A method for detecting the quality of musk pain relieving aerosol based on liquid chromatography

Abstract

This invention discloses a quality detection method for musk analgesic aerosol based on liquid chromatography, belonging to the field of traditional Chinese medicine component detection technology. It uses acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, employing gradient elution. The detection wavelength is 400 nm for the first 15 minutes and then switches to 210 nm for the next 55 minutes. The quality detection method for musk analgesic aerosol provided by this invention establishes an HPLC fingerprint of the aerosol and uses osthol as a reference to determine the content of hydroxysaffron yellow and dracoside B using a multi-analysis method. This facilitates more comprehensive monitoring of drug quality and provides a reference and basis for the quality control of musk analgesic aerosol. Furthermore, the method established in this invention has advantages such as simplicity, stability, high precision, and good reproducibility.

Description

Technical Field

[0001] This invention relates to a quality testing method for musk analgesic aerosol based on liquid chromatography, belonging to the field of traditional Chinese medicine component detection technology. Background Technology

[0002] Musk-based analgesic aerosol is primarily used for external treatment of injuries, bruises, swelling, and pain. Musk is the principal ingredient in the formula; its pungent and aromatic properties promote blood circulation, relieve pain, and quickly guide the medicinal effects directly to the affected area. The assistant ingredients, safflower, notoginseng, and dragon's blood, all have the effects of promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain, working synergistically with musk to enhance these effects. Borneol and menthol clear heat, reduce swelling, dispel wind, and relieve pain, while their cooling properties alleviate local inflammation. Angelica pubescens dispels wind and dampness, and relieves pain; Rehmannia glutinosa clears heat and cools the blood, preventing the warming and drying ingredients from further damaging yin, thus harmonizing the formula's properties. Menthol, the guiding ingredient, disperses pathogenic factors, guiding them out of the body, allowing blood stasis to dissipate and swelling and pain to subside. The combined effects of these ingredients promote blood circulation, remove blood stasis, relax muscles and tendons, reduce swelling, and relieve pain.

[0003] The preparation method of musk analgesic aerosol is as follows: Take artificial musk, Panax notoginseng, and safflower, and soak them in 10ml of 50% ethanol three times, each time for 7 days. Combine the soaking liquids, filter, and reserve the filtrate. Soak Rehmannia glutinosa in 100ml of 50% ethanol three times, each time for 7 days. Combine the soaking liquids, filter, and reserve the filtrate. Soak Dragon's Blood and Angelica pubescens in 10ml of ethanol three times, each time for 7 days. Combine the soaking liquids, filter, and reserve the filtrate. Add 100ml of ethanol to borneol and camphor, stir to dissolve, then add 700ml of 50% ethanol and mix well. Add the above soaking liquids and mix well. Dissolve menthol in an appropriate amount of 50% ethanol, add it to the above medicinal solution, add 50% ethanol to a total volume of 1000ml, mix well, let stand, filter, fill, seal, and fill with an appropriate amount of propellant to obtain the product.

[0004] A holistic evaluation of the quality of traditional Chinese medicine (TCM), establishing control items, methods, and indicators reflecting the overall quality of TCM based on key quality attributes and product characteristics, is a necessary means to ensure the safety, efficacy, and stable and controllable quality of TCM. The active ingredients of Musk Analgesic Aerosol are made from nine medicinal materials. Currently, the quality control standards for Musk Analgesic Aerosol in the Chinese Pharmacopoeia mainly include the following: ① gas chromatographic identification of muscone in musk; ② gas chromatographic identification of menthol, camphor, and borneol; ③ content determination of menthol, camphor, and borneol. That is, the control standards only reflect the quality control methods for four medicinal materials.

