A method for constructing HPLC characteristic chromatogram of Chinese herbal medicine slices of redbud bark, standard decoction and formula granules

Abstract

The application provides a construction method of HPLC characteristic spectrum of redbud bark medicinal material decoction pieces, standard decoction and formula granules, comprising the following steps: A) dissolving raw materials of the test sample by using a solvent, extracting, and obtaining a to-be-tested solution; B) determining the to-be-tested solution by using high performance liquid chromatography, and obtaining the HPLC characteristic spectrum of the redbud bark medicinal material decoction pieces, the standard decoction and the formula granules; and the high performance liquid chromatography chromatographic conditions are as follows: a C 18 column is used as the chromatographic column; a mobile phase A is acetonitrile solution; a mobile phase B is 0.2% formic acid aqueous solution; and gradient elution is adopted. The application adopts the high performance liquid chromatography, selects acetonitrile-0.2% formic acid solution as the mobile phase for gradient elution, takes procyanidin B2 as a reference substance, establishes the HPLC characteristic spectrum method of the redbud bark medicinal material decoction pieces, the standard decoction and the formula granules, and provides more scientific technical means for controlling the medicinal quality of the redbud bark medicinal material decoction pieces, the standard decoction and the formula granules.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and in particular to a construction method of HPLC characteristic spectrum of Chinese herbal medicine decoction pieces, standard decoction and formula granules of Cercidiphyllum japonicum. BACKGROUND

[0002] Cercidiphyllum japonicum (Schneid.) is the dried root bark of Kadsura longipedunculata Finet et Gagnep. of Magnoliaceae Kadsura, which has the effects of activating blood, unblocking channels, relieving swelling and pain, and detoxifying. It is used for blood stasis with amenorrhea, dysmenorrhea, sprains and injuries, rheumatism and arthralgia, and sore throat.

[0003] In order to ensure the uniformity and stability of the quality of Cercidiphyllum japonicum decoction pieces, standard decoction and formula granules, a new characteristic spectrum method needs to be established to control the quality. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a construction method of HPLC characteristic spectrum of Chinese herbal medicine decoction pieces, standard decoction and formula granules of Cercidiphyllum japonicum.

[0005] A construction method of HPLC characteristic spectrum of Chinese herbal medicine decoction pieces, standard decoction and formula granules of Cercidiphyllum japonicum, comprising:

[0006] A) dissolving and extracting the test sample raw material with a solvent to obtain a test solution;

[0007] B) determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic spectrum of Cercidiphyllum japonicum Chinese herbal medicine decoction pieces, standard decoction and formula granules;

[0008] The high performance liquid chromatography has the following chromatographic conditions: a C18 column is used as the chromatographic column; the mobile phase A is acetonitrile solution, the mobile phase B is 0.2% formic acid aqueous solution, and gradient elution is used.

[0009] The test sample raw material is one or more of Cercidiphyllum japonicum Chinese herbal medicine, Cercidiphyllum japonicum decoction pieces, Cercidiphyllum japonicum standard decoction and Cercidiphyllum japonicum formula granules.

[0010] The construction method of HPLC characteristic spectrum of Cercidiphyllum japonicum Chinese herbal medicine decoction pieces, standard decoction and formula granules first dissolves and extracts the test sample raw material with a solvent to obtain a test solution.

[0011] When the raw material is Chinese corktree bark medicinal material and Chinese corktree bark decoction pieces, step A) is to use water to decoct the Chinese corktree bark medicinal material or Chinese corktree bark decoction pieces, filter, then dissolve the residue with 30% methanol, filter to obtain the test solution; the ratio of the mass g of the test sample raw material, the volume mL of water and the volume mL of 30% methanol is 1:50:25; the decocting time is 30 min.

[0012] When the raw material is Chinese corktree bark standard decoction or Chinese corktree bark formula granules, step A) is to use 30% methanol to ultrasonically treat the Chinese corktree bark standard decoction or Chinese corktree bark formula granules, cool, filter to obtain the test solution;

[0013] The power of the ultrasonication is 600 W, the frequency is 40 kHz; the ultrasonic time is 30 min;

[0014] The ratio of the mass g of the test sample raw material and the volume mL of solvent is 0.2:25.

[0015] The above raw materials of the present application can be subjected to quality control and qualitative and quantitative detection by the method of the present application.

[0016] The present application also comprises preparing a control and a control medicinal material reference solution:

[0017] Take procyanidin B2, dissolve with 30% methanol to obtain the reference solution;

[0018] Take Chinese corktree bark control medicinal material, decoct with water, cool, centrifuge, dissolve the residue with 30% methanol by ultrasonication, filter to obtain the control medicinal material reference solution;

[0019] Determine the control and the control medicinal material reference solution by high performance liquid chromatography to obtain the chromatogram of the control and the control medicinal material reference solution respectively; and qualitatively determine the components of the HPLC characteristic chromatogram of Chinese corktree bark medicinal material, decoction pieces, standard decoction and formula granules according to the chromatogram of the control and the control medicinal material reference solution.

[0020] The concentration of the reference solution of the present application is preferably specifically: procyanidin B2 is 50 μg / mL.

[0021] Determine the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Chinese corktree bark medicinal material decoction pieces, standard decoction and formula granules.

[0022] The high performance liquid chromatography chromatography condition is: the chromatography column is a C18 column; the chromatography column is a column with a specification of 100*2.1mm 1.8 μm; the column temperature is 20 ℃; the chromatography peak shape of the present application is symmetrical at the above chromatography column temperature, the resolution is good and the peak is completely separated.

[0023] The mobile phase A is an acetonitrile solution, and the mobile phase B is a 0.2% formic acid aqueous solution, and gradient elution is adopted.

[0024] Specifically, the gradient elution is specifically as follows:

[0025] 0-18min, A phase: 2%-4%, B phase: 98-96%;

[0026] 18-20min, A phase: 4%, B phase: 96%-96%;

[0027] 20-26min, A phase: 4%-9%, B phase: 96%-91%;

[0028] 26-37min, A phase: 9%, B phase: 91%;

[0029] 37-42min, A phase: 9%-11%, B phase: 91%-89%.

[0030] The theoretical plate number calculated based on procyanidin B2 should be not less than 5000.

[0031] The present application has good baseline separation under the above elution gradient, and the separation degree of each peak is good, and the baseline is stable.

[0032] The flow rate of the mobile phase is 0.2mL / min.

[0033] It is found that the separation of each chromatographic peak is good under the above flow rate, and the separation degree is moderate, which is the most preferred scheme.

[0034] The injection amount is 1ul.

[0035] The detection wavelength is 202nm. It is found that the information amount of the chromatographic peak is larger at 202nm, the baseline of the chromatogram is more stable, and the peak area of each peak is larger.

[0036] The present application adopts the traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of the HPLC characteristic spectrum of the Chinese herbal medicine decoction piece, the standard decoction and the formula granule of redbud bark, and obtains the HPLC standard characteristic spectrum of the Chinese herbal medicine decoction piece, the standard decoction and the formula granule of redbud bark composed of 7 characteristic peaks, peak 6 (S): procyanidin B2; the relative retention time of each characteristic peak to the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified value is 0.49 (peak 1), 0.53 (peak 2), 0.82 (peak 3), 0.89 (peak 4), 0.92 (peak 5), 1.07 (peak 7).

[0037] The present application provides a characteristic spectrum identification method for Chinese herbal medicine decoction pieces, standard decoctions and formula granules, which adopts the above-mentioned method for detection and analysis of the detection results.

