Method for simultaneously determining phenolic acids and tanshinones in Danshen injection

Abstract

The application discloses a method for simultaneously determining phenolic acid and tanshinone components in Danshen injection, and according to the structure and property characteristics of the phenolic acid and tanshinone components, the best mobile phase composition, elution program, flow rate, chromatographic column and mass spectrometry condition are screened through a large number of experiments. Experimental verification shows that the UPLC-TQ-MS / MS method can simultaneously determine 11 different structure and type of phenolic acid and tanshinone component compounds, and the methodological verification shows that the detection method has good precision, repeatability and high accuracy, and has important significance for controlling the quality of Danshen injection and guaranteeing the clinical curative effect.

Description

Technical Field

[0001] This invention relates to a quality control method for traditional Chinese medicine preparations, specifically a method for determining phenolic acids and tanshinone components in Danshen injection. Background Technology

[0002] Danshen, as a traditional Chinese medicine, has a variety of effects and functions, mainly including the following:

[0003] Promoting blood circulation and removing blood stasis: Danshen can promote blood circulation, improve microcirculation, and reduce blood viscosity, thereby promoting blood circulation and removing blood stasis. It can also dilate coronary arteries, increase myocardial blood flow, and reduce myocardial oxygen consumption, thus protecting the heart.

[0004] Calming the mind and relieving irritability: Salvia miltiorrhiza has the effect of calming the mind and relieving the symptoms of irritability and insomnia.

[0005] Cooling the blood and reducing swelling: Danshen can eliminate excess free radicals in the body and prevent free radical peroxidation, thereby achieving the effect of lowering blood sugar. In addition, Danshen also has antibacterial and anti-inflammatory effects, and has an inhibitory effect on a variety of bacteria.

[0006] Liver protection: Danshen can promote liver cell regeneration and has anti-fibrotic effects. It can also inhibit inflammation and reduce liver cell degeneration and necrosis caused by inflammation.

[0007] Anti-tumor: Danshen has certain anti-tumor effects because it can inhibit the central nervous system and protect the body's health.

[0008] Lowering blood lipids: Danshen can lower blood lipids and prevent and treat hyperlipidemia.

[0009] Improving cardiovascular disease symptoms: Danshen (Salvia miltiorrhiza) has the effects of promoting blood circulation, lowering blood pressure and cholesterol, and improving cardiovascular disease symptoms. It can dilate blood vessels and improve blood circulation, having a good effect on patients with thrombosis caused by hyperlipidemia. In addition, Danshen can also inhibit coronary atherosclerosis and prevent myocardial infarction.

[0010] Delaying aging: Danshen contains bioactive substances that can improve the activity of body cells on the one hand, and remove excess free radicals in the body on the other hand, so as to prevent free radical peroxidation, thereby achieving the effect of delaying aging.

[0011] Modern research indicates that *Salvia miltiorrhiza* contains fat-soluble components, such as tanshinone I, IIA, IIB, cryptotanshinone, isocryptoshinone, tanshinol, and tanshinaldehyde. It also contains water-soluble components, such as tanshinone, tanshinone acid, protocatechuic acid, and protocatechuic aldehyde.

[0012] Currently marketed Danshen injections are clinically used to treat coronary heart disease, chest tightness, angina pectoris, and other conditions. However, due to variations in the active ingredients of Danshen injections produced by different origins and manufacturing processes, it is necessary to examine the key phenolic acids and tanshinone active ingredients to ensure efficacy and control the content of effective components. Existing reported detection methods, such as Chinese Patent 201510142320.5 (detecting six components: protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, lithospermic acid, caffeic acid, and isoflavone acid) and CN202311781089.5 (only simultaneously determining four components: sodium tanshinone, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B), are not comprehensive enough, have long analysis times, and are not very efficient. Summary of the Invention

[0013] Purpose of the Invention: The purpose of this invention is to address the shortcomings of existing technologies. Through extensive experimental screening, UHPLC-TQ-MS / MS was used to detect 11 components of Danshen injection. This method exhibits high sensitivity and good stability, enabling objective, comprehensive, and accurate evaluation of the quality of Danshen extracts and preparations, which is of great significance for quality control and ensuring efficacy.