[0005] In addition, the literature “Liu Jian, Yu Decai. Study on the quality standard of musk analgesic spray [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2011” uses TLC to qualitatively identify dragon's blood, angelica pubescens, menthol, borneol, and camphor in the prescription, and uses GC to determine the content of camphor, menthol, and borneol. The literature “Zhang Nan, Lei Chengkang. Study on improving the quality standard of musk analgesic liniment [J]. China Pharmacy, 2016” uses thin-layer chromatography (TLC) to qualitatively identify dragon's blood and rehmannia in the preparation, and determines the mass fraction of total solids and ether-soluble extracts in the preparation. It also uses gas chromatography to determine the content of muscone in the preparation. The literature “Yang Shuyan, Li Zhaodong. Determination of the content of four effective components of musk analgesic gel by gas chromatography [J]. Modern Biomedical Progress, 2006” uses gas chromatography and programmed temperature method to simultaneously determine the content of the main components of musk analgesic gel, muscone, camphor, menthol, and borneol.

[0006] Quality control standards for musk-based analgesic products often employ TLC (Transmission Linear Chromatography) to identify musk, Angelica pubescens, and dragon's blood, and gas chromatography (GC) to quantitatively detect volatile components such as muscone, menthol, borneol, and camphor. However, according to the manufacturing process, musk-based analgesic aerosols primarily use 50% ethanol to extract the alcohol- and water-soluble components of artificial musk, Panax notoginseng, safflower, and Rehmannia glutinosa, and ethanol is used to extract the alcohol-soluble components from dragon's blood and Angelica pubescens. Therefore, using GC to detect volatile components is too simplistic. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a quality detection method for musk analgesic aerosol based on liquid chromatography, which has the advantages of being simple, stable, highly precise and reproducible.

[0008] The present invention achieves the above objectives by adopting the following technical solutions: A quality detection method for musk analgesic aerosol based on liquid chromatography, including a method for establishing fingerprint spectra and / or a method for determining the content of multiple components; The method for establishing the fingerprint spectrum includes the following steps: S1. Determine the liquid chromatography conditions: The column is packed with octadecylsilane-bonded silica gel; acetonitrile is used as mobile phase A, and 0.05% phosphoric acid solution is used as mobile phase B. Gradient elution is performed with the following elution program: 0-15 min, 10-40% A; 15-40 min, 40% A; 40-52 min, 40-75% A; 52-55 min, 75% A; 55-56 min, 75-90% A; 56-65 min, 90% A; 65-66 min, 90-10% A; 66-70 min, 10% A; The detection wavelength is 400 nm for the first 15 min and then switched to 210 nm for the last 55 min. S2. Prepare a mixed reference solution and a test solution of osthol, hydroxysaffron yellow A and dracoside B; S3. Collect the chromatograms of the mixed reference solution and the test solution, identify the common peaks in the chromatogram of the test solution, and compare it with the chromatogram of the mixed reference solution to identify the characteristic peaks of osthol, hydroxysaffron yellow A and dracoside B; using osthol as a reference, calculate the relative retention times of the remaining characteristic peaks to obtain the fingerprint chromatogram.

[0009] Preferably, the mixed reference solution is prepared by taking appropriate amounts of reference standards of osthol, hydroxysaffron yellow A and dracoside B, dissolving and diluting them with 70% methanol to prepare a mixed reference solution containing 5 μg of osthol, 5 μg of hydroxysaffron yellow A and 2.5 μg of dracoside B per 1 ml.

[0010] Preferably, the test solution is prepared as follows: take 5 ml of musk analgesic aerosol solution, place it in a 10 ml volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate as the test solution.

[0011] The relative retention times of hydroxysaffron yellow pigment A and dracoside B were 0.17 and 0.86, respectively.

[0012] Preferably, the method for determining the content of multiple components further includes the following steps in addition to steps S1-S3: S4. Determine the relative correction factor: Chromatograms of mixed reference solutions of different concentrations were collected, and standard curves were plotted with peak area as the ordinate and concentration as the abscissa to obtain linear regression equations for osthol, hydroxysaffron yellow A, and dracothin B. Using osthol as a reference, the relative correction factors for hydroxysafflower yellow pigment A and dracoside B were calculated based on the linear regression equation. S5. Collect the chromatograms of the osthol reference solution and the test solution, locate the remaining characteristic peaks according to the relative retention time, and calculate the contents of osthol, hydroxysaffron yellow A and dracoside B in the test solution according to the relative correction factor, using the concentration and peak area of ​​osthol as references.

