Quality detection method of Kaihoujian spray

Abstract

The invention relates to the technical field of medicine detection, in particular to a quality detection method of Kaihoujian spray. The quality detection method of the Kaihoujian spray adopts gas chromatography to measure the content of menthol, and specifically comprises the following steps: preparing a test solution; preparing a reference substance solution; performing chromatographic condition and system adaptability test; and a measurement method. The method for determining the content of the Kaihoujian spray, established by the invention, has the advantages that the specificity is strong, the stability, repeatability, intermediate precision, accuracy and the like all meet the requirements, the durability of chromatographic conditions is good, and a technical support is provided for further improving the quality standard of the Kaihoujian spray.

Description

Technical Field

[0001] This invention relates to the field of drug testing technology, specifically to a quality testing method for Kaihoujian spray. Background Technology

[0002] Kaihoujian spray has the effects of clearing heat and detoxifying, reducing swelling and relieving pain. It is often used to treat acute and chronic pharyngitis, tonsillitis, sore throat, stomatitis, gingival swelling and pain, etc. It is favored by doctors and patients because of its significant clinical efficacy.

[0003] The prescription for Kaihoujian spray consists of: 313g of Cinnamon Root, 313g of Sophora Root, 250g of Cicada Slough, and 1g of Menthol. The preparation method is as follows: Except for menthol, the above four herbs (Cinnamon Root, Sophora Root, and Cinnamon Root) are decocted twice with water, the first time for 2 hours and the second time for 1 hour. The decoctions are combined, filtered, and the filtrate is concentrated to a clear extract with a relative density of 1.05-1.10 at 50℃. Ethanol is added to make the alcohol content reach 80%, and the mixture is allowed to stand for 24 hours. The extract is then filtered, and the ethanol is recovered under reduced pressure and concentrated to a clear extract with a relative density of 1.10-1.20 at 80℃. 1g of Menthol, 2.5g of Sodium Benzoate, 1g of Citric Acid, 5.5mL of Myrica Flavoring, 1.04g of Stevioside, and 803mL of Polysorbate are dissolved in 20mL of ethanol. The resulting solution is added to the clear extract, and water is added to 1000mL. The mixture is stirred until homogeneous and then filtered to obtain the final product.

[0004] The current quality testing standards for Kaihoujian spray include thin-layer chromatography identification of cinnabar root, thin-layer chromatography identification of sophora root, thin-layer chromatography identification of menthol, content determination of cinnabar root, and content determination of sophora root. However, a method for determining the content of menthol, one of the main components of this product, has not yet been established, which is not conducive to comprehensive quality control of this product.

[0005] In the existing technology, the published literature "Simultaneous Determination of Four Components in Kaihoujian Spray by GC-FID" provides a method for determining the menthol content in Kaihoujian spray using the internal standard method. The chromatographic conditions are as follows: HP-INNOWA column (30m × 0.25mm × 0.25μm); FID detector, injection port temperature 260℃, detector temperature 260℃; split injection, split ratio 5:1; temperature program: initial temperature 100℃, hold for 8 min, increase to 250℃ at a rate of 20℃ / min, hold for 20 min. However, when using the above-mentioned method to determine the menthol content in this product, the separation between the target peak and the impurity peak is relatively small, which easily leads to inaccurate quantification of menthol in Kaihoujian spray.

[0006] Therefore, in order to further improve the quality testing standards of Kaihoujian spray, it is necessary to establish a highly accurate, stable, and feasible method for determining menthol content. Summary of the Invention

