Quality detection method of ganoderma lucidum and ginseng oral liquid

Abstract

The quality detection method is characterized by comprising a thin-layer chromatography identification method for radix salviae miltiorrhizae and radix angelicae sinensis and / or a method for determining the content of radix salviae miltiorrhizae by adopting liquid chromatography. By optimizing the thin-layer chromatography method, efficient and specific identification of salvia miltiorrhiza and angelica sinensis in the ganoderma lucidum and ginseng oral liquid is realized, and a reliable technical scheme is provided for quality control of a traditional Chinese medicine preparation; the method passes methodology verification (specificity, durability and the like) and is stable in stability investigation, the quality safety of the ganoderma lucidum and ginseng oral liquid is further ensured, and evaluation is comprehensive.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a quality testing method for Ganoderma lucidum and ginseng oral liquid. Background Technology

[0002] Lingzhi Shuangshen Oral Liquid is a traditional Chinese medicine preparation composed of Lingzhi (Ganoderma lucidum), ginseng, salvia miltiorrhiza, eucommia ulmoides, wolfberry, prepared he shou wu (Polygonum multiflorum), stir-fried jujube seed, schisandra chinensis, prepared rehmannia glutinosa, angelica sinensis, and honey. It is a unique product in China and was approved for marketing by the China Food and Drug Administration (CFDA) in 2002. The main functions of Lingzhi Shuangshen Oral Liquid are to nourish qi and blood, and calm the mind. It is suitable for those with deficiency of both qi and blood, and restlessness of the mind, manifested as palpitations, anxiety, soreness and weakness of the lower back and knees, insomnia, and forgetfulness. It also has an auxiliary therapeutic effect on neurasthenia.

[0003] The Ganoderma lucidum and ginseng oral liquid of this invention is made from the following medicinal materials in the indicated weight ratios: 500g Ganoderma lucidum, 6g ginseng, 20g Eucommia ulmoides, 20g Lycium barbarum, 20g processed Polygonum multiflorum, 20g stir-fried Ziziphus jujuba var. spinosa, 20g Schisandra chinensis, 20g Rehmannia glutinosa, 15g Angelica sinensis, and 650g honey. Except for ginseng and honey, the remaining nine ingredients (Ganoderma lucidum, etc.) are pulverized into coarse powder and extracted three times by reflux. The first extraction uses 6 times the amount of 60% ethanol and is refluxed for 2 hours. The second extraction uses 50% ethanol... Extract with 4 times the volume of 40% ethanol under reflux for 2 hours. Then, extract again with 4 times the volume of 40% ethanol under reflux for 2 hours. Filter, combine the filtrates, recover the ethanol, and concentrate to 1.18-1.23 (50°C). Add an equal volume of freshly boiled water, filter, and reserve the filtrate. Ginseng is coarsely ground, soaked in 60% ethanol for 24 hours, then percolated, collecting 100 ml of the percolate. Then, percolate again with 40% ethanol, collecting 80 ml of the percolate. Combine the percolates, recover the ethanol, and concentrate to 1.02. 1.05 (50°C), refrigerate, filter, and reserve the filtrate. Boil honey, filter, combine the filtrate with the above filtrate and percolate, add 3g of sodium benzoate to dissolve, add water to 1000ml, refrigerate for 72 hours, filter, fill, and sterilize to obtain the final product. Currently, Lingzhi Shuangshen Oral Liquid has qualified thin-layer chromatography identification experiments for major medicinal materials such as Lingzhi, Ziziphus jujuba seed, and Lycium barbarum, and also has qualified experiments for the content determination of medicinal materials such as Salvia miltiorrhiza. However, it has the following technical defects: 1. Defects in the identification of Salvia miltiorrhiza: The original method uses protocatechuic aldehyde as a reference standard, but it is a degradation product of Salvia miltiorrhiza (not a native component), which requires multiple extraction steps (ether extraction, sodium carbonate solution back extraction, acid adjustment of pH, and then ether extraction). The operation is cumbersome, the sample loss rate is high, and the loss rate is >20%. Moreover, the content of protocatechuic aldehyde in Salvia miltiorrhiza is low (about 0.03%~0.16%), which is not representative enough, has insufficient sensitivity, and is not a major active ingredient.

[0004] 2. Lack of identification of Angelica sinensis: The original standard did not include identification methods for Angelica sinensis, resulting in a lack of quality control for this herb and failing to meet the requirement of the National Center for Drug Evaluation to "suggest the establishment of identification methods for Angelica sinensis". Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a quality testing method for Ganoderma lucidum and Ginseng oral liquid. This invention optimizes thin-layer chromatography to achieve efficient and specific identification of Salvia miltiorrhiza and Angelica sinensis in Ganoderma lucidum and Ginseng oral liquid, providing a reliable technical solution for the quality control of traditional Chinese medicine preparations. Furthermore, this method has passed methodological validation (specificity, robustness, etc.) and demonstrated stability in stability studies, further ensuring the quality and safety of Ganoderma lucidum and Ginseng oral liquid and enabling comprehensive evaluation.

