Determination of HPLC fingerprint of Guanxintai Pills

Abstract

The purpose of the present invention is to solve the problem that the traditional Chinese medicine ingredients of the existing Guanxintai pill preparations are complex and the effective ingredients are unclear. Determination method: 1. Grind Guanxintai pills, add methanol solution as the extraction solvent, heat and reflux, drop the methanol solution to the weight before heating and reflux, and shake well to obtain the test solution; 2. Take tannic acid B as the reference substance, add methanol solution to prepare the reference solution; 3. Use acetonitrile as the mobile phase A and a phosphoric acid solution with a volume concentration of 0.1% as the mobile phase B for gradient elution; 4. Take the test solution and the reference solution and inject them into the liquid chromatograph respectively. The present invention optimizes the chromatographic conditions, and the obtained fingerprint spectrum has high separation and good peak shape. The results of determining the fingerprint spectrum of Guanxintai pills are precise and have good repeatability and stability. By comparing the presence or absence of common peaks in the obtained fingerprint spectrum, the quality of Guanxintai pills can be comprehensively evaluated.

Description

Technical Field

[0001] The invention belongs to the field of analysis of traditional Chinese medicine preparations, and specifically relates to a method for establishing and determining a HPLC fingerprint of Guanxintai Pills and a fingerprint thereof. Background Art

[0002] Guanxintai Pills, formerly known as "Baoxin Pills", were developed by "Wang Mianzhi", a master of traditional Chinese medicine who has been engaged in TCM medical treatment, teaching and scientific research for more than 60 years. He is proficient in differentiation of viscera, qi and blood, and prescription of medicines. He is good at treating difficult diseases in internal medicine, gynecology and pediatrics and exogenous febrile diseases. It is composed of 14 herbs: ginseng, schisandra, chuanxiong, astragalus, frankincense (processed), fennel, rehmannia, myrrh (processed), calamus, ophiopogon, angelica, motherwort, cyathula, and salvia. It has the effects of invigorating qi and nourishing the heart, activating blood circulation and unblocking meridians. It is used for qi and yin deficiency and heart meridian stasis in chest pain (coronary heart disease, angina pectoris), with symptoms of chest tightness and shortness of breath, intermittent stabbing pain, palpitations and fatigue, restless sleep, and petechiae on the tongue.

