A method for quality control of a cold medicine composition

Abstract

The application discloses a quality control method for a cold medicine composition. The quality control method comprises the following steps: thin-layer identification of ephedra in the medicine composition, thin-layer identification of radix scrophulariae, thin-layer identification of ligusticum wallichii, thin-layer identification of mentha haplocalyx, and thin-layer identification of pueraria lobata; and content determination of ephedrine hydrochloride in the medicine composition. The application not only establishes qualitative identification of multiple index components, but also preliminarily establishes quantitative detection of ephedrine hydrochloride. The established thin-layer chromatography identification method has clear visible spots and no tailing phenomenon, and the quantitative detection method of ephedrine hydrochloride is stable and reliable. It is shown that the method has practical significance for internal quality monitoring of the medicine composition (cold soft capsules) for cold, and also provides a reference for subsequent in-depth research on quality standard improvement.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical science, and more particularly to a quality control method for cold medicine compositions. Background Technology

[0002] Cold and flu soft capsules are a product listed in the sixth volume of the Ministry of Health's Drug Standards for Traditional Chinese Medicine Preparations, with the standard number WS3-B-1256-92. The prescription is as follows: 30g of Notopterygium root, 60g of Ephedra, 45g of Cinnamon twig, 45g of Schizonepeta spike, 30g of Saposhnikovia root, 30g of Angelica dahurica root, 30g of Ligusticum chuanxiong rhizome, 15g of Acorus tatarinowii rhizome, 45g of Pueraria lobata root, 15g of Mentha haplocalyx, 60g of Bitter almond, 30g of Angelica sinensis root, 60g of Scutellaria baicalensis root, and 30g of Platycodon grandiflorus root. The preparation method is as follows: For the above fourteen herbs, add 7 times the amount of water to Schizonepeta spike and Mentha haplocalyx, distill for 5 hours, collect the volatile oil, and filter the decoction; for the remaining twelve herbs, add 8 times the amount of 80% ethanol, heat and reflux twice, the first time for 2 hours and the second time for 1 hour, filter, combine the filtrates, recover the ethanol until there is no alcohol taste, combine with the above filtrate, concentrate to a thick paste with a relative density of 1.30 (50℃), dry under reduced pressure, pulverize into fine powder, add vegetable oil and the above volatile oil, mix well, and make into soft capsules. Its effects are to dispel wind and relieve fever, and it is used for symptoms such as fever in the head and body, nasal congestion and runny nose, chills without sweating, joint pain, and sore throat caused by exogenous wind-cold.

[0003] The main active ingredients in cold and flu soft capsules include: ephedrine hydrochloride from Ephedra sinica, baicalin from Scutellaria baicalensis, ferulic acid from Ligusticum chuanxiong, puerarin from Pueraria lobata, and menthol from Mentha haplocalyx. However, current standards only provide thin-layer chromatography (TLC) identification for Saposhnikovia divaricata and ephedrine. The TLC identification of Ephedra sinica is as follows: Take 5g of the contents of this product, add 10mL of 1% hydrochloric acid solution, stir well, transfer to a separatory funnel, add 10mL of petroleum ether, shake and extract, separate the aqueous layer, add concentrated ammonia solution, adjust the pH to 11-12, add 20mL of diethyl ether, shake and extract, separate the ether layer, evaporate to dryness, dissolve the residue in 10mL of acidic ethanol (10mL ethanol and 0.5mL hydrochloric acid), evaporate to dryness, dissolve the residue in 1mL of ethanol to obtain the test solution. Separately, take ephedrine hydrochloride reference standard, add ethanol to prepare a solution containing 2mg per 1mL, as the reference solution. Perform thin-layer chromatography (TLC). Apply 5 μL of each of the two solutions to the same silica gel G TLC plate. Develop the plate using chloroform-methanol-concentrated ammonia solution (20:3.5:0.5) as the developing solvent. Remove the plate, air dry, spray with ninhydrin reagent, and bake at 105°C for about 5 minutes. The test sample chromatogram should show the same red spots at the corresponding positions as the reference sample chromatogram.

[0004] However, the current method has the following drawbacks: when products produced in large quantities are identified by this thin-layer chromatography method, problems such as spot tailing and insufficient separation occur.

[0005] Therefore, this invention modifies the thin-layer chromatography identification method for ephedra based on the original standard. However, since qualitative identification of only one or two indicator components is insufficient to reflect the quality of the product, in order to better conduct multi-component quality testing, further explore effective detection methods, and strengthen specific identification and quantitative detection methods, this invention preliminarily establishes a qualitative and quantitative detection method for multiple indicator components in a cold medicine composition (cold soft capsules). The results show that the method of this invention is stable and reliable, and has practical significance for the intrinsic quality monitoring of cold medicine compositions (cold soft capsules). It also provides a reference for further in-depth research on improving quality standards. Summary of the Invention

[0006] To address the problems in the prior art, the purpose of this invention is to provide a quality control method for cold medicine compositions. This method not only solves the problems of chromatographic spot tailing and insufficient separation in the thin-layer identification of ephedra in the prior art, but also adds the thin-layer qualitative identification of the active ingredients in four medicinal materials and the determination of the content of ephedrine hydrochloride, which can better reflect the quality of the medicine composition product.

[0007] This invention is achieved through the following technical solution:

[0008] The present invention discloses a quality control method for a cold medicine composition, comprising: thin-layer chromatography identification of ephedra, scutellaria, chuanxiong, peppermint, and kudzu root in the medicine composition; and determination of the content of ephedrine hydrochloride in the medicine composition, the specific method of which is as follows:

[0009] (1) Thin-layer chromatography identification of ephedra in drug composition

[0010] Take 4-6g of the contents of this product, add 10-15mL of 0.5-1.5% hydrochloric acid solution, sonicate for 20-40 minutes, filter, add concentrated ammonia test solution to the filtrate, adjust the pH to 11-12, extract with ether 2-4 times, 10-30mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5-1mL of ethanol to obtain the test solution; separately take an appropriate amount of ephedrine hydrochloride reference standard, add methanol to prepare a solution containing 1mg per 1mL as the test solution. Prepare the reference solution; according to the thin-layer chromatography method in General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, apply 2-8 μL of each of the above two solutions to the same silica gel G thin-layer plate. Develop the plate using a 6-8:1:2 ratio of n-butanol-glacial acetic acid-water as the developing solvent. Remove the plate, air dry, spray with 0.1-0.5% ninhydrin solution, and blow with hot air until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatogram of the reference sample.

[0011] (2) Thin-layer chromatography identification of Scutellaria baicalensis in pharmaceutical compositions

[0012] Take 2-4g of the contents of this product, add 20-30mL of water, sonicate for 20-40 minutes, filter with defatted cotton, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, extract with ethyl acetate 2-4 times, 10-30mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 0.5-1mL of ethanol to prepare the test solution; separately take an appropriate amount of baicalin reference standard, add methanol to prepare a solution containing 1mg per mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 1-4μL of each of the above two solutions, spot them separately on the same polyamide film, use the upper layer of ethyl acetate-butanone-acetic acid-water solution in a ratio of 10-12:7:5:3 as the developing solvent, develop, remove, air dry, and spray with 2-5% ferric chloride ethanol solution;

[0013] (3) Thin-layer chromatography identification of Ligusticum chuanxiong in pharmaceutical compositions

[0014] Take 4-6g of the contents of this product, add 20-30mL of water, sonicate for 20-40 minutes, filter with defatted cotton, extract the filtrate with water-saturated ether 2-4 times, 10-30mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5-1mL of ethanol to prepare the test solution; separately take an appropriate amount of ferulic acid reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2-8μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use benzene-ethyl acetate-formic acid in a ratio of 4.5-5.5:2:1 as the developing solvent, develop, remove, air dry, and spray with 1-2% ferric chloride solution and 1-2% potassium ferricyanide solution;

[0015] (4) Thin-layer identification of peppermint in pharmaceutical compositions

[0016] Take 2-4g of the contents of this product, add 5-15mL of methanol, sonicate for 20-40 minutes, filter, and concentrate the filtrate to 1-2mL at low temperature (35-40℃) as the test solution; separately take an appropriate amount of menthol reference standard, add methanol to prepare a solution containing 1mg per mL as the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2-8μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use petroleum ether (30-60℃)-ethyl acetate-benzene in a ratio of 8-10:1:2 as the developing solvent, develop, remove, air dry, spray with 5-10% vanillin sulfuric acid solution, and blow with hot air until the spots are clearly visible;

[0017] (5) Thin-layer chromatography identification of kudzu root in pharmaceutical compositions

[0018] Take 3-5g of the contents of this product, add 20-30mL of ethyl acetate, sonicate for 20-40 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5-1mL of methanol to prepare the test solution; separately take an appropriate amount of puerarin reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2-5μL of each of the above two solutions, spot them separately on the same silica gel H thin-layer plate, use n-butanol-anhydrous ethanol-glacial acetic acid-water in a ratio of 10:8:1:0.2-0.5 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm;

[0019] (6) Determination of the content of ephedrine hydrochloride in the pharmaceutical composition

[0020] 1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase. The mobile phase consisted of acetonitrile as mobile phase A, 0.2-0.3% phosphoric acid solution as mobile phase B, and methanol as mobile phase C. Gradient elution was performed with the following program: 0 min, 5% A, 90% B, 5% C; 7 min, 12% A, 83% B, 5% C; 15 min, 15% A, 80% B, 5% C; 24 min, 20% A, 75% B, 5% C; 32 min, 30% A, 65% B, 5% C; 40 min, 5% A, 90% B, 5% C; 45 min, 5% A, 90% B, 5% C; Flow rate: 0.8-1.2 mL / min; Detection wavelength: 210 nm; Column temperature: 25-35℃; Injection volume: 10-20 μL; The theoretical plate number, calculated based on the ephedrine hydrochloride peak, should not be less than 4000.

