A method for constructing an HPLC characteristic chromatogram of a traditional Chinese medicine composition for promoting blood circulation to remove blood stasis and unblocking meridians

Abstract

The application provides a construction method of HPLC characteristic atlas of a traditional Chinese medicine composition for activating blood and resolving stasis, promoting qi and unblocking collaterals. The application establishes an HPLC characteristic atlas common mode of the traditional Chinese medicine composition for activating blood and resolving stasis, promoting qi and unblocking collaterals, marks 10 characteristic peaks, and comprehensively reflects the types and quantities of the chemical components, so that the quality control of the product is avoided to be single and one-sided, and the product quality is controlled comprehensively. The application has the characteristics of convenience, rapidness, stability, high precision, good reproducibility and the like, and can effectively control the product quality.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a method for constructing HPLC characteristic chromatograms of a traditional Chinese medicine composition that promotes blood circulation, removes blood stasis, regulates qi, and unblocks meridians. Background Technology

[0002] Danshen, kudzu root, and chuanxiong have the effects of promoting blood circulation, removing blood stasis, dispelling wind and unblocking collaterals, and clearing heat and detoxifying. Therefore, traditional Chinese medicine compositions composed of these herbs can promote blood circulation, remove blood stasis, regulate qi, and unblock collaterals. However, currently, traditional Chinese medicine compositions containing these herbs are mainly identified through thin-layer chromatography and the content of puerarin and salvianolic acid B is determined. However, existing quality standards are insufficient to comprehensively control product quality. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC characteristic chromatograms of traditional Chinese medicine compositions that promote blood circulation, remove blood stasis, regulate qi, and unblock meridians. The characteristic chromatograms established by the present invention can comprehensively reflect the types and quantities of the chemical components contained therein, avoiding the singularity and one-sidedness of quality control of the product, and facilitating comprehensive control of product quality.

[0004] This invention provides a method for constructing the HPLC characteristic chromatogram of a traditional Chinese medicine composition that promotes blood circulation, removes blood stasis, regulates qi, and unblocks meridians, comprising the following steps:

[0005] A) Prepare the test solution and the reference solution separately;

[0006] The test sample is a traditional Chinese medicine composition that promotes blood circulation, removes blood stasis, regulates qi, and unblocks the meridians;

[0007] The reference material was selected from puerarin and salvianolic acid B;

[0008] B) The test solution and the reference solution were determined by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of the test sample and the chromatogram of the reference solution.

[0009] The chromatographic conditions for the high performance liquid chromatography method are as follows: C18 column, mobile phase A is acetonitrile, mobile phase B is 0.05% phosphoric acid solution, and gradient elution.

[0010] Preferably, the gradient elution is as follows:

[0011] 0–5 min, Phase A (25%–27%)–(28%–30%), Phase B (73%–75%)–(70%–72%);

[0012] 5–10 min, Phase A (28%–30%)–(29%–31%), Phase B (70%–72%)–(69%–71%);

[0013] 10–19 min, Phase A (29%–31%)–(35%–37%), Phase B (69%–71%)–(63%–65%);

[0014] 19–22 min, Phase A (35%–37%), Phase B (63%–65%);

[0015] 22–30 min, Phase A (35%–37%)–(42%–44%), Phase B (63%–65%)–(56%–58%);

[0016] 30–35 min, Phase A (42%–44%), Phase B (56%–58%).

[0017] Preferably, the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians includes: Salvia miltiorrhiza, Pueraria lobata, Angelica sinensis, Ligusticum chuanxiong, and Glycyrrhiza uralensis.

[0018] Preferably, the test solution is prepared according to the following method:

[0019] Mix the test sample powder with a 50% methanol solution, filter, and collect the filtrate to obtain the test sample solution.

[0020] The mass-to-volume ratio of the test sample powder to the methanol solution is 0.5~1.5g:25~50ml.

[0021] Preferably, the reference solution is prepared according to the following method:

[0022] Puerarin was mixed with methanol solution to obtain a puerarin solution with a concentration of 50~75μg / ml;

[0023] Tanshinone B was mixed with a methanol solution to obtain a tanshinone B solution with a concentration of 0.1~0.25 mg / ml.