[0007] The purpose of this invention is to provide a quality testing method for Kaihoujian spray.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The quality testing method for the throat-opening spray of the present invention involves determining the menthol content using gas chromatography, and the steps are as follows: S1. Preparation of the test solution: Accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 0-9 mL of water, and shake well; extract with 10-20 mL of ethyl acetate by shaking 1-2 times, and let stand to allow the layers to separate; collect and combine the upper ethyl acetate layer into a 25 mL volumetric flask, add ethyl acetate to the mark, filter with a 0.45 μm or 0.22 μm organic microporous membrane, and take the filtrate; S2. Preparation of the reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL; S3. Chromatographic conditions and system suitability test: The chromatographic column was a capillary column with cross-linked polyethylene glycol as the stationary phase, measuring 30 m × 0.32 mm × 0.5 μm. A programmed temperature ramp was used, starting at 60 °C, ramping to 120 °C at a rate of 2 °C / min, and then ramping to 246 °C at a rate of 5 °C / min. The injection port temperature was 198 °C–202 °C; the detector temperature was 278 °C–282 °C; splitless injection was used, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min; the theoretical plate number, calculated based on the menthol peak, should be no less than 10,000. S4. Determination Method: Accurately pipette 1 μL of the test solution and inject it into the gas chromatograph. Measure and calculate the result. Preferably, in the quality testing method of the Kaihoujian spray of the present invention, step S1 in the determination of menthol content specifically comprises: accurately measuring 1 mL of Kaihoujian spray into a separatory funnel, adding 4-9 mL of water, and shaking well; extracting with 10-20 mL of ethyl acetate by shaking 1-2 times, and allowing it to stand to separate into layers; collecting and combining the upper ethyl acetate solution into a 25 mL volumetric flask, adding ethyl acetate to make up to the mark, filtering with a 0.45 μm organic microporous membrane, and taking the filtrate to obtain the product.

[0009] In a further preferred embodiment, in the quality testing method of the Kaihoujian spray of the present invention, step S1 in the determination of menthol content specifically comprises: accurately measuring 1 mL of Kaihoujian spray into a separatory funnel, adding 4 mL of water, and shaking well; extracting once with 20 mL of ethyl acetate, allowing it to stand to separate into layers; collecting the upper ethyl acetate solution into a 25 mL volumetric flask, adding ethyl acetate to make up to the mark, filtering with a 0.45 μm organic microporous membrane, and taking the filtrate to obtain the final product.

[0010] Preferably, in the quality testing method of the throat-opening spray of the present invention, in the determination of menthol content, the injection port temperature in step S3 is 200℃-202℃.

[0011] In a further preferred embodiment, in the quality testing method for the throat-opening spray of the present invention, the temperature of the injection port in step S3 for determining the menthol content is 200°C.

[0012] Preferably, in the quality detection method of the throat-opening spray of the present invention, in the determination of menthol content, the temperature of the detector in step S3 is 280℃-282℃.

[0013] In a further preferred embodiment, in the quality detection method of the throat-opening spray of the present invention, the temperature of the detector in step S3 for determining the menthol content is 280°C.

[0014] Preferably, in the quality detection method of the throat-opening spray of the present invention, the detector in step S3 for determining the menthol content is an FID detector.

[0015] The beneficial effects of this invention are: 1. This invention establishes a method for determining the content of menthol in Kaihoujian spray. The method has strong specificity, and its stability, repeatability, intermediate precision, and accuracy all meet the requirements. Moreover, the chromatographic conditions are robust, providing technical support for further improving the quality standards of Kaihoujian spray.

[0016] 2. By investigating the extraction solvent volume, extraction times, and extraction water volume, the optimal extraction parameters for menthol in Kaihoujian spray were determined to be: one extraction with 20 mL of ethyl acetate and 4 mL of extraction water. Attached Figure Description

[0017] Figure 1 Chromatogram of the reference standard for specificity assessment; Figure 2 To investigate the specificity of the sample chromatogram; Figure 3 Chromatogram of a negative sample for specificity assessment; Figure 4 This is the standard curve for menthol. Detailed Implementation

[0018] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0019] Example 1 The method for determining the menthol content in Kaihoujian spray is as follows: (1) Preparation of the test solution: Accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 4 mL of water, and shake well; extract once with 20 mL of ethyl acetate, and let stand to allow the layers to separate; collect the upper ethyl acetate solution into a 25 mL volumetric flask, add ethyl acetate to make up to the mark, filter with a 0.45 μm organic microporous membrane, and take the filtrate to obtain the product. (2) Preparation of reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL; (3) Chromatographic conditions and system suitability test: The chromatographic column was a DB-WAX capillary column with cross-linked polyethylene glycol as the stationary phase, measuring 30 m × 0.32 mm × 0.5 μm. A programmed temperature ramp was used: an initial temperature of 60 °C, ramped to 120 °C at a rate of 2 °C / min, and then ramped to 246 °C at a rate of 5 °C / min. The injection port temperature was 200 °C. An FID detector was used, with a detector temperature of 280 °C. Splitless injection was employed, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min. The theoretical plate number, calculated based on the menthol peak, should be no less than 10,000. (4) Determination method: Accurately pipette 1 μL of the test solution and inject it into the gas chromatograph for determination.

