Identification method of ginseng polysaccharides in compound fluorouracil oral solution

Abstract

This invention provides a sample pretreatment method and identification method for identifying ginseng polysaccharides in compound fluorouracil oral solution, belonging to the field of pharmaceutical detection and analysis technology. The sample pretreatment method of this invention includes: centrifuging the compound fluorouracil oral solution and taking the clear solution; adding silica gel to the clear solution, mixing, and removing the solvent to obtain a solid mixture; adding ethanol solution or anhydrous ethanol to the obtained solid mixture, heating and refluxing, discarding the ethanol solution, and obtaining the residue; adding water to the obtained residue, sequentially extracting and centrifuging, taking the supernatant obtained by centrifugation, removing the solvent from the supernatant, and then redissolving it with water; the identification method of this invention is thin-layer chromatography analysis, and the developing solvent includes the following components in parts by volume: 6.8-9.2 parts of n-propanol, 1.76-2.24 parts of water, 0.75-1.24 parts of ethyl acetate, and 0.75-1.24 parts of glacial acetic acid. The sample pretreatment method and identification method for identifying ginseng polysaccharides in compound fluorouracil oral solution of this invention have high specificity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing and analysis technology, and in particular to a method for identifying ginseng polysaccharides in compound fluorouracil oral solution. Background Technology

[0002] Ginseng is a member of the Araliaceae family. Panax ginseng The dried roots and rhizomes of *Camellia sinensis* are a precious traditional Chinese medicine with unique pharmacological activities. Ginseng contains various active ingredients such as polysaccharides, saponins, volatile oils, and amino acids. Among them, ginseng polysaccharides, as one of the important active ingredients in ginseng, have pharmacological effects such as anti-tumor, antioxidant, and immunomodulatory effects, and are safe and non-toxic. The composition and structure of ginseng polysaccharides are very complex, making isolation, purification, and structural analysis extremely difficult, which has led to slow progress in the research on quality control methods and structure-activity relationships of ginseng polysaccharides.

[0003] Compound fluorouracil oral solution is a chemical preparation made from fluorouracil, ginseng polysaccharide, soybean lecithin, water, and suitable excipients. It is an anti-tumor drug mainly used for the treatment of digestive tract cancers (colon cancer, rectal cancer, gastric cancer), breast cancer, and primary liver cancer. Ginseng polysaccharide enhances the patient's immunity while reducing the clinical toxicity of fluorouracil. The current standard for compound fluorouracil oral solution is "National Standard for Chemical Drugs (Volume 7)". This standard was revised in "National Standard for Chemical Drugs (Volume 16)". The specific revisions are: "[Execution Standard] [Prescription] Added: Appropriate amount of excipients; [Drug Alias] Former name added: Compound fluorouracil emulsion". The original standard used silica gel thin-layer chromatography to identify ginseng polysaccharide. After scraping the silica gel from the origin and eluting with water, the aqueous solution reacted with phenol-sulfuric acid solution to form a yellow-brown ring. Verification revealed that this method has poor specificity; a negative result also showed the same color reaction.

[0004] In accordance with the opinions in the comprehensive opinion sheet of the research project "Compound Fluorouracil Oral Solution (Project No.: 2020H056)" of the Pharmacopoeia Commission of the People's Republic of China, which stated that "the original standard did not consider the quality control indicators of ginseng polysaccharide, another component of the compound preparation, and it is necessary to raise the standard" and "study the corresponding quality standards for ginseng polysaccharide and include them in the standard as appropriate," the quality control methods for ginseng polysaccharide were studied. The study confirmed that the interfering component was soybean lecithin, leading to frequent discrepancies between test results and actual findings. Existing research reports that quantitative analysis methods for ginseng polysaccharide and its preparations mostly employ ultraviolet-visible spectrophotometry and high-performance liquid chromatography (HPLC). However, during the actual research process, it was found that ultraviolet-visible spectrophotometry was not suitable because this preparation is a compound preparation; HPLC analysis of ginseng polysaccharide is limited to molecular weight analysis, and quantitative analysis requires the addition of a certain amount of derivatizing reagents, resulting in weak specificity of the analytical method after derivatization; qualitative analysis methods mostly employ chemical reactions, such as phenol-sulfuric acid reaction and carbazole-sulfuric acid reaction, which make it difficult to completely remove interfering components during the research process. The application of thin-layer chromatography (TLC) in the identification of polysaccharide components usually involves acid hydrolysis into monosaccharide components, followed by TLC analysis. However, research has shown that when ginseng polysaccharides are acid-hydrolyzed, they mostly yield glucose, rendering them insignificant for identification. Summary of the Invention

[0005] To address the issue of poor specificity in the existing physicochemical identification of ginseng polysaccharides in compound fluorouracil oral solution, this invention provides a sample pretreatment method for identifying ginseng polysaccharides in compound fluorouracil oral solution with good specificity.

[0006] Meanwhile, the present invention also provides a method for identifying ginseng polysaccharides in compound fluorouracil oral solution with high specificity and good stability.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a sample pretreatment method for identifying ginseng polysaccharides in compound fluorouracil oral solution, comprising the following steps: S1. After centrifuging the compound fluorouracil oral solution, take the clear solution; S2. Add silica gel to the clear solution obtained in step S1, mix, and remove the solvent from the system to obtain a solid mixture. S3. Add ethanol solution or anhydrous ethanol to the solid mixture obtained in step S2 and heat under reflux. After the heating and reflux are completed, discard the ethanol solution to obtain the residue. S4. Add water to the residue obtained in step S3, and perform extraction and centrifugation in sequence. Then take the supernatant obtained by centrifugation, remove the solvent from the supernatant, and add water to dissolve it again.

[0008] The compound fluorouracil oral solution is actually a liposomal formulation, but its encapsulation rate is less than 80%, so it cannot be managed as a liposomal formulation. Because fluorouracil has significant toxic side effects, a portion of it needs to be encapsulated. Therefore, soybean lecithin is used for encapsulation. To further reduce toxic side effects, ginseng polysaccharides are added, which can both reduce side effects and have anti-tumor activity. However, ginseng polysaccharides have a large molecular weight and are difficult to encapsulate with soybean lecithin, resulting in the formulation's characteristic of separating upon prolonged storage; when mixed, it resembles an emulsion.

