A quality control method for pine knot formula particles and its application

Abstract

The present invention provides a quality control method for pine knot formula particles and its application. The quality control method comprises: using ultra-high performance liquid chromatography to obtain a characteristic spectrum of pine knot formula particles, using the chromatographic peak of pine methyl ether as the reference peak S in the characteristic spectrum, calculating the relative retention time of each characteristic peak and the reference peak S, wherein the relative retention time of at least three characteristic peaks is within ±10% of a specified value, and the specified value is: 0.47, 0.81, 0.87, and the content of pine methyl ether is not less than 245 μg / g. Selecting pine methyl ether as an indicator component to control the quality of pine knot formula particles, based on ultra-high performance liquid chromatography using different wavelengths under the same gradient elution program, a content determination method and a characteristic spectrum method of pine methyl ether are established to achieve overall quality control of pine knot formula particles, and the evaluation method is more accurate and objective and meets the needs of clinical medication.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine detection and quality control, and particularly relates to a quality control method for pine knot formula particles and an application thereof. Background Art

[0002] The Chinese Pharmacopoeia (2020 edition) stipulates that pine knots are the dried nodular nodes or branch nodes of Pinus tabulieformis Carr. or Pinus massoniana Lamb. of the Pinaceae family. They have the aroma of turpentine and taste slightly bitter and acrid. They have the effects of dispelling wind and dampness, dredge meridians and relieve pain. They are used for wind-cold and dampness arthralgia, rheumatism, cramps and contusions. The earliest record of their use as medicine was in the "Famous Doctors' Records" of the Wei and Jin Dynasties. Pine knots are a component of many Chinese patent medicines for rheumatism, joint pain, and contusions, such as rheumatism joint wine, bone pain wine, Jinmao Gouji pills, injury-related Wanhua oil, and Tianma Zhuifeng ointment. Pine knots mainly contain cellulose, lignin, a small amount of volatile oil (turpentine) and resin.

[0003] The Chinese Pharmacopoeia (2020 edition) contains the quality standards for Chinese pine knot medicinal materials, which stipulates the content of its volatile oil and α-pinene. There are also documents that use gas chromatography to study the quality of Chinese pine knot medicinal materials with α-pinene as the index component. In the "Study on the Quality of Chinese Pine Knot Medicinal Materials", researchers analyzed the quality of Chinese pine knot samples from different origins across the country from different perspectives, and used α-terpineol as the index component to develop a qualitative identification standard for Chinese pine knot medicinal materials using thin-layer chromatography; the total volatile oil and the main component α-pinene of Chinese pine knot medicinal materials were quantitatively determined.

[0004] The clinical use of pine knot is mostly in the form of decoction. α-pinene is a volatile oil component and is almost not retained in the standard decoction. Therefore, the method disclosed in the prior art is only applicable to the detection of pine knot medicinal materials and semi-finished products, and its use as a quality indicator of pine knot formula granules remains to be discussed.

[0005] Silver pinoline methyl ether has analgesic and anti-inflammatory effects and is the main active ingredient of pine knot. HPLC was used to establish a method for determining the content of silver pinoline methyl ether in pine knot medicinal materials. In "HPLC Determination of the Content of Silver Pinoline Methyl Ether in Pine Twigs, Pine Needles and Rosin", researchers used high-performance liquid chromatography to establish a method for determining the content of pine knot medicinal materials. In the established content determination method, the preparation method of the test sample is: first defatting the pine knot with petroleum ether, adding methanol ultrasonic extraction for 60 minutes; the elution method is isocratic elution using a methanol-water system. However, the above method is for the determination of the content of silver pinoline methyl ether in pine knot medicinal materials, and its characteristic spectrum has not been studied, making it difficult to control the quality of the medicinal materials as a whole.

