Method for constructing characteristic chromatogram and determining content of Jingzhi Guanxin Oral Liquid

Abstract

The present invention belongs to the technical field of pharmaceutical analysis and detection, and particularly relates to a method for constructing a characteristic chromatogram and determining the content of Jingzhi Guanxin Oral Liquid. This method uses ultra-high performance liquid chromatography for detection, and can achieve the analysis of characteristic chromatogram and multi-component content determination under one chromatographic condition, greatly shortening the detection cycle and improving the analysis efficiency. Moreover, this method has good specificity, precision, stability, accuracy, etc., and is very suitable for controlling the overall quality of Jingzhi Guanxin Oral Liquid.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis and detection. More specifically, it relates to a method for constructing a characteristic fingerprint and determining the content of Jingzhi Guanxin Oral Liquid. Background Technique

[0002] Jingzhi Guanxin Oral Liquid is derived from the Guanxin No. 2 formula created by the veteran Chinese medicine doctor Mr. Guo Shikui of Xiyuan Hospital, China Academy of Chinese Medical Sciences. This formula consists of five medicinal materials: Salvia miltiorrhiza, Paeonia lactiflora, Ligusticum chuanxiong, Carthamus tinctorius, and Dalbergia odorifera. It is currently included in the first part of the Chinese Pharmacopoeia (2020 Edition) and is used for chest pain caused by stasis of blood, manifested as chest tightness and stabbing pain in the precordial area. It is applicable to patients with coronary heart disease angina pectoris presenting with the above syndromes.

[0003] The same series of drugs as Jingzhi Guanxin also include Jingzhi Guanxin Tablets and Jingzhi Guanxin Soft Capsules. The common point of the preparation methods of these two is to first extract the volatile oil from Dalbergia odorifera, and then combine and concentrate the aqueous solution after distillation with the 85% ethanol heated reflux extract of the other four medicinal materials, and mix it with the volatile oil. Jingzhi Guanxin Oral Liquid is prepared by decocting the four Chinese medicinal materials except Carthamus tinctorius, filtering, and then warm-soaking Carthamus tinctorius at 80°C and combining and concentrating it with the filtrate. Due to the differences in extraction methods and preparation processes, the chemical components and contents in Jingzhi Guanxin Tablets, soft capsules, and oral liquid are not the same. Tablets and soft capsules contain a large number of lipophilic molecules, while the oral liquid mainly includes water-soluble components such as phenolic acids and paeoniflorin.

[0004] Chinese Patent Application CN105738499A discloses a method for constructing a characteristic fingerprint of Jingzhi Guanxin Tablets and determining the content of 6 components. The characteristic fingerprint of the tablets includes common peaks such as paeoniflorin, rosmarinic acid, salvianolic acid B, ligustilide, cryptotanshinone, and tanshinone IIA; Li Shuang et al. ([1] Li Shuang, Yan Bixin, Zhang Ying, etc. Re-evaluation study on the quality control method of Jingzhi Guanxin Tablets [J]. Ginseng Research, 2021.) used high-performance liquid chromatography to determine the contents of salvianolic acid B, tanshinone IIA, and paeoniflorin in Jingzhi Guanxin Tablets. However, different from this, Jingzhi Guanxin Oral Liquid does not contain lipophilic components such as cryptotanshinone and tanshinone IIA, and the quality control method of Jingzhi Guanxin Tablets is not applicable to the detection of oral liquid; moreover, in addition to salvianolic acid B and paeoniflorin, there are also multiple water-soluble components with relatively high contents in Jingzhi Guanxin Oral Liquid for which there is no suitable quality control method.

[0005] Currently, the Chinese Pharmacopoeia only uses high-performance liquid chromatography to determine the contents of two components, salvianolic acid B and paeoniflorin, at a detection wavelength of 230 nm. However, this method only detects one component in Salvia miltiorrhiza and Paeonia lactiflora, and does not monitor the active components in Ligusticum chuanxiong, Carthamus tinctorius, and Dalbergia odorifera, and cannot comprehensively and effectively measure the quality of Jingzhi Guanxin Oral Liquid. Moreover, the content determination method of Jingzhi Guanxin Oral Liquid in the current Chinese Pharmacopoeia has a long detection time, low detection efficiency, and few chromatographic peak information in the detected spectrum, which is not conducive to actual production applications.

[0006] Therefore, there is an urgent need to provide a method for constructing a characteristic chromatogram and determining the content, which has a short detection time, more chromatographic peak information, and can comprehensively monitor the quality of Jingzhi Guanxin Oral Liquid. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defects and deficiencies that the existing methods for Jingzhi Guanxin Tablets are not applicable to oral liquids, have a long detection time, low detection efficiency, and few chromatographic peak information, and to provide a method for constructing a characteristic chromatogram and determining the content, which has a short detection time, more chromatographic peak information, and can comprehensively monitor the quality of Jingzhi Guanxin Oral Liquid.

[0008] Another object of the present invention is to provide the application of the above method in the quality control of Jingzhi Guanxin Oral Liquid.

[0009] The above objects of the present invention are achieved by the following technical solutions:

[0010] A method for constructing a characteristic chromatogram and determining the content of Jingzhi Guanxin Oral Liquid is detected by ultra-high performance liquid chromatography. The conditions of the ultra-high performance liquid chromatography are as follows:

[0011] Chromatographic column: octadecylsilane-bonded silica gel, detection wavelength: 230-290 nm, column temperature: 25-30 °C, flow rate 0.25-0.30 ml / min; mobile phase A: acetonitrile or methanol, mobile phase B: 0.1% phosphoric acid solution, and the detection is carried out under the following gradient elution conditions:

[0012]

[0013] Furthermore, when constructing the characteristic chromatogram of Jingzhi Guanxin Oral Liquid, the detection wavelength is 228-232 nm. Preferably, the detection wavelength is 230 nm.

[0014] Still further, when determining the content of Jingzhi Guanxin Oral Liquid, the detection wavelength for determining the content of phenolic acid components is 284-288 nm, and the detection wavelength for determining the content of paeoniflorin components is 228-232 nm. Preferably, the detection wavelength for determining the content of phenolic acid components is 286 nm, and the detection wavelength for determining the content of paeoniflorin components is 228-232 nm. More preferably, the phenolic acid components include danshensu, protocatechuic aldehyde, lithospermic acid, salvianolic acid B and salvianolic acid A; the paeoniflorin components include albiflorin and paeoniflorin.

[0015] Further, the chromatographic column is an octadecylsilyl-bonded silica gel column with an inner diameter of 2.0 - 2.1 mm, a column length of 100 mm - 150 mm, and a particle size of 1.7 - 2.0 μm. Preferably, the chromatographic column is Waters Acquity UPLCTM HSS T3 (2.1×150 mm, 1.8 μm) or Agilent RRHD Eclipse Plus C18 (2.1×150 mm, 1.8 μm).

[0016] Furthermore, in the conditions of the ultra-high performance liquid chromatography method, the injection volume is 1.0 - 1.5 μL.

[0017] Further, the refined Guanxin oral liquid is prepared into a test solution and then detected by the ultra-high performance liquid chromatography method. The preparation method of the test solution includes the following steps:

[0018] Dilute the refined Guanxin oral liquid with an alcohol solution, perform ultrasonic treatment, filter, and take the subsequent filtrate to obtain the test solution.

[0019] Preferably, the dilution multiple is 25 - 50 times; the alcohol solution is a methanol solution or an ethanol solution, and the volume concentration of the alcohol solution is 50 - 100%; the power of the ultrasonic treatment is 300 - 500 W, the frequency is 35 - 40 kHz, and the treatment time is 15 - 30 min.