[0021] 2) Preparation of reference solution: Take an appropriate amount of ephedrine hydrochloride reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.05 mg per 1 mL.

[0022] 3) Preparation of the test solution: Accurately weigh 0.5-1.5g of the contents of this product and place it in a stoppered conical flask. Accurately add 50mL of 0.5-1.5% hydrochloric acid solution, weigh, sonicate for 30-60 minutes, cool, weigh again, replenish the lost weight with 0.5-1.5% hydrochloric acid solution, shake well, filter, accurately measure 25mL of the filtrate, place it in a separatory funnel, adjust the pH to 11-12 with ammonia water, extract with ether 2-4 times, 20-30mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue with an appropriate amount of acidic ethanol, place in a 10mL volumetric flask, dilute to the mark with acidic ethanol, shake well, and the test solution is obtained.

[0023] 4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0024] Preferably, the thin-layer chromatography method for identifying ephedra in the pharmaceutical composition of the present invention is as follows:

[0025] Take 5g of the contents of this product, add 10mL of 1% hydrochloric acid solution, sonicate for 30 minutes, filter, add concentrated ammonia test solution to the filtrate, adjust the pH to 11-12, extract with ether three times, 20mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the test solution; separately take an appropriate amount of ephedrine hydrochloride reference standard, add methanol to prepare a solution containing 1mg per 1mL to obtain the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use n-butanol-glacial acetic acid-water in a ratio of 7:1:2 as the developing solvent, develop, remove, air dry, spray with 0.3% ninhydrin test solution, and blow with hot air until the spots are clearly visible.

[0026] Preferably, the thin-layer chromatography method for identifying Scutellaria baicalensis in the pharmaceutical composition of the present invention is as follows:

[0027] Take 3g of the contents of this product, add 25mL of water, sonicate for 30 minutes, filter with defatted cotton, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, extract with ethyl acetate three times by shaking, 20mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1mL of ethanol, and use as the test solution; separately take an appropriate amount of baicalin reference standard, add methanol to prepare a solution containing 1mg in 1mL, as the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2μL of each of the above two solutions, spot them separately on the same polyamide film, use the upper layer solution of ethyl acetate-butanone-acetic acid-water in a ratio of 10:7:5:3 as the developing solvent, develop, remove, air dry, and spray with 5% ferric chloride ethanol solution.

[0028] Preferably, the thin-layer chromatography method for identifying Ligusticum chuanxiong in the pharmaceutical composition of the present invention is as follows:

[0029] Take 5g of the contents of this product, add 25mL of water, sonicate for 30 minutes, filter with defatted cotton, extract the filtrate three times with 20mL of water-saturated ether, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to prepare the test solution; separately take an appropriate amount of ferulic acid reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, develop with benzene-ethyl acetate-formic acid in a ratio of 5:2:1, remove, air dry, and spray with a mixed solution of 1% ferric chloride solution and 1% potassium ferricyanide solution.

[0030] Preferably, the volume ratio of the mixed solution of 1% ferric chloride solution and 1% potassium ferricyanide solution in this invention is 1:1.

[0031] Preferably, the thin-layer chromatography method for identifying peppermint in the pharmaceutical composition of the present invention is as follows:

[0032] Take 3g of the contents of this product, add 10mL of methanol, sonicate for 30 minutes, filter, and concentrate the filtrate to 1mL at low temperature (37℃) as the test solution; separately take an appropriate amount of menthol reference standard, add methanol to prepare a solution containing 1mg in 1mL as the reference solution; according to the thin-layer chromatography method of General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate, use petroleum ether (30-60℃)-ethyl acetate-benzene in a ratio of 9:1:2 as the developing solvent, develop, remove, air dry, spray with 5% vanillin sulfuric acid solution, and blow with hot air until the spots are clearly visible.

[0033] Preferably, the thin-layer chromatography method for identifying kudzu root in the pharmaceutical composition of the present invention is as follows:

[0034] Take 4g of the contents of this product, add 25mL of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the test solution; separately take an appropriate amount of puerarin reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel H thin-layer plate, use n-butanol-anhydrous ethanol-glacial acetic acid-water in a ratio of 10:8:1:0.5 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm.

[0035] Preferably, the method for determining the content of ephedrine hydrochloride in the pharmaceutical composition of the present invention is as follows:

[0036] 1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase. The mobile phase consisted of acetonitrile as mobile phase A, 0.25% phosphoric acid solution as mobile phase B, and methanol as mobile phase C. Gradient elution was performed with the following program: 0 min, 5% A, 90% B, 5% C; 7 min, 12% A, 83% B, 5% C; 15 min, 15% A, 80% B, 5% C; 24 min, 20% A, 75% B, 5% C; 32 min, 30% A, 65% B, 5% C; 40 min, 5% A, 90% B, 5% C; 45 min, 5% A, 90% B, 5% C. The flow rate was 1 mL / min; the detection wavelength was 210 nm; the column temperature was 30℃; the injection volume was 10 μL; and the theoretical plate number, calculated based on the ephedrine hydrochloride peak, should be no less than 4000.

[0037] 2) Preparation of reference solution: Take an appropriate amount of ephedrine hydrochloride reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.05 mg per 1 mL.

[0038] 3) Preparation of the test solution: Take 1g of the contents of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 1% hydrochloric acid solution, weigh it, sonicate for 30 minutes, cool it, weigh it again, replenish the lost weight with 1% hydrochloric acid solution, shake well, filter it, accurately measure 25mL of the filtrate, place it in a separatory funnel, adjust the pH value to 11-12 with ammonia water, extract it 3 times with 25mL of ether each time, combine the ether extracts, evaporate to dryness, dissolve the residue with an appropriate amount of acidic ethanol, place it in a 10mL volumetric flask, dilute to the mark with acidic ethanol, shake well, and the test solution is obtained.

[0039] 4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0040] The acidic ethanol of the present invention is prepared by taking 10 mL of ethanol, adding 0.5 mL of hydrochloric acid, and mixing well.

[0041] The ultrasonic conditions described in this invention are: 400W, 40kHz.

[0042] The present invention has the following advantages:

[0043] 1. This invention revises the thin-layer chromatography identification method for ephedrine. The thin-layer chromatography yields ephedrine hydrochloride with a moderate Rf value, clearly visible spots without obvious tailing, solving the problems of spot tailing, insufficient separation, and pale spot color in the current standard.

[0044] 2. This invention adds a thin-layer chromatography method for identifying Scutellaria baicalensis, Ligusticum chuanxiong, Pueraria lobata, and Mentha haplocalyx in cold and flu soft capsules. The thin-layer chromatography yields moderate Rf values ​​for each component, with clearly visible spots and no obvious tailing phenomenon, further improving the product quality standard.

[0045] 3. This invention establishes an HPLC method for determining the content of ephedrine hydrochloride in cold and flu soft capsules. Methodological investigation showed that the precision, stability, reproducibility, and recovery rate of this method all met the requirements. Specifically: (1) Precision test: The calculated RSD value of the peak area of ​​ephedrine hydrochloride was 0.59%, indicating good instrument precision; (2) Stability test: The calculated RSD of the ephedrine hydrochloride content was 2.12%, indicating good stability of the test solution within 24 hours; (3) Repeatability test: The measured ephedrine hydrochloride content was 336.08 μg / capsule, 335.72 μg / capsule, 335.14 μg / capsule, 333.65 μg / capsule, 331.46 μg / capsule, and 3... 32.19 μg / particle, with an average value of 334.04 μg / particle and an RSD of 1.92%, indicating good repeatability of the method; (4) The recovery rate of the sample was investigated, and the results showed that the average recovery rate was 100.15% and the RSD was 0.91%; (5) The content of ephedrine hydrochloride in the sample was determined and verified, and the results showed that the content of ephedrine hydrochloride in the three batches of samples was 335.68, 333.14 and 336.85 μg / particle, with an average value of 335.22 and an RSD of 1.90%, indicating that the method of the present invention is stable and feasible.

[0046] 4. The thin-layer chromatography identification method and HPLC content determination method established in this invention are simple to operate, accurate, reliable, and do not require high-end instruments and equipment. They have good repeatability and provide a reference for the quality standards of cold soft capsules, enabling more comprehensive and effective control of product quality. Detailed Implementation

[0047] Example 1: Thin-layer chromatography identification of ephedra in cold medicine compositions

[0048] Take 5g of the contents of this product, add 10mL of 1% hydrochloric acid solution, sonicate for 30 minutes, filter, add concentrated ammonia test solution to the filtrate, adjust the pH to 11-12, extract with ether three times, 20mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the test solution; separately take an appropriate amount of ephedrine hydrochloride reference standard, add methanol to prepare a solution containing 1mg per 1mL to obtain the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use n-butanol-glacial acetic acid-water in a ratio of 7:1:2 as the developing solvent, develop, remove, air dry, spray with 0.3% ninhydrin test solution, and blow with hot air until the spots are clearly visible.

