Detection method of Wei'an granules

Abstract

The invention provides a method for detecting Wei'an granules, and relates to the technical field of Chinese patent medicine detection. The invention discloses a method for detecting the content of rutin in Wei'an granules, and a method for evaluating the quality of the Wei'an granules is established according to the method. Preferably, high performance liquid chromatography is adopted to detect the rutin content in the Fuean granules, and the requirements of the guiding principle of Chinese pharmacopoeia 9101 are met.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and relates to a detection method for Fu'an granules. Background Art

[0002] Fu'an Granules are a traditional Chinese medicine composed of Agrimony Root, Firewood, Amaranthus Root, Bidens Bilibilis, and Schizonepeta Tenuifolia. They clear heat and detoxify, dry dampness, and stop dysentery. They are used to treat various gastrointestinal conditions, including dysentery, acute gastroenteritis, diarrhea, and abdominal pain. Testing the active ingredients in Fu'an Granules is an effective method for product quality control. However, no methods for testing the active ingredients in Fu'an Granules have been reported. Summary of the Invention

[0003] The ingredients in Fu'an Granules—herb agrimony, radix pyracantha, amaranthus chinensis, and bidens pilosa—all contain rutin, a natural flavonoid glycoside. Applicants have discovered that by testing the rutin content in Fu'an Granules, they can effectively monitor their product quality. Based on this, the present invention provides a method for testing Fu'an Granules.

[0004] The technical solutions of the present invention are as follows:

[0005] A method for detecting Fu'an granules, comprising detecting the content of rutin in the Fu'an granules.

[0006] Preferably, the rutin is extracted using an alcohol solvent or a mixed solvent consisting of an alcohol solvent and water.

[0007] More preferably, the volume proportion of the alcohol solvent in the mixed solvent is not less than 60%.

[0008] Preferably, the detection adopts one of chromatography, electrochemical sensing, spectroscopy and electrophoresis.

[0009] More preferably, the detection is performed using high performance liquid chromatography.

[0010] Further preferably, the mobile phase of the HPLC is a combination of mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.05-0.15% phosphoric acid.

[0011] Still further preferably, the volume ratio of the acetonitrile to the 0.05-0.15% phosphoric acid aqueous solution is 10-18:82-90.

[0012] More preferably, the surface of the liquid chromatography column used in the high performance liquid chromatography is hydrophilic.

[0013] Still further preferably, the liquid chromatography column is selected from ZORBAX SB-Aq liquid chromatography column.

[0014] Further preferably, the chromatographic conditions of the HPLC are: flow rate 0.8-1.2 mL / min, column temperature 25-30° C., and detection wavelength 256 nm.

[0015] The beneficial effects of the present invention are:

[0016] (1) The present invention establishes an effective method for monitoring the product quality of Fu'an granules by detecting the rutin content in Fu'an granules.

[0017] (2) The present invention adopts the method of high performance liquid chromatography to detect the rutin content in Fu'an granules, which has the characteristics of high accuracy, good repeatability, and good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 3 is a comparison chart of the detection results of three different chromatographic columns in the embodiment.

[0019] Figure 2 4 is a graph showing the specificity test results in the embodiments.

[0020] Figure 3 Graph showing the linear relationship between the peak area and injection volume of the rutin reference solution in the examples. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further illustrated and described below through specific implementation methods.

[0022] In order to establish a method for monitoring the quality of Fu'an granules, the present invention proposes a method for detecting Fu'an granules to detect the content of rutin in Fu'an granules.

[0023] Rutin is a natural flavonoid glycoside with the structure shown in the following formula (1). It is a flavonol glycoside widely found in plants and has anti-inflammatory, antioxidant, anti-allergic, and antiviral effects.

[0024]

[0025] Four of the five ingredients in Fu'an granules contain rutin. The applicant has discovered that the quality of Fu'an granules can be monitored by detecting the rutin content in the granules.

