Method for detecting content of effective components in shangtong tincture

Abstract

This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, specifically relating to a method for detecting the content of active ingredients in pain-relieving tinctures. The detection method is as follows: a test solution is prepared using ethanol solution as a solvent, and injected into a liquid chromatograph to determine the content of bergenin and paeonol. The chromatographic conditions are: octadecylsilane-bonded silica gel as the packing material; gradient elution using 0.1% phosphoric acid solution and acetonitrile as the mobile phase; column temperature of 30–40℃; detection wavelength of 265–280 nm; and flow rate of 0.8–1.2 mL / min. This method can detect the content of bergenin and paeonol in pain-relieving tinctures, effectively monitoring the quality of the tincture. The method exhibits high accuracy, good linearity, high precision, good separation, and good repeatability.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, specifically relating to a method for detecting the content of effective components in pain relief tincture. Background Technology

[0002] The pain relief tincture is a traditional Chinese medicine compound preparation composed of eight herbs: hibiscus leaf, cynanchum paniculatum, nemesis chinensis, cinnamon root, agastache rugosa, menthol, camphor, and cinnamon oil. It has the effects of removing blood stasis, promoting blood circulation, reducing swelling and relieving pain, and is used for acute soft tissue injuries such as sprains, contusions, crush injuries, and tenosynovitis.

[0003] Existing drug standards only evaluate product quality based on sensory indicators such as appearance and odor of pain relief tinctures, without conducting systematic qualitative or quantitative studies on any of the medicinal flavors in the formula. This fails to meet the basic requirements of "safety, efficacy, stability, and controllability" for drugs. The efficacy of traditional Chinese medicine compound preparations is the result of the synergistic effect of multiple active ingredients. Controlling only a single property cannot guarantee batch-to-batch quality consistency, nor can it ensure the stability of clinical efficacy.

[0004] Therefore, it is particularly urgent and important to develop a method that can detect the content of the active ingredients in pain relief tincture. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting the content of the active ingredients in pain relief tincture, so as to achieve quantitative monitoring of the active ingredients in pain relief tincture.

[0006] The present invention provides a method for detecting the content of effective components in analgesic tincture to solve the above-mentioned technical problems. The detection method is as follows: a test solution is prepared using ethanol solution as a solvent, and injected into a liquid chromatograph to determine the content of bergenin and paeonol; the chromatographic conditions are: octadecylsilane-bonded silica gel as the packing material; gradient elution using 0.1% phosphoric acid solution and acetonitrile as the mobile phase; column temperature of 30–40℃; detection wavelength of 265–280 nm; and flow rate of 0.8–1.2 mL / min.

[0007] Furthermore, the gradient elution conditions for the mobile phase are as follows:

[0008] The time was 0–6 min, the mobile phase was 0.1% phosphoric acid solution with a concentration of 94–90%, and the mobile phase was acetonitrile with a concentration of 6–10%.

[0009] The time is 6–10 min, the mobile phase is 0.1% phosphoric acid solution with a concentration of 90–84%, and the mobile phase is acetonitrile with a concentration of 10–16%.

[0010] The time was 10–30 min, the mobile phase was 0.1% phosphoric acid solution with a concentration of 84–68%, and the mobile phase was acetonitrile with a concentration of 16–32%.

[0011] The time is 30-40 min, the mobile phase is 0.1% phosphoric acid solution with a concentration of 68-35%, and the mobile phase is acetonitrile with a concentration of 32-65%.

[0012] The time was 40.1–45.1 min, the mobile phase consisted of 94% 0.1% phosphoric acid solution and 6% acetonitrile.

[0013] Further, the test solution is prepared by taking 4-6 mL of pain tincture, diluting it to 100 mL with an ethanol solution of 70-85% by volume, filtering it, and taking the filtrate.

[0014] Preferably, the test solution is prepared by taking 5 mL of pain tincture, diluting it to 100 mL with 80% ethanol solution, filtering, and collecting the filtrate.

[0015] Preferably, the chromatographic conditions are as follows: column temperature 30°C; detection wavelength 274 nm; flow rate 1.0 mL / min.

