Detection method of traditional Chinese medicine granules

Abstract

The invention discloses a method for detecting traditional Chinese medicine granules, which comprises the following step of: carrying out qualitative identification and / or content determination on exocarpium citri rubrum in the traditional Chinese medicine granules by adopting high performance liquid chromatography, taking octadecylsilane chemically bonded silica as a filling agent and taking hesperidin as an index component. The method is used for carrying out qualitative identification and content determination on exocarpium citri rubrum in the traditional Chinese medicine granules by taking hesperidin as an index component, and is a detection method for the traditional Chinese medicine granules, which is strong in specificity, high in accuracy and good in reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and specifically relates to a detection method for traditional Chinese medicine granules. Background Technology

[0002] Traditional Chinese medicine granules are a new type of TCM preparation made from processed medicinal herbs through modern processes such as extraction, concentration, and granulation. They offer advantages such as accurate dosage, stable properties, and ease of carrying and administration, and have become an important direction for the modernization of TCM. However, compared to chemical drugs with clearly defined components, TCM granules have complex compositions, and the quality of their raw materials is greatly affected by factors such as origin, harvesting season, and processing methods, resulting in significant fluctuations. Therefore, establishing scientific, accurate, and specific quality control methods is a crucial step in ensuring the safety, efficacy, and stable quality of TCM granules, and also represents a technological bottleneck restricting the development of its industry.

[0003] Citrus reticulata peel, a commonly used traditional Chinese medicine, is derived from the outer peel of Citrus reticulata Blanco and its cultivated varieties. It has the effects of regulating qi, relieving chest congestion, and resolving phlegm and dampness. Hesperidin is the main flavonoid component in Citrus reticulata peel and is also an indicator component for evaluating the quality of Citrus reticulata peel in the Chinese Pharmacopoeia. Therefore, establishing a quantitative analytical method using hesperidin as an indicator for compound granules containing Citrus reticulata peel (such as Tongmai Huazhuo Granules) is of great significance for controlling the overall quality of this preparation.

[0004] Currently, high-performance liquid chromatography (HPLC) is commonly used to determine the content of hesperidin in traditional Chinese medicine preparations. However, existing technologies have the following shortcomings:

[0005] First, the method lacks specificity. The components of traditional Chinese medicine compound prescriptions are extremely complex. Some existing HPLC detection methods have poorly selected chromatographic conditions (such as mobile phase composition, elution program, detection wavelength, etc.), which may lead to insufficient separation between the hesperidin chromatographic peak and adjacent impurity peaks, or even co-elution, thereby interfering with the accuracy of qualitative identification and quantitative determination, and failing to truly reflect the quality of tangerine peel used.

[0006] Second, the system suitability requirements are unclear or too low. Many existing methods do not specify clear requirements for the performance of the chromatographic system (such as the theoretical plate number). A low theoretical plate number means insufficient column separation efficiency, making it difficult to achieve baseline separation of hesperidin in complex sample matrices, directly affecting the reliability of analytical results.

[0007] Third, the pretreatment steps are not optimized enough. The extraction solvent, extraction method (such as ultrasound or reflux), and extraction time of the sample solution directly affect the extraction efficiency of hesperidin. Immature methods may lead to incomplete extraction or component destruction, resulting in lower test results and making it impossible to accurately control the lower limit of product content.

[0008] Fourth, there is a lack of complete methodological validation. A reliable quality control method needs to undergo a series of methodological validations, including specificity, linearity, precision, accuracy (recovery rate), and stability. Some methods disclosed in the existing technology often lack sufficient validation data, and their robustness and reliability are questionable, making it difficult to meet the stringent requirements of drug quality standards.

[0009] Therefore, there is an urgent need in this field for an HPLC detection method that is highly specific, sensitive, reproducible, and fully methodologically validated, specifically for detecting citrus red (calculated as hesperidin) in traditional Chinese medicine granules, in order to solve the above-mentioned technical problems and provide reliable technical support for the quality control of related traditional Chinese medicine granules. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a detection method for traditional Chinese medicine granules that is highly specific, accurate, and reproducible. This method is used to qualitatively identify and determine the content of citrus peel in traditional Chinese medicine granules, using hesperidin as an indicator component.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A method for detecting traditional Chinese medicine granules, the method comprising: using high performance liquid chromatography, with octadecylsilane-bonded silica gel as the packing material and hesperidin as the indicator component, to qualitatively identify and / or determine the content of citrus red in the traditional Chinese medicine granules.

[0013] Preferably, in the chromatographic conditions of the high-performance liquid chromatography (HPLC), the mobile phase is an organic phase-acid-water solution, and the detection wavelength is 280-286 nm. Further, the organic phase is acetonitrile or methanol, and the acid-water solution is a 0.05%-0.2% (v / v) phosphoric acid solution or formic acid solution. More preferably, the mobile phase is acetonitrile-0.1% (v / v) phosphoric acid solution, with a volume ratio of (19:81) to (21:79). Most preferably, the volume ratio of acetonitrile to 0.1% (v / v) phosphoric acid solution in the mobile phase is 20:80, and isocratic elution is used.