[0049] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0050] Example 2: Thin-layer chromatography identification of Scutellaria baicalensis in cold medicine compositions

[0051] Take 3g of the contents of this product, add 25mL of water, sonicate for 30 minutes, filter with defatted cotton, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, extract with ethyl acetate three times by shaking, 20mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1mL of ethanol, and use as the test solution; separately take an appropriate amount of baicalin reference standard, add methanol to prepare a solution containing 1mg in 1mL, as the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2μL of each of the above two solutions, spot them separately on the same polyamide film, use the upper layer solution of ethyl acetate-butanone-acetic acid-water in a ratio of 10:7:5:3 as the developing solvent, develop, remove, air dry, and spray with 5% ferric chloride ethanol solution.

[0052] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0053] Example 3: Thin-layer chromatography identification of Ligusticum chuanxiong in cold medicine compositions

[0054] Take 5g of the contents of this product, add 25mL of water, sonicate for 30 minutes, filter with defatted cotton, extract the filtrate three times with 20mL of water-saturated ether, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to prepare the test solution; separately take an appropriate amount of ferulic acid reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia, Volume IV, and apply 5μL of each of the above two solutions to the same silica gel G thin-layer plate, develop with benzene-ethyl acetate-formic acid in a ratio of 5:2:1, remove, air dry, and spray with a mixed solution of 1% ferric chloride solution and 1% potassium ferricyanide solution (1:1).

[0055] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0056] Example 4: Thin-layer chromatography identification of peppermint in cold medicine compositions

[0057] Take 3g of the contents of this product, add 10mL of methanol, sonicate for 30 minutes, filter, and concentrate the filtrate to 1mL at low temperature (37℃) as the test solution; separately take an appropriate amount of menthol reference standard, add methanol to prepare a solution containing 1mg in 1mL as the reference solution; according to the thin-layer chromatography method of General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate, use petroleum ether (30-60℃)-ethyl acetate-benzene in a ratio of 9:1:2 as the developing solvent, develop, remove, air dry, spray with 5% vanillin sulfuric acid solution, and blow with hot air until the spots are clearly visible.

[0058] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0059] Example 5: Thin-layer chromatography identification of kudzu root in cold medicine compositions

[0060] Take 4g of the contents of this product, add 25mL of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the test solution; separately take an appropriate amount of puerarin reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel H thin-layer plate, use n-butanol-anhydrous ethanol-glacial acetic acid-water in a ratio of 10:8:1:0.5 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm.

[0061] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0062] Example 6: Thin-layer chromatography identification of ephedra in cold medicine compositions

[0063] Take 4g of the contents of this product, add 10mL of 0.5% hydrochloric acid solution, sonicate for 20 minutes, filter, add concentrated ammonia test solution to the filtrate, adjust the pH to 11, extract twice with 10mL of ether each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the test solution; separately take an appropriate amount of ephedrine hydrochloride reference standard, add methanol to prepare a solution containing 1mg per 1mL to obtain the reference solution; perform the thin-layer chromatography test according to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 0502, apply 5μL of each of the above two solutions to the same silica gel G thin-layer plate, develop with n-butanol-glacial acetic acid-water in a ratio of 6:1:2, remove, air dry, spray with 0.1% ninhydrin test solution, and blow with hot air until the spots are clearly visible.

[0064] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0065] Example 7: Thin-layer chromatography identification of Scutellaria baicalensis in cold medicine compositions

[0066] Take 2g of the contents of this product, add 20mL of water, sonicate for 20 minutes, filter with defatted cotton, adjust the pH of the filtrate to 2 with dilute hydrochloric acid, extract twice with ethyl acetate, 10mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1mL of ethanol, and use as the test solution; separately take an appropriate amount of baicalin reference standard, add methanol to prepare a solution containing 1mg in 1mL, and use as the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 1μL of each of the above two solutions, spot them separately on the same polyamide film, use the upper layer solution of ethyl acetate-butanone-acetic acid-water in a ratio of 10:7:5:3 as the developing solvent, develop, remove, air dry, and spray with 2% ferric chloride ethanol solution.

[0067] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0068] Example 8: Thin-layer chromatography identification of Ligusticum chuanxiong in cold medicine compositions

[0069] Take 4g of the contents of this product, add 20mL of water, sonicate for 20 minutes, filter with defatted cotton, extract the filtrate twice with 10mL of water-saturated ether, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to prepare the test solution; separately take an appropriate amount of ferulic acid reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use benzene-ethyl acetate-formic acid in a ratio of 4.5:2:1 as the developing solvent, develop, remove, air dry, and spray with a mixed solution of 1% ferric chloride solution and 1% potassium ferricyanide solution (1:1).

[0070] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0071] Example 9: Thin-layer chromatography identification of peppermint in cold medicine compositions

[0072] Take 2g of the contents of this product, add 5mL of methanol, sonicate for 20 minutes, filter, and concentrate the filtrate to 1mL at low temperature (35℃) as the test solution; separately take an appropriate amount of menthol reference standard, add methanol to prepare a solution containing 1mg in 1mL as the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use petroleum ether (30-60℃)-ethyl acetate-benzene in a ratio of 8:1:2 as the developing solvent, develop, remove, air dry, spray with 5% vanillin sulfuric acid solution, and blow with hot air until the spots are clearly visible.

[0073] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0074] Example 10: Thin-layer chromatography identification of kudzu root in cold medicine compositions

[0075] Take 3g of the contents of this product, add 20mL of ethyl acetate, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5mL of methanol to prepare the test solution; separately take an appropriate amount of puerarin reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2μL of each of the above two solutions, spot them separately on the same silica gel H thin-layer plate, use n-butanol-anhydrous ethanol-glacial acetic acid-water in a ratio of 10:8:1:0.2 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm.

[0076] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0077] Example 11: Thin-layer chromatography identification of ephedra in cold medicine compositions

[0078] Take 6g of the contents of this product, add 15mL of 1.5% hydrochloric acid solution, sonicate for 40 minutes, filter, add concentrated ammonia test solution to the filtrate, adjust the pH to 12, extract with ether 4 times, 30mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the test solution; separately take an appropriate amount of ephedrine hydrochloride reference standard, add methanol to prepare a solution containing 1mg per 1mL as the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use n-butanol-glacial acetic acid-water in a ratio of 8:1:2 as the developing solvent, develop, remove, air dry, spray with 0.5% ninhydrin test solution, and blow with hot air until the spots are clearly visible.

[0079] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0080] Example 12: Thin-layer chromatography identification of Scutellaria baicalensis in cold medicine compositions

[0081] Take 4g of the contents of this product, add 30mL of water, sonicate for 40 minutes, filter with defatted cotton, adjust the pH of the filtrate to 3 with dilute hydrochloric acid, extract with ethyl acetate 4 times, 30mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1mL of ethanol, and use as the test solution; separately take an appropriate amount of baicalin reference standard, add methanol to prepare a solution containing 1mg in 1mL, and use as the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 4μL of each of the above two solutions, spot them separately on the same polyamide film, use the upper layer solution of ethyl acetate-butanone-acetic acid-water in a ratio of 12:7:5:3 as the developing solvent, develop, remove, air dry, and spray with 5% ferric chloride ethanol solution.

[0082] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0083] Example 13: Thin-layer chromatography identification of Ligusticum chuanxiong in cold medicine compositions

[0084] Take 6g of the contents of this product, add 30mL of water, sonicate for 40 minutes, filter with defatted cotton, extract the filtrate with water-saturated ether 4 times, 30mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 1mL of ethanol to prepare the test solution; separately take an appropriate amount of ferulic acid reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 8μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use benzene-ethyl acetate-formic acid in a ratio of 5.5:2:1 as the developing solvent, develop, remove, air dry, and spray with a mixed solution of 2% ferric chloride solution and 2% potassium ferricyanide solution (1:1).

[0085] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0086] Example 14: Thin-layer chromatography identification of peppermint in cold medicine compositions

[0087] Take 4g of the contents of this product, add 15mL of methanol, sonicate for 40 minutes, filter, and concentrate the filtrate to 2mL at low temperature (40℃) as the test solution; separately take an appropriate amount of menthol reference standard, add methanol to prepare a solution containing 1mg in 1mL as the reference solution; according to the thin-layer chromatography method of General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 8μL of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate, use petroleum ether (30-60℃)-ethyl acetate-benzene in a ratio of 10:1:2 as the developing solvent, develop, remove, air dry, spray with 10% vanillin sulfuric acid solution, and blow with hot air until the spots are clearly visible.

[0088] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0089] Example 15: Thin-layer chromatography identification of kudzu root in cold medicine compositions

[0090] Take 5g of the contents of this product, add 30mL of ethyl acetate, sonicate for 40 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the test solution; separately take an appropriate amount of puerarin reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel H thin-layer plate, use n-butanol-anhydrous ethanol-glacial acetic acid-water in a ratio of 10:8:1:0.5 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm.

