Detection method for rose and reed acne eliminating cream

Abstract

The invention provides a detection method for rose and reed acne eliminating cream. The detection method comprises the following steps: carrying out gas phase identification on borneol and dementholized peppermint oil in the rose and reed acne eliminating cream, and determining the content of sophocarpidine in the rose and reed acne eliminating cream. The gas chromatography identification method is stable and reliable through specificity and durability experiment investigation; according to the method for determining the content of the sophocarpidine in the rose-luou acne eliminating cream, the results are verified to be abnormal through nine items including system applicability, specificity, linearity and range, accuracy, repeatability, intermediate precision, durability, solution stability and sample treatment investigation, and the method is stable and reliable. Compared with an original standard detection method, the method overcomes the defect that benzene-containing toxic reagents affect the health of experimenters; gas phase identification of borneol and dementholized peppermint oil is added, so that the product can be identified more accurately; and the high performance liquid chromatography is high in content detection sensitivity and more accurate in result, and human errors are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to a detection method for Meilu Xiaozao Cream. Background Art

[0002] Meilu Xiaozao Cream, a product of Guizhou Liangji Pharmaceutical Co., Ltd., clears heat and dampness, kills insects, and relieves itching. It is used for acne, itchy skin, eczema, and sunburn. It is included in the "National Compendium of Traditional Chinese Medicine Standards: Ophthalmology, Otolaryngology, and Dermatology Volume." Meilu Xiaozao Cream product, [Prescription] 450g fresh aloe juice, 250g rose, 200g Sophora flavescens, 200g Radix Corydalis, 2g borneol, 1g mint oil, 87g stearic acid, 60g octadecyl alcohol, 25g glycerol, 40g monoglyceride, 45g polysorbate-80; [Preparation Method] Take the above medicinal materials, take rose and use steam distillation to extract 250ml of aromatic water, set aside; take Sophora flavescens and Radix Corydalis, add water twice, each time for 2 hours, filter, combine the filtrate, and concentrate to a clear paste with a relative density of 1.01 (45℃~55℃), add ethanol to make the alcohol content reach 72%, let it stand for 12 hours, take the supernatant, recover the ethanol, add 1 times the amount of water, let it stand for 24 hours, filter separately to obtain Sophora flavescens and Radix Corydalis extracts, and concentrate to 20g. Mix with the above-mentioned rose aromatic water and fresh aloe vera juice, add borneol, peppermint oil, stearic acid, stearyl alcohol, glycerin, monoglyceride, polysorbate 80, mix well, and make 1000g.

[0003] The original quality standard test method for Meilu Xiaozao Cream included two steps: first, identification of Sophora flavescens using thin-layer chromatography (TLC) using a benzene-acetone-ethyl acetate-concentrated ammonia test solution (2:3:4:0.2) as the developing solvent for matrine identification; second, determination of matrine content in Sophora flavescens using TLC, scanning at wavelengths of λS = 480 nm and λR = 650 nm according to TLC (General Standard 0502 Thin-layer Chromatography Scanning Method), measuring the integrated absorbance of the test sample and the reference sample, and calculating the matrine content. Because the original standard developing solvent contained benzene, which was detrimental to the health of the test personnel, and the use of TLC for content measurement was subject to significant human error, the borneol and menthol oil in the Meilu Xiaozao Cream formula were transdermal preparations intended to promote transdermal drug absorption, yet these components were not identified in the original standard. Therefore, improvements to the original test method were necessary. Summary of the Invention

[0004] The present invention aims to provide a method for detecting Meilu Xiaozao Cream. This method eliminates the thin-layer chromatography (TLC) method used in the original standard and adds gas phase identification of borneol and menthol oil. It also optimizes the content determination method used in the original standard, replacing the original TLC method with high-performance liquid chromatography (HPLC). Compared to the original standard method, this method overcomes the health risks of benzene-containing toxic reagents, which can affect the health of experimenters. The addition of borneol and menthol oil gas phase identification allows for more accurate product identification. Furthermore, HPLC content detection offers high sensitivity, resulting in more accurate results and reduced human error.

[0005] In order to achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] A method for detecting Meiluxiaozao cream, comprising: gas phase identification of borneol and peppermint oil in Meiluxiaozao cream; and determination of the content of matrine in Meiluxiaozao cream; specifically as follows:

[0007] The gas phase identification of borneol and peppermint oil in the Meiluxiaozao cream is as follows:

[0008] 1) Preparation of test solution

[0009] Take 1-5g of this product, add 10-30ml of ethyl acetate, shake for 1-5min, take the supernatant and filter it, and use the filtrate as the test solution;

[0010] 2) Preparation of reference solution

[0011] Take menthol reference substance and borneol reference substance, add ethyl acetate to make a mixed solution containing 1 mg of menthol and 1 mg of borneol per 1 ml, as the reference solution;

[0012] 3) Gas phase identification

[0013] According to the General Methods for Gas Chromatography 0521, a capillary column with polyethylene glycol PEG-20M as the stationary phase was used: the column temperature was programmed, initially at 60-80°C, then raised at a rate of 8°C per minute to 180°C, held for 12-18 minutes, then raised at a rate of 8°C per minute to 210°C, held for 12-18 minutes; split injection was performed with a split ratio of 40-60:1; 1 μl of each reference solution and test solution was aspirated and injected into the gas chromatograph; the test solution chromatogram should show a peak with the same retention time as the reference solution peak.

[0014] The content of matrine in the Meilu Xiaozao cream is determined as follows:

[0015] S1 Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile-anhydrous ethanol-triethylamine-0.5% phosphoric acid solution was used as the mobile phase; the detection wavelength was 210 nm; the flow rate was 1.0 ml / min; the theoretical plate number calculated based on the matrine peak should be no less than 6000;

[0016] Preparation of S2 reference solution Take an appropriate amount of matrine reference solution, accurately weigh it, and add anhydrous ethanol to make a solution containing 40 μg per 1 ml.

