A method for constructing high-performance liquid chromatography (HPLC) characteristic spectra for the content determination of *Schisandra chinensis* bark (a type of schisandra chinensis) in medicinal materials, processed slices, standard decoctions, and formulated granules.

Abstract

This invention provides a method for determining the content of proanthocyanidin B2 in processed medicinal slices, standard decoctions, and formulation granules of Bauhinia purpurea bark (Schisandra chinensis). The method includes: determining the content of proanthocyanidin B2 in the test solution and reference solution using high-performance liquid chromatography (HPLC) under the same detection conditions; calculating the content of proanthocyanidin B2 by external standard method based on the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the areas of proanthocyanidin B2 in Bauhinia purpurea bark and the reference solution in the chromatogram; using a C18 column; mobile phase A being a 70:30 methanol-acetonitrile mixture, and mobile phase B being a 0.2% formic acid solution with gradient elution. This method not only enables quality control of Bauhinia purpurea bark raw materials but also provides effective detection and quality control methods for the entire production process of Bauhinia purpurea bark formulation granules, as well as the production of related derivatives and derivative products.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a method for constructing a high-performance liquid chromatography (HPLC) characteristic spectrum for the determination of the content of Bauhinia bark (Schisandra chinensis) medicinal material, processed slices, standard decoctions, and formulated granules. Background Technology

[0002] Bauhinia bark (Southern Schisandra) is the dried root bark of *Kadsura longipedunculata* Finet et Gagnep., a plant belonging to the genus *Kadsura* in the family Magnoliaceae. It possesses the effects of promoting blood circulation and regulating menstruation, reducing swelling and relieving pain, and detoxifying. It is used for amenorrhea due to blood stasis, dysmenorrhea, traumatic injuries, rheumatic pain, and sore throat. As a commonly used traditional Chinese medicine, the current quality control methods for Bauhinia bark (Southern Schisandra) are limited to identification based on its appearance and microscopic characteristics; there are currently no specific detection methods for its effective components. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for constructing a high-performance liquid chromatography (HPLC) characteristic spectrum for the determination of the content of Bauhinia bark (Schisandra chinensis) medicinal materials, processed slices, standard decoctions, and formulated granules. This method provides a stable and reproducible scientific and technological means for controlling the medicinal quality of Bauhinia bark (Schisandra chinensis) medicinal materials, processed slices, standard decoctions, and formulated granules.

[0004] This invention provides a method for constructing high-performance liquid chromatography (HPLC) characteristic spectra for the content determination of *Schisandra chinensis* bark (a type of schisandra chinensis), including the following steps:

[0005] A) Extract the Bauhinia bark raw material with methanol to obtain the test solution;

[0006] The proanthocyanidin B2 reference standard was mixed with methanol to obtain the reference standard solution;

[0007] B) The test solution and the reference solution were determined by high performance liquid chromatography, and chromatograms of proanthocyanidin B2 in the reference solution and the test solution were obtained under the same detection conditions.

[0008] The content of proanthocyanidin B2 in Bauhinia bark was calculated using the external standard method based on the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the area of ​​proanthocyanidin B2 in Bauhinia bark and the reference solution in the chromatogram.

[0009] The chromatographic conditions for the high performance liquid chromatography were as follows: the column was a C18 column; mobile phase A was a 70:30 methanol-acetonitrile mixed solvent; mobile phase B was a 0.2% formic acid solution; and gradient elution was used.

[0010] In this invention, methanol solution and 0.2% phosphoric acid solution are both volume fractions; 0.2% phosphoric acid solution is a 0.2% phosphoric acid aqueous solution.

[0011] This invention uses methanol to extract Bauhinia bark raw material to obtain a test solution.

[0012] This invention investigated the extraction solvents and found that there was little difference in extraction efficiency between pure methanol and 70% methanol. The preferred volume fraction of methanol in this invention is 68-73%, more preferably a 70 vol% methanol solution. The extraction method in this invention is ultrasonic extraction, with a power of 580-620 W and a frequency of 35-45 kHz. This invention verified ultrasonic extraction and reflux extraction, ultimately determining that ultrasonic extraction is more efficient than reflux extraction. The ultrasonic extraction time is 20-40 min, preferably 25-35 min; in a specific embodiment, the extraction time is 30 min, and this extraction time can be repeated.

[0013] This invention investigated the sampling amount. Research showed that a methanol volume to the mass ratio of *Bauhinia purpurea* bark (medicinal material or processed slices) of 25 mL : (0.48~0.53) g, and a methanol volume to the mass ratio of *Bauhinia purpurea* bark standard decoction or *Bauhinia purpurea* bark formula granules of 25 mL : (0.18~0.23) g, ensured complete extraction of the target component. In a specific embodiment, the methanol volume to the mass ratio of *Bauhinia purpurea* bark (medicinal material or processed slices) of 25 mL : 0.5 g, and the methanol volume to the mass ratio of *Bauhinia purpurea* bark standard decoction or *Bauhinia purpurea* bark formula granules of 25 mL : 0.2 g.

[0014] This invention involves mixing proanthocyanidin B2 reference standard with methanol to obtain a reference standard solution. The concentration of the reference standard solution is 45-55 μg / ml, preferably 48-52 μg / ml; in a specific embodiment, the concentration of the reference standard solution is 50 μg / ml. Proanthocyanidin B2, as a natural flavonoid, can regulate blood-brain barrier permeability, prevent infarct volume and cerebral edema caused by cerebral ischemia in rats, and reduce tight junction degeneration, mitochondrial depolarization, and intracellular oxidative stress caused by cerebral ischemia, thus exerting an antioxidant effect. It also has a certain effect on osteoarthritis, consistent with the blood-activating, menstruation-regulating, swelling-reducing, and pain-relieving effects of Bauhinia bark (Schisandra chinensis). This component is considered one of the active medicinal components of Bauhinia bark. Furthermore, proanthocyanidin B2, being a polyphenol, possesses good water solubility and thermal stability, which aligns with the characteristic of traditional Chinese medicine requiring decoction for extraction to exert its effects. Therefore, this component can be well transferred from the medicinal materials and processed slices to the standard decoction, resulting in a relatively high content of this component in the medicinal materials, processed slices, and standard decoction. It is feasible to use it as an indicator component for evaluating the quality of Bauhinia bark (Schisandra chinensis) and related preparations.

[0015] After obtaining the reference solution and the test solution, the present invention uses high performance liquid chromatography to determine the proanthocyanidin B2 in the test solution and the reference solution respectively, and obtains the chromatograms of proanthocyanidin B2 in the reference solution and the test solution respectively under the same detection conditions.

[0016] The present invention calculates the content of proanthocyanidin B2 in Bauhinia bark using the external standard method based on the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the area of ​​proanthocyanidin B2 in Bauhinia bark and the reference solution in the chromatogram.

