A method for constructing an hplc characteristic map of a tripterygium extract and a preparation thereof

Abstract

The application provides a HPLC characteristic spectrum construction method of tripterygium extract and preparation thereof, comprising the following steps: A) using solvent to extract the raw material of the test sample to obtain a test solution; B) using high performance liquid chromatography to determine the test solution to obtain the HPLC characteristic spectrum of tripterygium extract and preparation thereof; the high performance liquid chromatography chromatographic conditions are as follows: a C 18 column is used as the chromatographic column; mobile phase A is acetonitrile, mobile phase B is 0.5% phosphoric acid aqueous solution, and gradient elution is adopted. The application adopts high performance liquid chromatography, selects acetonitrile-0.5% phosphoric acid solution as the mobile phase for gradient elution, takes catechin and epicatechin as the reference substances, establishes the HPLC characteristic spectrum of tripterygium extract and preparation thereof, fully displays the chemical component characteristics of tripterygium extract, comprehensively reflects the quality information of tripterygium extract, and therefore can achieve the comprehensive and effective control of the quality of tripterygium extract and related preparation, guarantees the chemical composition stability and use safety of tripterygium extract and related preparation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis and detection technology, and in particular to a method for constructing HPLC characteristic chromatograms of Tripterygium wilfordii extract and its preparations. Background Technology

[0002] Tripterygium wilfordii Hook.f., a plant in the Celastraceae family, is the dried xylem of its root. It is harvested in summer and autumn, the outer bark is removed, and it is dried; alternatively, it can be sliced ​​while fresh and then dried. It has the effects of dispelling wind and dampness, relieving muscle tension and promoting blood circulation, and killing parasites and detoxifying. The extract is a freeze-dried powder prepared from the medicinal material using a fixed processing method.

[0003] Therefore, in order to ensure the uniformity and stability of the quality of Tripterygium wilfordii medicinal materials, processed slices and its extracts, it is necessary to establish a new characteristic spectral method to control its quality. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an HPLC characteristic spectrum of Tripterygium wilfordii extract and its preparations. The characteristic spectrum method constructed by the present invention is stable and reliable, and can control the quality of Tripterygium wilfordii extract, Tripterygium wilfordii extract and Aucklandia lappa, and can also identify the above-mentioned medicinal materials.

[0005] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0006] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0007] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0008] This invention discloses the construction and application of a high-performance liquid chromatography (HPLC) characteristic spectrum detection method for Tripterygium wilfordii extract. This method is applied to Tripterygium wilfordii extract and its related preparations, identifying nine indicator components, confirming 14 common characteristic peaks, and studying their relative retention times and relative peak areas. The relative retention times and relative peak areas are defined, and a control characteristic spectrum is established. This fully demonstrates the chemical composition characteristics of Tripterygium wilfordii extract, comprehensively reflecting its quality information. Therefore, it can comprehensively and effectively control the quality of Tripterygium wilfordii extract and its related preparations, ensuring the stability of its chemical composition and the safety of its use.

[0009] This invention provides a method for constructing HPLC characteristic chromatograms of Tripterygium wilfordii extract and its preparations, comprising:

[0010] A) Extract the sample raw material with a solvent to obtain the test solution;

[0011] B) The test solution was analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of Tripterygium wilfordii extract and its preparations;

[0012] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.5% phosphoric acid aqueous solution, and gradient elution is used.

[0013] The method for constructing HPLC characteristic chromatograms of Tripterygium wilfordii extract and its preparations provided by the present invention first involves solvent extraction of the test sample raw material to obtain the test solution.

[0014] The solvent is preferably 80% methanol.

[0015] The present invention utilizes the above-mentioned extraction solvent, which yields a large amount of chromatographic peak information and produces a better peak shape in the chromatogram.

[0016] The raw materials for the test samples described in this invention include one or more of Tripterygium wilfordii extract or Tripterygium wilfordii granule formulations. This invention does not limit the specific raw materials used; all of the above-mentioned raw materials can be subjected to quality control and qualitative testing using the methods of this invention.

[0017] The extraction method described in this invention is ultrasonic extraction; the ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 30-50min; more preferably 40min.

[0018] The ultrasonic extraction time of this invention is 30–50 min; preferably 40 min. Under different extraction time conditions, the chromatographic results are basically consistent. To ensure sufficient extraction, an extraction time of 40 min results in better peak shape and resolution of the chromatogram.

[0019] The present invention also includes the preparation of a reference solution: catechin and epicatechin reference standards are taken separately and dissolved in 80% methanol to obtain a reference solution;

[0020] The reference solution was analyzed by high-performance liquid chromatography (HPLC) to obtain a chromatogram of the reference substance. The components of the Tripterygium wilfordii extract and its preparations were then identified based on the chromatogram of the reference substance. Specifically, the concentrations of the reference solution were: catechin 25 μg / mL and epicatechin 25 μg / mL.