[0038] The application provides a construction method of HPLC characteristic spectrum of redbud bark medicinal material decoction pieces, standard decoction and formula granules, comprising the following steps: A) dissolving and extracting raw materials of a test sample to obtain a test solution; B) determining the HPLC characteristic spectrum of the redbud bark medicinal material decoction pieces, the standard decoction and the formula granules by using high performance liquid chromatography, wherein the high performance liquid chromatography is performed under the following chromatographic conditions: a C18 column is used as the chromatographic column; acetonitrile solution is used as mobile phase A, and 0.2% formic acid solution is used as mobile phase B, and gradient elution is performed. By using high performance liquid chromatography, acetonitrile-0.2% formic acid solution is selected as the mobile phase for gradient elution, and procyanidin B2 is used as a reference substance, the HPLC characteristic spectrum method of the redbud bark medicinal material decoction pieces, the standard decoction and the formula granules is established, and more scientific technical means for controlling the medicinal quality of the redbud bark medicinal material decoction pieces, the standard decoction and the formula granules is provided. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 3D chromatogram of redbud bark formula granules;

[0040] Figure 2 Chromatogram of redbud bark formula granules at different wavelengths;

[0041] Figure 3 Column temperature investigation;

[0042] Figure 4 Flow rate investigation;

[0043] Figure 5 Delay investigation;

[0044] Figure 6 Extraction solvent investigation;

[0045] Figure 7 Extraction time investigation;

[0046] Figure 8 Sample amount investigation;

[0047] Figure 9 Chromatographic peak identification;

[0048] Figure 10 Procyanidin B2 ultraviolet absorption spectrum;

[0049] Figure 11 Sample ultraviolet absorption spectrum;

[0050] Figure 12 Different instrument investigation;

[0051] Figure 13 Different chromatographic column investigation;

[0052] Figure 14 Characteristic spectrum of redbud bark medicinal material;

[0053] Figure 15 Herbal piece characteristic map;

[0054] Figure 16 Control characteristic map;

[0055] Figure 17 Cassia bark dispensing granules 3D chromatogram;

[0056] Figure 18 Cassia bark dispensing granules different wavelength chromatogram;

[0057] Figure 19 Column temperature investigation;

[0058] Figure 20 Flow rate investigation;

[0059] Figure 21 Delay investigation;

[0060] Figure 22 Extraction method investigation;

[0061] Figure 23 Extraction solvent investigation;

[0062] Figure 24 Extraction time investigation;

[0063] Figure 25 Solvent addition amount investigation;

[0064] Figure 26 Standard decoction specificity;

[0065] Figure 27 Control product UV absorption;

[0066] Figure 28 Sample UV absorption;

[0067] Figure 29 Different chromatographic column investigation;

[0068] Figure 30 Different instrument investigation;

[0069] Figure 31 Cassia bark standard decoction characteristic map;

[0070] Figure 32 Control characteristic map;

[0071] Figure 33 Cassia bark dispensing granules 3D chromatogram;

[0072] Figure 34 Cassia bark dispensing granules different wavelength chromatogram;

[0073] Figure 35 Column temperature investigation;

[0074] Figure 36 Flow rate investigation;

[0075] Figure 37 Delay investigation;

[0076] Figure 38 Extraction solvent investigation;

[0077] Figure 39 Extraction method investigation;

[0078] Figure 40 Extraction time investigation;

[0079] Figure 41 Sample amount investigation;

[0080] Figure 42 Chromatographic peak assignment;

[0081] Figure 43 Procyanidin B2 UV absorption graph;

[0082] Figure 44 Sample UV absorption graph;

[0083] Figure 45 Different chromatographic column investigation.

[0084] Figure 46 Different instrument investigation;

[0085] Figure 47 Chinese redbud bark formula granule characteristic chromatogram;

[0086] Figure 48 Control characteristic chromatogram;

[0087] Figure 49 Different mobile phase comparison;

[0088] Figure 50 Different gradient comparison;

[0089] Figure 51 Different gradient comparison;

[0090] Figure 52 Different gradient comparison. DETAILED DESCRIPTION

[0091] The application provides a construction method of HPLC characteristic spectrum of redbud bark medicinal material decoction pieces, standard decoction and formula granules, and those skilled in the art can refer to the content herein, and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they all belong to the protection range of the application. The method and application of the application have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application herein without departing from the content, spirit and range of the application, to realize and apply the technical field of the application.

[0092] In order to further illustrate the application, the construction method of HPLC characteristic spectrum of redbud bark medicinal material decoction pieces, standard decoction and formula granules provided by the application is described in detail below in combination with embodiments.

[0093] Example 1: HPLC characteristic spectrum of redbud bark medicinal material and decoction pieces

[0094] 1. Instruments and materials

[0095] High performance liquid chromatograph: Agilent ultra-high performance liquid chromatograph, Thermo ultra-high performance liquid chromatograph;

[0096] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0097] Ultra-pure water machine: CelluSafe 1810A (Shanghai Molier Scientific Instruments Co., Ltd.);

[0098] Ultrasonic cleaner: KQ5200DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0099] 2. Reagents and reagents

[0100] Methanol, acetonitrile and formic acid are chromatographically pure, water is ultra-pure water, and the rest of the reagents are analytically pure.

[0101] Proanthocyanidin B2 (Chengdu Manster Biological Technology Co., Ltd., batch number: MUST-23041401, purity: 98.03%) redbud bark (Longgang Nanwweizi) control medicinal material (China Food and Drug Inspection Research Institute, batch number: 121161-201803

[0102] Cortex Catalpae (Schisandrae Sphenantherae) (Sichuan Xinglv Pharmaceutical Science and Technology Development Co., Ltd., Batch No.: 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.

[0103] Cortex Catalpae (Schisandrae Sphenantherae) (Sichuan Xinglv Pharmaceutical Science and Technology Development Co., Ltd., Batch No.: 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.

[0104] 3. Chromatographic conditions and system suitability test

[0105] 3.1 Proposed chromatographic conditions

[0106] Octadecylsilane-bonded silica gel was used as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B, which was gradient eluted according to the provisions in the following table; the flow rate was 0.2 ml per minute; the column temperature was 20°C; the injection volume was 1 μL; and the detection wavelength was 202 nm. The theoretical plate number calculated according to the procyanidin B2 peak should not be less than 5000. The theoretical plate number calculated according to the procyanidin B2 peak should not be less than 5000.

[0107]

[0108] 3.2 Determination of detection wavelength

[0109] Based on the above proposed experimental conditions, the diode array detector was used for full-wave band scanning of the test solution, and the chromatograms of the test solution at 202 nm, 220 nm and 279 nm wavelengths were extracted, respectively. See Figure 1 and Figure 2 . Figure 1 3D chromatogram of Cortex Catalpae Dispensing Granules; Figure 2 Chromatograms of Cortex Catalpae Dispensing Granules at different wavelengths. The results showed that the amount of chromatographic peak information was larger at the detection wavelength of 202 nm, and the baseline of the chromatogram was more stable, so the detection wavelength was determined to be 202 nm.

[0110] 3.3 Column temperature investigation

[0111] Based on the above experimental conditions, the column temperature was 15℃, 20℃, 30℃ respectively. See Figure 3 . See Table 1-2. Figure 3 Column temperature investigation.

[0112] Table 1 Column temperature investigation - retention time

[0113]

[0114] Table 2 Column temperature investigation - relative retention time

[0115]

[0116] The results show that when the column temperature is 20℃, the chromatogram peak shape is more symmetrical, the separation degree is better, and the peak is more complete. Therefore, the column temperature is determined to be 20℃.

[0117] 3.4 Flow rate investigation

[0118] When the column temperature is 25℃, the flow rate is 0.2mL / min, 0.3ml, 0.4mL respectively. See Figure 4 , Table 3-4. Figure 4 Flow rate investigation.

[0119] Table 3 Flow rate investigation - retention time

[0120]

[0121] Table 4 Flow rate investigation - relative retention time

[0122]

[0123] The results show that when the flow rate is 0.2ml / min, the chromatogram peak shape is good, and the separation degree is moderate. Therefore, the flow rate is determined to be 0.2ml / min

[0124] 3.5 Delay investigation

[0125] Based on the above experimental conditions, the delay test was carried out. The results are shown in Figure 5 . Figure 5 Delay investigation; the results show that the sample has no chromatographic peak after 42 minutes, so the sample detection time is determined to be 42 minutes.