[0016] Furthermore, the detection method also includes the preparation of a reference solution.

[0017] Furthermore, the reference solution is prepared by accurately weighing bergenin reference standard and paeonol reference standard, adding methanol to prepare a mixed solution containing 20 μg of each per 1 mL.

[0018] The beneficial effects of this invention are:

[0019] This invention provides a method for detecting the content of active ingredients in pain-relieving tinctures. This method can detect the content of bergenin and paeonol in pain-relieving tinctures, effectively monitoring their quality. The method exhibits high accuracy, good linearity, high precision, good separation, and good repeatability. This invention solves the technical challenge of simultaneously determining polarity-differentiated components in traditional Chinese medicine compound tinctures, providing scientific, reliable, and efficient technical support for improving the quality standards, optimizing processes, and regulating the market of pain-relieving tinctures. Attached Figure Description

[0020] Figure 1 The chromatogram is for sample batch number 230201 in Example 1.

[0021] Figure 2 The chromatograms are for different column temperatures in Example 2.

[0022] Figure 3 The chromatograms are for different wavelengths in Example 2.

[0023] Figure 4 The chromatogram for Example 2 is a gradient elution of methanol-water according to the present invention (from top to bottom, the reference solution and the test solution with sample batch number 250501 are respectively).

[0024] Figure 5 The chromatogram for Example 2 is a gradient elution of methanol-0.1% phosphoric acid according to the present invention (from top to bottom, the reference solution and the test solution with sample batch number 250501 are respectively).

[0025] Figure 6 The chromatogram for Example 2 is as follows: the mobile phase is acetonitrile-water, and the chromatogram is obtained by gradient elution according to the present invention (from top to bottom, the reference solution and the test solution with sample batch number 250501 are respectively).

[0026] Figure 7 The chromatogram for Example 2 is as follows: the mobile phase is acetonitrile-0.1% phosphoric acid solution, and the chromatogram is obtained by gradient elution according to the present invention (from top to bottom, the reference solution and the test solution with sample batch number 250501 are respectively).

[0027] Figure 8 The chromatogram for Example 2 is the result of elution using acetonitrile-0.1% phosphoric acid solution as the mobile phase according to the gradient in Table 4 (from top to bottom: reference solution and test solution with sample batch number 250501).

[0028] Figure 9 This is the standard curve for the linear investigation of Bergenin in Example 3.

[0029] Figure 10 This is the standard curve for the linear investigation of paeonol in Example 3. Detailed Implementation

[0030] The present invention will be further described in detail below through specific embodiments. All drugs and reagents involved in the specific embodiments are commercially available.

[0031] Example 1: Determination of the content of active ingredients in different batches of pain relief tincture

[0032] 1. Instruments and Reagents

[0033] 1.1 Instruments

[0034] High-performance liquid chromatograph (Shimadzu, model LC-2030C Plus)

[0035] 1.2 Reagents

[0036] Methanol: HPLC grade, batch number 2025032101, manufacturer: Chengdu Kelong Chemical Co., Ltd.

[0037] Acetonitrile: HPLC grade, batch number 2025070603, manufacturer: Chengdu Kelong Chemical Co., Ltd.

[0038] Phosphoric acid: GR grade, batch number 240528A0, manufacturer is Xilong Scientific Co., Ltd.

[0039] 17 batches of samples, batch numbers: 230201, 230301, 230302, 230901, 240501, 240502, 240503, 250301, 250302, 250501, 250502, 250601, 250602, 250603, 250701, 250702, 250801 (Yunnan Shengke Pharmaceutical Co., Ltd.).

[0040] 1.3 Reference Standard

[0041] Bergenin reference standard (China National Institutes for Food and Drug Control; batch number 111532-202406; purity content calculated as 93.6%) and Paeonol reference standard (China National Institutes for Food and Drug Control; batch number 110708-202309; purity content calculated as 99.9%).