[0014] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0015] A method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection, characterized by comprising the following steps:

[0016] Step (1) Preparation of mixed reference solution

[0017] Accurately weigh salvianolic acid A, salvianolic acid B, salvianolic acid C, rosmarinic acid, protocatechuic acid, protocatechuic aldehyde, caffeic acid, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I, add methanol to prepare a mixed standard stock solution, and store it for later use.

[0018] Step (2) Preparation of Danshen Injection Test Solution

[0019] Take Danshen injection, dilute with methanol, filter through a 0.22μm microporous membrane, and set aside for later use;

[0020] Step (3) Establishing the linear regression equation

[0021] Take the mixed reference solution from step (1), dilute it 5 times in sequence, filter it through a 0.22 μm microporous membrane to obtain mixed reference solutions of different concentrations, and then inject it into UPLC-TQ-MS / MS in sequence. With the reference concentration as the abscissa X and the corresponding peak area as the ordinate Y, perform linear regression analysis on each reference component and establish a linear regression equation.

[0022] Step (4) Content determination

[0023] Take the tanshinone injection test solution from step (2) and inject it into UPLC-TQ-MS / MS for analysis. Based on the retention time, substitute the peak area in the chromatogram of the test sample into the linear regression equation of step (3) to calculate the content of various phenolic acids and tanshinone components in the test sample solution.

[0024] As a preferred embodiment, the method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection described above is characterized by...

[0025] The preparation method of the reference solution in step (1) is as follows:

[0026] Accurately weigh salvianolic acid A, salvianolic acid B, salvianolic acid C, rosmarinic acid, protocatechuic acid, protocatechuic aldehyde, caffeic acid, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I, and add methanol to prepare mixed standard stock solutions with concentrations of 1.2, 1.5, 3.0, 0.8, 2.5, 2.0, 4.0, 1.5, 0.5, 0.3, and 6.0 μg / mL, respectively. Store at 4℃ and filter through a 0.22 μm microporous membrane before injection.

[0027] As a preferred embodiment, the method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection described above is characterized by...

[0028] The chromatographic conditions for UPLC-TQ-MS / MS in steps (3) and (4) are as follows:

[0029] Chromatographic column: ACQUITYUPLC C18 column, 100×2.1mm, 1.7μm; column temperature: 30℃; flow rate: 0.50mL / min; injection volume: 1μL; mobile phase A: methanol, B: 0.1% phosphoric acid solution; gradient elution: 0–2 min 81% A, 2–4.5 min 81% → 43% A, 4.5–6.3 min 43% → 21% A, 6.3–8.6 min 21% → 18% A;

[0030] Mass spectrometry conditions

[0031] The triple quadrupole tandem mass spectrometer was configured with the following ionization source conditions: capillary voltage 3.0 kV; source temperature 155 °C; ambient temperature 450 °C; orifice gas flow rate 40 L / h. -1 Desolvation gas flow rate: 800 L / h -1 Multiple reaction monitoring (MRM) is used for the detection and quantification of analytes.

[0032] As a preferred method, the 11 reference quality spectral conditions for the method for simultaneously determining phenolic acids and tanshinone components in Danshen injection described above are shown in the table below:

[0033]

[0034] Methodological investigation experiment

[0035] 1. Precision Experiment Investigation

[0036] The intra-day and inter-day precision of the detection method of this invention were evaluated by testing six samples containing the above 11 mixed reference solutions within 24 hours and for three consecutive days. The results showed that the intra-day precision (RSD) of the 11 reference solutions was less than 4.12% and the inter-day precision (RSD) was less than 4.89%, indicating that the detection method established by this invention has good precision.