[0020] Example 2 The method for determining the menthol content in Kaihoujian spray is as follows: (1) Preparation of the test solution: Accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 4 mL of water, and shake well; extract once with 15 mL of ethyl acetate, and let stand to allow the layers to separate; collect the upper ethyl acetate solution into a 25 mL volumetric flask, add ethyl acetate to the mark, filter with a 0.22 μm organic microporous membrane, and collect the filtrate. (2) Preparation of reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL; (3) Chromatographic conditions and system suitability test: The chromatographic column was a DB-WAX capillary column with cross-linked polyethylene glycol as the stationary phase, measuring 30 m × 0.32 mm × 0.5 μm. A programmed temperature ramp was used: an initial temperature of 60 °C, ramped to 120 °C at a rate of 2 °C / min, and then ramped to 246 °C at a rate of 5 °C / min. The injection port temperature was 200 °C. An FID detector was used, with a detector temperature of 280 °C. Splitless injection was employed, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min. The theoretical plate number, calculated based on the menthol peak, should be no less than 10,000. (4) Determination method: Accurately pipette 1 μL of the test solution and inject it into the gas chromatograph for determination.

[0021] Example 3 The method for determining the menthol content in Kaihoujian spray is as follows: (1) Preparation of the test solution: Accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 4 mL of water, and shake well; extract twice with 10 mL of ethyl acetate, and let stand to allow the layers to separate; collect and combine the upper ethyl acetate layer into a 25 mL volumetric flask, add ethyl acetate to the mark, filter with a 0.45 μm organic microporous membrane, and take the filtrate to obtain the product. (2) Preparation of reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL; (3) Chromatographic conditions and system suitability test: The chromatographic column was a DB-WAX capillary column with cross-linked polyethylene glycol as the stationary phase, measuring 30 m × 0.32 mm × 0.5 μm. A programmed temperature ramp was used: an initial temperature of 60 °C, ramped to 120 °C at a rate of 2 °C / min, and then ramped to 246 °C at a rate of 5 °C / min. The injection port temperature was 200 °C. An FID detector was used, with a detector temperature of 280 °C. Splitless injection was employed, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min. The theoretical plate number, calculated based on the menthol peak, should be no less than 10,000. (4) Determination method: Accurately pipette 1 μL of the test solution and inject it into the gas chromatograph for determination.

[0022] Example 4 The method for determining the menthol content in Kaihoujian spray is as follows: (1) Preparation of the test solution: Accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 4 mL of water, and shake well; extract once with 20 mL of ethyl acetate, and let stand to allow the layers to separate; collect the upper ethyl acetate solution into a 25 mL volumetric flask, add ethyl acetate to make up to the mark, filter with a 0.45 μm organic microporous membrane, and take the filtrate to obtain the product. (2) Preparation of reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL; (3) Chromatographic conditions and system suitability test: The chromatographic column was a DB-WAX capillary column with cross-linked polyethylene glycol as the stationary phase, measuring 30 m × 0.32 mm × 0.5 μm. A programmed temperature ramp was used: an initial temperature of 60 °C, ramped to 120 °C at a rate of 2 °C / min, and then ramped to 246 °C at a rate of 5 °C / min. The injection port temperature was 198 °C. An FID detector was used, with a detector temperature of 278 °C. Splitless injection was employed, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min. The theoretical plate number, calculated based on the menthol peak, should be no less than 10,000. (4) Determination method: Accurately pipette 1 μL of the test solution and inject it into the gas chromatograph for determination.