[0017] Preparation of S3 test solution

[0018] Take 1-5g of this product, accurately weigh it, place it in a container, accurately add 50ml of hydrochloric acid solution, heat it in an 85℃ water bath for 10-30min to dissolve it, and place it at 0-4℃ for 0.5-1.5h to allow the matrix to solidify and precipitate. Filter it, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of anhydrous ethanol, transfer it to a 5ml volumetric flask, stopper it tightly, weigh it, and extract it in 60℃ warm water for 0.5-1.5h. Remove it, let it cool to room temperature, weigh it again, make up the loss with anhydrous ethanol, shake it well, filter it, and take the filtrate.

[0019] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0020] Preferably, the preparation of the test solution in step 1) of the present invention is as follows: 1-3 g of the product is added with 10-20 ml of ethyl acetate, shaken for 2-3 minutes, and the supernatant is filtered, and the filtrate is used as the test solution.

[0021] Preferably, the preparation of the test solution in step 1) of the present invention is as follows: take 2 g of the product, add 10 ml of ethyl acetate, shake for 2 minutes, take the supernatant and filter it, and use the filtrate as the test solution.

[0022] Preferably, the shaking according to the present invention may also be ultrasonic extraction.

[0023] Preferably, the step 3) gas phase identification of the present invention is as follows: according to the general rules of gas chromatography 0521 test, a capillary column with polyethylene glycol PEG-20M as the stationary phase is used: the column temperature is programmed, the initial temperature is 70-80°C, the temperature is increased to 180°C at a rate of 8°C per minute, maintained for 14-16 minutes, and then the temperature is increased to 210°C at a rate of 8°C per minute, maintained for 14-16 minutes; split injection, the split ratio is 45-55:1; 1 μl of the reference solution and the test solution are respectively drawn and injected into the gas chromatograph; the test sample chromatogram should show a chromatographic peak with the same retention time as the reference sample chromatographic peak.

[0024] Preferably, the step 3) gas phase identification of the present invention is as follows: the gas chromatography general rule 0521 test is performed, and a capillary column with polyethylene glycol PEG-20M as the stationary phase is used: the column temperature is programmed, the initial temperature is 80°C, the temperature is increased to 180°C at a rate of 8°C per minute, and the temperature is maintained for 15 minutes, and then the temperature is increased to 210°C at a rate of 8°C per minute and the temperature is maintained for 15 minutes; split injection, the split ratio is 50:1; 1 μl of the reference solution and the test solution are respectively drawn and injected into the gas chromatograph; the test solution chromatogram should show a chromatographic peak with the same retention time as the reference solution chromatographic peak.

[0025] Preferably, the mobile phase for determining the content of matrine in the Meiluxiaozao cream of the present invention is: acetonitrile-anhydrous ethanol-triethylamine-0.5% phosphoric acid solution = 75:20:0.1:5.

[0026] Preferably, the preparation of the test solution in step S3 of the present invention is as follows: take 1-3 g of the product, accurately weigh it, place it in a 100 ml beaker, accurately add 50 ml of hydrochloric acid solution, heat it in an 85 ° C water bath for 15-25 min to dissolve it, place it at 0-4 ° C for 0.5-1 h to allow the matrix to solidify and precipitate, filter it, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of anhydrous ethanol, transfer it to a 5 ml volumetric flask, seal it tightly, weigh it, extract it in warm water at 60 ° C for 0.5-1 h, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0027] Preferably, the preparation of the test solution in step S3 of the determination of the content of matrine in the Meilu Xiaozao cream of the present invention is as follows: take 2g of the product, accurately weigh it, place it in a 100ml beaker, accurately add 50ml of hydrochloric acid solution, heat it in an 85°C water bath for 20min to dissolve it, place it at 0-4°C for 1h to allow the matrix to solidify and precipitate, filter it, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of anhydrous ethanol, transfer it to a 5ml measuring flask, seal it tightly, weigh it, extract it in warm water at 60°C for 1h, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0028] Preferably, the hydrochloric acid solution of the present invention is prepared by adding 9 parts of hydrochloric acid to 200 parts of water and mixing.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. Compared with the original standard method, the present invention adds the gas phase identification of borneol and peppermint oil, and removes the thin layer identification of Sophora flavescens in the original standard. It not only improves the quality standard of Mei Lu Xiao Zao Cream, but also overcomes the defect of the toxic reagent containing benzene affecting the health of the experimenters.

[0031] 2. Compared with the original standard method, the present invention adopts high performance liquid chromatography to determine the content of matrine in Sophora flavescens, which has high detection sensitivity, more accurate results and reduces human errors.

[0032] 3. The analytical method for matrine content in Meilu Xiaozao Cream was verified by nine tests, including system applicability, specificity, linearity and range, accuracy, repeatability, intermediate precision, durability, solution stability, and sample handling. The results were all normal, indicating that the method of the present invention was stable and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Specificity investigation chromatogram of gas phase identification (reference solution);

[0034] Figure 2 : Specificity investigation chromatogram of gas phase identification (negative);

[0035] Figure 3 : Specificity investigation chromatogram of gas phase identification (borneol);

[0036] Figure 4 : Specificity investigation chromatogram of gas phase identification (menthol);

[0037] Figure 5 : Specificity investigation chromatogram of gas phase identification (borneol);

[0038] Figure 6 : Specificity investigation chromatogram of gas phase identification (isoborneol);

[0039] Figure 7 : Specificity investigation chromatogram of gas phase identification (sample);

[0040] Figure 8 : Gas phase identification chromatogram of Meiluxiaozao cream sample with batch number 20240301;

[0041] Figure 9 : Gas phase identification chromatogram of Meiluxiaozao cream sample with batch number 20240302;

[0042] Figure 10 : Gas phase identification chromatogram of Meiluxiaozao cream sample with batch number 20240303;