[0021] The solvent used was 80% methanol. Using 80% methanol as the extraction solvent resulted in a high amount of chromatographic peak information and good separation of each peak.

[0022] Mobile phase A is acetonitrile, and mobile phase B is 0.5% phosphoric acid aqueous solution, with gradient elution.

[0023] The gradient elution described in this invention specifically includes:

[0024] 0–9 min, Phase A: 5%–9%, ​​Phase B: 95–91%;

[0025] 9–23 min, Phase A: 9%–10%, Phase B: 91%–90%;

[0026] 23–38 min, Phase A: 10%–12%, Phase B: 90%–88%;

[0027] 38–60 min, Phase A: 12%–20%, Phase B: 88%–80%.

[0028] The present invention exhibits good baseline separation, good peak separation, and stable baseline under the above-mentioned elution gradient.

[0029] The chromatographic column of this invention is a C18 column; the column has a specification of 250×4.6mm 5μm.

[0030] The inventors have discovered that the C18 chromatographic columns of the above specifications can all meet the detection requirements of this invention.

[0031] For particularly preferred options, you can choose Acclaim™ 120C18 120A 4.6*250mm, 5μm (column 1) or YMC-Pack ODS-A 4.6*250mm, 5μm (column 2). C18 100A 4.6*250mm, 5μm (column 3).

[0032] The column temperature of this invention is 25°C. The theoretical plate number, calculated based on the catechin peak, should be no less than 3000.

[0033] The present invention produces chromatograms with relatively symmetrical peak shapes, good separation, and suitable retention time at a column temperature of 25°C.

[0034] The mobile phase flow rate described in this invention is 1.2 mL / min;

[0035] The present invention found that at a flow rate of 1.2 ml / min, the chromatographic peaks have good separation, good peak shape, symmetrical peak shape and appropriate retention time, which is the optimal solution.

[0036] The detection wavelength of this invention is 280nm;

[0037] The inventors have discovered that the chromatographic information at 280 nm is abundant, each component has good absorption, the response value is moderate, the amount of chromatographic peak information is large, the separation of each peak is good, and the baseline is stable.

[0038] The injection volume of this invention is 10 μL.

[0039] This invention also uses a reference solution and a costus root reference medicinal material solution to identify the characteristic chromatogram of Tripterygium wilfordii extract: peak 3 is catechin; peak 5 is epicatechin;

[0040] The preparation of the reference solution specifically involves: taking appropriate amounts of catechin and epicatechin reference standards, accurately weighing them, and adding 80% methanol to prepare a solution containing 25 μg per 1 ml, which serves as the reference solution.

[0041] This invention identifies the above six characteristic peaks.

[0042] This invention employs a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine to evaluate the similarity of HPLC characteristic chromatograms of Tripterygium wilfordii extract and its preparations, obtaining a standard HPLC characteristic chromatogram consisting of six characteristic peaks, where peak 3 represents catechin and peak 5 represents epicatechin. In the characteristic chromatograms of Tripterygium wilfordii extract and its preparations, using catechin as a reference peak (S peak), the relative retention times of each characteristic peak and peak S peak are calculated. These relative retention times are within ±10% of specified values, which are: 0.30 (peak 1), 0.52 (peak 2), 1.05 (peak 4), 1.57 (peak 5), and 1.63 (peak 6).

[0043] The method provided by this invention can effectively monitor the quality of different batches of Tripterygium wilfordii extract, ensuring its quality stability. The method features high precision and good reproducibility, which is beneficial for comprehensive monitoring of product quality.

[0044] This invention provides a method for constructing HPLC characteristic chromatograms of Tripterygium wilfordii extract and its preparations, comprising: A) extracting the test sample raw material with a solvent to obtain a test solution; B) determining the test solution using high-performance liquid chromatography (HPLC) to obtain HPLC characteristic chromatograms of Tripterygium wilfordii extract and its preparations; the HPLC chromatographic conditions are as follows: a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.5% phosphoric acid aqueous solution, and gradient elution is performed. This invention uses HPLC with acetonitrile-0.5% phosphoric acid solution as the mobile phase for gradient elution, and uses catechin and epicatechin as references to establish HPLC characteristic chromatograms that distinguish Tripterygium wilfordii extract and its preparations. This fully demonstrates the chemical composition characteristics of Tripterygium wilfordii extract, comprehensively reflects the quality information of Tripterygium wilfordii extract, and thus achieves comprehensive and effective quality control of Tripterygium wilfordii extract and its related preparations, ensuring the stability of its chemical composition and safety of use. Attached Figure Description

[0045] Figure 1 Identification of characteristic chromatographic peaks for Tripterygium wilfordii extract;

[0046] Figure 2 Spectrum of catechin reference standard;

[0047] Figure 3 The spectrum of catechins in the test sample;

[0048] Figure 4 Spectrum of epicatechin reference standard;

[0049] Figure 5 The spectrum of epicatechin in the test sample.