[0009] The sample pretreatment method of the present invention uses a physical method (high-speed centrifugation) to physically break the emulsion and remove most of the soybean lecithin, leaving ginseng polysaccharides in the solution layer; the clear solution is taken, adsorbed with silica gel, and then eluted with ethanol, since soybean lecithin is soluble in ethanol. After removing the remaining soybean lecithin, the ginseng polysaccharides in the silica gel are then extracted by shaking with water to obtain a solution containing only ginseng polysaccharide components.

[0010] The sample solution prepared using the above-described sample pretreatment method of the present invention for identifying ginseng polysaccharides exhibits good specificity.

[0011] In some embodiments, in step S2, the ratio of the mass of silica gel to the volume of the clarified solution taken is 1-1.5 g: 1 mL.

[0012] In some embodiments, the silica gel is thin-layer chromatography silica gel G. Using thin-layer chromatography silica gel G can effectively adsorb ginseng polysaccharides and prevents them from being lost during elution.

[0013] In some embodiments, in step S3, the volume percentage of ethanol in the ethanol solution is 95% or more.

[0014] In some embodiments, the heating reflux time is 40-60 minutes.

[0015] In some embodiments, in step S3, the volume percentage of ethanol in the ethanol solution is 95%, and the heating and reflux time is 40 minutes. Soybean lecithin has good solubility in a 95% (v / v) ethanol solution; the preparation process of ginseng polysaccharide in this invention is a water extraction and alcohol precipitation process, so ethanol can effectively extract soybean lecithin components while retaining ginseng polysaccharide.

[0016] In some embodiments, the compound fluorouracil oral solution comprises the following components: fluorouracil, ginseng polysaccharide, soybean lecithin, and water. In some embodiments, the compound fluorouracil oral solution also includes other excipients. The compound fluorouracil oral solution can be prepared according to the methods specified in the "National Standards for Chemical Drugs (Upgraded to National Standards, Volume VII)" and the "National Standards for Chemical Drugs (Upgraded to National Standards, Volume XVI)".

[0017] Secondly, the present invention provides a method for identifying ginseng polysaccharides in compound fluorouracil oral solution, comprising the following steps: The sample pretreatment was performed using the above method to obtain the test solution; Using ginseng as a reference herb, a ginseng reference herb solution was prepared. The test solution and the ginseng reference solution were spotted onto the same thin-layer plate, developed with a developing solvent, and then sprayed with a colorimetric reagent for thin-layer chromatographic analysis. The developing solvent consisted of the following components in parts by volume: 6.8-9.2 parts of n-propanol, 1.76-2.24 parts of water, 0.75-1.24 parts of ethyl acetate, and 0.75-1.24 parts of glacial acetic acid.

[0018] The applicant of this invention has discovered through extensive research that using a mixed solution comprising the above-mentioned n-propanol, water, ethyl acetate, and glacial acetic acid as a developing solvent results in better development and can effectively improve the R-value of the spots. f It has the value and can improve the morphology of spots and prevent spot tailing.

[0019] The thin-layer chromatography method described above for identifying ginseng polysaccharides in compound fluorouracil oral solution of the present invention exhibits good sample spot separation, no negative interference, and can specifically identify ginseng polysaccharide components. Furthermore, the robustness of the thin-layer chromatography method of the present invention was investigated through experimental examples, revealing that the detection process of the thin-layer chromatography identification method is not easily affected by changes in external conditions such as the thin-layer plate, temperature, and humidity, demonstrating that the method has low requirements for external environment and good robustness.

[0020] In some embodiments, the developing solvent comprises the following components in parts by volume: 8 parts n-propanol, 2 parts water, 1 part ethyl acetate, and 1 part glacial acetic acid. Using this developing solvent provides optimal development results, clear ginseng polysaccharide spots, good separation, and no interference from negative samples during thin-layer chromatography detection. It also exhibits strong specificity and good durability.

[0021] In some embodiments, the colorimetric agent is an anhydrous ethanol-sulfuric acid solution of carbazole; preferably, the concentration of carbazole in the anhydrous ethanol-sulfuric acid solution of carbazole is 0.05%-0.2% (g / mL).

[0022] In some embodiments, the concentration of carbazole in the anhydrous ethanol-sulfuric acid solution is 0.2% (g / mL). It can be prepared by weighing 0.2 g of carbazole, dissolving it in 100 mL of anhydrous ethanol, adding 10 mL of sulfuric acid, stirring constantly, and cooling to room temperature.

[0023] In some embodiments, the sampling method is strip-shaped sampling; preferably, the strip width is 6 mm. The sample is dried with hot air immediately after sampling.

[0024] In some embodiments, the volume of the spotting is 4-10 μL, preferably 6 μL.

[0025] In some embodiments, the developing agent is used to develop the material as follows: a double-groove developing tank is used, the developing agent is added to one side, and a thin film is placed on the other side. The tank is then sealed and the developing agent is used to saturate the thin film for about 30 minutes. Then, the developing tank is tilted so that the developing agent is poured into the other side. The material is developed to about 8cm-10cm, then removed and dried.

[0026] In some embodiments, the thin film is a silicone G thin film, preferably an HSG thin film.

[0027] In some embodiments, after spraying with a color developer, the sample is heated to 80°C until the spots are clearly visible and then examined under sunlight.

[0028] In some embodiments, the method for preparing the ginseng reference herb solution is as follows: ethanol solution is added to the ginseng reference herb and heated under reflux. After the heating and reflux are completed, the residue is removed. Then, water is added to the residue, and the decoction is prepared and the supernatant is removed. The supernatant is concentrated to obtain a concentrated solution. Ethanol is added to the concentrated solution, and the mixture is centrifuged. The precipitate obtained by centrifugation is removed, and water is added to the precipitate. After mixing, the supernatant is removed.