[0091] Results: In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0092] Example 16: Determination of ephedrine hydrochloride content in a cold medicine composition.

[0093] (1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase. The mobile phase was: acetonitrile as mobile phase A, 0.25% phosphoric acid solution as mobile phase B, and methanol as mobile phase C. Gradient elution was performed. The elution program was: 0 min, 5% A, 90% B, 5% C; 7 min, 12% A, 83% B, 5% C; 15 min, 15% A, 80% B, 5% C; 24 min, 20% A, 75% B, 5% C; 32 min, 30% A, 65% B, 5% C; 40 min, 5% A, 90% B, 5% C; 45 min, 5% A, 90% B, 5% C; Flow rate: 1 mL / min; Detection wavelength: 210 nm; Column temperature: 30 ℃; Injection volume: 10 μL; The theoretical plate number calculated based on the ephedrine hydrochloride peak should not be less than 4000.

[0094] (2) Preparation of reference solution: Take an appropriate amount of ephedrine hydrochloride reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.05 mg per 1 mL.

[0095] (3) Preparation of test solution: Take 1g of the contents of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 1% hydrochloric acid solution, weigh it, sonicate (400W, 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 1% hydrochloric acid solution, shake well, filter it, accurately measure 25mL of the filtrate, place it in a separatory funnel, adjust the pH value to 11-12 with ammonia water, extract it 3 times with 25mL of ether each time, combine the ether liquids, evaporate to dryness, add an appropriate amount of acidic ethanol to dissolve the residue, place it in a 10mL volumetric flask, add acidic ethanol to dilute to the mark, shake well, and the test solution is obtained.

[0096] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph. Inject the reference solution 5 times consecutively. The RSD% of the peak area of ​​the 5 injections shall not be greater than 2.0%. Inject the test solution once. The relative average deviation of the contents of the two test solutions shall not be greater than 2.0%. Measure and obtain the result.

[0097] Example 17: Determination of ephedrine hydrochloride content in a cold medicine composition.

[0098] (1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase. The mobile phase was: acetonitrile as mobile phase A, 0.25% phosphoric acid solution as mobile phase B, and methanol as mobile phase C. Gradient elution was performed. The elution program was: 0 min, 5% A, 90% B, 5% C; 7 min, 12% A, 83% B, 5% C; 15 min, 15% A, 80% B, 5% C; 24 min, 20% A, 75% B, 5% C; 32 min, 30% A, 65% B, 5% C; 40 min, 5% A, 90% B, 5% C; 45 min, 5% A, 90% B, 5% C; Flow rate: 0.8 mL / min; Detection wavelength: 210 nm; Column temperature: 25℃; Injection volume: 10 μL; The theoretical plate number, calculated based on the ephedrine hydrochloride peak, should not be less than 4000.

[0099] (2) Preparation of reference solution: Take an appropriate amount of ephedrine hydrochloride reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.05 mg per 1 mL.

[0100] (3) Preparation of test solution: Take 0.5g of the contents of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 0.5% hydrochloric acid solution, weigh it, sonicate (400W, 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 0.5% hydrochloric acid solution, shake well, filter it, accurately measure 25mL of the filtrate, place it in a separatory funnel, adjust the pH value to 11-12 with ammonia water, extract it twice with ether, 20mL each time, combine the ether liquids, evaporate to dryness, add an appropriate amount of acidic ethanol to dissolve the residue, place it in a 10mL volumetric flask, add acidic ethanol to dilute to the mark, shake well, and the test solution is obtained.

[0101] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph. Inject the reference solution 5 times consecutively. The RSD% of the peak area of ​​the 5 injections shall not be greater than 2.0%. Inject the test solution once. The relative average deviation of the contents of the two test solutions shall not be greater than 2.0%. Measure and obtain the result.

[0102] Example 18: Determination of ephedrine hydrochloride content in a cold medicine composition.

[0103] (1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase. The mobile phase was: acetonitrile as mobile phase A, 0.25% phosphoric acid solution as mobile phase B, and methanol as mobile phase C. Gradient elution was performed. The elution program was: 0 min, 5% A, 90% B, 5% C; 7 min, 12% A, 83% B, 5% C; 15 min, 15% A, 80% B, 5% C; 24 min, 20% A, 75% B, 5% C; 32 min, 30% A, 65% B, 5% C; 40 min, 5% A, 90% B, 5% C; 45 min, 5% A, 90% B, 5% C; Flow rate: 1.2 mL / min; Detection wavelength: 210 nm; Column temperature: 35℃; Injection volume: 10 μL; The theoretical plate number, calculated based on the ephedrine hydrochloride peak, should not be less than 4000.

[0104] (2) Preparation of reference solution: Take an appropriate amount of ephedrine hydrochloride reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.05 mg per 1 mL.

[0105] (3) Preparation of test solution: Take 1g of the contents of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 1.5% hydrochloric acid solution, weigh it, sonicate (400W, 40kHz) for 60 minutes, cool it, weigh it again, replenish the lost weight with 1.5% hydrochloric acid solution, shake well, filter it, accurately measure 25mL of the filtrate, place it in a separatory funnel, adjust the pH value to 11-12 with ammonia water, extract it with ether 4 times, 30mL each time, combine the ether liquids, evaporate to dryness, add an appropriate amount of acidic ethanol to dissolve the residue, place it in a 10mL volumetric flask, add acidic ethanol to dilute to the mark, shake well, and the test solution is obtained.

[0106] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph. Inject the reference solution 5 times consecutively. The RSD% of the peak area of ​​the 5 injections shall not be greater than 2.0%. Inject the test solution once. The relative average deviation of the contents of the two test solutions shall not be greater than 2.0%. Measure and obtain the result.

[0107] To further verify the effectiveness of the embodiments of the method of the present invention, the inventors conducted a series of verification experiments, some of which are excerpted below:

[0108] I. Screening Experiment for Thin-Layer Chromatography Identification Methods

[0109] 1.1 Reagents and reagents

[0110] The cold and flu soft capsules were provided by Guizhou Weilide Pharmaceutical Co., Ltd.; the silicone G thin-layer plates and self-made silicone G thin-layer plates were produced by Qingdao Ocean Chemical Plant Branch.

[0111] Some key reagents: Methanol, ethyl acetate, toluene, ethyl formate, formic acid, diethyl ether, ethanol, methanol, n-butanol, glacial acetic acid, ethyl acetate, butanone, acetic acid, benzene, formic acid, petroleum ether (30–60℃), and chloroform are all analytical grade. Reference standards: ephedrine hydrochloride reference standard, baicalin reference standard, ferulic acid reference standard, puerarin reference standard, and menthol reference standard.

[0112] Other reagents:

[0113] 0.5% hydrochloric acid solution: Take 0.5 mL of hydrochloric acid, add water to dissolve it to make 100 mL, and you have the solution.

[0114] 1% hydrochloric acid solution: Take 1 mL of hydrochloric acid, add water to dissolve it to make 100 mL, and you will get the solution.

[0115] 1.5% hydrochloric acid solution: Take 1.5 mL of hydrochloric acid, add water to dissolve it to make 100 mL, and you have the solution.

[0116] Concentrated ammonia test solution: Concentrated ammonia solution can be used.

[0117] 0.1% ninhydrin test solution: Take 0.1g of ninhydrin, add ethanol to dissolve it to make 100mL, and you will get the solution.

[0118] 0.3% ninhydrin test solution: Take 0.3g of ninhydrin, add ethanol to dissolve it to make 100mL, and you will get the solution.

[0119] 0.5% ninhydrin test solution: Take 0.5g of ninhydrin, add ethanol to dissolve it to make 100mL, and you will get the solution.

[0120] Dilute hydrochloric acid: Take 234 mL of hydrochloric acid and dilute it with water to 1000 mL.

[0121] 2% ferric chloride ethanol solution: Take 2g of ferric chloride, add ethanol to dissolve it to make 100mL, and you will get the solution.

[0122] 5% ferric chloride ethanol solution: Take 5g of ferric chloride, add ethanol to dissolve it to make 100mL, and you will get the solution.

[0123] 1% ferric chloride solution: Take 1g of ferric chloride, add water to dissolve it to make 100mL, and you will get the solution.

[0124] 2% ferric chloride solution: Take 2g of ferric chloride, add water to dissolve it to make 100mL, and you will get the solution.

[0125] 1% potassium ferricyanide solution: Take 1g of potassium ferricyanide, add water to dissolve it to make 100mL, and you will get the solution.

[0126] 2% potassium ferricyanide solution: Take 2g of potassium ferricyanide, add water to dissolve it to make 100mL, and you will get the solution.

[0127] 5% vanillin sulfuric acid solution: Take 5g of vanillin, add sulfuric acid to dissolve it to make 100mL.

[0128] 10% vanillin sulfuric acid solution: Take 10g of vanillin, add sulfuric acid to dissolve it to make 100mL.

[0129] 1.2 Main Instruments

[0130] Electronic balance, silicone G thin film, polyamide film, three-way ultraviolet analyzer, etc.