[0013] The relative correction factors for hydroxysaffron yellow pigment A and dragon's blood pigment B are 2.7 and 0.9, respectively.

[0014] The preparation method of the osthol reference solution is as follows: take the osthol reference standard, dissolve it in 70% methanol and quantitatively dilute it to a solution containing 5 μg of osthol per ml.

[0015] The beneficial effects of the present invention include, but are not limited to: This invention provides a quality detection method for musk analgesic aerosols based on liquid chromatography. An HPLC fingerprint of the musk analgesic aerosol is established, and the contents of hydroxysaffron yellow and dracoside B are determined using a single-analysis, multi-evaluation method with osthol as a reference. This facilitates more comprehensive monitoring of drug quality and provides a reference and basis for the quality control of musk analgesic aerosols. Furthermore, the method established in this invention has advantages such as simplicity, stability, high precision, and good reproducibility. Attached Figure Description

[0016] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 The liquid phase fingerprint spectrum of the musk analgesic aerosol established in this invention shows that peak 2 is the characteristic peak of hydroxysaffron yellow pigment A, peak 13 is dracosanol B, and peak 15 is osthol. Figure 2 The chromatogram obtained from Comparative Example 1; Figure 3 The chromatogram obtained from Comparative Example 2; Figure 4 The chromatogram obtained from Comparative Example 3; Figure 5 The chromatogram obtained from Comparative Example 4; Figure 6 The chromatogram obtained from Comparative Example 5; Figure 7 Chromatograms of different samples; Figure 8 Linear relationship diagram for hydroxysaffron yellow A; Figure 9 The linear relationship graph of dragon's blood B; Figure 10 Linear relationship diagram of osthol; Detailed Implementation

[0017] The present invention will be further described in detail below. However, it should be noted that the following specific embodiments are merely exemplary examples of the invention, and the scope of protection of the invention is not limited thereto. The scope of protection of the invention is defined only by the claims. It will be apparent to those skilled in the art that various other modifications and substitutions can be made to the embodiments of the invention within the scope of protection defined by the claims, and the same technical effects can still be achieved, thus achieving the ultimate technical objective of the invention.

[0018] Unless otherwise specified, all raw materials used in this manual were purchased commercially. The raw materials and instruments used are as follows: 1. Instruments and reagents 1.1 Instruments High-performance liquid chromatograph (Agilent 1260Ⅱ), chromatographic column (Waters Atlantis T3 250*4.6mm, 5μm), 0.001 g balance (Mettler XSR304 / A), 0.001 g balance (Mettler XSR105 / A), ultrasonic cleaner (Zhike UC-23). 1.2 Reagents and Test Chemicals Reference standards: Hydroxysaffron Yellow A reference standard (National Institutes for Food and Drug Control, batch number: 111637-202412, purity: 97.7%), Draconisin B reference standard (National Institutes for Food and Drug Control, batch number: 111558-202409, purity: 99.8%), Osthol reference standard (National Institutes for Food and Drug Control, batch number: 110822-202412, purity: 99.9%).

[0019] Reagents: Acetonitrile (TEDIA batch number: 25016198, grade: HPLC), phosphoric acid (Sinopharm Chemical Reagent Co., Ltd. batch number: 20180111, grade: AR), methanol (Shanghai Xingke High Purity Solvent Co., Ltd. batch number: 0234250601D, grade: HPLC).

[0020] Test sample: Musk analgesic aerosol (Zhejiang Sukean Pharmaceutical Co., Ltd., batch numbers: 22407012, 22408004, 22409009, 22410006, 22411005, 22412006, 22501004, 22502002, 22503005, 22504002).

