution and 40% methanol aqueous solution (see Table 14 for results).

[0119] Table 14 Results of the investigation of different rinsing agents

[0120]

Claims

1. A method for detecting ginsenoside Rbl, characterized by, It includes the following steps: The sample to be tested was detected using liquid chromatography. The preparation of the sample to be tested includes the following steps: extracting the Ginseng and Trichosanthes dual-core granules containing ginsenoside Rb1 with a solvent, separating them by solid-phase extraction column, and then detecting them; The detection conditions for the liquid chromatography method include: The chromatographic column is a C18 reversed-phase column; Mobile phase A is acetonitrile; Mobile phase B is water; The elution method of the liquid chromatography is linear gradient elution, and the elution procedure is as follows:

2. The detection method of claim 1, wherein, The preparation of the sample to be tested satisfies one or more of the following conditions: (1) The solvent is an alcohol solvent or a mixture of an alcohol solvent and water; (2) The extraction is performed by ultrasonic extraction, reflux extraction or soaking extraction; reflux extraction is preferred. (3) The ratio (g / mL) of the ginseng and trichosanthes dual-core granules to the solvent is 1:(20-80); (4) The solid phase extraction column is a C18 solid phase extraction column; (5) The separation includes the following steps: activation, loading, rinsing, and elution.

3. The detection method of claim 2, wherein, It satisfies one or more of the following conditions: (1) The solvent is a 25% methanol aqueous solution, a 50% methanol aqueous solution, a 70% methanol aqueous solution, or methanol; (2) The reflux extraction is performed by heating in a water bath to reflux; (3) The reflux extraction time is 30 to 90 minutes; (4) The ratio (g / mL) of the ginseng and trichosanthes dual-core granules to the solvent is 1:80, 1:40, or 1:20; (5) the solid phase extraction cartridge is HLB SPE cartridge, C18 SPE cartridge or SelectCore HR-C18; (6) The activation is performed using methanol and water; (7) The rinsing agent is water, 20% methanol aqueous solution, 30% methanol aqueous solution or 40% methanol aqueous solution; (8) The eluent used for elution is an 80% aqueous methanol solution or methanol.

4. The detection method as described in claim 2, characterized in that, It satisfies one or more of the following conditions: (1) The solvent is methanol; (2) The reflux extraction time is 60 min; (3) The ratio (g / mL) of the ginseng and trichosanthes dual-core granules to the solvent is 1:80; (4) The solid-phase extraction column is C18SPE column, 500mg / 6ml; (5) In the activation process, the volume ratio of methanol to water is 1:1; (6) The rinsing agent used in the rinsing is water; (7) The eluent used for elution is an 80% methanol aqueous solution.

5. The detection method as described in claim 1, characterized in that, The preparation of the sample to be tested includes the following steps: mixing the ginseng and halomena dual-core granule powder with methanol solution, reflux extraction, filtration, evaporation of the filtrate to dryness, dissolving in water, and separating the sample to be tested by solid phase extraction column.

6. The detection method as described in claim 1, characterized in that, The chromatographic column satisfies one or more of the following conditions: (1) The particle size of the chromatographic column packing is 5 μm; (2) The length of the chromatographic column is 250 mm; (3) The inner diameter of the chromatographic column is 4.6 mm; (4) the column type is Waters Xselect CSH TM or NanoChrom C18 or GL sciences AQ-C18; (5) The column temperature of the chromatographic column is 25-40℃.

7. The detection method as described in claim 6, characterized in that, The chromatographic column is a Waters Xselect CSH TM 4.6 mm x 250 mm, 5 μm or NanoChrom C18 250 x 4.6 mm, 5 μm; And / or, the column temperature of the chromatographic column is 30°C.

8. The detection method as described in claim 1, characterized in that, It satisfies one or more of the following conditions: (1) The detector in the detection method is an ultraviolet detector; the detection wavelength of the detector is preferably 203 nm; (2) The flow rate of the mobile phase is 1.0 mL / min; (3) The injection volume of the liquid chromatography method is 10 μL.

9. The detection method as described in claim 1, characterized in that, The detection method further includes the use of a reference solution, the preparation of which includes the following steps: mixing ginsenoside Rb1 with an organic solvent; The organic solvent is preferably an alcohol solvent, such as methanol; Preferably, the concentration of the reference solution is 5–95 μg / mL, more preferably 23–27 μg / mL, for example 23 μg / mL.

10. A method for determining the content of ginsenoside Rb1, characterized in that, It includes the following steps: Step 1: Detect the sample to be tested using the detection method described in any one of claims 1 to 9; Step 2: Calculate the content of ginsenoside Rb1 in the sample using the external standard method.

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