[0020] Furthermore, the method for constructing the characteristic chromatogram and determining the content of the refined Guanxin oral liquid also requires the preparation of a reference solution, which is prepared by dissolving the reference substance in a 50% - 90% methanol or ethanol solution.

[0021] Further, in the construction of the method characteristic chromatogram, 9 common chromatographic peaks are detected: peak 1 is gallic acid, peak 2 is danshensu, peak 3 is protocatechuic aldehyde, peak 4 is albiflorin glycoside, peak 5 is paeoniflorin, peak 6 is lithospermic acid, peak 7 is salvianolic acid B, peak 8 is salvianolic acid A, and peak 9 is (3R)-Sativanone (inferred by LC-MS technology); taking peak 7, salvianolic acid B, as the reference peak of the characteristic chromatogram, the relative retention times of other peaks are within ±10% of the specified values, and the specified values are respectively: peak 1: 0.05, peak 2: 0.10, peak 3: 0.19, peak 4: 0.42, peak 5: 0.47, peak 6: 0.94, peak 7: 1.00, peak 8: 1.07, peak 9: 1.31.

[0022] Preferably, the common chromatographic peaks are characteristic peaks. Among them, the characteristic peaks belonging to Salvia miltiorrhiza are peaks 2, 3, 6, 7, and 8; the characteristic peaks belonging to Paeonia lactiflora Pall. are peaks 1, 4, and 5; the characteristic peak belonging to Dalbergia odorifera T. Chen is peak 9.

[0023] Furthermore, the method can simultaneously determine the contents of danshensu, protocatechuic aldehyde, lithospermic acid, salvianolic acid B, salvianolic acid A, albiflorin, and paeoniflorin in the refined Guanxin oral liquid. Among them, gallic acid has poor specificity, and there is no reference substance for (3R)-Sativanone, so it is not determined in the content determination.

[0024] In addition, the present invention also provides a quality control method for the refined Guanxin oral liquid, which uses the characteristic fingerprint construction and content determination methods of the refined Guanxin oral liquid for quality control.

[0025] Furthermore, the results of the content determination of the refined Guanxin oral liquid are specified as follows: for every 1 mL of the refined Guanxin oral liquid, the total amount of danshensu, protocatechuic aldehyde, lithospermic acid, salvianolic acid B, and salvianolic acid A calculated as danshen should not be less than 7.5 mg; for every 1 mL of the refined Guanxin oral liquid, the total amount of albiflorin and paeoniflorin calculated as red peony root should not be less than 4.3 mg.

[0026] The present invention has the following beneficial effects:

[0027] The present invention provides a method for constructing the characteristic fingerprint and determining the content of the refined Guanxin oral liquid. The detection is carried out by ultra-high performance liquid chromatography, and the characteristic fingerprint and multi-component content determination analysis can be realized under one chromatographic condition, greatly shortening the detection period, improving the analysis efficiency, and the method has good specificity, precision, stability, accuracy, etc., and is very suitable for controlling the overall quality of the refined Guanxin oral liquid. Description of the Drawings

[0028] Figure 1 It is the control characteristic fingerprint of the refined Guanxin oral liquid in Example 1 (the characteristic peaks 1 to 9 are marked from left to right in the figure).

[0029] Figure 2 It is the comparative chromatogram of the refined Guanxin oral liquid and danshen medicinal material in Example 1 (the numbers 2, 3, 6, 7, and 8 in the figure are the peak numbers of the chromatographic peaks attributed to danshen in the refined Guanxin oral liquid).

[0030] Figure 3 It is the comparative chromatogram of the refined Guanxin oral liquid and red peony root medicinal material in Example 1 (the numbers 1, 4, and 5 in the figure are the peak numbers of the chromatographic peaks attributed to red peony root in the refined Guanxin oral liquid).

[0031] Figure 4 It is the comparative chromatogram of the refined Guanxin oral liquid and dalbergia odorifera medicinal material in Example 1 (the number 9 in the figure is the peak number of the chromatographic peak attributed to dalbergia odorifera in the refined Guanxin oral liquid).

[0032] Figure 5 It is the chromatogram for the determination of the content of phenolic acid components in the refined Guanxin oral liquid in Example 2 (detection wavelength: 286 nm).

[0033] Figure 6 It is the chromatogram of the reference substance for the content determination of Jingzhi Guanxin Oral Liquid in Example 2 (detection wavelength: 286 nm).

[0034] Figure 7 It is the chromatogram for the content determination of paeoniflorin components in Jingzhi Guanxin Oral Liquid in Example 2 (detection wavelength: 230 nm).

[0035] Figure 8 It is the chromatogram of the reference substance for the content determination of Jingzhi Guanxin Oral Liquid in Example 2 (detection wavelength: 230 nm).

[0036] Figure 9 It is the chromatogram for the content determination of Jingzhi Guanxin Oral Liquid in Chinese Pharmacopoeia in Comparative Example 1.

[0037] Figure 10 It is the chromatogram of Jingzhi Guanxin Oral Liquid analyzed by using ordinary high performance liquid chromatography in Comparative Example 2. Specific Embodiments

[0038] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0039] Reagents: Methanol (Tianjin Damao Chemical Reagent Factory, 20200901, analytical grade); Methanol (Honeywell, U8MG1H, chromatographic grade); Acetonitrile (Honeywell, TAQA1H, chromatographic grade); Phosphoric acid (Shanghai Aladdin Biochemical Technology Co., Ltd., D1923158, chromatographic grade).

[0040] Reference substances: Gallic acid (batch number: 110831-200803, purity 91.5%), Danshensu sodium (batch number: 110855-201614, purity 98.1%), Protocatechuic aldehyde (batch number: 110810-201608, purity 99.3%), Paeoniflorin (batch number: 110736-202044, purity 96.8%) and Salvianolic acid B (batch number: 111562-201716, purity 99.9%) were purchased from the National Institutes for Food and Drug Control. Paeonolide glycoside (batch number: SMB00082, purity 91.4%), Lithospermic acid (batch number: PHL80491, purity 99.6%) and Salvianolic acid A (batch number: 97599, purity 98.7%) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0041] Samples: 13 batches of Jingzhi Guanxin Oral Liquid (batch numbers: 6018010101, 6118060101, 6118070101, 6118070102, 6118100401, 6118110101, 6118110102, 6119030101, 6120040201, 6120040202, 6020080101, 6020090101, 6020090102) were all provided by Guangdong Litaipharm Co., Ltd. The raw medicinal materials of Salvia miltiorrhiza, Paeonia lactiflora, Ligusticum chuanxiong, Carthamus tinctorius and Aquilaria sinensis were provided by Guangdong Litaipharm Co., Ltd.; the negative controls of Salvia miltiorrhiza, Paeonia lactiflora, Ligusticum chuanxiong, Carthamus tinctorius and Aquilaria sinensis were prepared according to the prescription ratio and preparation method of Jingzhi Guanxin Oral Liquid in Chinese Pharmacopoeia.

[0042] Unless otherwise specified, the reagents and materials used in the following examples were all purchased commercially.

[0043] Example 1 Construction of the characteristic fingerprint of Jingzhi Guanxin Oral Liquid

[0044] 1. Solution preparation:

[0045] 1-1. Preparation of the reference substance solution: Appropriate amounts of reference substances of gallic acid, sodium danshensu, protocatechualdehyde, albiflorin, paeoniflorin, lithospermic acid, salvianolic acid B and salvianolic acid A were accurately weighed and dissolved in 75% methanol to prepare a mixed solution containing about 20 μg of each in 1 ml, and thus obtained.

