Analysis method of radix salviae miltiorrhizae injection, fingerprint spectrum of radix salviae miltiorrhizae injection and application of fingerprint spectrum

Abstract

The invention discloses an analysis method of a salvia miltiorrhiza injection, a fingerprint spectrum of the salvia miltiorrhiza injection and application of the fingerprint spectrum, and particularly relates to an analysis method of the salvia miltiorrhiza injection, an analysis method of components of the salvia miltiorrhiza injection, the fingerprint spectrum of the salvia miltiorrhiza injection and application of the fingerprint spectrum, and a quality control method of the salvia miltiorrhiza injection. The analysis method is carried out by adopting ultra-high performance liquid chromatography. Chromatographic conditions of the ultra-high performance liquid chromatography are as follows: a filler of a chromatographic column is octadecylsilane chemically bonded silica; the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.2%-0.5%; a gradient elution procedure is adopted. The analysis method can effectively separate components with similar structures in the salvia miltiorrhiza injection, the obtained fingerprint spectrum can simultaneously calibrate and identify multiple common peaks, and based on the same detection method, accurate quantitative detection can be further performed on the identified components in the fingerprint spectrum.

Description

Technical Field

[0001] The present invention relates to an analysis method of danshen injection, a fingerprint spectrum of danshen injection and its application, and specifically relates to an analysis method of danshen injection, an analysis method of components of danshen injection, a fingerprint spectrum of danshen injection and its application, and a quality control method of danshen injection. Background Art

[0002] Danshen injection is a sterilized aqueous solution prepared from danshen by modern processes such as water extraction and alcohol precipitation. It has the effects of promoting blood circulation to remove blood stasis and dredging the channels to nourish the heart. Because of its significant curative effect and rapid onset in the treatment of cardiovascular and cerebrovascular diseases, it is widely used clinically for the treatment of diseases such as coronary heart disease, chest tightness, and angina pectoris. Its main components are water-soluble salvianolic acids and oligosaccharides. Salvianolic acids are the main pharmacodynamic components and have pharmacological effects such as anti-atherosclerosis, anti-hyperlipidemia, anti-hypertension, anti-inflammatory, and cardiovascular protection.

[0003] In the current quality standard of danshen injection (standard number: WS3-B-3766-98-2011), only 4 components including sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B are specified in the content determination and fingerprint spectrum. In the quality standard of the first part of the 2020 edition of the Chinese Pharmacopoeia, the quality standard of danshen water extract (danshen total phenolic acid extract) is included. Among them, only rosmarinic acid (R9) and salvianolic acid B (R12) are specified in the fingerprint spectrum and content determination indexes, and there is no qualitative and quantitative research on other active components. The above standard index components are relatively limited and cannot meet the requirements of comprehensive quality control. Therefore, it is necessary to clarify the pharmacodynamic material basis of this preparation and then improve the quality control level.

[0004] Literature retrieval found that in the existing literature (Zhang Bei, Li Moying, Wu Tong, et al. Comparative study on the quality of commercially available Danshen injection [J]. Chinese Journal of Pharmaceutical Industry, 2021(011):052.), the contents of 3 saccharide components (D-fructose, D-glucose anhydrous, sucrose) and 6 phenolic acid components (sodium danshensu, protocatechuic aldehyde, salvianolic acid B, rosmarinic acid, lithospermic acid, salvianolic acid D) were determined. The fingerprint established in the existing literature (Zhang Han, Li Zhenhao, Fan Xiaohui. Application research of one-standard multi-detection UPLC quantitative fingerprint in the quality evaluation of Danshen injection [J]. China Journal of Chinese Materia Medica, 2019, 44(17):8.) calibrated 13 common peaks, identified the corresponding components of 11 common peaks by liquid chromatography-mass spectrometry technology, and simultaneously determined the contents of 6 components (danshensu, protocatechuic aldehyde, caffeic acid, rosmarinic acid, lithospermic acid, salvianolic acid B). The fingerprint established in the existing literature (Han Xiujuan, Feng Liang, Zhang Duoduo, et al. Analysis of the "sub-structure" of the material basis of Danshen injection from different manufacturers and quality comparison research [J]. China Journal of Chinese Materia Medica, 2016, 41(3):6.) calibrated 20 common peaks, identified and simultaneously determined 10 phenolic acid components, including danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, isoferulic acid, lithospermic acid, rosmarinic acid, salvianolic acid B, salvianolic acid A, salvianolic acid C).

[0005] In summary, the index components detected by the quality analysis methods of Danshen injection established in current research are still relatively limited, and all only reflect partial information of phenolic acid components or oligosaccharide components. For components with similar structures, such as salvianolic acid F and salvianolic acid D, etc., it is still difficult to effectively separate them, and the safety-related substance 5-hydroxymethylfurfural is rarely mentioned, which cannot comprehensively and fully reflect the overall quality of Danshen injection.

[0006] Therefore, it is urgent to establish a more comprehensive fingerprint of Danshen injection and an analysis method for the components of Danshen injection, improve the quality standard of this preparation, so as to conduct a systematic and comprehensive evaluation of the overall quality of this preparation. Summary of the Invention

[0007] To solve the technical defects such as incomplete results existing in the existing fingerprint and analysis methods of Danshen injection, the present invention proposes an analysis method for Danshen injection, an analysis method for the components of Danshen injection, a fingerprint of Danshen injection and its application, and a quality control method for Danshen injection. This analysis method can effectively separate components with similar structures in Danshen injection. The obtained fingerprint can simultaneously calibrate and identify multiple common peaks, and based on the same detection method, it can further quantitatively detect the components identified in the fingerprint, and can more comprehensively and accurately reflect the chemical composition information of Danshen injection, providing a more comprehensive scientific basis for effectively controlling the quality of Danshen injection.

[0008] To achieve the above object, the present invention adopts the following technical solutions.

[0009] The present invention provides an analytical method for danshen injection. The analytical method for danshen injection is carried out by ultra-high performance liquid chromatography. The chromatographic conditions of the ultra-high performance liquid chromatography include the following:

[0010] (1) The filler of the chromatographic column is octadecylsilyl-bonded silica gel;

[0011] (2) Mobile phase A is acetonitrile, and mobile phase B is an aqueous phosphoric acid solution with a volume fraction of 0.2% - 0.5%;

[0012] (3) The following gradient elution program is adopted:

[0013]

[0014] The percentages in the table are the volume percentages of each mobile phase in the total volume of mobile phase A and mobile phase B respectively.

[0015] In the present invention, through research, it is found that there are great difficulties in the actual detection of the components of danshen injection. Specifically: ① There are multiple chemically similar components, resulting in close elution positions, and the low-content components are easily interfered and masked by the peak signals of high-content components (especially danshensu and protocatechuic acid, salvianolic acid D and salvianolic acid F, salvianolic acid E, etc.); ② In addition to phenolic acid components, the injection safety-related substance 5-hydroxymethylfurfural contained is difficult to ensure effective separation and identification under the same detection conditions. In view of this, through various exploratory experiments on the chromatographic column, mobile phase and gradient elution program, the present invention finds that only when the above conditions are used can the effective separation of the above components with similar structures and 5-hydroxymethylfurfural be ensured as much as possible.

[0016] In some embodiments, the danshen injection is a qualified danshen injection or a danshen injection to be tested. Among them, the qualified danshen injection can refer to a danshen injection that meets the pharmaceutical grade; the danshen injection to be tested can refer to a danshen injection to be detected, the quality of which is uncertain whether it is qualified, and those skilled in the art can understand its specific meaning.

[0017] In some embodiments, the specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and packing particle size 1.8 μm, 1.9 μm or 2.6 μm.

[0018] In some specific embodiments, the chromatographic column is Waters ACQUITY HSS T3.

[0019] In some embodiments, mobile phase B is an aqueous phosphoric acid solution with a volume fraction of 0.3% - 0.5%.