[0024] Preferably, the C18 chromatographic column has a size of 1.7~1.9μm, 2.1*100mm, and a column temperature of 30~40℃.

[0025] Preferably, the flow rate of the mobile phase is 0.2~0.4 ml / min, and the detection wavelength is 275~287 nm. The theoretical plate number, calculated based on the puerarin peak, should be no less than 12000.

[0026] Preferably, an HPLC standard characteristic chromatogram consisting of 10 characteristic peaks is obtained based on the HPLC characteristic chromatogram of the test sample, wherein peak No. 3 is the puerarin peak and peak No. 6 is the salvianolic acid B peak.

[0027] Preferably, the peak corresponding to the puerarin peak is the S peak. The relative retention times of peaks 1 to 10 with the S peak are calculated. The relative retention times are within ±10% of a specified value. The specified values ​​are: 0.35 - peak 1, 0.51 - peak 2, 0.6 - peak 4, 0.75 - peak 5, 0.86 - peak 7, 0.98 - peak 8, 1.17 - peak 9, and 1.26 - peak 10.

[0028] Compared with existing technologies, this invention provides a method for constructing HPLC characteristic chromatograms of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians. The method includes the following steps: A) preparing a test sample solution and a reference solution; the test sample is a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians; the reference is selected from puerarin and salvianolic acid B; B) determining the test sample solution and the reference solution using high-performance liquid chromatography (HPLC) to obtain the HPLC characteristic chromatograms of the test sample and the reference chromatograms; the HPLC conditions are: C18 column, mobile phase A is acetonitrile, mobile phase B is 0.05% phosphoric acid solution, and gradient elution. This invention establishes a common pattern for the HPLC characteristic chromatograms of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians, identifying 10 characteristic peaks. The established characteristic chromatograms comprehensively reflect the types and quantities of the contained chemical components, avoiding the singularity and one-sidedness of quality control for this product, and facilitating comprehensive quality control. This invention is convenient, fast, stable, highly precise, and has good reproducibility, and can effectively control product quality. Attached Figure Description

[0029] Figure 1 Standard characteristic chromatograms of traditional Chinese medicine compositions for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians;

[0030] Figure 2 Characteristic spectra and common patterns of 10 batches of traditional Chinese medicine compositions for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians. Detailed Implementation

[0031] This invention provides a method for constructing the HPLC characteristic chromatogram of a traditional Chinese medicine composition that promotes blood circulation, removes blood stasis, regulates qi, and unblocks meridians, comprising the following steps:

[0032] A) Prepare the test solution and the reference solution separately;

[0033] The test sample is a traditional Chinese medicine composition that promotes blood circulation, removes blood stasis, regulates qi, and unblocks the meridians;

[0034] The reference material was selected from puerarin and salvianolic acid B;

[0035] B) The test solution and the reference solution were determined by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of the test sample and the chromatogram of the reference solution.

[0036] The chromatographic conditions for the high performance liquid chromatography method are as follows: C18 column, mobile phase A is acetonitrile, mobile phase B is 0.05% phosphoric acid solution, and gradient elution.

[0037] The present invention first prepares a test solution and a reference solution.

[0038] The test solution is prepared according to the following method:

[0039] Mix the test sample powder with a 50% methanol solution, filter, and collect the filtrate to obtain the test sample solution.

[0040] Specifically, the test sample is ground into a fine powder and mixed with a 50% methanol solution by volume. The mixture is weighed and ultrasonically treated for 20–40 minutes. After cooling, the mixture is weighed again. The weight loss is replenished with 50% methanol, the mixture is shaken well, filtered, and the filtrate is collected to obtain the test sample solution.

[0041] In this invention, the test sample, namely the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians, includes: Salvia miltiorrhiza, Pueraria lobata, Angelica sinensis, Ligusticum chuanxiong, and Glycyrrhiza uralensis.

[0042] In some specific embodiments of the present invention, the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians includes:

[0043] Salvia miltiorrhiza 14-18 parts by weight, Pueraria lobata 10-14 parts by weight, Angelica sinensis 10-14 parts by weight, Ligusticum chuanxiong 10-14 parts by weight, Glycyrrhiza uralensis 6-10 parts by weight

[0044] In addition to this, one or more of the following can also be added: Panax notoginseng, Paeonia lactiflora, Crataegus pinnatifida leaves, Boswellia carterii, Commiphora myrrha, Leonurus japonicus, Spatholobus suberectus, Cistanche deserticola, and Achyranthes bidentata. The specific dosage can be adjusted according to the actual situation.