[0029] In some embodiments, a 95% (v / v) ethanol solution is added to the ginseng reference material and heated under reflux; preferably, the mass of the ethanol solution is 10 times the mass of the ginseng reference material.

[0030] In some embodiments, when water is added to the dregs, the mass of the water is 15-20 times the mass of the ginseng reference material.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention not only provides a sample pretreatment method for identifying ginseng polysaccharides in compound fluorouracil oral solution with high specificity, but also establishes an identification method for ginseng polysaccharides by preparing a control solution using ginseng as a reference herb and the ginseng polysaccharide prototype. The identification method for ginseng polysaccharides in compound fluorouracil oral solution of this invention is simple and rapid to operate, produces clear spots, has high specificity and good stability, and is suitable for wide application.

[0032] This invention establishes a quality control method for ginseng polysaccharide components in compound fluorouracil oral solution, which has important practical significance for improving the quality standards and clinical use of compound fluorouracil oral solution. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 The following are thin-layer chromatograms of the test solution, ginseng reference material solution, and negative solution in the examples; wherein, 1-negative solution (A), 2-test solution (A1), 3-test solution (A2), 4-test solution (A3), 5-ginseng reference material solution, 6-test solution (A4), 7-test solution (A5), 8-test solution (A6), and 9-negative solution (B).

[0035] Figure 2 and Figure 3 The thin-layer chromatogram for the identification of ginseng polysaccharides in the current standard of compound fluorouracil oral solution in Experiment Example 1; Figure 2 In Chinese: 1-Blank reagent, 2-Test solution A1, 3-Negative solution A; Figure 3 In the middle: 1-Negative solution A, 2-Test solution A1, 3-Ginseng reference material solution, 4-Test solution A4, 5-Negative solution B.

[0036] Figure 4 and Figure 5 This is a thin-layer chromatogram of the test solution preparation method investigated in Example 2; Figure 4 In the examples, 1-negative solution A of comparative example 7, 2-test solution A1 of comparative example 6, 3-ginseng reference medicinal material solution, 4-test solution A1 of the example, and 5-negative solution A of the example; Figure 5 In the above, 1-negative solution A of comparative example 5, 2-test solution A1 of comparative example 4, 3-ginseng reference medicinal material solution, 4-test solution A1 of example, and 5-negative solution A of example.

[0037] Figure 6 The following are thin-layer chromatograms for the sample quantity investigation in Example 3; where 1-5 are ginseng reference solutions from the examples (1-2 μL, 2-4 μL, 3-6 μL, 4-8 μL, 5-10 μL); 6 is negative solution A from the example; and 7-11 are test solution A1 from Example 1 (7-2 μL, 8-4 μL, 9-6 μL, 10-8 μL, 11-10 μL).

[0038] Figure 7The image shows a thin-layer chromatogram for examining the width of the spotted bands in Example 4; where 1-3 are the test solution A1 (1-4mm, 2-6mm, 3-8mm) of the example, 4 is the ginseng reference material solution, and 5 is the negative solution A.

[0039] Figure 8 and Figure 9 The thin-layer chromatogram of the colorimetric reagent used in Experimental Example 5; Figure 8 The intermediate reagent is a 0.2% carbazole anhydrous ethanol-sulfuric acid (10:1) solution; Figure 9 The colorimetric reagent is a 0.2% anthrone anhydrous ethanol-sulfuric acid (10:1) solution. Figures 8-9 In the table, 1-test solution (A1), 2-test solution (A2), 3-ginseng reference material solution, and 4-negative solution A.

[0040] Figures 10-12 This is a thin-layer chromatogram for investigating the composition of the developing solvent in Experimental Example 6; Figure 10 The developing solvent was n-propanol-water-ethyl acetate-glacial acetic acid (8:2:1:1). Figure 11 The developing solvent is n-butanol-methanol-chloroform-glacial acetic acid-water (12.5:4.5:5:1.5:1.5). Figure 12 The developing solvent is n-butanol-methanol-formic acid-water-dichloromethane (13:5:1.5:2:5). Figures 10-12 In the table, 1-test solution (A1), 2-test solution (A2), 3-ginseng reference material solution, and 4-negative solution A.

[0041] Figure 13 The thin-layer chromatograms are for the specificity investigation in Example 7; where 1-negative solution (A), 2-test solution (A1), 3-ginseng reference material solution, 4-test solution (A4), and 5-negative solution (B).

[0042] Figures 14-17 The following are the thin-layer chromatograms of thin-layer plates from different sources used in Experimental Example 8; among them, Figure 14 For Merck silicone G thin-layer prefabricated panels, Figure 15 This is a silicone G thin-layer prefabricated panel for Yantai Huayang New Materials Co., Ltd. Figure 16 This is a Qingdao marine silicone G thin-layer precast panel. Figure 17 Silicone G thin-layer prefabricated board for Tianjin Slida Technology Co., Ltd.; Figures 14-17 In the table, 1-test solution (A1), 2-test solution (A2), 3-test solution (A3), and 4-ginseng reference material solution.

[0043] Figures 18-20 The thin-layer chromatogram for the temperature investigation in Example 9; Figure 18 The temperature is 30℃. Figure 19 The temperature is 8℃. Figure 20 The temperature is 22℃; Figures 18-20 In the table, 1-test solution (A1), 2-test solution (A2), 3-test solution (A3), and 4-ginseng reference material solution.

[0044] Figures 21-23 The thin-layer chromatogram for the relative humidity investigation in Example 10; Figure 21 The relative humidity is 18%. Figure 22 The relative humidity is 47%. Figure 23 The relative humidity was 72%. Figures 21-23 In the sample, 1-test solution (A1), 2-test solution (A2), 3-test solution (A3), 4-ginseng reference material solution.