[0021] 2. Liquid chromatography conditions: Chromatographic column: Waters Atlantis C18 T3 250*4.6mm, 5μm; acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution; detection wavelength 400nm for the first 15 min, then switched to 210nm for the next 55 min; flow rate 1.0ml / min, column temperature 25℃; theoretical plate number calculated based on the dragon's blood B peak should not be less than 3000. Determined according to high performance liquid chromatography (General Rule 0512).

[0022] The gradient elution procedure is shown in Table 1.

[0023] Table 1 Gradient elution program 3. Methods for establishing liquid phase fingerprint spectra The method for establishing the liquid phase fingerprint of the musk analgesic aerosol provided by this invention is as follows: 3.1 The specific methods for preparing the mixed reference solution and test solution of osthol, hydroxysaffron yellow A and dracoside B are as follows: Take appropriate amounts of osthol reference standard, hydroxysaffron yellow A reference standard and dracosanol B reference standard, accurately weigh them, dissolve and dilute them with 70% methanol to prepare a mixed reference solution containing 5 μg of osthol, 5 μg of hydroxysaffron yellow A and 2.5 μg of dracosanol B per 1 ml.

[0024] Accurately measure 5 ml of musk analgesic aerosol solution and place it in a 10 ml volumetric flask. Add 70% methanol to the mark, shake well, filter, and take the filtrate as the test solution.

[0025] 3.2 Accurately inject 20 μl of the reference solution and multiple batches of test solution into the liquid chromatograph, and collect the chromatograms of the mixed reference solution and test solution according to "2. Liquid Chromatography Conditions".

[0026] The chromatograms of multiple batches of test sample solutions were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System for analysis. The fingerprint spectrum was generated by the average value algorithm. The chromatogram of the test sample should show the same chromatographic peak retention time as the reference standard. According to the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, the similarity between the fingerprint spectrum of the test sample and the fingerprint spectrum of the reference standard should not be less than 0.90.

[0027] The chromatograms of the test samples were marked with common peaks and compared with the chromatograms of the mixed reference samples to identify the characteristic peaks of osthol, hydroxysaffron yellow A, and dracoside B.

[0028] Using osthol as a reference, the relative retention times of the remaining characteristic peaks were calculated. The same method was used to determine the relative retention times of the remaining characteristic peaks by changing different instruments and different chromatographic columns. The average relative retention times under each condition were taken, as shown in Table 2. The relative retention times should be within ±10% of the specified values.

[0029] like Figure 1 As shown, 16 common peaks were identified in the fingerprint spectrum. Among them, peak 2 is the characteristic peak of hydroxysaffron yellow pigment A, peak 13 is dracosanol B, and peak 15 is osthol.

[0030] 4. Methods for determining the content of multiple components The method for determining the content of multiple components in musk analgesic aerosol, in addition to items "3.1 and 3.2", also includes the following steps: 4.1 Determine the relative correction factor Prepare mixed reference solutions with concentrations equivalent to 10%, 50%, 100%, 150%, and 200% of the test sample solution. Accurately pipette 20 μl of each mixed reference solution of different concentrations and inject them into the liquid chromatograph to collect the chromatogram of the mixed reference solutions.

[0031] A standard curve was plotted with peak area as the ordinate and concentration as the abscissa to obtain the linear regression equations for osthol, hydroxysaffron yellow A, and dracosanol B. Using osthol as a reference, the relative correction factors for hydroxysaffron yellow A and dracosanol B were calculated based on the linear regression equations, as shown in Table 3.

[0032] 4.2 Collect chromatograms of osthol reference solution and test solution, locate the remaining characteristic peaks according to the relative retention time, and calculate the contents of osthol, hydroxysaffron yellow A and dracoside B in the test solution according to the relative correction factor, using the concentration and peak area of ​​osthol as references.