[0045] The mass-to-volume ratio of the test sample powder to the methanol solution is 0.5~1.5g:25~50ml, which can be any value between 0.5g:25ml, 1.0g:25ml, 2.0g:25ml, or 0.5~1.5g:25~50ml.

[0046] The reference solution was prepared according to the following method:

[0047] Puerarin is mixed with methanol solution to obtain puerarin solution with a concentration of 50~75μg / ml, which can be 50, 55, 60, 65, 70, 75, or any value between 50~75μg / ml.

[0048] Mix salvianolic acid B with a methanol solution to obtain a salvianolic acid B solution with a concentration of 0.1~0.25 mg / ml, which can be any value between 0.1, 0.15, 0.2, 0.25, or 0.1~0.25 mg / ml.

[0049] Then, the test solution and the reference solution were determined by high performance liquid chromatography.

[0050] Specifically, precisely pipette 1-5 μl of the reference solution and the test solution respectively, inject them into the ultra-high pressure liquid chromatograph, and perform high performance liquid chromatography for determination.

[0051] The chromatographic conditions for the high performance liquid chromatography method are as follows: a C18 column with a diameter of 1.7~1.9μm and a thickness of 2.1*100mm, and a column temperature of 30~40℃, which can be any value between 30, 35, 40, or 30~40℃.

[0052] Mobile phase A is acetonitrile, mobile phase B is 0.05% phosphoric acid solution, and gradient elution is performed. The gradient elution program is as follows:

[0053] 0–5 min, Phase A (25%–27%)–(28%–30%), Phase B (73%–75%)–(70%–72%);

[0054] 5–10 min, Phase A (28%–30%)–(29%–31%), Phase B (70%–72%)–(69%–71%);

[0055] 10–19 min, Phase A (29%–31%)–(35%–37%), Phase B (69%–71%)–(63%–65%);

[0056] 19–22 min, Phase A (35%–37%), Phase B (63%–65%);

[0057] 22–30 min, Phase A (35%–37%)–(42%–44%), Phase B (63%–65%)–(56%–58%);

[0058] 30–35 min, Phase A (42%–44%), Phase B (56%–58%).

[0059] In some specific embodiments of the present invention, the gradient elution procedure is as follows:

[0060] 0–5 min, Phase A 25%–28%, Phase B 75%–72%;

[0061] 5–10 min, phase A 28%–29%, phase B 72%–71%;

[0062] 10–19 min, Phase A 29%–35%, Phase B 71%–65%;

[0063] 19–22 min, Phase A 35%, Phase B 65%;

[0064] 22–30 min, phase A 35%–42%, phase B 65%–58%;

[0065] 30–35 min, Phase A 42%, Phase B 58%.

[0066] In some specific embodiments of the present invention, the gradient elution procedure is as follows:

[0067] 0–5 min, Phase A 27%–30%, Phase B 73%–70%;

[0068] 5–10 min, Phase A 30%–31%, Phase B 70%–69%;

[0069] 10–19 min, phase A 31%–37%, phase B 69%–63%;

[0070] 19–22 min, Phase A 37%, Phase B 63%;

[0071] 22–30 min, phase A 37%–44%, phase B 63%–56%;

[0072] 30-35 min, Phase A 44%, Phase B 56%.

[0073] The flow rate of the mobile phase is 0.2~0.4 ml / min, which can be 0.2, 0.3, 0.4, or 0.2~0.4 ml / min. The theoretical plate number, calculated based on the puerarin peak, should not be less than 12000.

[0074] The detection wavelength of the high-performance liquid chromatography is 275–287 nm. This invention selects a detection wavelength of 275 nm for measurement, which results in numerous peaks, good peak shape, easy identification, high similarity, good stability, and accuracy and reliability.