[0045] Figures 24-31 This is a thin-layer chromatogram for investigating the developing solvent ratio in Experimental Example 11; Figure 24 The developing solvent ratio of n-propanol:water:ethyl acetate:glacial acetic acid is 8:2:1:0.75; Figure 25 The ratio of n-propanol:water:ethyl acetate:glacial acetic acid in the developing solvent is 8:2:1:1.24. Figure 26 The developing solvent ratio of n-propanol:water:ethyl acetate:glacial acetic acid is 8:2:0.75:1; Figure 27 The developing solvent ratio of n-propanol:water:ethyl acetate:glacial acetic acid is 8:2:1.24:1. Figure 28 The developing solvent ratio of n-propanol:water:ethyl acetate:glacial acetic acid is 8:1.76:1:1. Figure 29 The developing solvent ratio of n-propanol:water:ethyl acetate:glacial acetic acid is 8:2.24:1:1. Figure 30 The ratio of n-propanol:water:ethyl acetate:glacial acetic acid in the developing solvent is 6.8:2:1:1; Figure 31 The developing solvent ratio of n-propanol:water:ethyl acetate:glacial acetic acid is 9.2:2:1:1. Figures 24-31 In the table, 1-test solution (A1), 2-test solution (A2), 3-test solution (A3), and 4-ginseng reference material solution. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0047] The present invention will be further described below through specific embodiments.

[0048] The instruments and reagents used in this invention are as follows: 1. Instruments: Linomat5 semi-automatic spotter (Karma, Switzerland), TG16-WS high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), BS323S electronic balance (Beijing Sartorius Instrument Balance Co., Ltd.).

[0049] 2. Reagents: n-Propanol, n-Butanol, Trichlorohexane, Dichloromethane, Formic acid, Methanol, Anhydrous ethanol, 95% ethanol, Ethyl acetate, Glacial acetic acid, Sulfuric acid, Anthrone, Carbazole, Silica G for thin-layer chromatography, and Diatomaceous earth were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. Unless otherwise specified in this invention, all reagents used are analytical grade. Silica G thin-layer prefabricated plates: Qingdao Marine Chemical Plant, 2020217; Yantai Huayang New Material Technology Co., Ltd., 2022-12-07; Merck KCaA, HX02024426; Tianjin Slida Technology Co., Ltd., 20241223.

[0050] 3. Test Drugs: Ginseng reference material (120917-201712) was purchased from the China National Institutes for Food and Drug Control; six batches of compound fluorouracil oral solution came from two companies. Both companies' compound fluorouracil oral solutions consisted of the following substances: fluorouracil, ginseng polysaccharide, soybean lecithin, cholesterol, vitamin E, sodium dihydrogen phosphate monohydrate, polysorbate 80, oleic acid, disodium hydrogen phosphate, povidone K30, and water. All tests met the requirements of the "National Chemical Drug Local Standard Upgrade to National Standard Volume VII". Sample information is shown in Table 1.

[0051] Table 1. Sample Information of Compound Fluorouracil Oral Solution

[0052] Example An embodiment of the method for identifying ginseng polysaccharides in compound fluorouracil oral solution of the present invention is as follows: (1) Preparation of the test solution Take 10 mL of each of the six batches of compound fluorouracil oral solution in Table 1, centrifuge at high speed (10000 r / min) for 10 minutes, discard the liposome layer, take 2 mL of the clear solution, add 2 g of thin-layer chromatography silica gel G, grind evenly, evaporate to dryness, place in a Soxhlet extractor, add an appropriate amount of 95% ethanol, reflux for 40 minutes, discard the ethanol solution; evaporate the solvent from the residue, place in an Erlenmeyer flask, add 20 mL of water, shake to extract for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 2 mL of water to dissolve, and obtain the test solution A1, A2, A3, A4, A5, and A6.

[0053] (2) Preparation of ginseng reference herb solution Take 3g of ginseng reference material, add 50mL of 95% ethanol, reflux for 30 minutes, remove the residue and evaporate until there is no alcohol taste, add 50mL of water and decoct for 1 hour, centrifuge, take the supernatant, concentrate to about 5mL, take 2mL of the aqueous solution, add 8mL of anhydrous ethanol, shake for 1 minute, centrifuge, take the precipitate, add 5mL of water, stir to dissolve, take the supernatant, and you will get the ginseng reference material solution.

[0054] (3) Preparation of negative solution Take the other components (which do not contain ginseng polysaccharide) from the compound fluorouracil oral solution provided by Company A and Company B in Table 1, respectively, and prepare negative solutions according to the sample preparation process. Take 10 mL of the above negative solution, centrifuge at high speed (10000 r / min) for 10 minutes, discard the liposome layer, take 2 mL of the clear solution, add 2 g of thin-layer chromatography silica gel G, grind evenly, evaporate to dryness, place in a Soxhlet extractor, add an appropriate amount of 95% ethanol, reflux for 40 minutes, discard the ethanol solution; evaporate the solvent from the residue, place it in an Erlenmeyer flask, add 20 mL of water, shake to extract for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 2 mL of water to dissolve, and obtain negative solution A (prepared from other components without ginseng polysaccharide provided by Company A) and negative solution B (prepared from other components without ginseng polysaccharide provided by Company B).

[0055] (4) Thin-layer chromatography detection According to the thin-layer chromatography method (General Chapter 0502 of the 2025 edition of the Chinese Pharmacopoeia), 6 μL each of the six batches (numbered A1, A2, A3, A4, A5, A6) of test solutions prepared in the preparation of the test solution, the two batches (A, B) of negative solutions prepared in the preparation of negative solutions, and the ginseng reference material solution were spotted onto the same silica gel G thin-layer plate in strip form, with a band width of 6 mm. The plates were dried immediately with hot air and then analyzed with n-propanol-water-ethyl acetate. - Using glacial acetic acid (in a volume ratio of 8:2:1:1 for n-propanol, water, ethyl acetate, and glacial acetic acid) as the developing solvent, develop in a double-tank developing tank until approximately 8 cm deep. Remove, air dry, and spray with 0.2% carbazole anhydrous ethanol-sulfuric acid solution (in a volume ratio of 10:1 for anhydrous ethanol and sulfuric acid). Heat at 80°C until the spots are clearly visible. Examine under sunlight. In the chromatogram of the test sample, a single identical blue-purple main spot should be detected at the corresponding position in the chromatogram of the ginseng reference material.