[0046] 1-2. Preparation of the test solution: 1 ml of Jingzhi Guanxin Oral Liquid was accurately measured and placed in a 50-ml volumetric flask, filled with 75% methanol to the scale, shaken well, sonicated (power 500 W, frequency 40 kHz) for 15 minutes, filtered, and the subsequent filtrate was taken, and thus obtained.

[0047] 1-3. Preparation of the reference crude drug solution: 22.8 g of Salvia miltiorrhiza, 11.4 g of Paeonia lactiflora, 11.4 g of Ligusticum chuanxiong, 11.4 g of Carthamus tinctorius and 7.6 g of Aquilaria sinensis were accurately weighed. Salvia miltiorrhiza, Paeonia lactiflora, Ligusticum chuanxiong and Aquilaria sinensis were decocted with water three times respectively and concentrated to an appropriate amount, and then filtered; Carthamus tinctorius was soaked with an appropriate amount of water at 80 °C twice and concentrated to an appropriate amount, and then filtered; the above filtrates were cooled and placed in 50-mL volumetric flasks respectively, filled with water to the scale, and shaken well; 1 ml of the above solutions was accurately measured respectively and placed in a 50-ml volumetric flask, filled with 75% methanol to the scale, shaken well, sonicated (power 500 W, frequency 40 kHz) for 15 minutes, filtered, and the subsequent filtrate was taken, and thus obtained.

[0048] 2. Chromatographic conditions:

[0049] Detection was carried out using ultra-high performance liquid chromatography. Chromatographic column: Waters Acquity UPLC™ HSS T3 (2.1×150 mm, 1.8 μm); column temperature was 25 °C, flow rate was 0.3 mL per minute, injection volume was 1 μL, and detection wavelength was 230 nm;

[0050] Mobile phase: Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was carried out according to Table 1.

[0051] Table 1 Elution gradient

[0052] Time (min) Mobile phase A (%) Mobile phase B (%) 0 4 96 30 12 88 35 12 88 65 24 76 72 24 76 95 47 53

[0053] 3. Determination of characteristic chromatogram standard

[0054] The refined Guanxin Koufuye with batch numbers 6018010101, 6118060101, 6118070101, 6118070102, 6118100401, 6118110101, 6118110102, 6119030101, 6120040201, and 6120040202 were respectively prepared into test solution, and were injected for detection according to the above chromatographic conditions. The results are shown in Table 2.

[0055] Table 2 Relative retention times (min) of characteristic peaks in the characteristic chromatograms of 10 batches of refined Guanxin Koufuye

[0056]

[0057]

[0058] It can be seen that the differences in the relative retention times of the 9 common peaks in the characteristic chromatograms of multiple batches of refined Guanxin Koufuye are small, and the RSDs are all within the range of ±10%, meeting the quality control requirements; the average value of the relative retention times can be selected as the measured value, and the measured values are: 0.05 (peak 1), 0.10 (peak 2), 0.19 (peak 3), 0.42 (peak 4), 0.47 (peak 5), 0.94 (peak 6), 1.00 (peak 7S), 1.07 (peak 8), 1.31 (peak 9).

[0059] The "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (2012 version) was used to carry out pattern recognition on 10 batches of refined Guanxin Koufuye, and the common pattern of the high performance liquid chromatography characteristic chromatogram of refined Guanxin Koufuye was obtained by the average method, and a reference characteristic chromatogram was established. The results are shown in Figure 1Peak 1 is gallic acid, Peak 2 is danshensu, Peak 3 is protocatechuic aldehyde, Peak 4 is albiflorin glycoside, Peak 5 is paeoniflorin, Peak 6 is lithospermic acid, Peak 7 is salvianolic acid B, Peak 8 is salvianolic acid A, and Peak 9 is inferred to be (3R)-Sativanone according to LC-MS technology; among them, Peak 7 has good stability and high resolution, and it is salvianolic acid B, the active ingredient in the monarch drug danshen, and is selected as the reference peak.

[0060] 4. Verification of characteristic chromatogram

[0061] Take the refined Guanxin Oral Liquid with batch numbers 6020080101, 6020090101, and 6020090102 (these 3 batches of oral liquid are samples after replacing the auxiliary material sucrose with sucralose in the production process) to prepare the test solution, and inject samples for detection according to the above chromatographic conditions. The results show that the relative retention times of each characteristic peak in the characteristic chromatograms of the 3 batches of refined Guanxin Oral Liquid samples all meet the above characteristic chromatogram standards.

[0062] 5. Identification and attribution of common peaks in characteristic chromatogram:

[0063] Use the PDA detector and TOF-MS / MS detection system to qualitatively identify the 9 characteristic peaks in the characteristic chromatogram, and determine the chromatographic peak attribution and peak purity. By comparing the retention times of the characteristic peaks and the ultraviolet absorption spectra in the reference substance solution, reference medicinal material solution and test solution of refined Guanxin Oral Liquid, and combining the fragmentation information of the quasi-molecular ion peaks of the mass spectra, the common peaks in the characteristic chromatogram are identified and attributed, and the results are shown in Figures 2 - 4 .

[0064] By comparing with the chromatogram of the reference medicinal material, it is concluded that the 9 common peaks are attributed to 3 medicinal materials, danshen, red peony root and dalbergia wood. Among them, Peaks 2, 3, 6, 7, and 8 are attributed to danshen, Peaks 1, 4, and 5 are attributed to red peony root, and Peak 9 is attributed to dalbergia wood.

[0065] Example 2 Determination of the contents of phenolic acids and paeoniflorin glycosides in refined Guanxin Oral Liquid

[0066] 1. Solution preparation:

[0067] 2-1. Preparation of reference substance solution: Take appropriate amounts of reference substances of danshensu sodium, protocatechuic aldehyde, albiflorin glycoside, paeoniflorin, lithospermic acid, salvianolic acid B and salvianolic acid A, weigh accurately, and make a mixed reference substance solution containing 54 μg of danshensu, 3 μg of protocatechuic aldehyde, 20 μg of albiflorin glycoside, 73 μg of paeoniflorin, 18 μg of lithospermic acid, 62 μg of salvianolic acid B and 23 μg of salvianolic acid A in 1 ml with 75% methanol, and you will get it.

[0068] 2-2. Preparation of the test solution: Accurately measure 1 ml of the refined Guanxin Oral Liquid, place it in a 50-ml volumetric flask, add 75% methanol to the scale, shake well, perform ultrasonic treatment (power 500 W, frequency 40 kHz) for 15 minutes, filter, and take the subsequent filtrate to obtain the solution.

[0069] 2-3. Preparation of the reference crude drug solution: Weigh accurately 22.8 g of Salvia miltiorrhiza Bunge, 11.4 g of Paeonia lactiflora Pall., 11.4 g of Ligusticum wallichii Franch., 11.4 g of Carthamus tinctorius L., and 7.6 g of Dalbergia odorifera T. Chen. Decoct Salvia miltiorrhiza Bunge, Paeonia lactiflora Pall., Ligusticum wallichii Franch., and Dalbergia odorifera T. Chen with water three times respectively, concentrate to an appropriate volume, and filter; Add an appropriate amount of water to Carthamus tinctorius L., perform warm soaking at 80 °C twice, concentrate to an appropriate volume, and filter; Let the above filtrates cool, place them in 50-mL volumetric flasks respectively, add water to the scale, and shake well; Accurately measure 1 mL of the above solutions respectively, place them in a 50-ml volumetric flask, add 75% methanol to the scale, shake well, perform ultrasonic treatment (power 500 W, frequency 40 kHz) for 15 minutes, filter, and take the subsequent filtrate to obtain the solution.