[0075] In some preferred embodiments of the present invention, the optimal method for constructing the HPLC characteristic chromatogram of the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians is as follows:

[0076] (1) Preparation of test solution: Take 0.5g of finely ground test sample powder, weigh it accurately, place it in a stoppered conical flask, add 25ml of 50% methanol accurately, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0077] (2) Preparation of reference solutions: Take appropriate amounts of puerarin, salvianolic acid B and other reference standards, weigh them accurately, and add 50% methanol to prepare solutions containing 75 μg of puerarin and 0.1 mg of salvianolic acid B per ml.

[0078] (3) Determination: Accurately pipette 1 μl of the reference solution and the test solution into an ultra-high pressure liquid chromatograph and determine them by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of a traditional Chinese medicine composition that promotes blood circulation, removes blood stasis, regulates qi and unblocks meridians.

[0079] The chromatographic conditions for the high-performance liquid chromatography determination are as follows:

[0080] The chromatographic column was a C18 column (1.7–1.9 μm, 2.1 × 100 mm). Mobile phase A was acetonitrile, and mobile phase B was 0.05% phosphoric acid solution. Gradient elution was used, and the ratio of mobile phases A and B changed as follows:

[0081] 0–5 min, Phase A 25%–28%, Phase B 75%–72%;

[0082] 5–10 min, phase A 28%–29%, phase B 72%–71%;

[0083] 10–19 min, Phase A 29%–35%, Phase B 71%–65%;

[0084] 19–22 min, Phase A 35%, Phase B 65%;

[0085] 22–30 min, Phase A 35%–42%, Phase B 65%–58%,

[0086] 30–35 min, Phase A 42%, Phase B 58%.

[0087] The flow rate was 0.3 ml / min, the column temperature was 30℃, and the detection wavelength was 275 nm. The theoretical plate number, calculated based on the puerarin peak, should be no less than 12,000.

[0088] This invention obtains a standard HPLC characteristic spectrum consisting of 10 characteristic peaks from the HPLC characteristic spectra of several batches of traditional Chinese medicine compositions for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians. Peak 3 is the puerarin peak, and peak 6 is the salvianolic acid B peak. The 10 characteristic peaks are assigned as follows: peaks 1, 2, and 5 are assigned to kudzu root; peaks 4 and 7 are assigned to angelica root; peaks 6 and 9 are assigned to chuanxiong rhizome; and peaks 8 and 10 are assigned to licorice root.

[0089] In the standard characteristic spectrum, the peak corresponding to the puerarin peak is the S peak. The relative retention times of peaks 1 to 10 with the S peak are calculated. The relative retention times are within ±10% of the specified values, which are: 0.35 - peak 1, 0.51 - peak 2, 0.6 - peak 4, 0.75 - peak 5, 0.86 - peak 7, 0.98 - peak 8, 1.17 - peak 9, and 1.26 - peak 10.

[0090] This invention establishes a common HPLC characteristic chromatogram pattern for a traditional Chinese medicine composition that promotes blood circulation, removes blood stasis, regulates qi, and unblocks meridians. Ten characteristic peaks were identified. The established characteristic chromatogram comprehensively reflects the types and quantities of the contained chemical components, avoiding the limitations and biases of quality control for this product, and facilitating comprehensive quality control. This invention is convenient, rapid, stable, highly precise, and reproducible, effectively controlling product quality.

[0091] To further understand the present invention, the following description, in conjunction with embodiments, illustrates the method for constructing the HPLC characteristic chromatogram of the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians provided by the present invention. The scope of protection of the present invention is not limited by the following embodiments.

[0092] In the following examples and comparative examples:

[0093] Puerarin reference standard: Puerarin reference standard (110752-201816, 95.6%, China National Institutes for Food and Drug Control).

[0094] Tanshinone B Reference Standard: Tanshinone B Reference Standard (111562-201917, 96.6%, China National Institutes for Food and Drug Control).

[0095] A traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians: This composition contains 18 parts of Salvia miltiorrhiza, 14 parts of Pueraria lobata, 14 parts of Angelica sinensis, 14 parts of Ligusticum chuanxiong, 8 parts of Panax notoginseng, 8 parts of Crataegus pinnatifida leaf, 8 parts of Boswellia carterii, 8 parts of Commiphora myrrha, 8 parts of Leonurus japonicus, 6 parts of Cistanche deserticola, 6 parts of Achyranthes bidentata, and 6 parts of Glycyrrhiza uralensis, provided by Xiuzheng Pharmaceutical Group Co., Ltd.