[0017] In this invention, the chromatographic conditions for high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is a 70:30 methanol-acetonitrile mixed solvent, mobile phase B is a 0.2% formic acid solution, and gradient elution is used.

[0018] In the methanol-acetonitrile mixed solvent, 70:30 is a volume ratio.

[0019] The gradient elution in this invention specifically refers to:

[0020] 0~20min, Phase A: 7~10%, Phase B: 93~90%.

[0021] The chromatographic column used in this invention is an Agilent ZORBAX SB-Aq C18 with dimensions of 100×2.1mm and 1.8μm.

[0022] This invention provides a methodological investigation into the determination of proanthocyanidin B2 content in Bauhinia bark using the above-mentioned high-performance liquid chromatography method:

[0023] 1) Specificity test: By using the test solution, reference solution and negative control solution, the test results showed that the negative solution chromatogram did not interfere with the determination of the target peak, indicating that the method has good specificity.

[0024] 2) Precision test: In this invention, the reference solution was continuously injected, the peak area of ​​proanthocyanidin B2 was recorded, and the RSD value was calculated. The results showed that the average RSD value of the peak area of ​​proanthocyanidin B2 was 0.9%, indicating that the injection precision of this method was good.

[0025] 3) Linearity: Proanthocyanidin B2 reference standard was adapted to prepare solutions with concentrations of 8.0541448 μg / mL, 16.1082896 μg / mL, 24.1624344 μg / mL, 40.2707240 μg / mL, 80.5414480 μg / mL, and 201.3536200 μg / mL. 1 μl of each solution was accurately injected into the liquid chromatograph, and the peak area was obtained. Response curves were plotted with concentration (X, μg / mL) on the x-axis and peak area (Y) on the y-axis. The results showed that when the proanthocyanidin B2 concentration ranged from 8.0541448 to 201.35362 μg / mL, the linear relationship was y = 22.406x + 9.1675, R0. 2 =0.9999, indicating a good linear relationship in the concentration range of 8.0541448~201.35362μg / mL.

[0026] 4) Repeatability: Take an appropriate amount of the test sample, accurately weigh 6 portions, prepare the test sample solution according to the determined method, calculate the proanthocyanidin B2 content, and the calculated RSD value is 1.4%~1.8%, indicating that the method has good repeatability.

[0027] 5) Recovery rate: Recovery rate = (quantity measured - content in sample) / amount added × 100%. The results showed that the RSD of proanthocyanidin B2 recovery rate was 1.5~2.9%. This method has good accuracy.

[0028] 6) Intermediate precision: Different instruments were used for the determination, all with an Agilent ZORBAX SB-Aq C18 100×2.1mm, 1.8μm column. The RSD values ​​were 0.3~1.4%, indicating that the instrument has good durability. Different personnel and time periods were used to investigate the same test sample (batch number: YC-21). Different personnel (A, B) prepared test sample solutions at different times (I, II) for analysis and calculated the content of proanthocyanidins B2 in the test sample. The results showed that the RSD value of proanthocyanidins B2 content was 0.8%~1.3%, and the intermediate precision of this method was good.

[0029] 7) Durability assessment, The column robustness was investigated by using different batches of C18 columns (100×2.1mm, 1.8μm) of the same brand to test the same sample (batch number: YC-21). The results showed that the analytical chromatographic parameters of different columns were good. The same test sample (batch number: YC-21) solution was used, and the peak area of ​​proanthocyanidin B2 was measured at 0h, 2h, 4h, 8h, 16h and 24h respectively. The results showed that the RSD value of the peak area of ​​proanthocyanidin B2 was 1.8%~2.0%, and the test sample solution had good stability within 24 hours.

[0030] The HPLC method for determining the content of Bauhinia bark (Schisandra chinensis) medicinal materials, standard decoctions, and formulation granules provided by this invention is a means to detect the content of proanthocyanidin B2, an indicator component of Bauhinia bark (Schisandra chinensis) medicinal materials, standard decoctions, and formulation granules, based on the effective component proanthocyanidin B2. It can not only control the quality of Bauhinia bark (Schisandra chinensis) medicinal materials and medicinal materials, but also provide an effective detection and quality control means for the entire production process of Bauhinia bark (Schisandra chinensis) formulation granules, as well as the production of related derivative preparations and derivative products. Attached Figure Description

[0031] Figure 1 The ultraviolet absorption spectrum of the reference standard proanthocyanidin B2 is shown.

[0032] Figure 2 The UV absorption spectrum of proanthocyanidin C2 in the sample medicinal material;

[0033] Figure 3 Chromatograms of medicinal materials at different flow rates;

[0034] Figure 4 Chromatograms of medicinal materials at different column temperatures;

[0035] Figure 5 For specificity assessment;

[0036] Figure 6 This is the ultraviolet absorption spectrum of the reference standard;

[0037] Figure 7 The ultraviolet absorption spectrum of Bauhinia bark is shown.

[0038] Figure 8 The standard curve for proanthocyanidin B2;

[0039] Figure 9 This is the UV absorption spectrum of the reference standard, proanthocyanidin B2.

[0040] Figure 10 The UV absorption spectrum of proanthocyanidin B2 in the sample;

[0041] Figure 11 shows the chromatograms of the standard decoction at different flow rates;

[0042] Figure 12 Chromatograms of the standard decoction at different column temperatures;

[0043] Figure 13 For the specificity of standard decoctions;

[0044] Figure 14 This is the ultraviolet absorption spectrum of the reference standard;

[0045] Figure 15 The image shows the ultraviolet absorption spectrum of the standard decoction sample.

[0046] Figure 16 A standard curve of proanthocyanidin B2 in standard decoction;

[0047] Figure 17 This is the UV absorption spectrum of the reference standard, proanthocyanidin B2.

[0048] Figure 18 This is the UV absorption spectrum of proanthocyanidin B2;

[0049] Figure 19 The results of column temperature investigation for the formulated particles;

[0050] Figure 20 The results of the formulation particle flow rate investigation;

[0051] Figure 21 For the investigation of the specificity of the formulation particles;

[0052] Figure 22 This is a standard curve of proanthocyanidin B2 in the formulation granules. Detailed Implementation

[0053] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a method for constructing high-performance liquid chromatography (HPLC) characteristic spectra for the content determination of Bauhinia bark (Schisandra chinensis) medicinal materials, processed slices, standard decoctions, and formulated granules provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1: HPLC content determination of medicinal materials and processed medicinal slices:

[0055] 1. Experimental instruments and materials

[0056] High-performance liquid chromatograph: Instrument 1, Instrument 2;

[0057] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0058] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0059] Ultrasonic cleaner: KQ600-DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0060] 2. Reagents and reagents

[0061] Methanol (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd.), methanol (analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.), formic acid (chromatographic grade, Tianjin Kemeio Chemical Reagent Co., Ltd.), and water used for the mobile phase were all laboratory-made ultrapure water, and the rest were laboratory-made pure water.