[0050] Figure 6 Different instruments were used to investigate the extract of Tripterygium wilfordii;

[0051] Figure 7 Investigation of different chromatographic columns for Tripterygium wilfordii extract;

[0052] Figure 8 Characteristic spectrum of Tripterygium wilfordii extract;

[0053] Figure 9 Characteristic spectrum of extract;

[0054] Figure 10 This is the ultraviolet absorption spectrum of catechins;

[0055] Figure 11 This is the ultraviolet absorption spectrum of epicatechin;

[0056] Figure 12 Chromatograms of Tripterygium wilfordii granules at different wavelengths;

[0057] Figure 13The chromatogram is for column temperature determination;

[0058] Figure 14 Chromatograms were used to investigate flow rate;

[0059] Figure 15 To examine the extraction method;

[0060] Figure 16 Figure showing the results of the solvent extraction investigation;

[0061] Figure 17 Extraction time consideration;

[0062] Figure 18 To investigate the amount of solvent added;

[0063] Figure 19 Identification of chromatographic peaks in the characteristic chromatogram of Tripterygium wilfordii product;

[0064] Figure 20 Spectrum of catechin reference standard;

[0065] Figure 21 The spectrum of the catechin sample;

[0066] Figure 22 Spectrum of epicatechin reference standard;

[0067] Figure 23 The spectrum of the epicatechin sample;

[0068] Figure 24 Comparison of characteristic spectra of Tripterygium wilfordii products using different instruments;

[0069] Figure 25 For column durability testing;

[0070] Figure 26 Characteristic spectrum verification diagrams of three batches of Tripterygium wilfordii formula granules;

[0071] Figure 27 Characteristic spectrum of Tripterygium wilfordii granules.

[0072] Figure 28 Chromatogram of Tripterygium wilfordii under comparative test conditions. Detailed Implementation

[0073] This invention provides a method for constructing HPLC characteristic chromatograms of Tripterygium wilfordii extract and its preparations. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0074] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing HPLC characteristic chromatograms of Tripterygium wilfordii extract and its preparations provided by the present invention.

[0075] 2.2.1 Source of Tripterygium wilfordii medicinal material

[0076] To ensure the reliability of the established quality standards for Tripterygium wilfordii, a total of 16 batches of Tripterygium wilfordii medicinal materials from Sichuan Province and Luoyang City, Henan Province were collected. The specific origin information is shown in Table 1.

[0077] Table 1 Summary of Tripterygium wilfordii medicinal material origin information

[0078]

[0079] 2.2.2 Preparation of Tripterygium wilfordii slices

[0080] Take the Tripterygium wilfordii medicinal material and remove impurities and debris. Process 16 batches of Tripterygium wilfordii medicinal material into 16 batches of Tripterygium wilfordii slices. See Table 2 for specific information.

[0081] Table 2 Correspondence Table of Processing Methods for 16 Batches of Tripterygium wilfordii

[0082]

[0083]

[0084] 2.2.3 Preparation of Tripterygium wilfordii extract

[0085] Take 100g of Tripterygium wilfordii slices, add water and decoct twice. For the first decoction, add 10 times the amount of water, soak for 30 minutes, bring to a boil, and simmer for 60 minutes. Filter through a 200-mesh sieve and immediately cool to room temperature. For the second decoction, add 8 times the amount of water, bring to a boil, and simmer for 40 minutes. Filter through a 200-mesh sieve. Combine the decoctions, immediately cool to room temperature, concentrate, freeze-dry, and package to obtain the final product.

[0086] Sixteen batches of Tripterygium wilfordii extract were prepared from 16 batches of Tripterygium wilfordii slices. The corresponding information is shown in the table below.

[0087] Table 3. Correspondence table for the preparation of Tripterygium wilfordii extract.

[0088]

[0089] High-performance liquid chromatographs: Agilent high-performance liquid chromatographs, Waters high-performance liquid chromatographs;

[0090] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0091] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0092] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0093] Column: Acclaim TM 120C18 120A4.6*250mm, 5μm, YMC-Pack ODS-A4.6*250mm, 5μm, C18 100A 4.6*250mm, 5μm.