[0006] Currently, there are methods for determining the content of pine knot medicinal materials, but there is no relevant research on the quality control of pine knot formula granules. Therefore, establishing a quality control method for the main components in pine knot formula granules has important application value in the overall quality control of pine knot formula granules. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a quality control method for pine knot formula particles and its application. The present invention selects argentopsis methyl ether as an indicator component to control the quality of pine knot formula particles, and establishes a content determination method and a characteristic spectrum method of argentopsis methyl ether based on ultra-high performance liquid chromatography (UPLC) technology using different wavelengths under the same gradient elution program, so as to achieve overall quality control of pine knot formula particles, and the evaluation method is more accurate, objective, and meets the needs of clinical medication.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a quality control method for pine knot formula particles, the quality control method comprising the following steps:

[0010] Ultra-high performance liquid chromatography is used to obtain a characteristic spectrum of pine knot formula particles, in which the chromatographic peak of pinus pyroligne methyl ether is used as the reference peak S, and the relative retention time of each characteristic peak and the reference peak S is calculated, wherein the relative retention time of at least three characteristic peaks is within ±10% of the specified value, and the specified value is: 0.47, 0.81, 0.87, and the content of pinus pyroligne methyl ether is not less than 245 μg / g.

[0011] Preferably, four characteristic peaks are presented in the characteristic spectrum of the pine knot formula particles, and the peak corresponding to the pine methyl ether reference peak is taken as the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: Peak 1: 0.47, Peak 2: 0.81, Peak 3: 0.87.

[0012] Preferably, the content of the argentopsis methyl ether is in the range of 0.245 to 24.5 mg / g.

[0013] Preferably, the step of obtaining the characteristic spectrum of pine knot formula particles by ultra-high performance liquid chromatography comprises:

[0014] (1) Mixing the pine knot formula particles and methanol aqueous solution, and ultrasonically treating them to prepare a test solution; preparing a reference medicinal material reference solution from the pine knot reference medicinal material, and mixing the pine methyl ether reference substance with methanol to prepare a reference substance reference solution;

[0015] (2) The test solution, the control medicinal material reference solution and the control reference solution were detected by ultra-high performance liquid chromatography to obtain the content of argentopsis methyl ether in the pine knot formula granules and the characteristic spectrum of the pine knot formula granules.

[0016] Preferably, in step (1), the volume fraction of methanol in the methanol aqueous solution is 30-100%, for example, it can be 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0017] Preferably, the mass ratio of the pine knot formula particles to the methanol aqueous solution is 1:(50-200), for example, it can be 1:50, 1:60, 1:100, 1:120, 1:150, 1:180 or 1:200, etc.

[0018] Preferably, the power of the ultrasonic treatment is 260-520 W, for example, 260 W, 300 W, 350 W, 400 W, 450 W, 500 W or 520 W, etc., the frequency is 30-50 kHz, for example, 30 kHz, 35 kHz, 40 kHz, 45 kHz or 50 kHz, etc., and the time is 10-40 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min or 40 min, etc.

[0019] Preferably, the mass concentration of pinopora methyl ether in the reference substance solution is 0.08-0.12 mg / mL, preferably 0.1 mg / mL.

[0020] Preferably, in step (1), the control medicinal material reference solution is prepared by a method comprising the following steps: mixing pine knot reference medicinal material powder with water, heating to reflux, filtering, taking the filtrate and drying it, and dissolving the dried residue with methanol-water solution to obtain a control medicinal material reference solution.

[0021] Preferably, the mass ratio of the Chinese pine knot control medicinal material powder to water is 1:(40-60), for example, it can be 1:40, 1:45, 1:50, 1:55 or 1:60.

[0022] Preferably, the heating reflux temperature is 98-100°C, for example, 98°C, 99°C or 100°C, and the heating reflux time is 30-60min, for example, 30min, 35min, 40min, 45min, 50min, 55min or 60min.

[0023] Preferably, the volume fraction of methanol in the methanol aqueous solution is 30-100%, for example, it can be 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0024] Preferably, the mass ratio of the Chinese pine knot control medicinal material powder to the methanol aqueous solution is 1:(25-100), for example, it can be 1:25, 1:50, 1:60, 1:75 or 1:100.

[0025] Preferably, in step (2), the chromatographic conditions of the ultra-high performance liquid chromatography are:

[0026] The chromatographic column of the ultra-high performance liquid chromatography is filled with octadecylsilane bonded silica gel, with 0.05-0.15% formic acid solution (for example, 0.05%, 0.08%, 0.1%, 0.12% or 0.15%) as mobile phase A, and with acetonitrile as mobile phase B, and gradient elution is performed according to the following elution procedure:

[0027]

[0028]

[0029] Preferably, the chromatographic column is a chromatographic column using octadecylsilane bonded silica gel as a filler, with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm.