[0003] Among them, ginseng tastes sweet and slightly bitter, is slightly warm but not hot, and can greatly replenish vital energy. Astragalus is sweet and slightly warm, and can replenish qi and consolidate the exterior. Therefore, ginseng and astragalus are both the main medicines, replenishing the ancestral qi to smooth the heart meridian. Shengdihuang, that is, dried rehmannia, tastes sweet and bitter and is cool in nature. It can nourish yin and blood, and can also promote blood circulation. It enters the heart, liver, and kidney meridians, replenishes yin and blood, clears blood heat, and can promote blood circulation. It is used as the main medicine together with ginseng and astragalus, and can also promote blood circulation while replenishing qi and blood. Ophiopogon japonicus tastes sweet, slightly bitter, and is cool in nature. It can nourish yin and moisten the lungs, clear the heart, relieve troubles, and produce body fluids. It enters the heart, lung, and stomach meridians, and has the function of "clearing the hidden fire in the lungs and calming the heart palpitations." Angelica sinensis tastes sweet, bitter, and warm in nature, and can replenish blood and promote blood circulation. It enters the heart, liver, and spleen meridians, replenishes blood and promotes blood circulation, and is named because it can lead all blood to its own meridian. Frankincense is bitter, bitter, and warm in nature, and can regulate qi, promote blood circulation, and relieve pain. Myrrh tastes bitter and is neutral in nature. It can disperse blood and remove blood stasis, reduce swelling and relieve pain. Medical Records of Chinese and Western Medicine: "Frankincense and myrrh are used together as the key medicines for clearing the internal organs and abdomen and circulating the meridians. Therefore, it can benefit all pains in the heart, stomach, flanks, abdomen, limbs and joints." Chuanxiong tastes pungent and warm in nature. It can promote qi and relieve depression, promote blood circulation and relieve pain. It is a qi medicine for the liver, gallbladder and pericardium. It goes up to the head and goes down to the sea of ​​blood. It is an important medicine for promoting blood circulation and qi. However, it is warm, dry and pungent in nature, so it is not suitable for large quantities. Therefore, it is used as a minister with Angelica, Myrrh and Ophiopogon. Salvia miltiorrhiza tastes bitter and slightly cold, can promote blood circulation and remove blood stasis, calm the mind and calm the heart. It enters the heart, liver and pericardium meridians, nourishes blood and harmonizes the body, and helps other medicines nourish blood and promote blood circulation, and is an adjuvant. Motherwort tastes pungent and slightly cold in nature, promotes blood circulation and removes blood stasis, and regulates menstruation. Achyranthes bidentata tastes bitter, sour and neutral in nature, can disperse blood stasis and eliminate carbuncle swelling. It enters the liver, kidney and pericardium meridians, descends blood and lowers qi, promotes water circulation and removes dampness, relieves pain and arthralgia, and can nourish the liver and kidney for pregnant women. It has no tonifying effect for Sichuan women, so it is used here to descend blood and lower qi, so Sichuan Achyranthes bidentata is used. Rhizoma Lulutong is pungent and warm in nature, can dispel wind and spread throughout the collaterals, and pass through the twelve meridians. "Compendium of Materia Medica" says: "Its nature is great and can pass through the twelve meridians and acupoints." Its pungent and dispersing qi and promoting qi can relax the meridians and eliminate arthralgia and pain. They are all adjuvants. When combined with ginseng and astragalus, it can nourish and promote qi. When combined with angelica, chuanxiong, milk vetch and melon, it can promote blood circulation and nourish the body. Schisandra chinensis is sour and slightly warm, and can restrain the lungs and produce body fluid, nourish the kidneys and astringe the essence. It is used as an adjuvant, and when combined with ginseng and ophiopogon japonicus, it is Shengmai Yin, which can replenish qi and produce yin. Acorus chinensis is pungent and warm in nature, and can open the orifices and disperse wind, regulate qi and promote blood circulation. It is "the key medicine for clearing the heart and spleen meridians" (Compendium of Materia Medica). It can dispel filth and resolve knots, clear numbness and open the heart (orifices). It is not only an adjuvant medicine, but also has the meaning of leading other medicines into the heart, so it is also a guiding medicine. The combination of other medicines can achieve the effect of nourishing essence, activating blood circulation, clearing meridians and nourishing the heart.

[0004] Guanxintai Pills are a traditional Chinese medicine compound preparation containing multiple Chinese medicines with complex chemical components. Effective quality control is an important basis for ensuring its clinical efficacy. At present, reports on the quality control of Guanxintai Pills are mainly quantitative analysis of single or several ingredients and qualitative identification of some ingredients. Its active ingredients are still unclear, and there is a lack of more comprehensive and comprehensive quality control research on Guanxintai Pills. In order to ensure and stabilize product quality, it is necessary to conduct a comprehensive study on its quality inspection and control methods.

[0005] As a quality control technology and method for traditional Chinese medicine, traditional Chinese medicine fingerprint has the characteristics of "integrity" and "fuzziness", and can more comprehensively reflect the overall quality of traditional Chinese medicine. At present, there are few patent disclosures and literature reports on the use of traditional Chinese medicine fingerprint to control the quality of Guanxintai Pills at home and abroad. In the existing quality control technology, the ginseng in Guanxintai Pills is quantitatively studied by thin layer scanning, which fails to achieve comprehensive evaluation and control of the preparation. Therefore, it is necessary to provide a new HPLC fingerprint establishment method for Guanxintai Pills, which can effectively reflect the integrity and complexity of the chemical components of Guanxintai Pills, comprehensively evaluate and control the quality of the preparation, and thus ensure the safety and effectiveness of clinical medication. Summary of the invention

[0006] The purpose of the present invention is to solve the problem that the traditional Chinese medicine ingredients of the existing Guanxintai pill preparations are complex and the effective ingredients are unclear, and to provide a method for determining the HPLC fingerprint of Guanxintai pills.