[0094] Phosphoric acid and acetonitrile were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0095] Catechins (China National Institutes for Food and Drug Control, batch number: 110877-202306, content calculated as 96.6%)

[0096] Epicatechin (China National Institutes for Food and Drug Control, batch number: 110878-201703, content calculated as 99.7%);

[0097] Tripterygium wilfordii extract (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: LGT-BT-230901, LGT-BT-230902, LGT-BT-230903, LGT-BT-230904, LGT-BT-230905, LGT-BT-230906, LGT-BT-230907, LGT-BT-230908, LGT-BT-230909, LGT-BT-230910, LGT-BT-230911, LGT-BT-230912, LGT-BT-230913, LGT-BT-230914, LGT-BT-230915, LGT-BT-230916).

[0098] Example 1: HPLC Characteristic Chromatography Method for Tripterygium wilfordii Extract

[0099] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.5% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.2 mL / min; column temperature 25 °C; detection wavelength 280 nm. The theoretical plate number, calculated based on the catechin peak, should be no less than 3000.

[0100]

[0101] Preparation of reference solution: Take appropriate amounts of catechin and epicatechin reference standards, accurately weigh them, and add 80% methanol to prepare a solution containing 25 μg per ml, which is used as the reference solution.

[0102] Preparation of the test solution: Weigh approximately 0.5 g of this product accurately, place it in a stoppered conical flask, accurately add 25 ml of 80% methanol, stopper tightly, weigh, sonicate (power 600 W, frequency 40 kHz) for 40 minutes, cool, weigh again, replenish the lost weight with 80% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0103] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0104] Example 2 Methodological Investigation

[0105] 2.1 Chromatographic Peak Identification

[0106] Preparation of test solution: Prepare the Tripterygium wilfordii extract test solution according to the experimental conditions proposed above.

[0107] Preparation of reference solution: Take appropriate amounts of catechin reference standard and epicatechin reference standard, accurately weigh them, and add 80% methanol solution to prepare a solution containing 25 μg per ml.

[0108] Preparation of negative control solution: A negative control solution lacking Tripterygium wilfordii extract was prepared according to the experimental conditions outlined above. The characteristic peaks of the Tripterygium wilfordii extract were located. See [link / reference]. Figures 1-5 .in Figure 1 Identification of characteristic chromatographic peaks for Tripterygium wilfordii extract; Figure 2 Spectrum of catechin reference standard; Figure 3 The spectrum of catechins in the test sample; Figure 4 Spectrum of epicatechin reference standard; Figure 5 The spectrum of epicatechin in the test sample.

[0109] The results showed that the retention times and spectra of catechin and epicatechin reference standards were consistent with those of the target peak in Tripterygium wilfordii extract. Figure 1The method exhibits good specificity, with a one-to-one correspondence and no interference from the negative solution.

[0110] 2.2 Repeatability Test

[0111] Six portions of Tripterygium wilfordii extract (batch number: LGT-BT-230905) were accurately weighed and prepared and measured according to the proposed experimental method. See Table 4.

[0112] Table 4 Repeatability Tests—Ratio of Relative Retention Time of Characteristic Peaks

[0113]

[0114]

[0115] The results show that the relative retention times of each characteristic peak are consistent, with a relative retention time RSD of 0.00%–0.77%. The method exhibits good repeatability.

[0116] 2.3 Intermediate Precision Examination

[0117] 2.3.1 Investigation with different instruments

[0118] Based on the above-planned experimental conditions, 12 portions of Tripterygium wilfordii extract were accurately weighed to prepare test solutions, which were then analyzed using Waters (1–6) and Agilent (7–12) high-performance liquid chromatographs, respectively. See Figure 6 And Table 5, Figure 6 Different instruments were used to investigate the extract of Tripterygium wilfordii.

[0119] Table 5. Ratio of relative retention time of characteristic peaks under different instrument studies.

[0120]

[0121] The results show that the method has good intermediate precision.

[0122] 2.4 Durability Test

[0123] 2.4.1 Column robustness test

[0124] Based on the above-planned experimental conditions, the chromatographic column was tested separately: Acclaim column. TM 120C18120A 4.6*250mm, 5μm (column 1), YMC-Pack ODS-A 4.6*250mm, 5μm (column 2) The study was conducted using a C18100A 4.6*250mm, 5μm column (Column 3). Results are shown below. Figure 7 Table 6. Figure 7 Different chromatographic columns were used to investigate the effects of Tripterygium wilfordii extract.

[0125] Table 6. Column robustness study—Ratio of relative retention times of characteristic peaks

[0126]

[0127] The results showed that the relative retention times were basically consistent when using different chromatographic columns, indicating that the method had good robustness.

[0128] 2.5 Durability Test

[0129] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h, respectively. See Table 7.

[0130] Table 7 Stability Study—Retention Time

[0131]

[0132] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.09% and 0.20%, and the sample solution was relatively stable within 24 hours.

