CN104749308A - A kind of quality control method of Danshen injection - Google Patents

Abstract

The invention relates to a quality control method of a salvia miltiorrhiza injection. The method comprises respectively determining fingerprint of Saviae Miltiorrhizae radix injection reference substance and product to be tested by HPLC or HPLC-MS, comparing the two, and determining the quality of the product to be tested to be qualified when the similarity between the two is greater than 0.900.

Description

A kind of quality control method of Danshen injection

technical field

The invention relates to the field of analytical chemistry, in particular to a quality control method for Danshen injection.

Background technique

The traditional Chinese medicine Salvia miltiorrhiza is the dry root and rhizome of Salvia miltiorrhiza Bge., a dicotyledonous plant in the family Labiatae. Modern medicine and clinical experiments have shown that Danshen can expand coronary arteries, increase coronary blood flow, slow down heart rate, improve myocardial hypoxia, improve acute symptoms and ischemic changes suggested by electrocardiogram, inhibit platelet aggregation, and inhibit platelet blood flow in cardiovascular aspects. Release reaction, reduce blood viscosity, lower blood lipids, resist red blood cell aggregation, reduce atherosclerosis, inhibit fibrinogen from converting into fibrin, improve microcirculation, central sedation, antibacterial and anti-inflammatory effects, etc. It can be used to treat angina pectoris of coronary heart disease, Chest tightness, palpitations, myocardial infarction, myocarditis and other diseases, used for antithrombotic and anticoagulant.

The main active ingredients of Danshen are fat-soluble tanshinone (diterpene quinone) and water-soluble salvianolic acid compounds. So far, more than seventy tanshinone compounds and more than thirty salvianolic acid compounds have been isolated from Danshen, including Tanshinone IIA (Tanshinone IIA), Tanshinone I (Tanshinone I), cryptotanshinone (cryptotanshinone), dihydro Tanshinone I (15,16-dihydrotanshinone I), danshensu, protocatechualdehyde, salvianolic acid A, salvianolic acid B (Salvianolic acid B), salvianolic acid C, rosmarinic acid, etc.

Danshen injection is a sterilized aqueous solution made from Danshen. It is recorded in Volume 20 of the Standards issued by the Ministry of Traditional Chinese Medicine, page number: Z20-34, standard number: WS3-B-3766-98. The standard stipulates that the preparation method is: take 1500g of Danshen, add water and decoct three times, the first time for 2 hours, the second and third times for 1.5 hours each, combine the decoction, filter, and concentrate the filtrate to 750ml under reduced pressure. Add ethanol for precipitation twice, the first time the alcohol content is 75%, the second time the alcohol content is 85%, each time it is refrigerated and then filtered. The filtrate recovers ethanol and concentrates it to about 250ml. Add water for injection to 400ml, mix well, refrigerate, filter, adjust the pH value to 6.8 with 10% sodium hydroxide solution, boil for half an hour, filter, add water for injection to 1000ml, potting, and sterilize to get ready. The product is a brown to brown-red clear liquid. The pH value is between 5.0 and 7.0. Every 1ml contains protocatechualdehyde (C ₇ H ₆ O ₃ ) not less than 0.2mg. The main functions are promoting blood circulation and removing blood stasis, dredging the pulse and nourishing the heart. For coronary heart disease chest tightness, angina pectoris. Both intramuscular and intravenous injections are available. During the preparation process of Danshen injection, after a series of processes such as heating, sterilization and acid-base treatment, its chemical composition and content inevitably changed, and the content of Danshen injection in different batches and different manufacturers The type and amount of chemical constituents may also vary considerably. Therefore, it is of great significance for the quality control and mechanism research of Danshen injection to systematically study the components of the water-soluble components of Danshen, identify its structure to the maximum extent, and study the content ratio of these compounds in the product.

Contents of the invention

The invention provides a quality control method of Danshen injection, which is used for controlling the quality of Danshen injection preparation. The method is to obtain a control fingerprint on the basis of a large number of fingerprints of Danshen injection, and then compare the fingerprint of the sample to be tested with it to evaluate the similarity, so as to control the quality of Danshen injection.

