Method for determining content of tetrahydrogastrodin in shengpiyin soft capsules

Abstract

This invention discloses a method for determining the content of codonopsis glycosides in Shengmai Yin soft capsules, belonging to the field of traditional Chinese medicine preparation quality testing technology. The method employs high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the packing material, methanol-water (40:60) as the mobile phase, a detection wavelength of 267 nm, and a column temperature of 40℃. The test sample is prepared by reflux of 30% methanol for 30 minutes. Method validation showed that codonopsis glycosides exhibited good linearity in the range of 0.02608 μg–0.28688 μg (r = 0.9999). The instrument precision, repeatability, stability, and recovery rate all met the requirements for quantitative analysis, and the negative control showed no interference. This method is highly specific, simple to operate, and provides accurate and reliable results. It enables precise control of the key components of the principal herb, Codonopsis pilosula, overcoming the shortcomings of the original standard which only determined schisandrol A. It is suitable for quality control, production process optimization, and product quality improvement of Shengmai Yin soft capsules.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, and specifically relates to a method for determining the content of codonopsis glycosides in Shengmai Yin soft capsules. Background Technology

[0002] Shengmai Yin Soft Capsules (Codonopsis Formula) are a preparation of Guizhou Didao Pharmaceutical Co., Ltd., and are a unique product nationwide. The formula of this product consists of Codonopsis, Ophiopogon japonicus, and Schisandra chinensis, and has the effects of invigorating qi, nourishing yin and promoting body fluid. It is used for deficiency of both qi and yin, palpitations, shortness of breath, and spontaneous sweating.

[0003] Existing quality standards only use schisandrol A as the content determination indicator, which is a single component and does not quantitatively control the characteristic effective components of the principal herb, Codonopsis pilosula, making it difficult to comprehensively reflect the intrinsic quality and process stability of the preparation. Codonopsis pilosula glycosides are specific indicator components of Codonopsis pilosula, directly related to the qi-tonifying and yin-nourishing effects of the preparation, and can be used as key markers for quality control.

[0004] Currently, there is no specific, accurate, and stable HPLC method for determining the content of codonopsis glycosides in Shengmai Yin soft capsules, which cannot meet the needs of quality improvement during production process optimization and post-marketing changes. According to the "Administrative Measures for Post-Marketing Changes of Drugs (Trial Implementation)," there is an urgent need to establish a content determination method that can accurately control the principal drug component, is easy to operate, and is reliable, in order to improve quality standards and achieve full life-cycle management of drugs. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining the content of codonopsis glycosides in Shengmai Yin soft capsules, filling the gap in the quality standard of Shengmai Yin soft capsules which does not include the determination of codonopsis glycoside content, thus improving the quality standard and enabling precise monitoring of key active ingredients.

[0006] To achieve the above objectives, the core technical solution adopted by this invention is as follows: A method for determining the content of codonopsis glycosides in Shengmai Yin soft capsules, using high performance liquid chromatography, includes the following steps: 1) Preparation of test solution: Take the contents of Shengmai Yin soft capsules, mix well, accurately weigh 1-5g, place in a stoppered conical flask, accurately add 15mL of 10%-70% methanol solution, weigh, heat under reflux for 15-60min, cool, replenish the lost weight with 10%-70% methanol solution, shake well, and filter; accurately measure 25mL of the filtrate, evaporate to dryness, dissolve the residue in 2mL of methanol, add to a neutral alumina solid phase extraction column, elute with methanol and 10%-70% methanol solution in sequence, collect the eluent and evaporate to dryness, add methanol to the residue to make up to 5mL, filter through a 0.45μm filter membrane, and take the filtrate to obtain the test solution; 2) Preparation of reference solution: Take codonopsis glycoside reference standard and add methanol to prepare a solution containing 26 μg per 1 mL; 3) Chromatographic conditions: The column was filled with octadecylsilane-bonded silica gel; the mobile phase was methanol-water at a volume ratio of 40:60; the detection wavelength was 267 nm; the column temperature was 40 °C; and the theoretical plate number, calculated based on the codonopsis glycoside peak, was not less than 3000. 4) Determination: Accurately pipette the reference solution and the test solution into the liquid chromatograph, determine and calculate the content.

