A quality control method for Shenshitong granules based on one-test-multiple-evaluation method

Abstract

The present invention belongs to the technical field of drug quality detection, and specifically relates to a quality control method for Shenshitong granules based on a one-test-multiple-evaluation method. The present invention uses a one-test-multiple-evaluation method to establish a method for simultaneously determining the content of three main components (Vaccaria flavonoid glycosides, salvianolic acid A, and salvianolic acid B) of two herbs (Vaccaria flavonoid glycosides, Salvia miltiorrhiza) in Shenshitong granules. When the content of Vaccaria flavonoid glycosides in Shenshitong granules is known, the content of salvianolic acid A and salvianolic acid B can be directly calculated based on relative correction factors. The present invention can simultaneously measure the content of the three main components in Shenshitong granules using only one component reference substance, Vaccaria flavonoid glycosides, thereby greatly reducing the use of reference substances and reducing detection costs. At the same time, detecting the content of multiple main components can more accurately reflect the quality of the preparation and is more conducive to quality control.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug quality detection, and in particular relates to a quality control method for Shenshitong granules based on a one-test-multiple-evaluation method. Background Art

[0002] Shenshitong Granules are composed of Lysimachia chinensis, Corydalis yanhusuo (prepared with vinegar), Gallus gallus domestica (scalded), Vaccaria segetalis (fried), Polygonum multiflorum, Dianthus chinensis, Achyranthes bidentata, Lygodium japonicum, Salvia miltiorrhiza, and Aucklandia lappa. They are known to clear heat and dampness, promote blood circulation and relieve pain, dissolve stones, and expel calculi. They are used to treat kidney stones, renal pelvic stones, bladder stones, and ureteral stones. The quality standard for Shenshitong Granules is included in the "Drug Standards of the Ministry of Health of the People's Republic of China - Traditional Chinese Medicine Formulas," which only covers properties and inspection items and does not specify the content of the main ingredients.

[0003] For the quality control of traditional Chinese medicine compound preparations, thin-layer chromatography and high-performance liquid chromatography (HPLC) are currently used for qualitative and quantitative identification of active ingredients in individual herbs. However, these methods cannot fully reflect the chemical components and their relative content in Shenshitong Granules. The single-test, multiple-evaluation method, proposed by Wang Zhimin, effectively addresses the lack of reference materials by measuring the content of a single representative ingredient and then determining the contents of multiple ingredients based on relative correction factors. This method can effectively address the lack of reference materials, reduce testing costs, and thus facilitate the quality control of multiple ingredients in traditional Chinese medicine and compound preparations. Currently, no quality control method exists that simultaneously measures the contents of multiple ingredients in Shenshitong Granules using the single-test, multiple-evaluation method. Existing methods often measure only one or two ingredients, lacking representativeness and making it difficult to effectively reflect the overall quality of the preparation. Alternatively, they require the use of multiple reference materials, which is costly. Therefore, it is necessary to develop a quality control method for Shenshitong Granules based on the single-test, multiple-evaluation method to more effectively reflect the overall quality of the preparation and reduce testing costs. Summary of the Invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the present invention proposes a quality control method for Shenshitong granules based on the one-test-multiple-evaluation method. This method can simultaneously obtain the contents of the three main components of the two herbs (Vaccaria segetalis and Salvia miltiorrhiza) in Shenshitong granules while saving reference substances and reducing quality control costs. It is more representative and can more accurately reflect the quality of the preparation.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a method for detecting Shenshitong granules based on a one-test-multiple-evaluation method, comprising the following steps:

[0007] S1. Dissolve Shenshitong granules in 75% methanol to make a 14-16 g / 50 mL solution to obtain the test solution.

[0008] S2. Dissolve the flavonoid glycosides of Vaccaria officinalis in methanol to prepare a reference stock solution of 390-410 μg / mL, and dilute it to 125-130 μg / mL before use to obtain the reference solution;

[0009] S3. The test solution and the reference solution were respectively injected for high performance liquid chromatography (HPLC) determination, and after calculating the peak area, the content of the flavonoid glycosides of Vaccaria segetalis was calculated using the external standard method;

[0010] S4. Calculate the contents of salvianolic acid B and salvianolic acid A, respectively, according to the formula fsi = fs / fi = (AS / wS) / (Ai / wi); wherein fsi is the relative correction factor, AS is the peak area of ​​vaccariae flavonoid glycosides in the test sample, wS is the concentration of vaccariae flavonoid glycosides in the test sample, Ai is the peak area of ​​a certain component to be tested in the test sample, and wi is the concentration of a certain component to be tested in the test sample; the fsi of salvianolic acid B and salvianolic acid A are 2.347 and 0.930, respectively.