[0006] To address the above issues, the following technical solution is provided: A quality testing method for Ganoderma lucidum and Angelica sinensis oral liquid, including a thin-layer chromatography method for identifying Salvia miltiorrhiza and Angelica sinensis and / or a method for determining the content of Salvia miltiorrhiza using liquid chromatography.

[0007] Furthermore, the thin-layer chromatography identification method for Danshen uses two reference materials: Danshen reference material and sodium tanshinone.

[0008] Furthermore, the thin-layer chromatography identification method includes any one or any two of the following identification methods; (1) Thin-layer chromatographic identification method of Salvia miltiorrhiza Preparation of test solution: Take 30-50 ml of Ganoderma lucidum and ginseng oral liquid sample, adjust the pH value to 1-2 with hydrochloric acid, add ether and shake to extract 3 times, 40 ml each time, combine the ether extracts, evaporate the solvent, and add 1 ml of methanol to dissolve the residue.

[0009] Preparation of control solution: Danshen control herb solution: Take 0.5g of Danshen control herb, add appropriate amount of water, heat under reflux for 2 hours, filter, add water to 20ml of filtrate, adjust pH to 1~2 with hydrochloric acid, add ether and shake to extract 3 times, 40ml each time, combine ether extracts, evaporate to dryness, add 1ml of methanol to dissolve the residue; Tanshinone sodium reference solution: Take an appropriate amount of tanshinone sodium reference standard and add methanol to prepare a solution containing 1 mg per 1 ml; Determination: Apply 5-10 μl of the test solution, 5 μl of the tanshinone reference material solution, and 2 μl of the sodium tanshinone reference solution to the same silica gel G thin-layer plate. Develop the plate using chloroform-acetone-acetic acid (25:10:4) as the developing solvent. After development, remove the plate, air dry it, fumigate it in ammonia vapor for about 10 minutes, remove it and let it stand for more than half an hour, then examine it under ultraviolet light (365 nm). If fluorescent spots of the same color appear at the corresponding positions in the chromatograms of the test sample, tanshinone reference material, and sodium tanshinone reference solution, it is considered a positive result for tanshinone identification.

[0010] (2) Thin-layer chromatographic identification method of Angelica sinensis Preparation of test solution: Take 30-50 ml of Ganoderma lucidum and ginseng oral liquid sample, extract with 30 ml of ether three times, combine the ether extracts, evaporate the solvent, and dissolve the residue in 1 ml of methanol.

[0011] Preparation of control solution: Take 0.1g of Angelica sinensis reference material, add 20ml of ether, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of methanol.

[0012] Determination: Apply 5-20 μl of the test solution and 2 μl of the Angelica sinensis reference material solution to the same silica gel G thin-layer plate; Development: Develop with n-hexane-ethyl acetate (9:1) as the developing solvent; After development, remove the thin-layer plate and examine it under ultraviolet light (365 nm); If fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the test sample and the Angelica sinensis reference material, it is considered a positive result for Angelica sinensis identification.

[0013] Furthermore, when determining the content of tanshinone using liquid chromatography, salvianolic acid B is used as a reference standard.

[0014] Furthermore, the determination of the content of *Salvia miltiorrhiza* using liquid chromatography includes the following steps: Preparation of reference solution: Weigh an appropriate amount of salvianolic acid B reference standard, accurately weigh it, dissolve it in 80% methanol and quantitatively dilute it to prepare a solution containing approximately 0.16 mg per 1 ml, which is used as the reference solution; Preparation of Danshen negative solution: Take 10 ml of Danshen negative sample (batch number: 22082501) into a 15 ml volumetric flask, dilute with methanol and make up to the mark, shake well, filter through a 0.45 μm filter membrane, and take the filtrate as Danshen negative solution. Preparation of test solution: Accurately measure 10 ml of this product (batch number: 230301) into a 15 ml volumetric flask, dilute with methanol and bring to volume, shake well, filter through a 0.45 μm filter membrane, and take the filtrate as the test solution; Determination: Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatograph, and record the chromatograms; the chromatographic column used is an Agilent ZORBAX SB-C18 5μm 4.6×250mm stainless steel column, and the mobile phase is: A. 0.05% phosphoric acid, B. acetonitrile.

[0015] Further, perform gradient elution as shown in the table below: .

[0016] Beneficial effects Identification of Salvia miltiorrhiza: 1. High specificity: Using Salvia miltiorrhiza reference material and sodium tanshinone reference standard as dual controls solves the problem of insufficient representativeness of protocatechuic aldehyde. 2. Simple operation: The preparation of the test solution is simplified, and the pretreatment is optimized, retaining only one step of acid adjustment for pH and ether extraction. Ether extraction is performed 3 times × 40ml, eliminating the alkaline washing step and improving efficiency by 50%. The developing solvent is optimized to be chloroform-acetone-acetic acid, with a ratio of 25:10:4. After fumigation with ammonia vapor and ultraviolet detection, clear separation of spots is achieved, significantly improving the clarity and specificity of the spots.