[0023] Example 5 The method for determining the menthol content in Kaihoujian spray is as follows: (1) Preparation of the test solution: Accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 4 mL of water, and shake well; extract once with 20 mL of ethyl acetate, and let stand to allow the layers to separate; collect the upper ethyl acetate solution into a 25 mL volumetric flask, add ethyl acetate to make up to the mark, filter with a 0.45 μm organic microporous membrane, and take the filtrate to obtain the product. (2) Preparation of reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL; (3) Chromatographic conditions and system suitability test: The chromatographic column was a DB-WAX capillary column with cross-linked polyethylene glycol as the stationary phase, measuring 30 m × 0.32 mm × 0.5 μm. A programmed temperature ramp was used: an initial temperature of 60 °C, ramped to 120 °C at a rate of 2 °C / min, and then ramped to 246 °C at a rate of 5 °C / min. The injection port temperature was 202 °C. An FID detector was used, with a detector temperature of 282 °C. Splitless injection was employed, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min. The theoretical plate number, calculated based on the menthol peak, should be no less than 10,000. (4) Determination method: Accurately pipette 1 μL of the test solution and inject it into the gas chromatograph for determination.

[0024] Example 6 The method for determining the menthol content in Kaihoujian spray is as follows: (1) Preparation of the test solution: Accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 4 mL of water, and shake well; extract twice with 10 mL of ethyl acetate, and let stand to allow the layers to separate; collect and combine the upper ethyl acetate layer into a 25 mL volumetric flask, add ethyl acetate to the mark, filter with a 0.45 μm organic microporous membrane, and take the filtrate to obtain the product. (2) Preparation of reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL; (3) Chromatographic conditions and system suitability test: The chromatographic column was a DB-WAX capillary column with cross-linked polyethylene glycol as the stationary phase, measuring 30 m × 0.32 mm × 0.5 μm. A programmed temperature ramp was used: an initial temperature of 60 °C, ramped to 120 °C at a rate of 2 °C / min, and then ramped to 246 °C at a rate of 5 °C / min. The injection port temperature was 198 °C. An FID detector was used, with a detector temperature of 278 °C. Splitless injection was employed, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min. The theoretical plate number, calculated based on the menthol peak, should be no less than 10,000. (4) Determination method: Accurately pipette 1 μL of the test solution and inject it into the gas chromatograph for determination.

[0025] Example 7 The method for determining the menthol content in Kaihoujian spray is as follows: (1) Preparation of the test solution: Accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 4 mL of water, and shake well; extract twice with 10 mL of ethyl acetate, and let stand to allow the layers to separate; collect and combine the upper ethyl acetate layer into a 25 mL volumetric flask, add ethyl acetate to the mark, filter with a 0.45 μm organic microporous membrane, and take the filtrate to obtain the product. (2) Preparation of reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL; (3) Chromatographic conditions and system suitability test: The chromatographic column was a DB-WAX capillary column with cross-linked polyethylene glycol as the stationary phase, measuring 30 m × 0.32 mm × 0.5 μm. A programmed temperature ramp was used: an initial temperature of 60 °C, ramped to 120 °C at a rate of 2 °C / min, and then ramped to 246 °C at a rate of 5 °C / min. The injection port temperature was 202 °C. An FID detector was used, with a detector temperature of 282 °C. Splitless injection was employed, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min. The theoretical plate number, calculated based on the menthol peak, should be no less than 10,000. (4) Determination method: Accurately pipette 1 μL of the test solution and inject it into the gas chromatograph for determination.

[0026] To further verify the reliability of the present invention and select the optimal solution, the inventors conducted a series of experiments, as follows: 1. Instruments and reagents Main instruments: Gas chromatograph (Agilent 8890); chromatographic column (DB-WAX, HP-5, both 30 m × 0.32 mm × 0.5 μm); Electronic analytical balance (EX225DZH, 0.0001 g).

[0027] Main reagents: purified water (Hangzhou Wahaha Group Co., Ltd.); ethyl acetate (analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.); menthol reference standard (batch number: 110728-202208, purity 99.6%, purchased from China National Institutes for Food and Drug Control).