[0020] In some embodiments, in the ultra-high performance liquid chromatography, the column temperature is 35-45°C, preferably 40-45°C.

[0021] In some embodiments, in the ultra-high performance liquid chromatography, the flow rate is 0.35-0.45 mL / min, preferably 0.40-0.45 mL / min.

[0022] In some embodiments, in the ultra-high performance liquid chromatography method, the injection volume is 1-5 μL, for example, 5 μL.

[0023] In some embodiments, in the ultra-high performance liquid chromatography, the detection wavelength is 254-326 nm, preferably 286 nm.

[0024] In some embodiments, the gradient elution program is shown in the following table:

[0025]

[0026] The percentages in the table are the volume percentages of each component in the total volume of mobile phase A and mobile phase B.

[0027] In some specific embodiments, the gradient elution program is shown in the following table:

[0028]

[0029] The percentages in the table are the volume percentages of each component in the total volume of mobile phase A and mobile phase B.

[0030] In some embodiments, the analysis method of Danshen injection comprises the following steps:

[0031] The test solution A containing qualified Danshen injection is tested by the ultra-high performance liquid chromatography method, and the test result is used to generate a fingerprint spectrum;

[0032] Preferably, the analysis method of Danshen injection further comprises the following steps:

[0033] The test solution B containing the Salvia miltiorrhiza injection to be tested is detected by the ultra-high performance liquid chromatography method, and the detection result is compared with the fingerprint spectrum.

[0034] In some preferred embodiments, the solvent in the test solutions A and B is methanol-water solution; wherein the volume fraction of methanol in the methanol-water solution is preferably 10%-30%, for example 10%.

[0035] In some preferred embodiments, the volume ratio of the qualified Danshen injection to the solvent in the test solution A is 1:(1-9), for example 1:4.

[0036] In some preferred embodiments, the volume ratio of the Danshen injection to be tested to the solvent in the test sample solution B is 1:(1 - 9), such as 1:4.

[0037] In some preferred embodiments, the preparation of the test sample solution A or B comprises the following steps: mixing the qualified Danshen injection or the Danshen injection to be tested with the solvent, and centrifuging to obtain the supernatant.

[0038] Among them, the rotation speed of the centrifugation is preferably 10000 - 20000 r·min -1 , such as 10000 r·min -1 , and the centrifugation time is preferably 10 - 30 min, such as 10 min.

[0039] In some preferred embodiments, the test sample solutions A and B further comprise a stabilizer; the stabilizer is preferably formic acid and / or acetic acid, more preferably acetic acid; the volume proportion of the stabilizer relative to the solvent is preferably 0.1% - 2%, such as 0.5%.

[0040] In some more preferred embodiments, the preparation of the test sample solution A or B comprises the following steps: mixing the stabilizer and the solvent first, then mixing with the qualified Danshen injection or the Danshen injection to be tested, and centrifuging to obtain the supernatant.

[0041] The present invention also provides a method for analyzing the components of Danshen injection. The method for analyzing the components of Danshen injection is carried out by using the ultra - high performance liquid chromatography method as described above, and comprises the following steps:

[0042] Substitute the peak area measured for the Danshen injection to be tested into the linear regression equation for calculation.

[0043] In some embodiments, the detection is carried out with a test sample solution containing the Danshen injection to be tested; the test sample solution containing the Danshen injection to be tested is defined as the test sample solution B as described above.

[0044] In some embodiments, the linear regression equation is obtained by detecting a reference substance and calculating; among them, the reference substance is selected from one or more of 5 - hydroxymethylfurfural, sodium danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, isoferulic acid, salvianolic acid F, salvianolic acid D, rosmarinic acid, salvianolic acid E, lithospermic acid, salvianolic acid B, salvianolic acid A, and salvianolic acid Y.

[0045] In some preferred embodiments, the detection is carried out with a reference substance solution containing a reference substance; the solvent in the reference substance solution is an aqueous methanol solution.

[0046] Among them, the volume fraction of methanol in the aqueous methanol solution is preferably 10% - 30%, such as 10%.

[0047] In some more preferred embodiments, the reference solution further comprises a stabilizer.

[0048] Wherein, the stabilizer is preferably formic acid and / or acetic acid, more preferably acetic acid.

[0049] Wherein, the volume percentage of the stabilizer relative to the solvent is preferably 0.1% - 2%, for example 0.5%.

[0050] In some specific embodiments, when the reference solution contains 5 - hydroxymethylfurfural, the concentration of 5 - hydroxymethylfurfural is preferably 1 - 4 μg / mL, for example 2.88 μg / mL.

[0051] In some specific embodiments, when the reference solution contains sodium danshensu, the concentration of sodium danshensu is preferably 200 - 300 μg / mL, for example 255.60 μg / mL.

[0052] In some specific embodiments, when the reference solution contains protocatechuic acid, the concentration of protocatechuic acid is preferably 1 - 3 μg / mL, for example 2.08 μg / mL.

[0053] In some specific embodiments, when the reference solution contains protocatechualdehyde, the concentration of protocatechualdehyde is preferably 50 - 70 μg / mL, for example 63.94 μg / mL.

[0054] In some specific embodiments, when the reference solution contains caffeic acid, the concentration of caffeic acid is preferably 1 - 3 μg / mL, for example 2.52 μg / mL.

[0055] In some specific embodiments, when the reference solution contains isoferulic acid, the concentration of isoferulic acid is preferably 0.5 - 2 μg / mL, for example 1.76 μg / mL.

[0056] In some specific embodiments, when the reference solution contains salvianolic acid F, the concentration of salvianolic acid F is preferably 0.5 - 2.5 μg / mL, for example 2.09 μg / mL.

[0057] In some specific embodiments, when the reference solution contains salvianolic acid D, the concentration of salvianolic acid D is preferably 10 - 25 μg / mL, for example 24.13 μg / mL.

[0058] In some specific embodiments, when the reference solution contains rosmarinic acid, the concentration of rosmarinic acid is preferably 30 - 50 μg / mL, for example 39.55 μg / mL.

[0059] In some specific embodiments, when salvianolic acid E is contained in the reference solution, the concentration of salvianolic acid E is preferably 10 - 25 μg / mL, such as 24.57 μg / mL.

[0060] In some specific embodiments, when lithospermic acid is contained in the reference solution, the concentration of lithospermic acid is preferably 10 - 30 μg / mL, such as 29.94 μg / mL.

[0061] In some specific embodiments, when salvianolic acid B is contained in the reference solution, the concentration of salvianolic acid B is preferably 30 - 80 μg / mL, such as 76.13 μg / mL.

[0062] In some specific embodiments, when salvianolic acid A is contained in the reference solution, the concentration of salvianolic acid A is preferably 30 - 65 μg / mL, such as 63.03 μg / mL.

[0063] In some specific embodiments, when salvianolic acid Y is contained in the reference solution, the concentration of salvianolic acid Y is preferably 1 - 6 μg / mL, such as 5.43 μg / mL.

[0064] In some embodiments, the linear regression equation is selected from one or more of the following linear regression equations:

[0065] For 5 - hydroxymethylfurfural, y = 1.6319x + 0.0016;

[0066] For sodium danshensu, y = 0.1156x + 0.5447;

[0067] For protocatechuic acid, y = 0.3657x - 0.0077;

[0068] For protocatechualdehyde, y = 0.7971x + 3.3397;

[0069] For caffeic acid, y = 0.5059x - 0.0145;

[0070] For isoferulic acid, y = 0.5239x - 0.0108;

[0071] For salvianolic acid F, y = 1.2665x + 0.0031;

[0072] For salvianolic acid D, y = 0.2439x - 0.0218;

[0073] For rosmarinic acid, y = 0.4387x - 0.1533;

[0074] For salvianolic acid E, y = 0.3463x - 0.0635;

[0075] Lithospermic acid, y = 0.2742x - 0.0588;

[0076] Salvianolic acid B, y = 0.2758x - 0.2044;

[0077] Salvianolic acid A, y = 0.5954x - 0.5462;

[0078] Salvianolic acid Y, y = 0.2361x - 0.0335;

[0079] Wherein, the x are the concentrations of the respective components, and the y are the peak areas corresponding to the respective components.