[0026] There is no particular limitation on the extraction method of rutin in Fu'an granules. A commonly used method for extracting the active ingredients of traditional Chinese medicine and Chinese patent medicine is solvent extraction, or solvent extraction with the assistance of ultrasound. Due to the high polarity of rutin, a solvent with a higher polarity can be used for extraction. In some embodiments, the extraction method of rutin is to use an alcohol solvent or a mixed solvent consisting of an alcohol solvent and water. For alcohol solvents, methanol, ethanol, isopropanol, etc. can be used. For mixed solvents, a combination of methanol and water, a combination of ethanol and water, a combination of isopropanol and water, etc. can be used.

[0027] In some embodiments, the volume proportion of the alcohol solvent in the above-mentioned mixed solvent is not less than 60%. If the volume proportion of the alcohol solvent is too low, the polarity of the mixed solvent is too high, which will affect the extraction of rutin. For example, the volume proportion of the alcohol solvent in the above-mentioned mixed solvent can be 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc. For example, the mixed solvent can be a combination of methanol and water in a volume ratio of 6:4, a combination of methanol and water in a volume ratio of 7:3, a combination of methanol and water in a volume ratio of 8:2, a combination of ethanol and water in a volume ratio of 6:4, a combination of ethanol and water in a volume ratio of 7:3, a combination of ethanol and water in a volume ratio of 8:2, etc. Preferably, the mixed solvent can be a combination of methanol and water in a volume ratio of 7:3.

[0028] In some embodiments, there is no particular limitation on the method for detecting rutin, and one of chromatography, electrochemical sensing, spectroscopy, and electrophoresis can be used. The electrochemical sensing method is to use an electrochemical sensor for detection, such as the electrochemical sensors and detection methods disclosed in Chinese patents CN113433180A and CN118896986A.

[0029] In some embodiments, rutin is detected by high performance liquid chromatography (HPLC), which has the advantages of fast detection speed, high sensitivity, high separation efficiency, and reduced interference.

[0030] In certain embodiments, the mobile phase of high performance liquid chromatography is a combination of mobile phase A and mobile phase B, mobile phase A is acetonitrile, and mobile phase B is a phosphoric acid aqueous solution with a volume ratio of 0.05-0.15% of phosphoric acid. For the concentration of phosphoric acid, the present invention has no particular restrictions. For example, concentrated phosphoric acid can be used, and the concentration is not less than 85wt%. Rutin has a high molecular polarity and has multiple phenolic hydroxyl groups with certain acidity. The mobile phase of the present invention adopts a combination of mobile phase A and mobile phase B, acetonitrile is mobile phase A, and phosphoric acid aqueous solution is mobile phase B. The mobile phase has a high polarity, and a small amount of phosphoric acid in mobile phase B can inhibit the dissociation of phenolic hydroxyl groups and improve the separation effect. For the volume ratio of phosphoric acid in the phosphoric acid aqueous solution, there is no particular restriction. For example, it can be any value among 0.05%, 0.07%, 0.08%, 0.1%, 0.12%, 0.13%, 0.15%, etc. Further, the volume ratio of phosphoric acid in the phosphoric acid aqueous solution can be 0.1%. There is no particular limitation on the volume ratio of acetonitrile to the aqueous phosphoric acid solution. In some embodiments, the volume ratio may be 10-18:82-90. For example, the volume ratio may be any value of 10:90, 12:88, 13:87, 14:86, 15:85, 16:84, 17:83, 18:82, etc.

[0031] Rutin has a certain acidity in its structure, making separation more difficult. Besides certain requirements for the mobile phase, the chromatographic column also has certain requirements, especially the properties of the column surface (i.e., the surface of the column packing). In some embodiments, the surface of the liquid chromatography column used in high-performance liquid chromatography is hydrophilic, which provides stronger retention than hydrophobic liquid chromatography columns (such as C18 HPLC), and also has good reproducibility and peak shape.

[0032] In some embodiments, the liquid chromatography column is selected from a ZORBAX SB-Aq liquid chromatography column, which is suitable for efficient separation of rutin and suitable for the highly polar, acidic mobile phase used in the present invention.

[0033] In some embodiments, the chromatographic conditions of the high performance liquid chromatography are: a flow rate of 0.8-1.2 mL / min, a column temperature of 25-30° C., and a detection wavelength of 256 nm. For example, the flow rate of the mobile phase can be 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, etc., preferably 0.8 mL / min; the column temperature can be 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., etc., preferably 30° C.