[0028] The samples used in the study were produced by Guizhou Sanli Pharmaceutical Co., Ltd., with batch numbers 250101, 241221, 241222, 241223, 241224, 241225, 241226, 250102, 250201, 250202, 250203, 250204, 250205, C251206, C251207, and C251208.

[0029] 2. Exploration of methods for determining menthol content Preparation of the test solution: Accurately measure 1 mL of this product (batch number 250101) into a separatory funnel, add 4 mL of water, and shake well; extract twice with ethyl acetate, 10 mL each time, and let stand to allow the layers to separate; collect and combine the ethyl acetate solution (upper layer) into a 25 mL volumetric flask, add ethyl acetate to the mark, filter with a 0.45 μm or 0.22 μm organic microporous membrane, and take the filtrate to obtain the test solution.

[0030] Preparation of reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL.

[0031] Chromatographic conditions and system suitability test: The chromatographic column was a gas capillary column (DB-WAX, 30 m × 0.32 mm × 0.5 μm) with cross-linked polyethylene glycol as the stationary phase; programmed temperature rise was used, with an initial temperature of 60℃, increased to 120℃ at a rate of 2℃ / min, and then increased to 246℃ at a rate of 5℃ / min; the injection port temperature was 200℃; the FID detector temperature was 280℃; splitless injection was used, with an injection volume of 1 μL; the carrier gas was high-purity nitrogen, with a carrier gas flow rate of 1 mL / min; the theoretical plate number, calculated based on the menthol peak, should not be less than 10,000.

[0032] 2.1 Selection of Chromatographic Column The test solution and reference solution were prepared according to the method under "2. Determination of Menthol Content". Gas chromatography analysis was performed using a capillary column with cross-linked polyethylene glycol as the stationary phase (DB-WAX, 30 m × 0.32 mm × 0.5 μm) and a capillary column with cross-linked 5% phenylmethyl polysiloxane as the stationary phase (HP-5, 30 m × 0.32 mm × 0.5 μm), respectively. The menthol content was calculated, and the results are shown in Table 1.

[0033] .

[0034] The results show that there was no significant difference in the determination of menthol content when using a DB-WAX capillary column with cross-linked polyethylene glycol as the stationary phase and an HP-5 capillary column with cross-linked 5% phenylmethyl polysiloxane as the stationary phase. However, when using the HP-5 capillary column with cross-linked 5% phenylmethyl polysiloxane as the stationary phase, a large impurity peak appeared before the sample peak, and the resolution did not meet the requirements. Therefore, a capillary column with cross-linked polyethylene glycol as the stationary phase (DB-WAX) was selected for chromatographic analysis.

[0035] 2.2 Preparation and Investigation of the Test Solution (1) Investigation on the amount of extraction solvent used Accurately measure 1 mL of this product into a separatory funnel, add 4 mL of water, and shake well. Prepare three samples using the same method. Extract each sample once with 10 mL, 15 mL, and 20 mL of ethyl acetate, respectively. Allow the samples to stand to separate into layers. Collect the ethyl acetate solution (superior layer) into a 25 mL volumetric flask, add ethyl acetate to the mark, and filter through a 0.45 μm organic microporous membrane. Collect the filtrate to obtain the test solution. Perform gas chromatography analysis according to the chromatographic conditions under "2. Determination of Menthol Content" and calculate the menthol content. The results are shown in Table 2.

[0036] .

[0037] The results showed that the menthol content was higher when the ethyl acetate extraction volume was 20 mL than when the extraction volumes were 10 mL and 15 mL. Therefore, the optimal extraction volume for ethyl acetate was determined to be 20 mL.

[0038] (2) Examination of the number of extractions Accurately measure 1 mL of this product into a separatory funnel, add 4 mL of water, and shake well. Prepare two samples using the same method, and treat them as follows: Sample 1 is extracted once with 20 mL of ethyl acetate by shaking; Sample 2 is extracted twice with 10 mL of ethyl acetate each time. After standing to allow the layers to separate, collect the ethyl acetate solution (supernatant), transfer it to a 25 mL volumetric flask and dilute to volume. Filter through a 0.45 μm organic microporous membrane, and collect the filtrate to obtain the test solution. Perform gas chromatography analysis according to the chromatographic conditions under "2. Determination of Menthol Content" to calculate the menthol content. The results are shown in Table 3.