[0030] Preferably, the flow rate of the mobile phase in the ultra-high performance liquid chromatography is 0.23-0.27 mL per minute, for example, it can be 0.23 mL, 0.24 mL, 0.25 mL, 0.26 mL or 0.27 mL, etc., the column temperature is 33-37°C, for example, it can be 33°C, 34°C, 35°C, 36°C or 37°C, etc., the detection wavelength is 275-290 nm, and the number of theoretical plates calculated based on the argentopsis methyl ether peak should be not less than 5000.

[0031] In a second aspect, the present invention provides application of the quality control method of pine knot formula particles described in the first aspect in quality inspection of pine knot formula particles.

[0032] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0033] In summary, the present invention controls the quality of pine knot formula particles through the following technical solutions:

[0034] (1) The characteristic spectrum method described in the present invention is determined according to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0035] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.7 μm); 0.05-0.15% formic acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is performed according to the following regulations; flow rate is 0.23-0.27 mL per minute; column temperature is 33-37°C; detection wavelength is 280 nm. The number of theoretical plates calculated based on the argentopsis methyl ether peak should be no less than 5000.

[0036]

[0037] Preparation of reference solution: Take about 0.5g of pine knot reference medicinal material powder (passed through No. 3 sieve), accurately weigh, place in a stoppered conical flask, accurately add 25mL of water, weigh, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with water, shake well, filter, take 10mL of the filtrate, evaporate to dryness, dissolve the residue with 70% methanol, transfer to a 5mL volumetric flask, and dilute to the scale with 70% methanol, shake well, filter, take the filtrate to obtain the reference medicinal material reference solution.

[0038] Take an appropriate amount of argentopsis methyl ether reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg per 1 mL to obtain the reference substance solution.

[0039] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.5g, accurately weigh it, accurately add 25mL of 70% methanol, seal it, weigh it, ultrasonically treat it (power 390W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0040] Determination method: Accurately pipette 2 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0041] There should be 4 characteristic peaks in the chromatogram of the test product. The peak corresponding to the reference peak of argentopsis methyl ether is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.47 (peak 1), 0.81 (peak 2), 0.87 (peak 3), 1.00 (peak 4S).

[0042] (2) The content detection method described in the present invention is determined according to the high performance liquid chromatography method (General Rules 0512 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia).

[0043] Chromatographic conditions and system suitability test: the detection wavelength is 308 nm, and the rest are the same as the characteristic spectrum items.

[0044] Preparation of reference solution: Take an appropriate amount of argentopsis methyl ether reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg per 1 mL.

[0045] Preparation of test solution: same as characteristic spectrum item.

[0046] Determination method: Accurately pipette 2 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention establishes an evaluation method for the main components in the pine knot formula granules, and establishes a content determination and characteristic spectrum method for the medicinal ingredient argentopsis methyl ether, which meets the quality control requirements of clinical drug use, can ensure the effectiveness of clinical drug use, and has significant progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the characteristic spectrum chromatogram of pine knot formula particles;

[0050] Figure 2 This is a comparison chart for the confirmation of the characteristic peaks of pine knot formula particles;

[0051] Figure 3 It is the chromatogram result of Experiment 2 in the experimental example;

[0052] Figure 4 It is the chromatogram result of experiment 3 in the experimental example;

[0053] Figure 5 This is the chromatogram result of Experiment 4 in the experimental example. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0055] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0056] The sources of materials and instruments in the following specific embodiments are as follows:

[0057] 1. Reagents and test drugs

[0058] Silver pinotrin methyl ether (batch number: AF21061204, purity: ≥98%), Chengdu Aifa Biotechnology Co., Ltd.;

[0059] Chinese pine knot formula granules (batch numbers: 20022171, 20022191, 20210515), prepared by Beijing Kangrentang Pharmaceutical Co., Ltd.;

[0060] Chinese pine knot medicinal materials (batch numbers: 202106-5-1, 202106-5-2, 202106-5-3) were provided by Beijing Kangrentang Pharmaceutical Co., Ltd.;

[0061] Reagents: Acetonitrile was of chromatographic grade; methanol, formic acid and glacial acetic acid were of analytical grade; water was purified water.