[0017] Determination of Salvia miltiorrhiza content: 1. Indicator substitution: Protocatechuic aldehyde was abandoned, and salvianolic acid B was selected. 2. Breakthrough in chromatographic conditions: Mobile phase: 0.05% phosphoric acid (A)-acetonitrile (B) gradient elution (35℃ column temperature, 286nm detection), the resolution is better than the original method. 3. Simplified sample processing: direct methanol dilution and filtration.

[0018] Identification of Angelica sinensis: A new identification method using Angelica sinensis reference material as a reference has been added, which only requires extraction of the test sample with ether, reducing the time by 50%; the developing solvent is n-hexane-ethyl acetate (9:1), and direct ultraviolet detection is simple to operate and produces obvious spots; the new identification method for Angelica sinensis covers the flavor of prescription herbs, and there is no interference from negative samples.

[0019] The method of this invention can withstand changes in the brand of thin-layer plates and temperature and humidity, and the spots are clear and stable.

[0020] This invention addresses the specificity deficiencies of existing detection methods, ensuring drug safety and efficacy while meeting modern quality control requirements, and guaranteeing product safety, effectiveness, and quality control. It resolves the shortcomings of the original standard, complies with the "Technical Guidelines for Pharmaceutical Change Research of Marketed Traditional Chinese Medicines," and is applicable to the post-marketing quality standard upgrade of Ganoderma lucidum and ginseng oral liquid. Attached Figure Description

[0021] Figure 1 The image shows a thin-layer chromatogram of Salvia miltiorrhiza, where 1: test sample 230501; 2: Salvia miltiorrhiza reference material; 3: test sample 230502; 4: sodium tanshinone reference standard; 5: test sample 230503; 6: negative for Salvia miltiorrhiza. Figure 2 The image shows a thin-layer chromatogram of Angelica sinensis, where 1: test sample 230501; 2: test sample 230501; 3: test sample 230501; 4: negative chromatogram of Angelica sinensis; 5: reference material of Angelica sinensis. Figure 3The figures shown are thin-layer chromatograms comparing different spotting amounts of *Salvia miltiorrhiza*. In A, 1: sodium tanshinone reference standard; 2: *Salvia miltiorrhiza* reference material; 3: test solution (2 μl); 4: test solution (5 μl); 5: test solution (10 μl); 6: test solution (20 μl). In B, 1: sodium tanshinone reference standard; 2: *Salvia miltiorrhiza* reference material (1 μl); 3: *Salvia miltiorrhiza* reference material (2 μl); 4: *Salvia miltiorrhiza* reference material (5 μl); 5: *Salvia miltiorrhiza* reference material (10 μl); 6: test solution. Figure 4 The figures shown are thin-layer chromatograms comparing different spotting amounts of Angelica sinensis; in A, 1: test solution (5 μl); 2: test solution (10 μl); 3: test solution (20 μl); 4: test solution (30 μl); 5: Angelica sinensis reference material; in B, 1: test solution; 2: Angelica sinensis reference material (1 μl); 3: Angelica sinensis reference material (2 μl); 4: Angelica sinensis reference material (5 μl); 5: Angelica sinensis reference material (10 μl). Figure 5 The figures shown are comparisons of different developing solvents for *Salvia miltiorrhiza*. In Figure A, the developing solvent is chloroform-acetone-formic acid (8:1:1), with 1 representing *Salvia miltiorrhiza* reference material (5 μl), 2 representing sodium tanshinone reference standard, and 3 representing *Salvia miltiorrhiza* reference material (10 μl). In Figure B, the developing solvent is chloroform-acetone-acetic acid (25:10:4), with 1 representing *Salvia miltiorrhiza* reference material (5 μl), 2 representing sodium tanshinone reference standard, and 3 representing *Salvia miltiorrhiza* reference material (10 μl). C is the developing solvent: chloroform-ethyl acetate-acetone-acetic acid (20:10:15:4), where 1: *Salvia miltiorrhiza* reference material (5 μl); 2: sodium tanshinone reference standard; and 3: *Salvia miltiorrhiza* reference material (10 μl). D is the developing solvent: toluene-ethyl acetate-anhydrous ethanol-acetic acid (10:3:3:1.5), where 1: *Salvia miltiorrhiza* reference material (5 μl); 2: sodium tanshinone reference standard; and 3: *Salvia miltiorrhiza* reference material (10 μl). Figure 6The figure shows a comparison of different developing solvents for Angelica sinensis; where: A is the developing solvent: n-hexane-ethyl acetate (4:1), 1: 1 μl; 2: 2 μl; 3: 5 μl; 4: 10 μl; B is the developing solvent: cyclohexane-ethyl acetate (9:1), 2: 2 μl; 3: 5 μl; 4: 10 μl; C is the developing solvent: petroleum ether-ethyl acetate (17:3), figure... In the figure, 1: 1 μl sample volume; 2: 2 μl sample volume; 3: 5 μl sample volume; 4: 10 μl sample volume; D is the developing solvent: n-hexane-ethyl acetate (9:1). In the figure, 1: 1 μl sample volume; 2: 2 μl sample volume; 3: 5 μl sample volume; 4: 10 μl sample volume; E is the developing solvent: petroleum ether-ethyl acetate (10:1). In the figure, 1 μl sample volume; 2: 2 μl sample volume; 3: 5 μl sample volume; 4: 10 μl sample volume.