[0011] The present invention establishes a method for detecting Shenshitong granules using a one-test, multiple-evaluation method. When the content of vaccaria flavonoid glycosides in Shenshitong granules is known, the contents of salvianolic acid A and salvianolic acid B can be directly calculated based on relative correction factors, thereby enabling the determination of the three main components (vaccaria flavonoid glycosides, salvianolic acid A, and salvianolic acid B) of the two medicinal herbs (vaccaria flavonoid glycosides and salvia miltiorrhiza) in Shenshitong granules. While saving reference substances and reducing quality control costs, the present invention can simultaneously determine the contents of the three main components in Shenshitong granules, providing a more representative and accurate reflection of the quality of the preparation, and has broad application prospects.

[0012] Preferably, the chromatographic column used for the high performance liquid chromatography determination is ULtimate XB-C18, with specifications of 4.6×250 mm and 5 μm.

[0013] Preferably, the injection volume of the high performance liquid chromatography determination is 15-25 μL, and the detection wavelength is 350-370 nm.

[0014] Preferably, the flow rate of the HPLC determination is 0.9-1 mL / min, more preferably 1 mL / min.

[0015] Preferably, the column temperature of the HPLC determination is 25-30° C. More preferably, the column temperature is 30° C.

[0016] Preferably, the mobile phase for the high performance liquid chromatography determination is: acetonitrile (B)-0.2% phosphoric acid aqueous solution (A), and the gradient elution program is: 0-30 min 5% B-20% B, 95% A-80% A; 30-60 min 20%-45% B, 80% A-55% A; 60-65 min 45%-5% B, 55% A-95% A; 65-70 min 5% B, 95% A.

[0017] Preferably, in step S1, the Shenshitong granules are dissolved in 75% methanol by ultrasound, and the ultrasound time is 45-60 minutes, and more preferably the ultrasound time is 60 minutes.

[0018] Preferably, the test solution is filtered through a 0.22-0.45 μm microporous membrane before being subjected to high performance liquid chromatography.

[0019] The second aspect of the present invention provides the application of the Shenshitong granule detection method described in the first aspect in the quality control of Shenshitong granules.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] Most existing quality control methods for Shenshitong granules only measure the content of one or two components in Shenshitong granules, lacking representativeness and making it difficult to effectively reflect the overall quality of the preparation. To this end, the present invention utilizes a one-measurement-multiple-evaluation method to establish a method for simultaneously measuring the three main components (vabuliuxing flavonoid glycosides, salvianolic acid A, and salvianolic acid B) of two herbs (Vaccaria segetalis and Salvia miltiorrhiza) in Shenshitong granules. When the content of vabuliuxing flavonoid glycosides in Shenshitong granules is known, the contents of salvianolic acid A and salvianolic acid B can be directly calculated based on relative correction factors. The present invention can simultaneously measure the contents of the three main components in Shenshitong granules using only one component reference substance, vabuliuxing flavonoid glycosides, greatly reducing the use of reference substances and reducing testing costs. At the same time, detecting the contents of multiple main components can more accurately reflect the quality of the preparation and is more conducive to quality control. In addition, the present invention also measures the salvianolic acid A component, but there is currently no method for measuring the content of salvianolic acid A in Shenshitong granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the specificity test results (from top to bottom in the figure: fenugreek flavonoid glycosides, salvianolic acid A, salvianolic acid B, mixed standard, Shenshitong granules, and methanol). DETAILED DESCRIPTION

[0023] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0025] Example: Construction of a quality control method for Shenshitong granules based on the one-test-multiple-evaluation method

[0026] 1. Experimental Materials

[0027] (1) Instruments: High performance liquid chromatograph (Waters e2695, with 2998PDA Detector), chromatographic column (Welch XB-C18, 4.6×250 mm, 5 μm), 100,000th analytical balance.

[0028] (2) Reagents: Vaccaria segetalis flavonoid glycosides (Aladdin), salvianolic acid B, salvianolic acid A (McLean) reference substances, ultrapure water, Shenshitong granules (230602), produced by Guangdong Zaitian Pharmaceutical Co., Ltd.

[0029] 2. Preparation of test solution

[0030] (1) Prepare 75% methanol solution.