[0007] The determination method of the HPLC fingerprint of Guanxintai Pills of the present invention is implemented according to the following steps:

[0008] 1. Grind Guanxintai pills into powder, add methanol solution as extraction solvent, weigh the weight, heat and reflux, take out, weigh again, add methanol solution dropwise to the weight before heating and reflux, and shake well to obtain the test solution;

[0009] 2. Take salvianolic acid B as the reference substance and add methanol solution to prepare the reference solution;

[0010] 3. Liquid chromatography conditions: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% by volume phosphoric acid solution as mobile phase B, flow rate of 1.0-1.5 mL per minute, detection wavelength of 210 nm, gradient elution;

[0011] The gradient elution process is as follows: from 0 to 20 minutes, the volume concentration of mobile phase A is 8%→26%, and the volume concentration of mobile phase B is 92%→74%; from 20 to 25 minutes, the volume concentration of mobile phase A is 26%→27%, and the volume concentration of mobile phase B is 74%→73%; from 25 to 33 minutes, the volume concentration of mobile phase A is 27%→51%, and the volume concentration of mobile phase B is 73%→49%; from 33 to 49 minutes, the volume concentration of mobile phase A is 51%→55%, and the volume concentration of mobile phase B is 49%→45%; from 49 to 56 minutes, the volume concentration of mobile phase A is 55%→75%, and the volume concentration of mobile phase B is 45%→25%; from 56 to 80 minutes, the volume concentration of mobile phase A is 75%→100%, and the volume concentration of mobile phase B is 25%→0%; from 80 to 100 minutes, the volume concentration of mobile phase A is 100%, and the volume concentration of mobile phase B is 0%;

[0012] 4. Separately aspirate the test solution and reference solution and inject them into the liquid chromatograph to obtain the fingerprint spectrum of Guanxintai Pills.

[0013] The present invention optimizes the chromatographic conditions, and the results of determining the fingerprint spectrum of Guanxintai Pills are precise, and have good repeatability and stability. The present invention optimizes the optimal HPLC conditions according to the composition characteristics of the prescription components of Guanxintai Pills, solves the problem that the characteristic peaks are difficult to separate and the impurity peaks have large interference, and the obtained fingerprint spectrum has high separation degree and good peak shape, and each characteristic chromatographic peak achieves good baseline separation, good stability, and many characteristic peaks. The quality of Guanxintai Pills can be comprehensively and accurately evaluated, and is suitable for controlling the quality of Guanxintai Pills products.

[0014] The present invention identified 14 common peaks as the common peaks constituting the fingerprint of Guanxintai Pills, and identified Salvia miltiorrhiza, Schisandra chinensis, Olibanum and Acorus calamus, which can be used as the quality evaluation index of Guanxintai Pills. The UPLC-Q-TOF-MS method was used for the first time to analyze the chemical components of the chromatographic peaks in the fingerprint, and a total of 25 compounds were identified.

[0015] The HPLC fingerprint spectrum of Guanxintai Pills established by the present invention overcomes the shortcomings of the prior art that the detection index is single and cannot reflect the intrinsic quality. The provided method for establishing the HPLC fingerprint spectrum of Guanxintai Pills has high precision and good reproducibility. By comparing the presence or absence of common peaks in the obtained fingerprint spectrum, the quality of Guanxintai Pills can be comprehensively evaluated, effectively ensuring the quality of the finished product.