[0062] Proanthocyanidin B2 reference standard (Chengdu Manster Biotechnology Co., Ltd., batch number: MUST-23041401, purity: 98.03%).

[0063] Bauhinia bark (Southern Schisandra chinensis) medicinal materials: YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17, YC-18, YC-19, YC-20, YC-21.

[0064] Bauhinia bark (Schisandra chinensis) decoction pieces: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10, YP- 11. YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, YP-20, YP-21, YP-22, YP-23.

[0065] 3. Investigation of chromatographic conditions

[0066] 3.1 Proposed chromatographic conditions

[0067] Octadecylsilane-bonded silica gel was used as the packing material; methanol:acetonitrile (70:30) was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 0.4 ml / min; the column temperature was 30℃; and the detection wavelength was 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

[0068] Table 1

[0069] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0~20 7→10 93→90

[0070] 3.2 Determination of detection wavelength

[0071] Based on the above-specified experimental conditions, a full-band scan of proanthocyanidin B2 was performed, as shown in the figure. Figure 1 , Figure 2 The results showed that, through analysis of the spectrum, proanthocyanidin B2 had strong absorption at a wavelength of 202 nm, so the detection wavelength of 202 nm was selected.

[0072] 3.3 Flow velocity investigation

[0073] Based on the above-established experimental conditions, flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min were investigated, with proanthocyanidin B2 resolution and theoretical plate number used as evaluation indicators. Results are shown below. Figure 3 Table 2:

[0074] Table 2 Analysis results for different flow velocities

[0075]

[0076] The results showed that the proanthocyanidin B2 peak did not appear at a flow rate of 0.2 ml / min; the proanthocyanidin B2 peak could be effectively detected at flow rates of 0.3 ml / min and 0.4 ml / min, and the flow rate of 0.4 ml / min was finally determined to be the optimal flow rate.

[0077] 3.4 Column Temperature Investigation

[0078] Based on the above-specified experimental conditions, column temperatures of 20℃, 30℃, and 40℃ were investigated, with proanthocyanidin B2 resolution and theoretical plate number used as evaluation indicators. Results are shown below. Figure 4 Table 3:

[0079] Table 3 Analytical results at different column temperatures

[0080]

[0081] The results showed that proanthocyanidin B2 could be effectively detected at column temperatures ranging from 20℃ to 40℃, and 30℃ was selected as the detection column temperature.

[0082] In summary, the chromatographic conditions and system suitability test for the determination of Bauhinia bark granules are as follows: Octadecylsilane-bonded silica gel is used as the packing material; methanol-acetonitrile (70:30) is used as mobile phase A, and 0.2% formic acid solution is used as mobile phase B, with gradient elution performed according to the specifications in Table 1 above; the detection wavelength is 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

[0083] 4. Preparation and investigation of the test solution

[0084] 4.1 Examination of Extraction Methods

[0085] Accurately weigh approximately 0.5g of this product (batch number: YC-21) powder and place it in a stoppered conical flask. Accurately add 25ml of 30% methanol, seal tightly, and weigh. Perform extraction under both reflux and ultrasonic methods for 30 minutes each. After cooling, weigh again, replenish the lost weight with 30% methanol, shake well, filter, and collect the filtrate. Analyze and calculate the proanthocyanidin B2 content under different extraction methods; the results are shown in Table 4.

[0086] Table 4. Analytical results of different extraction solvents

[0087]

[0088] The results showed that ultrasonic extraction was more efficient than reflux extraction, and ultrasonic extraction was selected as the extraction method.

[0089] 4.2 Investigation of Extraction Solvents

[0090] Accurately weigh approximately 0.5 g of this product (batch number: YC-21) powder and place it in a stoppered conical flask. Investigate the extraction with ethanol, methanol, 70% methanol, 50% methanol, 30% methanol, and water respectively. Accurately add 25 ml of each solvent, seal tightly, weigh, and sonicate (600 W, 40 kHz) for 30 minutes. Cool, weigh again, and replenish the lost weight with the corresponding solvent. Shake well, filter, and collect the filtrate. Accurately inject 1 μl of each test solution into the liquid chromatograph and calculate the content of proanthocyanidin B2 extracted with different solvents. The results are shown in Table 5.

[0091] Table 5 Results of the investigation of different extraction solvents

[0092]

[0093] The results showed that there was little difference in extraction efficiency between 70% methanol and pure methanol. The medicinal material samples were prepared in the same manner as the granules, and the extraction solvent for the medicinal materials was tentatively set as 70% methanol solution.

[0094] 4.3 Examination of extraction time

[0095] Accurately weigh approximately 0.5g of this product (batch number: YC-21) powder and place it in a stoppered conical flask. Accurately add 25ml of 30% methanol solution, seal tightly, and weigh. Extract using ultrasonic treatment (600W power, 40kHz frequency) for 20, 30, and 40 minutes respectively. After cooling, weigh again, replenish the lost weight with 30% methanol, shake well, filter, and collect the filtrate. The content of proanthocyanidin B2 at different extraction times was analyzed and calculated; the results are shown in Table 6.

[0096] Table 6. Analysis results of different extraction times

[0097]

[0098] The results showed that a extraction time of 30 minutes was sufficient for complete extraction. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0099] 4.4 Sampling Quantity Examination

[0100] Take approximately 0.2g, 0.5g, and 0.7g of this product (batch number: YC-21), accurately weigh them, and place them in stoppered conical flasks. Accurately add 25ml of 30% methanol to each flask for testing. Seal tightly, weigh, and extract using ultrasonic treatment (600W power, 40kHz frequency) for 30 minutes. Cool, weigh again, and replenish the lost weight with 30% methanol. Shake well, filter, and collect the filtrate. Calculate the proanthocyanidin B2 content; the results are shown in Table 7.

[0101] Table 7 Results of the study with different sampling sizes

[0102]

[0103] The results showed that when the sample size was 0.5g and the solvent volume was 25ml, the target component could be completely extracted. Therefore, when the solvent volume was 25ml, the sample size was tentatively set at 0.5g.

[0104] In summary, the preparation method for the test sample for determining the content of Bauhinia bark granules is as follows: Take an appropriate amount of this product, about 0.5g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, seal tightly, weigh it, extract by ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the product.

[0105] 5. Methodological Investigation

[0106] 5.1 Specificity Experiment

[0107] Preparation of the test solution: Prepare the test solution of Bauhinia bark granules according to the experimental conditions proposed above.

[0108] Take appropriate amounts of each of the proanthocyanidin B2 reference standards, weigh them accurately, and add 70% methanol to prepare a solution containing 50 μg per ml.

[0109] Preparation of negative control solution: A negative control solution lacking Bauhinia bark granules was prepared according to the experimental conditions outlined above. The test chromatograms are shown in Figures 5-7. The results indicate that the negative control solution chromatogram did not interfere with the determination of the target peak, demonstrating the good specificity of this method.