[0070] 2-4. Preparation of the negative control solution: Refer to the preparation of the reference crude drug solution in 2-3. The difference is that Salvia miltiorrhiza Bunge, Paeonia lactiflora Pall., Ligusticum wallichii Franch., Carthamus tinctorius L., and Dalbergia odorifera T. Chen are removed from the components respectively to prepare the Salvia miltiorrhiza Bunge negative control solution, Paeonia lactiflora Pall. negative control solution, Ligusticum wallichii Franch. negative control solution, Carthamus tinctorius L. negative control solution, and Dalbergia odorifera T. Chen negative control solution; Take 5 g of sucrose and 0.1 g of sodium benzoate as excipients, add an appropriate amount of water, boil to dissolve and filter, let the filtrate cool and place it in a 50-mL volumetric flask, add water to the scale, and shake well to obtain the excipient negative control solution. Accurately measure 1 mL of the Salvia miltiorrhiza Bunge, Paeonia lactiflora Pall., Ligusticum wallichii Franch., Carthamus tinctorius L., Dalbergia odorifera T. Chen, and excipient negative control solutions respectively, place them in a 50-ml volumetric flask, add 75% methanol to the scale, shake well, perform ultrasonic treatment (power 500 W, frequency 40 kHz) for 15 minutes, filter, and take the subsequent filtrate to obtain the solution.

[0071] 2. Chromatographic conditions: The chromatographic conditions refer to Example 1. The difference is that when determining the content of phenolic acid components, the detection wavelength is 286 nm, and when detecting the content of paeoniflorin components, the detection wavelength is 230 nm.

[0072] 3. Content determination:

[0073] Take 13 batches of refined Guanxin Oral Liquid samples (10 batches of samples, namely 6018010101, 6118060101, 6118070101, 6118070102, 6118100401, 6118110101, 6118110102, 6119030101, 6120040201, and 6120040202, are refined Guanxin Oral Liquid with unchanged excipients; 3 batches of samples, namely 6020080101, 6020090101, and 6020090102, are oral liquids with the excipient changed from sucrose to sucralose) and prepare them according to the method under item 2-2 Preparation of Test Solution. Prepare the reference substance according to the method under item 2-1 Preparation of Reference Solution, and determine the content using the above chromatographic conditions. Calculate the contents of danshensu, protocatechuic aldehyde, albiflorin glycoside, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A in 13 batches of refined Guanxin Oral Liquid. When the concentration of the test solution is within the range of ±10% of the concentration of the reference solution, calculate the content using the external standard single-point method. If it is not within the range of ±10%, calculate the content using the reference substance dilution method or the sample dilution method.

[0074] The results show that the content of danshensu in 10 batches of the original refined Guanxin Oral Liquid is between 2.0464 and 3.3084 mg / mL, with an average content of 2.8310 mg / mL; the content of protocatechuic aldehyde is between 0.0903 and 0.2662 mg / mL, with an average content of 0.1632 mg / mL; the content of albiflorin glycoside is between 1.3341 and 2.7912 mg / mL, with an average content of 2.3498 mg / mL; the content of paeoniflorin is between 2.6653 and 4.5714 mg / mL, with an average content of 3.8833 mg / mL; the content of lithospermic acid is between 0.8848 and 1.4382 mg / mL, with an average content of 1.1886 mg / mL; the content of salvianolic acid B is between 2.8241 and 6.6906 mg / mL, with an average content of 4.1792 mg / mL; the content of salvianolic acid A is between 1.8063 and 2.9681 mg / mL, with an average content of 2.3395 mg / mL.

[0075] If the content limit is set by decreasing 30% based on the average content of 10 batches, then for this product, per 1 mL, the total amount of danshen calculated as the total of danshensu, protocatechuic aldehyde, lithospermic acid, salvianolic acid B, and salvianolic acid A shall not be less than 7.5 mg; the total amount of red peony root calculated as the total of albiflorin glycoside and paeoniflorin shall not be less than 4.3 mg.

[0076] Detect the 13 batches of samples according to the above standards. Except for the batch 6119030101 sample with 4.0 mg of albiflorin glycoside and paeoniflorin being unqualified, the rest of the samples are qualified.

[0077] Example 3 Optimization of Conditions for Characteristic Chromatogram and Content Determination

[0078] 1. Optimization of detection wavelength:

[0079] (1) Optimization of detection wavelength of characteristic spectrum: Using the PDA detector, the test solution sample was scanned at 210-400 nm under the detection conditions of Example 1. The 3D ultraviolet absorption spectrum showed that there were more absorption peaks at 228-232 nm and the baseline was stable. Therefore, 228-232 nm was selected as the detection wavelength to detect as many components in the refined Guanxin oral liquid as possible.

[0080] (2) Optimal detection wavelength for content determination:

[0081] When determining the content of phenolic acid components, methanol solutions of sodium danshensu, protocatechuic aldehyde, lithospermic acid, salvianolic acid B and salvianolic acid A reference substances were taken to determine the ultraviolet absorption spectrum; among them, the maximum absorption wavelength of the salvianolic acid B reference substance was 286.9nm, and according to the national metrological verification procedures, the maximum allowable error of the wavelength indication of the diode array detector is ±2nm. Combined with the "Chinese Pharmacopoeia" 2020 edition, page 78 of the Danshen Medicinal Materials [Content Determination], 286nm was selected as the detection wavelength for salvianolic acid B, and 284-288nm was selected as the detection wavelength.

[0082] When detecting the content of paeoniflorin components, methanol solutions of paeoniflorin lactone glycosides and paeoniflorin reference substances were taken to determine the ultraviolet absorption spectrum; among them, the maximum absorption wavelength of paeoniflorin reference substance was 232.3nm, and according to the national metrological verification procedures, the maximum allowable error of the wavelength indication of the diode array detector is ±2nm. Combined with the "Chinese Pharmacopoeia" 2020 edition, page 1845, the refined Guanxin Oral Liquid [Content Determination] selected 230nm as the detection wavelength for paeoniflorin, and 228-232nm was selected as the detection wavelength.

[0083] 2. Optimization of chromatographic column: Refer to the detection conditions of Example 1, except that the chromatographic column is replaced with AgilentRRHD Eclipse Plus C18 (2.1×150mm, 1.8μm) or Waters ACQUITY BEH C18 (2.1×150mm, 1.7μm), and other parameters or operations are referred to Example 1, and the test solution and the reference solution are tested. It was found that the peak shape of each chromatographic peak in the obtained chromatogram was good, and basic separation could be achieved, indicating that conventional C18 chromatographic columns are suitable for the detection of the characteristic spectrum of the present invention.

[0084] 3. Optimization of column temperature: Refer to the detection conditions of Example 1, except that the column temperature is changed to 30°C, and other parameters or operations are referred to Example 1. The test solution is tested, and the results are shown in Table 3. The results show that the column temperature has little effect on the separation of chromatographic peaks, and a column temperature of 25 to 30°C is applicable.

[0085] Table 3 Effect of different column temperatures on chromatographic peak separation

[0086]

[0087] 4. Optimization of the mobile phase: Referring to the detection conditions of Example 1, except that mobile phase A is changed to methanol, and other parameters or operations refer to Example 1, the test solution is detected. It is found that the peaks in the obtained chromatogram have good peak shapes and the baseline is stable, indicating that either acetonitrile or methanol can be used as the organic phase for the detection of the characteristic chromatogram of the present invention.

[0088] 5. Optimization of the flow rate: Referring to the detection conditions of Example 1, except that the flow rate is changed to 0.25 mL / min, and other parameters or operations refer to Example 1, the test solution is detected. It is found that different flow rates have little effect on the resolution of chromatographic peaks, and flow rates of 0.25 - 0.30 mL / min are all applicable.