[0126] In summary, the chromatographic conditions and system suitability test of purpleking bark granules characteristic spectrum are as follows: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, gradient elution according to the following table; detection wavelength is 202nm. The theoretical plate number should be not less than 5000 according to the procyanidin B2 peak.

[0127]

[0128]

[0129] 4 Preparation of Test Solution

[0130] 4.1 Investigation of Extraction Solvent

[0131] Take 0.2 g of the powder of the medicinal material (batch number: YC-01), grind finely, and place in a conical flask with a stopper. Investigate the test solution by adding 25 ml of each of the following extraction solvents: methanol, 30% methanol, 50% methanol, 70% methanol, ethanol, and water. Seal the flask tightly, and perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes. Allow to cool, shake well, filter, and take the filtrate, which is the test solution. See Figure 6 . Figure 6 Investigation of extraction solvent;

[0132] The results show that when the extraction solvent is 30% methanol, the peak shape is good and the separation degree is moderate. Therefore, the extraction solvent is temporarily determined to be 30% methanol.

[0133] 4.2 Investigation of Extraction Time

[0134] Take 0.2 g of the powder of the medicinal material (batch number: YC-01), grind finely, and place in a conical flask with a stopper. Add 25 ml of 30% methanol, seal the flask tightly, and perform ultrasonic treatment. Investigate the test solution by adding 25 ml of each of the following extraction solvents: methanol, 30% methanol, 50% methanol, 70% methanol, ethanol, and water. Seal the flask tightly, and perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes. Allow to cool, shake well, filter, and take the filtrate, which is the test solution. See Figure 7 . Figure 7 Investigation of extraction time; the results show that when the extraction time is 30 minutes, the peak shape and separation degree are better. Therefore, the extraction time is determined to be 30 minutes.

[0135] 4.3 Investigation of Sample Amount

[0136] Take 0.1 g, 0.2 g, and 0.3 g of the powder of the medicinal material (batch number: YC-01), grind finely, and place in a conical flask with a stopper. Investigate the sample amount by adding 25 ml of 30% methanol, seal the flask tightly, and perform ultrasonic treatment for 30 minutes. Allow to cool, shake well, filter, and take the filtrate, which is the test solution. See Figure 8 . Figure 8 Investigation of sample amount; the results show that when the sample amount is 0.2 g and the solvent addition amount is 25 ml, the peak shape and separation degree are better. Therefore, the sample amount is temporarily determined to be 0.2 g and the solvent addition amount is temporarily determined to be 25 ml.

[0137] Based on the above, the preparation method of the characteristic chromatogram of Zijingpi Jiaotang is determined as follows: take 0.2 g of the product, grind finely, place in a conical flask with a stopper, add 25 ml of 30% methanol, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, allow to cool, shake well, filter, and take the filtrate, which is the test solution.

[0138] 5 Methodology Investigation

[0139] 5.1 Chromatographic Peak Assignment

[0140] Preparation of Test Solution: The test solution of Cacumen Tetracenthinae was prepared according to the experimental conditions determined in 4.1-4.3 above.

[0141] Preparation of Reference Solution: 1 g of the reference material of Cacumen Tetracenthinae was added to a conical flask with a stopper, 50 ml of water was added, and decoction was performed for 30 minutes. After cooling, centrifugation was performed for 10 minutes at a speed of 2000 rpm per minute. The supernatant was taken, evaporated to dryness, and the residue was added to 30% methanol and ultrasonically treated for 30 minutes. Filtration was performed, and the filtrate was taken as the reference solution of the reference material. An appropriate amount of procyanidin B2 reference substance was precisely weighed and added to 70% methanol to prepare a mixed solution containing 50 μg per 1 ml, which was taken as the reference solution of the reference substance.

[0142] Preparation of Negative Control Solution: The negative control solution of Cacumen Tetracenthinae was prepared according to the experimental conditions determined above.

[0143] The characteristic peaks of Cacumen Tetracenthinae were located. See Figures 9-11 . Figure 9 Chromatographic peak assignment; Figure 10 Procyanidin B2 UV absorption spectrum; Figure 11 Sample UV absorption spectrum; the results showed that peak 6 was procyanidin B2.

[0144] 5.2 Precision Investigation

[0145] The test solution of Cacumen Tetracenthinae (batch number: YC-01) was taken, and 6 injections of 1 μl each were successively performed according to the experimental method determined. The relative retention times of the characteristic peaks were calculated. See Table 5.

[0146] Table 5 Precision Investigation - Retention Time

[0147]

[0148]

[0149] The results showed that the retention time RSD of each characteristic peak of the sample was in the range of 0.04%-0.30%, indicating that the injection precision of the method was good.

[0150] 5.3 Investigation of Different Instruments

[0151] Based on the experimental conditions determined above, two portions of Cacumen Tetracenthinae (batch number: YC-01) were weighed, and the test solutions were prepared. The determination was performed on Agilent 1290 and Thermo Vanquish Flex ultra-high performance liquid chromatographs, respectively. See Figure 12 , Table 6.Figure 12 Different instruments were investigated.

[0152] Table 6 Instrument robustness investigation - relative retention time

[0153]

[0154] The results showed that when the above two instruments were used to detect the test sample, the RSD of the relative retention time of each characteristic peak was 0.32% to 2.34%, indicating that the instrument precision of the method was good.

[0155] 5.4 Different personnel and time investigation

[0156] Based on the above proposed experimental conditions, two batches of Chinese herbal medicine (batch number: YC-01) were respectively weighed by different personnel (A, B) at different times (T1, T2), and test samples were prepared for determination. See Table 7.

[0157] Table 7 Personnel and time investigation - relative retention time

[0158]

[0159] The results showed that when the same sample was determined by different personnel at different times, the RSD of the relative retention time of each characteristic peak was 0.11% to 0.79%, indicating that the intermediate precision of the method was good.

[0160] 5.5 Column robustness investigation

[0161] Based on the above proposed experimental conditions, different serial numbers of C18 100 x 2.1 mm, 1.8 μm chromatographic columns were used for analysis investigation, and the results are shown in Table 8. Figure 13 . Figure 13 Different chromatographic columns were investigated.

[0162] Table 8 Column robustness investigation - relative retention time

[0163]

[0164] The results showed that when the above three chromatographic columns were used to detect the sample, the RSD of the relative retention time of the characteristic peak was 0% to 7.44%, indicating that the column robustness was good.

[0165] 5.6 Stability investigation

[0166] Based on the above proposed experimental conditions, the same test sample solution was measured at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. See Table 9.

[0167] Table 9 Stability investigation - retention time

[0168]

[0169] The results showed that the RSD of the retention time of the characteristic peaks was 0.04%-0.81%, and the sample solution was stable within 24 hours.

[0170] In summary, the RSD of the relative retention time of each characteristic peak met the requirements in the above investigations, and the method was good. The above 7 characteristic peaks were included in the subsequent investigations.

[0171] 6. Verification of Cortex Catalpae (Schisandrae Sphenantherae) and Establishment of HPLC Characteristic Chromatogram Method

[0172] 6. Verification results of 121 batches of Cortex Catalpae (Schisandrae Sphenantherae).

[0173] The Cortex Catalpae was tested, and the ratio of the relative retention time was calculated. The results are shown in Table 10. Figure 14 Figure 14 Characteristic chromatogram of Cortex Catalpae; (S1-S21 are 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, respectively)

[0174] Table 10 Relative retention time of characteristic chromatogram of Cortex Catalpae

[0175]

[0176]

[0177] 23 batches of Cortex Catalpae decoction pieces were determined, and the results are shown in Table 11. Figure 15 Figure 15 Characteristic chromatogram of decoction pieces. Peak 6 (S): Proanthocyanidin B2; (S1-S21 are 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, respectively)

[0178] Table 11 Relative retention time of characteristic chromatogram of decoction pieces

[0179]

[0180] ​​According to the principle of relative retention time stability and the detection of each batch of medicinal material sample, decoction piece sample, marker soup sample, intermediate sample, total mixed powder sample and granule sample with relatively high peak, a total of 7 peaks with good repeatability were selected as characteristic peaks.