[0043] Figure 11 :Chromatogram of content determination of sample with batch number 20221104;

[0044] Figure 12 :Chromatogram of content determination of sample with batch number 20230412;

[0045] Figure 13 :Chromatogram of content determination of sample with batch number 20230712;

[0046] Figure 14 :Chromatogram of content determination of sample with batch number 20230910;

[0047] Figure 15 :Chromatogram of content determination of sample with batch number 20231008;

[0048] Figure 16 :Chromatogram of content determination of sample with batch number 20231101;

[0049] Figure 17 :Chromatogram of content determination of sample with batch number 20231108;

[0050] Figure 18 : Chromatogram of content determination of sample with batch number 20231109;

[0051] Figure 19 :Chromatogram of content determination of sample with batch number 20231201;

[0052] Figure 20 :Chromatogram of content determination of sample with batch number 20231202;

[0053] Figure 21 : Chromatogram of content determination of sample with batch number 20240301;

[0054] Figure 22 : Chromatogram of content determination of sample with batch number 20240302. DETAILED DESCRIPTION

[0055] Example 1 Gas Phase Identification of Borneol and Menthol Oil

[0056] Take 2g of this product, add 10ml of ethyl acetate, shake for 2 minutes, filter the supernatant, and use the filtrate as the test solution. Separately, take a menthol reference substance and a borneol reference substance, add ethyl acetate to create a mixed solution containing 1mg of each menthol and borneol per 1ml. This will serve as the reference solution. Perform the test according to the gas chromatography method (General Rule 0521): initially at 80°C, raise the temperature to 180°C at a rate of 8°C / min, hold for 15 minutes, then raise the temperature to 210°C at a rate of 8°C / min, hold for 15 minutes. Perform split injection with a split ratio of 50:1. Pipette 1μl each of the reference solution and the test solution into the gas chromatograph. The test solution should show a peak with the same retention time as the reference solution.

[0057] Example 2 Determination of matrine content

[0058] Determined according to high performance liquid chromatography (General Rule 0512).

[0059] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; acetonitrile-anhydrous ethanol-triethylamine-0.5% phosphoric acid solution (75:20:0.1:5) as the mobile phase; detection wavelength at 220 nm; flow rate at 1.0 ml / min. The theoretical plate number, calculated based on the matrine peak, should be no less than 5000.

[0060] Preparation of reference solution: Take an appropriate amount of matrine reference substance, accurately weigh it, and add anhydrous ethanol to make a solution containing 40 μg per 1 ml.

[0061] Preparation of test solution: Take about 2 g of the product, accurately weigh it, place it in a 100 ml beaker, accurately add 50 ml of hydrochloric acid (9→200), heat it in an 85 ° C water bath to dissolve it for about 20 minutes, place it at 0-4 ° C for about 1 hour to allow the matrix to solidify and precipitate, filter it, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of anhydrous ethanol, transfer it to a 5 ml volumetric flask, stopper it, weigh it, soak it in warm water at 60 ° C for 1 hour, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0062] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0063] Each 1g of this product contains matrine (C 15 H 24 N2O), it shall not be less than 0.21 mg.

[0064] Example 3 Gas Phase Identification of Borneol and Menthol Oil

[0065] Take 1g of this product and add 10ml of ethyl acetate. Shake for 1 minute, filter the supernatant, and use the filtrate as the test solution. Separately, take a menthol reference substance and a borneol reference substance and add ethyl acetate to create a mixed solution containing 1mg of each menthol and borneol per 1ml. This will serve as the reference solution. Follow the gas chromatography method (General Method 0521) using a capillary column with polyethylene glycol (PEG-20M) as the stationary phase: program the column temperature from 60°C, then increase the temperature at a rate of 10°C / min to 180°C, hold for 12 minutes, then increase the temperature at a rate of 8°C / min to 210°C, hold for 12 minutes. Use split injection with a split ratio of 40:1. Pipette 1μl each of the reference solution and the test solution into the gas chromatograph. The test solution should show a peak with the same retention time as the reference solution.

[0066] Example 4 Gas Phase Identification of Borneol and Menthol Oil

[0067] Take 5g of this product, add 30ml of ethyl acetate, shake for 5 minutes, filter the supernatant, and use the filtrate as the test solution. Separately, take a menthol reference substance and a borneol reference substance, add ethyl acetate to make a mixed solution containing 1mg of each menthol and borneol per 1ml. This will serve as the reference solution. Follow the gas chromatography method (General Method 0521) using a capillary column with polyethylene glycol (PEG-20M) as the stationary phase: program the column temperature from 70°C, then increase the temperature to 180°C at a rate of 8°C / min, hold for 18 minutes, then increase the temperature to 210°C at a rate of 8°C / min, hold for 18 minutes. Use split injection with a split ratio of 60:1. Pipette 1μl each of the reference solution and the test solution into the gas chromatograph. The test solution should show a peak with the same retention time as the reference solution.

[0068] Example 5 Determination of matrine content

[0069] Determined according to high performance liquid chromatography (General Rule 0512).

[0070] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; acetonitrile-anhydrous ethanol-triethylamine-0.5% phosphoric acid solution (75:20:0.1:5) as the mobile phase; detection wavelength at 220 nm; flow rate at 1.0 ml / min. The theoretical plate number, calculated based on the matrine peak, should be no less than 5000.

[0071] Preparation of reference solution: Take an appropriate amount of matrine reference substance, accurately weigh it, and add anhydrous ethanol to make a solution containing 40 μg per 1 ml.

[0072] Preparation of test solution: Take about 3 g of the product, accurately weigh it, place it in a 100 ml beaker, accurately add 50 ml of hydrochloric acid (9→200), heat it in an 85 ° C water bath to dissolve it for about 30 minutes, place it at 0-4 ° C for about 1.5 hours, filter it, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of anhydrous ethanol, transfer it to a 5 ml volumetric flask, stopper it, weigh it, soak it in warm water at 60 ° C for 1.5 hours, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it well, filter it, and take the filtrate.