[0056] Test results are shown Figure 1The results showed that in the chromatogram of the test sample, a single blue-purple main spot was detected at the corresponding position in the chromatogram of the ginseng reference material. Other components without ginseng polysaccharides provided by companies A and B did not interfere with the identification of ginseng polysaccharide components in the compound fluorouracil oral solution.

[0057] Comparative Example 1: Preparation of the test solution Take more than 1.5 mL of compound fluorouracil oral solution and spot it in a strip on a silica gel G thin-layer plate. Use methanol-chloroform (3:7) as the developing solvent. After development, dry the plate, scrape off the original spot strip, wash with 3-4 mL of water, and filter.

[0058] Preparation of negative solution in Comparative Example 2 Prepare a negative solution by taking other components provided by the company that do not contain ginseng polysaccharides. Take more than 1.5 mL of the negative solution and process it in the same way as the test solution of Comparative Example 1.

[0059] Comparative Example 3: Preparation of Ginseng Control Herb Solution Take 1g of ginseng reference material, add 20mL of ethanol, sonicate at 80℃ for 1 hour, discard the ethanol solution, evaporate the solvent from the material, add 20mL of water, sonicate under the same conditions for 1 hour, place at room temperature, centrifuge, take the supernatant, concentrate to 10mL, and obtain the final product.

[0060] Example 1: Identification of ginseng polysaccharides in the current standard of Compound Fluorouracil Oral Solution (National Drug Standard of the State Food and Drug Administration: WS-10001-(HD-0634)-2002). Samples from Company A (No. A1) and Company B (No. A4) were processed according to the preparation method of the test solution in Comparative Example 1 to obtain test solutions A1 and A4. Other components without ginseng polysaccharides provided by Company A and Company B were processed according to the preparation method of the negative solution in Comparative Example 2 to obtain negative solution A (prepared from other components without ginseng polysaccharides provided by Company A) and negative solution B (prepared from other components without ginseng polysaccharides provided by Company B). Test solution A1 and negative solution A of this experimental example were added to 2 mL of 5% phenol aqueous solution, mixed well, and 2 mL of sulfuric acid was slowly added along the wall. 2 mL of water was used as a reagent blank and treated in the same way as a control.

[0061] Test results are shown Figure 2 The results showed that the current standard (standard number: ws-10001-(HD-0634)-2002) for compound fluorouracil oral solution was significantly interfered with by negative results.

[0062] Further, take 5 μL each of the test solutions A1 and A4, negative solutions A and B, and the ginseng control solution of Comparative Example 3, and spot them on the same silica gel G thin-layer plate. Use methanol-chloroform (volume ratio 3:7) as the developing solvent and develop to about 8 cm. Remove and air dry. Then use n-butanol-methanol-chloroform-glacial acetic acid-water (volume ratio of n-butanol, methanol, chloroform, glacial acetic acid, and water is 12.5:4.5:5:1.5:1.5) as the developing solvent for a second development. Develop to about 8 cm. Remove and air dry. Spray with 0.2% anthrone anhydrous ethanol-sulfuric acid (10:1) solution and heat at 80°C until the spots are clear.

[0063] See results Figure 3 The results showed that, in this experimental case, the other components provided by companies A and B that did not contain ginseng polysaccharides showed spots of the same color at the corresponding positions in the chromatogram of the ginseng reference material; the polysaccharide components extracted by ultrasound from the ginseng reference material showed two main blue spots under the same conditions.

[0064] Comparative Example 4: Preparation of the test solution Take 2 mL of the compound fluorouracil oral solution, shake well, add 2 g of thin-layer chromatography silica gel G, grind evenly, evaporate to dryness, place in a Soxhlet extractor, add an appropriate amount of 95% ethanol, soak for 2 hours, then reflux for 2 hours, discard the ethanol solution, evaporate the solvent from the silica gel and drug residue, place in an Erlenmeyer flask, add 20 mL of water, shake and extract for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 2 mL of water to dissolve, and the product is obtained.

[0065] Preparation of negative solution in Comparative Example 5 Prepare a negative solution by taking other components provided by the company that do not contain ginseng polysaccharides. Take 2 mL of the negative solution, add 2 g of thin-layer chromatography silica gel G, grind evenly, evaporate to dryness, place in a Soxhlet extractor, add an appropriate amount of 95% ethanol, soak for 2 hours, then reflux for 2 hours, discard the ethanol solution, evaporate the solvent from the silica gel and residue, place in an Erlenmeyer flask, add 20 mL of water, shake and extract for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 2 mL of water to dissolve, and the product is obtained.

[0066] Comparative Example 6: Preparation of the test solution Shake the compound fluorouracil oral solution well, measure 2 mL, add an appropriate amount of diatomaceous earth, grind evenly, evaporate to dryness, place in a Soxhlet extractor, add an appropriate amount of ether, reflux for 1 hour; evaporate the solvent, add an appropriate amount of 95% ethanol, reflux for another 1 hour, evaporate the solvent from the diatomaceous earth and residue, place in an Erlenmeyer flask, add 20 mL of water, shake to extract for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 2 mL of water to dissolve, and the product is obtained.

[0067] Preparation of negative solution in Comparative Example 7 Prepare a negative solution by taking other components provided by the company that do not contain ginseng polysaccharides. Take 2 mL of the negative solution, add an appropriate amount of diatomaceous earth, grind evenly, evaporate to dryness, place in a Soxhlet extractor, add an appropriate amount of ether, and reflux for 1 hour. Evaporate the solvent, add an appropriate amount of 95% ethanol, reflux for another 1 hour, discard the ethanol solution, evaporate the solvent of the diatomaceous earth and residue, place in an Erlenmeyer flask, add 20 mL of water, shake and extract for 30 minutes, centrifuge, take the supernatant, evaporate to dryness, add 2 mL of water to dissolve, and the product is obtained.