[0022] Figure 7 The chromatogram shown is the specific identification chromatogram of Salvia miltiorrhiza (temperature 25℃, relative humidity 45%), where 1. Sodium tanshinone reference standard 2. Test sample (220901) 3. Salvia miltiorrhiza reference material 4. Salvia miltiorrhiza negative; Figure 8 The chromatogram of the salvianolic acid B reference solution is shown below. Figure 9 The image shows the chromatogram of a negative solution of Danshen. Figure 10 The chromatogram of the test sample solution is shown below. Figure 11 The graph shown is a linear relationship graph for the determination of salvianolic acid B content. Figure 12 The following is a specific chromatogram for the identification of Angelica sinensis (temperature 25℃, relative humidity 45%): 1. Angelica sinensis negative; 2. Angelica sinensis reference material; 3. Test sample (220901). Detailed implementation method: To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. The described embodiments are only some embodiments of this application, and not all of them. The present invention is not limited to the following embodiments.

[0023] I. Thin-layer chromatography identification method 1. Thin-layer chromatographic identification method for Salvia miltiorrhiza Preparation of test solution: Take 40 ml of Ganoderma lucidum and ginseng oral liquid sample, adjust the pH value to 1-2 with hydrochloric acid, add ether and shake to extract 3 times, 40 ml each time, combine the ether extracts, evaporate the solvent, and add 1 ml of methanol to dissolve the residue.

[0024] Preparation of reference herb solution: Take 0.5g of Salvia miltiorrhiza reference herb, add appropriate amount of water, heat under reflux for 2 hours, filter, add water to 20ml of filtrate, adjust pH to 1-2 with hydrochloric acid, add ether and shake to extract 3 times, 40ml each time, combine ether extracts, evaporate to dryness, add 1ml of methanol to dissolve residue.

[0025] Tanshinone sodium reference solution: Take an appropriate amount of tanshinone sodium reference standard and add methanol to prepare a solution containing 1 mg per 1 ml.

[0026] Spotting: Take 5-10 μl of the test solution, 5 μl of the tanshinone reference material solution, and 2 μl of the sodium tanshinone reference solution, and spot them separately on the same silica gel G thin-layer plate.

[0027] Development: The development was carried out using chloroform-acetone-acetic acid (25:10:4) as the developing solvent.

[0028] Color development and detection: After development, remove the thin-layer plate, air dry it, fumigate it in ammonia vapor for about 10 minutes, remove it and let it stand for more than half an hour, then examine it under ultraviolet light (365nm).

[0029] Result determination: If the test sample shows fluorescent spots of the same color at the corresponding positions in the chromatograms of the reference herb *Salvia miltiorrhiza* and sodium tanshinone, it is considered a positive result for *Salvia miltiorrhiza* identification.

[0030] 2. Thin-layer chromatographic identification method for Angelica sinensis Preparation of test solution: Take 50 ml of Ganoderma lucidum and ginseng oral liquid sample, extract with 30 ml of ether three times, combine the ether extracts, evaporate the solvent, and dissolve the residue in 1 ml of methanol.

[0031] Preparation of reference herb solution: Take 0.1g of Angelica sinensis reference herb, add 20ml of ether, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of methanol.

[0032] Spotting: Take 5-20 μl of the test solution and 2 μl of the Angelica sinensis reference material solution and spot them separately on the same silica gel G thin layer plate.

[0033] Development: The development was carried out using n-hexane-ethyl acetate (9:1) as the developing solvent.

[0034] Testing: After unfolding, remove the thin-layer plate and examine it under a UV lamp (365nm).

[0035] Result determination: If the test sample shows fluorescent spots of the same color at the corresponding positions as the reference chromatogram of Angelica sinensis, it is considered a positive result for Angelica sinensis identification.

[0036] 3. Basis for Key Parameter Optimization Test sample dosage and extraction times: 30ml, 40ml, and 50ml of Ganoderma lucidum and Codonopsis pilosula oral liquid (batch number: 220901) were taken sequentially, and extracted with the corresponding extraction solvents for different numbers of extractions (1-3 times) to prepare test sample solutions. The results showed that the more the amount of test sample and the more extraction times increased, the clearer the colored spots of the test sample became. In the identification of Codonopsis pilosula, a test sample dosage of 40ml and 3 extractions with ether were used; in the identification of Angelica sinensis, a dosage of 50ml and 3 extractions with ether were used to ensure clear spots.

[0037] Table 1. Results of Sample Dosage and Extraction Times - Salvia miltiorrhiza

[0038] Table 2 Results of Sample Dosage and Extraction Times - Angelica sinensis

[0039] Spotting amounts: For the identification of Salvia miltiorrhiza, the spotting amounts were 5-10 μl (test sample) and 5 μl (reference material); for the identification of Angelica sinensis, the spotting amounts were 5-20 μl (test sample) and 2 μl (reference material), optimized based on the spot separation effect. The identification method for Salvia miltiorrhiza used the following four developing solvents for comparison: chloroform-acetone-formic acid (8:1:1), chloroform-acetone-acetic acid (25:10:4), chloroform-ethyl acetate-acetone-acetic acid (20:10:15:4), and toluene-ethyl acetate-anhydrous ethanol-acetic acid (10:3:3:1.5). The results showed that the developing solvent chloroform-acetone-acetic acid (25:10:4) was the most effective.