Quality control method described in the present invention comprises the steps:

(1) Establishment of control fingerprint of Danshen injection

Preparation of Danshen injection reference solution:

Take Danshen injection and dilute it 5 times with water, or filter it with PVDF hydrophilic filter membrane or centrifuge and add water to dilute it 5 times for later use;

Determination of Danshen Injection Control Fingerprint:

Draw the above-mentioned reference substance solution and inject it into a liquid chromatograph, and use the HPLC method for determination to obtain the control fingerprint of Danshen injection. Chromatographic conditions: the chromatographic column is an octadecylsilane bonded silica gel column, and the mobile phase is acetonitrile-0.03% phosphoric acid aqueous solution , the detection wavelength is 270-280nm, the flow rate is 1.0mL/min, the column temperature is 25-30°C, and the gradient elution procedure is as follows,

(2) Determination of fingerprints of Danshen injection product to be tested

Get Danshen injection need testing solution, measure fingerprint according to the steps and conditions described in above-mentioned (1);

(3) Compare the fingerprints of the Danshen injection product to be tested with the control fingerprints of the Danshen injection to identify the similarity, so as to determine whether the product quality is qualified.

By comparing with the chromatogram, ultraviolet spectrum and mass spectrometry behavior of the reference substance, the inventor confirmed that the nine fingerprint peaks of the control fingerprint of Danshen injection (see Figure 1) are: peak No. 1 is Danshen and peak No. 2 is protocatechu , Peak No. 3 is protocatechu, peak No. 4 is caffeic acid, peak No. 5 is isoferulic acid, peak No. 6 is salvianolic acid D, peak No. 7 is rosmarinic acid, peak No. 8 is shikonic acid, Peak No. 9 is salvianolic acid B.

In view of the characteristics of spectral collection fingerprinting technology lies in its integrity and ambiguity, but under the condition of lack of reference substances, the chromatographic peaks, especially the unknown component peaks, have defects to the greatest extent. Therefore, the inventors further supplementarily solved the above-mentioned problems by using liquid chromatography-mass spectrometry technology.

Quality control method described in the present invention comprises the steps:

(1) Establishment of control fingerprint of Danshen injection

Preparation of Danshen injection reference solution:

Take Danshen injection and dilute it 5 times with water, or filter it with PVDF hydrophilic filter membrane or centrifuge and add water to dilute it 5 times for later use;

Determination of Danshen Injection Control Fingerprint:

Draw the above-mentioned reference substance solution and inject it into a liquid chromatograph, use LC-MS method to measure, and obtain the control fingerprint of Danshen injection, chromatographic conditions: the chromatographic column is an octadecylsilane-bonded silica gel column, and the mobile phase is acetonitrile-0.03% Formic acid aqueous solution, the detection wavelength is 270-280nm, the flow rate is 1.0mL/min, the column temperature is 25-30°C, the gradient elution procedure is as follows,

Mass spectrometry parameters: ESI(-), ultra scan 50～1200m/z, Nebulizer 40psi, Dry gas 9l/min, Dry Temp350℃, split mode into mass spectrometer, split ratio is about 1:3;

(2) Determination of fingerprints of Danshen injection product to be tested

Get Danshen injection need testing solution, measure fingerprint according to the steps and conditions described in above-mentioned (1);

(3) Compare the fingerprints of the Danshen injection product to be tested with the control fingerprints of the Danshen injection to identify the similarity, so as to determine whether the product quality is qualified.

From the detected main peaks, 15 fingerprint peaks of the control fingerprint of Danshen injection (Fig. Protocatechuic acid, peak 3 is protocatechualdehyde, peak 4 is caffeic acid, peak 5 is desalvianolic acid E1, peak 6 is desalvianolic acid E4, peak 7 is desalvianolic acid Danshensu salvianolic acid E2, peak No. 9 is salvianolic acid D, peak No. 10 is salvianolic acid E, peak No. 11 is rosmarinic acid, peak No. 12 is shikoninic acid, peak No. 15 is salvianolic acid B, Peak No. 16 is iso-salvianolic acid B, peak No. 17 is salvianolic acid L, and peak No. 18 is iso-salvianolic acid A.

Studies have shown that: when the octadecylsilane bonded silica gel column is a Shiseido Capcell pak MG C18 column or a Phenomenex Luna C18 column, preferably the Shiseido Capcell pak MG C18 column, the obtained chromatogram has a stable baseline, and the separation of each main chromatographic peak reaches 1.5; when the detection wavelength is 270-280nm, preferably 286nm, it is suitable for chromatographic analysis of salvianolic acid components, with a stable baseline, high response value of main components, and comprehensive spectral information; when the column temperature is 25-30°C, each The separation of the main peaks is good, and the analysis time is short. When the column temperature is 20°C, the separation of protocatechuic acid and isoferulic acid is not good. At 35°C, salvianolic acid D cannot be separated from the previous impurity peak. Among them, the octadecylsilane bonded silica gel column can be further protected with ShiseidoMG C18 column.