[0096] Example 1: A method for constructing HPLC characteristic chromatograms of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians.

[0097] Instruments: Vanquish Duo U HPLC system, MS205DU analytical balance

[0098] Test reagents: Puerarin reference standard, Danshensu B reference standard, acetonitrile for liquid chromatography analysis was of chromatographic grade, the other reagents were of analytical grade, water was ultrapure water, and the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi and unblocking meridians was provided by Xiuzheng Pharmaceutical Group Co., Ltd.

[0099] Preparation of the test solution: Take 0.5g of the fine powder of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0100] Preparation of reference solutions: Take appropriate amounts of puerarin and salvianolic acid B reference standards, weigh them accurately, and add methanol to prepare solutions containing 75 μg of puerarin and 0.1 mg of salvianolic acid B per 1 ml.

[0101] Determination: Chromatographic conditions: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1*100 mm) column; mobile phase A: acetonitrile; mobile phase B: 0.05% phosphoric acid solution; gradient elution; flow rate: 0.2 mL / min; column temperature: 30℃; detection wavelength: 275 nm. The theoretical plate number, calculated based on the puerarin peak, should be no less than 12000. The volume ratio concentrations of the gradient elution program are as follows:

[0102] Table 1

[0103]

[0104] Accurately pipette 1 μl each of the reference solution and the test solution into the high-performance liquid chromatograph (HPLC), and determine the HPLC characteristic chromatogram of the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, and unblocking meridians. For example... Figure 1 As shown, Figure 1 This is an HPLC characteristic chromatogram of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians, as determined by the present invention.

[0105] HPLC characteristic chromatogram of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians was obtained, consisting of 10 characteristic peaks. Peak 3 is puerarin, peak 6 is salvianolic acid B peak, and the peak corresponding to the puerarin reference peak is peak S. The relative retention times of peaks 1-10 and peak S were calculated, and the relative retention times were within ±10% of a specified value, which were: 0.35 for peak 1, 0.51 for peak 2, 0.62 for peak 4, 0.75 for peak 5, 0.86 for peak 7, 0.98 for peak 8, 1.17 for peak 9, and 1.26 for peak 10.

[0106] Example 2: A method for constructing HPLC characteristic chromatograms of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians.

[0107] Instruments: Vanquish Duo U HPLC system, MS205DU analytical balance

[0108] Test reagents: Puerarin reference standard, Danshensu B reference standard, acetonitrile for liquid chromatography analysis was of chromatographic grade, the other reagents were of analytical grade, water was ultrapure water, and the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi and unblocking meridians was provided by Xiuzheng Pharmaceutical Group Co., Ltd.

[0109] Preparation of the test solution: Take 1.0 g of the fine powder of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, sonicate for 40 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0110] Preparation of reference solutions: Take appropriate amounts of puerarin and salvianolic acid B reference standards, weigh them accurately, and add methanol to prepare solutions containing 75 μg of puerarin and 0.1 mg of salvianolic acid B per 1 ml.

[0111] Determination: Chromatographic conditions: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1*100 mm) column; mobile phase A: acetonitrile; mobile phase B: 0.05% phosphoric acid solution; gradient elution; flow rate: 0.2 mL / min; column temperature: 30℃; detection wavelength: 275 nm. The theoretical plate number, calculated based on the puerarin peak, should be no less than 12000. The volume ratio concentrations of the gradient elution program are as follows:

[0112] Table 2

[0113]

[0114] Accurately pipette 2 μl each of the reference solution and the test solution into the high-performance liquid chromatograph (HPLC), and determine the HPLC characteristic chromatogram of the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians. For example... Figure 1 As shown.

[0115] HPLC characteristic chromatogram of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians was obtained, consisting of 10 characteristic peaks. Peak 3 is puerarin, peak 6 is salvianolic acid B peak, and the peak corresponding to the puerarin reference peak is peak S. The relative retention times of peaks 1-10 and peak S were calculated, and the relative retention times were within ±10% of a specified value, which were: 0.35 for peak 1, 0.51 for peak 2, 0.62 for peak 4, 0.75 for peak 5, 0.86 for peak 7, 0.98 for peak 8, 1.17 for peak 9, and 1.26 for peak 10.