[0040] The identification method for Angelica sinensis used the following five developing solvents for comparison: n-hexane-ethyl acetate (4:1), cyclohexane-ethyl acetate (9:1), petroleum ether-ethyl acetate (17:3), n-hexane-ethyl acetate (9:1), and petroleum ether-ethyl acetate (10:1). The results showed that the developing solvent n-hexane-ethyl acetate (9:1) was the most effective.

[0041] Example 1: Identification of Salvia miltiorrhiza Sample: Ganoderma lucidum and ginseng oral liquid (batch number 220901).

[0042] Steps: Prepare the test solution according to the above-described method for identifying Danshen.

[0043] Spotting: Spot 10 μl of the test solution, 5 μl of the tanshinone reference material solution, and 2 μl of the sodium tanshinone reference solution onto a silica gel G thin-layer plate.

[0044] Expand: chloroform-acetone-acetic acid (25:10:4)

[0045] Detection: UV 365nm examination after ammonia vapor fumigation.

[0046] Results: In the chromatogram of the test sample, clear blue fluorescent spots appeared at the same positions as the reference medicinal material and reference standard. The negative sample (without Danshen) showed no interference, indicating that the method has strong specificity.

[0047] Example 2: Identification of Angelica sinensis Sample: Ganoderma lucidum and ginseng oral liquid (batch number 220901).

[0048] Steps: Prepare the test solution according to the above-described method for identifying Angelica sinensis.

[0049] Spotting: Spot 20 μl of the test solution and 2 μl of the Angelica sinensis reference material solution onto a silica gel G thin-layer plate.

[0050] Expand: n-hexane-ethyl acetate (9:1)

[0051] Detection: Direct inspection using 365nm ultraviolet light.

[0052] Results: In the chromatogram of the test sample, a yellow fluorescent spot appeared at the same position as the main spot of the Angelica sinensis reference material. The negative sample (without Angelica sinensis) showed no interference, indicating that the method was accurate and reliable.

[0053] Example 3: Methodological Investigation of the Identification of Salvia miltiorrhiza with Ganoderma lucidum and Ginseng Oral Liquid Exclusivity Take 40 ml of Ganoderma lucidum and Ginseng oral liquid (batch number: 220901), adjust the pH to 1-2 with hydrochloric acid, add ether and shake to extract 3 times, 40 ml each time. Combine the ether extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to prepare the test solution. Take a negative sample of Salvia miltiorrhiza (batch number: 22082501) and prepare a negative solution using the same method as the test sample. Take 0.5 g of Salvia miltiorrhiza reference material, add an appropriate amount of water, heat under reflux for 2 hours, filter, add water to 20 ml of the filtrate, and prepare the reference material solution using the same method starting from "adjust the pH to 2 with hydrochloric acid". Take sodium tanshinone reference standard and prepare a solution containing 1 mg per ml with methanol to prepare the reference solution. Take 10 μl each of the test solution and the Danshen negative solution, 5 μl of the reference herb solution, and 2 μl of the reference standard solution, and spot them separately on the same silica gel G thin-layer plate. Develop the plate using chloroform-acetone-formic acid (25:10:4) as the developing solvent. Remove the plate, air dry, fumigate with ammonia vapor, and then place it under ultraviolet light (365 nm) for 10 minutes. The results show that the Danshen negative solution did not contain any spots identical to those in the Danshen reference herb, indicating that the Danshen negative solution did not interfere with the determination, and the method has good specificity. See [link to detailed results] Figure 7 ; Durability The test solution, reference herb solution, and reference standard solution under the specificity section were used for a durability test. The identification performance of the test sample was examined by varying the silica gel G plates from different manufacturers and by applying different temperatures and humidity levels. The results showed that this method is not affected by the brand of the silica gel G plate or the test environment, and exhibits good durability. Specific results are shown in Table 2.

[0054] Table 3. Durability Test Table for Salvia miltiorrhiza .

[0055] Methodological content of Angelica sinensis Exclusivity Take 50 ml of Ganoderma lucidum and Ginseng oral liquid (batch number: 220901), extract three times with 30 ml of ether, combine the extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to prepare the test solution. Take a negative sample of Angelica sinensis (batch number: 22081101) and treat it in the same way as the test sample to prepare the negative solution of Angelica sinensis. Take 0.1 g of Angelica sinensis, add 20 ml of ether, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to prepare the control solution. Take 20 μl each of the negative solution of Angelica sinensis and the test solution, and 2 μl of the control solution, and spot them separately on the same silica gel G thin-layer plate. Develop with n-hexane-ethyl acetate (9:1) as the developing solvent, remove the plate, and examine under a UV lamp (365 nm). The results show that there are no spots in the negative solution of Angelica sinensis that are consistent with the main spot of the control sample of Angelica sinensis, and the negative solution of Angelica sinensis does not interfere with the determination, indicating good method specificity.