[0014] Preferably, the detection wavelength is 283nm.

[0015] Preferably, the system suitability requirement for the high performance liquid chromatography method is that the theoretical plate number, calculated based on the hesperidin peak, should be no less than 6000.

[0016] Preferably, the method includes the following steps:

[0017] (1) Preparation of reference solution: Take an appropriate amount of hesperidin reference standard, accurately weigh it, dissolve and dilute it with methanol to prepare a solution containing 45-55 μg per 1 mL;

[0018] (2) Preparation of test solution: Take an appropriate amount of the Chinese herbal medicine granules, grind them into a fine powder, take about 2.0g of the fine powder, weigh it accurately, put it in a stoppered conical flask, add 50mL of methanol accurately, stopper tightly, weigh it, heat and reflux for 30-40min, cool it, weigh it again, make up the lost weight with methanol, shake well, filter it, and take the filtrate.

[0019] (3) Determination method: Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph and determine them.

[0020] Furthermore, the procedure also includes the step of preparing a negative control solution: preparing a negative sample without tangerine peel according to the prescription and process of the traditional Chinese medicine granules, and then preparing a negative control solution in the same way as the test sample solution for specificity testing.

[0021] This invention also provides the application of the above method in the quality detection of Tongmai Huazhuo granules. Preferably, for controlling the quality of Tongmai Huazhuo granules, it is specified that the content of citrus red, calculated as hesperidin (CHO), in each 1g of granules shall not be less than 0.50mg.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. High specificity: The chromatographic conditions provided by this invention enable the hesperidin peak to achieve baseline separation from the chromatographic peaks of other components in the sample, with a resolution greater than 1.5, and there is no interference from the negative control, indicating that the method has good specificity and can accurately identify and determine tangerine peel.

[0024] 2. High accuracy: Through optimized sample pretreatment (heating and reflux extraction) and chromatographic conditions, the target components can be fully extracted and accurately separated. The results of the spiking recovery test show that the average recovery rate is between 98% and 102%, and the RSD is less than 2.0%, proving that the method has extremely high accuracy.

[0025] 3. Good reproducibility: The RSD of the method precision test (intra-day and inter-day) is less than 2.0%, indicating that the method has good stability and high reproducibility, and can meet the requirements of routine quality inspection.

[0026] 4. Clear system applicability requirements: The system clearly stipulates a high standard of no less than 6000 theoretical plates, ensuring that the chromatographic system maintains high separation efficiency throughout the entire analysis process, which is an important guarantee for obtaining reliable data.

[0027] 5. Strong applicability: This invention not only provides a general methodological framework, but also has a specific application in the formulation of quality standards for Tongmai Huazhuo Granules, providing a scientific and quantitative basis for quality control, which is of great significance for ensuring the efficacy and safety of the product. Attached Figure Description

[0028] Figure 1 This is the HPLC chromatogram of the hesperidin reference solution.

[0029] Figure 2 This is the HPLC chromatogram of the test solution of Tongmai Huazhuo Granules (the peak of hesperidin is marked in the figure).

[0030] Figure 3 This is the HPLC chromatogram of the negative control solution that does not contain orange-red. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art.

[0032] Example 1: Chromatographic conditions and system suitability test

[0033] An Agilent 1260 high-performance liquid chromatograph was used. The chromatographic column was an Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile-0.1% phosphoric acid solution (20:80), isocratic elution; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the detection wavelength was 283 nm; and the injection volume was 10 μL.

[0034] The hesperidin reference solution (preparation method as described in Example 2) was injected six times consecutively, and the chromatograms were recorded. The theoretical plate number calculated based on the hesperidin peak was higher than 6000, and the peak area RSD was less than 1.0%, indicating that the instrument precision was good and the system suitability met the requirements.

[0035] Example 2: Solution Preparation

[0036] I. Preparation of Tongmai Huazhuo Granules for Detection:

[0037] Weigh out 60g of peach kernel, 60g of safflower, 72g of prepared pinellia, 90g of tangerine peel, 90g of vinegar-processed corydalis, 90g of angelica, 90g of salvia miltiorrhiza, 60g of cyperus rotundus, 90g of poria cocos, 150g of prepared astragalus membranaceus, and 36g of prepared licorice root. Soak these eleven herbs in water for 30 minutes, then decoct twice. For the first decoction, add 10 times the amount of water and decoct for 1.5 hours. For the second decoction, add 8 times the amount of water and decoct for 1.5 hours. Combine the decoctions, filter, and concentrate the filtrate under reduced pressure at -0.05 MPa to a clear extract with a relative density of 1.25 at 65℃. Centrifuge the extract, add an appropriate amount of dextrin, mix well, granulate using a 16-mesh sieve, dry at 70℃, and then granulate again using a 16-mesh sieve to produce 1000g of granules, which are then packaged.