Furthermore, in the quality control method of the present invention, wherein the control fingerprint of Danshen injection is shown in Figure 1 or Figure 5 .

Still further, in the quality control method of the present invention, wherein the fingerprint of the Danshen injection to be tested is compared with the control fingerprint of the Danshen injection, and when the similarity of the two fingerprints is greater than 0.900, preferably 0.950, it is considered that the The quality of the tested product of Danshen injection is qualified.

The quality control method of the present invention has the following advantages:

(1) The HPLC or LC-MS fingerprints established based on the main active components of Danshen injection, phenolic acids, represent most of the pharmacologically active components of Danshen injection, and can effectively characterize the quality of Danshen injection, so as to determine the quality of Danshen injection. Each component of the injection is tested to the greatest extent, which is conducive to the overall control of its quality;

(2) The present invention realizes the identification of the chromatographic peaks of Danshen injection, especially the peaks of unknown components, to the greatest extent under the condition that the reference substance is lacking;

(3) The quality control method of the present invention has the characteristics of simplicity, stability, high precision, good reproducibility and easy mastery.

Description of drawings

Fig. 1 is the control fingerprint of the Danshen injection of embodiment 2.

Fig. 2 is the control fingerprint of the Danshen injection of embodiment 3.

Fig. 3 is the control fingerprint of the Danshen injection of Example 4.

Fig. 4 is the control fingerprint of the Danshen injection of Example 5.

Fig. 5 is the control fingerprint of the Danshen injection of Example 6.

Fig. 6 is the fingerprint of Danshen injection of Example 7.

specific implementation plan

The present invention will be further described below in conjunction with the examples, the following examples are only used to illustrate the present invention and not limit the present invention, any form of equivalent replacement all falls within the protection scope of the present invention.

The preparation of embodiment 1 Danshen injection

According to No. WS3-B-3766-98 standard, 200 batches of Danshen injection were prepared, wherein the dosage of Danshen was 132.5g.

Example 2 Establishment of Fingerprint of Danshen Injection Control Solution

1. Instruments and reagents

High performance liquid chromatography system: Agilent 1100 Agilent Technologies, USA (configured with vacuum degasser, autosampler, quaternary pump, VWD G1314A, DAD G1315B; Agilent ChemStation A 10.02 chromatographic workstation).

Danshensu sodium reference substance, protocatechuic acid reference substance, and protocatechualdehyde reference substance were all purchased from China Institute for the Control of Pharmaceutical and Biological Products, rosmarinic acid reference substance, shikoninic acid reference substance, salvianolic acid B reference substance, caffeic acid The reference substance and the isoferulic acid reference substance were purchased from Shanghai Yousi Biotechnology Co., Ltd., and the salvianolic acid D reference substance was isolated from Salvia miltiorrhiza (self-made, with a purity of about 70%); phosphoric acid (85%), formic acid (96% ), acetic acid were purchased from Tedia (USA), methanol and acetonitrile were purchased from Burdick & Jackson (Honeywell, USA), and water was prepared by Mili-Q ultrapure water apparatus.

2. Chromatographic conditions

Chromatographic column: Octadecylsilane bonded silica gel as filler, Shiseido Capcell pak MG C18 (4.6×250mm, 5μm) column and Shiseido MG C18 (4.6×12.5mm, 5μm) guard column; mobile phase: acetonitrile- 0.03% phosphoric acid aqueous solution; detection wavelength: 286nm; flow rate: 1.0mL/min; column temperature: 30°C; gradient elution program: see Table 1; chromatography integration parameters: see Table 2.

Table 1 Gradient elution program

Table 2 Fingerprint Integration Parameters

3. Solution preparation

Preparation of the test solution: take each batch of Danshen injection in Example 1 and dilute it 5 times with water, or filter it with a PVDF hydrophilic membrane or add water and dilute it 5 times after centrifugation, to obtain final product.

4. Fingerprint chromatography and peak identification

The fingerprints of 200 batches of Danshen injection were analyzed, and the similarity was above 90%. By comparing with the chromatogram, ultraviolet spectrum and mass spectrometry behavior of the reference substance, it is confirmed that the 9 fingerprint peaks are: peak No. 1 is Salvia miltiorrhiza, peak No. 2 is protocatechu, peak No. 3 is protocatechu, peak No. 4 is caffeic acid , Peak No. 5 is isoferulic acid, peak No. 6 is salvianolic acid D, peak No. 7 is rosmarinic acid, peak No. 8 is shikoninic acid, and peak No. 9 is salvianolic acid B.