[0056] Durability A robustness test was conducted using a negative control solution of Angelica sinensis, the test sample solution, and a control solution from the specificity test. The identification performance of the test sample was examined by varying the silica gel G plates from different manufacturers and by applying different temperatures and humidity levels. The results showed that this method is not affected by the brand of the silica gel G plate or the test environment, and exhibits good robustness. Specific results are shown in Table 4.

[0057] Table 4. Test Table for the Durability of Angelica Sinensis

[0058] II. Determination of Salvia miltiorrhiza content Determination of salvianolic acid B content Based on the "Technical Guidelines for Validation of Analytical Methods for Quality Control of Chemical Drugs", the "Technical Guidelines for Standardized Processes in the Establishment of Quality Standards for Chemical Drugs", and the relevant guidelines in the appendix of the current edition of the "Pharmacopoeia of the People's Republic of China", the methodological validation for the determination of salvianolic acid B content was carried out.

[0059] Table 5. Results of Method Validation for Determination of Danshenol B Content

[0060] Chromatographic conditions Column: Agilent ZORBAX SB-C18 5μm 4.6×250mm stainless steel column Mobile phase: A. 0.05% phosphoric acid B. Acetonitrile Perform gradient elution according to the table below:

[0061] (1) Specificity test Weigh an appropriate amount of salvianolic acid B reference standard accurately, dissolve it in 80% methanol and dilute quantitatively to prepare a solution containing approximately 0.16 mg per ml, as the reference solution; take 10 ml of the salvianolic acid negative sample (batch number: 22082501) into a 15 ml volumetric flask, dilute with methanol and bring to the mark, shake well, filter through a 0.45 μm filter membrane, and take the filtrate as the salvianolic acid negative solution; accurately measure 10 ml of this product (batch number: 230301) into a 15 ml volumetric flask, dilute with methanol and bring to the mark, shake well, filter through a 0.45 μm filter membrane, and take the filtrate as the test solution.

[0062] Accurately measure 10 μl of each of the above solutions and inject them separately into the liquid chromatograph, recording the chromatograms. The results showed that the negative solution for Danshen did not interfere with the content determination; the Danshen acid B in the test solution was completely separated from adjacent peaks, with a symmetry factor of 1.24 and a theoretical plate number of 73531. Figures 8-10 .

[0063] (2) Linearity and Range Test Accurately weigh approximately 40 mg of salvianolic acid B reference standard and place it in a 25 ml volumetric flask. Add an appropriate amount of 80% methanol solution, shake to completely dissolve salvianolic acid B, and dilute to the mark with 80% methanol. This is the stock solution of salvianolic acid B reference standard. Accurately measure 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml, and 2.0 ml of the stock solution and place them in separate 10 ml volumetric flasks. Dilute to the mark with 80% methanol solution and shake well. These are the solutions for linearity tests 1–6. Inject 10 μl of each solution under the chromatographic conditions described in section 3.2.3.2 and record the chromatograms. Use the chromatograms from the limit of quantitation section for calculations. Perform linear regression on the peak area (A) against the concentration (C, μg / ml). The results are shown in Table 6. Figure 11 .

[0064] Table 36 Linearity and Range Data for the Determination of Tanshinone B Content

[0065] (3) System precision test Weigh an appropriate amount of salvianolic acid B reference standard accurately, dissolve it in 80% methanol, and quantitatively dilute it to prepare a solution containing approximately 0.16 mg per ml. This solution serves as the reference standard solution (concentration: 0.16 mg / ml). Inject the solution five times consecutively under the chromatographic conditions described above, and record the chromatograms. The results show that the instrument system has good precision. Data obtained from the chromatograms are shown in Table 7.

[0066] Table 7 Precision test data of the salvianolic acid B content determination system

[0067] (4) Determination of the limit of quantitation Take the linear relationship experiment linear 1 # A suitable amount of solution was gradually diluted with 80% methanol solution. Under the chromatographic conditions described above, 10 μl of the diluted solution was accurately measured and injected into the chromatograph, and the chromatogram was recorded. The signal-to-noise ratio was calculated; when the peak height was approximately 10 times the baseline noise (s / n≈10), this concentration of the diluted solution was considered the limit of quantitation (LOQ). The LOQ solution was injected six times consecutively under the chromatographic conditions described above, and the chromatograms were recorded. The LOQ for the determination of salvianolic acid B was 0.50 µg / ml (1.11% of the limit concentration of the test sample), and the RSD of the LOQ precision was 3.54% (<10%), indicating good precision. Data obtained from the chromatograms are shown in Table 8.

[0068] Table 8. Precision results of the determination of salvianolic acid B content within the limit of quantitation.