[0007] Preferably, the contents of the Shengmai Yin soft capsules of the present invention are weighed in the amount of 3g.

[0008] Preferably, the methanol solution with a volume fraction of 10%-70% in step 1) of the present invention is a methanol solution with a volume fraction of 20%-50%.

[0009] Preferably, the methanol solution with a volume fraction of 20%-50% is a methanol solution with a volume fraction of 30%-40%.

[0010] Preferably, the methanol solution with a volume fraction of 30%-40% is a methanol solution with a volume fraction of 30%.

[0011] Preferably, the heating reflux time in step 1) of the present invention is 20-45 min.

[0012] Preferably, the heating reflux time in step 1) of the present invention is 30-45 min.

[0013] Preferably, the heating reflux time in step 1) of the present invention is 30 min.

[0014] The codonopsis glycosides of this invention showed a good linear relationship between the injection amount and the peak area in the range of 0.02608μg-0.28688μg, with the regression equation being Y=1603727.5416X-361.4524 and r=0.9999.

[0015] The method for determining the content of codonopsis glycosides in Shengmai Yin soft capsules of the present invention is applicable to the quality evaluation and process screening of Shengmai Yin soft capsules produced by different processes.

[0016] The beneficial effects of this invention are: 1. This invention establishes for the first time a method for determining the content of codonopsis glycosides in Shengmai Yin soft capsules, filling the gap in the quality standard of Shengmai Yin soft capsules which lacks a codonopsis glycoside content determination item, improving the quality control system, and achieving precise monitoring of key active ingredients.

[0017] 2. Good linear relationship: In the range of 0.02608-0.28688 μg, the regression equation of codonopsis glycosides is Y=1603727.5416X-361.4524, r=0.9999.

[0018] 3. High specificity: No interference from negative controls, symmetrical peak shape, and resolution meets quantitative requirements.

[0019] 4. High precision: The instrument's precision RSD is 1.0%.

[0020] 5. Good repeatability: The average content of the sample was 0.04717 mg / g, and the RSD was 0.8%.

[0021] 6. Good stability: The test solution is stable at room temperature for 10 hours, with an RSD of 0.6%.

[0022] 7. High accuracy: average recovery rate 100.8%, RSD=1.3%. Attached Figure Description

[0023] Figure 1 High-performance liquid chromatogram of Codonopsis pilosula glycosides; A. Negative chromatogram of Codonopsis pilosula (without Codonopsis pilosula); B. Codonopsis pilosula glycosides reference standard chromatogram; C. Sample image of Shengmai Yin soft capsules.

[0024] Figure 2 High-performance liquid chromatogram of ultrasonic extraction for 30 min.

[0025] Figure 3 High-performance liquid chromatogram after reflux extraction for 20 min.

[0026] Figure 4 High-performance liquid chromatogram after reflux extraction for 30 min.

[0027] Figure 5 High-performance liquid chromatogram after reflux extraction for 45 min.

[0028] Figure 6 High-performance liquid chromatogram after reflux extraction for 60 min. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0030] The following examples illustrate the instruments, reagents, and reagents used: 1. Main instruments used in the experiment: High performance liquid chromatograph: Shimadzu LC-20A high performance liquid chromatograph; Electronic balance AUW-220D (Shimadzu, Japan); Electronic balance AB135-S (Mettler); HH-4 digital display constant temperature water bath (Changzhou Putian Instrument Manufacturing Co., Ltd.); Ultrasonic cleaner (Shanghai Shangyi).

[0031] 2. Main reagents used in the experiment: Shengmai Yin soft capsules (batch numbers: 20240101, 20240501, 20240502, 20240503, 20240504, 20240505, 20240506, 20240507, 20240508, 20240509) are provided by Guizhou Didao Pharmaceutical Co., Ltd.

[0032] 3. Main reagents used in the experiment: Codonopsis glycoside reference standard (batch number: 111732-201908) was provided by the National Institutes for Food and Drug Control. Methanol and ethanol (Chongqing Chuandong Chemical (Group) Co., Ltd.) were analytical grade; methanol (Thermo Fisher Scientific Co., Ltd.) was chromatographic grade; water (Wahaha purified water), etc.