[0131] 1.3 Establishment of Thin-Layer Chromatography Identification Method

[0132] Previously, our company's testing program for cold medicine compositions (cold soft capsules) only involved thin-layer chromatography (TLC) identification of ephedra, without including TLC identification of scutellaria baicalensis, chuanxiong rhizome, kudzu root, and peppermint. To effectively improve product quality and enhance the quality standards of cold soft capsules, we have re-established the TLC identification method for ephedra and added TLC identification methods for scutellaria baicalensis, chuanxiong rhizome, kudzu root, and peppermint in cold soft capsules, further improving product quality standards.

[0133] 1.3.1 Thin-layer chromatography identification of ephedra

[0134] First, the processing and developing solvent of ephedra samples were tested according to the varieties listed in Volume VI of the Ministry of Health's Drug Standards for Traditional Chinese Medicine Compound Preparations:

[0135] Take 5g of the contents of this product, add 10mL of 1% hydrochloric acid solution, stir well, transfer to a separatory funnel, add 10mL of petroleum ether and shake to extract, separate the aqueous layer, add concentrated ammonia test solution, adjust the pH to 11-12, add 20mL of diethyl ether, shake to extract, separate the diethyl ether layer, evaporate to dryness, dissolve the residue in 10mL of acidic ethanol (10mL ethanol and 0.5mL hydrochloric acid), evaporate to dryness, dissolve the residue in 1mL of ethanol to obtain the test solution. Separately, take ephedrine hydrochloride reference standard, add ethanol to prepare a solution containing 2mg per 1mL as the reference solution. Perform thin-layer chromatography, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use chloroform-methanol-concentrated ammonia solution (20:3.5:0.5) as the developing solvent, develop, remove, air dry, spray with ninhydrin test solution, and dry at 105℃ for about 5 minutes. In the chromatogram of the test sample, red spots appear at the same positions as those in the chromatogram of the reference sample.

[0136] Results: In thin-layer chromatography, the spots showed severe tailing, moved close to the starting line, had low Rf values, poor separation, and pale spot color.

[0137] Therefore, the sample needs to be reprocessed and the developing solvent needs to be re-examined.

[0138] 1.3.1.1 Investigation of the developing solvent

[0139] Using ephedrine hydrochloride as a control, chloroform-methanol-concentrated ammonia solution (15:5:0.5), cyclohexane-chloroform-isopropanol (5:4:1), n-butanol-glacial acetic acid-water (7:1:2), and cyclohexane-chloroform-glacial acetic acid (5:5:4) were used as developing solvents. After development, the samples were removed, dried, sprayed with 0.3% ninhydrin solution, and heated with hot air until the spots were clearly visible.

[0140] Results: Using n-butanol-glacial acetic acid-water (7:1:2) as the developing solvent, the Rf value was moderate, there was no tailing phenomenon, the separation effect was good, and the toxicity was low. Therefore, "n-butanol-glacial acetic acid-water (7:1:2)" was selected as the developing solvent.

[0141] 1.3.1.2 Examination of Extraction Methods

[0142] Take three portions of the contents of this product, each 5g, add 10mL of 1% hydrochloric acid solution, and extract by shaking for 0.5h, ultrasonic extraction for 0.5h, and reflux extraction for 0.5h respectively. Filter, add concentrated ammonia test solution to the filtrate, adjust the pH to 11-12, and extract by shaking with 20mL of ether three times each time. Combine the ether extracts, evaporate to dryness, and dissolve the residue in 0.5mL of ethanol to obtain the test solution.

[0143] Result: The spots in the test solution obtained by ultrasonic extraction were clearer, thus confirming that the extraction method was "ultrasonic extraction".

[0144] 1.3.1.3 Examination of extraction time

[0145] Take three portions of the contents of this product, each 5g, add 10mL of 1% hydrochloric acid solution, and extract by ultrasonication for 20min, 30min and 40min respectively. Filter, add concentrated ammonia test solution to the filtrate, adjust the pH to 11-12, and extract by shaking with ether 3 times, 20mL each time. Combine the ether extracts, evaporate to dryness, and dissolve the residue in 0.5mL of ethanol to obtain the test solution.

[0146] Results: The ultrasonic extraction time of 30 min was just right, and the spots were clearer than those of the test sample solution. Therefore, the extraction time of 30 min was finally selected.

[0147] 1.3.1.4 Establishment of a thin-layer chromatography method for identifying ephedra

[0148] Take 5g of the contents of this product, add 10mL of 1% hydrochloric acid solution, sonicate for 30 minutes, filter, add concentrated ammonia test solution to the filtrate, adjust the pH to 11-12, extract with ether three times, 20mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the test solution; separately take an appropriate amount of ephedrine hydrochloride reference standard, add methanol to prepare a solution containing 1mg per 1mL to obtain the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use n-butanol-glacial acetic acid-water in a ratio of 7:1:2 as the developing solvent, develop, remove, air dry, spray with 0.3% ninhydrin test solution, and blow with hot air until the spots are clearly visible.

[0149] 1.3.2 Thin-layer chromatography identification of Scutellaria baicalensis

[0150] First, the sample processing and developing solvent were determined according to the thin-layer chromatography identification method for Scutellaria baicalensis in the 2020 edition of the Chinese Pharmacopoeia:

[0151] Take 1 g of the contents of this product, add 30 mL of a mixed solution of ethyl acetate and methanol (3:1), heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol, and use the supernatant as the test solution. Separately, take baicalin reference standard, add methanol to prepare a solution containing 0.5 mg per 1 mL, as the reference solution. Perform thin-layer chromatography (General Rule 0502), apply 2 μL each of the above test solution and reference solution to the same polyamide film, use toluene-ethyl acetate-methanol-formic acid (10:3:1:2) as the developing solvent, pre-saturate for 30 minutes, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0152] Results: In thin-layer chromatography, the spots showed severe tailing, did not move at the starting line, had poor resolution, and were not visible under ultraviolet light.

[0153] Since the spots cannot be observed under ultraviolet light, this invention uses a display reagent. After reviewing a large number of relevant literature and combining chemical composition analysis, this invention uses 5% ferric chloride ethanol solution as the display reagent. In addition, due to the spot tailing phenomenon and insufficient separation, the sample needs to be reprocessed and the developing solvent needs to be re-examined.

[0154] 1.3.2.1 Sample Preparation

[0155] Take 3g of the contents of this product, add 25mL of water, heat under reflux for 30min, filter with defatted cotton, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, extract with ethyl acetate three times by shaking, 20mL each time, combine the ethyl acetate extracts, evaporate to dryness, add 1mL of ethanol to dissolve the residue, and use it as the test solution.

[0156] Results: In thin-layer chromatography, the spots showed tailing, the separation was insufficient, and the spots were small and light in color.

[0157] 1.3.2.2 Investigation of the developing solvent

[0158] Using baicalin as a control, chloroform-methanol-ethyl acetate (10:2:1), methanol-ethyl acetate-methanol-formic acid (5:4:1:0.5), ethyl acetate-butanone-water (10:7:3), and ethyl acetate-butanone-acetic acid-water (10:7:5:3) were used as developing solvents. The samples were then removed, dried, and sprayed with 5% ferric chloride ethanol solution.

[0159] Results: Using the upper layer of ethyl acetate-butanone-acetic acid-water (10:7:5:3) as the developing solvent, the spots were clearly displayed, the Rf value was moderate, there was no tailing phenomenon, the separation effect was good, and the toxicity was low. Therefore, the upper layer of ethyl acetate-butanone-acetic acid-water (10:7:5:3) was selected as the developing solvent.

[0160] 1.3.2.3 Examination of Extraction Methods

[0161] Take three portions of the contents of this product, each 3g, add 25mL of water, and extract by shaking for 30min, ultrasonic extraction for 30min, and reflux extraction for 30min respectively. Filter with defatted cotton, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, and extract by shaking with ethyl acetate 3 times, 20mL each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1mL of ethanol to obtain the test solution.

[0162] Result: The spots in the test solution obtained by ultrasonic extraction were clearer, thus confirming that the extraction method was "ultrasonic extraction".

[0163] 1.3.2.4 Examination of extraction time

[0164] Take three portions of the contents of this product, each 3g, add 25mL of water, and sonicate for 20min, 30min and 40min respectively. Filter with defatted cotton, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, and extract with ethyl acetate three times, 20mL each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1mL of ethanol to obtain the test solution.

[0165] Results: The spots from ultrasonic extraction for 30 min and 40 min were clearer than those from ultrasonic extraction for 20 min. However, the difference in spot clarity between ultrasonic extraction for 30 min and 40 min was not significant. Therefore, the ultrasonic extraction time of 30 min was ultimately selected.

[0166] 1.3.2.5 Establishment of a thin-layer chromatography method for identifying Scutellaria baicalensis

[0167] Take 3g of the contents of this product, add 25mL of water, sonicate for 30 minutes, filter with defatted cotton, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, extract with ethyl acetate three times by shaking, 20mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1mL of ethanol, and use as the test solution; separately take an appropriate amount of baicalin reference standard, add methanol to prepare a solution containing 1mg in 1mL, as the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2μL of each of the above two solutions, spot them separately on the same polyamide film, use the upper layer solution of ethyl acetate-butanone-acetic acid-water in a ratio of 10:7:5:3 as the developing solvent, develop, remove, air dry, and spray with 5% ferric chloride ethanol solution.