[0133] Example 3: Determination of characteristic peaks and establishment of reference spectra

[0134] 3.1 Validation results of 16 batches of Tripterygium wilfordii extract

[0135] Using this method, characteristic spectral analysis was performed on 16 batches of samples, and relative retention times and relative peak areas were calculated. See Figure 8 Table 8. Figure 8 Characteristic chromatograms of Tripterygium wilfordii extract (batch numbers from bottom to top: LGT-BT-230901, LGT-BT-230902, LGT-BT-230903, LGT-BT-230904, LGT-BT-230905, LGT-BT-230906, LGT-BT-230907, LGT-BT-230908, LGT-BT-230909, LGT-BT-230910, LGT-BT-230911, WFG-BT-210912, LGT-BT-230913, LGT-BT-230914, LGT-BT-230915, LGT-BT-230916).

[0136] Table 8. Ratio of relative retention time of Tripterygium wilfordii extract

[0137]

[0138]

[0139] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peaks, a total of 6 peaks with good repeatability were selected as characteristic peaks.

[0140] 3.2 Establishment of Limits for Relative Retention Time

[0141] Table 9 summarizes the methodological examination items and validation results:

[0142] Table 9 Summary of Methodological Results (RSD%) – Relative Retention Time

[0143]

[0144] The results show that, except for instrument durability, the retention times or RSD values ​​of the relative retention times of each characteristic peak meet the requirements in all the above tests, indicating that the method is effective. The above six characteristic peaks will be included in subsequent investigations.

[0145] The final specification stipulates that the chromatogram of the test sample should show 6 characteristic peaks, with peak 3 corresponding to the retention time of the reference peak. The peak corresponding to the catechin reference peak is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.30 (peak 1), 0.52 (peak 2), 1.05 (peak 4), 1.57 (peak 5), and 1.63 (peak 6). The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize 16 batches of Tripterygium wilfordii extract, and a reference chromatogram of the characteristic chromatogram of Tripterygium wilfordii extract was established. Figure 9 The above, Figure 9 This is a characteristic spectrum of the extract as a reference.

[0146] 3.3 Characteristic chromatographic method for determining Tripterygium wilfordii extract

[0147] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.5% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.2 mL / min; column temperature 25 °C; detection wavelength 280 nm. The theoretical plate number, calculated based on the catechin peak, should be no less than 3000.

[0148]

[0149] Preparation of reference solution: Take an appropriate amount of catechin reference standard, accurately weigh it, and add 80% methanol to prepare a solution containing 25 μg per ml, which is used as the reference solution.

[0150] Preparation of the test solution: Weigh approximately 0.5 g of this product accurately, place it in a stoppered conical flask, accurately add 25 ml of 80% methanol, stopper tightly, weigh, sonicate (power 600 W, frequency 40 kHz) for 40 minutes, cool, weigh again, replenish the lost weight with 80% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0151] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0152] Example 4: HPLC Characteristic Chromatography of Tripterygium wilfordii Formulation Granules

[0153] 4.1 Experimental Instruments and Materials

[0154] High-performance liquid chromatographs: Agilent high-performance liquid chromatographs, Waters high-performance liquid chromatographs;

[0155] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0156] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0157] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0158] Column: Acclaim TM 120C18 120A4.6*250mm, 5μm, YMC-Pack ODS-A4.6*250mm, 5μm, C18 100A 4.6*250mm, 5μm.

[0159] 4.2 Reagents and reagents

[0160] Phosphoric acid and acetonitrile were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0161] Catechins (China National Institutes for Food and Drug Control, batch number: 110877-202306, content calculated as 96.6%)

[0162] Epicatechin (China National Institutes for Food and Drug Control, batch number: 110878-201703, content calculated as 99.7%);

[0163] Tripterygium wilfordii formula granules (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: 2309048, 2309049, 2309050).

[0164] 4.3 Chromatographic conditions

[0165] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material; acetonitrile as mobile phase A and 0.5% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 280 nm.

[0166]

[0167] Preparation of reference solution: Take appropriate amounts of catechin and epicatechin reference standards, accurately weigh them, and add 80% methanol to prepare a solution containing 25 μg per ml, which is used as the reference solution.

[0168] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 80% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 80% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0169] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0170] 4.4 Chromatographic conditions and system suitability test

[0171] 4.4.1 Wavelength Selection

[0172] Based on the above-planned experimental conditions, a diode array detector was used to perform full-band scanning of catechin and epicatechin solutions, and chromatograms of the test solutions were extracted at wavelengths of 260 nm, 270 nm, 280 nm, 290 nm, and 300 nm, respectively. (See attached figures.) Figures 10-12 . Figure 10 This is the ultraviolet absorption spectrum of catechins; Figure 11 This is the ultraviolet absorption spectrum of epicatechin; Figure 12 The images show chromatograms of Tripterygium wilfordii granules at different wavelengths. The results indicate that the chromatographic peak information content is greater and the chromatographic baseline is more stable at a detection wavelength of 280 nm; therefore, the detection wavelength was determined to be 280 nm.