[0069] (5) Recovery rate test Accurately measure 100 ml of a negative sample of *Salvia miltiorrhiza* (batch number: 22082501), quantitatively add 50 ml of methanol, shake to mix, and use as the negative solution. Take an appropriate amount of the negative solution, filter, and use the filtrate as the blank solution. Accurately measure three portions each of 0.2 ml, 0.5 ml, 1.0 ml, and 1.5 ml of the stock solution of *Salvia miltiorrhiza* B reference standard in the linearity test, place them in 10 ml volumetric flasks, dilute to the mark with the negative solution, shake to mix, filter, and use the filtrate as the recovery test solution. Take the linearity test... # The solution was used as a reference solution. 10 μL each of the test solution, reference solution, and blank solution were accurately measured and injected into the liquid chromatograph, and the chromatograms were recorded. The recovery rate was calculated based on the measured amount and the amount added. The results showed that the average recovery rate was 98.99%, and the RSD was less than 2% (n=12). The recovery results were good. The data from the chromatograms are shown in Table 9.

[0070] Table 9 Results of the recovery test for salvianolic acid B content determination.

[0071] (6) Solution stability test Take the linear relationship experiment linear 5 # The solution was used as a reference solution. Accurately measure 15 ml of this product (batch number: 230503) into a 25 ml volumetric flask, dilute with methanol and bring to the mark, shake well, filter through a 0.45 μm filter membrane, and use the filtrate as the test solution. Inject 10 μl of the above solution into the liquid chromatograph at 0 h, 4 h, 8 h, 12 h, and 24 h, and record the chromatograms. The results showed that the peak areas of both the reference solution and the test solution did not change significantly over 24 h, and the peak area RSD was within 2%, remaining stable for at least 24 h. Data from the chromatograms are shown in Table 10.

[0072] Table 10 Results of stability test of solution for determination of salvianolic acid B content

[0073] (7) Repeatability test (same as intermediate precision test I) Take an appropriate amount of this product (batch number: 230503), and prepare a reference solution and 6 test solutions according to the method under the specificity section. Inject 10 μl of each solution into the liquid chromatograph and record the chromatograms. Calculate the content based on peak area using the external standard method. The results show that the method has good repeatability. Data obtained from the chromatograms are shown in Table 11.

[0074] Table 11 Results of repeatability test for the determination of salvianolic acid B content

[0075] (8) Intermediate precision test Take an appropriate amount of this product (batch number: 230503), and prepare a reference solution and 6 test solutions according to the method under the specificity section. Another analyst will conduct the test at different times using different instruments. Inject 10 μl into the liquid chromatograph according to the method under the content determination section, and record the chromatogram. 9 data points are obtained from the chromatogram. Determine the content according to the chromatographic conditions under the specificity section, calculate the content by peak area using the external standard method, and calculate the relative standard deviation of the content determination for 12 samples from the two analysts. The results show that the intermediate precision test results for the determination of salvianolic acid B content are good. Data obtained from the chromatogram are shown in Table 12.

[0076] Table 12 Results of intermediate precision test for determination of salvianolic acid B content

[0077] (9) Filter membrane adsorption test Accurately measure 10 ml of this product (batch number: 220901) into a 15 ml volumetric flask, dilute with methanol and bring to the mark, shake well, filter through a 0.45 μm filter membrane, and use the filtrate as the test solution. Using the filter membrane, discard 0 ml, 1 ml, 2 ml, 3 ml, and 4 ml of the initial filtrate, respectively, and inject 10 μl of each subsequent filtrate into the liquid chromatograph. Record the chromatograms, and obtain the data in Table 13 from the chromatograms. After investigation, it was found that after discarding more than 1 ml of the initial filtrate, there was no significant adsorption on the filter membrane.

[0078] Table 13 Filter membrane adsorption test data

[0079] (10) Durability test Prepare reference solution and test solution (batch number 230301) according to the method under the specificity section, and conduct a robustness test. By changing the column temperature, flow rate, mobile phase ratio and different chromatographic columns, the system suitability and the determination of the content of the test sample were examined.

[0080] By changing the test conditions, the durability range of the chromatographic conditions was confirmed. The specific methods are shown in Tables 14 and 15.

[0081] Table 14 Durability of Chromatographic Conditions for Content Determination

[0082] Table 15 System suitability under different chromatographic conditions

[0083] Table 16 Content determination under different chromatographic conditions

[0084] The durability test showed that minor variations in chromatographic conditions such as column temperature, mobile phase flow rate, and column type do not affect the system's suitability or the content determination results.

[0085] In summary, this invention establishes a method for identifying and determining the content of Danshen (Salvia miltiorrhiza) in Ganoderma lucidum and Salvia miltiorrhiza oral liquid, thereby comprehensively controlling the quality of the preparation. Simultaneously, a detection method using gradient elution with 0.05% phosphoric acid (A)-acetonitrile (B) as the mobile phase was discovered.