[0039] .

[0040] The results showed no significant difference in menthol content between extraction with 20 mL ethyl acetate once and extraction with 10 mL ethyl acetate twice. Therefore, extraction with 20 mL ethyl acetate once was selected.

[0041] (3) Investigation of water extraction volume Accurately measure 1 mL of this product into a separatory funnel, and prepare three samples using the same method, setting the water addition volumes to 0 mL, 4 mL, and 9 mL, respectively. After shaking well, extract once with 20 mL of ethyl acetate, allow to stand to separate the layers, collect the ethyl acetate solution (superior solution) into a 25 mL volumetric flask, add ethyl acetate to the mark, filter through a 0.45 μm organic microporous membrane, and collect the filtrate to obtain the test solution. Perform gas chromatography analysis according to the chromatographic conditions under "2. Determination of Menthol Content", calculate the menthol content, and the results are shown in Table 4.

[0042] .

[0043] The results showed no significant difference in menthol content when the extraction water volume was 0 mL, 4 mL, and 9 mL. To better observe the stratification phenomenon, an extraction water volume of 4 mL was selected.

[0044] 3. Preliminary determination of content determination method After a series of condition investigation experiments, the preliminary determination method for the content of menthol in Kaihoujian spray is as follows: Preparation of the test solution: Accurately measure 1 mL of this product into a separatory funnel, add 4 mL of water, and shake well; extract once with 20 mL of ethyl acetate, and let stand to allow the layers to separate; collect the ethyl acetate solution (upper layer) into a 25 mL volumetric flask, add ethyl acetate to the mark, filter with a 0.45 μm organic microporous membrane, and collect the filtrate to obtain the test solution.

[0045] Preparation of reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL.

[0046] Chromatographic conditions and system suitability test: The chromatographic column was a gas capillary column (DB-WAX, 30 m × 0.32 mm × 0.5 μm) with cross-linked polyethylene glycol as the stationary phase; programmed temperature rise was used, with an initial temperature of 60℃, increased to 120℃ at a rate of 2℃ / min, and then increased to 246℃ at a rate of 5℃ / min; the injection port temperature was 200℃; the FID detector was used, with a detector temperature of 280℃; splitless injection was used, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min; the theoretical plate number, calculated based on the menthol peak, should not be less than 10,000.

[0047] 4. Methodological Examination 4.1 Specificity test To investigate whether other medicinal ingredients and excipients interfere with the determination of menthol in Kaihoujian spray, medicinal ingredients and excipients were weighed according to the prescription ratio to prepare menthol-deficient negative samples using the same method. The menthol-deficient negative sample solution was then prepared according to the treatment method for the test sample and injected. The chromatogram was recorded (e.g., ...). Figure 1-3 (As shown). The results showed that in the menthol-deficient negative samples, there were no chromatographic peaks at the retention times corresponding to menthol, indicating that other medicinal ingredients and excipients did not interfere with the determination of menthol, and that this method is specific for determining the menthol content in the finished product.

[0048] 4.2 Examination of Linear Relationships Accurately weigh 25.03 mg of menthol reference standard and place it in a 50 mL volumetric flask. Dissolve and dilute to the mark with ethyl acetate, and shake well to obtain a reference standard stock solution with a concentration of 0.5006 mg / mL. Accurately measure 0.1 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, and 5 mL of the stock solution and place them in 10 mL volumetric flasks. Dilute to the mark with ethyl acetate, shake well, and prepare reference standard solutions with concentrations of 5.01 μg / mL, 25.03 μg / mL, 50.06 μg / mL, 100.12 μg / mL, 150.18 μg / mL, and 250.30 μg / mL, respectively. Accurately pipette 1 μL of each of the above reference standard solutions and inject it into the gas chromatograph. Analyze the solution under the gas chromatographic conditions described above. Plot a standard curve with the injected concentration as the x-axis and the peak area as the y-axis. Calculate the regression equation: Menthol Y = 17.231X + 14.007, correlation coefficient: =1.000. The results show that when the menthol injection concentration is between 5.01 μg / mL and 250.30 μg / mL, the linear relationship between injection concentration and peak area is good. The results of the linearity study of menthol are shown in Table 5, and the menthol standard curve is shown in... Figure 4 As shown.