[0042] 2. Methods and Results

[0043] 2.1 Chromatographic conditions

[0044] The chromatographic column was packed with octadecylsilane-bonded silica gel (WondaSil C18 column, 5 μm, 4.6 × 150 mm); gradient elution was performed using 0.1% phosphoric acid solution and acetonitrile as the mobile phase, as specified in Table 1; the column temperature was 30 °C; the detection wavelength was 274 nm; and the flow rate was 1.0 mL / min.

[0045]

[0046] 2.2 Preparation of the test solution

[0047] Take 5 mL of the pain tincture, dilute it to 100 mL with 80% ethanol solution, filter it, and collect the filtrate.

[0048] 2.3 Preparation of reference solution

[0049] Accurately weigh bergenin and paeonol reference standards, add methanol to prepare a mixed solution containing 20 μg of each per 1 mL.

[0050] 2.4 Determination Method

[0051] Accurately pipette 5 µl each of the reference solution and the test solution into the liquid chromatograph and determine the results. See Table 2 and... Figure 1 .

[0052]

[0053] Example 2: Screening of chromatographic conditions

[0054] 2.1 Column Temperature Investigation

[0055] The separation was investigated at column temperatures of 30℃, 35℃, and 40℃. The separation was best at 30℃, and the baseline position of the paeonol main peak was better than that at 35℃. (See...) Figure 2 .

[0056] 2.2 Wavelength Examination

[0057] The detection was examined at different wavelengths of 265 nm, 274 nm, and 280 nm. The absorbance values ​​of the reference standard and the sample showed maximum absorption at 274 nm. The results are shown in Table 3. Figure 3 .

[0058]

[0059] 2.3 Investigation of the mobile phase

[0060] 2.3.1 Investigations were conducted using different mobile phases according to the gradient of this invention.

[0061] The mobile phases methanol-water, methanol-0.1% phosphoric acid solution, acetonitrile-water, and acetonitrile-0.1% phosphoric acid solution were investigated using the mobile phase gradient according to the present invention. The results showed that gradient elution with acetonitrile-water and acetonitrile-0.1% phosphoric acid solution had no effect on the mixed reference solution. In the sample chromatogram, there was an impurity peak preceding bergenin. With gradient elution with acetonitrile-water, the impurity peak was not separated from bergenin; with gradient elution with acetonitrile-0.1% phosphoric acid solution, the impurity peak was separated from bergenin. Using acetonitrile-water as the mobile phase, the resolution between paeonol and the subsequent impurity peak was 2.33; using acetonitrile-0.1% phosphoric acid solution as the mobile phase, the resolution was 2.40. Therefore, acetonitrile-0.1% phosphoric acid was selected as the mobile phase in this invention. See [link to relevant documentation]. Figures 4-7 .

[0062] 2.3.2 Elution according to different gradients

[0063] Using 0.1% phosphoric acid solution and acetonitrile as the mobile phase, gradient elution was performed according to the specifications in Table 4. The results showed that using the mobile phase gradient specified in Table 1 of this invention resulted in better separation of bergenin from impurity peaks and paeonol from impurity peaks compared to using Table 4. (See Table 4 for details.) Figure 8 .

[0064]

[0065] Example 3 Methodological Validation

[0066] 3.1 System Suitability Test

[0067] According to the method of this invention, the mixed reference solution was injected into the liquid chromatograph, with six consecutive injections. The peak areas were recorded, and the RSD values ​​were calculated. If the RSD values ​​of the peak areas of each component were within 2%, it indicates good system suitability. The results are shown in Table 5.

[0068]

[0069] 3.2 Linearity Examination

[0070] Take appropriate amounts of bergenin and paeonol reference standards, accurately weigh them, and add methanol to prepare a mixed reference solution containing 0.4830696 mg / mL bergenin and 1.0277712 mg / mL paeonol per 1 mL.

[0071] Accurately measure 1 mL, 2 mL, 25 mL, 30 mL, and 50 mL of the mixed reference solution into 50 mL volumetric flasks, respectively. Dilute to the mark with methanol, shake well, filter, and inject 5 μL of methanol (blank) and the above reference solution into the high-performance liquid chromatograph (HPLC). Record the chromatograms, determine the peak areas, and plot the standard curves for each reference solution with the reference concentration as the x-axis and the peak area as the y-axis. Calculate the linear regression equation, correlation coefficient, and linear range. The results are shown in Table 6. Figures 9-10 .