[0031] (2) Preparation of reference solution: Accurately weigh the reference substances Vaccariae flavonoid glycosides (protect from light, actual weight 9.99 mg), salvianolic acid B (actual weight 9.96 mg), and salvianolic acid A (10.04 mg), dissolve them in methanol (analytical grade), and dilute to volume in a 25 mL volumetric flask to prepare the reference stock solution. Store at 4°C. The concentration of each solution is approximately 400 μg / mL. Before use, take 8 mL of each solution and add it to a 25 mL volumetric flask and dilute to volume with methanol (analytical grade) to obtain a mixed standard solution with a concentration of approximately 128 μg / mL.

[0032] (3) Preparation of test solution: Take an appropriate amount of Shenshitong granules, grind them into powder, accurately weigh about 15 g, place it in a 250 mL stoppered conical flask, accurately add 50 mL of 75% methanol, accurately weigh the weight, ultrasonicate for 60 min, let it stand and cool to room temperature, weigh it again, make up the weight with 75% methanol, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution.

[0033] 3. HPLC chromatography analysis

[0034] HPLC analysis was performed by injecting the test solution and the reference solution under the following chromatographic conditions: ULtimate XB-C18 column (4.6×250 mm, 5 μm), mobile phase: acetonitrile (B)-0.2% phosphoric acid aqueous solution (A), flow rate: 1.0 mL / min, column temperature: 30°C, injection volume: 20 μL, detection wavelength: 360 nm. The elution gradient program is shown in Table 1.

[0035] Table 1 Gradient elution program

[0036]

[0037] 4. Methodological Investigation

[0038] (1) Specificity

[0039] According to the HPLC chromatographic analysis conditions of step 3, the chromatograms were obtained. Figure 1 As shown in the figure, from top to bottom, they are: Vaccaria flavonoid glycosides, salvianolic acid A, salvianolic acid B, mixed standard, Shenshitong granules, and methanol. It can be seen that the separation and tailing factors of each component meet the requirements.

[0040] (2) Linear

[0041] The mixed standard was analyzed under the HPLC chromatographic conditions of step 3, with injection volumes of 20 μL, 15 μL, 10 μL, 5 μL, 2.5 μL, and 1 μL, respectively. The peak area (Y) was used to regress the injection volume of the measured component (X, μg / mL).

[0042] According to Table 2, regression analysis was performed to obtain the regression equations of flavonoid glycosides of Vaccaria segetalis, salvianolic acid A, and salvianolic acid B, respectively: y = 955248x + 139803 (R 2 =0.9992);y=410379x+56573(R 2 =0.9995);y=1000000x+142592(R 2 =0.9996).

[0043] Table 2 Peak area (Y) and injection volume (X, μg / mL) for regression analysis

[0044]

[0045]

[0046] (3) Precision

[0047] The mixed standard solution was injected and measured 6 times, the average peak area of ​​each component was measured (Table 3), and the RSD (relative standard deviation) was calculated (n=6).

[0048] Finally, the RSD of flavonoid glycosides from Vaccaria segetalis was 0.37%, the RSD of salvianolic acid B was 0.35%, and the RSD of salvianolic acid A was 0.50%, indicating that the method of the present invention has good precision.

[0049] Table 3 Peak area of ​​each component

[0050]

[0051] (4) Stability

[0052] The test solution (batch number 230602) was allowed to stand for 0, 2, 4, 6, and 8 hours. The content of each component was determined, and the peak areas were recorded (Table 4). The RSDs for vaccariae flavonoid glycosides were 1.30%, 0.68% for salvianolic acid B, and 1.64% for salvianolic acid A. This indicates that the content of each component remained essentially stable within 8 hours.

[0053] Table 4 Peak area of ​​each component

[0054]

[0055]

[0056] (5) Repeatability

[0057] Six test sample solutions were prepared in parallel from the same batch of test samples (batch number: 230602) for the experiment. The content of each component was determined, the peak area was recorded (Table 5), and the RSD was calculated.

[0058] Finally, the RSD of flavonoid glycosides from Vaccaria segetalis was 2.20%, the RSD of salvianolic acid B was 1.18%, and the RSD of salvianolic acid A was 0.95%, indicating that the method of the present invention has good repeatability.