[0033] Example 1: Determination of Codonopsis glycoside content in Shengmai Yin soft capsules Step 1: Preparation of the test solution for Shengmai Yin soft capsules Take the contents of this product from the "Variation in Content" section, mix well, accurately weigh 3 g, place in a stoppered conical flask, accurately add 50 mL of 30% methanol, weigh, heat under reflux for 30 minutes, cool, replenish the lost weight with 30% methanol, shake well, and filter; accurately measure 25 mL of the filtrate, evaporate to dryness, dissolve the residue in 2 mL of methanol, add to a neutral alumina solid-phase extraction column, elute with methanol and 30% methanol sequentially, collect the eluent and evaporate to dryness, add methanol to the residue to a final volume of 5 mL, filter through a 0.45 μm filter membrane, and collect the filtrate to obtain the final product.

[0034] Step 2: Preparation of the reference solution Take an appropriate amount of Codonopsis pilosula glycoside reference standard, accurately weigh it, and add methanol to prepare a solution containing 26 μg per ml.

[0035] Step 3, Chromatographic and Mass Spectrometric Conditions Chromatographic column: octadecylsilane-bonded silica gel as packing material; methanol-water volume ratio of 40:60 as mobile phase; detection wavelength of 267 nm; column temperature of 40℃; theoretical plate number calculated based on the codonopsis glycoside peak of not less than 3000; Step 4, Content determination Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, determine the concentration, and record the chromatogram. (See...) Figure 4 .

[0036] Example 2: Determination of Codonopsis glycoside content in Shengmai Yin soft capsules The difference between Example 2 and Example 1 is that in the sample pretreatment step of this example, the solvent is pure methanol, while the rest are the same.

[0037] Example 3: Determination of Codonopsis glycoside content in Shengmai Yin soft capsules The difference between Example 3 and Example 1 is that in the sample pretreatment step of this example, the solvent is 70% methanol, while the rest are the same.

[0038] Example 4: Determination of Codonopsis glycoside content in Shengmai Yin soft capsules The difference between Example 4 and Example 1 is that in the sample pretreatment step of this example, the solvent is ethanol, while the rest are the same.

[0039] Comparing the chromatographic effects of Examples 1 to 4, the results are as follows: Using pure methanol as the solvent system (Example 2): Severe interference from impurity peaks resulted in high baseline noise. Using 70% methanol as the solvent system (Example 3): Fewer response peaks and a low target peak response value. Using ethanol as the solvent system (Example 4): Numerous impurity peaks and a low target peak response value. Compared to the pure methanol, 70% methanol, and ethanol solvent systems, Example 1, using 30% methanol (v / v) as the extraction and elution solvent, showed the best detection performance. The sample solution exhibited symmetrical peak shapes, no impurity peak interference, a stable baseline, and good theoretical plate number and resolution. Therefore, 30% methanol (v / v) is the optimal extraction and elution solvent and is suitable as the pretreatment solvent for the determination of the content of Codonopsis pilosula glycosides in this variety.

[0040] Example 5

[0041] The difference between Example 5 and Example 1 is that in the sample pretreatment step, 1g of the contents is taken, while the rest are the same.

[0042] Example 6

[0043] The difference between Example 6 and Example 1 is that in the sample pretreatment step, 5g of the contents is taken, while the rest are the same.

[0044] Based on the response peaks and the number of interference peaks near codonopsis glycosides in Examples 1, 5, and 6, when the sample amount is 3g, the response peaks are better than those in Example 1 (sample amount 1g), and the number of interference peaks is less than that in Example 6.

[0045] Example 7

[0046] Accurately weigh approximately 3g of the contents of Shengmai Yin soft capsules (batch number: 20240101), place them in a stoppered conical flask, accurately add 15mL of 30% methanol, seal tightly, weigh, and sonicate (250W power, 40kHz frequency) for 30 minutes. Cool, weigh again, and replenish the lost weight with 30% methanol. Shake well and filter. Accurately measure 25mL of the filtrate, evaporate to dryness, dissolve the residue in 2mL of methanol, and add to a neutral alumina solid-phase extraction column. Elute sequentially with methanol and 30% methanol, collect the eluent and evaporate to dryness, dilute the residue to 5mL with methanol, filter through a 0.45 μm filter membrane, and collect the filtrate to obtain the test solution. Accurately pipette 10µL of the codonopsis glycoside reference solution and the test solution, inject into the liquid chromatograph, and determine the chromatogram according to the chromatographic conditions under "Example 1". See [link to chromatographic description]. Figure 2 .