Claims

1. A quality detection method of a Ganoderma lucidum and Radix Pseudostellariae oral liquid, characterized in that, The method comprises a thin layer chromatography identification method for salvia miltiorrhiza and angelica and / or a method for determining the content of salvia miltiorrhiza by using liquid chromatography. 2.The quality detection method of Ganoderma lucidum and Radix Pseudostellariae oral liquid according to claim 1, characterized in that, The thin layer chromatography identification method for salvia miltiorrhiza adopts two controls of salvia miltiorrhiza control medicinal materials and sodium danshensu. 3.The quality detection method of Ganoderma lucidum and Radix Pseudostellariae oral liquid according to claim 2, characterized in that, The thin layer chromatography identification method comprises any one or any two of the following identification methods: (1) the thin layer chromatography identification method for salvia miltiorrhiza Preparation of test sample solution: 30-50 ml of lingzhi shuangshen oral liquid sample is taken, the pH value is adjusted to 1-2 by hydrochloric acid, and ether is added for shaking extraction for three times, 40 ml each time, the ether liquid is combined, the solvent is evaporated, and the residue is dissolved in 1 ml of methanol. Preparation of control solution: 0.5 g of salvia miltiorrhiza control medicinal materials is taken, water is added, heated for reflux for 2 hours, filtered, the filtrate is diluted with water to 20 ml, the pH value is adjusted to 1-2 by hydrochloric acid, and ether is added for shaking extraction for three times, 40 ml each time, the ether liquid is combined, evaporated, and the residue is dissolved in 1 ml of methanol; Preparation of sodium danshensu control solution: an appropriate amount of sodium danshensu control is taken, and methanol is added to prepare a solution containing 1 mg per 1 ml; Determination: 5-10 μl of test sample solution, 5 μl of salvia miltiorrhiza control medicinal material solution, and 2 μl of sodium danshensu control solution are taken and spotted on the same silica gel G thin layer plate; chloroform-acetone-acetic acid 25:10:4 is used as a developing agent for development; after development, the thin layer plate is taken out, dried, smoked in ammonia vapor for about 10 minutes, taken out and placed for more than half an hour, and observed under ultraviolet light 365 nm; in the test sample chromatogram, the same color fluorescent spots are shown at the corresponding positions of the salvia miltiorrhiza control medicinal material chromatogram and the sodium danshensu control solution chromatogram, which is regarded as positive identification of salvia miltiorrhiza. (2) the thin layer chromatography identification method for angelica Preparation of test sample solution: 30-50 ml of lingzhi shuangshen oral liquid sample is taken, the pH value is adjusted to 1-2 by hydrochloric acid, and ether is added for shaking extraction for three times, 40 ml each time, the ether liquid is combined, the solvent is evaporated, and the residue is dissolved in 1 ml of methanol. Preparation of control solution: 0.5 g of salvia miltiorrhiza control medicinal materials is taken, water is added, heated for reflux for 2 hours, filtered, the filtrate is diluted with water to 20 ml, the pH value is adjusted to 1-2 by hydrochloric acid, and ether is added for shaking extraction for three times, 40 ml each time, the ether liquid is combined, evaporated, and the residue is dissolved in 1 ml of methanol; Determination: 5-10 μl of test sample solution, 5 μl of salvia miltiorrhiza control medicinal material solution, and 2 μl of sodium danshensu control solution are taken and spotted on the same silica gel G thin layer plate; chloroform-acetone-acetic acid 25:10:4 is used as a developing agent for development; after development, the thin layer plate is taken out, dried, smoked in ammonia vapor for about 10 minutes, taken out and placed for more than half an hour, and observed under ultraviolet light 365 nm; in the test sample chromatogram, the same color fluorescent spots are shown at the corresponding positions of the salvia miltiorrhiza control medicinal material chromatogram and the sodium danshensu control solution chromatogram, which is regarded as positive identification of salvia miltiorrhiza. When the content of salvia miltiorrhiza is determined by using liquid chromatography, danshenshan B is used as a control. The determination of the content of salvia miltiorrhiza by using liquid chromatography comprises the following steps:

4. The quality detection method of Ganoderma lucidum and Radix Pseudostellariae oral liquid according to claim 1, characterized in that, Preparation of control solution: an appropriate amount of danshenshan B control is weighed and accurately measured, dissolved in 80% methanol and diluted to prepare a solution containing about 0.16 mg per 1 ml as a control solution; 5. The quality detection method of the Ganoderma lucidum and Radix Pseudostellariae oral liquid according to claim 4, characterized in that, Preparation of negative solution of salvia miltiorrhiza: 10 ml of negative sample of salvia miltiorrhiza is taken into a 15 ml volumetric flask, diluted with methanol and diluted to the mark, shaken well, filtered through a 0.45 μm filter membrane, and the filtered solution is taken as a negative solution of salvia miltiorrhiza; ​ ​ Preparation of test solution: precisely take 10 ml of the product into a 15 ml volumetric flask, dilute with methanol and make up to the mark, shake well, filter through a 0.45 μm filter membrane, take the filtrate as the test solution; Determination: precisely take 10 μl of each of the above solutions, inject into the liquid chromatograph respectively, record the chromatogram; the chromatographic column is Agilent ZORBAX SB-C18 5 μm 4.6*250 mm stainless steel column, the mobile phase is A. 0.05% phosphoric acid, B. acetonitrile.

6. The quality detection method of the Ganoderma lucidum and Radix Pseudostellariae oral liquid according to claim 5, characterized in that, Gradient elution is carried out according to the following table: 。

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