[0116] Example 3: Method for constructing HPLC characteristic chromatograms of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians.

[0117] Instruments: Vanquish Duo U HPLC system, MS205DU analytical balance

[0118] Test reagents: Puerarin reference standard, Danshensu B reference standard, acetonitrile for liquid chromatography analysis was of chromatographic grade, the other reagents were of analytical grade, water was ultrapure water, and the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi and unblocking meridians was provided by Xiuzheng Pharmaceutical Group Co., Ltd.

[0119] Preparation of the test solution: Take 2.0 g of the fine powder of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0120] Preparation of reference solutions: Take appropriate amounts of puerarin and salvianolic acid B reference standards, weigh them accurately, and add methanol to prepare solutions containing 75 μg of puerarin and 0.1 mg of salvianolic acid B per 1 ml.

[0121] Determination: Chromatographic conditions: Xtimate C18 column (5μm, 4.6×250mm), mobile phase A was acetonitrile, mobile phase B was 0.05% phosphoric acid solution, gradient elution, flow rate 0.2ml / min, column temperature 30℃, detection wavelength 275nm. The theoretical plate number, calculated based on the puerarin peak, should not be less than 12000. The volume ratio concentration configuration of the gradient elution program is as follows:

[0122] Table 3

[0123]

[0124] Accurately pipette 1 μl each of the reference solution and the test solution into a high-performance liquid chromatograph (HPLC), and determine the HPLC characteristic chromatogram of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians. For example... Figure 1 As shown.

[0125] HPLC characteristic chromatogram of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians was obtained, consisting of 10 characteristic peaks. Peak 3 is puerarin, peak 6 is salvianolic acid B peak, and the peak corresponding to the puerarin reference peak is peak S. The relative retention times of peaks 1-10 and peak S were calculated, and the relative retention times were within ±10% of a specified value, which is: 0.35 for peak 1, 0.51 for peak 2, 0.62 for peak 4, 0.75 for peak 5, 0.86 for peak 7, 0.98 for peak 8, 1.17 for peak 9, and 1.26 for peak 10.

[0126] Example 4: Establishment of HPLC standard characteristic chromatogram of a traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians.

[0127] Instruments: Vanquish Duo U HPLC system, MS205DU analytical balance

[0128] Test reagents: Puerarin reference standard, Tanshinone B reference standard, acetonitrile for liquid chromatography analysis was chromatographic grade, other reagents were analytical grade, water was ultrapure water, and the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi and unblocking meridians (batch numbers: 220408, 220428, 230527, 230530, 230612, 230618, 230619, 230620, 230722, 230822) were provided by Xiuzheng Pharmaceutical Group Co., Ltd.

[0129] Preparation of the test solution: Take 1.0 g of the fine powder of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0130] Preparation of reference solutions: Take appropriate amounts of puerarin and salvianolic acid B reference standards, weigh them accurately, and add methanol to prepare solutions containing 75 μg of puerarin and 0.1 mg of salvianolic acid B per 1 ml.

[0131] Chromatographic conditions: A Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1*100 mm) was used. Mobile phase A was acetonitrile, and mobile phase B was 0.05% phosphoric acid solution. Gradient elution was used at a flow rate of 0.2 mL / min, a column temperature of 30 °C, and a detection wavelength of 275 nm. The theoretical plate number, calculated based on the puerarin peak, should be no less than 12000. The volume ratio concentrations of the gradient elution program were configured as follows:

[0132] Table 4

[0133]

[0134] Determination: Accurately pipette 1 μl of the reference solution and the test solution into the high performance liquid chromatograph and determine them by high performance liquid chromatography.