[0049] .

[0050] 4.3 Detection Limit Accurately pipette appropriate amounts of the 5.01 μg / mL standard solution, dilute successively with ethyl acetate, and inject for determination. The results showed that the limit of detection (LOD) was 0.05 μg / mL (S / N=3).

[0051] 4.4 Precision Test Accurately measure 1 mL of this product and operate according to the method under "3. Preliminary Determination of Content Determination Method". Inject 6 consecutively to examine the instrument precision. The results are shown in Table 6. The results show that the peak area RSD value of 0.83% after 6 parallel injections indicates that the instrument has good precision.

[0052] .

[0053] 4.5 Repeatability Test Accurately measure 1 mL of this product, and take 6 portions. Perform the procedure according to the method under "3. Preliminary Determination of Content Determination Method". Calculate the menthol content and examine the repeatability of the method. The results are shown in Table 7. The results show that the peak area RSD value of the 6 parallel tests is 0.82%, indicating that the method has good repeatability.

[0054] .

[0055] 4.6 Stability Test Accurately measure 1 mL of this product and prepare the test solution according to the method under "3. Preliminary Determination of Content Determination Method". Perform gas chromatography analysis at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, record the peak area and calculate the RSD value to evaluate the stability of the method. The results are shown in Table 8. The results show that the test solution has good stability within 24 h.

[0056] .

[0057] 4.7 Accuracy Test The accuracy of the method was evaluated using the spiked recovery method. Specifically, 0.5 mL of the sample was accurately measured into a separatory funnel, and three portions were taken. 1 mL, 2 mL, and 3 mL of the prepared menthol reference stock solution (concentration 89 μg / mL) were accurately added to each portion, respectively. The test solutions were then prepared according to the method described in section "3. Preliminary Determination of Content Determination Method," and gas chromatography analysis was performed. Three replicates were set up for each group to determine the menthol content, and the recovery rate was calculated to evaluate the accuracy of the method. The results are shown in Table 9. The results show that the recovery rate ranged from 97.00% to 103.37%, with an average recovery rate of 99.90% and an RSD of 2.09%, indicating good accuracy of the method.

[0058] .

[0059] 4.8 Intermediate Precision Different determination times and different personnel were selected for the operation. 1 mL of the sample was accurately measured, and test solutions were prepared according to the method described in section "3. Preliminary Determination of Content Determination Method," and gas chromatography analysis was performed to determine and calculate the menthol content. The results are shown in Table 10. The results show that the average menthol content in samples A and B was 0.354 mg / mL, with an RSD of 1.91%, indicating good intermediate precision of this method.

[0060] .

[0061] 4.9 Durability Test Gas chromatography analysis was performed according to the method described in section 3, "Preliminary Determination of Content Determination Method." The effects of different detector temperatures (278℃, 280℃, 282℃) and injection port temperatures (198℃, 200℃, 202℃) on the chromatographic analysis were investigated to evaluate the robustness of the method. The results are shown in Table 11. The results show that the menthol content determination results were basically consistent under various conditions, with an RSD value <3%. The menthol peak in the chromatogram was sharp and symmetrical, indicating that the method has good robustness to different detector temperatures and injection port temperatures.

[0062] 5. Content determination of multiple batches of samples Following the method described in section 3, "Preliminary Determination of Content Determination Method", the menthol content of 15 batches of preparations was determined, and the results are shown in Table 11.

[0063] .

[0064] 6. Summary and Analysis The above results indicate that the method for determining the menthol content in Kaihoujian spray established in this study is highly specific, and its stability, repeatability, intermediate precision, and accuracy all meet the requirements. Furthermore, the chromatographic conditions are robust, providing technical support for further improving the quality standards of Kaihoujian spray.