[0073] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0074] Each 1g of this product contains matrine (C 15 H 24 N2O), it shall not be less than 0.21 mg.

[0075] Example 6 Determination of matrine content

[0076] Determined according to high performance liquid chromatography (General Rule 0512).

[0077] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; acetonitrile-anhydrous ethanol-triethylamine-0.5% phosphoric acid solution (75:20:0.1:5) as the mobile phase; detection wavelength at 220 nm; flow rate at 1.0 ml / min. The theoretical plate number, calculated based on the matrine peak, should be no less than 5000.

[0078] Preparation of reference solution: Take an appropriate amount of matrine reference substance, accurately weigh it, and add anhydrous ethanol to make a solution containing 40 μg per 1 ml.

[0079] Preparation of test solution: Take about 1g of the product, accurately weigh it, place it in a 100ml beaker, accurately add 50ml of hydrochloric acid (9→200), heat it in an 85℃ water bath to dissolve it for about 20 minutes, place it at 0-4℃ for about 0.5 hour, filter it, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of anhydrous ethanol, transfer it to a 5ml volumetric flask, stopper it, weigh it, soak it in warm water at 60℃ for 0.5 hour, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it well, filter it, and take the filtrate.

[0080] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0081] Each 1g of this product contains matrine (C 15 H 24 N2O), it shall not be less than 0.21 mg.

[0082] In order to further verify the feasibility and effectiveness of the present invention and screen out the best solution, the present invention conducted a series of experiments, some of which are excerpted as follows:

[0083] 1. Test materials

[0084] Sample: Mei Lu Xiao Zao Cream (Guizhou Liangji Pharmaceutical Co., Ltd.), batch numbers 20221104, 20230412, 20230712, 20230910, 20231008, 20231101, 20231108, 20231109, 20231201, 20231202, 20240301, 20240302, 20240303.

[0085] Reference substances and reference medicinal materials: borneol reference substance (China Food and Drug Inspection Institute, batch number: 110743-201706, for content determination, purity 97.6%); menthol reference substance (China Food and Drug Inspection Institute, batch number: 110728-202208, for content determination, purity 99.6%); matrine reference substance (China Food and Drug Inspection Institute, batch number: 110805-202010, for content / limit determination, purity 98.7%).

[0086] Reagents: Methanol and acetonitrile were of chromatographic grade, and all other reagents were of analytical grade.

[0087] Instruments: Agilent 1260Ⅱ high-performance liquid chromatograph (including quaternary gradient pump, vacuum degasser, autosampler, column oven, DAD detector, and Agilent OpenLAB CDS chromatography workstation); ultrasonic cleaner (500 W, 53 kHz; Kunshan Ultrasonic Instrument Co., Ltd.); Shimadzu GC-2014CAF / SPL, Shimadzu GC-2010PLUS, and Zhejiang Fuli GC9790 gas chromatograph (including injection septum, glass liner, gas filter, autosampler, column oven, FID detector, and workstation).

[0088] 2. Identification of borneol and menthol oil and gas phase

[0089] Refer to the identification and content determination method of peppermint oil in the Chinese Pharmacopoeia 2020 edition:

[0090] (1) Refer to the identification method of peppermint oil in plant oils and extracts in the Chinese Pharmacopoeia 2020 edition, specifically:

[0091] Take 2g of this product and dissolve it in 15ml of anhydrous ethanol to prepare the test solution. Take menthol reference substance and borneol reference substance and add anhydrous ethanol to prepare 1mg menthol and borneol solution per ml respectively as the reference solution. According to the thin layer chromatography method (general rule 0502), take 5μl of each of the above two solutions and spot them on the same silica gel GF 254 The thin layer plate was developed with toluene-ethyl acetate (19:1), taken out, dried, and examined under ultraviolet light (254 nm).

[0092] Results: Sprayed with anisaldehyde test solution and heated at 105°C until the spots became blurred. The test sample chromatogram showed no spots of the same color at the corresponding positions in the control sample chromatogram. Examination under ultraviolet light (365nm) also revealed no fluorescent spots of the same color. Therefore, this thin-layer identification method was unable to identify borneol and peppermint oil in the product of this invention.

[0093] (2) Based on the gas chromatography method for determining the menthol content in peppermint oil using fingerprints in the 2020 edition of the Chinese Pharmacopoeia, the test was carried out according to the General Chapter of Gas Chromatography 0521, specifically:

[0094] Take 2g of this product, add 10ml of ethyl acetate, shake for 2 minutes, filter the supernatant, and use the filtrate as the test solution. Take a menthol reference substance and a borneol reference substance, add ethyl acetate to make a mixed solution containing 1mg of menthol and borneol per 1ml, which serves as the reference solution. According to the General Guide to Gas Chromatography 0521, use a capillary column with polyethylene glycol PEG-20M as the stationary phase: program the column temperature from 60-80°C, increase the temperature at a rate of 8°C / min to 180°C, hold for 15 minutes, then increase the temperature at a rate of 8°C / min to 210°C, hold for 15 minutes; perform split injection with a split ratio of 50:1; aspirate 1μl of each reference solution and test solution into the gas chromatograph.

[0095] Results: In the chromatogram of the test sample, the sample solution showed a chromatographic peak with the same retention time as the chromatographic peak of borneol and menthol reference substances.

[0096] 2.1 Specificity (Shimadzu GC-2014CAF / SPL)

[0097] Preparation of reference solution: Take menthol reference substance and borneol reference substance, add ethyl acetate to make a mixed solution containing 1 mg of menthol and borneol per 1 ml, as the reference solution.

[0098] Preparation of test solution: Take 2 g of this product, add 10 ml of ethyl acetate, shake for 2 minutes, take the supernatant and filter it, and the filtrate is used as the test solution.