[0062] 2. Instruments

[0063] Analytical balance (Sartorius, SQP QUINTIX224-1CN, Germany);

[0064] Waters high performance liquid chromatograph (equipped with DAD detector, temperature-controlled column oven, temperature-controlled autosampler, USA);

[0065] High-speed desktop centrifuge (SIGMA, 1-14, Germany);

[0066] Ultrasonic cleaning apparatus (Tianjin Aotesense, AS20500BT, China).

[0067] Chromatographic column: ACQUITY BEH Shield RP C18 (2.1 mm×100 mm, 1.7 μm).

[0068] Example 1: Method and results of establishing characteristic spectrum

[0069] 1. Chromatographic conditions and system suitability test

[0070] Octadecylsilane bonded silica gel was used as the filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.7 μm); 0.1% formic acid solution was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 0.25 mL per minute; the column temperature was 35°C; the detection wavelength was 280 nm. The theoretical plate number calculated based on the argentopsis methyl ether peak should not be less than 5000.

[0071] Table 1

[0072] Time (minutes) A(％) B(％) 0～4 88→79 12→21 4～8 79→75 21→25 8～10 75→70 25→30 10～11 70 30 11～15 70→65 30→35 15～17 65→55 35→45 17～22 55→45 45→55 22～25 45→35 55→65 25～30 35 65

[0073] 2. Preparation of reference solution

[0074] Take about 0.5g of pine knot reference medicinal material powder (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of water, weigh it, heat and reflux for 30min, cool, weigh it again, make up the lost weight with water, shake well, filter, take 10mL of the filtrate, evaporate to dryness, dissolve the residue with 70% methanol, transfer it to a 5mL volumetric flask, and dilute it to the scale with 70% methanol, shake well, filter, and take the filtrate to obtain the reference medicinal material reference solution.

[0075] Take an appropriate amount of argentopsis methyl ether reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg per 1 mL to obtain the reference substance solution.

[0076] 3. Preparation of test solution

[0077] Take an appropriate amount of this product, grind it into powder, take about 0.5g, weigh it accurately, add 25mL of 70% methanol accurately, plug it tightly, weigh it, treat it ultrasonically (power 390W, frequency 40kHz) for 30min, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0078] 4. Establishment and identification of feature maps

[0079] 4.1. Obtaining reference spectra and identifying common peaks

[0080] Take different batches of pine knot formula granule samples and obtain their liquid phase spectra according to the above method. The characteristic spectrum chromatogram of pine knot formula granule is as follows: Figure 1 As shown, Figure 1 Peak 4 (S) is the characteristic peak of argentopsis methyl ether. Using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.1 version), the S1 liquid phase spectrum was used as the reference spectrum, and the median was calculated to obtain the reference spectrum, and the common peaks were identified, and a total of 4 common peaks were identified.

[0081] 4.2 Determination of characteristic peaks

[0082] Take the test solution and the reference solution, and perform detection and comparison according to the chromatographic conditions in the text to confirm the characteristic peaks in the characteristic spectrum of the test solution. The results are as follows Figure 2 As shown, Figure 2 This is a comparison chart for the confirmation of the characteristic peaks of pine knot formula particles. By comparing the characteristic peaks in the reference solution and the test solution, it was analyzed and determined that peak 4 is the methyl ether peak of pinopora.

[0083] 5. Methodological Validation

[0084] 5.1 Precision

[0085] Take the same test solution and inject it 6 times continuously to obtain its characteristic spectrum. Take peak 4 as the reference peak to calculate its relative peak area and relative retention time. And calculate RSD. The results show that the relative retention time RSD of each characteristic peak is in the range of 0.0% to 0.2%, and the relative peak area RSD is in the range of 0.0% to 0.4%, indicating that the precision of the characteristic spectrum is good.

[0086] 5.2 Repeatability

[0087] Take 6 portions of the test solution to obtain its characteristic spectrum, and use peak 4 as the reference peak to calculate its relative peak area and relative retention time. And calculate RSD. The results show that the relative retention time RSD of each characteristic peak is in the range of 0.0% to 0.2%, and the relative peak area RSD is in the range of 0.0% to 1.1%, indicating that the repeatability of the characteristic spectrum is good.