[0068] Experimental Example 2: Investigation of the preparation method of the test solution Take sample A (number A1) from Company A and other components that do not contain ginseng polysaccharide, and process them according to the preparation methods of test solution and negative solution in the examples to obtain test solution A1 and negative solution A. Process them in the same way as comparative examples 4, 5, 6 and 7 to obtain test solution A1 of comparative example 4, negative solution A of comparative example 5, test solution A1 of comparative example 6 and negative solution A of comparative example 7. Take 6 μL of each of the above solutions and the ginseng reference medicinal material solution in the examples, spot them on the same silica gel G thin layer plate in strip form with a width of 6 mm, and dry them with hot air in time. Use n-propanol-water-ethyl acetate-glacial acetic acid (volume ratio of n-propanol, water, ethyl acetate and glacial acetic acid is 8:2:1:1) as the developing solvent, and develop in a double tank. Develop to about 8 cm, remove, air dry, spray with 0.2% carbazole anhydrous ethanol-sulfuric acid (10:1) solution, heat at 80°C until the spots are clear, and examine under sunlight.

[0069] See results Figures 4-5 The results showed that although the same blue-purple main spot was detected at the corresponding position in the thin-layer chromatography of the ginseng reference material in the examples and comparative examples 4 and 6, the same blue-purple spot was also detected at the corresponding position in the negative solution chromatograms prepared in comparative examples 5 and 7. In the negative solution prepared in comparative example 5, the interfering streak was invisible when the spotting band width reached 8 mm, while the interfering streak was still present in the negative solution prepared in comparative example 7. This indicates that the test solution preparation method provided by the present invention has good specificity.

[0070] Experimental Example 3: Investigation of Sample Size Take 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of the test solution A1 from the example, and 6 μL of the ginseng reference solution from the example, and spot them separately on the same silica gel G thin-layer plate, and dry them immediately with hot air. Spot the samples in strip form with a bandwidth of 2 mm. Use n-propanol-water-ethyl acetate-glacial acetic acid (volume ratio of n-propanol, water, ethyl acetate, and glacial acetic acid is 8:2:1:1) as the developing solvent in a double-tank developing tank. Develop to approximately 8 cm, remove, air dry, spray with 0.2% carbazole anhydrous ethanol-sulfuric acid (10:1) solution, and heat at 80°C until the spots are clear. Examine under sunlight.

[0071] See results Figure 6 The results showed that when the sample volume of the ginseng reference solution was 2–10 μL, and when the sample volume of the test solution was 4–10 μL, the spots were clear. It was verified that the sample volume should not be too large, as this would affect the shape of the spots. Therefore, the sample volume for both the test solution and the ginseng reference solution was 6 μL, and a strip-like spotting method was chosen.

[0072] Experimental Example 4: Investigation of the Width of the Spotted Strip Take 6 μL each of the negative solution A from Example 1, the ginseng reference herb solution from Example 2, and the test solution A1 from Example 3, and spot them separately on the same silica gel G thin-layer plate. The spot widths of the test solution are 4 mm, 6 mm, and 8 mm, respectively, while the spot widths of the ginseng reference herb solution and negative solution A are 6 mm. Dry them immediately with hot air. Use n-propanol-water-ethyl acetate-glacial acetic acid (volume ratio of n-propanol, water, ethyl acetate, and glacial acetic acid is 8:2:1:1) as the developing solvent and develop in a double-tank developing tank until the spots are about 8 cm deep. Remove the plate, air dry it, spray it with 0.2% carbazole anhydrous ethanol-sulfuric acid (10:1) solution, and heat it at 80°C until the spots are clear. Examine them under sunlight.

[0073] See results Figure 7 The width of the spotting strip affects the visualization of ginseng polysaccharide components. Compared with the chromatogram of ginseng reference material solution, a spotting strip width of 6 mm is more suitable, and there is no interference from negative samples. Therefore, a spotting strip width of 6 mm was selected for subsequent studies in this invention.

[0074] Experimental Example 5: Investigation of the Colorimetric Reagent Two batches (numbered A1 and A2) of test solution prepared in the examples, each 6 μL, along with 6 μL of ginseng reference material solution from the examples and 6 μL of negative solution A from the examples, were spotted separately onto the same silica gel G thin-layer plate in strip form, with a width of 6 mm. The plates were immediately dried with hot air. Developed in a double-tank developing tank using n-propanol-water-ethyl acetate-glacial acetic acid (volume ratio of n-propanol, water, ethyl acetate, and glacial acetic acid was 8:2:1:1) as the developing solvent. After development to approximately 8 cm, the plates were removed, air-dried, and sprayed with 0.2% carbazole anhydrous ethanol-sulfuric acid (volume ratio 10:1) solution and 0.2% anthrone anhydrous ethanol-sulfuric acid (volume ratio 10:1) solution, respectively. The plates were then heated to 80°C until the spots were clear and examined under sunlight.

[0075] See results Figure 8 and 9 The results showed that using a 0.2% carbazole anhydrous ethanol-sulfuric acid (volume ratio 10:1) solution as the colorimetric reagent yielded richer thin-layer chromatograms, detecting other spots in addition to the main spot, and with negative interference.

[0076] Experimental Example 6: Investigation of the composition of the developing solvent Two batches (numbered A1 and A2) of test solution prepared in the examples, each 6 μL, along with 6 μL of ginseng reference solution from the examples and 6 μL of negative solution A from the examples, were spotted separately onto the same silica gel G thin-layer plate in strip form, with a width of 6 mm. The plates were immediately dried with hot air. Using three different developing systems as the developing solvent, the plates were developed in a double-barrel developing tank until approximately 8 cm in diameter. After development, the plates were removed, air-dried, sprayed with a 0.2% carbazole anhydrous ethanol-sulfuric acid (10:1) solution, and heated at 80°C until the spots were clear. The results are shown in the figure. Figures 10-12 .