[0110] 5.2 Precision test

[0111] The reference solution was injected six times consecutively, and the peak area of ​​proanthocyanidin B2 was recorded. The RSD value was calculated, and the results are shown in Table 8.

[0112] Table 8 Precision test results

[0113]

[0114] The results showed that the peak area RSD of proanthocyanidin B2 was 0.9% in the precision study. The injection precision of this method is good.

[0115] 5.3 Linear Relationship

[0116] Take an appropriate amount of proanthocyanidin B2 reference standard (purity 98.03%), place it in a 50 ml volumetric flask, and dissolve it in 70% methanol to prepare a solution with a concentration of 201.35362 μg per ml. Then dilute it to solutions with concentrations of 8.0541448 μg, 16.1082896 μg, 24.1624344 μg, 40.2707240 μg, 80.5414480 μg, and 201.3536200 μg per ml, respectively.

[0117] Accurately pipette 1 μl of each sample and inject it into the liquid chromatograph. Obtain the peak area and plot the response curve with concentration (X, μg / ml) on the x-axis and peak area (Y) on the y-axis. The results are shown in Table 9 and [Table data would be inserted here]. Figure 8 :

[0118] Table 9. Results of the standard curve analysis of proanthocyanidin B2

[0119]

[0120] The results showed that when the concentration of proanthocyanidin B2 was in the range of 8.0541448~201.35362 μg / mL, the linear relationship was y = 22.406x + 9.1675, R0 2 =0.9999, indicating a good linear relationship in the concentration range of 8.0541448~201.35362μg / mL.

[0121] 5.4 Repeatability

[0122] Take an appropriate amount of this product (batch number: YC-21), approximately 0.5g, and accurately weigh 6 portions. Prepare the test solution using the same operator according to the established method. Calculate the proanthocyanidin B2 content of the 6 test samples. The results are shown in Table 10:

[0123] Table 10 Results of Repeatability Experiments

[0124]

[0125] The RSD value of proanthocyanidin B2 content in the 6 repeatability tests was 1.4%, indicating that the method has good repeatability.

[0126] 5.5 Recovery rate

[0127] Six portions of a known-content test sample (batch number: YC-21, proanthocyanidin B2 content 0.533%) were accurately weighed. 25 ml of a control solution containing proanthocyanidin B2 (purity: 98.03%) was accurately added to each portion. The test solutions were prepared and analyzed according to the prescribed method. The recovery rate was calculated, and the results are shown in Table 11. The calculation formula is as follows:

[0128] Recovery rate (%) = (Measured amount - Content in sample) / Amount added × 100%;

[0129] Table 11 Results of the recovery experiment of proanthocyanidin B2

[0130]

[0131] The results showed that the recovery rate RSD of proanthocyanidin B2 was 2.9%. The method had good accuracy.

[0132] 5.6 Intermediate Precision

[0133] 5.6.1 Investigation with different instruments

[0134] According to the determined method, accurately weigh 0.5 g of the powder (batch number: YC-21) into two portions and analyze them separately on an Agilent or Waters ultra-high performance liquid chromatograph (both columns were Agilent ZORBAX SB-AqC18 100×2.1 mm, 1.8 μm). Calculate the total content of proanthocyanidins B2. The results are shown in Table 12.

[0135] Table 12 Exploration using different instruments

[0136]

[0137] The results showed that the RSD value of the results measured by the Waters and Agilent 1290 high performance liquid chromatograph was 1.4%, indicating that the instrument has good durability.

[0138] 5.6.2 Investigations by different personnel and at different times

[0139] The same test sample (batch number: YC-21) was prepared into test solution by different personnel (A, B) at different times (Ⅰ, Ⅱ) for analysis. The content of proanthocyanidin B2 in the test sample was calculated, and the results are shown in Table 13.

[0140] Table 13 Results of the surveys of different personnel and time periods

[0141]

[0142] The results showed that the RSD value of proanthocyanidin B2 was 0.8%, indicating good intermediate precision of this method.

[0143] 5.7 Durability Test

[0144] 5.7.1 Column robustness test

[0145] The same test sample (batch number: YC-21) was detected using different batches of C18 columns (100×2.1mm, 1.8μm) of the same brand. The results are shown in Table 14.

[0146] Table 14 Durability test results

[0147]

[0148] The results showed that the analytical chromatographic parameters of different chromatographic columns were good.

[0149] 5.7.2 Stability Test

[0150] The same test sample solution (batch number: YC-21) was used, and the peak area of ​​proanthocyanidin B2 was determined at 0h, 2h, 4h, 8h, 16h, and 24h, respectively. The results are shown in Table 15.

[0151] Table 15 Results of the stability experiment of proanthocyanidin B2

[0152]

[0153] The results showed that the RSD value of the peak area of ​​proanthocyanidin B2 was 1.8%, and the test solution had good stability within 24 hours.

[0154] 6. Validation of Bauhinia bark (Schisandra chinensis) medicinal materials and decoction pieces, and establishment of HPLC method for content determination.

[0155] 6.1 Verification results of 21 batches of Bauhinia bark (Schisandra chinensis) medicinal materials and processed slices are shown in Tables 16 and 17:

[0156] Table 16 Verification of the content of 21 batches of Bauhinia bark (Schisandra chinensis) medicinal materials

[0157]

[0158] Table 17 Verification of the content of 23 batches of Bauhinia bark (Schisandra chinensis) slices

[0159]

[0160] Conclusion: The method for determining the content of Bauhinia bark (Schisandra chinensis) in medicinal materials and processed slices can effectively detect the content of Bauhinia bark (Schisandra chinensis) in medicinal materials and processed slices, proving that the method is feasible.

[0161] 7. Determination of the content of Bauhinia bark (Schisandra chinensis) in medicinal materials and processed slices

[0162] [Content Determination] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0163] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; methanol:acetonitrile (70:30) as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in Table 1 above; flow rate was 0.4 mL / min; column temperature was 30℃; and detection wavelength was 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

[0164] Preparation of the reference solution: Take an appropriate amount of proanthocyanidin B2 reference standard, accurately weigh it, and add 70% methanol to prepare a mixed solution containing 50 μg of each in 1 ml.

[0165] Preparation of the test solution: Weigh approximately 0.5 g of the powder (passed through a No. 4 sieve) accurately, place it in a stoppered conical flask, add 25 ml of 70% methanol accurately, stopper tightly, weigh, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0166] The assay involves precisely pipetting 1 µl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0167] Example 2: HPLC method for determining the content of standard decoctions

[0168] 1. Drug testing

[0169] Standard decoction of Bauhinia bark (Schisandra chinensis): BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20, BT-21, BT-22, BT-23;

[0170] 2. Investigation of chromatographic conditions

[0171] 2.1 Proposed chromatographic conditions

[0172] Octadecylsilane-bonded silica gel was used as the packing material; methanol:acetonitrile (70:30) was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1 above; the flow rate was 0.4 ml per minute; the column temperature was 30℃; and the detection wavelength was 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

[0173] 2.2 Determination of detection wavelength

[0174] Based on the above-specified experimental conditions, a full-band scan of proanthocyanidin B2 was performed, as shown in the figure. Figure 9 , Figure 10 The results showed that proanthocyanidin B2 had strong absorption at a wavelength of 202 nm, so the detection wavelength of 202 nm was selected.