[0089] 6. Optimization of the preparation of the test solution: Referring to the detection conditions of Example 1, except that the ultrasonic treatment time of the test solution is changed to 30 minutes, and other parameters or operations refer to Example 1, the test solution is detected. It is found that the difference in the peak area between the ultrasonic treatment time of 15 minutes and that of 30 minutes is not significant, and ultrasonic treatment for 15 - 30 minutes is all applicable.

[0090] Methodology investigation of the characteristic chromatogram in Example 4

[0091] 1. Specificity

[0092] Using 75% methanol solution as the blank solvent, taking the test solution of Jingzhi Guanxin Oral Liquid (batch number: 6118110101) and the negative control solution of excipients, detecting and analyzing under the chromatographic conditions of Example 1. The results show that the identification of 9 common chromatographic peaks in Jingzhi Guanxin Oral Liquid is not interfered by the blank solvent and excipient factors, and it has good specificity.

[0093] 2. Precision

[0094] Precisely measure the test solution of the same batch of Jingzhi Guanxin Oral Liquid (batch number: 6118110101), detect and analyze by injecting samples continuously for 6 times under the chromatographic conditions of Example 1, and record the chromatogram; using the 7th peak as the reference peak, calculate the relative retention time and relative peak area of each characteristic peak, as well as the RSD. The results show that the RSD of the relative retention time of each characteristic peak is in the range of 0.0% - 0.4%, and the RSD of the relative peak area is in the range of 0.0% - 1.4%, indicating that the precision of this characteristic chromatogram is good.

[0095] 3. Intermediate precision

[0096] Referring to the method and conditions of Example 1, the effects of different analysts, different dates, and different equipment on the precision of the same batch of Refined Guanxin Oral Liquid (batch number: 6118110101) were investigated. Two parallel samples were used. Taking peak 7 as the reference peak, the relative retention time and relative peak area of each characteristic peak were calculated, as well as the RSD. It was found that the RSD of the relative retention time of each characteristic peak among different analysts was in the range of 0.0% - 0.5%, and the RSD of the relative peak area was in the range of 0.0% - 1.2%; the RSD of the relative retention time of each characteristic peak on different dates was in the range of 0.0% - 0.7%, and the RSD of the relative peak area was in the range of 0.0% - 0.4%; the RSD of the relative retention time of each characteristic peak on different equipment was in the range of 0.0% - 1.0%, and the RSD of the relative peak area was in the range of 0.0% - 1.3%, indicating that the intermediate precision of this characteristic chromatogram is good.

[0097] 4. Stability

[0098] The test solution of Refined Guanxin Oral Liquid (batch number: 6118110101) was injected at 0, 2, 8, 24, 36, and 48 hours respectively, and analyzed and detected according to the chromatographic conditions of Example 1. The chromatogram was recorded. Taking peak 7 as the reference peak, the relative retention time and relative peak area of each characteristic peak were calculated, as well as the RSD. It was found that the RSD of the relative retention time of each characteristic peak was in the range of 0.0% - 0.5%, and the RSD of the relative peak area was in the range of 0.0% - 1.9%, indicating that the characteristic components in the test solution of Refined Guanxin Oral Liquid have good stability within 48 hours.

[0099] 5. Repeatability

[0100] Referring to the preparation of the test solution, 6 test solutions of Refined Guanxin Oral Liquid (batch number: 6118110101) were prepared in parallel, analyzed and detected according to the chromatographic conditions of Example 1. The chromatogram was recorded. Taking peak 7 as the reference peak, the relative retention time and relative peak area of each characteristic peak were calculated, as well as the RSD. It was found that the RSD of the relative retention time of each characteristic peak was in the range of 0.0% - 0.9%, and the RSD of the relative peak area was in the range of 0.0% - 1.9%, indicating that the repeatability of this characteristic chromatogram is good.

[0101] 6. Robustness

[0102] The test solution of Jingzhi Guanxin Oral Liquid (batch number: 6118110101) was separately injected using two different types of chromatographic columns, Waters AcquityUPLCTM HSS T3 (2.1×150 mm, 1.8 μm) and Agilent RRHD Eclipse Plus C18 (2.1×150 mm, 1.8 μm), and the chromatograms were recorded according to the chromatographic conditions of Example 1. It was found that the relative retention time RSD of each characteristic peak was in the range of 0.0% - 0.4%, and the relative peak area RSD was in the range of 0.0% - 1.8%, indicating that the durability of this characteristic chromatogram was good.

[0103] Methodology Investigation of Content Determination in Example 5

[0104] 1. System Suitability:

[0105] The reference substance solution and the test solution (batch number: 6120040201) were separately injected into the liquid chromatograph and determined under the conditions of Example 2. It was found that danshensu, protocatechuic aldehyde, albiflorin, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A were all baseline separated from other components, and the resolution R > 1.5. The results are shown in Figures 5 - 8 .

[0106] 2. Specificity:

[0107] The 2-1 reference substance solution, 2-2 test solution (batch number: 6120040201), 2-3 reference medicinal material solution, and 2-4 negative control solution were separately injected into the liquid chromatograph and determined under the conditions of Example 2. The results showed that the content determination of danshensu, protocatechuic aldehyde, albiflorin, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A was not interfered by factors such as solvents, other medicinal materials, and excipients, and had good specificity.

[0108] 3. Linear Relationship and Range:

[0109] The reference substance solutions of danshensu with concentrations of 21.33 μg / mL, 42.65 μg / mL, 53.32 μg / mL, 106.64 μg / mL, 170.62 μg / mL, and 213.27 μg / mL were separately injected and determined at 286 nm under the conditions of Example 2. The regression analysis was performed with the integrated peak area value Y of danshensu against the concentration X of the danshensu reference substance, and the regression equation was: Y = 1939.2X - 17019, and the correlation coefficient r = 0.9998. The results showed that danshensu had a good linear relationship between the concentration X (μg / mL) and the peak area Y value in the range of 21.33 - 213.27 μg / mL.

[0110] Inject the protocatechuic aldehyde reference substance solutions with concentrations of 1.02 μg / mL, 2.03 μg / mL, 2.54 μg / mL, 5.08 μg / mL, 8.13 μg / mL, and 10.16 μg / mL respectively, and determine them at 286 nm under the conditions of Reference Example 2. Perform regression analysis on the integrated peak area value Y of protocatechuic aldehyde against the concentration X of the protocatechuic aldehyde reference substance. The regression equation is: Y = 14156X - 6237.1, and the correlation coefficient r = 0.9998. The results show that within the range of 1.02 - 8.13 μg / mL of protocatechuic aldehyde, there is a good linear relationship between the concentration X (μg / mL) and the peak area Y value.

[0111] Inject the albiflorin glycoside reference substance solutions with concentrations of 9.90 μg / mL, 19.81 μg / mL, 24.76 μg / mL, 49.52 μg / mL, 79.24 μg / mL, and 99.05 μg / mL respectively, and determine them at 230 nm under the conditions of Reference Example 2. Perform regression analysis on the integrated peak area value Y of albiflorin glycoside against the concentration X of the albiflorin glycoside reference substance. The regression equation is: Y = 1810.9X - 5209, and the correlation coefficient r = 0.9997. The results show that within the range of 9.90 - 99.05 μg / mL of albiflorin glycoside, there is a good linear relationship between the concentration X (μg / mL) and the peak area Y value.