[0016] In summary, the present invention provides a detection method for the fingerprint of Guanxintai Pills and its fingerprint, so as to ensure the safety and effectiveness of the preparation, control the content of its ingredients, and establish a characteristic fingerprint that can comprehensively reflect the integrity of the chemical components of Guanxintai Pills, thereby ensuring and stabilizing the quality of Guanxintai Pills products. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The original fingerprints of fifteen batches of Guanxintai Pills samples in the embodiment;

[0018] Figure 2 The fingerprints of fifteen batches of Guanxintai pills samples after matching in the embodiment (refer to the reference spectrum: 2005709);

[0019] Figure 3 This is the control fingerprint of the Guanxintai Pill sample in the embodiment;

[0020] Figure 4 This is the positive ion mode ion current spectrum of Guanxintai Pills in the embodiment;

[0021] Figure 5 The negative ion mode ion current spectrum of Guanxintai Pills in the embodiment;

[0022] Figure 6 The HPLC chromatogram of Guanxintai Pills with a detection wavelength of 210 nm in the embodiment;

[0023] Figure 7 for Figure 6 The local chromatogram at A in the middle;

[0024] Figure 8 for Figure 6 The local chromatogram at B in the middle;

[0025] Fig. 9 This is the full spectrum of compounds identified by UPLC-Q-TOF-MS in the examples;

[0026] Fig.10 for Fig. 9 The local map at A in the middle;

[0027] Fig.11 for Fig. 9 The local map at B in the middle;

[0028] Fig.12 for Fig. 9 The local map at C in the middle;

[0029] Fig.13 for Fig. 9 The local map at D in the middle;

[0030] Fig.14 This is a test chart of the chromatographic peak identification results of the reference substance in the embodiment;

[0031] Fig.15 This is the result of identifying the chromatographic peak of the reference substance in the embodiment;

[0032] Fig.16 This is the positive fingerprint of ginseng in the embodiment;

[0033] Fig.17 This is the positive fingerprint of Astragalus in the embodiment;

[0034] Fig.18 It is the positive fingerprint of Rehmannia root in the embodiment;

[0035] Fig.19 This is the positive fingerprint of Ophiopogon japonicus in the embodiment;

[0036] Fig. 20 This is the positive fingerprint of Schisandra chinensis in the embodiment;

[0037] Fig.21 It is the positive fingerprint of frankincense in the embodiment;

[0038] Fig. 22 It is the positive fingerprint of myrrh in the embodiment;

[0039] Fig.23 This is the positive fingerprint of Angelica sinensis in the embodiment;

[0040] Fig.24 This is the positive fingerprint of Ligusticum chuanxiong in the embodiment;

[0041] Fig.25 It is the positive fingerprint of Tonglutong in the embodiment;

[0042] Fig.26 This is the positive fingerprint of Cyathula capitata in the embodiment;

[0043] Fig. 27 It is the positive fingerprint of Danshen in the embodiment;

[0044] Fig.28 The positive fingerprint of Acorus tatarinowii in the embodiment;

[0045] Fig.29 This is the positive fingerprint of Leonurus japonicus in the embodiment;

[0046] Fig.30 Figure A is the test result of the investigation of the attribution of the Chinese medicinal ingredients of the chromatographic peaks of the fingerprint of Guanxintai Pills in the embodiment;

[0047] Fig.31 Figure B is the test result of the investigation of the attribution of the Chinese medicinal ingredients to the chromatographic peaks of the fingerprint of Guanxintai Pills in the embodiment. DETAILED DESCRIPTION

[0048] Specific implementation method 1: The method for determining the HPLC fingerprint of Guanxintai Pills in this implementation method is implemented according to the following steps:

[0049] 1. Grind Guanxintai pills into powder, add methanol solution as extraction solvent, weigh the weight, heat and reflux, take out, weigh again, add methanol solution dropwise to the weight before heating and reflux, and shake well to obtain the test solution;

[0050] 2. Take salvianolic acid B as the reference substance and add methanol solution to prepare the reference solution;