[0080] The present invention also provides a fingerprint of Danshen injection, and the fingerprint has at least 10 common peaks: sodium danshensu peak, protocatechuic aldehyde peak, caffeic acid peak, isoferulic acid peak, salvianolic acid D peak, rosmarinic acid peak, salvianolic acid E peak, lithospermic acid peak, salvianolic acid B peak and salvianolic acid A peak; taking the rosmarinic acid peak as the reference peak, the information of each common peak is as follows:

[0081] Sodium danshensu peak, relative retention time is 0.088 - 0.093, such as 0.091; relative peak area is 1.8662 - 1.8764, such as 1.8685;

[0082] Protocatechuic aldehyde peak, relative retention time is 0.173 - 0.179, such as 0.176; relative peak area is 3.5576 - 3.5751, such as 3.5616;

[0083] Caffeic acid peak, relative retention time is 0.288 - 0.293, such as 0.291; relative peak area is 0.0735 - 0.0738, such as 0.0735;

[0084] Isoferulic acid peak, relative retention time is 0.536 - 0.559, such as 0.547; relative peak area is 0.0592 - 0.0605, such as 0.0597;

[0085] Salvianolic acid D peak, relative retention time is 0.853 - 0.871, such as 0.870; relative peak area is 0.3191 - 0.3231, such as 0.3193;

[0086] Rosmarinic acid peak, relative retention time is 1.000; relative peak area is 1.0000;

[0087] Salvianolic acid E peak, relative retention time is 1.077 - 1.083, such as 1.080; relative peak area is 0.3235 - 0.3264, such as 0.3257;

[0088] The lithospermic acid peak has a relative retention time of 1.170 - 1.181, such as 1.180; and a relative peak area of 0.1273 - 0.1316, such as 0.1288;

[0089] The salvianolic acid B peak has a relative retention time of 1.301 - 1.396, such as 1.352; and a relative peak area of 1.1482 - 1.1602, such as 1.1585;

[0090] The salvianolic acid A peak has a relative retention time of 1.381 - 1.510, such as 1.447; and a relative peak area of 1.2691 - 1.3208, such as 1.3029.

[0091] In some embodiments, the fingerprint also includes 4 common peaks: 5 - hydroxymethylfurfural peak, protocatechuic acid peak, salvianolic acid F peak, and salvianolic acid Y peak; taking the rosmarinic acid peak as the reference peak, the information of each common peak is as follows:

[0092] The 5 - hydroxymethylfurfural peak has a relative retention time of 0.075 - 0.078, such as 0.077; and a relative peak area of 0.2782 - 0.2801, such as 0.2789;

[0093] The protocatechuic acid peak has a relative retention time of 0.106 - 0.113, such as 0.110; and a relative peak area of 0.0487 - 0.0503, such as 0.0492;

[0094] The salvianolic acid F peak has a relative retention time of 0.851 - 0.873, such as 0.853; and a relative peak area of 0.0847 - 0.0873, such as 0.0865;

[0095] The salvianolic acid Y peak has a relative retention time of 1.390 - 1.529, such as 1.460; and a relative peak area of 0.0434 - 0.0449, such as 0.0445.

[0096] The present invention also provides a fingerprint of salvia miltiorrhiza injection, which is obtained by detecting a qualified salvia miltiorrhiza injection according to the analysis method of salvia miltiorrhiza injection as described above.

[0097] In some embodiments, the fingerprint of salvia miltiorrhiza injection is as defined above.

[0098] The present invention also provides an application of the fingerprint of salvia miltiorrhiza injection as described above in the quality control of salvia miltiorrhiza injection.

[0099] The present invention also provides a detection method for salvia miltiorrhiza injection, which comprises the following steps:

[0100] (1)Establish the fingerprint of the Danshen injection to be tested by referring to the analysis method of Danshen injection as described above;

[0101] (2)Compare the fingerprint of the Danshen injection to be tested with the fingerprint of the Danshen injection as described above, calculate the similarity. When the similarity is not less than 0.9, the Danshen injection to be tested is qualified.

[0102] In some embodiments, the similarity is calculated by the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines.

[0103] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0104] The reagents and raw materials used in the present invention are all commercially available.

[0105] The positive and progressive effects of the present invention are as follows:

[0106] 1. The analysis method provided by the present invention can obtain the fingerprint of Danshen injection, and ensure that the chromatographic peaks of each index component in the fingerprint have good peak shapes, stable elution times, good resolution without interference, and have good precision, stability and repeatability. It can more comprehensively and accurately reflect the chemical composition information of Danshen injection and is successfully used for the quality verification of different batches of Danshen injection; specifically:

[0107] In the fingerprint of the present invention, at least 10 common peaks can be calibrated, namely: sodium danshensu peak, protocatechualdehyde peak, caffeic acid peak, isoferulic acid peak, salvianolic acid D peak, rosmarinic acid peak, salvianolic acid E peak, lithospermic acid peak, salvianolic acid B peak and salvianolic acid A peak; taking the rosmarinic acid peak as the reference peak; at most 14 common peaks can be calibrated among 33 common peaks simultaneously, specifically including: 5-hydroxymethylfurfural peak, protocatechuic acid peak, salvianolic acid F peak and salvianolic acid Y peak; taking the rosmarinic acid peak.

[0108] 2. The component analysis method provided by the present invention can further determine the contents of various chemical components identified in the fingerprint of Danshen injection by using the same ultra-high performance liquid chromatography method, and has good system suitability, linear relationship, detection limit and quantitative limit investigation, precision, stability, repeatability, etc.; without the need to rely on additional analysis methods, and can more comprehensively and systematically reflect the quality of Danshen injection than the determination of single component content, which is more meaningful for guiding industrial production to control the quality of drugs and better guarantees the quality of products. Description of the Drawings

[0109] Figure 1 The chromatographic results obtained in Comparative Example 1-1.

[0110] Figure 2 is Figure 1 A partial enlarged view of the chromatographic peaks in the retention time range of 20 - 38 min in

[0111] Figure 3 The chromatographic result obtained from Comparative Example 1-2.

[0112] Figure 4 is Figure 3 A partial enlarged view of the chromatographic peaks in the retention time range of 8 - 28 min in

[0113] Figure 5 The chromatographic result obtained from Comparative Example 1-3.

[0114] Figure 6 The chromatographic result obtained from Example 1-1.

[0115] Figure 7 Chromatograms of different mobile phase systems, from bottom to top are the results with 0.5% phosphoric acid aqueous solution, 0.5% acetic acid aqueous solution, and 0.5% formic acid aqueous solution as mobile phase B.

[0116] Figure 8 Chromatograms when the dosages of phosphoric acid in the phosphoric acid aqueous solution of mobile phase B are 0.1%, 0.2%, and 0.5% respectively, from bottom to top are 0.1%, 0.2%, and 0.5%.

[0117] Figure 9 Chromatograms at detection wavelengths of 254, 286, and 326 nm respectively, from bottom to top are 254, 286, and 326 nm.

[0118] Figure 10 Chromatograms when using different chromatographic columns, from bottom to top are Waters ACQUITY HSS T3 chromatographic column (a), Thermo Hypersi1 Gold chromatographic column (b), and Thermo Accucore aQ (c).

[0119] Figure 11 Chromatograms at different flow rates, from bottom to top are 0.35 mL / min, 0.40 mL / min, and 0.45 mL / min.

[0120] Figure 12 Chromatograms at different column temperatures, from bottom to top are 40 °C, 35 °C, and 45 °C.