[0112] Inject the paeoniflorin reference substance solutions with concentrations of 20.57 μg / mL, 41.14 μg / mL, 51.42 μg / mL, 102.84 μg / mL, 164.54 μg / mL, and 205.68 μg / mL respectively, and determine them at 230 nm under the conditions of Reference Example 2. Perform regression analysis on the integrated peak area value Y of paeoniflorin against the concentration X of the paeoniflorin reference substance. The regression equation is: Y = 2933.9X - 19114, and the correlation coefficient r = 0.9998. The results show that within the range of 20.57 - 205.68 μg / mL of paeoniflorin, there is a good linear relationship between the concentration X (μg / mL) and the peak area Y value.

[0113] Inject the lithospermic acid reference substance solutions with concentrations of 14.79 μg / mL, 29.59 μg / mL, 36.99 μg / mL, 74.97 μg / mL, 118.36 μg / mL, and 147.95 μg / mL respectively, and determine them at 286 nm under the conditions of Reference Example 2. Perform regression analysis on the integrated peak area value Y of lithospermic acid against the concentration X of the lithospermic acid reference substance. The regression equation is: Y = 5075.3X - 33418, and the correlation coefficient r = 0.9998. The results show that within the range of 14.79 - 147.95 μg / mL of lithospermic acid, there is a good linear relationship between the concentration X (μg / mL) and the peak area Y value.

[0114] Solutions of salvianolic acid B reference substance with concentrations of 20.18 μg / mL, 40.36 μg / mL, 50.44 μg / mL, 100.89 μg / mL, 161.42 μg / mL, and 201.78 μg / mL were injected respectively. The measurements were carried out at 286 nm under the conditions of Reference Example 2. Regression analysis was performed on the integrated peak area value Y of salvianolic acid B against the concentration X of the salvianolic acid B reference substance. The regression equation was: Y = 4226.8X - 37349, and the correlation coefficient r = 0.9998. The results showed that in the range of 20.18 - 201.78 μg / mL of salvianolic acid B, there was a good linear relationship between the concentration X (μg / mL) and the peak area Y value.

[0115] Solutions of salvianolic acid A reference substance with concentrations of 10.33 μg / mL, 20.66 μg / mL, 25.83 μg / mL, 51.65 μg / mL, 82.65 μg / mL, and 103.31 μg / mL were injected respectively. The measurements were carried out at 286 nm under the conditions of Reference Example 2. Regression analysis was performed on the integrated peak area value Y of salvianolic acid A against the concentration X of the salvianolic acid A reference substance. The regression equation was: Y = 10018X - 42953, and the correlation coefficient r = 0.9998. The results showed that in the range of 10.33 - 103.31 μg / mL of salvianolic acid A, there was a good linear relationship between the concentration X (μg / mL) and the peak area Y value.

[0116] 4. Precision:

[0117] Take the 2-1 reference substance solution. Referring to the chromatographic conditions of Reference Example 2, inject it continuously 6 times on a high-performance liquid chromatograph, and record the peak areas of danshensu, protocatechuic aldehyde, albiflorin glycoside, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A, and calculate the RSD value. The results showed that the RSD% of the peak area of the reference substance danshensu was 0.33%, the RSD% of the peak area of the reference substance protocatechuic aldehyde was 0.27%, the RSD% of the peak area of the reference substance albiflorin glycoside was 0.24%, the RSD% of the peak area of the reference substance paeoniflorin was 0.34%, the RSD% of the peak area of the reference substance lithospermic acid was 0.28%, the RSD% of the peak area of the reference substance salvianolic acid B was 0.27%, and the RSD% of the peak area of the reference substance salvianolic acid A was 0.21%, indicating that the precision of this method was good.

[0118] 5. Intermediate precision:

[0119] Refer to the method and conditions of Reference Example 2 to investigate the influence of different analysts, different dates, and different equipment on the precision of the same batch of refined Guanxin Oral Liquid (batch number: 6118110101), and calculate the relative average deviation RAD% of the contents of danshensu, protocatechuic aldehyde, albiflorin glycoside, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A.

[0120] The results showed that the contents of danshensu in the test samples under different date conditions were 3.1401 mg / mL, 3.1383 mg / mL, and the RAD% was 0.03%; the contents of protocatechuic aldehyde in the test samples were 0.1499 mg / mL, 0.1506 mg / mL, and the RAD% was 0.23%; the contents of albiflorin in the test samples were 2.0547 mg / mL, 2.0531 mg / mL, and the RAD% was 0.04%; the contents of paeoniflorin in the test samples were 3.8584 mg / mL, 3.8693 mg / mL, and the RAD% was 0.14%; the contents of lithospermic acid in the test samples were 0.8192 mg / mL, 0.8237 mg / mL, and the RAD% was 0.27%; the contents of salvianolic acid B in the test samples were 2.9243 mg / mL, 2.9295 mg / mL, and the RAD% was 0.09%; the contents of salvianolic acid A in the test samples were 1.1861 mg / mL, 1.1814 mg / mL, and the RAD% was 0.19%.

[0121] Under different analyst conditions, the contents of danshensu in the test samples were 3.1321 mg / mL, 3.1500 mg / mL, and the RAD% was 0.28%; the contents of protocatechuic aldehyde in the test samples were 0.1495 mg / mL, 0.1482 mg / mL, and the RAD% was 0.43%; the contents of albiflorin in the test samples were 2.0636 mg / mL, 2.0539 mg / mL, and the RAD% was 0.24%; the contents of paeoniflorin in the test samples were 3.8545 mg / mL, 3.8310 mg / mL, and the RAD% was 0.31%; the contents of lithospermic acid in the test samples were 0.8145 mg / mL, 0.8213 mg / mL, and the RAD% was 0.41%; the contents of salvianolic acid B in the test samples were 2.9317 mg / mL, 2.9098 mg / mL, and the RAD% was 0.37%; the contents of salvianolic acid A in the test samples were 1.1843 mg / mL, 0.1894 mg / mL, and the RAD% was 0.22%.

[0122] Under different equipment conditions, the contents of danshensu in the test samples were 3.1349 mg / mL and 3.1028 mg / mL, and the RAD% was 0.51%; the contents of protocatechuic aldehyde in the test samples were 0.1501 mg / mL and 0.1482 mg / mL, and the RAD% was 0.61%; the contents of albiflorin in the test samples were 2.0623 mg / mL and 2.0476 mg / mL, and the RAD% was 0.36%; the contents of paeoniflorin in the test samples were 3.8553 mg / mL and 3.9015 mg / mL, and the RAD% was 0.60%; the contents of lithospermic acid in the test samples were 0.8277 mg / mL and 0.8185 mg / mL, and the RAD% was 0.56%; the contents of salvianolic acid B in the test samples were 2.9173 mg / mL and 2.9401 mg / mL, and the RAD% was 0.39%; the contents of salvianolic acid A in the test samples were 1.1843 mg / mL and 1.1931 mg / mL, and the RAD% was 0.37%.

[0123] The results showed that under different analysts, different dates, and different equipment conditions, the relative average deviation values of the contents of danshensu, protocatechuic aldehyde, albiflorin, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A in Jingzhi Guanxin Oral Liquid were within a reasonable range, indicating good intermediate precision for this method.

[0124] 6. Stability:

[0125] Inject the reference substance solution 2-1 and the test sample solution 2-2 (Jingzhi Guanxin Oral Liquid, batch number: 6118110101) at 0, 2, 8, 24, 36, and 48 hours respectively, and analyze and detect according to the chromatographic conditions in Example 2. Determine the peak areas of danshensu, protocatechuic aldehyde, albiflorin, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A and calculate the RSD values.