[0051] 3. Liquid chromatography conditions: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% by volume phosphoric acid solution as mobile phase B, flow rate of 1.0-1.5 mL per minute, detection wavelength of 210 nm, gradient elution;

[0052] The gradient elution process is as follows: from 0 to 20 minutes, the volume concentration of mobile phase A is 8%→26%, and the volume concentration of mobile phase B is 92%→74%; from 20 to 25 minutes, the volume concentration of mobile phase A is 26%→27%, and the volume concentration of mobile phase B is 74%→73%; from 25 to 33 minutes, the volume concentration of mobile phase A is 27%→51%, and the volume concentration of mobile phase B is 73%→49%; from 33 to 49 minutes, the volume concentration of mobile phase A is 51%→55%, and the volume concentration of mobile phase B is 49%→45%; from 49 to 56 minutes, the volume concentration of mobile phase A is 55%→75%, and the volume concentration of mobile phase B is 45%→25%; from 56 to 80 minutes, the volume concentration of mobile phase A is 75%→100%, and the volume concentration of mobile phase B is 25%→0%; from 80 to 100 minutes, the volume concentration of mobile phase A is 100%, and the volume concentration of mobile phase B is 0%;

[0053] 4. Separately aspirate the test solution and reference solution and inject them into the liquid chromatograph to obtain the fingerprint spectrum of Guanxintai Pills.

[0054] Specific implementation method 2: The difference between this implementation method and specific implementation method 1 is that the extraction solvent described in step 1 is methanol with a volume concentration of 80%.

[0055] Specific implementation method three: This implementation method is different from specific implementation methods one or two in that the heating reflux time in step one is 30 to 40 minutes.

[0056] Specific embodiment 4: This embodiment is different from specific embodiments 1 to 3 in that in step 2, each 1 mL of the reference solution contains 0.15 to 0.18 mg of salvianolic acid B.

[0057] Specific implementation mode 5: This implementation mode is different from specific implementation mode 4 in that in step 2, each 1 mL of the reference solution contains 0.16 mg of salvianolic acid B.

[0058] Specific implementation method 6: The difference between this implementation method and any one of specific implementation methods 1 to 5 is that the column temperature of the chromatographic column in step 3 is 30°C.

[0059] Specific implementation method 7: This implementation method is different from any one of specific implementation methods 1 to 6 in that the flow rate in step 3 is 1.0 mL per minute.

[0060] Specific embodiment eight: This embodiment is different from specific embodiments one to seven in that the filler in step three is Kromasil 100-5-C18 (4.6*250 mm, 5 μm).

[0061] Specific implementation method 9: The difference between this implementation method and specific implementation methods 1 to 8 is that in step 4, 10 μL of the test solution and the reference solution are respectively aspirated and injected into the liquid chromatograph.

[0062] Specific implementation method ten: The difference between this implementation method and specific implementation methods one to nine is that in step four, the UPLC-Q-TOF-MS method is used to analyze the chemical composition of the chromatographic peaks in the fingerprint of Guanxintai Pills.

[0063] Embodiment: The determination method of the HPLC fingerprint of Guanxintai Pills in this embodiment is implemented according to the following steps:

[0064] 1. Grind 1g of Guanxintai pills into powder, put it into a stoppered conical flask, add 80% methanol solution (extraction solvent) to seal it, weigh it, heat it under reflux for 30 minutes, take it out, weigh it again, let it cool, add 80% methanol solution to make up for the lost weight, shake it well and filter it to get the test solution;

[0065] 2. Take salvianolic acid B as the reference substance, weigh it accurately, and add 80% methanol by volume to prepare a reference solution containing 0.16 mg salvianolic acid B per 1 ml;

[0066] 3. Liquid chromatography conditions: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% volume concentration of phosphoric acid solution as mobile phase B, column temperature of 30°C, flow rate of 1.0 mL per minute, detection wavelength of 210 nm, theoretical plate number calculated based on the salvianolic acid B peak should be no less than 10,000, and gradient elution was performed;

[0067] The gradient elution process is shown in the following table:

[0068]

[0069]

[0070] 4. Respectively aspirate 10 μL of the test solution and the reference solution and inject them into the liquid chromatograph to obtain the fingerprint of Guanxintai Pills.