[0173] 4.4.2 Column Temperature Investigation

[0174] Based on the above-specified experimental conditions, the results were investigated at column temperatures of 20℃, 25℃, and 30℃. (See...) Figure 13 . Figure 13 The column temperature was used to investigate the chromatogram. The results showed that at 25℃, the chromatogram peaks were more symmetrical and the retention time was more suitable; therefore, 25℃ was selected as the column temperature.

[0175] 4.4.3 Flow velocity assessment

[0176] Based on the above-established experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated. (See...) Figure 14 , Figure 14 The chromatogram was used to investigate the flow rate.

[0177] The results showed that the chromatogram peaks were more symmetrical and the retention time was appropriate at a flow rate of 1.2 ml / min, so a flow rate of 1.2 ml / min was selected.

[0178] In summary, the chromatographic conditions and system suitability test results for the characteristic chromatogram of Tripterygium wilfordii formula granules were determined as follows: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.5% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in the table below; the flow rate was 1.2 ml / min; the column temperature was 25℃; and the detection wavelength was 280 nm. The theoretical plate number, calculated based on the catechin peak, should not be less than 3000.

[0179]

[0180] 4.5 Preparation of the test solution

[0181] 4.5.1 Examination of Extraction Methods

[0182] Take an appropriate amount of this product (batch number: 2309049), grind it into a fine powder, accurately weigh about 0.5g, place it in a stoppered conical flask, accurately add 25ml of 80% methanol, seal tightly, and weigh. Examine the extraction methods for reflux and ultrasonication (600W power, 40kHz frequency) respectively. Extract for 30 minutes, cool, weigh again, replenish the lost weight with 80% methanol, shake well, filter, and collect the filtrate. See [link to product description]. Figure 15 . Figure 15 To examine the extraction method.

[0183] The results showed that the chromatographic effects were basically the same when using ultrasonic extraction and reflux extraction. Ultrasonic extraction was chosen as the extraction method for the test sample in this experiment.

[0184] 4.5.2 Investigation of Extraction Solvents

[0185] Take approximately 0.5g of the finely ground granules (batch number: 2309049) and accurately weigh it. Place it in a stoppered conical flask and extract with water, 30% methanol, 50% methanol, 80% methanol, methanol, and ethanol, respectively. Accurately add 25ml of each extract to the flask, seal tightly, weigh, and sonicate (600W, 40kHz) for 30 minutes. Cool, weigh again, and replenish the lost weight with the extraction solvent. Shake well, filter, and collect the filtrate. See [link to product description]. Figure 16 , Figure 16 The figure shows the results of the solvent extraction investigation.

[0186] The results showed that when the extraction solvent was 80% methanol, the chromatographic peak information was greater and the peak shape was better. Therefore, the extraction solvent for the test sample was determined to be 80% methanol.

[0187] 4.5.3 Examination of extraction time

[0188] Take an appropriate amount of this product (batch number: 2309049), grind it into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of 80% methanol, seal tightly, and test the sample by ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, 40 minutes, and 50 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the product. See Figure 17 . Figure 17 Extraction time was examined.

[0189] The results showed that the chromatograms were basically consistent under different extraction time conditions. To ensure sufficient extraction, the extraction time for the test sample was determined to be 30 minutes.

[0190] 4.5.4 Investigation of Solvent Addition Amount

[0191] Take an appropriate amount of this product (batch number: 2309049), grind it into a fine powder, and take about 0.5g. Place it in a stoppered conical flask, and add 10ml, 25ml, and 50ml of 80% methanol respectively. Seal the flasks tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate. See [link to product details]. Figure 18 . Figure 18 The amount of solvent added was investigated.

[0192] In summary, the preparation method of the test solution of Tripterygium wilfordii formula granules is determined as follows: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of 80% methanol solution, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0193] 4.6 Methodological Examination

[0194] 4.6.1 Chromatographic Peak Identification

[0195] Preparation of the test solution: Prepare the Tripterygium wilfordii formula granule test solution according to the experimental conditions proposed above.

[0196] Preparation of reference solution: Take appropriate amounts of catechin and epicatechin reference standards, weigh them accurately, and add 80% methanol to prepare a solution containing 25 μg of each per ml.

[0197] Preparation of negative control solution: Prepare a negative control solution for Tripterygium wilfordii granules according to the experimental conditions proposed above.