[0126] The results found that the RSD% of the peak areas of danshensu in the reference substance solution and the test sample solution were 0.48% and 0.87% respectively; the RSD% of the peak areas of protocatechuic aldehyde in the reference substance solution and the test sample solution were 0.77% and 1.12% respectively; the RSD% of the peak areas of albiflorin in the reference substance solution and the test sample solution were 0.69% and 0.88% respectively; the RSD% of the peak areas of paeoniflorin in the reference substance solution and the test sample solution were 0.60% and 0.82% respectively; the RSD% of the peak areas of lithospermic acid in the reference substance solution and the test sample solution were 0.51% and 0.76% respectively; the RSD% of the peak areas of salvianolic acid B in the reference substance solution and the test sample solution were 0.79% and 0.85% respectively; the RSD% of the peak areas of salvianolic acid A in the reference substance solution and the test sample solution were 0.64% and 0.73% respectively. The results indicated that the reference substance solutions and the test sample solutions of danshensu, protocatechuic aldehyde, albiflorin, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A had good stability within 48 hours.

[0127] 7. Repeatability:

[0128] Referring to the preparation of the test solution in 2-2, prepare 6 portions of the refined Guanxin Oral Liquid (batch number: 6120040201) in parallel. Analyze and detect according to the chromatographic conditions in Example 2, and calculate the average contents and RSD% of danshensu, protocatechuic aldehyde, albiflorin glycoside, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A.

[0129] The average content of danshensu in the 6 portions of the test solution was measured to be 2.9895 mg / mL, and the RSD% value was 2.87%; the average content of protocatechuic aldehyde in the 6 portions of the test solution was measured to be 0.1480 mg / mL, and the RSD% value was 2.81%; the average content of albiflorin glycoside in the 6 portions of the test solution was measured to be 2.0904 mg / mL, and the RSD% value was 2.67%; the average content of paeoniflorin in the 6 portions of the test solution was measured to be 3.6403 mg / mL, and the RSD% value was 2.68%; the average content of lithospermic acid in the 6 portions of the test solution was measured to be 0.8901 mg / mL, and the RSD% value was 1.74%; the average content of salvianolic acid B in the 6 portions of the test solution was measured to be 3.1067 mg / mL, and the RSD% value was 1.55%; the average content of salvianolic acid A in the 6 portions of the test solution was measured to be 1.1716 mg / mL, and the RSD% value was 2.02%. It indicates that the repeatability of this content determination method is good.

[0130] 8. Sample addition recovery rate:

[0131] Precisely measure an appropriate amount of the refined Guanxin Oral Liquid (batch number: 6120040201, average content 2.9895 mg / mL) with the measured danshensu content, and prepare 9 portions in parallel. Respectively and precisely add danshensu sodium reference substances with low, medium, and high concentrations. After processing according to the method under the preparation of the test solution in 2-2, refer to the chromatographic conditions in Example 2 for content determination, and calculate the recovery rate. The formula for calculating the sample addition recovery rate:

[0132]

[0133] Original amount (mg) = Sampling amount (g) × Average content (mg / g)

[0134] The recovery rates of danshensu for the 9 portions of the test solution were 98.07%, 99.84%, 101.46%, 98.31%, 103.95%, 97.85%, 94.21%, 97.49%, and 99.72% respectively. The average recovery rate was 98.99%, and the RSD value of the recovery rate was 2.76%. It indicates that the recovery rate of this method is good and the accuracy is high.

[0135] Accurately measure an appropriate amount of the refined Guanxin Oral Liquid (batch number: 6120040201, average content 0.1480 mg / mL) with the measured protocatechuic aldehyde content, in 9 parallel portions. Respectively and accurately add reference substances of sodium danshensu at low, medium, and high concentrations. After treating according to the method under item 2-2 Preparation of Test Solution, determine the content with reference to the chromatographic conditions in Example 2, and calculate the recovery rate. The results show that the recovery rates of protocatechuic aldehyde for the 9 test samples are 93.56%, 94.24%, 95.07%, 98.70%, 98.47%, 95.89%, 99.80%, 100.49%, and 96.38% respectively. The average recovery rate is 96.96%, and the RSD value of the recovery rate is 2.58%, indicating that this method has good recovery rate and high accuracy.

[0136] Accurately measure an appropriate amount of the refined Guanxin Oral Liquid (batch number: 6120040201, average content 2.0904 mg / mL) with the measured albiflorin content, in 9 parallel portions. Respectively and accurately add reference substances of albiflorin at low, medium, and high concentrations. After treating according to the method under item 2-2 Preparation of Test Solution, determine the content with reference to the chromatographic conditions in Example 2, and calculate the recovery rate. The results show that the recovery rates of albiflorin for the 9 test samples are 98.46%, 99.75%, 102.59%, 100.22%, 102.20%, 101.75%, 103.12%, 102.34%, and 101.69% respectively. The average recovery rate is 101.35%, and the RSD value of the recovery rate is 1.51%, indicating that this method has good recovery rate and high accuracy.

[0137] Accurately measure an appropriate amount of the refined Guanxin Oral Liquid (batch number: 6120040201, average content 3.6403 mg / mL) with the measured paeoniflorin content, in 9 parallel portions. Respectively and accurately add reference substances of paeoniflorin at low, medium, and high concentrations. After treating according to the method under item 2-2 Preparation of Test Solution, determine the content with reference to the chromatographic conditions in Example 2, and calculate the recovery rate. The results show that the recovery rates of paeoniflorin for the 9 test samples are 96.84%, 101.48%, 102.80%, 96.80%, 98.01%, 98.37%, 98.92%, 101.14%, and 101.93% respectively. The average recovery rate is 99.59%, and the RSD value of the recovery rate is 2.29%, indicating that this method has good recovery rate and high accuracy.

[0138] Precisely measure an appropriate amount of the refined Guanxin Oral Liquid (batch number: 6120040201, average content 0.8901 mg / g) with the measured lithospermic acid content, in 9 parallel portions. Respectively and precisely add lithospermic acid reference substances at low, medium, and high concentrations. After treatment according to the method under the preparation of test solution in 2-2, conduct the content determination with reference to the chromatographic conditions in Example 2, and calculate the recovery rate. The results show that the recovery rates of lithospermic acid for the 9 test samples are 101.34%, 95.18%, 94.57%, 100.40%, 102.80%, 98.82%, 96.86%, 97.58%, and 97.22% respectively. The average recovery rate is 98.31%, and the RSD value of the recovery rate is 2.82%, indicating that this method has good recovery rate and high accuracy.

[0139] Precisely measure an appropriate amount of the refined Guanxin Oral Liquid (batch number: 6120040201, average content 3.1067 mg / mL) with the measured salvianolic acid B content, in 9 parallel portions. Respectively and precisely add salvianolic acid B reference substances at low, medium, and high concentrations. After treatment according to the method under the preparation of test solution in 2-2, conduct the content determination with reference to the chromatographic conditions in Example 2, and calculate the recovery rate. The results show that the recovery rates of salvianolic acid B for the 9 test samples are 95.86%, 94.97%, 94.22%, 97.17%, 95.54%, 94.68%, 92.64%, 90.44%, and 90.03% respectively. The average recovery rate is 93.95%, and the RSD value of the recovery rate is 2.59%, indicating that this method has good recovery rate and high accuracy.

[0140] Precisely measure an appropriate amount of the refined Guanxin Oral Liquid (batch number: 6120040201, average content 1.1716 mg / mL) with the measured salvianolic acid A content, in 9 parallel portions. Respectively and precisely add salvianolic acid A reference substances at low, medium, and high concentrations. After treatment according to the method under the preparation of test solution in 2-2, conduct the content determination with reference to the chromatographic conditions in Example 2, and calculate the recovery rate. The results show that the recovery rates of salvianolic acid A for the 9 test samples are 106.05%, 101.18%, 102.75%, 101.18%, 99.37%, 102.84%, 106.55%, 102.58%, and 99.44% respectively. The average recovery rate is 102.44%, and the RSD value of the recovery rate is 2.48%, indicating that this method has good recovery rate and high accuracy.