[0121] Figure 13 Fingerprint spectra of 9 batches of Danshen injection samples (S1 - S9) and reference spectrum (R).

[0122] Figure 14Chromatograms of the mixed reference solution (upper figure) and the test solution (lower figure). Among them, each peak is as follows: Peak 1, 5-hydroxymethylfurfural (R1); Peak 2, sodium danshensu (R2); Peak 3, protocatechuic acid (R3); Peak 5, protocatechualdehyde (R4); Peak 7, caffeic acid (R5); Peak 15, isoferulic acid (R6); Peak 20, salvianolic acid F (R7); Peak 21, salvianolic acid D (R8); Peak 22, rosmarinic acid (R9); Peak 23, salvianolic acid E (R10); Peak 24, lithospermic acid (R11); Peak 27, salvianolic acid B (R12); Peak 28, salvianolic acid A (R13); Peak 29, salvianolic acid Y (R14).

[0123] Figure 15 These are the chemical structures of the 14 chemical components identified in the fingerprint of the Danshen injection of the present invention. Detailed implementation manners

[0124] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0125] In the following examples and comparative examples, the information of the instruments and reagents used is as follows:

[0126] Instruments: Thermo Vanquish ultra-high performance liquid chromatograph (Thermofisher, USA); MS105DU analytical balance (Mettler Toledo, Switzerland). KQ250-DE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); H2500R low-temperature high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); Milli-Q ultrapure water instrument (Millipore, USA).

[0127] Reagents: Acetonitrile is of chromatographic purity, water is ultrapure water, and other reagents are of analytical purity.

[0128] Reference substances: 5-Hydroxymethylfurfural (R1) (batch number ST00720120; content ≥ 98.0%), Sodium Danshensu (R2) (batch number ST01440120; content ≥ 98.0%), Protocatechuic acid (R3) (batch number 14855; content ≥ 98.0%), Rosmarinic acid (R9) (batch number ST00500120; content ≥ 98.0%), Salvianolic acid B (R12) (batch number ST00050120; content ≥ 98.0%), Salvianolic acid A (R13) (batch number ST05570120; content ≥ 98.0%), Salvianolic acid Y (R14) (batch number ST55350205; content ≥ 95.0%) reference substances were all purchased from Shanghai Standard Technology Service Co., Ltd. of Shidande; Caffeic acid (R5) (batch number DSTDK001301; content ≥ 98.0%), Salvianolic acid D (R8) (batch number DST230928 - 230; content ≥ 95%) reference substances were all purchased from Chengdu Lemeitian Medical Technology Co., Ltd.; Isoferulic acid (R6) (batch number 21111103; content 99.58%), Lithospermic acid (R11) (batch number 22031806; content 99.75%) reference substances were all purchased from Chengdu Gelipu Biotechnology Co., Ltd.; Salvianolic acid F (R7) (batch number PS010137; content ≥ 98.0%), Salvianolic acid E (R10) (batch number PS013716; content > 95.0%) reference substances were all purchased from Chengdu Pusi Biotechnology Co., Ltd.; Protocatechualdehyde (R4) (batch number 110810 - 201909; content 99.60%) reference substance was purchased from the National Institutes for Food and Drug Control.

[0129] Samples: Danshen injection (standard number WS3 - B - 3766 - 98 - 2011) (batch numbers: 240401, 240402, 240403, 24020121, 24020221, 24030111, 190828211, 190829211, 190830211, numbered S1 - S9, specification: 10 mL / ampoule) were all provided by Guoyao Yibin Pharmaceutical Co., Ltd.

[0130] Selection of Fingerprint and Component Analysis Chromatographic Conditions for Danshen Injection

[0131] 1. Selection of Gradient Elution Program

[0132] Example 1 - 1

[0133] 1.1 Chromatographic conditions: Chromatographic column: Waters Acquity uplc HSS T3 (2.1 * 100 mm, 1.8 μm) (the corresponding packing material is octadecylsilyl silica gel); Mobile phase: acetonitrile (A) - 0.5% phosphoric acid aqueous solution (B); Flow rate 0.4 mL / min; Column temperature 40 °C; Detection wavelength 286 nm; Injection volume 5 μL.

[0134] 1.2 Preparation of blank solution: Precisely add a certain amount of acetic acid to an aqueous solution of 10% methanol to obtain a 10% methanol solution containing 0.1% acetic acid, and mix well.

[0135] 1.3 Preparation of mixed reference solution: Weigh an appropriate amount of reference substances R1 - R14 precisely, and add them to the blank solution to prepare single reference stock solutions with certain concentrations respectively. Precisely transfer an appropriate amount of the above solutions and dilute them with a 10% methanol solution containing 0.1% acetic acid to obtain a mixed reference solution of R1 - R14, and their concentrations are 2.88, 255.60, 2.08, 63.94, 2.52, 1.76, 2.09, 24.13, 39.55, 24.57, 29.94, 76.13, 63.03, 5.43 μg / mL in sequence.

[0136] 1.4 Test solution: Take 1.0 mL of Danshen injection into a 5 mL volumetric flask, dilute it to the mark with the blank solution (aqueous solution of 10% methanol), and centrifuge at 10000 r·min -1 for 10 min, and take the supernatant for direct injection.

[0137] Perform gradient elution according to the regulations in Table 1 below.

[0138] Table 1 Mobile phase gradient table of Example 1 - 1

[0139]

[0140] Comparative Example 1 - 1

[0141] The difference from Example 1 - 1 is only that: the gradient elution program adopted is shown in Table 2.

[0142] Table 2 Mobile phase gradient table of Comparative Example 1 - 1

[0143]

[0144] Comparative Example 1 - 2

[0145] The difference from Example 1 - 1 is only that: the gradient elution program adopted is shown in Table 3.

[0146] Table 3 Mobile phase gradient table of Comparative Example 1 - 2

[0147]

[0148] Comparative Examples 1 - 3

[0149] They are different from Example 1 - 1 only in that: the gradient elution program used is as shown in Table 4.

[0150] Table 4 Mobile Phase Gradient Table of Comparative Examples 1 - 3

[0151]

[0152] The results are respectively as Figures 1-6 shown: in the fingerprint obtained from Comparative Example 1 - 1 ( Figure 1 and Figure 2 ), the number of chromatographic peaks in the first 20 min is less, the chromatographic peaks are dense from 20 - 38 min, peak 3 protocatechuic acid (R3), peak 29 salvianolic acid Y (R14) are not detected, and peak 23 salvianolic acid E (R10) is not completely separated, failing to effectively separate the components in the test sample; in the fingerprint obtained from Comparative Example 1 - 2 ( Figure 3 and 4 ), the chromatographic peaks are still dense at the retention time of 8 - 28 min, peak 3 protocatechuic acid (R3) is not detected, and peak 2 danshensu (R2), peak 7 caffeic acid (R5), peak 21 salvianolic acid D (R8) are not completely separated; in the fingerprint obtained from Comparative Example 1 - 3 ( Figure 5 ), peak 3 protocatechuic acid (R3) is not detected, and at the retention time of about 6 min, peak 2 danshensu (R2) and at 39 min, peak 28 salvianolic acid A (R13) and peak 29 salvianolic acid Y (R14) are not completely separated; while in the fingerprint obtained from Example 1 ( Figure 6 ), the overall chromatographic behavior is improved, the baseline is stable, and the separation effect of each target peak is good.

[0153] 2. Selection of the mobile phase system (selection of acidic additives in mobile phase B)

[0154] Comparative Example 2 - 1

[0155] It is different from Example 1 - 1 only in that: mobile phase B is 0.5% formic acid aqueous solution.

[0156] Comparative Example 2 - 2

[0157] It is different from Example 1 - 1 only in that: mobile phase B is 0.5% acetic acid aqueous solution.