5. Validation of fingerprint analysis method

(1) Inspection of precision

The samples of Danshen injection were taken and analyzed according to the established HPLC conditions, and the samples were injected continuously for 6 times, each injection was 10 μl. The chromatograms were imported into the Chinese medicine chromatographic fingerprint similarity evaluation software promulgated by the National Pharmacopoeia Commission, and the similarity of each chromatogram was calculated. The results are shown in Table 3. The results showed that the similarity of the chromatograms obtained by continuous injection for 6 times was above 0.993, and the precision met the requirements.

Table 3 Fingerprint precision inspection similarity results

(2) Repeated inspection

Take the same bottle of Danshen injection sample, prepare 6 test solutions in parallel, and analyze according to the established HPLC fingerprint conditions. The results are shown in Table 4. The results showed that the similarity of the chromatograms obtained from six parallel samples was 1.000, and the repeatability met the requirements.

Table 4 Similarity Results of Fingerprint Repeatability Inspection

(3) Stability inspection

The same batch of Danshen injection samples were taken, placed at room temperature, injected at 0, 3, 6, 9, 12, 15, 18, 21, and 24 hours, and analyzed according to the established HPLC fingerprint analysis method. The results are shown in Table 5. The results showed that the similarity of the chromatograms obtained within 24 hours was greater than 0.998, and the stability met the requirements.

Table 5 Similarity Results of Fingerprint Stability Inspection

The above test results show that the method has high accuracy, good reproducibility and qualified stability, which meets the method validation requirements of fingerprints.

Example 2 Establishment of Danshen Injection Control Fingerprint

Replace the detection wavelength in the chromatographic conditions of Example 2 with 270 or 280nm, and obtain the fingerprint of Salvia Miltiorrhiza Injection (see Figure 2) according to its method steps, investigate the precision, repeatability and stability, the results show that this conforms to the fingerprint of the fingerprint Method validation requirements.

Example 3 Establishment of Danshen Injection Control Fingerprint

Replace the column temperature in the chromatographic conditions of Example 2 with 25°C, and follow the method steps to obtain the fingerprint of Danshen injection (see Figure 3), and investigate the precision, repeatability and stability. The results show that this method conforms to the fingerprint Authentication required.

Example 4 Establishment of Danshen Injection Control Fingerprint

Replace the chromatographic column in the chromatographic conditions of Example 2 with a Phenomenex Luna C18 (4.6 × 250mm, 5 μm) chromatographic column, and obtain the fingerprint of Danshen injection according to the method steps (see Figure 4), and investigate the precision, repeatability and stability The results show that it meets the method verification requirements for fingerprints.

Example 5 Establishment of Danshen Injection Control Fingerprint

1. Instruments and reagents

Liquid chromatography-mass chromatography: Agilent 1200RRLC liquid phase system and Bruker Esquire HCT ion trap mass spectrometer.

2. Chromatographic conditions

Chromatographic column: Shiseido Capcell pak MG C18 (4.6×250mm, 5μm) column and Shiseido MG C18 (4.6×12.5mm, 5μm) guard column; mobile phase: acetonitrile-0.3% formic acid aqueous solution; detection wavelength: 286nm; flow rate: 1.0 ml/min; column temperature: 30°C; gradient elution program: see Table 6.

Table 6 Gradient elution program

Mass spectrometry parameters: ESI(-), ultra scan 50~1200m/z, Nebulizer 40psi, Dry gas 9l/min, Dry Temp 350°C, split mode into mass spectrometer, split ratio is about 1:3.

3. Carry out the identification of the chromatographic peaks of the control fingerprint according to the similar method and steps of Example 2, the results are shown in Figure 5 and Table 7.

Table 7 Fingerprint Identification Data of Danshen Injection

4. Validation of fingerprint analysis method

According to the method steps of Example 2, the precision, repeatability and stability were investigated, and the results are shown in Table 8-10.

Table 8 Fingerprint precision inspection similarity results

Table 9 Fingerprint repeatability inspection similarity results

Table 10 Fingerprint Stability Inspection Similarity Results

The above test results show that the method has high accuracy, good reproducibility and qualified stability, which meets the method validation requirements of fingerprints.