[0141] 9. Durability:

[0142] The test solution of Jingzhi Guanxin Oral Liquid (batch number: 6120040201) was separately injected and determined for the contents of danshensu, protocatechuic aldehyde, albiflorin glycoside, paeoniflorin, lithospermic acid, salvianolic acid B, and salvianolic acid A using two different types of chromatographic columns, Waters AcquityUPLCTM HSS T3 (2.1×150 mm, 1.8 μm) and Agilent RRHD Eclipse Plus C18 (2.1×150 mm, 1.8 μm), with reference to the chromatographic conditions in Example 2, and the relative standard deviation RSD was calculated. The RSD value of danshensu in the oral liquid was 1.19%, the RSD value of protocatechuic aldehyde was 1.15%, the RSD value of albiflorin glycoside was 1.03%, the RSD value of paeoniflorin was 1.25%, the RSD value of lithospermic acid was 0.96%, the RSD value of salvianolic acid B was 0.81%, and the RSD value of salvianolic acid A was 1.42%. The results showed that the method had good durability.

[0143] Comparative Example 1 Content determination of Jingzhi Guanxin Oral Liquid in the 2020 Edition of the Chinese Pharmacopoeia

[0144] Jingzhi Guanxin Oral Liquid was determined according to the conditions under [Content Determination] in the 2020 Edition of the Chinese Pharmacopoeia. The results are shown in Figure 9 . As can be seen from the figure, except for good separation of salvianolic acid B and paeoniflorin, the resolution of many other components was poor and there was little chromatographic peak information.

[0145] Comparative Example 2 Content determination of Jingzhi Guanxin Oral Liquid by ordinary high performance liquid chromatography

[0146] High performance liquid chromatography was used for detection. Chromatographic column: Welch AQ-C18 (4.6×250 mm, 5 μm); column temperature was 25 °C, flow rate was 1 mL per minute, injection volume was 10 μL, detection wavelength was 230 nm; mobile phase: acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was carried out according to Table 4.

[0147] Table 4 Elution gradient

[0148] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5 95 110 26 74 140 52 48

[0149] The results are shown in Figure 10 . As can be seen from the figure, the detection time required was longer, and the resolution of peak 1 gallic acid, peak 2 danshensu, and peak 8 salvianolic acid A was poor, with low separation efficiency.

[0150] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for constructing a characteristic fingerprint of Jingzhi Guanxin Oral Liquid, characterized in that, Detection was carried out by ultra - performance liquid chromatography, and the conditions of the ultra - performance liquid chromatography were as follows: Chromatographic column: octadecylsilyl bonded silica gel with an inner diameter of 2.0 - 2.1 mm, a column length of 100 - 150 mm, and a particle size of 1.7 - 2.0 μm; detection wavelength: 230 - 290 nm; column temperature: 25 - 30 °C; flow rate: 0.25 - 0.30 ml / min; mobile phase A: acetonitrile or methanol; mobile phase B: 0.1% phosphoric acid solution. Detection was carried out under the following gradient elution conditions: In the construction of the characteristic chromatogram, 9 common chromatographic peaks were detected: peak 1 was gallic acid, peak 2 was danshensu, peak 3 was protocatechuic aldehyde, peak 4 was albiflorin glycoside, peak 5 was paeoniflorin, peak 6 was lithospermic acid, peak 7 was salvianolic acid B, peak 8 was salvianolic acid A, and peak 9 was (3R)-Sativanone.

2. The method for constructing the characteristic chromatogram of the refined coronary heart oral liquid according to claim 1, characterized in that, In the construction of the characteristic chromatogram, salvianolic acid B at peak 7 was used as the reference peak of the characteristic chromatogram, and the relative retention times of other peaks were within ±10% of the specified values. The specified values were as follows: peak 1: 0.05; peak 2: 0.10; peak 3: 0.19; peak 4: 0.42; peak 5: 0.47; peak 6: 0.94; peak 7: 1.00; peak 8: 1.07; peak 9: 1.

31.

3. The method for constructing the characteristic chromatogram of the refined coronary heart oral liquid according to claim 1, wherein When constructing the characteristic chromatogram of Jingzhi Guanxin Oral Liquid, the detection wavelength was 228 - 232 nm.

4. The method for constructing the characteristic chromatogram of the refined coronary heart oral liquid according to claim 1, wherein In the conditions of the ultra - performance liquid chromatography, the injection volume was 1.0 - 1.5 μL.

5. The method for constructing the characteristic chromatogram of the refined coronary heart oral liquid according to claim 1, wherein Jingzhi Guanxin Oral Liquid was prepared into a test solution and then detected by ultra - performance liquid chromatography. The preparation method of the test solution included the following steps: Dilute Jingzhi Guanxin Oral Liquid with an alcohol solution, perform ultrasonic treatment, filter, and take the subsequent filtrate to obtain the test solution.

6. A method for determining the contents of multiple components in refined coronary heart oral liquid, characterized in that, Detection was carried out by ultra - performance liquid chromatography, and the conditions of the ultra - performance liquid chromatography were as follows: Chromatographic column: octadecylsilyl bonded silica gel with an inner diameter of 2.0 - 2.1 mm, a column length of 100 - 150 mm, and a particle size of 1.7 - 2.0 μm; detection wavelength: 230 - 290 nm; column temperature: 25 - 30 °C; flow rate: 0.25 - 0.30 ml / min; mobile phase A: acetonitrile or methanol; mobile phase B: 0.1% phosphoric acid solution. Detection was carried out under the following gradient elution conditions: The method can simultaneously determine the contents of danshensu, protocatechuic aldehyde, lithospermic acid, salvianolic acid B, salvianolic acid A, albiflorin glycoside, and paeoniflorin in Jingzhi Guanxin Oral Liquid.

7. The multi-component content determination method of Jingzhi Guanxin Oral Liquid according to claim 6, wherein, The detection wavelength for determining the content of phenolic acid components was 284 - 288 nm, and the detection wavelength for determining the content of paeoniflorin components was 228 - 232 nm.

8. The method for determining the contents of multiple components in the refined coronary heart oral liquid according to claim 6, wherein In the conditions of the ultra - performance liquid chromatography, the injection volume was 1.0 - 1.5 μL.

9. The multi-component content determination method of the refined coronary heart oral liquid according to claim 6, characterized in that, Jingzhi Guanxin Oral Liquid was prepared into a test solution and then detected by ultra - performance liquid chromatography. The preparation method of the test solution included the following steps: Dilute Jingzhi Guanxin Oral Liquid with an alcohol solution, perform ultrasonic treatment, filter, and take the subsequent filtrate to obtain the test solution.

10. A quality monitoring method for Jingzhi Guanxin Oral Liquid, characterized in that, The quality control is carried out by using the characteristic fingerprint construction method for refined Guanxin oral liquid described in any one of claims 1 to 5 or the multi-component content determination method for refined Guanxin oral liquid described in any one of claims 6 to 9.

11. The quality control method of the refined coronary heart oral liquid according to claim 10, characterized in that, The regulations for the determination results of the content of refined Guanxin oral liquid are as follows: Each 1 mL contains not less than 7.5 mg of Salvia miltiorrhiza in terms of the total amount of danshensu, protocatechuic aldehyde, lithospermic acid, salvianolic acid B and salvianolic acid A; each 1 mL contains not less than 4.3 mg of Paeonia lactiflora Pall. in terms of the total amount of albiflorin and paeoniflorin.

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