[0047] The content determination results of Examples 1 and 7 are shown in Table 1: ; As shown in Table 1, the content of codonopsis glycosides extracted by reflux extraction is higher than that extracted by ultrasound, therefore reflux extraction was chosen as the extraction method.

[0048] Example 8

[0049] The difference between Example 8 and Example 1 is that in this example, the sample pretreatment step involves reflux for 20 minutes; all other steps are the same. See the chromatogram below. Figure 3 .

[0050] Example 9

[0051] The difference between Example 9 and Example 1 is that in this example, the sample pretreatment step involves reflux for 45 minutes; all other steps are the same. See the chromatogram below. Figure 5 .

[0052] Example 10

[0053] The difference between Example 10 and Example 1 is that in this example, the sample pretreatment step involves reflux for 60 min; all other steps are the same. See the chromatogram below. Figure 6 .

[0054] The measurement results obtained from Examples 1 and 8 to 10 are shown in Table 2: ; Based on the content values ​​of codonopsis glycosides in Examples 1, 8 to 10, the content of codonopsis glycosides after reflux extraction for 20 min is significantly lower than that after reflux extraction for 30 min, but the content at 30 min, 45 min and 60 min is not significantly different. Therefore, the preferred extraction time is 30 min.

[0055] In summary, Embodiment 1 of the present invention is a preferred embodiment, and the following methodological investigation test was conducted on its determination method.

[0056] Experimental Example 1: Specificity Test Take the contents of Shengmai Yin soft capsules (batch number: 20240101) and negative samples, and prepare sample solutions and negative sample solutions according to "Example 1". According to the chromatographic conditions of Example 1, inject the mixed reference solution, sample solution and negative sample solution into the liquid chromatograph respectively.

[0057] Results: The sample solution chromatogram showed corresponding peaks at the same positions as the reference solution chromatogram, with no negative interference, as indicated by specific chromatogram. Figure 1 .

[0058] Experimental Example 2: Examination of Linear Relationship Under the chromatographic conditions described in "Example 1", 1 μL, 3 μL, 5 μL, 7 μL, 9 μL, and 11 μL of the codonopsis glycoside reference solution were accurately pipetted into the liquid chromatograph sequentially, and the peak area was measured. Linear regression was performed with the injection volume X (μg) as the abscissa and the peak area Y as the ordinate.

[0059] The regression equation was obtained as: Y = 1603727.5416X - 361.4524, r = 0.9999. The results show that the injection amount of codonopsis glycosides has a good linear relationship with the peak area in the range of 0.02608 μg-0.28688 μg.

[0060] Test Example 3, Precision Test Accurately pipette 10 µL of the reference solution from "Example 1" and inject it 6 times under the chromatographic conditions described in "Example 1".

[0061] Results: The RSD of the peak area of ​​codonopsis glycosides was 1.0%, indicating that the instrument precision was good.

[0062] Test Example 4, Stability Test Take approximately 3g of the contents of Shengmai Yin soft capsules (batch number: 20240101) and prepare the test solution according to "Example 1". Inject 10µL of the solution at room temperature for 0, 2, 4, 6, 8 and 10 hours respectively, and determine the peak area and record the chromatogram according to the chromatographic conditions in "Example 1".

[0063] Results: The RSD of the peak area of ​​codonopsis glycosides was 0.6%, indicating that the sample was stable within 10 hours.

[0064] Test Example 5, Repeatability Test Take about 3g of the contents of Shengmai Yin soft capsules (batch number: 20240101), prepare 6 test solutions according to "Example 1", and determine the content of Codonopsis pilosula glycosides according to the chromatographic conditions of "Example 1".

[0065] Results: The average content of the samples was 0.04717 mg / g, and the RSD was 0.8%, indicating that the method has good repeatability.