[0158] The results are as Figure 7As shown, when the mobile phase B is an aqueous solution of 0.5% acetic acid, the resolution of peaks 1-2 and 27-29 is poor; when the mobile phase B is an aqueous solution of 0.5% formic acid, there are impurity peaks interfering only in front of peak 23, and baseline separation is not achieved; when the mobile phase B is an aqueous solution of 0.5% phosphoric acid, the shapes of all target peaks are good and the resolution is good.

[0159] 3. Selection of the amount of phosphoric acid in the aqueous phosphoric acid solution of mobile phase B

[0160] Example 3-1

[0161] The difference from Example 1-1 is only that: the mobile phase B is an aqueous solution of 0.2% phosphoric acid.

[0162] Comparative Example 3-1

[0163] The difference from Example 1-1 is only that: the mobile phase B is an aqueous solution of 0.1% phosphoric acid.

[0164] The results are as Figure 8 shown. When the amount of phosphoric acid in the mobile phase B is 0.1%, only peaks 20 and 21 do not reach baseline separation; when the amount of phosphoric acid is between 0.2% and 0.5%, all components can be detected.

[0165] 4. Selection of different detection wavelengths

[0166] Example 4-1

[0167] The difference from Example 1-1 is only that: the detection wavelength is 254 nm.

[0168] Example 4-2

[0169] The difference from Example 1-1 is only that: the detection wavelength is 326 nm.

[0170] The results are as Figure 9 shown. At wavelengths of 254 nm and 286 nm, more peaks appear, and the responses of all chromatographic peaks are higher at a wavelength of 286 nm.

[0171] 5. Selection of chromatographic column

[0172] Example 5-1

[0173] The difference from Example 1-1 is only that: the chromatographic column is Thermo Hypersi1 Gold (100 mm * 2.1 mm, 1.9 µm).

[0174] Example 5-2

[0175] The difference from Example 1-1 is only that: the chromatographic column is Thermo Accucore aQ (100 mm * 2.1 mm, 2.6 µm).

[0176] The results are as follows Figure 10 shown. When using Hypersi1 Gold and Thermo Accucore aQ chromatographic columns, peaks 1-3 were eluted relatively quickly, and the chromatographic peaks were piled up; the resolution of peaks 20 and 21 was poor; while when using Waters ACQUITY HSS T3 (100mm*2.1 mm, 1.8 µm) chromatographic column, the overall performance was relatively better.

[0177] 6. Selection of flow rate

[0178] Example 6-1

[0179] The difference from Example 1-1 is only that the flow rate is 0.35 mL / min.

[0180] Example 6-2

[0181] The difference from Example 1-1 is only that the flow rate is 0.45 mL / min.

[0182] The results are as follows Figure 11 shown. When the flow rate is 0.35 mL / min, only the resolution of peaks 28 and 29 is poor; when the flow rate is between 0.40 and 0.45 mL / min, all components can be detected.

[0183] 7. Selection of column temperature

[0184] Example 7-1

[0185] The difference from Example 1-1 is only that the column temperature is 35°C.

[0186] Example 7-2

[0187] The difference from Example 1-1 is only that the column temperature is 45°C.

[0188] The results are as follows Figure 12 shown. When the column temperature is 35°C, only the resolution of peaks 28 and 29 is poor; when the column temperature is between 40 and 45°C, all components can be detected.

[0189] 8. Selection of different blank solvents (selection of stabilizers)

[0190] Example 8-1

[0191] The difference from Example 1-1 is only that 0.1% acetic acid is added to the blank solution.

[0192] Example 8-2

[0193] The difference from Example 1-1 is only that 0.5% acetic acid is added to the blank solution.

[0194] Example 8-3

[0195] It is only different from Example 1-1 in that: 2% acetic acid is further added to the blank solution.

[0196] Example 8-4

[0197] It is only different from Example 1-1 in that: 0.1% formic acid is further added to the blank solution.

[0198] Example 8-5

[0199] It is only different from Example 1-1 in that: 0.5% formic acid is further added to the blank solution.

[0200] Example 8-6

[0201] It is only different from Example 1-1 in that: 2% formic acid is further added to the blank solution.

[0202] As above, 10% methanol solutions with different concentrations of acid added (0.1%, 0.5%, 2% acetic acid and formic acid solutions) were used as blank solvents respectively, and samples were injected for detection at 0, 6, 12, 18, 48, and 72 h to investigate the stability of each target peak. The results are shown in Table 5. When the above-mentioned acids with various concentrations were added as stabilizers, the RSD values of the peak areas of each target component within 72 h were less than 3%, and all could be used to improve the sample stability. Among them, considering the stability of each peak comprehensively, the 10% methanol solution with 0.1% acetic acid as the blank solvent had relatively the best effect.

[0203] Table 5 RSD values of peak areas of each target peak within 72 h when different concentrations of acid were added as stabilizers (%)

[0204]

[0205] Example 9 Establishment of fingerprint

[0206] 1. Solution preparation

[0207] 1.1 Preparation of blank solution

[0208] A certain amount of acetic acid was precisely added to 10% methanol aqueous solution to make a 10% methanol solution containing 0.1% acetic acid, and it was fully mixed evenly to obtain.

[0209] 1.2 Preparation of mixed reference substance solution

[0210] Weigh appropriate amounts of the reference substances of R1~R14 accurately, and add them to blank solutions to prepare single reference substance stock solutions with certain concentrations respectively. Pipette appropriate amounts of the above solutions accurately and dilute them with 10% methanol solution containing 0.1% acetic acid to obtain a mixed reference substance solution of R1~R14, with concentrations of 2.88, 255.60, 2.08, 63.94, 2.52, 1.76, 2.09, 24.13, 39.55, 24.57, 29.94, 76.13, 63.03, 5.43 μg / mL in sequence.

[0211] 1.3 Preparation of test solution

[0212] Take 1.0 mL of Danshen injection of different batches into a 5 mL volumetric flask, dilute to the mark with blank solution, mix well, centrifuge at 10000 r·min -1 for 10 min, and take the supernatant as the test solution.

[0213] 2. Chromatographic conditions

[0214] Use a Waters Acquity uplc HSS T3 chromatographic column (2.1*100 mm, 1.8 μm), mobile phase acetonitrile (A) - 0.5% phosphoric acid water (B), gradient elution: 0 - 10 min, 5% - 14% A; 10 - 29 min, 14% - 18% A; 29 - 37 min, 18% - 25% A; 37 - 42 min, 25% - 40% A; 42 - 45 min, 40% - 95% A; 45 - 50 min, 95% A; 50.1 - 55 min, 5% A; flow rate 0.4 mL / min; column temperature 40°C; detection wavelength 286 nm; injection volume 5 μL.

[0215] 3. Methodology investigation of fingerprint

[0216] 3.1 Precision test

[0217] Take Danshen injection (batch number 240401), prepare the test solution according to the method under item "1.2", inject 6 needles continuously under the chromatographic conditions under item "2", and record the chromatogram. The retention times and peak areas of the common peaks in the precision test are shown in Tables 6 and 7. Taking rosmarinic acid at peak 22 as the reference peak (S), calculate the relative retention time RSD and relative peak area RSD of each common peak. The results are shown in Table 12, which are 0.01% - 0.16% and 0.06% - 3.43% respectively, indicating that the instrument precision of this method is good.

[0218] Table 6 Results of precision test (retention time, min)

[0219]

[0220] Table 7 Results of Precision Test (Peak Area)

[0221]

[0222] 3.2 Stability Test: Take Danshen Injection (batch number 240401), prepare the test solution according to the method under "1.2", and inject samples for detection at 0, 2, 4, 8, 12, 24, and 48 h respectively according to the chromatographic conditions under "2", and record the chromatograms. The retention times and peak areas of the common peaks in the stability test are shown in Table 8 and Table 9. Taking rosmarinic acid at peak 22 as the reference peak (S), the relative standard deviations (RSDs) of the relative retention times and relative peak areas of the common peaks are calculated, and the results are shown in Table 12, which are 0.02% - 0.15% and 0.07% - 5.36% respectively, indicating that the test solution has good stability within 48 h.