[0175] 2.3 Flow velocity investigation

[0176] Based on the above-established experimental conditions, flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min were investigated, with proanthocyanidin B2 resolution and theoretical plate number used as evaluation indicators. Results are shown below. Figure 11 Table 18:

[0177] Table 18 Analysis results at different flow velocities

[0178]

[0179] The results showed that the proanthocyanidin B2 peak did not appear at a flow rate of 0.2 ml / min; the proanthocyanidin B2 peak could be effectively detected at flow rates of 0.3 ml / min and 0.4 ml / min, and the flow rate of 0.4 ml / min was finally determined to be the optimal flow rate.

[0180] 2.4 Column Temperature Investigation

[0181] Based on the above-specified experimental conditions, column temperatures of 20℃, 30℃, and 40℃ were investigated, with proanthocyanidin B2 resolution and theoretical plate number used as evaluation indicators. Results are shown below. Figure 12 Table 19:

[0182] Table 19 Analytical results at different column temperatures

[0183]

[0184] The results showed that proanthocyanidin B2 could be effectively detected at column temperatures ranging from 20℃ to 40℃, and 30℃ was selected as the detection column temperature.

[0185] In summary, the chromatographic conditions and system suitability test for the determination of Bauhinia bark granules are as follows: Octadecylsilane-bonded silica gel is used as the packing material; methanol-acetonitrile (70:30) is used as mobile phase A, and 0.2% formic acid solution is used as mobile phase B, with gradient elution performed according to the specifications in Table 1 above; the detection wavelength is 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

[0186] 3. Preparation and investigation of the test solution

[0187] 3.1 Examination of Extraction Methods

[0188] Accurately weigh approximately 0.2 g of the powder (batch number: BT-21) and place it in a stoppered conical flask. Accurately add 25 ml of 70% methanol, seal tightly, and weigh. Perform extraction under both reflux and ultrasonic methods for 30 minutes each. After cooling, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate. Analyze and calculate the proanthocyanidin B2 content under different extraction methods; the results are shown in Table 20.

[0189] Table 20 Analysis results of different extraction methods

[0190]

[0191] The results showed that ultrasonic extraction was more efficient than reflux extraction, and ultrasonic extraction was selected as the extraction method.

[0192] 3.2 Investigation of Extraction Solvents

[0193] Accurately weigh approximately 0.2 g of this product (batch number: BT-21) powder and place it in a stoppered conical flask. Investigate the extraction solvents ethanol, methanol, 70% methanol, 50% methanol, 30% methanol, and water, adding 25 ml of each solvent precisely. Seal the flask tightly, weigh, and sonicate (600 W, 40 kHz) for 30 minutes. Cool, weigh again, and replenish the lost weight with the corresponding solvent. Shake well, filter, and collect the filtrate. Accurately inject 1 μl of each test solution into the liquid chromatograph and calculate the content of proanthocyanidin B2 extracted with different solvents. The results are shown in Table 21.

[0194] Table 21 Results of the investigation of different extraction solvents

[0195]

[0196] The results showed that there was little difference in extraction efficiency between 70% methanol and pure methanol. To maintain consistency with the particle size distribution, 70% methanol solution was tentatively chosen as the extraction solvent.

[0197] 3.3 Examination of extraction time

[0198] Accurately weigh approximately 0.2g of this product (batch number: BT-21) powder and place it in a stoppered conical flask. Accurately add 25ml of 30% methanol solution, seal tightly, and weigh. Extract using ultrasonic treatment (600W power, 40kHz frequency) for 20, 30, and 40 minutes respectively. After cooling, weigh again, replenish the lost weight with 30% methanol, shake well, filter, and collect the filtrate. The content of proanthocyanidin B2 at different extraction times was analyzed and calculated; the results are shown in Table 22.

[0199] Table 22 Analysis results of different extraction times

[0200]

[0201] The results showed that a extraction time of 30 minutes was sufficient for complete extraction. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0202] 3.4 Sampling Quantity Examination

[0203] Take approximately 0.1g, 0.2g, and 0.3g of this product (batch number: BT-21), accurately weigh them respectively, and place them in stoppered conical flasks. Accurately add 25ml of 30% methanol to each flask for further investigation. Seal tightly, weigh, and extract using ultrasonic treatment (600W power, 40kHz frequency) for 30 minutes. Cool, weigh again, and replenish the lost weight with 30% methanol. Shake well, filter, and collect the filtrate. Calculate the proanthocyanidin B2 content; the results are shown in Table 22.

[0204] Table 22 Analysis results of different extraction times

[0205]

[0206] The results showed that when the sample size was 0.2g and the solvent volume was 25ml, the target component could be completely extracted. Therefore, when the solvent volume was 25ml, the sample size was tentatively set at 0.2g.

[0207] In summary, the preparation method for the test sample for determining the content of Bauhinia bark granules is as follows: Take an appropriate amount of this product, about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, seal tightly, weigh, and extract by ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes. Cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the product.

[0208] 4. Methodological Examination

[0209] 4.1 Specificity Experiment

[0210] Preparation of the test solution: Prepare the test solution of the Bauhinia bark standard decoction according to the experimental conditions proposed above.

[0211] Preparation of reference solution: Take an appropriate amount of proanthocyanidin B2 reference standard, accurately weigh it, and add 70% methanol to prepare a mixed solution containing 50 μg of each in 1 ml.

[0212] Preparation of negative control solution: A negative control solution for the standard decoction lacking Bauhinia bark was prepared according to the experimental conditions outlined above. See the results below. Figures 13-15 The results showed that the negative solution chromatogram did not interfere with the determination of the target peak, indicating that the method has good specificity.

[0213] 4.2 Precision test

[0214] The reference solution was injected six times consecutively, and the peak area of ​​proanthocyanidin B2 was recorded. The RSD value was calculated, and the results are shown in Table 24.

[0215] Table 24 Precision test results

[0216]

[0217] The results showed that the peak area RSD of proanthocyanidin B2 was 0.9% in the precision study. The injection precision of this method is good.

[0218] 4.3 Linear Relationship

[0219] Take an appropriate amount of proanthocyanidin B2 reference standard (purity 98.03%), place it in a 50 ml volumetric flask, and dissolve it in 70% methanol to prepare a solution with a concentration of 201.35362 μg per ml. Then dilute it to solutions with concentrations of 8.0541448 μg, 16.1082896 μg, 24.1624344 μg, 40.2707240 μg, 80.5414480 μg, and 201.3536200 μg per ml, respectively.