[0038] II. Preparation of the detection solution:

[0039] Preparation of reference solution: Weigh approximately 12.5 mg of hesperidin reference standard accurately, place it in a 250 mL brown volumetric flask, dissolve and dilute to the mark with methanol, shake well, and prepare a solution containing 50 μg per mL.

[0040] Preparation of the test solution: Take an appropriate amount of the above-prepared Tongmai Huazhuo granules, grind them into a fine powder, weigh about 2.0 g of the fine powder accurately, place it in a 100 mL stoppered conical flask, accurately add 50 mL of methanol, stopper tightly, weigh, heat under reflux for 30 min, cool, weigh again, replenish the lost weight with methanol, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate.

[0041] Preparation of negative control solution: Weigh the remaining ten herbs (excluding tangerine peel) according to the prescription of Tongmai Huazhuo Granules (60g peach kernel, 60g safflower, 72g prepared pinellia, 90g vinegar-processed corydalis, 90g angelica, 90g salvia miltiorrhiza, 60g cyperus rotundus, 90g poria cocos, 150g prepared astragalus membranaceus, 36g prepared licorice). Prepare a negative sample lacking tangerine peel according to the method described in "I. Preparation of Tongmai Huazhuo Granules for Detection" above. Then prepare the test solution according to the same method as in "Preparation of Test Solution".

[0042] Example 3: Specificity Test

[0043] Accurately pipette 10 μl each of the above-mentioned reference solution, test solution and negative control solution, and inject them into the liquid chromatograph under the chromatographic conditions of Example 1, and record the chromatograms.

[0044] The results are as follows Figure 1-3 As shown. Figure 1 The peak shape of hesperidin in the reference solution was good; Figure 2 The test sample solution showed a chromatographic peak at the same position as the reference standard at the same retention time, with no surrounding interference. Figure 3 The negative control solution showed no chromatographic peak at this retention time. The results indicate that the method is highly specific and does not interfere with negative control responses.

[0045] Example 4: Content determination method and quality standard setting

[0046] Take the test solution and the reference solution, and analyze them according to the chromatographic conditions of Example 1. Calculate the content of hesperidin in the test solution by peak area using the external standard method, and then convert it into the amount of hesperidin (CHO) in each 1g of granules.

[0047] The three batches of pilot samples (hereinafter referred to as batch 1, batch 2 and batch 3) were tested, and the results were 1.301 mg / g, 1.275 mg / g and 1.288 mg / g, respectively (converted to 13.01 mg / bag, 12.75 mg / bag and 12.88 mg / bag for a specification of 10g / bag).

[0048] Based on the above test results, the content limits for this product are established as follows:

[0049] The lowest hesperidin content in the samples was 12.21 mg / bag (1.221 mg / g), and the highest was 13.87 mg / bag (1.387 mg / g). According to the content limit of hesperidin under the Citrus reticulata (not less than 1.7%) in the 2015 edition of the Chinese Pharmacopoeia, the minimum theoretical content of hesperidin in this product should be no less than 15.3 mg per bag (no less than 1.53 mg per 1g).

[0050] Taking the following factors into consideration:

[0051] (1) Solubility of hesperidin in water and its transfer rate during preparation;

[0052] (2) Potential process losses during actual large-scale production;

[0053] (3) The quality differences between batches of Chinese medicinal materials themselves;

[0054] (4) The actual measured values ​​of the above three batches of samples.

[0055] To better control the quality of this product and ensure its stability and efficacy, the content limit is ultimately set as follows: each bag (10g) of this product shall contain no less than 5.0mg of tangerine peel as hesperidin (CHO) (i.e., no less than 0.50mg per 1g of granules).

[0056] Example 5: Methodological Validation

[0057] The method of this invention was fully validated using batch 1 samples.

[0058] Linearity assessment: A series of hesperidin reference solutions at precise concentrations (approximately 10, 20, 40, 60, 80, and 100 μg / mL) were prepared and analyzed under the chromatographic conditions of Example 1. A standard curve was plotted with concentration (μg / mL) on the x-axis and peak area on the y-axis. The results showed that hesperidin exhibited good linearity in the concentration range of 10–100 μg / mL, with a regression equation of Y = 35.21X + 12.47 and r = 0.9999.

[0059] Precision test: Take the same test solution (batch 1) and inject it 6 times consecutively under the chromatographic conditions of Example 1. The RSD of the peak area of ​​hesperidin was calculated to be 0.85% (intra-day precision); the same test solution was measured once a day for 3 consecutive days. The RSD of the hesperidin content was calculated to be 1.32% (inter-day precision), indicating good precision.