[0223] Table 8 Results of Stability Test (Retention Time, min)

[0224]

[0225] Table 9 Results of Stability Test (Peak Area)

[0226]

[0227] 3.3 Repeatability Test: Take the same batch of Danshen Injection (batch number 240401), prepare 6 test solutions in parallel according to the method under "1.2", inject samples for detection according to the chromatographic conditions under "2", and record the chromatograms. The retention times and peak areas of the common peaks in the repeatability test are shown in Table 10 and Table 11. Taking rosmarinic acid at peak 22 as the reference peak (S), the relative standard deviations (RSDs) of the relative retention times and relative peak areas of the common peaks are calculated, and the results are shown in Table 12, which are 0.01% - 0.14% and 0.07% - 2.33% respectively, indicating that the method has good repeatability.

[0228] Table 10 Results of Repeatability Test (Retention Time, min)

[0229]

[0230] Table 11 Results of Repeatability Test (Peak Area)

[0231]

[0232] Table 12 RSDs of Relative Retention Times (%) and Relative Peak Areas (%) of Common Peaks

[0233]

[0234] 4. Establishment of fingerprint and similarity analysis and evaluation

[0235] Take 9 batches of Danshen injection samples to prepare test solution according to the method under "1.2", inject and detect according to the chromatographic conditions under "2", record the chromatogram, import the data into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprint (Version 2012.130723)", take the chromatogram of sample S1 as the reference chromatogram, adopt the median method, with a time window of 0.1 min, generate the superimposed chromatogram and reference chromatogram after multi-point calibration, and a total of 33 common peaks are determined, as shown in Figure 13 . The retention times of peaks 1 - 33 in the reference chromatogram are 2.04, 2.42, 2.94, 4.35, 4.69, 5.09, 7.75, 8.77, 9.84, 11.06, 11.37, 11.75, 12.42, 13.24, 14.57, 16.31, 17.21, 18.54, 18.95, 22.71, 23.16, 26.60, 28.73, 31.39, 32.88, 33.62, 35.99, 38.53, 38.88, 39.98, 40.19, 41.06, 41.42 min respectively.

[0236] Take the reference substance as the reference ( Figure 14 ), identify 14 known compounds, and the results are shown in Table 13. The structural formulas of the above compounds are shown in Figure 15 . Among them, the separation degree of rosmarinic acid at peak 22 is better, the retention time and peak area are relatively stable and of moderate size, and the reference substance is easy to obtain. Therefore, rosmarinic acid at peak 22 is used as the reference peak (S), with the relative retention time of rosmarinic acid being 1.00, and the relative retention times of other peaks are calculated to be 0.08, 0.09, 0.11, 0.16, 0.18, 0.19, 0.29, 0.33, 0.37, 0.42, 0.43, 0.44, 0.47, 0.50, 0.55, 0.61, 0.65, 0.70, 0.71, 0.85, 0.87, 1.00, 1.08, 1.18, 1.24, 1.26, 1.35, 1.45, 1.46, 1.50, 1.51, 1.54, 1.56.

[0237] Use the cosine of the included angle algorithm to calculate the similarities between each batch of samples and the reference chromatogram, which are 0.998, 0.997, 0.997, 0.981, 0.996, 0.999, 0.986, 0.992, 0.986 respectively, all higher than 0.98, indicating that the quality uniformity of 9 batches of Danshen injection is good.

[0238] Table 13 Results of 14 common peaks identified by reference substances

[0239]

[0240] Example 10 Detection of Component Content

[0241] 1. Solution Preparation

[0242] 1.1 Preparation of blank solution: Accurately add a certain amount of acetic acid to an aqueous solution of 10% methanol to make a 10% methanol solution containing 0.1% acetic acid, and mix well to obtain.

[0243] 1.2 Preparation of reference solution

[0244] Take appropriate amounts of R1 - R14 reference substances, accurately weigh them, and add them to the blank solution to prepare single reference stock solutions with concentrations of 0.719, 9.129, 0.519, 7.993, 0.629, 0.220, 0.261, 1.207, 9.888, 2.047, 7.486, 9.516, 5.253, 1.359 mg / mL respectively. Accurately pipette appropriate amounts of the above solutions and dilute them with the blank solution to obtain a stock solution of mixed reference solution for R1 - R14, with concentrations of 14.39, 1278.02, 10.39, 319.72, 12.58, 8.80, 10.43, 120.65, 197.76, 122.84, 149.72, 380.63, 315.17, 27.17 μg / mL in sequence. Accurately pipette 1.0 mL of the above stock solution of mixed reference solution into a 5 - mL volumetric flask, and dilute it to the mark with the blank solution to obtain a mixed reference solution, with concentrations of 2.88, 255.60, 2.08, 63.94, 2.52, 1.76, 2.09, 24.13, 39.55, 24.57, 29.94, 76.13, 63.03, 5.43 μg / mL in sequence. Other mixed reference solutions with different concentrations are all obtained by diluting the stock solution of mixed reference solution with the blank solution.

[0245] 1.3 Preparation of test solution

[0246] Take 1.0 mL of Danshen injection of different batches respectively into 5 - mL volumetric flasks, dilute them to the mark with the blank solution, mix well, centrifuge at 10000 r·min -1 for 10 min, and take the supernatant as the test solution.

[0247] 2. Chromatographic conditions

[0248] The chromatographic column used was Waters Acquity uplc HSS T3 (2.1*100 mm, 1.8 μm). The mobile phase consisted of acetonitrile (A) and 0.5% phosphoric acid in water (B). Gradient elution was performed as follows: from 0 to 10 min, 5% - 14% A; from 10 to 29 min, 14% - 18% A; from 29 to 37 min, 18% - 25% A; from 37 to 42 min, 25% - 40% A; from 42 to 45 min, 40% - 95% A; from 45 to 50 min, 95% A; from 50.1 to 55 min, 5% A. The flow rate was 0.4 mL / min, the column temperature was 40 °C, the detection wavelength was 286 nm, and the injection volume was 5 μL.

[0249] The chromatograms of the mixed reference substance and the test sample solution are as Figure 14 .

[0250] 3. Methodology investigation for the determination of component content

[0251] 3.1 System suitability

[0252] Inject according to the chromatographic conditions under "2". Inject the mixed reference substance solution under "1.2" continuously for 6 injections, and inject the test sample solution under "1.3" continuously for 3 injections. As can be seen from Table 14, for the mixed reference substance solution injected continuously for 6 times, the RSD% of the peak areas of 14 target compounds are all less than 2%. As can be seen from Table 15, the theoretical plate numbers of 14 target compounds are all above 9000.

[0253] Table 14 Peak areas of the mixed reference substance solution injected continuously for 6 times

[0254]

[0255] Table 15 Results of the tailing factor, resolution, signal-to-noise ratio and theoretical plate number of 14 target compounds in the test sample solution

[0256]

[0257] 3.1 Investigation of linear relationship, detection limit and quantitation limit

[0258] Precisely pipette appropriate amounts of each single reference substance stock solution under item "1.2", and dilute with the blank solution to obtain the stock solution of the mixed reference substance solution for R1 - R14, with their concentrations being 14.39, 1278.02, 10.39, 319.72, 12.58, 8.80, 10.43, 120.65, 197.76, 122.84, 149.72, 380.63, 315.17, 27.17 μg / mL in sequence. Dilute them by 1, 2.5, 5, 25, and 50 times respectively, inject samples for detection under the chromatographic conditions in item "2", use the concentration of the reference substance solution as the abscissa and the peak area as the ordinate to plot the standard curve, calculate the limit of detection (LOD) with the signal-to-noise ratio S / N = 3 and the limit of quantitation (LOQ) with S / N = 10 respectively, and the results are shown in Table 16.