[0181] The final provision is that 7 characteristic peaks should appear in the chromatogram of the test sample, and the retention time of the 7 characteristic peaks in the chromatogram of the control medicinal material reference should correspond, and the peak corresponding to the procyanidin B2 control reference peak is the S peak. The relative retention time of each characteristic peak to the S peak should be within ±10% of the specified value. The specified value is 0.49 (peak 1), 0.53 (peak 2), 0.82 (peak 3), 0.89 (peak 4), 0.92 (peak 5), 1.07 (peak 7).

[0182] The chromatographic fingerprint of 21 batches of purple bark was synthesized by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control characteristic chromatogram of purple bark was established. See Figure 16 . Figure 16 Control characteristic chromatogram; Peak 6 (S): Procyanidin B2; Column: Agilent ZORBAX SB-Aq 2.1*100mm, 1.8μm; Conclusion: The characteristic chromatogram method of purple bark (Schisandra chinensis) medicinal material and decoction piece can effectively detect purple bark (Schisandra chinensis) medicinal material and decoction piece, which proves that the method is feasible.

[0183] 7 Characteristic chromatogram method of purple bark (Schisandra chinensis) medicinal material

[0184]

Characteristic chromatogram

[0185] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler (column length 100mm, inner diameter 2.1mm, particle size 1.8μm); acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, gradient elution according to the following table; flow rate is 0.2ml per minute; column temperature is 20℃; detection wavelength is 202nm. The theoretical plate number calculated according to procyanidin B2 peak should not be less than 5000. The theoretical plate number calculated according to procyanidin B2 peak should not be less than 5000.

[0186]

[0187] Preparation of reference solution: 1 g of reference material of Cacumen Platycladi was placed in a conical flask with a stopper, 50 ml of water was added, and decoction was performed for 30 minutes. After cooling, centrifugation was performed (at a speed of 2,000 revolutions per minute) for 10 minutes. The supernatant was taken, and was evaporated to dryness. The residue was dissolved in 25 ml of 30% methanol, and ultrasonic treatment was performed (at a power of 600 W and a frequency of 40 kHz) for 30 minutes. Filtration was performed, and the filtrate was taken as the reference solution of the reference material. An appropriate amount of procyanidin B2 reference substance was precisely weighed and dissolved in 70% methanol to prepare a mixed solution containing 50 μg of procyanidin B2 per 1 ml, which was used as the reference solution of the reference substance.

[0188] Preparation of test solution: 0.7 g of Cacumen Platycladi powder (passed through a No. 4 sieve) was dissolved in the reference solution of the reference material to prepare the test solution.

[0189] Determination: 1 μl of the reference solution and the test solution was precisely taken and injected into the liquid chromatograph for determination.

[0190] Example 2: Standard decoction HPLC characteristic chromatogram

[0191] 1. Experimental instruments and materials

[0192] High-performance liquid chromatograph: Agilent ultra-high-performance liquid chromatograph, Thermo ultra-high-performance liquid chromatograph;

[0193] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0194] Ultrapure water machine: CelluSafe 1810A (Shanghai Molier Scientific Instruments Co., Ltd.);

[0195] Ultrasonic cleaner: KQ5200DB (600 W, 40 kHz; Kunshan Ultrasonic Instrument Co., Ltd.)

[0196] Methanol (chromatographically pure, Sigma-Aldrich Shanghai Trading Co., Ltd.), methanol (analytically pure, Chengdu Kolon Chemical Reagent Co., Ltd.), formic acid (chromatographically pure, Tianjin Kemio Chemical Reagent Co., Ltd.), acetonitrile (chromatographically pure, Sigma-Aldrich Shanghai Trading Co., Ltd.), and water (laboratory-made ultrapure water) were used as the mobile phase.

[0197] Procyanidin B2 (Chengdu Manster Biological Technology Co., Ltd., batch number: MUST-23041401, purity: 98.03%);

[0198] Reference material of Cacumen Platycladi (China Institute for Drug Control, batch number: 121161-201803)

[0199] Cortex Catalpae (Schisandra sphenanthera) (Sichuan New Green Pharmaceutical Science and Technology Development Co., Ltd., Batch No. 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.

[0200] 3. Investigation of chromatographic conditions

[0201] 3.1 Proposed chromatographic conditions

[0202] Octadecylsilane-bonded silica gel was used as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B, which was gradient eluted according to the provisions in the following table; the flow rate was 0.2 ml per minute; the column temperature was 20°C; the detection wavelength was 202 nm. The theoretical plate number calculated according to the procyanidin B2 peak should not be less than 5000. The theoretical plate number calculated according to the procyanidin B2 peak should not be less than 5000.

[0203]

[0204] 3.2 Determination of detection wavelength

[0205] Based on the above proposed experimental conditions, the diode array detector was used for full wave band scanning of the test solution, and the chromatograms of the test solution at 202 nm, 220 nm, 279 nm wavelengths were extracted respectively. See Figure 17 ,18. Figure 17 Cortex Catalpae Formula Granules 3D chromatogram; Figure 18 Cortex Catalpae Formula Granules chromatogram at different wavelengths.

[0206] The results showed that when the detection wavelength was 202 nm, the amount of chromatographic peak information was larger, and the baseline of the chromatogram was more stable, so the detection wavelength was determined as 202 nm.

[0207] 3.3 Column temperature investigation

[0208] Based on the above proposed experimental conditions, the column temperature was investigated at 15°C, 20°C, 30°C respectively. See Figure 19 . See Tables 12-13. Figure 19 Investigation of column temperature.

[0209] Table 12 Investigation of column temperature- retention time

[0210]

[0211] Table 13 Investigation of column temperature- relative retention time

[0212]

[0213] The results showed that at a column temperature of 20℃, the chromatogram peaks were more symmetrical, the separation was better, and the peaks were more complete. Therefore, the column temperature was determined to be 20℃.

[0214] 3.4 Flow velocity investigation

[0215] The experiment was conducted at a column temperature of 25°C and flow rates of 0.2 mL, 0.3 mL, and 0.4 mL per minute. See [link to relevant documentation]. Figure 20 Tables 14-15. Figure 20 Flow velocity study.

[0216] Table 14 Flow velocity study - retention time

[0217]

[0218] Table 15 Flow velocity study - relative retention time

[0219]

[0220]

[0221] The results showed that a flow rate of 0.2 ml / min resulted in good peak shape and moderate resolution. Therefore, the flow rate was determined to be 0.2 ml / min.

[0222] 3.5 Delayedness Assessment

[0223] Based on the above-specified experimental conditions, a delay test was conducted. The results are shown below. Figure 21 . Figure 21 Delayed investigation.

[0224] The results showed that the sample had virtually no chromatographic peaks after 42 minutes, so the sample detection time was set at 42 minutes.

[0225] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of Bauhinia bark particles were determined as follows: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 202 nm. The theoretical plate number, calculated based on the proanthocyanidin B2 peak, should not be less than 5000.

[0226]

[0227] 4. Preparation of the test solution

[0228] 4.1 Examination of Extraction Methods

[0229] Take the standard decoction (batch number: BT-01) 0.1 g, put it in a conical flask with a plug, add 30% methanol 25 ml, tightly plug, and respectively investigate the extraction method of the test sample by refluxing and ultrasonic. The extraction time is 30 minutes, cool down, shake well, filter, and take the filtrate, which is the test sample. See Figure 22 . Figure 22 Extraction method investigation. The results show that there is little difference in the effect of refluxing and ultrasonic extraction of the test sample, and the ultrasonic method is fast and simple, so the extraction method of the test sample is determined to be ultrasonic extraction.