[0220] Accurately pipette 1 μl of each sample and inject it into the liquid chromatograph. Obtain the peak area and plot the response curve with concentration (X, μg / ml) on the x-axis and peak area (Y) on the y-axis. The results are shown in Table 25. Figure 16 :

[0221] Table 25 Standard Curve Analysis Results

[0222]

[0223] 4.4 Repeatability

[0224] Take approximately 0.2g of this product (batch number: BT-21) powder and prepare a test solution using the same operator according to the established method. Calculate the proanthocyanidin B2 content of 6 test samples. The results are shown in Table 26:

[0225] Table 26 Results of Repeatability Experiments

[0226]

[0227] The RSD value of proanthocyanidin B2 content in the 6 repeatability tests was 1.7%, indicating that the method has good repeatability.

[0228] 4.5 Recovery rate

[0229] Six portions of a known-content test sample (batch number: BT-21, proanthocyanidin B2 content 5.887 mg / g) were accurately weighed. Each portion was precisely added to 25 ml of a control solution containing proanthocyanidin B2 (purity: 98.03%). The test solutions were prepared and analyzed according to the prescribed method. The recovery rate was calculated, and the results are shown in Table 27. The calculation formula is as follows:

[0230] Recovery rate (%) = (Measured amount - Content in sample) / Amount added × 100%

[0231] Table 27 Results of the Proanthocyanidin B2 Spiking Recovery Experiment

[0232]

[0233] The results showed that the recovery rate RSD of proanthocyanidin B2 was 2.5%. The method had good accuracy.

[0234] 4.6 Intermediate Precision

[0235] 4.6.1 Investigation with different instruments

[0236] According to the determined method, accurately weigh 0.2 g of the powder (batch number: BT-21) into two portions and analyze them separately on an Agilent or Waters ultra-high performance liquid chromatograph (both columns were Agilent ZORBAX SB-AqC18 100×2.1 mm, 1.8 μm). Calculate the total content of proanthocyanidins B2. The results are shown in Table 28.

[0237] Table 28 Experimental Results with Different Instruments

[0238]

[0239] The results showed that the RSD value of the results measured by the Waters e2695 and Agilent Inifity 1260 high performance liquid chromatographs was 0.5%, indicating that the instruments used in this method have good durability.

[0240] 4.6.2 Investigations by different personnel and at different times

[0241] The same test sample (batch number: BT-21) was used, and different personnel (A, B) prepared test sample solutions at different times (I, II) for analysis. The content of proanthocyanidins B2 in the test sample was calculated, and the results are shown in Table 29:

[0242] Table 29 Results of the survey for different personnel and time periods

[0243]

[0244] The results showed that the RSD value of proanthocyanidin B2 was 1.9%, indicating good intermediate precision of this method.

[0245] 4.7 Durability Test

[0246] 4.7.1 Column robustness test

[0247] The same test sample (batch number: BT-21) was tested using C18 columns (100×2.1mm, 1.8μm) from different batches of the same brand. The results are shown in Table 30.

[0248] Table 30 Durability Test Results

[0249]

[0250] The results showed that the analytical chromatographic parameters of different chromatographic columns were good.

[0251] 4.7.2 Stability Test

[0252] Take the same test sample (batch number: BT-21) solution and determine the chromatographic peak area of ​​proanthocyanidin B2 at 0h, 2h, 4h, 8h, 16h and 24h respectively. The results are shown in Table 31.

[0253] Table 31 Stability test results

[0254]

[0255] The results showed that the RSD value of the peak area of ​​proanthocyanidin B2 was 1.8%, and the test solution had good stability within 24 hours.

[0256] 5. Validation of the standard decoction of Bauhinia bark (Schisandra chinensis) and establishment of an HPLC method for its content determination.

[0257] Table 32 Verification of the content of Bauhinia bark (Schisandra chinensis) in standard decoctions of 23 batches.

[0258]

[0259] Conclusion: The method for determining the content of Bauhinia bark (Schisandra chinensis) in the standard decoction can effectively detect the content of Bauhinia bark (Schisandra chinensis) in the standard decoction, proving that the method is feasible.

[0260] 6. Determination of the content of Bauhinia bark (Schisandra chinensis) in standard decoction

[0261] [Content Determination] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0262] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; methanol:acetonitrile (70:30) as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in Table 1 above; flow rate was 0.4 ml / min; column temperature was 30℃; and detection wavelength was 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

[0263] Preparation of the reference solution: Take an appropriate amount of proanthocyanidin B2 reference standard, accurately weigh it, and add 70% methanol to prepare a mixed solution containing 50 μg of each in 1 ml.

[0264] Preparation of the test solution: Weigh approximately 0.2 g of the powder, place it in a stoppered conical flask, add 25 ml of 70% methanol, seal tightly, weigh, sonicate (600 W, 40 kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0265] The assay involves precisely pipetting 1 µl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0266] Example 3: HPLC content determination of the formulated granules:

[0267] 1. Drug testing

[0268] Bauhinia bark (Schisandra chinensis) formula granules: KL-01, KL-02, KL-03

[0269] 2. Investigation of chromatographic conditions

[0270] 2.1 Proposed chromatographic conditions

[0271] Octadecylsilane-bonded silica gel was used as the packing material; methanol-acetonitrile (70:30) was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 1 above; the flow rate was 0.4 ml per minute; the column temperature was 30℃; and the detection wavelength was 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

[0272] 2.2 Determination of detection wavelength

[0273] Based on the above-specified experimental conditions, a full-band scan of proanthocyanidin B2 was performed, as shown in the figure. Figure 17 and Figure 18 The results showed that proanthocyanidin B2 had strong absorption at a wavelength of 202 nm, so the detection wavelength of 202 nm was selected.

[0274] 2.3 Column Temperature Investigation

[0275] Based on the above-specified experimental conditions, column temperatures of 20℃, 30℃, and 40℃ were investigated, with proanthocyanidin B2 resolution and theoretical plate number used as evaluation indicators. Results are shown below. Figure 19 And Table 33:

[0276] Table 33 Analytical results at different column temperatures

[0277]

[0278] The results showed that proanthocyanidin B2 could be effectively detected at column temperatures ranging from 20℃ to 40℃, and 30℃ was selected as the detection column temperature.

[0279] 2.4 Flow velocity investigation

[0280] Based on the above-established experimental conditions, flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min were investigated, with proanthocyanidin B2 resolution and theoretical plate number used as evaluation indicators. Results are shown below. Figure 20 And Table 34:

[0281] Table 34 Analysis results at different flow velocities

[0282]

[0283] The results showed that the proanthocyanidin B2 peak did not appear at a flow rate of 0.2 ml / min; the proanthocyanidin B2 peak could be effectively detected at flow rates of 0.3 ml / min and 0.4 ml / min, and the flow rate of 0.4 ml / min was finally determined to be the optimal flow rate.