[0088] 5.3 Stability

[0089] Take the test solution and measure it according to the above method at 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h, and 24h to obtain its characteristic spectrum. Take peak 4 as the reference peak to calculate its relative peak area and relative retention time. And calculate RSD. The results show that the relative retention time RSD of each characteristic peak is in the range of 0.0% to 0.3%, and the relative peak area RSD is in the range of 0.0% to 0.5%, indicating that the characteristic components in the solution have good stability within 24 hours.

[0090] 6. Determination of characteristic spectrum standards

[0091] Three batches of pine knot formula particles were taken for determination, and the relative retention time results are shown in Table 2:

[0092] Table 2

[0093]

[0094] From the above results, it can be seen that there are 4 common peaks in the chromatogram of the test sample, and the relative retention time of each characteristic peak is slightly different, all within the range of ±10%, which meets the quality control requirements. The average value of the relative retention time is selected as the measured value; the measured values ​​are: 0.47 (peak 1), 0.81 (peak 2), 0.87 (peak 3), 1.00 (peak 4S).

[0095] Example 2 Content Determination Method

[0096] 1. Chromatographic conditions and system suitability test

[0097] Octadecylsilane bonded silica gel was used as the filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.7 μm); 0.1% formic acid solution was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 0.25 mL per minute; the column temperature was 35°C; the detection wavelength was 308 nm. The theoretical plate number calculated based on the argentopsis methyl ether peak should be no less than 5000.

[0098] 2. Preparation of reference solution

[0099] Take an appropriate amount of argentopsis methyl ether reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg per 1 mL.

[0100] 3. Preparation of test solution

[0101] Take an appropriate amount of this product, grind it into powder, take about 0.5g, weigh it accurately, add 25mL of 70% methanol accurately, plug it tightly, weigh it, treat it ultrasonically (power 390W, frequency 40kHz) for 30min, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0102] 4. Determination method

[0103] Accurately pipette 2 μL of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography, and measure to obtain the result.

[0104] 5. Methodological Validation

[0105] 5.1 Accuracy

[0106] Take pine knot formula granules, add reference substances at a ratio of 0.5:1, 1:1, and 1:1.5 to the amount of the component to be determined in the test sample, respectively (n=9), prepare the test sample, and perform liquid phase analysis according to the chromatographic conditions in the determination of argentopsis methyl ether content. The test results are averaged. The results show that in the sample recovery test, the average recovery rate of argentopsis methyl ether is 98.7%, and the RSD is 1.08%, indicating that the accuracy of this method meets the requirements.

[0107] 5.2 Precision

[0108] 5.2.1 Repeatability: Take 6 portions of the test solution and determine the content of argentopsis methyl ether. The repeatability test results are shown in Table 3. The average content of argentopsis methyl ether measured is 5.04 mg / g, and its RSD is 0.6%, which meets the repeatability requirements of the methodology validation.

[0109] Table 3

[0110] serial number Content of argentopsis methyl ether mg / g 1 5.07 2 5.07 3 5.04 4 5.01 5 5.06 6 5.01 RSD / % 0.6

[0111] 5.2.2 Intermediate precision: Different analysts conducted intermediate precision tests at different times using different instruments. Six samples of the test solution were taken to determine the content of argentopsis methyl ether. Results The average content of argentopsis methyl ether measured by the intermediate precision test was 4.91 mg / g, with an RSD value of 0.7%, which was 1.8% compared with the RSD of the repeatability test result, meeting the precision requirements of the methodology validation.

[0112] 5.3 Linear

[0113] Take an appropriate amount of argentopsis methyl ether reference substance, weigh it accurately, add methanol to make a reference substance mother solution containing 490 μg of argentopsis methyl ether per 1 mL, as linear 1; dilute the reference substance mother solution with methanol by 2 times, 10 times, 50 times, and 100 times, respectively, as linear 2, linear 3, linear 4, and linear 5. Accurately draw 2 μL of the above filtrate, inject it into the ultra-high performance liquid chromatograph, measure the peak area of ​​the argentopsis methyl ether chromatographic peak, draw a standard curve with the peak area of ​​the argentopsis methyl ether chromatographic peak as the ordinate and the concentration as the abscissa, and obtain the regression equation of argentopsis methyl ether as y=0.5072x+6.1658, R 2 =0.9991, and its linear range is 4.9~490μg / mL.