[0168] 1.3.3 Thin-layer chromatography identification of Ligusticum chuanxiong

[0169] First, the sample processing and developing solvent were determined according to the thin-layer chromatography identification method for Ligusticum chuanxiong in the 2020 edition of the Chinese Pharmacopoeia:

[0170] Take 1g of the contents of this product, add 20mL of diethyl ether, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 2mL of ethyl acetate to obtain the test solution. Separately, prepare a reference solution by dissolving ferulic acid reference standard in ethyl acetate at a concentration of 0.1mg / mL. Perform thin-layer chromatography (General Rule 0502), applying 10μL of each of the above two solutions separately to the same silica gel GF254 thin-layer plate. Develop the plate using n-hexane-ethyl acetate (3:1) as the developing solvent. Remove the plate, air dry, and examine under ultraviolet light (254nm).

[0171] Results: In thin-layer chromatography, the spots showed severe tailing and poor separation. No obvious spots were visible under ultraviolet light (254nm).

[0172] Since no obvious spots can be observed under ultraviolet light, this invention uses a display reagent. After reviewing a large amount of relevant literature and combining chemical composition analysis, this invention uses a mixed solution of 1% ferric chloride and 1% potassium ferricyanide as the display reagent. Furthermore, due to spot tailing and insufficient separation, the sample needs to be reprocessed and the developing solvent re-evaluated.

[0173] 1.3.3.1 Sample Preparation

[0174] Take 5g of the contents of this product, add 25mL of water, heat under reflux for 1 hour, filter with defatted cotton, extract the filtrate three times with 20mL of water-saturated ether, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol, and use as the test solution.

[0175] Results: In thin-layer chromatography, the spots showed slight tailing, the Rf value was small, and the spots were small and light in color.

[0176] 1.3.3.2 Investigation of the developing solvent

[0177] Using ferulic acid as a control, chloroform-methanol-ethyl acetate (10:2:1), methanol-ethyl acetate-methanol-formic acid (10:4:1:0.5), benzene-ethyl acetate-formic acid (5:2:1), and methanol-ethyl acetate-acetic acid-water (10:3:1:1) were used as developing solvents. After development, the samples were removed, dried, and sprayed with a mixed solution of 1% ferric chloride solution and 1% potassium ferricyanide solution (1:1).

[0178] Results: Using benzene-ethyl acetate-formic acid (5:2:1) as the developing solvent, the spots were clearly displayed, the Rf value was moderate, and there was no tailing phenomenon, indicating good separation effect. Therefore, benzene-ethyl acetate-formic acid (5:2:1) was selected as the developing solvent.

[0179] 1.3.3.3 Examination of Extraction Methods

[0180] Take three portions of the contents of this product, each 3g, add 25mL of water, and extract by shaking for 1h, ultrasonic extraction for 1h, and reflux extraction for 1h respectively. Filter with defatted cotton, and extract the filtrate by shaking with 20mL of water-saturated ether three times. Combine the ether extracts, evaporate to dryness, and dissolve the residue in 0.5mL of ethanol to obtain the test solution.

[0181] Result: The spots in the test solution obtained by ultrasonic extraction were clearer, thus confirming that the extraction method was "ultrasonic extraction".

[0182] 1.3.3.4 Examination of extraction time

[0183] Take three portions of the contents of this product, each 5g, add 25mL of water, and sonicate for 20min, 30min and 60min respectively. Filter with defatted cotton, extract the filtrate three times with 20mL of water-saturated ether each time, combine the ether extracts, evaporate to dryness, and dissolve the residue in 0.5mL of ethanol to obtain the test solution.

[0184] Results: The spots from ultrasonic extraction for 30 min and 60 min were clearer than those from ultrasonic extraction for 20 min. However, the difference in spot clarity between ultrasonic extraction for 30 min and 60 min was not significant. Therefore, the ultrasonic extraction time of 30 min was ultimately selected.

[0185] 1.3.3.5 Establishment of a thin-layer chromatography identification method for Ligusticum chuanxiong

[0186] Take 5g of the contents of this product, add 25mL of water, sonicate for 30 minutes, filter with defatted cotton, extract the filtrate three times with 20mL of water-saturated ether, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to prepare the test solution; separately take an appropriate amount of ferulic acid reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, develop with benzene-ethyl acetate-formic acid in a ratio of 5:2:1, remove, air dry, and spray with a mixed solution of 1% ferric chloride solution and 1% potassium ferricyanide solution.

[0187] 1.3.4 Thin-layer chromatography identification of kudzu root

[0188] First, the sample processing and developing solvent were determined according to the thin-layer chromatography identification method for kudzu root in the 2020 edition of the Chinese Pharmacopoeia:

[0189] Take 1g of the contents of this product, add 10mL of methanol, let stand for 2 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5mL of methanol to prepare the test solution. Separately, take puerarin reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution. Perform thin-layer chromatography (General Rule 0502), take 10μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate to form strips, use chloroform-methanol-water (7:2.5:0.25) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365nm).

[0190] Results: The spots in the thin-layer chromatography were severely tailed and did not move at the starting line. The spots could be detected under ultraviolet light (365nm).

[0191] Due to the presence of spot tailing and insufficient separation, the sample needs to be reprocessed and the developing solvent re-examined.

[0192] 1.3.4.1 Sample Solvent Investigation

[0193] Take 4g of the contents of this product, add 25mL each of ethanol, ethyl acetate and diethyl ether, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the test solution.

[0194] Results: When ethyl acetate was used as the sample extraction solvent, the spots in the thin-layer chromatography were clearer.

[0195] 1.3.4.2 Investigation of the developing solvent

[0196] Using puerarin as a control, chloroform-methanol-ethyl acetate (7:2:1), chloroform-methanol-water (7:2.5:0.25), n-butanol-anhydrous ethanol-glacial acetic acid-water (10:8:1:0.5), and ethyl acetate-acetic acid-water (9:1:0.5) were used as developing solvents. After development, the samples were removed, dried, and sprayed with 5% ferric chloride ethanol solution.

[0197] Results: Using n-butanol-anhydrous ethanol-glacial acetic acid-water (10:8:1:0.5) as the developing solvent, the spots were clearly displayed, the Rf value was moderate, there was no tailing phenomenon, the separation effect was good, and the toxicity was low. Therefore, the developing solvent "n-butanol-anhydrous ethanol-glacial acetic acid-water (10:8:1:0.5)" was selected.

[0198] 1.3.4.3 Examination of Extraction Methods

[0199] Take three portions of the contents of this product, each 4g, add 25mL of ethyl acetate, and extract by shaking for 30min, ultrasonic extraction for 30min, and reflux extraction for 30min respectively. Filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of methanol to prepare the test solution.

[0200] Result: The spots in the test solution obtained by ultrasonic extraction were clearer, thus confirming that the extraction method was "ultrasonic extraction".

[0201] 1.3.4.4 Examination of extraction time

[0202] Take three portions of the contents of this product, each 4g, add 25mL of ethyl acetate, and sonicate for 20min, 30min and 40min respectively. Filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of methanol to prepare the test solution.

[0203] Results: The ultrasonic extraction time of 30 min was just right, and the spots were clearer than those of the test sample solution. Therefore, the extraction time of 30 min was finally selected.

[0204] 1.3.4.5 Establishment of a thin-layer chromatography identification method for kudzu root

[0205] Take 4g of the contents of this product, add 25mL of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the test solution; separately take an appropriate amount of puerarin reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel H thin-layer plate, use n-butanol-anhydrous ethanol-glacial acetic acid-water in a ratio of 10:8:1:0.5 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365nm.

[0206] 1.3.5 Thin-layer chromatography identification of peppermint

[0207] First, the sample processing and developing solvent were determined according to the thin-layer chromatography identification method for peppermint in the 2020 edition of the Chinese Pharmacopoeia:

[0208] Take 1g of the contents of this product, add 10mL of anhydrous ethanol, sonicate for 20 minutes, filter, and use the filtrate as the test solution. Separately, take menthol reference standard, add anhydrous ethanol to prepare a solution containing 2mg per 1mL, and use it as the reference solution. Perform thin-layer chromatography (General Rule 0502), take 5-10μL of each of the above two solutions, and spot them separately on the same silica gel G thin-layer plate. Use toluene-ethyl acetate (9:1) as the developing solvent, develop, remove, air dry, spray with 2% p-dimethylaminobenzaldehyde in 40% sulfuric acid ethanol solution, heat at 80℃ until the spots are clearly visible, and examine under ultraviolet light (365nm).

[0209] Results: In thin-layer chromatography, the spots showed severe tailing and did not move at the starting line, resulting in insufficient separation and the spots were not visible under ultraviolet light.

[0210] Since the spots are not visible under ultraviolet light, this invention directly uses a colorimetric reagent. After reviewing a large amount of relevant literature and combining chemical composition analysis, this invention uses a 5% vanillin-sulfuric acid solution, dried with hot air. Furthermore, due to spot tailing and insufficient separation, the sample needs to be reprocessed and the developing solvent re-evaluated.