[0037] 2. Repeatability and stability test

[0038] The reproducibility of the method was assessed by measuring six Danshen injection solutions prepared from the same batch using a detection and analysis method that could not be established previously. The stability of the detection method was examined by analyzing three copies of the Danshen injection solution placed at room temperature for 0, 4, 8, 12, 24, and 48 hours. The results showed that the reproducibility (RSD) of the six samples was less than 4.45%, and the RSD of the six measurements within 48 hours was less than 4.30%. This indicates that the reproducibility and stability of the present invention are good.

[0039] 3. Spike recovery experiment

[0040] Eleven known quantities of standard samples were added to a certain amount of the test solution, prepared in triplicate. The recovery rate of each test sample was calculated to evaluate the accuracy of the detection method established in this invention. The RSDs of the calculated RSDs for salvianolic acid A, salvianolic acid B, salvianolic acid C, rosmarinic acid, protocatechuic acid, protocatechuic aldehyde, caffeic acid, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I were 3.62%, 2.84%, 2.56%, 3.72%, 4.13%, 3.95%, 3.51%, 4.24%, 2.77%, 3.17%, and 4.02%, respectively. The spiking recovery test showed that the accuracy of the method established in this invention is good.

[0041] Beneficial effects: The method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection provided by this invention has the following advantages compared with the prior art:

[0042] Among the active ingredients detected in this invention, seven phenolic acids—tanshinone A, Tanshinone B, Tanshinone C, Rosmarinic acid, Protocatechuic acid, Protocatechuic aldehyde, and Caffeic acid—have very similar polarities, making them difficult to separate on a chromatographic column. Similarly, four tanshinone compounds—tanshinone I, Tanshinone IIA, Cryptotanshinone, and Dihydrotanshinone I—have very similar polarities, also making them difficult to separate on a chromatographic column. Compared to existing detection methods, the difficulty of separating each additional phenolic acid or tanshinone compound on a chromatographic column increases significantly.

[0043] Optimization of chromatographic conditions: This invention investigated the effects of different mobile phase compositions, such as methanol-water, acetonitrile-water, methanol-acetonitrile, methanol-formic acid water, acetonitrile-formic acid water, methanol-phosphoric acid water, and acetonitrile-phosphoric acid water, on the separation of phenolic acids and tanshinone components in Danshen injection. The results showed that using methanol-0.1% phosphoric acid water as the elution solvent could achieve good separation of seven phenolic acids and four tanshinone components with very similar polarities on a single chromatographic column.

[0044] After determining the mobile phase, this invention further screened gradient elution methods, finding that different elution methods yielded significantly different resolutions. Because the seven phenolic acid components (tanshinone A, B, C, rosmarinic acid, protocatechuic acid, protocatechuic aldehyde, and caffeic acid) have very similar polarities, and the four tanshinone components (tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I) have very similar polarities, separation on the chromatographic column was challenging. This invention, through extensive experimentation and screening of mobile phase compositions at different time points, determined the optimal gradient elution method, achieving good resolution, establishing a standard curve, and obtaining excellent technical results.

[0045] Based on the structure and properties of 11 components, including tanshinone compounds and tanshinone derivatives, this invention, through extensive experimentation, screened the optimal mobile phase composition, elution program, flow rate, chromatographic column, and mass spectrometry conditions. Experimental verification shows that this invention can simultaneously detect 11 compounds of different structures and types.

[0046] Furthermore, methodological verification has shown that the method established in this invention has high detection sensitivity, good stability and accuracy, and is of great significance for controlling the quality of Danshen injection and ensuring clinical efficacy. Detailed Implementation

[0047] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0048] The instruments and materials used in the following examples are: ACQUITY UPLC system, TQ mass spectrometer (Waters Corporation, USA), Mill-Q ultrapure water system (Millipore Corporation, USA), and methanol (Merck, Germany, mass spectrometry grade).