[0077] The three developing agents are: Developing solvent 1: n-propanol-water-ethyl acetate-glacial acetic acid (the volume ratio of n-propanol, water, ethyl acetate, and glacial acetic acid is 8:2:1:1) Developing solvent 2: n-Butanol-Methanol-Chloroform-Glacial Acetic Acid-Water (the volume ratio of n-butanol, methanol, chloroform, glacial acetic acid, and water is 12.5:4.5:5:1.5:1.5) Developing solvent 3: n-Butanol-Methanol-Formic acid-Water-Dichloromethane (the volume ratio of n-butanol, methanol, formic acid, water, and dichloromethane is 13:5:1.5:2:5) The results showed that the Rf value of the main spot obtained by developing solvent 1 was about 0.54, that obtained by developing solvent 2 was about 0.13, and that obtained by developing solvent 3 was about 0.21. When developing solvent 1 was used, the clarity of the spots in the chromatogram of the test sample was moderate, the Rf value was moderate, and there was no interference from negative samples, indicating that the developing system used in this invention can specifically identify ginseng polysaccharides in compound fluorouracil oral solution.

[0078] Experimental Example 7: Specificity Investigation Take 6 μL each of the test solutions (A1 and A4) and the ginseng reference solution prepared according to the examples, and 6 μL each of the negative solutions (A and B) prepared according to the examples from the other components without ginseng polysaccharides provided by Company A and Company B. Spot these solutions separately onto the same silica gel G thin-layer plate in strip form, 6 mm wide, and dry them immediately with hot air. Develop the plate in a double-tank environment using n-propanol-water-ethyl acetate-glacial acetic acid (volume ratio of n-propanol, water, ethyl acetate, and glacial acetic acid is 8:2:1:1) as the developing solvent. Develop the plate to approximately 8 cm, remove it, air dry, spray with 0.2% carbazole ethanol-sulfuric acid (10:1) solution, and heat at 80°C until the spots are clear. Examine the plate under sunlight. The results are shown in the figure. Figure 13 .

[0079] The results showed that in the chromatograms of representative samples from different sources, a single blue-purple main spot was detected at the same position as the chromatogram of ginseng reference material, and there was no negative interference. This indicates that the thin-layer chromatography identification method provided by this invention has good robustness and specificity.

[0080] Experimental Example 8: Investigation of Thin-Layer Plates The following products were examined: Merck silicone G thin-layer precast panels, Yantai Huayang New Material Co., Ltd. silicone G thin-layer precast panels, Qingdao Ocean silicone G thin-layer precast panels, and Tianjin Slida Technology Co., Ltd. silicone G thin-layer precast panels.

[0081] Three batches (numbered A1, A2, and A3) of test sample solutions and the ginseng reference herb solution prepared in the examples, each 6 μL, were spotted onto Merck silica gel G thin-layer plates (Germany, Yantai Huayang New Material, Qingdao Haiyang, and Tianjin Slida), respectively. The spots were spotted in strip form, 6 mm wide, and dried immediately with hot air. The plates were developed in a double-tank developing tank using a solution of n-propanol-water-ethyl acetate-glacial acetic acid (volume ratio of n-propanol, water, ethyl acetate, and glacial acetic acid 8:2:1:1) as the developing solvent. The plates were developed to approximately 8 cm, removed, and air-dried. They were then sprayed with a 0.2% carbazole anhydrous ethanol-sulfuric acid solution (volume ratio of anhydrous ethanol to sulfuric acid 10:1) and heated at 80°C until the spots were clearly visible. Upon examination under sunlight, the same blue-purple main spot was detected at the corresponding position in the chromatograms of the test sample and the ginseng reference herb. Results are shown below. Figures 14-17 .

[0082] The results showed that all four brands of thin-layer prefabricated plates could specifically identify the ginseng polysaccharide component in this product, indicating that the invention is durable and has wide applicability. Subsequent research in this invention will utilize thin-layer prefabricated plates from Tianjin Slida Technology Co., Ltd.

[0083] Experimental Example 9: Investigation of Temperature Take 6 μL each of the three batches (numbered A1, A2, and A3) of test sample solution prepared in the examples and the ginseng reference herb solution from the examples, and spot them separately on the same silica gel G thin-layer plate in strip form, with a width of 6 mm. Dry the plates immediately with hot air. Use n-propanol-water-ethyl acetate-glacial acetic acid (volume ratio of n-propanol, water, ethyl acetate, and glacial acetic acid is 8:2:1:1) as the developing solvent. Develop the plates in a double-tank developing tank at different temperature environments (low temperature 8℃, room temperature 22℃, high temperature 30℃) until the spots reach approximately 8 cm. Remove the plates, air dry them, and spray them with a 0.2% carbazole anhydrous ethanol-sulfuric acid (volume ratio of anhydrous ethanol to sulfuric acid is 10:1). Heat the plates at 80℃ until the spots are clearly visible. Examine the plates under sunlight. In the chromatogram of the test sample, a single identical blue-purple main spot should be detected at the corresponding position in the chromatogram of the ginseng reference herb. Results are shown below. Figures 18-20 .

[0084] The results showed that the spot visualization effect of the chromatogram of the test sample did not differ significantly at different temperatures. The thin-layer chromatography identification method provided by this invention is less affected by temperature changes and has good robustness.

[0085] Experimental Example 10: Investigation of Relative Humidity Take 6 μL each of the three batches (numbered A1, A2, and A3) of test sample solution prepared in the examples and the ginseng reference herb solution from the examples, and spot them separately on the same silica gel G thin-layer plate in strip form with a width of 6 mm. Dry the plates immediately with hot air. Use n-propanol-water-ethyl acetate-glacial acetic acid (volume ratio of n-propanol, water, ethyl acetate, and glacial acetic acid 8:2:1:1) as the developing solvent. Develop the plates in a double-tank developing tank under different humidity conditions (relative humidity 18%, 47%, and 72%) until the spots reach approximately 8 cm. Remove the plates, air dry them, and spray them with a 0.2% carbazole anhydrous ethanol-sulfuric acid (volume ratio of anhydrous ethanol to sulfuric acid 10:1). Heat the plates at 80°C until the spots are clearly visible. Examine the plates under sunlight. In the chromatogram of the test sample, a single identical blue-purple main spot should be detected at the corresponding position in the chromatogram of the ginseng reference herb. Results are shown below. Figures 21-23 .