[0211] 1.3.5.1 Investigation of Sample Extraction Solvents

[0212] Take three portions of the contents of this product, each 3g, add 10mL each of water, methanol and ethyl acetate, sonicate for 20min, filter, and concentrate the filtrate to 1mL at low temperature (35-40℃) to obtain the test solution.

[0213] Results: When methanol was used as the sample extraction solvent, the spots in the thin-layer chromatography were clearer.

[0214] 1.3.5.2 Investigation of the developing solvent

[0215] Using menthol as a control, chloroform-methanol-ethyl acetate (10:2:1), chloroform-ethyl acetate-formic acid (8:2:0.5), petroleum ether (30-60℃)-ethyl acetate-benzene (9:1:2), and ethyl acetate-acetic acid-water (5:5:1) were used as developing solvents. After development, the samples were removed, dried, sprayed with 5% vanillin-sulfuric acid solution, and blown with hot air until the spots were clearly visible.

[0216] Results: Using petroleum ether (30-60℃)-ethyl acetate-benzene (9:1:2) as the developing solvent, the spots were clearly displayed, the Rf value was moderate, and there was no tailing phenomenon, indicating good separation effect. Therefore, the developing solvent "petroleum ether (30-60℃)-ethyl acetate-benzene (9:1:2)" was selected.

[0217] 1.3.5.3 Examination of Extraction Methods

[0218] Take three portions of the contents of this product, each 3g, add 10mL of methanol, and extract by shaking for 20min, ultrasonic extraction for 20min, and reflux extraction for 20min respectively. Filter, and concentrate the filtrate to 1mL at low temperature (35-40℃) to obtain the test solution.

[0219] Result: The spots in the test solution obtained by ultrasonic extraction were clearer, thus confirming that the extraction method was "ultrasonic extraction".

[0220] 1.3.5.4 Examination of extraction time

[0221] Take three portions of the contents of this product, each 3g, add 10mL of methanol, and sonicate for 20min, 30min and 40min respectively. Filter, and concentrate the filtrate to 1mL at low temperature (35-40℃) to obtain the test solution.

[0222] Results: Ultrasonic extraction for 30 min and 40 min resulted in clearer spots in the test solution compared to ultrasonic extraction for 20 min. However, the results of ultrasonic extraction for 30 min and 40 min were not significantly different. Therefore, the extraction time of 30 min was ultimately selected.

[0223] 1.3.5.5 Establishment of a thin-layer chromatography method for identifying peppermint

[0224] Take 3g of the contents of this product, add 10mL of methanol, sonicate for 30 minutes, filter, and concentrate the filtrate to 1mL at low temperature (35-40℃) as the test solution; separately take an appropriate amount of menthol reference standard, add methanol to prepare a solution containing 1mg in 1mL as the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use petroleum ether (30-60℃)-ethyl acetate-benzene in a ratio of 9:1:2 as the developing solvent, develop, remove, air dry, spray with 5% vanillin sulfuric acid solution, and blow with hot air until the spots are clearly visible.

[0225] II. Determination of ephedrine hydrochloride content in cold medicine compositions (cold soft capsules)

[0226] 2.1 Determination of chromatographic conditions

[0227] 2.1.1 Determination of wavelength

[0228] In this experiment, the reference standard of ephedrine hydrochloride was scanned under ultraviolet light. The maximum absorption was found at around 210 nm. Therefore, 210 nm was used as the detection wavelength in this experiment.

[0229] 2.1.2 Investigation of column temperature

[0230] This experiment investigated 25℃, 30℃, and 35℃. The results showed that the separation effect of ephedrine hydrochloride reference standard was the best at 30℃, with complete peak shape. Therefore, 30℃ was chosen as the detection temperature.

[0231] 2.1.3 Examination of Flow Velocity

[0232] This experiment investigated flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min. The results showed that the separation effect of ephedrine hydrochloride reference standard was good and the peak shape was symmetrical when the flow rate was 1.0 mL / min. Therefore, 1.0 mL / min was selected as the flow rate.

[0233] 2.1.4 Investigation of the mobile phase

[0234] The effects of acetonitrile-water, acetonitrile-0.1% phosphoric acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution, acetonitrile-0.15% phosphoric acid aqueous solution, acetonitrile-0.25% phosphoric acid solution-methanol, methanol-acetonitrile, and acetonitrile-0.1% glacial acetic acid solution as mobile phases on the separation efficiency and baseline were investigated. Ultimately, acetonitrile (A)-0.25% phosphoric acid solution (B)-methanol (C) was determined as the optimal mobile phase, with the gradient elution program being: 0 min, 5% A, 90% B, 5%... Gradient elution was performed for the following steps: 7 min, 12% A, 83% B, 5% C; 15 min, 15% A, 80% B, 5% C; 24 min, 20% A, 75% B, 5% C; 32 min, 30% A, 65% B, 5% C; 40 min, 5% A, 90% B, 5% C; 45 min, 5% A, 90% B, 5% C. This resulted in the best separation effect, symmetrical peak shape, and stable baseline.

[0235] In summary, the optimal chromatographic conditions are as follows: C18 column (250 nm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A) - 0.25% phosphoric acid solution (B) - methanol (C); gradient elution; elution program: 0 min, 5% A, 90% B, 5% C; 7 min, 12% A, 83% B, 5% C; 15 min, 15% A, 80% B, 5% C; 24 min, 20% A, 75% B, 5% C; 32 min, 30% A, 65% B, 5% C; 40 min, 5% A, 90% B, 5% C; 45 min, 5% A, 90% B, 5% C; flow rate: 1.0 mL / min; detection wavelength: 210 nm; column temperature: 30 °C; injection volume: 10 μL.

[0236] 2.2 Preparation of the test sample

[0237] 2.2.1 Investigation of Sample Extraction Methods

[0238] Take three portions of the contents of this product, each approximately 1g, accurately weigh them, place them in a stoppered conical flask, accurately add 50mL of 1% hydrochloric acid solution, sonicate (400W, 40kHz) for 30min, cool, accurately measure 25mL of the filtrate, place it in a separatory funnel, adjust the pH to 11-12 with ammonia, add ether and extract by shaking, sonication, and reflux three times, 25mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in an appropriate amount of acidic ethanol, place it in a 10mL volumetric flask, add acidic ethanol (take 10mL of ethanol, add 0.5mL of hydrochloric acid, mix well) and dilute to the mark, shake well, and the product is obtained;

[0239] The content of ephedrine hydrochloride was determined according to the chromatographic conditions in section "2.1". The results showed that the peak area of ​​the shake-extracted sample was relatively large and the information was richer, while the number of impurity peaks was relatively small. Therefore, shake-extraction was chosen as the extraction method in this experiment.

[0240] 2.2.2 Investigation of extraction solvent

[0241] This experiment investigated diethyl ether, water, methanol, and anhydrous ethanol. The results showed that diethyl ether as the extraction solvent produced more chromatographic peaks and richer information, and its extraction efficiency was superior to other solvents. Therefore, diethyl ether was chosen as the solvent for the extraction of ephedrine hydrochloride.

[0242] 2.2.3 Examination of the number of extractions

[0243] This experiment examined the number of extractions: 2, 3, and 4. The results showed that the extraction efficiency of 3 and 4 extractions was comparable, so 3 extractions were chosen as the number of extractions for the experiment.

[0244] 2.2.4 Determination of the preparation method for the test sample

[0245] Accurately weigh 1g of the contents of this product and place it in a stoppered conical flask. Accurately add 50mL of 1% hydrochloric acid solution, weigh the flask, sonicate for 30 minutes, cool, weigh the flask again, replenish the lost weight with 1% hydrochloric acid solution, shake well, filter, accurately measure 25mL of the filtrate, place it in a separatory funnel, adjust the pH to 11-12 with ammonia, extract with 25mL of ether three times, combine the ether extracts, evaporate to dryness, dissolve the residue in an appropriate amount of acidic ethanol, place it in a 10mL volumetric flask, dilute to the mark with acidic ethanol, shake well, and the product is obtained.

[0246] 2.3 Preparation of reference solution

[0247] Accurately weigh an appropriate amount of ephedrine hydrochloride reference standard, place it in a volumetric flask, and add methanol to prepare a solution containing 0.05 mg per 1 mL.

[0248] 2.4 System Adaptability Experiment

[0249] Under the chromatographic conditions described in section “2.1”, methanol, reference solution and test solution were injected into the liquid chromatograph respectively. The results showed that the resolution between ephedrine hydrochloride and adjacent peaks was greater than 1.5, and the theoretical plate number calculated based on the ephedrine hydrochloride peak was not less than 4000.

[0250] 2.5 Examination of Linear Relationships

[0251] Accurately pipette 5, 10, 15, 20, and 25 μL of ephedrine hydrochloride reference solution (50.2 μg / mL) into the sample and inject. Record the chromatograms. Plot the peak area on the ordinate and the injection volume of ephedrine hydrochloride reference solution (μg) on ​​the abscissa. The linear equation obtained is Y = 605822X - 517.4, R0 2 =0.9998, indicating a good linear relationship in the determination of ephedrine hydrochloride content between 0.251 μg and 1.255 μg. See Table 1.