[0071] In this example, 15 batches of Guanxintai pill samples were subjected to fingerprint analysis (such as Figure 1 and Figure 2 The similarity is above 0.98. The method for generating the reference map adopts the average (time window: 0.10). The reference fingerprint map is shown in Figure 3 As shown, 14 common peaks were identified, among which peak 5 is the chromatographic peak of salvianolic acid B, represented by "S", as the reference chromatographic peak, and each chromatographic peak is represented by a sequence number from 1 to 14. The horizontal axis of each fingerprint in the attached figure is time (min).

[0072] Table 1 Attribution investigation results of chromatographic peaks of Guanxintai Pills fingerprint

[0073]

[0074] In this example, the UPLC-Q-TOF-MS method was used to perform a qualitative analysis of the chemical components of the chromatographic peaks in the fingerprint of Guanxintai Pills. A total of 28 compounds were identified by combining the ion fragment information with literature reports. The identification results are shown in Table 2 below, and the total ion current spectrum is shown in Table 2. Figure 4 and Figure 5 , HPLC chromatogram see Figure 6 As shown, Figure 7 and Figure 8 for Figure 6 Local chromatogram of .

[0075] Table 2 UPLC-IT-TOF data of Guanxintai pill extract

[0076]

[0077]

[0078]

[0079] According to the analysis results of Guanxintai Pills UPLC-Q-TOF-MS, the chromatographic peaks were identified using existing reference substances. The identification results of the chromatographic peaks of the reference substances are shown in Table 3.

[0080] Table 3 Chromatographic peak identification results of reference substances

[0081]

[0082]

[0083] Chromatographic peak correlation study of single herb positive control:

[0084] According to the “Guanxintai Pill Preparation Process”, single-drug positive samples of 14 medicinal materials including ginseng, astragalus, rehmannia, ophiopogon, schisandra, frankincense (processed), myrrh (processed), angelica, chuanxiong, fennel, cyathula, salvia miltiorrhiza, calamus, and motherwort were prepared respectively, and then the positive test solution of each medicinal material was prepared according to the “Test Solution Preparation Method”. The positive fingerprints of the 14 medicinal materials are as follows: Figure 16-Figure 29 As shown. Under the determined chromatographic conditions, each medicinal material positive test solution and finished product test solution were injected separately to determine the correlation of each chromatographic peak in the control fingerprint. Fig.30 and Fig.31The results showed that the fingerprint of Guanxintai Pills with batch number 2005709 was selected, the integration time was 4 to 100 min, and the integration parameters were: threshold value 10; peak width 5; minimum peak area 500, and minimum peak height 2000. A total of 14 chromatographic peaks were annotated, of which 4 were exclusively for Schisandra chinensis medicinal materials; 3 were exclusively for Salvia miltiorrhiza medicinal materials; 2 were exclusively for Frankincense (processed) medicinal materials; and 1 was exclusively for Acorus tatarinowii.

[0085] Precision test:

[0086] Take this product (batch number: 2005709), prepare it according to the preparation method of the test solution, inject it continuously for 6 times, detect the fingerprint spectrum, calculate the similarity, and the results are shown in Table 4. The results show that the precision is good.

[0087] Table 4 Precision test results

[0088]

[0089] Stability test:

[0090] Take a sample (batch number: 2005709), prepare it according to the preparation method of the test solution, detect the fingerprint spectrum at 0, 3, 6, 9, 15, and 24 hours, calculate the similarity, and the results are shown in Table 5. The results show that the test solution has good stability within 24 hours and meets the detection requirements of the fingerprint spectrum.