The HPLC fingerprint analysis of embodiment 6 Danshen injection samples

Eleven batches of Danshen injection samples (provided by Shineway Pharmaceutical Group Co., Ltd.) were analyzed according to the HPLC conditions established in Example 2. The investigation results are shown in Table 11. The comparison of the obtained fingerprints with the control fingerprints is shown in Figure 6. The results showed that the similarity of 11 batches of Danshen injection samples was between 0.970 and 1.000, which was a good similarity, which proved that the quality of 11 batches of samples was qualified.

Table 11 Results of Fingerprint of Danshen Injection

Claims (9)

1. a quality control method of Danshen injection, is characterized in that comprising the steps: (1) Establishment of control fingerprint of Danshen injection Preparation of Danshen injection reference solution: Take Danshen injection and dilute it 5 times with water, or filter it with PVDF hydrophilic filter membrane or centrifuge and add water to dilute it 5 times for later use; Determination of Danshen Injection Control Fingerprint: Draw the above-mentioned reference substance solution and inject it into a liquid chromatograph, use the HPLC method to measure, and obtain the control fingerprint of Danshen injection, chromatographic conditions: the chromatographic column is an octadecylsilane bonded silica gel column, and the mobile phase is acetonitrile-0.03% phosphoric acid aqueous solution , the detection wavelength is 270-280nm, the flow rate is 1.0mL/min, the column temperature is 25-30°C, and the gradient elution procedure is as follows, (2) Determination of fingerprints of Danshen injection product to be tested Get Danshen injection need testing solution, measure fingerprint according to the steps and conditions described in above-mentioned (1); (3) Compare the fingerprints of the Danshen injection product to be tested with the control fingerprints of the Danshen injection to identify the similarity, so as to determine whether the product quality is qualified. 2. A quality control method for Danshen injection, characterized in that it comprises the steps: (1) Establishment of control fingerprint of Danshen injection Preparation of Danshen injection reference solution: Take Danshen injection and dilute it 5 times with water, or filter it with PVDF hydrophilic filter membrane or centrifuge and add water to dilute it 5 times, set aside; Determination of Danshen Injection Control Fingerprint: Draw the above-mentioned reference substance solution and inject it into a liquid chromatograph, use LC-MS method to measure, and obtain the control fingerprint of Danshen injection, chromatographic conditions: the chromatographic column is an octadecylsilane-bonded silica gel column, and the mobile phase is acetonitrile-0.03% Formic acid aqueous solution, the detection wavelength is 270-280nm, the flow rate is 1.0mL/min, the column temperature is 25-30°C, the gradient elution procedure is as follows, Mass spectrometry parameters: ESI(-), ultra scan 50~1200m/z, Nebulizer 40psi, Dry gas 9l/min, Dry Temp 350℃, split mode into mass spectrometer, split ratio is about 1:3; (2) Determination of fingerprints of Danshen injection product to be tested Get Danshen injection need testing solution, measure fingerprint according to the steps and conditions described in above-mentioned (1); (3) Compare the fingerprints of the Danshen injection product to be tested with the control fingerprints of the Danshen injection to identify the similarity, so as to determine whether the product quality is qualified. 3. The quality control method according to claim 1 or 2, characterized in that the octadecylsilane bonded silica gel column is a Shiseido Capcell pak MG C18 post or a Phenomenex Luna C18 post, and is protected with a Shiseido MG C18 post, preferably Shiseido Capcell pak MG C18 column. 4. The quality control method according to claim 1 or 2, characterized in that the octadecylsilane bonded silica gel column is further protected with a Shiseido MG C18 column. 5. The quality control method according to claim 1 or 2, characterized in that the detection wavelength is 270nm, 280nm or 286nm, preferably 286nm. 6. The quality control method according to claim 1 or 2, characterized in that the column temperature is 25°C or 30°C, preferably 30°C. 7. The quality control method according to claim 1, characterized in that the control fingerprint of Danshen injection is as shown in Figure 1. 8. The quality control method according to claim 2, characterized in that the control fingerprint of Danshen injection is as shown in Figure 5. 9. The quality control method according to claim 1 or 2, characterized in that, the fingerprint of the Danshen injection product to be tested is compared with the comparison fingerprint of the Danshen injection, and the similarity of the two fingerprints is greater than 0.900, preferably 0.950, it is considered that the quality of the product to be tested of the Danshen injection is qualified.