[0072]

[0073] 3.3 Repeatability

[0074] Take this product (batch number: 250501), and prepare 6 test solutions according to the test solution preparation method. Calculate the content of bergenin and paeonol and their RSD values. The results are shown in Table 7. The RSDs of bergenin and paeonol content in all 6 test solutions are less than 2.0%, which meets the requirements, indicating good repeatability.

[0075]

[0076] 3.4 Spike Recovery Test

[0077] The contents of paeonol and bergenin in the test solution were determined based on repeatability. Spike recovery was performed at 80%, 100%, and 120% of their contents, respectively. Three test solutions were prepared in parallel for each concentration.

[0078]

[0079]

[0080] 3.5 Intermediate Precision Test

[0081] The study investigated the determination of the same batch of test sample (batch number: 250501) by different analysts at different times using different instruments. The results are shown in Table 10. There was no significant difference in the content results, indicating that the method has good intermediate precision.

[0082]

[0083] 3.6 Intermediate precision (stability of the test solution)

[0084] Three test solutions were prepared according to the sample solution processing method. The test solutions were dispensed into vials, sealed with sealing film, and placed in the sample tray of the liquid chromatograph. The control temperature was set to 15℃, and the samples were injected after the set time. The content results of each injection were compared with the content results at 0h. A relative deviation exceeding 2.0% was considered unqualified. The results are shown in Table 11. The data show that under the conditions of this invention, the content deviations of the test solutions at 24h and 0h were all within 2.0%.

[0085]

Claims

1. A method for detecting the content of effective components in Shangtong tincture, characterized in that: The detection method is as follows: the test solution is prepared using ethanol solution as solvent, and injected into a liquid chromatograph to determine the contents of bergenin and paeonol; the chromatographic conditions are: octadecylsilane-bonded silica gel as the stationary phase; Gradient elution was performed using 0.1% phosphoric acid solution and acetonitrile as the mobile phase; the column temperature was 30–40℃; the detection wavelength was 265–280 nm; and the flow rate was 0.8–1.2 mL / min.

2. The content detection method of claim 1, wherein: The gradient elution conditions for the mobile phase are as follows: The time was 0–6 min, the mobile phase was 0.1% phosphoric acid solution with a concentration of 94–90%, and the mobile phase was acetonitrile with a concentration of 6–10%. The time is 6–10 min, the mobile phase is 0.1% phosphoric acid solution with a concentration of 90–84%, and the mobile phase is acetonitrile with a concentration of 10–16%. The time was 10–30 min, the mobile phase was 0.1% phosphoric acid solution with a concentration of 84–68%, and the mobile phase was acetonitrile with a concentration of 16–32%. The time is 30-40 min, the mobile phase is 0.1% phosphoric acid solution with a concentration of 68-35%, and the mobile phase is acetonitrile with a concentration of 32-65%. The time was 40.1–45.1 min, the mobile phase consisted of 94% 0.1% phosphoric acid solution and 6% acetonitrile.

3. The content detection method of claim 2, wherein: The test solution is prepared by taking 4-6 mL of pain tincture, diluting it to 100 mL with ethanol solution of 70-85% (v / v), filtering, and collecting the filtrate.

4. The content detection method according to claim 3, characterized in that: The test solution is prepared by taking 5 mL of pain tincture, diluting it to 100 mL with 80% ethanol solution, filtering, and collecting the filtrate.

5. The content detection method of claim 1, wherein: The column temperature was 30℃; the detection wavelength was 274nm; and the flow rate was 1.0mL / min.

6. The content detection method of claim 1, wherein: The detection method also includes the preparation of a reference solution.

7. The content detection method of claim 6, wherein: The reference solution is prepared by accurately weighing bergenin reference standard and paeonol reference standard, adding methanol to prepare a mixed solution containing 20 μg of each per 1 mL.

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