[0059] Table 5 Peak area of ​​each component

[0060] Vaccaria segetalis flavonoid glycosides Peak area Salvianolic acid B Peak area Salvianolic acid A Peak area 1 162245 1 343271 1 470737 2 161374 2 339393 2 473685 3 162891 3 334643 3 469544 4 158693 4 333509 4 460798 5 153559 5 339301 5 471426 6 161844 6 342518 6 470382

[0061] (6) Spike recovery

[0062] Accurately weigh about 15 g of the sample from the same batch (batch number: 230602) with a known content (calculated in the above experiment), a total of 6 portions, and place them in a 250 mL stoppered conical flask. Accurately add the reference solution according to the addition values ​​in Tables 6-8, make up to 50 mL with 75% methanol, accurately weigh the weight, ultrasonicate for 60 minutes, let it stand and cool to room temperature, weigh again, make up to the weight with 75% methanol, shake well, filter through a 0.22 μm filter membrane, take the filtrate, and analyze it according to the above HPLC chromatographic conditions to calculate the recovery rate.

[0063] Finally, the average recovery rates of flavonoid glycosides from Vaccaria segetalis were 93.19%, the average recovery rates of salvianolic acid B were 99.34%, and the average recovery rates of salvianolic acid A were 98.23%, indicating that the method of the present invention has good accuracy and precision.

[0064] Table 6: Recovery rate of spiked flavonoid glycosides of Vaccaria segetalis

[0065]

[0066]

[0067] Table 7: Data on recovery of spiked salvianolic acid B

[0068]

[0069] Table 8: Recovery of salvianolic acid A

[0070]

[0071]

[0072] 5. Correction factor calculation

[0073] The linear relationship (Table 2) was used to investigate the chromatograms obtained after analysis of 6 different mixed standard injection volumes. The relative correction factors fsi between salvianolic acid B, salvianolic acid A, and vaccariae flavonoid glycosides were calculated according to the formula fsi = fs / fi = (AS / wS) / (Ai / wi) (where AS is the peak area of ​​the vaccariae flavonoid glycosides reference substance in the mixed standard, wS is the concentration of the vaccariae flavonoid glycosides reference substance in the mixed standard, Ai is the peak area of ​​the reference substance of a certain component to be tested in the mixed standard, and wi is the concentration of the reference substance of a certain component to be tested in the mixed standard).

[0074] The final calculated relative correction factor fsi of salvianolic acid B was 2.347, and the relative correction factor fsi of salvianolic acid A was 0.930 (Table 9).

[0075] Table 9 Relative correction factor fsi

[0076]

[0077] 6. Durability investigation of relative correction factor

[0078] Under the HPLC chromatographic analysis conditions of step 3, the effects of different flow rates and column temperatures on the relative correction factor were also analyzed, including the relative correction factor and relative retention time at 0.9 mL / min and at 25°C. The injections were repeated three times, the peak areas of the three components were recorded, and the RSD values ​​of the relative correction factors were calculated, indicating that different conditions did not significantly affect the relative correction factor (P>0.05). At the same time, a comparison of retention times revealed significant differences (P<0.05), indicating that changes in flow rate and column temperature can cause a certain shift in the relative retention time, but do not affect the relative correction factor.

[0079] Table 10 Relative correction factors at a flow rate of 0.9 mL / min

[0080] fsi 1 2 3 average value RSD (%) Salvianolic acid B 2.347 2.348 2.350 2.348 0.07 Salvianolic acid A 0.931 0.930 0.931 0.931 0.01

[0081] Table 11 Relative retention time at a flow rate of 0.9 mL / min

[0082]

[0083] Table 12 Relative correction factors at 25°C column temperature

[0084]

[0085] Table 13 Relative retention time at 25℃ column temperature

[0086]

[0087] 7. Comparison of the determination results between the external standard method and the single measurement multiple evaluation method

[0088] Five other batches (231002, 230801, 220901, 220801, and 220403) of Shenshitong Granules (produced by Guangdong Zaitian Pharmaceutical Co., Ltd.) were prepared into test solutions, and the content of each tablet was measured separately. First, the content (mg / bag) of vaccaria flavonoid glycosides, salvianolic acid B, and salvianolic acid A in the samples was determined using the external standard method, and then the calculation was performed using the previously constructed single-measurement, multiple-evaluation method. The results are shown in Table 14. The results showed that the two groups of measurement results were similar, with no significant difference, indicating that the single-measurement, multiple-evaluation method constructed in the present invention can be used to simultaneously quantify these three components in Shenshitong Granules. When the one-measurement-multiple-evaluation method of the present invention is used for calculation, the content of vaccaria flavonoid glycosides is quantified using the external standard method of the vaccaria flavonoid glycosides reference substance, and the contents of salvianolic acid B and salvianolic acid A are calculated based on the above-mentioned correction factors. Finally, it is concluded that the contents of the above-mentioned components in the sample should be no less than 0.368 mg / bag (vaccaria flavonoid glycosides); 1.267 mg / bag (salvianolic acid B); and 0.705 mg / bag (salvianolic acid A), respectively.