[0198] The characteristic peaks of the Tripterygium wilfordii formula granules were located. See [link / reference needed]. Figures 19-23 ,in Figure 19 Identification of chromatographic peaks in the characteristic chromatogram of Tripterygium wilfordii product; Figure 20 Spectrum of catechin reference standard; Figure 21 The spectrum of the catechin sample; Figure 22 Spectrum of epicatechin reference standard; Figure 23 This is the spectrum of the epicatechin sample.

[0199] 4.6.2 Repeatability Test

[0200] Six portions of the Tripterygium wilfordii formula granules were accurately weighed and prepared and measured according to the proposed experimental method. See Table 10.

[0201] Table 10 Repeatability Tests—Relative Retention Times of Characteristic Peaks

[0202]

[0203]

[0204] The results showed that the RSD of the retention times of each characteristic peak was 0.00%–0.78%. The instrument exhibited good repeatability.

[0205] 4.6.3 Intermediate Precision Examination

[0206] Based on the above-specified experimental conditions, 12 portions of Tripterygium wilfordii granules were accurately weighed to prepare test solutions. These solutions were then analyzed using Waters (1-6) and Agilent (7-12) high-performance liquid chromatographs, respectively. (See attached figures.) Figure 24 Table 11. Figure 24 Comparison of characteristic spectra of Tripterygium wilfordii products using different instruments.

[0207] Table 11 Intermediate Precision-Relative Retention Time Ratio

[0208]

[0209] The results show that the method has good intermediate precision.

[0210] 4.6.4 Durability Assessment

[0211] 4.6.4.1 Column robustness test

[0212] Based on the above-planned experimental conditions, the chromatographic column was tested separately: Acclaim column. TM 120C18120A 4.6*250mm, 5μm (column 1), YMC-Pack ODS-A 4.6*250mm, 5μm (column 2) The study was conducted using a C18100A 4.6*250mm, 5μm column (Column 3). See Table 12. Figure 25 , Figure 25 For the purpose of investigating the durability of the chromatographic column.

[0213] Table 12 Column robustness study—Relative retention times of characteristic peaks

[0214]

[0215]

[0216] The results showed that the relative retention time (RSD) values ​​differed little when using different chromatographic columns, indicating that the method was robust.

[0217] 4.6.5 Stability Test

[0218] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h. See Table 13.

[0219] Table 13 Stability Study—Retention Time of Characteristic Peaks

[0220]

[0221] The results showed that the RSD values ​​of the corresponding characteristic peak retention times were 0.10% to 0.21%, and the sample solution was relatively stable within 24 hours.

[0222] 4.7 Determination of characteristic peaks and establishment of reference spectra

[0223] 4.7.1 Validation results of three batches of Tripterygium wilfordii granule formulation

[0224] The characteristic spectra of three batches of this product were determined using the proposed method, and the relative retention times and relative peak areas were calculated. (See attached image.) Figure 26 Table 14. Figure 26 Characteristic spectrum verification diagrams of three batches of Tripterygium wilfordii formula granules.

[0225] Table 14 Relative retention times of three batches of Tripterygium wilfordii granules

[0226]

[0227] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peaks, a total of 6 peaks with good repeatability were selected as characteristic peaks.

[0228] 4.7.2 Establishment of Limits for Relative Retention Time

[0229] Table 15 summarizes the methodological examination items and validation results:

[0230] Table 15 Summary of RSD% for Methodological Results – Relative Retention Time

[0231]

[0232] The results show that, except for different flow rates and column temperatures, the retention times or relative retention times (RSDs) of each characteristic peak meet the requirements in all the above investigations, indicating that the method is effective. The above six characteristic peaks will be included in subsequent investigations.

[0233] The final specification stipulates that the chromatogram of the test sample should show 6 characteristic peaks, of which peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the catechin reference peak is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.30 (peak 1), 0.52 (peak 2), 1.05 (peak 4), 1.57 (peak 5), and 1.63 (peak 6).

[0234] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize 16 batches of Tripterygium wilfordii formula granules, and a reference chromatogram of the characteristic chromatogram of Tripterygium wilfordii formula granules was established. See [link / reference]. Figure 27 . Figure 27 Characteristic spectrum of Tripterygium wilfordii formula granules; Peak 3 (S): catechin; Peak 5: epicatechin.

[0235] 4.8 Determination of the Characteristic Spectrum of Tripterygium wilfordii Formulation Granules by

[0236] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.5% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.2 mL / min; column temperature 25 °C; detection wavelength 280 nm. The theoretical plate number, calculated based on the catechin peak, should be no less than 3000.