[0230] 4.2 Extraction solvent investigation

[0231] Take the standard decoction (batch number: BT-01) 0.2 g, put it in a conical flask with a plug, and respectively investigate the extraction solvent of the test sample by adding methanol, 30% methanol, 50% methanol, 70% methanol, ethanol, and water, each 25 ml. Ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool down, shake well, filter, and take the filtrate, which is the test sample. See Figure 23 . Figure 23 Extraction solvent investigation; the results show that when the extraction solvent is 30% methanol, the peak shape of each characteristic peak is good and the separation degree is moderate, so the extraction solvent is temporarily determined to be 30% methanol.

[0232] 4.3 Extraction time investigation

[0233] Take the standard decoction (batch number: BT-01) 0.2 g, put it in a conical flask with a plug, add 30% methanol 25 ml, tightly plug, and respectively investigate the extraction time of the test sample by ultrasonic treatment for 20 minutes, 30 minutes, and 30 minutes. Cool down, shake well, filter, and take the filtrate, which is the test sample. See Figure 24 . Figure 24 Extraction time investigation; the results show that when the extraction time is 30 minutes, the peak shape and separation degree of the chromatogram are better. Therefore, the extraction time is determined to be 30 minutes.

[0234] 4.4 Sample amount investigation

[0235] Take the standard decoction (batch number: BT-01) 0.1 g, 0.2 g, and 0.3 g, respectively, put them in conical flasks with plugs, and respectively add 30% methanol 25 ml for investigation. Ultrasonic treatment for 30 minutes, cool down, shake well, filter, and take the filtrate, which is the test sample. See Figure 25 . Figure 25 Solvent addition amount investigation; the results show that when the sample amount is 0.2 g, the peak shape and separation degree of each chromatographic peak are better, so the sample amount is selected to be 0.2 g.

[0236] In summary, the preparation method of the characteristic map of the test solution of the standard decoction of the bark of Caesalpinia decapetala is determined as follows: 0.2 g of the product is placed in a conical flask with a stopper, 25 ml of 30% methanol is added, and ultrasonic treatment (power 600 W, frequency 40 kHz) is performed for 30 minutes. After cooling, the mixture is shaken and filtered. The filtrate is collected, and the standard decoction of the bark of Caesalpinia decapetala is obtained.

[0237] 5 Methodological investigation

[0238] 5.1 Chromatographic peak identification

[0239] Preparation of the test solution: The test solution of the standard decoction of the bark of Caesalpinia decapetala is prepared according to the experimental conditions determined above.

[0240] Preparation of the reference solution: 1 g of the control medicinal material of the bark of Caesalpinia decapetala (S. chinensis Baill. var. vilmorinii Dode) is placed in a conical flask with a stopper, 50 ml of water is added, and decoction is performed for 30 minutes. After cooling, centrifugation (at a speed of 2000 rpm per minute) is performed for 10 minutes. The supernatant is collected, evaporated to dryness, and the residue is added with 30% methanol and ultrasonically treated for 30 minutes. The filtrate is collected, and the reference solution of the control medicinal material is obtained. In addition, an appropriate amount of procyanidin B2 reference substance is precisely weighed and measured, and 70% methanol is added to prepare a mixed solution containing 50 μg per 1 ml, which is used as the reference substance reference solution.

[0241] Preparation of the negative control solution: The negative control solution of the formula granules without the bark of Caesalpinia decapetala is prepared according to the experimental conditions determined above.

[0242] The characteristic peaks of the standard decoction of the bark of Caesalpinia decapetala are located. See Figures 26-28 .Specificity of the standard decoction; Figure 26 Ultraviolet absorption of the control substance;Ultraviolet absorption of the sample. The results show that peak 6 is procyanidin B2. In the following methodological investigation, 7 characteristic peaks in the sample are investigated. Figure 27 Figure 28 5.2 Precision investigation

[0243] The test solution of the standard decoction of the bark of Caesalpinia decapetala (batch number: BT-01) is taken, and 6 injections of 1 μl each are continuously performed according to the experimental method determined. The retention time of each characteristic peak is calculated. See Table 16.

[0244] Table 16 Precision investigation-retention time

[0245]

[0246] The results show that the RSD of the retention time of each characteristic peak is 0.0%-0.63%, indicating that the precision of the instrument is good.

[0247] 5.3 Reproducibility investigation

[0248]

[0249] Precisely weigh 6 portions of Redbud Bark Standard Decoction (Batch No. BT-01), prepare and determine according to the proposed experimental method, and calculate the relative retention time of each characteristic peak. See Table 17.

[0250] Table 17 Reproducibility Investigation - Relative Retention Time

[0251]

[0252] The results show that the relative retention time RSD of the 6 samples is 0% to 0.61%, indicating that the method has good reproducibility.

[0253] 5.4 Different Personnel and Time Investigation

[0254] On the basis of the above proposed experimental conditions, two portions of Redbud Bark Standard Decoction (Batch No. BT-01) were precisely weighed by different personnel (A, B) at different times (T1, T2), respectively, to prepare the test sample, determine, and calculate the relative retention time of each characteristic peak. See Table 18.

[0255] Table 18 Personnel and Time Investigation - Relative Retention Time

[0256]

[0257]

[0258] The results show that the relative retention time RSD of each characteristic peak is 0% to 0.61% when the same sample is determined by different personnel at different times, indicating that the method has good stability.

[0259] 5.5 Durability Investigation

[0260] 5.5.1 Column Durability Investigation

[0261] On the basis of the above proposed experimental conditions, C18 100 x 2.1 mm, 1.8 μm columns with different serial numbers were used for analysis investigation, and the relative retention time of each characteristic peak was calculated. The results are shown in Table 19. Figure 29 . Figure 29 Different column investigation.

[0262] Table 19 Column Durability Investigation - Relative Retention Time

[0263]

[0264] The results show that the RSD of the relative retention time of the characteristic peaks is 0% to 9.14% when the sample is detected by the above three columns, indicating that the column has good durability.

[0265] 5.5.2 Different Instrument Investigation

[0266] On the basis of the above experimental conditions, two portions of the standard decoction of Cercis siliquastrum L. (Batch No. ZJP-BT-2307011) were precisely weighed, and the test sample solutions were prepared. The relative retention times of the characteristic peaks were calculated on Agilent 1290 and Thermo Vanquish Flex ultra-high performance liquid chromatographs, respectively. See Table 20. Figure 30 . Figure 30 Different instruments were investigated.

[0267] Table 20 Investigation of instrument robustness-Relative retention time

[0268]

[0269] The results showed that when the test sample was detected by the above two instruments, the RSD of the relative retention time of each characteristic peak was 0% to 0.68%, indicating that the instrument robustness was good.

[0270] 5.6 Stability investigation

[0271] On the basis of the above experimental conditions, the same test sample solution was measured at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively. See Table 21.

[0272] Table 21 Stability investigation-Retention time

[0273]

[0274]

[0275] The results showed that the RSD of the retention time of the characteristic peaks was 0.05% to 0.74%, and the sample solution was stable within 24 hours.

[0276] In summary, the RSD of the relative retention time of each characteristic peak met the requirements in the above investigations, and the method was good. The above seven characteristic peaks were included in the subsequent investigation.

[0277] 6. Verification of Cercis siliquastrum L. (Schisandra chinensis) standard decoction and establishment of HPLC content determination method

[0278] 6. Verification results of 21 batches of Cercis siliquastrum L. (Schisandra chinensis) standard decoction, see Tables 12 and 13.

[0279] Using this method, 21 batches of samples were analyzed for characteristic chromatograms, and the relative retention times were calculated. The results are shown in Figure 1 , Table 22. Figure 31Cercis siliquastrum L. standard decoction characteristic chromatogram (S1-S23 are: 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)

[0280] Table 22 Cercis siliquastrum L. standard decoction relative retention time

[0281]

[0282] According to the principles of relative retention time stability and the detection of each batch sample, 7 peaks with better durability were selected as characteristic peaks. According to the results of methodological investigation and 23 batches of standard decoction verification, the theoretical plate number was temporarily determined to be not less than 5000 calculated by procyanidin B2.