[0089] Table 14 Determination results of external standard method and one-measurement-multiple-evaluation method

[0090]

[0091] In summary, the present invention successfully constructs a quality control method for Shenshitong granules based on the one-test-multiple-evaluation method, specifically: the test solution and the flavonoid glycoside reference solution are respectively injected according to the following HPLC conditions: ULtimateXB-C18 (4.6×250 mm, 5 μm), mobile phase: acetonitrile (B)-0.2% phosphoric acid aqueous solution (A), flow rate: 1.0 mL / min, column temperature: 30°C, injection volume: 20 μL, detection wavelength: 360 nm; gradient elution program: 0-30 min 5% B-20% B, 95% A-80% A; 30-60 min 20%-45% B, 80% A-55% A; 60-65 min 45%-5% B, 55% A-95% A; 65-70 min 5% B, 95% A. The peak area is calculated, and the content of vaccaria flavonoid glycosides is calculated by the external standard method. Then, according to the formula fsi=fs / fi=(AS / wS) / (Ai / wi), the contents of salvianolic acid B and salvianolic acid A are calculated respectively. Among them, AS is the peak area of ​​vaccaria flavonoid glycosides in the test sample, wS is the concentration of vaccaria flavonoid glycosides in the test sample, Ai is the peak area of ​​a certain component to be measured in the test sample, and wi is the concentration of a certain component to be measured in the test sample; the relative correction factors of salvianolic acid B and salvianolic acid A are 2.347 and 0.930, respectively. By the method of the present invention, the contents of the three main components (vaccaria flavonoid glycosides, salvianolic acid A, and salvianolic acid B) in Shenshitong granules can be simultaneously measured when only the vaccaria flavonoid glycosides reference substance is used, which can save the use of reference substances and reduce quality control costs. At the same time, the contents of multiple main components can more accurately reflect the quality of the preparation.

[0092] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A method for detecting Shenshitong granules based on a one-test-multiple-evaluation method, characterized in that: The following steps are involved: S1. Dissolve Shenshitong granules in 75% methanol by ultrasonication to make a 14-16 g / 50 mL solution for 60 min to obtain the test solution. S2. Dissolve the flavonoid glycosides of Vaccaria officinalis in methanol to prepare a 390-410 μg / mL reference substance stock solution, and dilute it to 125-130 μg / mL before use to obtain the reference substance solution; S3. The test solution and the reference solution were respectively injected for high performance liquid chromatography determination. After calculating the peak area, the content of flavonoid glycosides of Vaccaria segetalis was calculated using the external standard method. The chromatographic column used for the high performance liquid chromatography determination was ULtimateXB-C18, with a specification of 4.6×250 mm, 5 μm, and a detection wavelength of 360 nm. The mobile phase for the high performance liquid chromatography determination was acetonitrile (B)-0.2% phosphoric acid aqueous solution (A), and the gradient elution program was: 0-30 min 5%B-20%B, 95%A-80%A; 30-60 min 20%-45%B, 80%A-55%A; 60-65 min 45%-5%B, 55%A-95%A; 65-70 min 5%B, 95%A; S4. Calculate the contents of salvianolic acid B and salvianolic acid A according to the formula fsi=fs / fi=(AS / wS) / (Ai / wi); where fsi is the relative correction factor, AS is the peak area of ​​vaccariae flavonoid glycosides in the test sample, wS is the concentration of vaccariae flavonoid glycosides in the test sample, Ai is the peak area of ​​a certain component to be tested in the test sample, and wi is the concentration of a certain component to be tested in the test sample; the fsi of salvianolic acid B and salvianolic acid A are 2.347 and 0.930, respectively.

2. The method for detecting the Shenshitong particles based on the one-test-multiple-evaluation method according to claim 1, wherein: The injection volume of the HPLC assay was 15-25 μL.

3. A method for detecting Shenshitong granules based on a one-test-multiple-evaluation method according to claim 1, characterized in that: The flow rate of the HPLC measurement was 0.9-1 mL / min.

4. The method for detecting the Shenshitong particles based on a one-test-multiple-evaluation method according to claim 1, wherein: The column temperature of the high performance liquid chromatography measurement is 25-30°C.

5. The method for detecting Shenshitong granules based on a one-test-multiple-evaluation method according to claim 1, wherein: The test solution was filtered through a 0.22-0.45 μm microporous membrane before HPLC determination.

6. Application of the Shenshitong granule detection method according to any one of claims 1 to 5 in the quality control of Shenshitong granules.

Images