[0284] In summary, the method for determining the content of Bauhinia bark (Schisandra chinensis) granules is as follows: Octadecylsilane-bonded silica gel is used as the packing material; methanol-acetonitrile (70:30) is used as mobile phase A, and 0.2% formic acid solution is used as mobile phase B, with gradient elution performed according to the specifications in Table 1 above; the detection wavelength is 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

[0285] 3. Preparation and investigation of the test solution

[0286] 3.1 Examination of Extraction Methods

[0287] Take an appropriate amount of this product (batch number: KL-01), grind it into a fine powder, accurately weigh about 0.2g, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, seal tightly, and weigh. Perform extraction under both reflux and ultrasonic methods, with an extraction time of 30 minutes for each. After cooling, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate. Analyze and calculate the proanthocyanidin B2 content under different extraction methods; the results are shown in Table 35.

[0288] Table 35 Analysis results of different extraction methods

[0289]

[0290] The results showed that ultrasonic extraction was more efficient than reflux extraction, and ultrasonic extraction was selected as the extraction method.

[0291] 3.2 Examination of extraction time

[0292] Take an appropriate amount of this product (batch number: KL-01), grind it into a fine powder, accurately weigh about 0.2g, place it in a stoppered conical flask, accurately add 25ml of 30% methanol solution, seal tightly, weigh, and extract using ultrasonic treatment (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. After cooling, weigh again, replenish the lost weight with 30% methanol, shake well, filter, and collect the filtrate. Analyze and calculate the content of proanthocyanidin B2 at different extraction times. The results are shown in Table 36.

[0293] Table 36 Analysis results of different extraction times

[0294]

[0295] The results showed that a extraction time of 30 minutes was sufficient for complete extraction. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0296] 3.3 Sampling Quantity Examination

[0297] Take an appropriate amount of this product (batch number: KL-01), grind it into a fine powder, and accurately weigh approximately 0.1g, 0.2g, and 0.3g respectively. Place each powder in a stoppered conical flask, and accurately add 25ml of 30% methanol for each flask. Seal the flask tightly, weigh the flask, and extract using ultrasonic treatment (600W power, 40kHz frequency) for 30 minutes. After cooling, weigh the flask again, and replenish the lost weight with 30% methanol. Shake well, filter, and collect the filtrate. Calculate the proanthocyanidin B2 content; the results are shown in Table 37.

[0298] Table 37 Analytical results for different sampling amounts

[0299]

[0300] The results showed that when the sample size was 0.2g and the solvent volume was 25ml, the target component could be completely extracted. Therefore, when the solvent volume was 25ml, the sample size was tentatively set at 0.2g.

[0301] 3.4 Investigation of Extraction Solvents

[0302] Take an appropriate amount of this product (batch number: KL-01), grind it into a fine powder, and accurately weigh about 0.2g. Place it in a stoppered conical flask and test it with extraction solvents ethanol, methanol, 70% methanol, 50% methanol, 30% methanol, and water. Accurately add 25ml of each solvent, seal tightly, weigh, and sonicate (600W power, 40kHz frequency) for 30 minutes. Cool, weigh again, and replenish the lost weight with the corresponding solvent. Shake well, filter, and collect the filtrate. Accurately pipette 1μl of each test solution and inject it into the liquid chromatograph. Calculate the content of proanthocyanidin B2 extracted with different solvents. The results are shown in Table 38.

[0303] Table 38 Results of the investigation of different extraction solvents

[0304]

[0305] The results showed that there was little difference in extraction efficiency between 70% methanol and pure methanol, and 70% methanol solution was tentatively chosen as the extraction solvent.

[0306] In summary, the preparation method for the test sample for determining the content of Bauhinia bark granules is as follows: Take an appropriate amount of the product, grind it into a fine powder, take about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, seal tightly, weigh it, extract by ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the product.

[0307] 4. Methodological Examination

[0308] 4.1 Specificity test

[0309] Preparation of the test solution: Prepare the test solution of Bauhinia bark granules according to the experimental conditions proposed above.

[0310] Preparation of reference solution: Take appropriate amounts of proanthocyanidin B2 reference standards, accurately weigh them, and add 70% methanol to prepare a solution containing 50 μg per ml.

[0311] Preparation of negative control solution: A negative control solution lacking Bauhinia bark granules was prepared according to the experimental conditions outlined above. See the results below. Figure 21 The results showed that the negative solution chromatogram did not interfere with the determination of the target peak, indicating that the method has good specificity.

[0312] 4.2 Precision test

[0313] The reference solution was injected six times consecutively, and the peak area of ​​proanthocyanidin B2 was recorded. The RSD value was calculated, and the results are shown in Table 39.

[0314] Table 39 Precision test results

[0315]

[0316] The results showed that the peak area RSD of proanthocyanidin B2 was 0.9% in the precision study. The injection precision of this method is good.

[0317] 4.3 Linear Relationship

[0318] Take an appropriate amount of proanthocyanidin B2 reference standard (purity 98.03%), place it in a 50 ml volumetric flask, and dissolve it in 70% methanol to prepare a solution with a concentration of 201.35362 μg per ml. Then dilute it to solutions with concentrations of 8.0541448 μg, 16.1082896 μg, 24.1624344 μg, 40.2707240 μg, 80.5414480 μg, and 201.3536200 μg per ml, respectively.

[0319] Accurately pipette 1 μl of each sample and inject it into the liquid chromatograph. Obtain the peak area and plot the response curve with concentration (X, μg / ml) on the x-axis and peak area (Y) on the y-axis. The results are shown in Table 40 and 41. Figure 22 :

[0320] Table 40 Standard Curve Analysis Results

[0321]

[0322] The results showed that the linear relationship of proanthocyanidin B2 was y = 22.406x + 9.1675, with R² = 0.9999, within the concentration range of 8.0541448–201.35362 μg / mL. This indicates a good linear relationship within the concentration range of 8.0541448–201.35362 μg / mL.

[0323] 4.4 Repeatability

[0324] Take an appropriate amount of this product (batch number: KL-01), grind it into a fine powder, and accurately weigh approximately 0.2g into 6 portions. Prepare the test solution using the same operator according to the established method. Calculate the proanthocyanidin B2 content of the 6 test samples. The results are shown in Table 41.

[0325] Table 41 Results of Repeatability Experiments

[0326]

[0327] The results showed that the RSD value of proanthocyanidin B2 content in the six repeatability tests was 1.8%, indicating that the method had good repeatability.