[0135] Methodological investigation

[0136] Precision test: The same test sample (batch number: 230822) solution was injected six times consecutively, and the chromatograms were recorded. The relative retention times of each characteristic peak were compared. The results showed that the RSD values ​​of the relative retention times of all characteristic peaks were less than 3.0%, indicating good instrument precision. Detailed results are as follows:

[0137] Table 5

[0138]

[0139] Repeatability test: Six different samples from the same batch of test sample (batch number: 230822) were taken, and test solutions were prepared according to the test solution preparation method. The solutions were then injected, and the chromatograms were recorded. The results showed that the RSD values ​​of the relative retention times of all characteristic peaks were less than 3.0%, indicating good repeatability of the method. Detailed results are as follows:

[0140] Table 6

[0141]

[0142] Stability test: The same test sample (batch number: 230822) solution was injected at 0, 2, 6, 8, 12, 18, and 24 hours, and the chromatograms were recorded. The results showed that the RSD values ​​of the relative retention times of all characteristic peaks were less than 3.0%, indicating good stability of the solution over 24 hours. Detailed results are as follows:

[0143] Table 7

[0144]

[0145] Characteristic spectrum determination

[0146] The above method was used to determine the characteristics of 10 batches of samples (batch numbers: 220408, 220428, 230527, 230530, 230612, 230618, 230619, 230620, 230722, 230822), and the characteristic spectra were recorded. (See attached image). Figure 2 , Figure 2 Characteristic chromatograms and common patterns of 10 batches of traditional Chinese medicine compositions for promoting blood circulation, removing blood stasis, regulating qi, and unblocking meridians were obtained. The relative retention times were compared, and the RSD values ​​of the relative retention times of all 10 batches of test samples were less than 3.0%. Detailed results are as follows:

[0147] Table 8

[0148]

[0149] Comparative Example 1:

[0150] Instruments: U-3000 high-performance liquid chromatograph, MS205DU analytical balance

[0151] Test reagents: Puerarin reference standard, Tanshinone B reference standard, acetonitrile for liquid chromatography analysis was chromatographic grade, other reagents were analytical grade, water was ultrapure water, and the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi and unblocking meridians (batch numbers: 220408, 220428, 230527, 230530, 230612, 230618, 230619, 230620, 230722, 230822) were provided by Xiuzheng Pharmaceutical Group Co., Ltd.

[0152] Preparation of the test solution: Take 1.0 g of the fine powder of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0153] Preparation of reference solutions: Take appropriate amounts of puerarin and salvianolic acid B reference standards, weigh them accurately, and add methanol to prepare solutions containing 75 μg of puerarin and 0.1 mg of salvianolic acid B per 1 ml.

[0154] Chromatographic conditions: An Agilent (5 μm, 4.6*250 mm) column was used; mobile phase A was acetonitrile; mobile phase B was 0.1% phosphoric acid solution; gradient elution was used; the flow rate was 1 mL / min; the column temperature was 30 °C; and the detection wavelength was 230 nm. The volume ratio concentrations of the gradient elution program were configured as follows:

[0155] Table 9

[0156]

[0157] Determination: Accurately pipette 10 μl of the reference solution and the test solution into the high performance liquid chromatograph and determine them by high performance liquid chromatography.

[0158] Characteristic spectrum determination

[0159] The above method was used to determine the characteristics of 10 batches of samples, and the relative retention times were recorded. The detailed results are as follows:

[0160] Table 10

[0161]

[0162] The results showed that the relative retention times (RSD) of peaks 1, 4, 8, 9, and 10 in 10 batches of test samples were greater than 3.0%, which did not meet the requirements.

[0163] Comparative Example 2:

[0164] Instruments: U-3000 high-performance liquid chromatograph, MS205DU analytical balance

[0165] Test reagents: Puerarin reference standard, Tanshinone B reference standard, acetonitrile for liquid chromatography analysis was chromatographic grade, other reagents were analytical grade, water was ultrapure water, and the traditional Chinese medicine composition for promoting blood circulation, removing blood stasis, regulating qi and unblocking meridians (batch numbers: 220408, 220428, 230527, 230530, 230612, 230618, 230619, 230620, 230722, 230822) were provided by Xiuzheng Pharmaceutical Group Co., Ltd.

[0166] Preparation of the test solution: Take 1.0 g of the fine powder of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0167] Preparation of reference solutions: Take appropriate amounts of puerarin and salvianolic acid B reference standards, weigh them accurately, and add methanol to prepare solutions containing 75 μg of puerarin and 0.1 mg of salvianolic acid B per 1 ml.