[0049] Danshen Injection (produced by Changshu Lei Yunshang Pharmaceutical Co., Ltd., National Drug Approval Number Z32020236, Production Batch Number 202462~202464).

[0050] Example 1

[0051] 1. A method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection, characterized by comprising the following steps:

[0052] Step (1) Preparation of mixed reference solution

[0053] Accurately weigh salvianolic acid A, salvianolic acid B, salvianolic acid C, rosmarinic acid, protocatechuic acid, protocatechuic aldehyde, caffeic acid, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I, and add methanol to prepare mixed standard stock solutions with concentrations of 1.2, 1.5, 3.0, 0.8, 2.5, 2.0, 4.0, 1.5, 0.5, 0.3, and 6.0 μg / mL, respectively. Store at 4℃ and filter through a 0.22 μm microporous membrane before injection.

[0054] Step (2) Preparation of Danshen Injection Test Solution

[0055] Take 3 portions of Danshen Injection (production batches 202462-202464) produced by Changshu Lei Yunshang Pharmaceutical Co., Ltd., dilute it 5 times with methanol, filter it through a 0.22μm microporous membrane, and set it aside for later use;

[0056] Step (3) Establishing the linear regression equation

[0057] Take the mixed reference solution from step (1), dilute it 5 times in sequence, filter it through a 0.22 μm microporous membrane to obtain mixed reference solutions of different concentrations, and then inject it into UPLC-TQ-MS / MS in sequence. With the reference concentration as the abscissa X and the corresponding peak area as the ordinate Y, perform linear regression analysis on each reference component and establish a linear regression equation as shown in Table 1 below.

[0058] Table 1

[0059] Reference components Linear equations <![CDATA[r 2 ]]> Tanshinone A y = 105.42x + 1204.21 0.9992 Tanshinone B y = 315.23x + 636.25 0.9994 Tanshinone C y = 254.75x + 324.25 0.9995 Rosemary acid y = 632.51x + 252.21 0.9990 Protocatechuic acid y = 722.96x + 265.12 0.9985 Protocatechuic acid y = 250.25x + 592.52 0.9991 caffeic acid y = 965.20x + 385.59 0.9989 Tanshinone I y = 298.62x + 592.20 0.9991 Tanshinone IIA y = 150.26x + 195.22 0.9999 Cryptotanshinone y = 252.46x + 295.87 0.9997 Dihydrotanshinone I y = 144.69x + 321.24 0.9989

[0060] Step (4) Content determination

[0061] Take three batches of Danshen injection test solutions from step (2), inject them into UPLC-TQ-MS / MS for analysis, and substitute the peak areas in the chromatogram of the test samples into the linear regression equation of step (3) based on the retention time to calculate the content of various phenolic acids and tanshinone components in the test sample solution.

[0062] The chromatographic conditions for UPLC-TQ-MS / MS in steps (3) and (4) are as follows:

[0063] Chromatographic column: ACQUITY UPLC, C18 column, 100×2.1mm, 1.7μm; column temperature: 30℃; flow rate: 0.50mL / min; injection volume: 1μL; mobile phase A: methanol, B: 0.1% phosphoric acid solution; gradient elution: 0–2 min 81% A, 2–4.5 min 81→43% A, 4.5–6.3 min 43→21% A, 6.3–8.6 min 21→18% A;

[0064] Mass spectrometry conditions

[0065] The triple quadrupole tandem mass spectrometer was configured with the following ionization source conditions: capillary voltage 3.0 kV; source temperature 155 °C; ambient temperature 450 °C; orifice gas flow rate 40 L / h. -1 Desolvation gas flow rate: 800 L / h -1 Multiple reaction monitoring (MRM) is used for the detection and quantification of analytes.