[0033] The preparation method of osthol reference solution is as follows: Take osthol reference standard, dissolve it in 70% methanol and quantitatively dilute it to a solution containing 5 μg of osthol per ml.

[0034] 4.3 Content determination results Measurements showed that each 1 ml of the test solution contained hydroxysaffron yellow A (C). 27 H 32 O 16 The concentration was 6.3~7.9 μg, and the concentration of dragon's blood element B (C) was 6.3~7.9 μg. 18 H 20 O5) was 1.7~3.3 μg, and osthol (C 15 H 16 The concentration of O3 was 4.2~4.7 μg.

[0035] 5. Investigation of chromatographic conditions Comparative Example 1: The difference between this comparative example and the test solution preparation methods in "2. Liquid Chromatography Conditions" and "3.1" is as follows: Preparation method of the test solution: Take the musk analgesic aerosol solution, filter it, and take the filtrate to obtain the test solution.

[0036] The elution procedure is shown in Table 5; the detection wavelength is 203 nm.

[0037] The prepared test solution was injected and detected using the chromatographic conditions in this comparative example. The results are shown in the figure. Figure 2Observation shows that the peak separation effect is average, and hydroxysaffron yellow pigment A is not located in the chromatogram.

[0038] Comparative Example 2: The difference between this comparative example and the test solution preparation methods in "2. Liquid Chromatography Conditions" and "3.1" is as follows: Preparation method of test solution: Take 5ml of musk analgesic aerosol solution, add 5ml of methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0039] The elution procedure is shown in Table 6; the detection wavelength is 400 nm for the first 15 minutes and then changed to 203 nm.

[0040] The prepared test solution was injected and detected using the chromatographic conditions in this comparative example. The results are shown in the figure. Figure 3 Observations show that the peak separation effect is average, and the dragon's blood B is not completely separated from the adjacent peaks.

[0041] Comparative Example 3: The difference between this comparative example and the test solution preparation methods in "2. Liquid Chromatography Conditions" and "3.1" is as follows: Preparation method of test solution 1 (n-butanol extraction): Take 25ml of musk analgesic aerosol solution, add 50ml of water, mix well, and then extract twice with water-saturated n-butanol, 50ml each time. Combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 4ml of ethanol, filter, and take the filtrate to obtain the test solution.

[0042] Preparation method of test solution 2 (direct injection): Take the musk analgesic aerosol solution, filter it, and take the filtrate to obtain the solution.

[0043] The elution procedure is shown in Table 7; the detection wavelength is 400 nm for the first 15 minutes and then 203 nm after 15 minutes.

[0044] The prepared test solution was injected and detected using the chromatographic conditions in this comparative example. The results are shown in the figure. Figure 4 Observation shows that the peak shape of the chromatogram of test solution 1 is poor and it is not applicable. The chromatogram of test solution 2 has a stable baseline and a better peak shape, but the dragon's blood B peak is not separated from the adjacent peak.

[0045] Comparative Example 4: The difference between this comparative example and the test solution preparation methods in "2. Liquid Chromatography Conditions" and "3.1" is as follows: Column: Agilent XDB-C18 4.6*250mm 5μm.

[0046] The elution procedure is shown in Table 8; the detection wavelength is 400 nm for the first 15 minutes and then switched to 203 nm for the next 55 minutes.

[0047] The prepared test solution was injected and detected using the chromatographic conditions in this comparative example. The results are shown in the figure. Figure 5 Observation shows that the peak of hydroxysaffron yellow pigment A is bifurcated, indicating that this chromatographic column is not suitable.

[0048] Comparative Example 5: The difference between this comparative example and the test solution preparation methods in "2. Liquid Chromatography Conditions" and "3.1" is as follows: The elution procedure is shown in Table 9; the detection wavelength is 400 nm for the first 15 minutes and then switched to 203 nm for the next 55 minutes.

[0049] The prepared test solution was injected and detected using the chromatographic conditions in this comparative example. The results are shown in the figure. Figure 6 Observation shows that the peak of dragon's blood element B is located at the gradient and has a poor peak shape.