[0099] Preparation of negative solution lacking borneol and menthol oil: Take 2 g of negative sample lacking borneol and menthol oil, add 10 ml of ethyl acetate, shake for 2 minutes, take the supernatant and filter, and the filtrate is used as the negative solution lacking borneol and menthol oil.

[0100] According to the gas chromatography method (General Rule 0521), 1 μl of the reference solution and the test solution were respectively taken and injected into the gas chromatograph to record the chromatogram. Figures 1 to 7 .

[0101] Results: As can be seen from the figure, there are corresponding chromatographic peaks at the corresponding positions of the test solution and the reference solution, and there is no interference from the negative results of borneol and peppermint oil.

[0102] 2.2 Durability

[0103] Different gas chromatographs from different manufacturers were used to identify borneol and menthol in Meilu Xiaozao Cream (Batch No. 20240303). The effects of different gas chromatograph models and manufacturers were investigated. Following the gas chromatography method (General Rule 0521), 1 μl of each reference solution and test solution was injected into the gas chromatograph, and the chromatograms were recorded. Durability results are shown in Table 1.

[0104] Table 1 Identification results of gas chromatographs of different models and manufacturers

[0105]

[0106] 2.3 Sample gas phase identification (Shimadzu GC-2014CAF / SPL)

[0107] Take 3 batches of Meilu Xiaozao Cream samples and test them according to the gas chromatography method (General Rule 0521). Take 1μl of the reference solution and the test solution respectively, inject them into the gas chromatograph, and record the chromatogram. The identification results are shown in Table 2 and the chromatogram is shown in Figures 8 to 10 .

[0108] Table 2 Identification results

[0109] Serial number batch number Identification results 1 20240301 The chromatogram of the test solution shows a chromatographic peak with the same retention time as the chromatographic peak of the reference solution. 2 20240302 The chromatogram of the test solution shows a chromatographic peak with the same retention time as the chromatographic peak of the reference solution. 3 20240303 The chromatogram of the test solution shows a chromatographic peak with the same retention time as the chromatographic peak of the reference solution.

[0110] 2.4 Determine the gas phase identification method of borneol and menthol in Meiluxiaozao cream

[0111] Based on the test results, the gas phase identification method for borneol and menthol oil in Meiluxiaozao Cream is as follows: 2g of the product is added to 10ml of ethyl acetate and shaken for 2 minutes. The supernatant is filtered and used as the test solution. Separately, a menthol reference substance and a borneol reference substance are added to ethyl acetate to create a mixed solution containing 1mg of each menthol and borneol per 1ml, which serves as the reference solution. According to the gas chromatography method (General Method 0521), a capillary column with polyethylene glycol (PEG-20M) as the stationary phase is used. The column temperature is programmed from 80°C, then increased at a rate of 8°C / min to 180°C, held for 15 minutes, then increased at a rate of 8°C / min to 210°C, held for 15 minutes. Split injection is used with a split ratio of 50:1. 1μl of each of the reference solution and the test solution is injected into the gas chromatograph. The test solution should show a peak with the same retention time as the reference solution.

[0112] 3. Content determination method

[0113] Sophora flavescens is the dried root of the leguminous plant Sophora flavescens Ait. It is harvested in spring and autumn, the root heads and wheat roots removed, washed, and dried, or sliced ​​and dried while still fresh.

[0114] This study first determined the total amount of matrine and oxymatrine according to the chromatographic conditions under the content determination of Sophora flavescens in the first part of the 2020 edition of the Chinese Pharmacopoeia. As a result, no oxymatrine was detected in Meilu Xiaozao Cream, so matrine was used as its content determination indicator. Due to the large number of impurity peaks and poor separation, coupled with an unstable baseline, the present invention has undergone multiple trials and adjustments in the early stage, and finally established an HPLC method for the determination of Sophora flavescens in this product, specifically:

[0115] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; acetonitrile-anhydrous ethanol-triethylamine-0.5% phosphoric acid solution (75:20:0.1:5) as the mobile phase; detection wavelength at 220 nm; flow rate at 1.0 ml / min. The theoretical plate number, calculated based on the matrine peak, should be no less than 5000.

[0116] Preparation of reference solution: Take an appropriate amount of matrine reference substance, accurately weigh it, and add anhydrous ethanol to make a solution containing 40 μg per 1 ml.

[0117] Preparation of test solution: Take about 2 g of the product, accurately weigh it, place it in a 100 ml beaker, accurately add 50 ml of hydrochloric acid (9→200), heat it in an 85 ° C water bath to dissolve it for about 20 minutes, place it at 0-4 ° C for about 1 hour to allow the matrix to solidify and precipitate, filter it, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of anhydrous ethanol, transfer it to a 5 ml volumetric flask, stopper it, weigh it, soak it in warm water at 60 ° C for 1 hour, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0118] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0119] 3.1 System Applicability

[0120] Blank solution: anhydrous ethanol.

[0121] Preparation of reference solution: Take an appropriate amount of matrine reference substance, accurately weigh it, and add anhydrous ethanol to make a solution containing 40 μg per 1 ml.

[0122] Test according to high performance liquid chromatography (General Rule 0512):

[0123] Accurately pipette 10 μl of each reference solution into the liquid chromatograph. Inject reference solution 1 five times and reference solution 2 once, and record the chromatogram. System suitability results are shown in Table 3.

[0124] Table 3 System suitability results

[0125]

[0126]

[0127] Results: As shown in Table 3, in the chromatogram of the reference solution, the theoretical plate number of the matrine peak was greater than 5000; the RSD of the peak area of ​​the reference solution 1 after 5 repeated injections was less than 2.0%, and the content of the reference solution 2 relative to the reference solution 1 was between 98.0% and 102.0%.

[0128] 3.2 Exclusivity

[0129] Blank solution: anhydrous ethanol.