[0283] The relative retention time of each characteristic peak is stable and within the average value ± 10%, so the relative retention time of each peak is temporarily set to ± 10%, and finally it is specified that the test product chromatogram should present 7 characteristic peaks, which should correspond to the 7 characteristic peaks in the reference chromatogram of the control medicinal material, and the peak corresponding to the procyanidin B2 reference peak is S peak. The relative retention time of each characteristic peak to S peak should be within ± 10% of the specified value. The specified value is 0.49 (peak 1), 0.48 (peak 2), 0.77 (peak 3), 0.90 (peak 4), 0.93 (peak 5), 1.05 (peak 7).

[0284] The similarity evaluation system of traditional Chinese medicine chromatographic fingerprint (2012 edition) was used to synthesize 21 batches of Cercis siliquastrum L. standard decoction, and the control chromatogram of Cercis siliquastrum L. standard decoction characteristic chromatogram was established. See Figure 32 . Figure 32 Control characteristic chromatogram; Peak 6 (S): procyanidin B2; Column: Agilent ZORBAX SB-Aq 2.1*100mm, 1.8μm. Conclusion: The characteristic chromatogram method of Cercis siliquastrum L. (Schisandra sphenanthera) standard decoction can effectively detect Cercis siliquastrum L. (Schisandra sphenanthera) standard decoction, and prove that the method is feasible.

[0285] 7 Cercis siliquastrum L. (Schisandra sphenanthera) standard decoction characteristic chromatogram method

[0286]

Characteristic chromatogram

[0287] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler (column length 100 mm, internal diameter 2.1 mm, particle size 1.8 μm); acetonitrile as the mobile phase A, 0.2% formic acid solution as the mobile phase B, gradient elution according to the provisions in the following table; flow rate was 0.2 ml per minute; column temperature was 20 °C; detection wavelength was 202 nm. Theoretical plate number should not be less than 5000 calculated by procyanidin B2 peak.

[0288]

[0289] Preparation of reference solution: 1 g of reference material of Cornus officinalis Sieb. et Zucc. (Schizophragma multiflorum) was placed in a conical flask with a plug, 50 ml of water was added, decocted for 30 minutes, cooled, centrifuged (speed was 2000 rpm per minute) for 10 minutes, the supernatant was taken, evaporated to dryness, the residue was dissolved with 25 ml of 30% methanol by ultrasonic treatment (power was 600 W, frequency was 40 kHz) for 30 minutes, filtered, and the filtrate was taken as the reference solution of the reference material. In addition, procyanidin B2 reference substance was accurately weighed, 70% methanol was added to prepare a mixed solution containing 50 μg per 1 ml, which was taken as the reference solution of the reference substance.

[0290] Preparation of test solution: 0.2 g of standard decoction powder was finely ground, placed in a conical flask with a plug, 25 ml of 30% methanol was added, ultrasonic treatment (power was 600 W, frequency was 40 kHz) was carried out for 30 minutes, cooled, shaken uniformly, filtered, and the filtrate was taken.

[0291] Determination method: 1 μl of the reference solution and the test solution was precisely taken respectively, injected into the liquid chromatograph, and determined, and then the HPLC characteristic spectrum of the granules was obtained.

[0292] Example 3 HPLC characteristic spectrum of formula granules

[0293] 1 Experimental instruments and materials

[0294] High performance liquid chromatograph: Agilent ultra-high performance liquid chromatograph, Thermo ultra-high performance liquid chromatograph;

[0295] Electronic balance: ME204E / 02, MS205DΜ, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0296] Ultra-pure water machine: cell type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0297] Ultrasonic cleaner: KQ5200DB type (600 W, 40 KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0298] 2 Reagents and reagents

[0299] Methanol, acetonitrile, formic acid were chromatographically pure, water was ultra-pure water, and the rest of the reagents were analytically pure.

[0300] Procyanidin B2 (Chengdu Manster Biotechnology Co., Ltd., batch number: MUST-23041401, purity: 98.03%)

[0301] Cortex Catalpae (Kadsura longipedunculata) control medicinal material (China Institute for Drug Control, batch number: 121161-201803)

[0302] Cortex Catalpae formula granules (Sichuan New Green Medicine Technology Development Co., Ltd., batch numbers: KL-01, KL-02, KL-03.)

[0303] 3. Chromatographic condition investigation

[0304] 3.1 Proposed chromatographic conditions

[0305] Octadecylsilane-bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, gradient elution according to the provisions in the following table; flow rate is 0.2 ml per minute; column temperature is 20°C; detection wavelength is 202 nm. Theoretical plate number should not be less than 5000 according to procyanidin B2 peak. Theoretical plate number should not be less than 5000 according to procyanidin B2 peak.

[0306]

[0307] 3.2 Determination of detection wavelength

[0308] On the basis of the above proposed experimental conditions, the diode array detector was used to scan the full wave band of the test solution, and the chromatograms of the test solution at 202 nm, 220 nm and 279 nm wavelengths were extracted respectively. See Figure 33 ,34. Figure 33 Cortex Catalpae formula granules 3D chromatogram; Figure 34 Cortex Catalpae formula granules chromatogram at different wavelengths.

[0309] The results showed that when the detection wavelength was 202 nm, the amount of chromatographic peak information was larger, and the baseline of the chromatogram was more stable, so the detection wavelength was determined as 202 nm.

[0310] 3.3 Column temperature investigation

[0311] On the basis of the above proposed experimental conditions, the column temperature was investigated at 15°C, 20°C and 30°C respectively. See Figure 34 . See Tables 23-24. Figure 35 Column temperature investigation.

[0312] Table 23 Column temperature investigation-retention time

[0313]

[0314] Table 24 Column temperature investigation - relative retention time

[0315]

[0316] The results show that when the column temperature is 20°C, the chromatogram peak shape is relatively symmetrical, the resolution is good, and the peak is complete. Therefore, the column temperature is determined to be 20°C.

[0317] 3.4 Flow rate investigation

[0318] When the column temperature is 25°C, the flow rates of 0.2 mL / min, 0.3 mL / min and 0.4 mL / min were investigated, respectively. See Tables 25-26. Figure 36 Figure 36 Flow rate investigation.

[0319] Table 25 Flow rate investigation - retention time

[0320]

[0321]

[0322] Table 26 Flow rate investigation - relative retention time

[0323]

[0324] The results show that when the flow rate is 0.2 mL / min, the chromatogram peak shape is good, and the resolution is moderate. Therefore, the flow rate is determined to be 0.2 mL / min.

[0325] 3.5 Delayed investigation

[0326] Based on the above proposed experimental conditions, the delayed test was carried out. The results are shown in Table 27. Figure 37 Figure 37 Delayed investigation.

[0327] The results show that the sample has no chromatographic peak after 42 minutes, so the sample detection time is determined to be 42 minutes.

[0328] In summary, the chromatographic conditions and system suitability test of purple katsura bark granules characteristic map are determined as follows: octadecylsilane bonded silica gel is used as the filler; acetonitrile is used as the mobile phase A, and 0.2% formic acid solution is used as the mobile phase B, which is gradient eluted according to the following table; the detection wavelength is 202 nm. The theoretical plate number calculated by procyanidin B2 peak should be not less than 5000.

[0329]

[0330] 4 Investigation of preparation of test solution

[0331] 4.1 Investigation of extraction solvent ​​

[0332] Take the Redbud bark formula granules (batch number: KL-01) 0.2 g, fine, placed in a conical flask with a plug, respectively, the test sample extraction solvent is methanol, 30% methanol, 50% methanol, 70% methanol, ethanol, water, 25 ml were investigated, tightly sealed, ultrasonic treatment (power 600 W, frequency 40 kHz) 30 minutes, cool, shake, filter, take the filter, that is. See Figure 38 . Figure 38 Extraction solvent investigation; results show that the extraction solvent is 30% methanol, the peak shape is good, the separation degree is moderate, and the extraction solvent is temporarily determined as 30% methanol.