[0114] 5.4 Stability

[0115] Prepare the test solution and measure it according to the above method at 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h, and record the change of the peak area of ​​argentopsis methyl ether peak. The RSD value of the peak area of ​​argentopsis methyl ether within 24h is 0.3%, indicating that the test solution is stable within 24h.

[0116] 6. Sample determination

[0117] Different batches of pine knot formula particles were taken respectively, and the content of silver pinot methyl ether was determined according to the above method. The results of the determination of silver pinot methyl ether content are shown in Table 4:

[0118] Table 4

[0119] batch number Content of argentopsis methyl ether (mg / g) 20022171 5.04 20022191 4.50 20210515 4.13

[0120] According to the above test results, the contents of pinopora methyl ether in the three batches of pine knot formula granules were 5.04 mg / g, 4.50 mg / g and 4.13 mg / g respectively.

[0121] Example 3: Study on the durability of column temperature

[0122] Referring to the quality control method provided in Example 2, the content of the test solution was measured at different column temperatures (33°C, 35°C, and 37°C) to investigate the durability of the experimental method for column temperature. The test results are shown in Table 5 below:

[0123] Table 5

[0124] Column temperature Content of argentopsis methyl ether (mg / g) 33℃ 5.23 35℃ 5.04 37℃ 5.14 Mean 5.14 RSD(%) 1.9

[0125] From Table 5, it can be seen that the RSD value of the content of argentopsis methyl ether measured at different column temperatures (33°C, 35°C, and 37°C) is less than 2. The results show that the method has good durability within the column temperature range of 33 to 37°C.

[0126] Example 4: Durability of flow rate

[0127] Referring to the quality control method provided in Example 2, different flow rates (0.23 mL / min, 0.25 mL / min, 0.27 mL / min) were used to measure the test solution provided in Example 2 to investigate the durability of the experimental method for different flow rates. The test results are shown in Table 6 below:

[0128] Table 6

[0129] Flow rate Content of argentopsis methyl ether (mg / g) 0.23mL / min 5.17 0.25mL / min 5.04 0.27mL / min 5.12 Mean 5.11 RSD(%) 1.3

[0130] From Table 6, it can be seen that the RSD value of the content of argentopsis methyl ether measured at different flow rates (0.23 mL / min, 0.25 mL / min, 0.27 mL / min) is less than 2%. The results show that the method has good durability within the flow rate range of 0.23-0.27 mL / min.

[0131] Example 5 Investigating the effect of different column temperatures on content

[0132] Take the pine knot formula particles, add the reference substance to the amount of the component to be determined in the sample in a ratio of 1:1, add the reference substance for sampling (n=9), prepare the sample, and perform liquid phase analysis according to the chromatographic conditions in the determination of the content of silver pinot methyl ether in Example 2. The only difference from Example 2 is the column temperature, which is 29°C, 35°C, and 41°C respectively. The test results are averaged, and the test results are shown in Table 7 below:

[0133] Table 7

[0134]

[0135] From Table 7, it can be seen that when the column temperature is 29°C, the chromatographic peak separation effect is poor and the recovery rate does not meet the requirements. The results show that if the column temperature is too low, it will affect the chromatographic separation effect and cause inaccurate measurement results. Combined with the results of the characteristic spectrum investigation, the column temperature range needs to be controlled.

[0136] Example 6 Investigating the influence of different column temperatures on characteristic spectra

[0137] Test method:

[0138] Referring to the quality control method provided in Example 2, the content of the test solution provided in Example 2 was determined. The only difference from Example 2 is the column temperature, which is 29°C, 35°C, and 41°C, respectively. The 4S peak is used as the reference peak S to calculate the relative retention time of characteristic peaks 1 to 3; each column temperature is repeated twice, and the average value and RSD of the two measurements are calculated. The test results are shown in Table 8:

[0139] Table 8

[0140]

[0141] From the data in Table 8, it can be seen that the chromatographic peak separation effect is poor when the column temperature is 29°C, and the RSD value of the relative retention time of peak 1 is large under different column temperature conditions. The results show that the column temperature is too high or too low, and the durability is difficult to meet the requirements.