[0050] 6. Methodological Examination 6.1 Specificity Examination Deodorized Dragon's Blood Negative Sample Solution: Take 0.33g of artificial musk, 0.33g of Panax notoginseng, and 1g of safflower, and sonicate them separately with 10ml of 50% ethanol for 60 minutes, filter, and reserve the filtrate; add 20g of Rehmannia glutinosa to 100ml of 50% ethanol, sonicate for 60 minutes, filter, and reserve the filtrate; add 1g of Angelica pubescens to 10ml of ethanol, sonicate for 60 minutes, filter, and reserve the filtrate; add borneol and camphor to 100ml of ethanol, stir to dissolve, then add 700ml of 50% ethanol and mix well; add the above filtrates and mix well; dissolve menthol in an appropriate amount of 50% ethanol, add it to the above medicinal solution, and add 50% ethanol to a total volume of 1000ml.

[0051] Safflower negative sample solution (without flavor): Take 0.33g of artificial musk and 0.33g of Panax notoginseng, sonicate with 10ml of 50% ethanol for 60 minutes, filter, and reserve the filtrate; add 20g of Rehmannia glutinosa to 100ml of 50% ethanol, sonicate for 60 minutes, filter, and reserve the filtrate; add 0.33g of dragon's blood and 1g of Angelica pubescens to 10ml of ethanol, sonicate for 60 minutes, filter, and reserve the filtrate; add borneol and camphor to 100ml of ethanol, stir to dissolve, then add 700ml of 50% ethanol and mix well; add the above filtrates and mix well; dissolve menthol in an appropriate amount of 50% ethanol, add to the above medicinal solution, and add 50% ethanol to a total volume of 1000ml.

[0052] For the negative sample solution of Angelica pubescens with deodorized ingredients: Take 0.33g of artificial musk, 0.33g of Panax notoginseng, and 1g of safflower, and sonicate them separately with 10ml of 50% ethanol for 60 minutes, filter, and set aside the filtrate; Add 20g of Rehmannia glutinosa to 100ml of 50% ethanol, sonicate for 60 minutes, filter, and set aside the filtrate; Add 0.33g of dragon's blood to 10ml of ethanol, sonicate for 60 minutes, filter, and set aside the filtrate; Add borneol and camphor to 100ml of ethanol, stir to dissolve, then add 700ml of 50% ethanol and mix well; Add the above filtrates and mix well; Dissolve menthol in an appropriate amount of 50% ethanol, add it to the above medicinal solution, and add 50% ethanol to a total volume of 1000ml.

[0053] Deodorized Dragon's Blood Negative Sample Solution: Take about 5 ml of deodorized dragon's blood negative sample, place it in a 10 ml volumetric flask, add 70% methanol to the mark, shake well, filter, and the solution is obtained.

[0054] Safflower negative sample solution with deodorized flavor: Take about 5 ml of safflower negative sample with deodorized flavor, put it in a 10 ml volumetric flask, add 70% methanol to the mark, shake well, filter, and the solution is obtained.

[0055] Deodorized Angelica pubescens negative sample solution: Take about 5 ml of deodorized Angelica pubescens negative sample, place it in a 10 ml volumetric flask, add 70% methanol to the mark, shake well, filter, and the solution is obtained.

[0056] Blank solvent, mixed reference solution, test solution, negative sample solutions of safflower (without flavor), dragon's blood (without flavor), and angelica pubescens (without flavor) were tested. The results are shown in Table 10. Figure 7 As can be seen, the negative samples showed no interference, indicating that the method has good specificity.

[0057] 6.2 Examination of Linear Relationships Osthol reference standard stock solution: Weigh 10 mg of osthol reference standard accurately, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with 70% methanol, and shake well to obtain the solution.

[0058] Hydroxysaffron Yellow A reference standard stock solution: Weigh 10 mg of hydroxysaffron Yellow A reference standard accurately, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with 70% methanol, and shake well to obtain the solution.