[0091] Table 5 Stability test results

[0092]

[0093] Repeatability test:

[0094] Take the sample (batch number: 2005709), prepare 6 samples according to the preparation method of the sample solution, detect the fingerprint, calculate the similarity, and the results are shown in Table 6. The results show that the method has good repeatability and meets the detection requirements of the fingerprint.

[0095] Table 6 Repeatability test results

[0096]

[0097] Intermediate precision test:

[0098] This product (batch number: 2005709) was used to prepare test sample and reference solution according to the preparation method of test sample solution and reference solution by different analysts on different dates. The three types of analytical instruments, Agilent, Shimadzu and Watera, were used for detection. The similarity results of the three instruments are shown in Table 7.

[0099] Table 7 Intermediate precision test results

[0100]

[0101]

Claims

1. The method for determining the HPLC fingerprint of Guanxintai Pills is characterized in that The determination method is implemented according to the following steps:

1. Grind Guanxintai pills into powder, add 80% methanol solution as the extraction solvent, weigh the weight, heat and reflux, take out, weigh again, add methanol solution dropwise to the weight before heating and reflux, and shake well to obtain the test solution; 2. Take salvianolic acid B as the reference substance and add methanol solution to prepare the reference solution; 3. Liquid chromatography conditions: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% volume concentration of phosphoric acid solution as mobile phase B, flow rate of 1.0-1.5 mL per minute, detection wavelength of 210 nm, gradient elution; The gradient elution process is as follows: from 0 to 20 minutes, the volume concentration of mobile phase A is 8%→26%, and the volume concentration of mobile phase B is 92%→74%; from 20 to 25 minutes, the volume concentration of mobile phase A is 26%→27%, and the volume concentration of mobile phase B is 74%→73%; from 25 to 33 minutes, the volume concentration of mobile phase A is 27%→51%, and the volume concentration of mobile phase B is 73%→49%; from 33 to 49 minutes, the volume concentration of mobile phase A is 51%→55%, and the volume concentration of mobile phase B is 49%→45%; from 49 to 56 minutes, the volume concentration of mobile phase A is 55%→75%, and the volume concentration of mobile phase B is 45%→25%; from 56 to 80 minutes, the volume concentration of mobile phase A is 75%→100%, and the volume concentration of mobile phase B is 25%→0%; from 80 to 100 minutes, the volume concentration of mobile phase A is 100%, and the volume concentration of mobile phase B is 0%; 4. Separately aspirate the test solution and reference solution and inject them into the liquid chromatograph to obtain the fingerprint spectrum of Guanxintai Pills.

2. The method for determining the HPLC fingerprint of Guanxintai Pills according to claim 1, characterized in that The heating reflux time in step 1 is 30 to 40 minutes.

3. The method for determining the HPLC fingerprint of Guanxintai Pills according to claim 1, characterized in that In step 2, each 1 mL of the reference solution contains 0.15-0.18 mg of salvianolic acid B.

4. The method for determining the HPLC fingerprint of Guanxintai Pills according to claim 3, characterized in that In step 2, each 1 mL of the reference solution contains 0.16 mg of salvianolic acid B.

5. The method for determining the HPLC fingerprint of Guanxintai Pills according to claim 1, characterized in that The column temperature of the chromatographic column in step 3 is 30°C.

6. The method for determining the HPLC fingerprint of Guanxintai Pills according to claim 1, characterized in that The flow rate in step 3 is 1.0 mL per minute.

7. The method for determining the HPLC fingerprint of Guanxintai Pills according to claim 1, characterized in that In step 3, the filler used is Kromasil 100-5-C18.

8. The method for determining the HPLC fingerprint of Guanxintai Pills according to claim 1, characterized in that In step 4, 10 μL of the test solution and the reference solution were respectively injected into the liquid chromatograph.

9. The method for determining the HPLC fingerprint of Guanxintai Pills according to claim 1, characterized in that Step 4: The UPLC-Q-TOF-MS method was used to analyze the chemical components of the chromatographic peaks in the fingerprint of Guanxintai Pills.

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