[0142] Example 7 Investigating the Effect of Different Flow Rates on Content

[0143] Take the pine knot formula particles, add the reference substance to the amount of the component to be determined in the sample in a ratio of 1:1, add the reference substance for sampling (n=9), prepare the sample, and perform liquid phase analysis according to the chromatographic conditions in the determination of the content of silver pinot methyl ether in Example 2. The only difference from Example 2 is the flow rate, which is 0.16mL / min, 0.25mL / min, and 0.34mL / min respectively. The test results are averaged, and the test results are shown in Table 9 below:

[0144] Table 9

[0145]

[0146] From Table 9, it can be seen that when the flow rate is 0.34mL / min, the chromatographic peak separation effect is poor and the recovery rate does not meet the requirements. The results show that if the flow rate is too high, the separation effect will be affected, resulting in inaccurate measurement results. If the flow rate is too low, the chromatographic peak shape will become wider. Combined with the characteristic spectrum inspection results, the flow rate range needs to be controlled.

[0147] Example 8 Investigating the influence of different flow rates on characteristic spectra

[0148] Test method:

[0149] Referring to the quality control method provided in Example 2, the content of the test solution provided in Example 2 was determined. The only difference from Example 2 is the flow rate, which is 0.16mL / min, 0.25mL / min, and 0.34mL / min, respectively. The 4S peak is used as the reference peak S to calculate the relative retention time of characteristic peaks 1 to 3; each column temperature is repeated twice, and the average value and RSD of the two measurements are calculated. The test results are shown in Table 10:

[0150] Table 10

[0151]

[0152] From Table 10, we can see that the flow rates are 0.16mL / min, 0.25mL / min, and 0.34mL / min, respectively, and the RSD values ​​of the relative retention time at different flow rates vary greatly. The results show that when the flow rate is too high or too low, the durability is difficult to meet the requirements.

[0153] Experimental example: Comparative experiment on elution gradient

[0154] 1. Effect of elution gradient on characteristic spectrum and content determination of pine knot formula particles

[0155] Experimental methods:

[0156] Take the pine knot formula particles with batch number 20022171, prepare a test solution according to the preparation method provided in Example 2, and measure and obtain its characteristic spectrum according to the method provided in Example 2. The only difference from Example 2 is that the elution is performed according to the elution gradients described in Experiments 1 to 4.

[0157] Experiment 1: The mobile phases used in the ultra-high performance liquid chromatography were: 0.1% formic acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution was performed according to Table 1.

[0158] The results of Experiment 1 are as follows Figure 1 shown.

[0159] Experiment 2: The mobile phase used in the ultra-high performance liquid chromatography is: 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution is performed according to the following Table 11:

[0160] Table 11

[0161] Time (minutes) A(％) B(％) 0～10 95→80 5→20 10～24 80→62 20→38 24～30 62→35 38→65 30～32 35→95 65→5

[0162] The results of Experiment 2 are as follows Figure 3 shown.

[0163] Experiment 3: The mobile phase used in the ultra-high performance liquid chromatography is: 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution is performed according to the following Table 12:

[0164] Table 12

[0165]

[0166]

[0167] The results of Experiment 3 are as follows Figure 4 shown.

[0168] Experiment 4: The mobile phase used in the ultra-high performance liquid chromatography is: 0.1% formic acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution is performed according to the following Table 13:

[0169] Table 13

[0170] Time (minutes) A(％) B(％) 0～10 95→80 5→20 10～45 80→45 20→55 45～47 45→95 55→5 47～50 95 5

[0171] The results of Experiment 4 are as follows Figure 5 shown.

[0172] From the above chromatograms, it can be seen that the chromatographic peak shape of Experiment 1 is better and the separation effect is better.

[0173] In summary, the present invention selects pinopora methyl ether as an indicator component to carry out quality control of pine knot formula granules, and establishes a content determination method and a characteristic spectrum method of pinopora methyl ether based on ultra-high performance liquid chromatography technology using different wavelengths under the same gradient elution program, thereby achieving overall quality control of pine knot formula granules, and the evaluation method is more accurate, objective, and meets clinical medication needs.