[0130] Preparation of test solution: Take about 2g of this product (batch number: 20240303), accurately weigh it, place it in a 100ml beaker, accurately add 50ml of hydrochloric acid (9→200), heat and stir in an 85℃ water bath to dissolve it for about 20 minutes, place it at 0-4℃ for about 1 hour to allow the matrix to solidify and precipitate, filter it, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of anhydrous ethanol, transfer it to a 5ml volumetric flask, stopper it, weigh it, soak it in warm water at 60℃ for 1h, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it well, filter it, and take the filtrate.

[0131] Preparation of reference solution: Take an appropriate amount of matrine reference substance, accurately weigh it, and add anhydrous ethanol to make a solution containing 40 μg per 1 ml.

[0132] Preparation of negative solution for Sophora flavescens deficiency: Take an appropriate amount of negative sample for Sophora flavescens deficiency and prepare negative solution for Sophora flavescens deficiency according to the preparation method of the test solution.

[0133] According to the high performance liquid chromatography method (General Rule 0512), accurately aspirate 10 μl of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0134] Results: As can be seen from the figure, there is no interference at the peak position of matrine in the chromatograms of the blank solution and the negative solution lacking sophora flavescens.

[0135] 3.3 Linearity and range

[0136] Preparation of reference substance stock solution: Accurately weigh 0.0123 g of matrine reference substance, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the scale, shake well, and calculate based on the reference substance purity of 98.7% that each 1 ml contains 242.8020 μg of matrine.

[0137] Preparation of linear solution: Accurately measure 0.5 ml, 0.8 ml, 1.0 ml, 1.5 ml, and 2.0 ml of the reference stock solution and place them in 5 ml volumetric flasks, dilute to the scale with anhydrous ethanol, and shake well to prepare solutions with reference concentrations of 24.2802 μg / ml, 38.8483 μg / ml, 48.5604 μg / ml, 72.8406 μg / ml, and 97.1208 μg / ml, respectively.

[0138] 10 μl of each solution was injected into the chromatograph, and the chromatogram was recorded. The standard curve was obtained by performing a linear regression of the peak area against the injected concentration using the least squares method. The linearity results are shown in Table 4.

[0139] Table 4 Linearity results

[0140]

[0141] Results: As shown in Table 4, the matrine solution showed a good linear relationship in the concentration range of 24.2802μg / ml to 97.1208μg / ml, with a correlation coefficient of r 2 is 0.9998.

[0142] 3.4 Accuracy

[0143] Preparation of reference substance stock solution: Accurately weigh 0.0123 g of matrine reference substance, place it in a 50 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the scale, shake well, and calculate based on the reference substance purity of 98.7% that each 1 ml contains 242.8020 μg of matrine.

[0144] Sample solution: Prepare three samples in parallel according to the preparation method of the test solution under "3".

[0145] Accuracy solution:

[0146] ①②③ Limit (80%): Take the sample solution, add 0.8ml of the reference substance stock solution and transfer to a 5ml volumetric flask. Dilute to the mark with anhydrous ethanol, shake well, filter, and collect the filtrate. Prepare three replicates.

[0147] ④⑤⑥ Limit (100%): Take the sample solution, add 1.0ml of the reference substance stock solution and transfer to a 5ml volumetric flask. Dilute to the mark with anhydrous ethanol, shake well, filter, and collect the filtrate. Prepare three replicates.

[0148] ⑦⑧⑨Limit (120%): Take the sample solution, add 1.2ml of the reference substance stock solution and transfer to a 5ml volumetric flask. Dilute to the mark with anhydrous ethanol, shake well, filter, and collect the filtrate. Prepare three replicates.

[0149] Take 10 μl of accuracy solution respectively and inject it into the chromatograph, and record the chromatogram.

[0150] The accuracy results are shown in Table 5.

[0151] Table 5 Accuracy results

[0152]

[0153] Results: As shown in Table 5, the average recovery of 9 accuracy solutions was 99.4%, ranging from 98.0% to 102.0%, and the RSD was 0.9%, which was less than 2.0%.

[0154] 3.5 Repeatability

[0155] Preparation of test solution: Prepare 6 portions in parallel according to the test solution preparation method under "3".

[0156] Take 10 μl of the reproducible solution and inject it into the chromatograph, and record the chromatogram.

[0157] The repeatability results are shown in Table 6.

[0158] Table 6 Repeatability results

[0159]

[0160]

[0161] Results: From Table 6, we can see that the matrine content in the six test solutions is 0.58 mg / g, and the RSD is 0.9%, which is less than 2.0%.

[0162] 3.6 Intermediate precision

[0163] Another experimenter shall conduct the test on the same batch of test products on a different date, using a different instrument and a different chromatographic column, following the operating procedures under "3.5 Repeatability".

[0164] Take 10 μl of the intermediate precision solution and inject it into the chromatograph, and record the chromatogram. The intermediate precision results are shown in Tables 7 and 8.

[0165] Table 7 Intermediate precision results (n=6)

[0166]

[0167] Table 8 Intermediate precision results (n=12)

[0168]

[0169]

[0170] Results: From Table 8, it can be seen that the content of matrine in the 12 test solutions is 0.58 mg / g, and the RSD is 0.9%, which is less than 2.0%.

[0171] 3.7 Durability

[0172] The analytical method for determining matrine content in Meilu Xiaozao Cream should be tested for its robustness. Parameters examined for minor variations include column temperature, flow rate, and chromatographic column. By varying one parameter while keeping the others constant, reference and test solutions were prepared according to the analytical method, and matrine content was determined using the table of analytical condition variations. Robustness conditions are shown in Table 9, and robustness results are shown in Table 10.

[0173] Table 9 Durability conditions

[0174]

[0175] Table 10 Durability results

[0176] Chromatographic conditions Theoretical plate number Content (mg / g) Content change (mg / g) Initial conditions 17079 0.574 -- Column temperature: 25°C 15920 0.559 0.01 Column temperature: 35°C 17912 0.564 0.01 Flow rate: 0.9ml / min 17798 0.565 0.01 Flow rate: 1.1 ml / min 16702 0.570 0 Chromatographic column: Shiseido 17438 0.571 0

[0177] Note: The initial condition results were obtained from replicate solution 1, and the results for different columns were obtained from intermediate precision results.