[0328] 4.5 accuracy

[0329] Take approximately 0.1 g of the test sample (batch number: KL-01, proanthocyanidin B2 content 5.021 mg / g), a total of 6 portions, accurately weigh them, and accurately add 25 ml of a control solution containing proanthocyanidin B2 (purity: 98.03%) to each portion. Prepare and determine the test solution according to the prescribed method, calculate the recovery rate, and the results are shown in Table 42. The calculation formula is as follows:

[0330] Recovery rate (%) = (Measured amount - Content in sample) / Amount added × 100%.

[0331] Table 42 Results of the Proanthocyanidin B2 Recovery Experiment

[0332]

[0333] The results showed that the recovery rate RSD of proanthocyanidin B2 was 1.5%. The method had good accuracy.

[0334] 4.6 Intermediate Precision

[0335] 4.6.1 Investigation with different instruments

[0336] According to the determined method, accurately weigh 0.2 g of the powder (batch number: KL-01) into two portions and examine them separately on Instrument 1 and a Waters ultra-high performance liquid chromatograph (both columns are Agilent ZORBAX SB-Aq C18100×2.1 mm, 1.8 μm). Calculate the total content of proanthocyanidins B2. The results are shown in Table 43.

[0337] Table 43 Experimental Results with Different Instruments

[0338]

[0339] The results showed that the RSD value of the results measured by the Waters and Agilent 1290 high performance liquid chromatograph was 0.3%, indicating that the instrument has good durability.

[0340] 4.6.2 Investigations by different personnel and at different times

[0341] The same test sample (batch number: KL-01) was analyzed by different personnel (A, B) at different times (I, II) to prepare test solutions. The content of proanthocyanidins B2 in the test sample was calculated, and the results are shown in Table 44:

[0342] Table 44 Results of the surveys conducted by different personnel and at different times

[0343]

[0344] The results showed that the RSD value of proanthocyanidin B2 content was 1.3%, indicating good intermediate precision of this method.

[0345] 4.7 Durability Test

[0346] 4.7.1 Column robustness test

[0347] The same test sample (batch number: KL-01) was detected using C18 columns (100×2.1mm, 1.8μm) from different batches of the same brand. The results are shown in Table 45.

[0348] Table 45 Durability Test Results

[0349]

[0350] The results showed that the analytical chromatographic parameters of different chromatographic columns were good.

[0351] 4.7.2 Stability Test

[0352] The same test sample (batch number: KL-01) solution was used, and the peak area of ​​proanthocyanidin B2 was determined at 0h, 2h, 4h, 8h, 16h, and 24h, respectively. The results are shown in Table 46.

[0353] Table 46 Results of the stability test of proanthocyanidin B2

[0354]

[0355] The results showed that the RSD value of the peak area of ​​proanthocyanidin B2 was 2.0%, and the test solution had good stability within 24 hours.

[0356] 5. The verification results of three batches of Bauhinia bark (Schisandra chinensis) formula granules are shown in Table 47.

[0357] Table 47 Verification of the content of Bauhinia bark (Schisandra chinensis) in 3 batches of formula granules

[0358]

[0359] Conclusion: The method for determining the content of Bauhinia bark (Schisandra chinensis) in formula granules can effectively detect the content of Bauhinia bark (Schisandra chinensis) in formula granules, proving that the method is feasible.

[0360] 6. Determination of the content of Bauhinia bark (Schisandra chinensis) in formula granules

[0361] [Content Determination] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0362] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase (Agilent ZORBAX SB-Aq C18, 100 mm × 2.1 mm, 1.6 μm or similar performance column); methanol-acetonitrile (70:30) as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in Table 1 above; flow rate 0.4 mL / min; column temperature 30 °C; detection wavelength 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should be no less than 5000.

[0363] Preparation of the reference solution: Take an appropriate amount of proanthocyanidin B2 reference standard, accurately weigh it, and add 70% methanol to prepare a mixed solution containing 50 μg of each in 1 ml.

[0364] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 70% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0365] The assay involves precisely pipetting 1 µl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0366] As can be seen from the above embodiments, the HPLC content determination method for Bauhinia bark (Schisandra chinensis) medicinal slices, standard decoctions, and formulation granules provided by the present invention is a means to detect the content of proanthocyanidin B2, an indicator component of Bauhinia bark (Schisandra chinensis) medicinal slices, standard decoctions, and formulation granules, based on the effective component proanthocyanidin B2. It can not only control the quality of Bauhinia bark (Schisandra chinensis) medicinal slices, but also provide an effective detection and quality control means for the entire production process of Bauhinia bark (Schisandra chinensis) formulation granules, as well as the production of related derivatives and derivative products.

[0367] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a high-performance liquid chromatography (HPLC) characteristic spectrum for the content determination of Bauhinia bark medicinal materials, processed slices, standard decoctions, and formulated granules, comprising the following steps: A) Extract the Bauhinia bark raw material with methanol to obtain the test solution; the Bauhinia bark raw material is the dried root bark of Kadsura longipedunculata Finet et Gagnep., a plant of the genus Kadsura in the family Magnoliaceae; The proanthocyanidin B2 reference standard was mixed with methanol to obtain the reference standard solution; B) The test solution and the reference solution were determined by high performance liquid chromatography, and chromatograms of proanthocyanidin B2 in the reference solution and the test solution were obtained under the same detection conditions. The content of proanthocyanidin B2 in Bauhinia bark was calculated using the external standard method based on the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the area of ​​proanthocyanidin B2 in Bauhinia bark and the reference solution in the chromatogram. The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: the column is an Agilent ZORBAX SB-Aq C18 column with dimensions of 100×2.1 mm and 1.8 μm; mobile phase A is a 70:30 methanol-acetonitrile mixed solvent, mobile phase B is a 0.2% formic acid solution, and gradient elution is used; the detection wavelength is 200~205 nm. The gradient elution specifically refers to: 0~20min, Phase A: 7~10%, Phase B: 93~90%.

2. The construction method according to claim 1, characterized in that, The volume fraction of methanol is 68-73%; The volume ratio of methanol to the mass ratio of Bauhinia bark or processed medicinal material is 25 mL: (0.48~0.53) g; The volume ratio of methanol to the mass ratio of Bauhinia bark standard decoction or Bauhinia bark formula granules is 25 mL: (0.18~0.23) g.

3. The construction method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.2~0.4 ml / min; the column temperature is 20~40℃; the theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

4. The construction method according to claim 1, characterized in that, The extraction method was ultrasonic extraction, with an ultrasonic extraction power of 580~620W, an ultrasonic extraction frequency of 35~45kHz, and an ultrasonic extraction time of 25~35min.

5. The construction method according to claim 1, characterized in that, The concentration of the reference solution is 45~55 μg / ml.

6. The construction method according to claim 1, characterized in that, The injection concentration of proanthocyanidin B2 solution ranged from 8.0541448 to 201.3536200 μg / mL. The regression equation for proanthocyanidin B2 was y = 22.406x + 9.1675, R0. 2 =0.9999.

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