[0174] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A quality control method for pine knot formula particles, It is characterized in that The quality control method comprises the following steps: The characteristic spectrum of the pine knot formula particles is obtained by ultra-high performance liquid chromatography, wherein the chromatographic peak of pinus pyroligne methyl ether in the characteristic spectrum is used as the reference peak S, and the relative retention time of each characteristic peak and the reference peak S is calculated, wherein the relative retention time of at least three characteristic peaks is within ±10% of the specified value, and the specified value is: 0.47, 0.81, 0.87, and the content of the pinus pyroligne methyl ether is not less than 245 μg / g; The steps of obtaining the characteristic spectrum of pine knot formula particles by ultra-high performance liquid chromatography include: (1) Mixing pine knot formula particles and methanol aqueous solution, wherein the volume fraction of methanol in the methanol aqueous solution is 30-100%, and the mass ratio of the pine knot formula particles to the methanol aqueous solution is 1:(50-200); ultrasonic treatment at 260-520W and a frequency of 30-50kHz for 10-40min to prepare a test solution; preparing a reference medicinal material reference solution from pine knot control medicinal materials, and mixing pine methyl ether reference material with methanol to prepare a reference material reference solution; the mass concentration of pine methyl ether in the reference material reference solution is 0.08-0.12mg / mL; (2) using ultra-high performance liquid chromatography to detect the test solution, the control medicinal material reference solution and the control reference solution, and obtaining the content of argentopsis methyl ether in the pine knot formula granules and the characteristic spectrum of the pine knot formula granules; The chromatographic conditions of the ultra-high performance liquid chromatography method are as follows: the chromatographic column of the ultra-high performance liquid chromatography uses octadecylsilane bonded silica gel as a filler; uses 0.05-0.15% formic acid solution as mobile phase A, uses acetonitrile as mobile phase B, and performs gradient elution according to the following elution procedure: The chromatographic column is a chromatographic column using octadecylsilane bonded silica gel as a filler, with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm.

2. The quality control method of pine knot formula particles according to claim 1, It is characterized in that In the characteristic spectrum of the pine knot formula particles, four characteristic peaks are presented. The peak corresponding to the reference peak of pinopora methyl ether is the S peak, and the characteristic peaks are peak 1, peak 2, peak 3, and peak 4S respectively. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified value is: peak 1: 0.47, peak 2: 0.81, peak 3: 0.

87.

3. The quality control method of pine knot formula particles according to claim 1, It is characterized in that The content of the argentopsis methyl ether is in the range of 0.245 to 24.5 mg / g.

4. The quality control method of pine knot formula particles according to claim 1, It is characterized in that In step (1), the control medicinal material reference solution is prepared by a method comprising the following steps: mixing pine knot reference medicinal material powder with water, heating to reflux, filtering, taking the filtrate and evaporating it to dryness, and dissolving the dried residue with methanol aqueous solution to obtain a control medicinal material reference solution.

5. The quality control method of pine knot formula particles according to claim 4, It is characterized in that The mass ratio of the Chinese pine knot reference medicinal material powder to water is 1:(40-60).

6. The quality control method of pine knot formula particles according to claim 4, It is characterized in that The heating reflux temperature is 98-100° C., and the heating reflux time is 30-60 min.

7. The quality control method of pine knot formula particles according to claim 4, It is characterized in that The volume fraction of methanol in the methanol aqueous solution is 30-100%.

8. The quality control method of pine knot formula particles according to claim 4, It is characterized in that The mass ratio of the Chinese pine knot reference medicinal material powder to the methanol aqueous solution is 1:(25-100).

9. The quality control method of pine knot formula particles according to claim 1, It is characterized in that The flow rate of the mobile phase in the ultra-high performance liquid chromatography is 0.23-0.27 mL per minute, the column temperature is 33-37° C., the detection wavelength is 275-290 nm, and the theoretical plate number calculated based on the argentopsis methyl ether peak should be no less than 5000.

10. Use of the quality control method for pine knot formula particles according to any one of claims 1 to 9 in quality inspection of pine knot formula particles.

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