[0065] 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 testing method for a throat-opening spray, wherein the prescription composition of the throat-opening spray is: 313g of cinnabar root, 313g of sophora root, 250g of cicada slough, and 1g of menthol. The preparation method is as follows: Except for menthol, the above four medicinal materials, cinnabar root and the other two, are decocted twice with water, the first time for 2 hours and the second time for 1 hour. The decoctions are combined, filtered, and the filtrate is concentrated to a clear extract with a relative density of 1.05~1.10 at 50℃. Ethanol is added to make the alcohol content reach 80%, and the mixture is allowed to stand for 24 hours. After filtration, the ethanol is recovered from the filtrate under reduced pressure and concentrated to a clear extract with a relative density of 1.10~1.20 at 80℃. 1g of menthol, 2.5g of sodium benzoate, 1g of citric acid, 5.5mL of myrica flavoring, 1.04g of steviol glycosides, and 803mL of polysorbate are dissolved in 20mL of ethanol by stirring. The above clear extract is then added, and water is added to 1000ml. mL, stir well, filter, and the product is obtained; Its features are, The quality testing method for the Kaihoujian spray is to determine the menthol content using gas chromatography, and the steps are as follows: S1. Preparation of the test solution: Accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 0-9 mL of water, and shake well; extract with 10-20 mL of ethyl acetate by shaking 1-2 times, and let stand to allow the layers to separate; collect and combine the upper ethyl acetate layer into a 25 mL volumetric flask, add ethyl acetate to the mark, filter with a 0.45 μm or 0.22 μm organic microporous membrane, and take the filtrate; S2. Preparation of the reference solution: Take an appropriate amount of menthol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 50 μg per 1 mL; S3. Chromatographic conditions and system suitability test: The chromatographic column was a capillary column with cross-linked polyethylene glycol as the stationary phase, measuring 30 m × 0.32 mm × 0.5 μm. A programmed temperature ramp was used, starting at 60℃, ramping to 120℃ at a rate of 2℃ / min, and then ramping to 246℃ at a rate of 5℃ / min. The injection port temperature was 198℃-202℃; the detector temperature was 278℃-282℃; splitless injection was used, with high-purity nitrogen as the carrier gas at a flow rate of 1 mL / min; the theoretical plate number, calculated based on the menthol peak, should be no less than 10,000. S4. Determination Method: Accurately pipette 1 μL of the test solution and inject it into the gas chromatograph. Measure and calculate the result.

2. The quality testing method for the throat-opening spray according to claim 1, characterized in that, The specific steps S1 for the determination of menthol content are as follows: accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 4-9 mL of water, and shake well; extract with 10-20 mL of ethyl acetate by shaking 1-2 times, and let it stand to separate into layers; collect and combine the upper ethyl acetate layer into a 25 mL volumetric flask, add ethyl acetate to make up to the mark, filter with a 0.45 μm organic microporous membrane, and take the filtrate to obtain the final product.

3. The quality testing method for the throat-opening spray according to claim 2, characterized in that, The specific steps of step S1 in the determination of menthol content are as follows: accurately measure 1 mL of Kaihoujian spray into a separatory funnel, add 4 mL of water, and shake well; extract once with 20 mL of ethyl acetate, and let it stand to separate into layers; collect the upper ethyl acetate solution into a 25 mL volumetric flask, add ethyl acetate to make up to the mark, filter with a 0.45 μm organic microporous membrane, and take the filtrate to obtain the product.

4. The quality testing method for the throat-opening spray according to claim 1, characterized in that, In the determination of menthol content, the injection port temperature in step S3 is 200℃-202℃.

5. The quality testing method for the throat-opening spray according to claim 4, characterized in that, In the determination of menthol content, the injection port temperature in step S3 is 200℃.

6. The quality testing method for the throat-opening spray according to claim 1, characterized in that, In the determination of menthol content, the temperature of the detector in step S3 is 280℃-282℃.

7. The quality testing method for the throat-opening spray according to claim 6, characterized in that, In the determination of menthol content, the temperature of the detector in step S3 is 280°C.

8. The quality testing method for the throat-opening spray according to claim 1, characterized in that, In the determination of menthol content, the detector in step S3 is an FID detector.

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