[0034] The technical solution of the present invention is further described and illustrated below based on various embodiments.

[0035] Example

[0036] Rutin extraction: Accurately weigh about 5 g of ground Fu'an granules and add them to a stoppered volumetric flask. Add 25 ml of a mixed solvent consisting of methanol and water in a volume ratio of 7:3. Ultrasonicate (power 500 W, frequency 40 kHz) for 30 minutes, cool to room temperature, weigh the mixture, make up the lost weight with the above mixed solvent, shake well, filter, and obtain the filtrate, which is the rutin extract.

[0037] High performance liquid chromatography was used under the following chromatographic conditions: mobile phase, acetonitrile-0.1% phosphoric acid aqueous solution (the volume ratio of concentrated phosphoric acid in the phosphoric acid aqueous solution was 0.1%) (volume ratio of acetonitrile to phosphoric acid aqueous solution was 14:86); flow rate was 0.8 mL / min, column temperature was 30° C., and detection wavelength was 256 nm.

[0038] Comparison of different chromatographic columns, Agilent 5-HC C18 (2) (packing surface treated with octadecylsilane), Agilent SB-C18 (packing surface treated with octadecylsilane) and Agilent ZORBAX SB-AQ (packing surface treated with hydrophilicity) Figure 1 As shown, Figure a) shows the test results using 5-HC C18, showing that the baseline on the left side of the rutin main peak (gray-filled peak) drifts upward, resulting in poor peak shape; Figure b) shows the test results using SB-C18, showing that the baseline on the left side of the rutin main peak (gray-filled peak) drifts upward, resulting in poor peak shape, and the impurity peak shape on the left side of the main peak is poor; Figure c) shows the test results using SB-AQ, showing that the baseline on the left side of the rutin main peak (gray-filled peak) is stable and meets the peak shape requirements, and the separation of impurities on the left and right of the main peak meets the requirements. Therefore, compared to chromatographic columns using octadecylsilane as the filler surface, the chromatographic columns using hydrophilic filler surface treatment of the present invention are more suitable for HPLC detection of rutin.

[0039] Therefore, the chromatographic conditions for the following test contents in this example are as follows: an Agilent ZORBAX SB-Aq (4.6 mm × 250 mm, 5 μm) column, acetonitrile-0.1% phosphoric acid aqueous solution (the volume ratio of concentrated phosphoric acid in the phosphoric acid aqueous solution is 0.1%) (the volume ratio of acetonitrile to phosphoric acid aqueous solution is 14:86) as the mobile phase; a flow rate of 0.8 mL / min, a column temperature of 30°C, and a detection wavelength of 256 nm.

[0040] Preparation of reference solution: Take an appropriate amount of rutin reference substance, accurately weigh it, add it to a stoppered volumetric flask, and add methanol to make a solution containing 10 μg of rutin per 1 ml.

[0041] Preparation of test solution: Prepare according to the above rutin extraction method.

[0042] Determination: Accurately aspirate 5 μl of reference solution and 10 μl of test solution respectively, inject into high performance liquid chromatography instrument, and determine.

[0043] 1.1 Accuracy test

[0044] For the same sample, 6 portions of 100% solubility were prepared and recovered. The recovery rates were measured and calculated. As shown in Table 1, the average recovery rate was 100.01% and the RSD was 1.93%, indicating that the method of this embodiment has good accuracy.

[0045] Table 1 Sample recovery test results

[0046]

[0047] 1.2 Repeatability test

[0048] Six samples of rutin with the same concentration (0.0025%) were tested, with the rutin content as the test object. The results are shown in Table 2, with an RSD of 1.78%, indicating that the method of this example has good repeatability.

[0049] Table 2 Repeatability test results

[0050]

[0051]

[0052] Six samples of rutin with the same concentration were tested based on the peak area of ​​the chromatogram. The results are shown in Table 3. The RSD was 1.40%, indicating that the method of this embodiment has good repeatability.