[0237]

[0238] Preparation of reference solution: Take appropriate amounts of catechin and epicatechin reference standards, accurately weigh them, and add 80% methanol to prepare a solution containing 25 μg per ml, which is used as the reference solution.

[0239] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 80% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 80% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0240] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0241] Comparative Example 1

[0242] 1. Test results of chromatographic conditions for comparison documents

[0243] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 ml per minute; column temperature 35 °C; detection wavelength 218 nm.

[0244]

[0245] Preparation of the test solution: Accurately weigh 0.5 g of the powder and place it in a 25 mL volumetric flask. Add about 20 mL of anhydrous ethanol, seal tightly, and sonicate (120 W, 40 kHz) for 60 min. Cool to room temperature, add anhydrous ethanol to the mark, shake well, and filter. Accurately measure 15 mL of the filtrate and place it on a neutral alumina column [neutral alumina 1.0 g, column inner diameter 0.8–1.0 cm, pre-washed with 5 mL of petroleum ether (60–90 °C)–ethyl acetate (1:4)]. Elute with 30 mL of petroleum ether–ethyl acetate (1:4). Collect the eluent and evaporate it to dryness under reduced pressure in a water bath at 45 °C. Accurately add 1 mL of methanol–water (80:20) to dissolve the residue. Filter through a microporous membrane (0.45 μm) and use the filtrate as the test solution.

[0246] The assay involves precisely pipetting 10 μl of the test solution and injecting it into the liquid chromatograph for determination.

[0247] The results are as follows Figure 28 As shown. Figure 28 Chromatograms of the comparative test conditions.

[0248] 2. Conclusion

[0249] from Figure 27 , Figure 28 The comparison clearly shows that, compared with the comparison method, the chromatogram of this patent has better separation and a more stable baseline. In addition, it has a huge advantage in overall aesthetics and is more suitable as a characteristic chromatogram of Tripterygium wilfordii extract.

[0250] 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 an HPLC characteristic chromatogram of an extract of Tripterygium wilfordii and its preparation, comprising: A) extracting a test sample with a solvent to obtain a test solution; the solvent is 80% methanol; the extraction is ultrasonic extraction; B) determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of the extract of Tripterygium wilfordii and its preparation; preparing a reference solution: taking catechin and epicatechin reference substances respectively, dissolving them with 80% methanol to obtain the reference solution; the high performance liquid chromatography conditions are: a C 18 column; mobile phase A is acetonitrile, mobile phase B is 0.5% phosphoric acid aqueous solution, gradient elution; the gradient elution is specifically: 0-9 min, A phase: 5%-9%, B phase: 95-91%; 9-23 min, A phase: 9%-10%, B phase: 91%-90%; 23-38 min, A phase: 10%-12%, B phase: 90%-88%; 38-60 min, A phase: 12%-20%, B phase: 88%-80%; the detection wavelength is 280 nm. 2.The method of claim 1, wherein: the reference solution is determined by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the HPLC characteristic chromatogram of the extract of Tripterygium wilfordii and its preparation are qualitatively determined according to the chromatogram of the reference substance.

3. The method of claim 2, wherein, The concentration of the reference solution is specifically: catechin 25 μg / mL, epicatechin 25 μg / mL.

4. The method of claim 1, wherein, The column specifications are 250×4.6 mm 5 μm; the column temperature is 25 ℃; the theoretical plate number calculated according to the catechin peak should not be less than 3000.

5. The method of claim 1, wherein, The flow rate of the mobile phase is 1.2 mL / min; the injection volume is 10 μL.

6. The method of claim 1, wherein, The reference solution is used to identify the characteristic chromatogram of the extract of Tripterygium wilfordii, and the similarity of the HPLC characteristic chromatogram of the extract of Tripterygium wilfordii and its preparation is evaluated by a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain an HPLC standard characteristic chromatogram composed of six characteristic peaks, wherein peak 3 is catechin; peak 5 is epicatechin.

7. The method of claim 6, wherein, In the characteristic chromatogram of the extract of Tripterygium wilfordii and its preparation, catechin is used as a reference peak S peak to calculate the relative retention time of each characteristic peak to the S peak, and the relative retention time is within ±10% of the specified value, and the specified value is respectively: 0.30 (peak 1), 0.52 (peak 2), 1.05 (peak 4), 1.57 (peak 5), and 1.63 (peak 6).

8. The method of claim 1, wherein, In step A), the ultrasonic power is 600 W, the frequency is 40 kHz, and the ultrasonic time is 30-50 min.

9. The method of claim 8, wherein, The mass-volume ratio of the test sample to 80% methanol is (0.4-0.6) g:25 mL; the test sample is one or more of the extract of Tripterygium wilfordii or Tripterygium wilfordii formula granules.

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