[0178] Results: From Table 10, it can be seen that when any of the chromatographic conditions, such as column temperature, flow rate, or chromatographic column, is changed, the content of the test solution changes less than 0.03 mg / g compared with the initial conditions.

[0179] 3.8 Solution stability

[0180] Place the test solution under the specificity item at room temperature, inject the sample once at 0h, 12h, 24h, and 48h respectively, and determine the matrine content in the test solution.

[0181] The last time point that meets the above requirements is used as the maximum time of solution stability. If a time point does not meet the above requirements, the time point before it is used as the maximum time of solution stability. The stability results are shown in Table 11.

[0182] Table 11 Stability results

[0183] time name Peak area Content (mg / g) Content change (mg / g) 0h Test solution 1071.581 0.578 - 12h Test solution 1067.999 0.576 0 24h Test solution 1083.685 0.591 0.01 48h Test solution 1079.588 0.575 0

[0184] Results: From Table 11, we can see that when the test solution is placed at room temperature for 48 hours, the maximum change in its content is 0.01 mg / ml, which is less than 0.03 mg / ml.

[0185] 3.9 Sample processing inspection

[0186] Regarding the treatment of the sample solution for matrine content in Meilu Xiaozao Cream, the effects of water bath heating, dissolution time, and extraction solvent dosage on its content should be examined. The matrine content should be determined according to the sample treatment investigation conditions table. The sample treatment investigation conditions are shown in Table 12, and the sample treatment investigation results are shown in Table 13.

[0187] Table 12 Sample processing conditions

[0188] Serial number Sample processing conditions 1 Dissolution time in water bath: 10min 2 Dissolution time in water bath: 15min 3 Dissolution time in water bath: 20min 4 Dissolution time in water bath: 25min 5 Extraction solvent dosage: 10ml 6 Extraction solvent dosage: 15ml 7 Extraction solvent dosage: 20ml

[0189] Table 13 Sample processing investigation results

[0190]

[0191]

[0192] Results: As shown in Table 13, there was no difference in the content of the sample when the water bath dissolution time was 15min, 20min, and 25min, so the water bath dissolution time was set to about 20 minutes; there was no difference in the content of the extraction solvent dosage of 15ml and 20ml, so the extraction solvent dosage was set to 15ml.

[0193] The analytical method for matrine content in Meiluxiaozao Cream passed the validation (system suitability, specificity, linearity and range, accuracy, repeatability, intermediate precision, durability, solution stability and sample handling inspection) and the results of 9 items were normal.

[0194] 4. Sample Measurement

[0195] Twelve batches of Meilu Xiaozao cream samples were assayed. Test solution and reference solution were prepared according to the assay conditions. Samples were injected, chromatograms were recorded, and the content was calculated according to the following formula (results are shown in Table 14, chromatograms are shown in Table 14). Figures 11 to 22 ):

[0196]

[0197] Where:

[0198] W 对 : The sample weight of matrine reference solution, unit: g;

[0199] P 对 : represents the purity of matrine reference substance;

[0200] A 样 : is the peak area of ​​matrine in the test solution;

[0201] A 对 : is the average peak area of ​​the main peak in 5 injections of matrine reference solution 1;

[0202] V 样: The dilution volume for preparing the test solution, in ml;

[0203] V 对 : The dilution volume for preparing the reference solution, in ml;

[0204] W 样 : The amount of sample to prepare the test solution, unit is g.

[0205] Table 14 Determination of matrine content in Meilu Xiaozao cream

[0206]

[0207]

[0208] According to the results of the determination of matrine content in several batches of Meilu Xiaozao Cream samples, the highest was 0.44 mg / g and the lowest was 0.26 mg / g. Taking into account the uncontrollable factors of large-scale production, it is proposed to use matrine (C 15 H 24 N2O), it should not be less than 0.21mg. Therefore, the content determination method of Meilu Xiaozao Cream is proposed as follows:

[0209] The content was determined according to the high performance liquid chromatography method (General Rule 0512).

[0210] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; acetonitrile-anhydrous ethanol-triethylamine-0.5% phosphoric acid solution (75:20:0.1:5) as the mobile phase; detection wavelength at 220 nm; flow rate at 1.0 ml / min. The theoretical plate number, calculated based on the matrine peak, should be no less than 5000.

[0211] Preparation of reference solution: Take an appropriate amount of matrine reference substance, accurately weigh it, and add anhydrous ethanol to make a solution containing 40 μg per 1 ml.

[0212] Preparation of test solution: Take about 2 g of the product, accurately weigh it, place it in a 100 ml beaker, accurately add 50 ml of hydrochloric acid (9→200), heat it in an 85 ° C water bath to dissolve it for about 20 minutes, place it at 0-4 ° C for about 1 hour, filter it, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of anhydrous ethanol, transfer it to a 5 ml volumetric flask, stopper it, weigh it, soak it in warm water at 60 ° C for 1 hour, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it well, filter it, and take the filtrate.

[0213] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0214] Each 1g of this product contains matrine (C 15 H24 N2O), it shall not be less than 0.21 mg.

[0215] According to the proposed method for determining the content of Meiluxiaozao Cream, the contents of all twelve batches of Meiluxiaozao Cream were in compliance with the regulations. Although the present invention has been described in detail above using general instructions, specific embodiments, and tests, it is obvious to those skilled in the art that modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements, made without departing from the spirit of the present invention, all fall within the scope of protection claimed in the present invention.