[0053] Table 3 Repeatability test results

[0054]

[0055] 1.3 Specificity Test

[0056] 70% methanol was used as blank reagent and treated in the same manner as the rutin reference substance to analyze the specificity of the detection method of this embodiment. The results are shown in the attached figure. Figure 2 As shown in the figure, a) is the liquid chromatography spectrum of 70% methanol, and b) is the liquid chromatography spectrum of rutin reference substance. Figure 2 It can be seen that there is no spectrum at the same retention time position as the rutin reference substance in the blank reagent spectrum, indicating that the detection method of this embodiment has good specificity.

[0057] 1.4 Linearity test of the detection method

[0058] The rutin reference substance solution was tested with different volume injections (corresponding to different rutin weights), and linear regression was performed using peak area and sample size. The results are shown in Table 4 and the accompanying Figure 3 As shown, the linear equation is y = 2178.5x + 1.8773, and the correlation coefficient is R 2 =0.9994, indicating that the detection method of this embodiment has good linearity in the range of rutin content of 0.0000000ug-0.1089531ug.

[0059] Table 4 Linearity test results

[0060]

[0061] 1.5 Range Testing

[0062] Three samples of sample batch 20230501, each at 0.8, 1, and 1.2 times the sample volume, were taken for determination. The results are shown in Table 5 below. The RSD of the content was 1.36%, indicating that the precision of the high and low concentration determinations was good and the linear range was also covered.

[0063] Table 5 Range test results

[0064]

[0065] 1.6 Column durability test

[0066] The designated chromatographic column model is Agilent ZORBAX SB-Aq (4.6 mm × 250 mm, 5 μm). The test results of different batches of chromatographic columns on the same batch of Fu'an granules are shown in Table 6, indicating that the detection method of this embodiment has good durability for the chromatographic column.

[0067] Table 6 Test results of different batches of chromatographic columns

[0068]

[0069] 1.7 Stability test of Fu'an granules

[0070] A portion of Fu'an granules was prepared into a solution according to the above rutin extraction method. Samples were injected and tested 0, 4, 8, 12, 16, and 24 hours after the solution was prepared. The results are shown in Table 7. RSD = 1.85%, indicating that the sample solution was stable within 24 hours.

[0071] Table 7 Solution stability test results

[0072]

[0073]

[0074] Therefore, based on the above tests and results, the accuracy, repeatability, specificity, linearity, range, stability, and durability of the detection method for Fu'an Granules of the present invention all meet the requirements of the "Chinese Pharmacopoeia" 9101 guideline. Therefore, the present invention establishes a method for determining the content of rutin in Fu'an Granules, providing an evaluation basis for the quality standard of Fu'an Granules.

[0075] As described above, the basic principles, main features, and advantages of the present invention are shown and described. Those skilled in the art should understand that the present invention is not limited to the above-described embodiments. The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting Fu'an granules, characterized in that: The content of rutin in the Fu'an granules was detected.

2. The method for detecting Fu'an granules according to claim 1, wherein The rutin extraction method adopts an alcohol solvent or a mixed solvent consisting of an alcohol solvent and water.

3. The method for detecting Fu'an granules according to claim 2, wherein The volume proportion of the alcohol solvent in the mixed solvent is not less than 60%.

4. The method for detecting Fu'an granules according to claim 1, wherein The detection adopts one of chromatography, electrochemical sensing, spectroscopy and electrophoresis.

5. The method for detecting Fu'an granules according to claim 4, wherein: The detection is performed using high performance liquid chromatography.

6. The method for detecting Fu'an granules according to claim 5, characterized in that: The mobile phase of the high performance liquid chromatography is a combination of mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile and the mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.05-0.15% of phosphoric acid.

7. The method for detecting Fu'an granules according to claim 6, characterized in that: The volume ratio of the acetonitrile to the phosphoric acid aqueous solution is 10-18:82-90.

8. The method for detecting Fu'an granules according to claim 5, characterized in that: The surface of the liquid chromatography column used in the high performance liquid chromatography is hydrophilic.

9. The method for detecting Fu'an granules according to claim 8, characterized in that: The liquid chromatography column is selected from ZORBAX SB-Aq liquid chromatography column.

10. The method for detecting Fu'an granules according to claim 5, characterized in that: The chromatographic conditions of the high performance liquid chromatography are: flow rate 0.8-1.2 mL / min, column temperature 25-30° C., and detection wavelength 256 nm.

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