[0060] Repeatability test: Take 6 samples from the same batch (batch 1) and perform parallel operation according to the test solution preparation method described in Example 2. Prepare 6 test solutions and measure them sequentially. The RSD of hesperidin content was calculated to be 1.56%, indicating that the method has good repeatability.

[0061] Recovery test: Approximately 1.0 g of fine powder from the same batch of samples with known content (batch number: TMHZ230101, content 1.301 mg / g) was accurately weighed and divided into 9 portions. Hesperidin reference standard equivalent to 80%, 100%, and 120% of the hesperidin content in the sample was accurately added to each of the 3 groups. The test solution preparation method described in Example 2 was followed and the results were measured. The average recovery rate was calculated to be 99.7%, and the RSD was 1.23% (n=9), indicating that the method has good accuracy.

[0062] Stability test: The same test solution (batch 1) was placed at room temperature and injected for determination at 0, 2, 4, 8, 12 and 24 hours according to the chromatographic conditions of Example 1. The RSD of the hesperidin peak area was calculated to be 1.48%, indicating that the test solution had good stability within 24 hours.

[0063] The results showed that the hesperidin chromatographic peak in Tongmai Huazhuo granules was not interfered with by other chromatographic peaks and could be well separated from other components. The retention time of hesperidin in the test sample was consistent with that of the reference standard, and the theoretical plate number calculated based on the hesperidin peak was not less than 4000. Furthermore, the negative control solution showed no interference, meeting the requirements for content determination. (See attached results) Figure 1-3 .

[0064] The above verification results show that all indicators of the detection method established in this invention meet the requirements of the Chinese Pharmacopoeia's guidelines on the verification of analytical methods for drug quality standards, and the method is reliable and effective.

[0065] In summary, this invention provides a detection method with strong specificity, high accuracy, and good stability, which can be effectively used for the quality control of traditional Chinese medicine granules containing tangerine peel, and is particularly suitable for Tongmai Huazhuo granules.

[0066] 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 detecting traditional Chinese medicine granules, characterized in that, The method includes: using high performance liquid chromatography, with octadecylsilane-bonded silica gel as the packing material and hesperidin as the indicator component, to qualitatively identify and / or determine the content of tangerine peel in the traditional Chinese medicine granules.

2. The method for detecting traditional Chinese medicine granules according to claim 1, characterized in that, In the chromatographic conditions of the high-performance liquid chromatography method, the mobile phase is an organic phase-acid aqueous solution, and the detection wavelength is 280-286 nm.

3. The method for detecting traditional Chinese medicine granules according to claim 2, characterized in that, The organic phase is acetonitrile or methanol, and the acidic aqueous solution is a phosphoric acid solution or formic acid solution with a volume concentration of 0.05%-0.2%.

4. The method for detecting traditional Chinese medicine granules according to claim 3, characterized in that, The mobile phase is acetonitrile-phosphoric acid solution with a volume concentration of 0.1%, and the volume ratio is (19:81) to (21:79).

5. The method for detecting traditional Chinese medicine granules according to claim 4, characterized in that, The mobile phase contains acetonitrile in a volume ratio of 20:80 to a 0.1% phosphoric acid solution, and isocratic elution is employed.

6. The method for detecting traditional Chinese medicine granules according to claim 2, characterized in that, The detection wavelength is 283nm.

7. The method for detecting traditional Chinese medicine granules according to claim 1, characterized in that, The system suitability requirement for the high performance liquid chromatography method is that the theoretical plate number, calculated based on the hesperidin peak, should be no less than 6000.

8. The method for detecting traditional Chinese medicine granules according to claim 1, characterized in that, Includes the following steps: (1) Preparation of reference solution: Take an appropriate amount of hesperidin reference standard, accurately weigh it, dissolve and dilute it with methanol to prepare a solution containing 45-55 μg per 1 mL; (2) Preparation of test solution: Take an appropriate amount of the Chinese herbal medicine granules, grind them into a fine powder, take about 2.0g of the fine powder, weigh it accurately, put it in a stoppered conical flask, add 50mL of methanol accurately, stopper tightly, weigh it, heat and reflux for 30-40min, cool it, weigh it again, make up the lost weight with methanol, shake well, filter it, and take the filtrate. (3) Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine them.

9. The method for detecting traditional Chinese medicine granules according to claim 8, characterized in that, In step (1), a hesperidin reference solution containing 50 μg per 1 mL is prepared.

10. The method for detecting traditional Chinese medicine granules according to claim 1, characterized in that, It also includes the step of preparing a negative control solution: prepare a negative sample without tangerine peel according to the prescription and process of the Chinese herbal granules, and then prepare a negative control solution in the same way as the test sample solution for specificity testing.

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