[0333] 4.2 Extraction method investigation

[0334] Take the Redbud bark formula granules (batch number: KL-01) 0.1 g, fine, placed in a conical flask with a plug, add 30% methanol 25 ml, tightly sealed, respectively, the test sample extraction method is reflux, ultrasonic, and the extraction time is 30 minutes, cool, shake, filter, take the filter, that is. See Figure 39 . Figure 39 Extraction method investigation.

[0335] The results show that there is little difference between reflux and ultrasonic extraction of the test sample, and the ultrasonic method is fast and simple, so the test sample extraction method is determined as ultrasonic extraction.

[0336] 4.3 Extraction time investigation

[0337] Take the Redbud bark formula granules (batch number: KL-01) 0.2 g, fine, placed in a conical flask with a plug, add 30% methanol 25 ml, tightly sealed, ultrasonic treatment, respectively, the test sample extraction time is 20 minutes, 30 minutes, 40 minutes, cool, shake, filter, take the filter, that is. See Figure 40 . Figure 40 Extraction time investigation; results show that the extraction time is 30 minutes, the chromatogram peak shape and separation degree are better. Therefore, the extraction time is determined as 30 minutes.

[0338] 4.4 Sample size investigation

[0339] Take the Redbud bark formula granules (batch number: KL-01) 0.1 g, 0.2 g, 0.3 g, respectively, fine, placed in a conical flask with a plug, add 30% methanol 25 ml to investigate the sample size, tightly sealed, ultrasonic treatment 30 minutes, cool, shake, filter, take the filter, that is. See Figure 41 . Figure 41 Sample size investigation; results show that the sample size is 0.2 g, the solvent addition amount is 25 ml, the peak shape and separation degree of each chromatographic peak are better, so the sample size is temporarily determined as 0.2 g, and the solvent addition amount is 25 ml.

[0340] In summary, the preparation method of the test solution for the characteristic chromatogram of Bauhinia bark decoction is determined as follows: Take 0.2g of this product, grind it into a fine powder, place it in a stoppered conical flask, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0341] 5. Methodological Investigation

[0342] 5.1 Chromatographic Peak Identification

[0343] Preparation of the test solution: Prepare the test solution of Bauhinia bark granules according to the experimental conditions determined above.

[0344] Preparation of reference solution: Take 1g of Bauhinia bark (Schisandra chinensis long-stemmed) reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, cool, centrifuge (2000 rpm) for 10 minutes, collect the supernatant, evaporate to dryness, add 30% methanol to the residue, sonicate for 30 minutes, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of proanthocyanidin B2 reference standard, accurately weigh it, add 70% methanol to prepare a mixed solution containing 50μg of each per ml, as the reference solution.

[0345] Preparation of negative control solution: Prepare negative control solution of Bauhinia bark-deficient formula granules according to the experimental conditions proposed above.

[0346] The characteristic spectral peaks of the Bauhinia bark formulation granules were located. (See...) Figures 42-44 . Figure 42 Chromatographic peak identification; Figure 43 UV absorption spectrum of proanthocyanidin B2; Figure 44 UV absorption spectrum of the sample. The results show that peak 6 is proanthocyanidin B2.

[0347] 5.2 Precision test

[0348] Take the test solution of Bauhinia bark granules (batch number: KL-01) and inject it 6 times consecutively according to the proposed experimental method, 10 μl each time, and calculate the relative retention time of each characteristic peak. See Table 27.

[0349] Table 27 Precision Examination - Retention Time

[0350]

[0351] The results showed that the retention time RSD of each characteristic peak of the sample ranged from 0.04% to 0.30%, indicating that the method has good injection precision.

[0352] 5.3 Repeatability Test

[0353] Take 6 parts of Chinese redbud bark granules (batch number: KL-01), and prepare and determine according to the proposed experimental method. See Table 28.

[0354] Table 28 Reproducibility Investigation-Relative Retention Time

[0355]

[0356] The results show that the relative retention time RSD of the 6 samples is 0.08% to 0.86%, indicating that the method has good reproducibility.

[0357] 5.4 Different Personnel and Time Investigation

[0358] On the basis of the above proposed experimental conditions, two samples of Chinese redbud bark granules (batch number: KL-01) were prepared by different personnel (A, B) at different times (T1, T2), and the test samples were determined. See Table 29.

[0359] Table 29 Personnel and Time Investigation-Relative Retention Time

[0360]

[0361] The results show that the RSD of the relative retention time of each characteristic peak is 0.11% to 0.79% when the same sample is determined by different personnel at different times, indicating that the method has good intermediate precision.

[0362] 5.5 Intermediate Precision

[0363] 5.5.1 Column Durability Investigation

[0364] On the basis of the above proposed experimental conditions, different serial numbers of C18 100 x 2.1 mm, 1.8 μm chromatographic columns were used for analysis investigation, and the results are shown in Table 30. Figure 45 Figure 45 Different chromatographic column investigation.

[0365] Table 30 Column Durability Investigation-Relative Retention Time

[0366]

[0367] The results show that the RSD of the relative retention time of the characteristic peaks is 0.08% to 1.09% when the sample is detected by the above three chromatographic columns, indicating that the method has good column durability.

[0368] 5.5.2 Different Instrument Investigation

[0369] ​On the basis of the above experimental conditions, two batches of Chinese redbud bark formula granules (batch number: KL-01) were weighed and test sample solutions were prepared, which were determined on Agilent 1290 and Thermo Vanquish Flex ultra-high performance liquid chromatographs, respectively. See Figure 46 Table 31. Figure 46 Different instruments were investigated.

[0370] Table 31 Instrument durability investigation-Relative retention time

[0371]

[0372] The results showed that when the test sample was detected by the above two instruments, the RSD of the relative retention time of each characteristic peak was 0.32% to 2.34%, indicating that the instrument precision of the method was good.

[0373] 5.6 Stability investigation

[0374] On the basis of the above experimental conditions, the same test sample solution was taken and determined at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. See Table 32.

[0375] Table 32 Stability investigation-Retention time

[0376]

[0377] The results showed that the RSD of the retention time of the characteristic peaks was 0.46% to 1.99%, indicating that the sample solution was stable within 24 hours.

[0378] In summary, the RSD of the relative retention time of each characteristic peak met the requirements in the above investigations, and the method was good.

[0379] The above 7 characteristic peaks were included in the subsequent investigation

[0380] 5.7 Determination of characteristic peaks and establishment of control chromatogram

[0381] 5.7.13 Verification results of 3 batches of Chinese redbud bark (Kadsura longipedunculata) formula granules

[0382] Using this method, 3 batches of samples were analyzed for characteristic chromatograms, and the relative retention time was calculated. See Figure 47 Table 33. Figure 47 Characteristic chromatogram of Chinese redbud bark formula granules.

[0383] Table 33 Relative retention time of Chinese redbud bark formula granules

[0384]

[0385] According to the principle of relative retention time stability and the detection of each batch sample, 7 peaks with better durability were selected as characteristic peaks, including peak 1, peak 2, peak 3, peak 4, peak 5, peak 6 and peak 7. According to the results of methodological investigation and verification of 3 batches of granules, the theoretical plate number calculated based on procyanidin B2 peak should not be less than 5000.

[0386] The results of each investigation item and verification of the method are shown in Table 34:

[0387] Table 34 Results of each item of the method RSD% summary standard - relative retention time / retention time

[0388]

[0389] The relative retention time of each characteristic peak is stable and within the average value ± 10%, so the relative retention time of each peak is tentatively set to ± 10%.

[0390] Finally, the test sample chromatogram should show 7 characteristic peaks, which should correspond to the retention time of the 7 characteristic peaks in the reference chromatogram of the control medicinal material, and the peak corresponding to the procyanidin B2 reference peak is the S peak. The relative retention time of the remaining characteristic peaks to the S peak should be within ± 10% of the specified value. The specified value is: 0.49 (peak 1), 0.53 (peak 2), 0.82 (peak 3), 0.89 (peak 4), 0.92 (peak 5), 1.07 (peak 7).