[0059] Dragon's Blood B Reference Standard Stock Solution: Accurately weigh 10 mg of dragon's blood B reference standard, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with 70% methanol, and shake well to obtain the solution.

[0060] Mixed reference stock solution 1: Accurately measure 5 ml of osthol reference stock solution, 5 ml of hydroxysaffron yellow A reference stock solution, and 2.5 ml of dracosanol B reference stock solution, place them in the same 100 ml volumetric flask, dilute to the mark with 70% methanol, and shake well to obtain the solution.

[0061] The preparation of linear solutions is shown in the table below: Determination: Inject each linear solution separately and record the chromatogram. Obtain the peak area of ​​the reference standard at different concentrations, and calculate the RSD of the response factor at different concentrations, as shown in Tables 12-14. Plot a standard curve with concentration (μg / ml) on the x-axis and peak area (A) on the y-axis. Calculate the regression equation and correlation coefficient for each analyte. The results are as follows: Figures 8-10 As shown, each analyte exhibits good linearity within its respective concentration range.

[0062] 6.3 Repeatability Test Reference solution: Take appropriate amounts of osthol reference standard, hydroxysaffron yellow A reference standard and dracosanol B reference standard, accurately weigh them, dissolve and dilute them with 70% methanol to prepare a mixed solution containing 5 μg of osthol, 5 μg of hydroxysaffron yellow A and 2.5 μg of dracosanol B per 1 ml.

[0063] Test solution: Take 5 ml of the drug solution, accurately measure it, place it in a 10 ml volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate. Prepare 6 parallel portions.

[0064] Determination: Inject the above reference solution and test solution separately, record the chromatograms, and calculate the RSD of each analyte.

[0065] The results are shown in Table 15. As can be seen, the RSD of each analyte is less than 0.4%, indicating that the method has good repeatability.

[0066] 6.4 Recovery Test Osthol reference standard stock solution: Weigh 10 mg of osthol reference standard accurately, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with 70% methanol, and shake well to obtain the solution.

[0067] Hydroxysaffron Yellow A reference standard stock solution: Weigh 10 mg of hydroxysaffron Yellow A reference standard accurately, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with 70% methanol, and shake well to obtain the solution.

[0068] Dragon's Blood B Reference Standard Stock Solution: Accurately weigh 10 mg of dragon's blood B reference standard, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with 70% methanol, and shake well to obtain the solution.

[0069] Mixed reference stock solution 2: Accurately measure 5 ml of osthol reference stock solution, 5 ml of hydroxysaffron yellow A reference stock solution, and 2.5 ml of dracosanol B reference stock solution, place them in the same 20 ml volumetric flask, dilute to the mark with 70% methanol, and shake well to obtain the solution.

[0070] Test solution: Accurately measure 5 ml of this product and place it in a 20 ml volumetric flask. Dilute to the mark with 70% methanol, shake well, filter, and collect the filtrate.

[0071] 50% recovery solution: Accurately measure 5 ml of this product and place it in a 20 ml volumetric flask. Accurately add 1 ml of mixed reference stock solution 2, dilute to the mark with 70% methanol, shake well, filter, and take the filtrate. Prepare 3 parallel portions.

[0072] 100% recovery solution: Accurately measure 5 ml of this product and place it in a 20 ml volumetric flask. Accurately add 2 ml of mixed reference stock solution 2, dilute to the mark with 70% methanol, shake well, filter, and take the filtrate. Prepare 3 parallel portions.

[0073] 150% recovery solution: Accurately measure 5 ml of this product and place it in a 20 ml volumetric flask. Accurately add 3 ml of mixed reference stock solution 2, dilute to the mark with 70% methanol, shake well, filter, and take the filtrate. Prepare 3 parallel portions.

[0074] Determination: Take the above reference solution and recovery solution, inject them directly, record the chromatogram, and calculate the recovery rate and RSD.