Claims

1. A detection method for Meilu Xiaozao Cream, characterized in that: The detection method includes: gas phase identification of borneol and peppermint oil in Meilu Xiaozao Cream; and determination of the content of matrine in Meilu Xiaozao Cream; specifically as follows: The gas phase identification of borneol and peppermint oil in the Meiluxiaozao cream is as follows: 1) Preparation of test solution: Take 1-5 g of the product, add 10-30 ml of ethyl acetate, shake for 1-5 minutes, take the supernatant and filter it, and use the filtrate as the test solution; 2) Preparation of reference solution: Take menthol reference substance and borneol reference substance, add ethyl acetate to make a mixed solution containing 1 mg of menthol and 1 mg of borneol per 1 ml, which is used as the reference solution; 3) Gas Chromatography Identification: Follow the test in accordance with General Methods for Gas Chromatography 0521, using a capillary column with polyethylene glycol PEG-20M as the stationary phase: program the column temperature from 60-80°C, then increase the temperature to 180°C at a rate of 8°C per minute, hold for 12-18 minutes, then increase the temperature to 210°C at a rate of 8°C per minute, hold for 12-18 minutes; perform split injection with a split ratio of 40-60:1; aspirate 1 μl of each of the reference solution and the test solution into the gas chromatograph; the test solution should show a peak with the same retention time as the reference solution. The content of matrine in the Meiluxiaozao cream is determined as follows: S1 Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile-anhydrous ethanol-triethylamine-0.5% phosphoric acid solution was used as the mobile phase; the detection wavelength was 210 nm; the flow rate was 1.0 ml / min; the theoretical plate number calculated based on the matrine peak should be no less than 6000; Preparation of S2 reference solution: Take an appropriate amount of matrine reference solution, accurately weigh it, and add anhydrous ethanol to make a solution containing 40μg per 1ml; Preparation of S3 test solution: Take 1-5g of this product, accurately weigh it, place it in a container, accurately add 50ml of hydrochloric acid solution, heat it in an 85℃ water bath for 10-30min to dissolve it, place it at 0-4℃ for 0.5-1.5h, filter it, evaporate the filtrate to dryness, dissolve the residue in appropriate amount of anhydrous ethanol, transfer it to a 5ml volumetric flask, stopper it, weigh it, soak it in warm water at 60℃ for 0.5-1.5h, take it out, cool it to room temperature, weigh it again, make up the loss with anhydrous ethanol, shake it well, filter it, and take the filtrate to obtain the product; Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

2. The detection method according to claim 1, wherein The preparation of the test solution in step 1) is as follows: 1-3 g of the product is added with 10-20 ml of ethyl acetate, and the mixture is shaken for 2-3 minutes. The supernatant is filtered and the filtrate is used as the test solution.

3. The detection method according to claim 2, characterized in that The preparation of the test solution in step 1) is as follows: 2 g of the product is added to 10 ml of ethyl acetate, shaken for 2 minutes, and the supernatant is filtered, and the filtrate is used as the test solution.

4. The detection method according to any one of claims 1, 2 or 3, characterized in that The shaking may also be ultrasonic extraction.

5. The detection method according to claim 1, wherein Step 3) gas phase identification: According to the general method of gas chromatography 0521, a capillary column with polyethylene glycol PEG-20M as the stationary phase is used: the column temperature is programmed, with an initial temperature of 70-80°C, then increased to 180°C at a rate of 8°C per minute, maintained for 14-16 minutes, and then increased to 210°C at a rate of 8°C per minute, maintained for 14-16 minutes; split injection with a split ratio of 45-55:1; 1 μl of the reference solution and the test solution are respectively aspirated and injected into the gas chromatograph; the test solution chromatogram should show a chromatographic peak with the same retention time as the reference solution chromatographic peak.

6. The detection method according to claim 5, characterized in that Step 3) Gas phase identification: According to the general principle of gas chromatography 0521, a capillary column with polyethylene glycol PEG-20M as the stationary phase was used: the column temperature was programmed, with an initial temperature of 80°C, then raised to 180°C at a rate of 8°C per minute, held for 15 minutes, and then raised to 210°C at a rate of 8°C per minute, held for 15 minutes; split injection was performed with a split ratio of 50:1; 1 μl of the reference solution and the test solution were respectively aspirated and injected into the gas chromatograph; the test solution chromatogram should show a chromatographic peak with the same retention time as the reference solution chromatographic peak.

7. The detection method according to claim 1, characterized in that The mobile phase for determining the content of matrine in the Meiluxiaozao cream is: acetonitrile-anhydrous ethanol-triethylamine-0.5% phosphoric acid solution = 75:20:0.1:

5.

8. The detection method according to claim 1, wherein The preparation of the test solution in step S3 is as follows: take 1-3 g of the product, accurately weigh it, place it in a 100 ml beaker, accurately add 50 ml of hydrochloric acid solution, place it in an 85°C water bath and heat it for 15-25 minutes to dissolve it, place it at 0-4°C for 0.5-1 hour, filter it, evaporate the filtrate to dryness, add an appropriate amount of anhydrous ethanol to dissolve the residue, transfer it to a 5 ml volumetric flask, stopper it, weigh it, extract it in 60°C warm water for 0.5-1 hour, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it evenly, filter it, and take the filtrate to obtain it.

9. The detection method according to claim 8, characterized in that The preparation of the test solution in step S3 of the determination of the content of matrine in the Meilu Xiaozao cream is as follows: take 2 g of the product, accurately weigh it, place it in a 100 ml beaker, accurately add 50 ml of hydrochloric acid solution, heat it in an 85°C water bath for 20 minutes to dissolve it, place it at 0-4°C for 1 hour, filter it, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of anhydrous ethanol, transfer it to a 5 ml measuring flask, seal it tightly, weigh it, extract it in warm water at 60°C for 1 hour, take it out, cool it to room temperature, weigh it again, make up the reduced weight with anhydrous ethanol, shake it well, filter it, and take the filtrate to obtain it.

10. The detection method according to any one of claims 1, 8 or 9, characterized in that: The hydrochloric acid solution is prepared by adding 9 parts of hydrochloric acid to 200 parts of water.

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