[0168] Chromatographic conditions: An Agilent (5 μm, 4.6*250 mm) column was used; mobile phase A was acetonitrile, mobile phase B was methanol, and mobile phase C was 0.05% phosphoric acid solution. Gradient elution was used at a flow rate of 1 mL / min, a column temperature of 30 °C, and a detection wavelength of 230 nm. The volume ratio concentrations of the gradient elution program were configured as follows:

[0169] Table 11

[0170]

[0171] Determination: Accurately pipette 10 μl of the reference solution and the test solution into the high performance liquid chromatograph and determine them by high performance liquid chromatography.

[0172] Characteristic spectrum determination

[0173] The above method was used to determine the characteristics of 10 batches of samples, and the relative retention times were recorded. The detailed results are as follows:

[0174] Table 12

[0175]

[0176] The results showed that the relative retention time RSD values ​​of peaks 2, 4, 7, 8, 9, and 10 were greater than 3.0% in all 10 batches of test samples, which did not meet the requirements.

[0177] 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 HPLC characteristic chromatogram of a traditional Chinese medicine composition for activating blood and resolving stasis, promoting qi and unblocking collaterals, characterized in that, Comprising the following steps: A) separately preparing a test sample solution and a reference solution; The test sample is a traditional Chinese medicine composition for activating blood and resolving stasis, promoting qi and unblocking collaterals, which comprises Danshen, Gegen, Dangui, Chuanxiong and Gancao, and the extraction solvent for preparing the test sample solution is a methanol solution with a volume fraction of 50%; The reference is selected from Gegen and Danshensuan B; B) separately determining the test sample solution and the reference solution by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the test sample and the chromatogram of the reference; The chromatographic conditions of the high performance liquid chromatography are as follows: a C18 chromatographic column, a detection wavelength of 275-287 nm, a mobile phase A of acetonitrile, a mobile phase B of 0.05% phosphoric acid solution, and gradient elution; the gradient elution is as follows: 0-5 min, A phase (25%-27%)~(28%-30%), B phase (73%-75%)~(70%-72%); 5-10 min, A phase (28%-30%)~(29%-31%), B phase (70%-72%)~(69%-71%); 10-19 min, A phase (29%-31%)~(35%-37%), B phase (69%-71%)~(63%-65%); 19-22 min, A phase (35%-37%), B phase (63%-65%); 22-30 min, A phase (35%-37%)~(42%-44%), B phase (63%-65%)~(56%-58%); 30-35 min, A phase (42%-44%), B phase (56%-58%); According to the HPLC characteristic spectrum of the test sample, an HPLC standard characteristic spectrum composed of 10 characteristic peaks is obtained, wherein the No. 3 peak is the Gegen peak and the No. 6 peak is the Danshensuan B peak.

2. The construction method of claim 1, wherein, The test sample solution is prepared as follows: Mix the test sample powder with a methanol solution with a volume fraction of 50%, filter, and take the filtrate to obtain the test sample solution; The mass-volume ratio of the test sample powder to the methanol solution is 0.5-1.5 g: 25-50 ml.

3. The construction method of claim 1, wherein, The reference solution is prepared as follows: Mix Gegen with a methanol solution to obtain a Gegen solution with a concentration of 50-75 μg / ml; Mix Danshensuan B with a methanol solution to obtain a Danshensuan B solution with a concentration of 0.1-0.25 mg / ml.

4. The construction method of claim 1, wherein, The specifications of the C18 chromatographic column are 1.7-1.9 μm, 2.1*100 mm, and the column temperature is 30-40 ℃.

5. The construction method of claim 1, wherein, The flow rate of the mobile phase is 0.2-0.4 ml / min; the theoretical plate number calculated according to the Gegen peak should not be less than 12000.

6. The construction method of claim 1, wherein, The peak corresponding to the Gegen peak is the S peak, the relative retention times of peaks 1-10 and the S peak are calculated, and the relative retention times are within ±10% of the specified values, which are 0.35-peak 1, 0.51-peak 2, 0.6-2 peak 4, 0.75-peak 5, 0.86-peak 7, 0.98-peak 8, 1.17-peak 9, and 1.26-peak 10, respectively.

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