[0252] Table 1. Linearity of Ephedrine Hydrochloride

[0253]

[0254] 2.6 Methodological Examination

[0255] 2.6.1 Precision Experiment

[0256] Accurately pipette 10 μL of the reference solution and inject it 6 times, recording the peak area of ​​ephedrine hydrochloride. Results: The calculated RSD value of the peak area of ​​ephedrine hydrochloride was 0.59%, indicating good instrument precision. See Table 2.

[0257]

[0258] 2.6.2 Stability Test

[0259] The same test solution was injected and analyzed at 0, 2, 4, 8, 12, and 24 hours, and the peak area of ​​ephedrine hydrochloride was recorded. Results: The RSD of the ephedrine hydrochloride content was calculated to be 2.12%, indicating that the test solution had good stability within 24 hours. See Table 3.

[0260] Table 3 Stability Experiment

[0261]

[0262] 2.6.3 Repeatability Experiment

[0263] Take approximately 1g of the same batch of samples, accurately weigh each portion, and prepare the test solution according to the method in section "2.2". Determine the content under the chromatographic conditions in section "2.1". Results: The measured ephedrine hydrochloride contents were 336.08 μg / capsule, 335.72 μg / capsule, 335.14 μg / capsule, 333.65 μg / capsule, 331.46 μg / capsule, and 332.19 μg / capsule, with an average value of 334.04 μg / capsule and an RSD of 1.92%. See Table 4.

[0264] Table 4 Repeatability Experiments

[0265]

[0266] 2.6.4 Recovery Experiment

[0267] Take approximately 0.5 g of the contents of each cold and flu soft capsule and place it in a round-bottom flask. Add an appropriate amount of ephedrine hydrochloride reference standard stock solution, accurately weighed, and prepare the test solution according to the method in section "2.2". Determine the recovery rate under the chromatographic conditions in section "2.1". Results: The average recovery rate was 100.15%, and the RSD was 0.91%. See Table 5.

[0268] Table 5. Sample recovery rate experiment

[0269]

[0270] 2.7 Determination of ephedrine hydrochloride content in samples

[0271] Three batches of test solutions were prepared according to the method described in section "2.2". 10 μL of each of the reference solution and the test solution were precisely pipetted into the liquid chromatograph for determination. The content of the analyte, ephedrine hydrochloride, in the test sample was calculated based on the peak area of ​​ephedrine hydrochloride in the chromatograms of the reference solution and the test solution. Results: The contents of ephedrine hydrochloride in the three batches of samples were 335.68, 333.14, and 336.85 μg / particle, respectively, with an average value of 335.22 μg / particle and an RSD of 1.90%, indicating that the method of the present invention is stable and feasible. See Table 6.

[0272] Table 6. Sample Content Determination Data

[0273]

[0274] In summary, this invention establishes an HPLC method for determining the content of ephedrine hydrochloride in cold and flu soft capsules. Methodological evaluation results show that the precision, stability, reproducibility, and recovery rate of this method all meet the requirements. Furthermore, the method is simple to operate, requires minimal equipment, and exhibits good repeatability. It provides a reference for the quality standards of cold and flu soft capsules and can comprehensively and effectively control product quality.

[0275] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A quality control method for a cold medicine composition, the medicine composition comprising 30 parts of Notopterygium root, 60 parts of Ephedra, 45 parts of Cinnamon twig, 45 parts of Schizonepeta spike, 30 parts of Saposhnikovia root, 30 parts of Angelica dahurica root, 30 parts of Ligusticum chuanxiong rhizome, 15 parts of Acorus tatarinowii rhizome, 45 parts of Pueraria lobata root, 15 parts of Mentha haplocalyx, 60 parts of bitter almond, 30 parts of Angelica sinensis root, 60 parts of Scutellaria baicalensis root, and 30 parts of Platycodon grandiflorus root, characterized in that... The quality control methods include, in sequence: (1) thin-layer chromatography identification of ephedra; (2) thin-layer chromatography identification of scutellaria baicalensis; (3) thin-layer chromatography identification of chuanxiong rhizome; (4) thin-layer chromatography identification of peppermint; (5) thin-layer chromatography identification of kudzu root; (6) determination of the content of ephedrine hydrochloride in the drug composition; wherein: (1) Thin-layer chromatography identification of ephedra: Take 5g of the contents of this product, add 10mL of 1% hydrochloric acid solution, sonicate for 30 minutes, filter, add concentrated ammonia test solution to the filtrate, adjust the pH to 11-12, extract with ether three times, 20mL each time, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to obtain the test solution; separately take an appropriate amount of ephedrine hydrochloride reference standard, add methanol to prepare a solution containing 1mg per 1mL to obtain the reference solution; perform thin-layer chromatography, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use n-butanol-glacial acetic acid-water in a ratio of 7:1:2 as the developing solvent, develop, remove, air dry, spray with 0.3% ninhydrin test solution, and blow with hot air until the spots are clearly visible; (2) Thin-layer chromatography identification of Scutellaria baicalensis: Take 3g of the contents of this product, add 25mL of water, sonicate for 30 minutes, filter with defatted cotton, adjust the pH of the filtrate to 2-3 with dilute hydrochloric acid, extract with ethyl acetate three times by shaking, 20mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1mL of ethanol, and use as the test solution; separately take an appropriate amount of baicalin reference standard, add methanol to prepare a solution containing 1mg in 1mL, and use as the reference solution; perform thin-layer chromatography, take 2μL of each of the above two solutions, spot them separately on the same polyamide film, use the upper layer solution of ethyl acetate-butanone-acetic acid-water in a ratio of 10:7:5:3 as the developing solvent, develop, remove, air dry, and spray with 5% ferric chloride ethanol solution; (3) Thin-layer chromatography identification of Ligusticum chuanxiong: Take 5g of the contents of this product, add 25mL of water, sonicate for 30 minutes, filter with defatted cotton, extract the filtrate three times with 20mL of water-saturated ether, combine the ether extracts, evaporate to dryness, dissolve the residue in 0.5mL of ethanol to prepare the test solution; separately take an appropriate amount of ferulic acid reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform thin-layer chromatography, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, develop with benzene-ethyl acetate-formic acid in a ratio of 5:2:1, remove, air dry, and spray with a solution of 1% ferric chloride solution and 1% potassium ferricyanide solution in a volume ratio of 1:1; (4) Thin-layer chromatography identification of peppermint is as follows: Take 3g of the contents of this product, add 10mL of methanol, sonicate for 30 minutes, filter, and concentrate the filtrate to 1mL at a low temperature of 37℃ to obtain the test solution; separately take an appropriate amount of menthol reference standard, add methanol to prepare a solution containing 1mg in 1mL to obtain the reference solution; perform thin-layer chromatography, take 5μL of each of the above two solutions, spot them separately on the same silica gel G thin-layer plate, use petroleum ether at 30-60℃-ethyl acetate-benzene in a ratio of 9:1:2 as the developing solvent, develop, remove, air dry, spray with 5% vanillin sulfuric acid solution, and blow with hot air until the spots are clearly visible; (5) Thin-layer chromatography identification of kudzu root is as follows: Take 4g of the contents of this product, add 25mL of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the test solution; separately take an appropriate amount of puerarin reference standard, add methanol to prepare a solution containing 1mg per 1mL to prepare the reference solution; perform the thin-layer chromatography test according to the General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 5μL of each of the above two solutions, spot them separately on the same silica gel H thin-layer plate, use n-butanol-anhydrous ethanol-glacial acetic acid-water in a ratio of 10:8:1:0.5 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365 nm; (6) The determination of ephedrine hydrochloride content is performed by high performance liquid chromatography, including: 1) Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase. The mobile phase consisted of acetonitrile (Mobile Phase A), 0.25% phosphoric acid solution (Mobile Phase B), and methanol (Mobile Phase C). Gradient elution was performed using the following program: 0 min, 5% A, 90% B, 5% C; 7 min, 12% A, 83% B, 5% C; 15 min, 15% A, 80% B, 5% C; 24 min, 20% A, 75% B, 5% C; 32 min, 30% A, 65% B, 5% C; 40 min, 5% A, 90% B, 5% C; 45 min, 5% A, 90% B, 5% C. The flow rate was 1 mL / min; the detection wavelength was 210 nm; the column temperature was 30℃; the injection volume was 10 μL; and the theoretical plate number, calculated based on the ephedrine hydrochloride peak, should be no less than 4000. 2) Preparation of reference solution: Take an appropriate amount of ephedrine hydrochloride reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.05 mg per 1 mL. 3) Preparation of the test solution: Accurately weigh 1g of the contents of this product and place it in a stoppered conical flask. Accurately add 50mL of 1% hydrochloric acid solution, weigh the solution, sonicate for 30 minutes, cool, weigh the solution again, replenish the lost weight with 1% hydrochloric acid solution, shake well, filter, accurately measure 25mL of the filtrate, place it in a separatory funnel, adjust the pH to 11-12 with ammonia water, extract with 25mL of ether three times, combine the ether extracts, evaporate to dryness, dissolve the residue in an appropriate amount of acidic ethanol, place it in a 10mL volumetric flask, dilute to the mark with acidic ethanol, and shake well; the acidic ethanol is prepared by mixing 10mL of ethanol with 0.5mL of hydrochloric acid. 4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

2. The quality control method according to claim 1, characterized in that, The ultrasonic conditions are: 400W, 40kHz.

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