[0259] Table 16 Regression equations, correlation coefficients, linear ranges, limits of detection, and limits of quantitation of 14 components

[0260]

[0261] 3.2 Precision test

[0262] Take the mixed reference substance solution under item "1.2", continuously inject samples for determination 6 times under the chromatographic conditions in item "2", record the chromatogram, and calculate that the RSDs of the peak areas of the above components R1 - R14 are 0.07%, 0.05%, 0.55%, 0.03%, 0.17%, 0.11%, 0.44%, 0.05%, 0.02%, 0.04%, 0.13%, 0.05%, 0.19%, 0.12% (n = 6) respectively. The RSD < 2%, indicating good instrument precision.

[0263] 3.3 Stability test

[0264] Take the mixed reference substance solution and the test solution (batch number 240401) under item “1”, inject samples at 0, 2, 4, 8, 12, 24, 48, and 72 h respectively according to the chromatographic conditions under item “2”, record the chromatograms, and calculate the RSD of the peak areas of components R1 - R14 above. The RSDs of the peak areas of each compound in the mixed reference substance solution are 0.05%, 0.06%, 0.73%, 0.07%, 0.21%, 0.23%, 3.94%, 0.12%, 0.19%, 0.07%, 0.07%, 0.08%, 3.06%, 0.22% (n = 9), RSD < 4%. The RSDs of the peak areas of each compound in the test solution are 0.21%, 0.07%, 1.02%, 0.09%, 0.13%, 0.77%, 1.25%, 0.27%, 0.06%, 0.22%, 0.67%, 0.02%, 2.25%, 1.11% (n = 9), RSD < 3%. It shows that the mixed reference substance solution and the test solution have good stability within 72 h.

[0265] 3.4 Repeatability test

[0266] Take 6 portions of test solutions prepared in parallel from the same batch of Danshen injection (batch number 240401) according to the method under item “1.2”, inject samples for detection according to the chromatographic conditions under item “2”, record the chromatograms, and calculate that the RSDs of the contents of components R1 - R14 above are 0.34%, 0.43%, 0.74%, 0.38%, 0.38%, 0.47%, 0.72%, 0.44%, 0.41%, 0.42%, 0.86%, 0.38%, 0.55%, 0.64% (n = 6), RSD < 2%. It shows that this method has good repeatability.

[0267] 3.5 Recovery test

[0268] Six samples of the same batch (batch number 240401) were taken for the repeatability experiment, each 0.5 mL, placed in a 5 mL volumetric flask. Appropriate amounts of the stock solution of the mixed reference substance solution were added according to the repeatability results, diluted to the mark with the blank solution, and solutions equivalent to 100% of the amount of the components to be determined in the test sample were prepared. They were injected for detection under the chromatographic conditions described in item "2". The results are shown in Table 17. The average recoveries of the above 14 components were calculated to be 96.45%, 97.10%, 96.54%, 97.28%, 95.30%, 115.34%, 89.34%, 97.42%, 97.10%, 97.17%, 98.19%, 96.52%, 94.00%, 96.90% respectively, and the RSDs were 0.52%, 0.42%, 1.15%, 0.41%, 0.44%, 0.48%, 2.38%, 0.66%, 0.71%, 0.53%, 0.82%, 0.54%, 1.99%, 0.27% (n = 6), indicating that the method has good accuracy.

[0269] Table 17 Results of spike recoveries (n = 6)

[0270]

[0271]

[0272] 4. Determination of sample content

[0273] Nine batches of samples were taken, two portions for each batch. The test sample solutions were prepared according to the method described in item "1.3", injected for determination under the chromatographic conditions described in item "2", and the contents were calculated by the external standard method. The results are shown in Table. The results are shown in Table 18. The contents of the above components R1 - R14 in 9 batches of Danshen injection were 6.38 - 22.19, 1077.17 - 1313.18, 6.73 - 11.86, 258.34 - 315.21, 5.35 - 9.20, 2.54 - 8.60, 3.24 - 5.31, 70.26 - 92.02, 173.07 - 282.47, 49.20 - 106.14, 34.16 - 150.39, 143.55 - 526.82, 153.05 - 192.24, 5.00 - 25.47 μg / mL respectively. The total amount of the 14 compounds was 2206.20 - 2615.30 μg / mL, the average content was 2422.95 μg / mL, and the RSD was 5.81%.

[0274] Table 18 Results of determination of each component content (μg / mL)

[0275]

[0276] In summary, the analysis method of Salvia miltiorrhiza injection provided by the present invention (specifically, the fingerprint construction method) and the analysis method of the components of Salvia miltiorrhiza injection can comprehensively and clearly detect Salvia miltiorrhiza injection. Through the methodological verification of the fingerprint detection method and the content determination method respectively, it is proved that this method has good precision, good repeatability, good stability and high accuracy.

[0277] Under the optimal conditions, the fingerprint of the Salvia miltiorrhiza injection obtained by the present invention can simultaneously calibrate up to 33 common peaks at most, and identify the chemical structure names of 14 common peaks. The fingerprint of the Salvia miltiorrhiza injection provided by the present invention can be reflected in a single spectrum at the same wavelength, and can evaluate the quality of Salvia miltiorrhiza injection more comprehensively, accurately and reliably. On the basis of the fingerprint evaluation, the present invention further determines the contents of 14 identified components. The total amount of 14 compounds in 9 batches of Salvia miltiorrhiza injection samples is 2206.20 - 2615.30 μg / mL, with an average content of 2422.95 μg / mL and an RSD of 5.81%. The contents of each index component in different batches fluctuate within a certain range, which may be related to the medicinal materials and production processes of different batches. This method can more comprehensively and systematically reflect the quality of Salvia miltiorrhiza injection than the single-component content determination, and is more meaningful for guiding industrial production to control the drug quality and better ensuring the product quality.

[0278] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An analytical method for Salvia miltiorrhiza injection, characterized in that, The analytical method of the Danshen injection is carried out by ultra-high performance liquid chromatography; the chromatographic conditions of the ultra-high performance liquid chromatography are as follows: (1) The filler of the chromatographic column is octadecylsilane-bonded silica gel; (2) Mobile phase A is acetonitrile, and mobile phase B is an aqueous phosphoric acid solution with a volume fraction of 0.2% - 0.5%; (3) The following gradient elution program is adopted: ； The percentages in the table are the volume percentages of each mobile phase in the total volume of mobile phase A and mobile phase B respectively.

2. The analytical method of the Danshen injection as described in claim 1, characterized in that, The analytical method of the Danshen injection meets one or more of the following conditions: (1) The Danshen injection is a qualified Danshen injection or a Danshen injection to be tested; (2) Mobile phase B is an aqueous phosphoric acid solution with a volume fraction of 0.3% - 0.5%; (3) In the ultra-high performance liquid chromatography, the column temperature is 35 - 45°C, preferably 40 - 45°C; (4) In the ultra-high performance liquid chromatography, the flow rate is 0.35 - 0.45 mL / min, preferably 0.40 - 0.45 mL / min; (5) In the ultra-high performance liquid chromatography, the injection volume is 1 - 5 μL, such as 5 μL; (6) In the ultra-high performance liquid chromatography, the detection wavelength is 254 - 326 nm, preferably 286 nm; (7) The specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and the filler particle size is 1.8 μm, 1.9 μm or 2.6 μm; preferably, the chromatographic column is Waters ACQUITY HSS T3; (8) The gradient elution program is shown in the following table: ； The percentages in the table are the volume percentages of each component in the total volume of mobile phase A and mobile phase B respectively.

3. The analysis method of the Danshen injection according to claim 1 or 2, characterized in that, The analytical method of the Danshen injection includes the following steps: Detect the test solution A containing the qualified Danshen injection by the ultra-high performance liquid chromatography, and generate a fingerprint spectrum with the detection result; Preferably, the analytical method of the Danshen injection further includes the following steps: Detect the test solution B containing the Danshen injection to be tested by the ultra-high performance liquid chromatography, and compare the detection result with the fingerprint spectrum.