[0066] The quality profile conditions for 11 control samples are shown in the table below:

[0067] Table 2

[0068]

[0069] 2. Sample content determination

[0070] The calculated contents and average values ​​of 11 components, including salvianolic acid A, salvianolic acid B, salvianolic acid C, rosmarinic acid, protocatechuic acid, protocatechuic aldehyde, caffeic acid, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I, are shown in Table 3.

[0071] Table 3. Results of content determination of 11 components (mg·mL) -1 (3 batches)

[0072]

[0073]

[0074] This invention quantitatively detected 11 active ingredients (7 phenolic acid components and 4 tanshinone components) in Danshen injection using UPLC-TQ-MS / MS. This method can comprehensively, objectively and accurately detect the effective components in Danshen injection, providing a scientific basis for ensuring its clinical safety and efficacy.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection, characterized in that, Includes the following steps: Step (1) Preparation of mixed reference solution Accurately weigh salvianolic acid A, salvianolic acid B, salvianolic acid C, rosmarinic acid, protocatechuic acid, protocatechuic aldehyde, caffeic acid, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I, add methanol to prepare a mixed standard stock solution, and store it for later use. Step (2) Preparation of Danshen Injection Test Solution Take Danshen injection, dilute with methanol, filter through a 0.22μm microporous membrane, and set aside for later use; Step (3) Establishing the linear regression equation Take the mixed reference solution from step (1), dilute it 5 times in sequence, filter it through a 0.22 μm microporous membrane to obtain mixed reference solutions of different concentrations, and then inject it into UPLC-TQ-MS / MS in sequence. With the reference concentration as the abscissa X and the corresponding peak area as the ordinate Y, perform linear regression analysis on each reference component and establish a linear regression equation. Step (4) Content determination Take the tanshinone injection test solution from step (2) and inject it into UPLC-TQ-MS / MS for analysis. Based on the retention time, substitute the peak area in the chromatogram of the test sample into the linear regression equation of step (3) to calculate the content of various phenolic acids and tanshinone components in the test sample solution.

2. The method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection according to claim 1, characterized in that, The preparation method of the reference solution in step (1) is as follows: Accurately weigh salvianolic acid A, salvianolic acid B, salvianolic acid C, rosmarinic acid, protocatechuic acid, protocatechuic aldehyde, caffeic acid, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I, and add methanol to prepare mixed standard stock solutions with concentrations of 1.2, 1.5, 3.0, 0.8, 2.5, 2.0, 4.0, 1.5, 0.5, 0.3, and 6.0 μg / mL, respectively. Store at 4℃ and filter through a 0.22 μm microporous membrane before injection.

3. The method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection according to claim 1, characterized in that, The chromatographic conditions for UPLC-TQ-MS / MS in steps (3) and (4) are as follows: Chromatographic column: ACQUITY UPLC, C18 column, 100×2.1mm, 1.7μm; column temperature: 30℃; flow rate: 0.50mL / min; injection volume: 1μL; mobile phase A: methanol, mobile phase B: 0.1% phosphoric acid solution; gradient elution; Mass spectrometry conditions The triple quadrupole tandem mass spectrometer was configured with the following ionization source conditions: capillary voltage 3.0 kV; source temperature 155 °C; ambient temperature 450 °C; orifice gas flow rate 40 L / h. -1 Desolvation gas flow rate: 800 L / h -1 Multiple reaction monitoring (MRM) is used for the detection and quantification of analytes.

4. The method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection according to claim 3, characterized in that, The specific gradient elution conditions are: 0–2 min 81% A, 2–4.5 min 81 → 43% A, 4.5–6.3 min 43 → 21% A, 6.3–8.6 min 21 → 18% A.

5. The method for simultaneously determining phenolic acids and tanshinone components in tanshinone injection according to claim 3, characterized in that, The quality profile conditions for 11 control samples are shown in the table below:

6. The application of the method according to any one of claims 1 to 5 in the quality evaluation of Danshen injection.

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