[0391] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprint (2012 edition) was used to synthesize 3 batches of acacia cortex formula granules, and the control chromatogram of acacia cortex formula granule characteristic chromatogram was established. See Figure 48 . Figure 48 Control characteristic chromatogram; peak 6 (S): procyanidin B2; chromatographic column: Agilent ZORBAX SB-Aq 2.1*100mm, 1.8μm. Conclusion: The characteristic chromatogram method of acacia cortex (Schisandra chinensis) formula granules can effectively detect acacia cortex (Schisandra chinensis) formula granules, which proves that the method is feasible.

[0392] 6 Determination of acacia cortex (Schisandra chinensis) formula granule characteristic chromatogram method

[0393]

Characteristic chromatogram

[0394] Chromatographic conditions and system suitability test with octadecylsilane-bonded silica gel as the filler (100 mm x 2.1 mm, 1.8 μm); acetonitrile as the mobile phase A, 0.2% formic acid solution as the mobile phase B, gradient elution according to the provisions in the following table; the flow rate was 0.2 ml per minute; the column temperature was 20°C; the detection wavelength was 202 nm. The theoretical plate number should not be less than 5000 calculated by the procyanidin B2 peak.

[0395]

[0396] Preparation of reference solution: 1 g of reference material of Chinese herbal medicine of Cornus officinalis was placed in a conical flask with a plug, 50 ml of water was added, decocted for 30 minutes, cooled, centrifuged (at a speed of 2000 revolutions per minute) for 10 minutes, the supernatant was taken, evaporated to dryness, the residue was dissolved with 25 ml of 30% methanol by ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, filtered, and the filtrate was taken as the reference solution of the reference material of Chinese herbal medicine. In addition, an appropriate amount of procyanidin B2 reference substance was accurately weighed and dissolved in 70% methanol to prepare a mixed solution containing 50 μg per 1 ml, which was used as the reference solution of the reference substance.

[0397] Preparation of test solution: 0.2 g of Cornus officinalis formula granules was finely ground and placed in a conical flask with a plug, 25 ml of 30% methanol was added, ultrasonic treatment (power 600 W, frequency 40 kHz) was carried out for 30 minutes, cooled, shaken uniformly, filtered, and the filtrate was taken as the test solution.

[0398] Determination: 1 μl of the reference solution and the test solution was precisely taken and injected into the liquid chromatograph for determination.

[0399] Comparative Example 1

[0400] Using acetonitrile-0.2% formic acid, methanol-0.2% formic acid, and acetonitrile-0.2% phosphoric acid as different mobile phases, respectively, the rest was the same as in Example 1. The results showed that the elution ability of methanol-0.2% formic acid and acetonitrile-0.2% phosphoric acid was poor, and the elution ability of acetonitrile-0.2% formic acid was the best. Therefore, acetonitrile-0.2% formic acid was used as the mobile phase. Figure 49 Comparison of different mobile phases.

[0401] Comparative Example 2

[0402] Using methanol-0.2% formic acid as the mobile phase, gradient 1, gradient 2, and gradient 3 were used for elution, respectively, and the rest was the same as in Example 1. The results showed that the separation ability of gradient 2 and gradient 3 was poorer than that of gradient 1, and the elution effect and separation ability were poor. Gradient 3 had a long time and high time cost. Gradient 1 had the best elution ability and separation effect, and the time was appropriate. Therefore, gradient 1 was used as the gradient for this implementation. Figures 50-52 Comparison of different gradients.

[0403] Gradient 1

[0404]

[0405] Gradient 2

[0406]

[0407] Gradient 3

[0408]

[0409] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the scope of the present application.

Claims

1. A method for constructing HPLC characteristic chromatograms of Bauhinia bark medicinal slices, standard decoctions, and formulated granules, comprising: A) The test sample raw material is extracted with solvent to obtain the test solution; the test sample raw material is the dried root bark of *Kadsura longipedunculata* Finet et Gagnep., a plant of the genus *Kadsura* in the family Magnoliaceae; the test sample raw material is one or more of the following: *Bauhinia purpurea* bark medicinal material, *Bauhinia purpurea* bark slices, *Bauhinia purpurea* bark standard decoction, or *Bauhinia purpurea* bark formula granules. When the raw materials are Bauhinia bark medicinal materials and Bauhinia bark slices, step A) is to decoct the Bauhinia bark medicinal materials or Bauhinia bark slices in water, filter, dissolve the residue in 30% methanol, filter, and obtain the test solution; When the raw material is Bauhinia bark standard decoction or Bauhinia bark formula granules, step A) is to ultrasonically treat the Bauhinia bark standard decoction or Bauhinia bark formula granules with 30% methanol, cool, filter, and obtain the test solution. B) The test solution was determined by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of Bauhinia bark medicinal slices, standard decoction, and formula granules; The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: a C18 column with dimensions of 100 × 2.1 mm × 1.8 μm; a detection wavelength of 202 nm; mobile phase A being acetonitrile solution and mobile phase B being 0.2% formic acid aqueous solution, with gradient elution; the gradient elution specifically involves: 0–18 min, Phase A: 2%–4%, Phase B: 98–96%; 18–20 min, Phase A: 4%, Phase B: 96%; 20–26 min, Phase A: 4%–9%, ​​Phase B: 96%–91%; 26–37 min, Phase A: 9%, Phase B: 91%; 37~42min, phase A: 9%~11%, phase B: 91%~89%.

2. The method according to claim 1, characterized in that, This also includes the preparation of reference standards and reference drug solutions: Proanthocyanidin B2 was dissolved in 30% methanol to obtain a reference solution; The reference herb, Bauhinia bark, was decocted in water, cooled, centrifuged, and the residue was dissolved by sonication with 30% methanol and filtered to obtain a reference herb solution. The reference standard and reference medicinal material solutions were analyzed by high performance liquid chromatography to obtain chromatograms of the reference standard and reference medicinal material, respectively; and the components of the Bauhinia bark, decoction pieces, standard decoction and its formula granules were qualitatively determined based on the chromatograms of the reference standard and reference medicinal material.

3. The method according to claim 2, characterized in that, The specific concentration of the reference solution was: proanthocyanidin B2 was 50 μg / mL.

4. The method according to claim 1, characterized in that, The column temperature is 20℃; the theoretical plate number, calculated based on the proanthocyanidin B2 peak, should be no less than 5000.

5. The method according to claim 1, characterized in that, The flow rate of the mobile phase was 0.2 mL / min; the injection volume was 1 μL.

6. The method according to claim 1, characterized in that, The similarity of HPLC characteristic chromatograms of Bauhinia bark decoction pieces, standard decoction, and formula granules was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The results showed that the HPLC standard characteristic chromatograms of Bauhinia bark decoction pieces, standard decoction, and formula granules consisted of 7 characteristic peaks, with peak 6S representing proanthocyanidin B2. Using the peak corresponding to the reference peak of proanthocyanidin B2 as the S peak, calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value, which is 0.49 (peak 1), 0.53 (peak 2), 0.82 (peak 3), 0.89 (peak 4), 0.92 (peak 5), and 1.07 (peak 7).

7. The method according to claim 1, characterized in that, Step A) The ratio of the mass (g) of the test sample raw material, the volume (mL) of water, and the volume (mL) of 30% methanol is 1:50:25; the decoction time is 30 min.

8. The method according to claim 1, characterized in that, The ultrasound power is 600W, the frequency is 40kHz, and the ultrasound time is 30min. The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.2:

25.

9. A method for identifying characteristic chromatograms of Bauhinia bark medicinal slices, standard decoctions, and formulated granules, characterized in that, The detection is performed using the method described in any one of claims 1 to 8, and the detection results are analyzed.

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