[0086] The results show that the spot visualization of the test sample chromatogram is similar under different relative humidity conditions, and the spots become clearer as the relative humidity increases. This indicates that the thin-layer chromatography identification method provided by this invention is less affected by relative humidity and has good robustness.

[0087] Experimental Example 11: Investigation of the Proportion of Developing Solvent Take 6 μL each of the three batches (numbered A1, A2, and A3) of test sample solution prepared in the examples and the ginseng reference material solution of the examples, and spot them separately on the same silica gel G thin-layer plate in strip form with a width of 6 mm. Dry them with hot air immediately. Use the following eight developing solvents (all eight developing solvents are composed of n-propanol, water, ethyl acetate, and glacial acetic acid) as developing solvents. Develop in a double-tank developing tank until the spots are about 8 cm deep. Remove the plate, air dry it, spray it with 0.2% carbazole anhydrous ethanol-sulfuric acid (volume ratio 10:1), and heat it at 80°C until the spots are clearly visible. Examine it under sunlight. In the chromatogram of the test sample, a single blue-purple main spot is detected at the corresponding position in the chromatogram of the ginseng reference material.

[0088] The proportions of the eight developing agents are shown in Table 2 below.

[0089] Composition of the developing solvent in Table 2.8

[0090] See results Figures 24-31 The results showed that after fine-tuning the proportions of the four-phase solvents, the Rf values ​​of the blue-violet main spots were within the range of 0.49–0.55, and the corresponding spots were detected in the chromatograms of the test samples. The proportions of n-propanol were within ±10% of the absolute value, and the proportions of water, ethyl acetate, and glacial acetic acid were within ±2% of the absolute value, all meeting the experimental requirements. This indicates that the thin-layer chromatography identification method provided by this invention has good robustness.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sample pretreatment method for identifying ginseng polysaccharides in compound fluorouracil oral solution, characterized in that, Includes the following steps: S1. After centrifuging the compound fluorouracil oral solution, take the clear solution; S2. Add silica gel to the clear solution obtained in step S1, mix, and remove the solvent from the system to obtain a solid mixture. S3. Add ethanol solution or anhydrous ethanol to the solid mixture obtained in step S2 and heat under reflux. After the heating and reflux are completed, discard the ethanol solution to obtain the residue. S4. Add water to the residue obtained in step S3, and perform extraction and centrifugation in sequence. Then take the supernatant obtained by centrifugation, remove the solvent from the supernatant, and add water to dissolve it again.

2. The sample pretreatment method according to claim 1, characterized in that, In step S2, the ratio of the mass of silica gel to the volume of the clarified solution is 1-1.5 g: 1 mL. And / or, the silica gel is thin-layer chromatography silica gel G.

3. The sample pretreatment method according to claim 1 or 2, characterized in that, In step S3, the volume percentage of ethanol in the ethanol solution is 95% or more; and / or, the heating and reflux time is 40-60 minutes. Preferably, in step S3, the volume percentage of ethanol in the ethanol solution is 95%, and the heating and reflux time is 40 minutes.

4. The sample pretreatment method according to any one of claims 1-3, characterized in that, The compound fluorouracil oral solution comprises the following components: fluorouracil, ginseng polysaccharide, soybean lecithin, and water.

5. A method for identifying ginseng polysaccharides in compound fluorouracil oral solution, characterized in that, Includes the following steps: The sample pretreatment is performed using the method described in any one of claims 1-4 to obtain the test solution; Using ginseng as a reference herb, a ginseng reference herb solution was prepared. The test solution and the ginseng reference solution were spotted onto the same thin-layer plate, developed with a developing solvent, and then sprayed with a colorimetric reagent for thin-layer chromatography analysis. The developing solvent consisted of the following components in parts by volume: 6.8-9.2 parts of n-propanol, 1.76-2.24 parts of water, 0.75-1.24 parts of ethyl acetate, and 0.75-1.24 parts of glacial acetic acid.

6. The method for identifying ginseng polysaccharides in the compound fluorouracil oral solution according to claim 5, characterized in that, The developing solvent comprises the following components in parts by volume: 8 parts n-propanol, 2 parts water, 1 part ethyl acetate and 1 part glacial acetic acid.

7. The method for identifying ginseng polysaccharides in compound fluorouracil oral solution according to claim 5 or 6, characterized in that, The colorimetric agent is an anhydrous ethanol-sulfuric acid solution of carbazole; preferably, the concentration of carbazole in the anhydrous ethanol-sulfuric acid solution of carbazole is 0.05%-0.2% (g / mL).

8. The method for identifying ginseng polysaccharides in compound fluorouracil oral solution according to any one of claims 5-7, characterized in that, The dotting method is strip dotting; preferably, the strip width of the dotting is 6mm; And / or, the volume of the spotting is 4-10 μL, preferably 6 μL.

9. The method for identifying ginseng polysaccharides in compound fluorouracil oral solution according to any one of claims 5-8, characterized in that, The thin-layer plate is a silicone G thin-layer plate, preferably an HSG thin-layer plate.

10. The method for identifying ginseng polysaccharides in compound fluorouracil oral solution according to any one of claims 5-9, characterized in that, The method for preparing the ginseng reference herb solution is as follows: add ethanol solution to the ginseng reference herb and heat under reflux. After the heating and reflux are completed, take the residue. Then add water to the residue, decoct and take the supernatant. Concentrate the supernatant to obtain a concentrated solution. Add ethanol to the concentrated solution, mix and centrifuge. Take the precipitate obtained by centrifugation and add water to the precipitate. Mix and take the supernatant.

Images