4. The analysis method of the Danshen injection according to claim 3, wherein, The analytical method of the Danshen injection meets one or more of the following conditions: (1) The solvents in the test solutions A and B are both methanol aqueous solutions; Among them, the volume fraction of methanol in the methanol aqueous solution is preferably 10% - 30%, such as 10%; (2) The volume ratio of the qualified Danshen injection to the solvent in the test solution A is 1:(1 - 9), such as 1:4; (3) The volume ratio of the Danshen injection to be tested to the solvent in the test solution B is 1:(1 - 9), such as 1:4; (4) The preparation of the test solution A or B includes the following steps: mix the qualified Danshen injection or the Danshen injection to be tested with the solvent, and centrifuge to take the supernatant; Among them, the rotation speed of the centrifugation is preferably 10,000 - 20,000 r·min -1 , for example, 10,000 r·min -1 , and the centrifugation time is preferably 10 - 30 min, for example, 10 min; (5) The test solutions A and B also include a stabilizer; Among them, the stabilizer is preferably formic acid and / or acetic acid, more preferably acetic acid; Among them, the volume ratio of the stabilizer to the solvent is preferably 0.1% - 2%, such as 0.5%; Among them, the preparation of the test solution A or B preferably includes the following steps: the stabilizer and the solvent are first mixed, then mixed with the qualified Danshen injection or the Danshen injection to be tested, and the supernatant is taken by centrifugation.

5. A method for analyzing the components of Salvia miltiorrhiza injection, characterized in that, The analysis method of the components of the Danshen injection is carried out by the ultra-high performance liquid chromatography method described in claim 1 or 2, and includes the following steps: Just substitute the peak area measured from the Danshen injection to be tested into the linear regression equation for calculation.

6. The analysis method of the composition of the Salvia miltiorrhiza injection according to claim 5, characterized in that The analysis method of the components of the Danshen injection satisfies one or more of the following conditions: (1) The detection is carried out with the test solution containing the Danshen injection to be tested; The test solution containing the Danshen injection to be tested is defined as the test solution B in claim 3 or 4; (2) The linear regression equation is obtained by detecting the reference substance and calculating; Among them, the reference substance is selected from one or more of 5-hydroxymethylfurfural, sodium danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, isoferulic acid, salvianolic acid F, salvianolic acid D, rosmarinic acid, salvianolic acid E, lithospermic acid, salvianolic acid B, salvianolic acid A and salvianolic acid Y; Preferably, the detection is carried out with a reference solution containing the reference substance; the solvent in the reference solution is an aqueous methanol solution; Among them, the volume fraction of methanol in the aqueous methanol solution is preferably 10%-30%, such as 10%; More preferably, the reference solution also includes a stabilizer; Among them, the stabilizer is preferably formic acid and / or acetic acid, and more preferably acetic acid; Among them, the volume ratio of the stabilizer to the solvent is preferably 0.1%-2%, such as 0.5%; (3) The linear regression equation is selected from one or more of the following linear regression equations: For 5-hydroxymethylfurfural, y = 1.6319x + 0.0016; For sodium danshensu, y = 0.1156x + 0.5447; For protocatechuic acid, y = 0.3657x - 0.0077; For protocatechualdehyde, y = 0.7971x + 3.3397; For caffeic acid, y = 0.5059x - 0.0145; For isoferulic acid, y = 0.5239x - 0.0108; For salvianolic acid F, y = 1.2665x + 0.0031; For salvianolic acid D, y = 0.2439x - 0.0218; For rosmarinic acid, y = 0.4387x - 0.1533; For salvianolic acid E, y = 0.3463x - 0.0635; For lithospermic acid, y = 0.2742x - 0.0588; For salvianolic acid B, y = 0.2758x - 0.2044; For salvianolic acid A, y = 0.5954x - 0.5462; For salvianolic acid Y, y = 0.2361x - 0.0335; Among them, the x are the concentrations of each component respectively, and the y are the peak areas corresponding to each component respectively.

7. A fingerprint spectrum of a Salvia miltiorrhiza injection, characterized in that, The fingerprint of the Danshen injection has at least 10 common peaks: sodium danshensu peak, protocatechuic aldehyde peak, caffeic acid peak, isoferulic acid peak, salvianolic acid D peak, rosmarinic acid peak, salvianolic acid E peak, lithospermic acid peak, salvianolic acid B peak and salvianolic acid A peak; taking the rosmarinic acid peak as the reference peak, the information of each common peak is as follows: Sodium danshensu peak, relative retention time is 0.088 - 0.093, such as 0.091; relative peak area is 1.8662 - 1.8764, such as 1.8685; Protocatechuic aldehyde peak, relative retention time is 0.173 - 0.179, such as 0.176; relative peak area is 3.5576 - 3.5751, such as 3.5616; Caffeic acid peak, relative retention time is 0.288 - 0.293, such as 0.291; relative peak area is 0.0735 - 0.0738, such as 0.0735; Isoferulic acid peak, relative retention time is 0.536 - 0.559, such as 0.547; relative peak area is 0.0592 - 0.0605, such as 0.0597; Salvianolic acid D peak, relative retention time is 0.853 - 0.871, such as 0.870; relative peak area is 0.3191 - 0.3231, such as 0.3193; Rosmarinic acid peak, relative retention time is 1.000; relative peak area is 1.0000; Salvianolic acid E peak, relative retention time is 1.077 - 1.083, such as 1.080; relative peak area is 0.3235 - 0.3264, such as 0.3257; Lithospermic acid peak, relative retention time is 1.170 - 1.181, such as 1.180; relative peak area is 0.1273 - 0.1316, such as 0.1288; Salvianolic acid B peak, relative retention time is 1.301 - 1.396, such as 1.352; relative peak area is 1.1482 - 1.1602, such as 1.1585; Salvianolic acid A peak, relative retention time is 1.381 - 1.510, such as 1.447; relative peak area is 1.2691 - 1.3208, such as 1.3029; Preferably, the fingerprint also includes 4 common peaks: 5 - hydroxymethylfurfural peak, protocatechuic acid peak, salvianolic acid F peak and salvianolic acid Y peak; taking the rosmarinic acid peak as the reference peak, the information of each common peak is as follows: 5 - Hydroxymethylfurfural peak, relative retention time is 0.075 - 0.078, such as 0.077; relative peak area is 0.2782 - 0.2801, such as 0.2789; Protocatechuic acid peak, relative retention time is 0.106 - 0.113, such as 0.110; relative peak area is 0.0487 - 0.0503, such as 0.0492; Salvianolic acid F peak, relative retention time is 0.851 - 0.873, such as 0.853; relative peak area is 0.0847 - 0.0873, such as 0.0865; The salvianolic acid Y peak has a relative retention time of 1.390 - 1.529, such as 1.460; and a relative peak area of 0.0434 - 0.0449, such as 0.0445.

8. A fingerprint spectrum of a Salvia miltiorrhiza injection, characterized in that, The fingerprint of the Danshen injection is obtained by detecting a qualified Danshen injection according to the analysis method of the Danshen injection described in any one of claims 1 - 4.

9. Use of the fingerprint of the Danshen injection described in claim 7 or 8 in the quality control of Danshen injection.

10. A quality control method for Danshen injection, characterized in that, The quality control method of the Danshen injection comprises the following steps: (1) Establish the fingerprint of the Danshen injection to be tested with reference to the analysis method of the Danshen injection described in any one of claims 1 - 4; (2) Compare the fingerprint of the Danshen injection to be tested with the fingerprint of the Danshen injection described in claim 7 or 8, calculate the similarity, and when the similarity is not less than 0.9, the Danshen injection to be tested is qualified.

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