[0075] The results are shown in Tables 16-18. It can be seen that the average recoveries (RSDs) of hydroxysaffron yellow A, dracoside B, and osthol were 100.5% (0.7%), 99.4% (0.6%), and 100.4% (0.9%), respectively, indicating good accuracy of the method.

[0076] 6.5 Solution stability test Test solution: Take 5 ml of the drug solution, accurately measure it, place it in a 10 ml volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0077] Determination: The test solution was placed at room temperature and injected at different times. The chromatograms were recorded, and the peak area and RSD of each analyte were calculated.

[0078] See Table 19. It can be seen that the RSD of the peak area of ​​each analyte does not exceed 0.7%, indicating that the test solution has good stability within 45 hours at room temperature.

[0079] The above specific embodiments should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, any alternative improvements or modifications made to the embodiments of the present invention shall fall within the scope of protection of the present invention.

[0080] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A method for detecting the quality of musk analgesic aerosol based on liquid chromatography, characterized in that, This includes methods for establishing fingerprint spectra and / or methods for determining the content of multiple components; The method for establishing the fingerprint spectrum includes the following steps: S1. Determine the liquid chromatography conditions: The column is packed with octadecylsilane-bonded silica gel; acetonitrile is used as mobile phase A, and 0.05% phosphoric acid solution is used as mobile phase B. Gradient elution is performed with the following elution program: 0-15 min, 10-40% A; 15-40 min, 40% A; 40-52 min, 40-75% A; 52-55 min, 75% A; 55-56 min, 75-90% A; 56-65 min, 90% A; 65-66 min, 90-10% A; 66-70 min, 10% A; The detection wavelength is 400 nm for the first 15 min and then switched to 210 nm for the last 55 min. S2. Prepare a mixed reference solution and a test solution of osthol, hydroxysaffron yellow A and dracoside B; the test solution is prepared as follows: take 5 ml of musk analgesic aerosol solution, put it in a 10 ml volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate as the test solution. S3. Collect chromatograms of the mixed reference solution and the test solution, identify common peaks in the chromatogram of the test solution, and compare it with the chromatogram of the mixed reference solution to identify the characteristic peaks of osthol, hydroxysaffron yellow A and dracoside B; using osthol as a reference, calculate the relative retention times of the remaining characteristic peaks to obtain the fingerprint spectrum. S4. Determine the relative correction factor: Chromatograms of mixed reference solutions of different concentrations were collected, and standard curves were plotted with peak area as the ordinate and concentration as the abscissa to obtain linear regression equations for osthol, hydroxysaffron yellow A, and dracothin B. Using osthol as a reference, the relative correction factors for hydroxysafflower yellow pigment A and dracoside B were calculated based on the linear regression equation. S5. Collect the chromatograms of the osthol reference solution and the test solution, locate the remaining characteristic peaks according to the relative retention time, and calculate the contents of osthol, hydroxysaffron yellow A and dracoside B in the test solution according to the relative correction factors, using the concentration and peak area of ​​osthol as references; the relative correction factors of hydroxysaffron yellow A and dracoside B are 2.7 and 0.9, respectively.

2. The quality detection method according to claim 1, characterized in that, The mixed reference solution is prepared as follows: Take appropriate amounts of reference standards of osthol, hydroxysaffron yellow A and dracoside B, dissolve and dilute them with 70% methanol to prepare a mixed reference solution containing 5 μg of osthol, 5 μg of hydroxysaffron yellow A and 2.5 μg of dracoside B per 1 ml.

3. The quality inspection method according to claim 1, characterized in that, The relative retention times of hydroxysaffron yellow pigment A and dracoside B were 0.17 and 0.86, respectively.

4. The quality inspection method according to claim 1, characterized in that, The preparation method of osthol reference solution is as follows: Take osthol reference standard, dissolve it in 70% methanol and quantitatively dilute it to a solution containing 5 μg of osthol per ml.

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