[0066] Experimental Example 6: Recovery Test Take approximately 1.5g of the contents of Shengmai Yin soft capsules (batch number: 20240101, average content of codonopsis glycosides 0.04717 mg / g), make 6 portions, accurately weigh them, place them in stoppered conical flasks, accurately add 3mL of codonopsis glycoside reference solution (0.02608mg / ml), accurately add 15ml of 30% methanol, prepare the sample solution according to "Example 1", and determine the content of codonopsis glycosides according to the chromatographic conditions of "Example 1", and calculate the recovery rate.

[0067] The results are shown in Table 3. The average recovery rate of codonopsis glycoside was 100.8%, and the RSD was 0.4%, indicating a good recovery rate.

[0068] ; Example 7: Determination of Samples from Different Manufacturing Processes of Shengmai Yin Soft Capsules Nine batches of Shengmai Yin soft capsules were taken, and the content of Codonopsis glycosides was determined according to the method in "Example 1".

[0069] The results are shown in Table 4. The content of codonopsis glycosides was 36-99 μg / capsule.

[0070] ; Test Example 8: Method Durability Test The effects of mobile phase ratio, column temperature, detection wavelength, flow rate, and different chromatographic columns on the determination results were investigated, and the RSD of the peak area of ​​codonopsis glycosides was calculated.

[0071] The results are shown in Table 5. ; Conclusion: Under all conditions, the peak area RSD was less than 2.0%, and the resolution and theoretical plate number met the requirements, indicating that the method of the present invention has good robustness.

[0072] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for determining the content of codonopsis glycosides in Shengmai Yin soft capsules, characterized in that, The determination was performed using high-performance liquid chromatography, including the following steps: 1) Preparation of test solution Take the contents of Shengmai Yin soft capsules, mix well, accurately weigh 1-5g, place in a stoppered conical flask, accurately add 15mL of 10%-70% methanol solution, weigh, heat under reflux for 15-60min, cool, replenish the lost weight with 10%-70% methanol solution, shake well, and filter; accurately measure 25mL of the filtrate, evaporate to dryness, dissolve the residue in 2mL of methanol, add to a neutral alumina solid-phase extraction column, elute successively with methanol and 10%-70% methanol solution, collect the eluent and evaporate to dryness, add methanol to the residue to make up to 5mL, filter through a 0.45μm filter membrane, and collect the filtrate to obtain the product; 2) Preparation of reference solution Prepare a solution containing 26 μg per 1 mL by dissolving a reference standard of codonopsis glycosides in methanol. 3) Chromatographic conditions The column was filled with octadecylsilane-bonded silica gel; the mobile phase was methanol-water at a volume ratio of 40:60; the detection wavelength was 267 nm; the column temperature was 40 °C; and the theoretical plate number, calculated based on the codonopsis glycoside peak, was not less than 3000. 4) Measurement Accurately pipette the reference solution and the test solution into the liquid chromatograph, determine and calculate the content.

2. The content determination method according to claim 1, characterized in that, The contents of the Shengmai Yin soft capsules weigh 3g.

3. The content determination method according to claim 1, characterized in that, The methanol solution with a volume fraction of 10%-70% mentioned in step 1) is a methanol solution with a volume fraction of 20%-50%.

4. The content determination method according to claim 3, characterized in that, The methanol solution with a volume fraction of 20%-50% is a methanol solution with a volume fraction of 30%-40%.

5. The content determination method according to claim 4, characterized in that, The 30%-40% methanol solution is a 30% methanol solution by volume.

6. The content determination method according to claim 1, characterized in that, The heating reflux time mentioned in step 1) is 20-45 min.

7. The content determination method according to claim 6, characterized in that, The heating reflux time mentioned in step 1) is 30-45 min.

8. The content determination method according to claim 7, characterized in that, The heating reflux time mentioned in step 1) is 30 minutes.

9. The content determination method according to claim 1, characterized in that, Codonopsis glycosides showed a good linear relationship with peak area in the range of 0.02608 μg to 0.28688 μg, with the regression equation being Y = 1603727.5416X - 361.4524 and r = 0.9999.

10. The content determination method according to claim 1, characterized in that, This method is applicable to the quality evaluation and process screening of Shengmai Yin soft capsules produced using different processes.

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