In-vitro release test method for desonide ointment

Abstract

The invention provides an in-vitro release test method for desonide ointment. The in-vitro release test method comprises the following steps: (1) releasing a desonide component in the desonide ointment by using an in-vitro transdermal instrument; (2) determining through high performance liquid chromatography; and (3) calculating the release amount of the desonide ointment by using a standard curve method. The step (1) is specifically as follows: a test device is based on a Franz diffusion cell method; a proper inert and commercialized artificial film is installed in the Franz diffusion cell system; setting in-vitro release test parameters; and testing an in-vitro release test of the model medicine.

Description

Technical Field

[0001] This invention relates to the field of chemical drug analysis technology, and in particular to an in vitro release assay method for desonide ointment. Background Technology

[0002] Desonide is a synthetic, non-halogenated glucocorticoid, classified as a category VI glucocorticoid in potency, indicating it is a weak glucocorticoid. Similar to other topical glucocorticoids, desonide possesses anti-inflammatory, antipruritic, and vasoconstrictive activities. A 0.05% solution of desonide has been used to treat glucocorticoid-sensitive skin diseases for over 30 years, demonstrating reliable therapeutic efficacy and safety in clinical applications. Currently, no pharmacopoeia in any country includes an in vitro release assay method for desonide ointment.

[0003] In vitro release assays are one of the important methods for evaluating the dosage forms and formulations of semi-solid preparations (such as creams, ointments, and ointments). The extent and rate of in vitro release are a comprehensive reflection of drug properties (including the solubility of the active ingredient, particle size, and the rheological properties of the dosage form), and are one of the standard methods for characterizing the formulation properties of topical medications. They can reflect the physicochemical changes in semi-solid drugs. In vitro release assays are characterized by high operability, high reproducibility, and relatively high sensitivity.

[0004] Chinese patent CN117054556A provides a method for detecting the in vitro release rate of desonide cream. However, this method uses liquid chromatography-mass spectrometry (LC-MS), which requires sophisticated laboratory equipment. Furthermore, this patent provides an in vitro release method for creams, which has significant differences in characteristics compared to ointment formulations, such as dispersion systems, matrix structures, and drug states, directly leading to significant differences in in vitro release behavior and method design.

[0005] In summary, there is a need to develop an in vitro release assay method for desonide ointment. Summary of the Invention

[0006] The present invention proposes an in vitro release test method for desonide ointment, which aims to fill the above-mentioned deficiencies in the existing technology and achieve effective detection of the in vitro release rate of desonide ointment.

[0007] The technical solution of the present invention: an in vitro release test method for desonide ointment, comprising: (1) releasing the desonide component in the desonide ointment using an in vitro transdermal device; (2) determining by high performance liquid chromatography; and (3) calculating the release amount of desonide ointment using a standard curve method.

[0008] Specifically, the steps of step (1) above are as follows: the test apparatus is based on the Franz diffusion cell method; an appropriate inert and commercially available artificial membrane is installed in the Franz diffusion cell system; in vitro release test parameters are set; and the in vitro release test of the model drug is performed.

[0009] Preferably, the Franz diffusion cell system is a diffusion cell system with a standard open-cap frosted glass surface, the system diameter is 15-20 mm, and the receiving cell volume is 12-18 ml; more preferably, the Franz diffusion cell system is a diffusion cell system with a standard open-cap frosted glass surface, the system diameter is 20 mm, and the receiving cell volume is 18 ml.

[0010] Preferably, the Franz diffusion cell system uses either a manual transdermal diffusion tester or a fully automatic transdermal diffusion tester; more preferably, a manual transdermal diffusion tester is used; and most preferably, a TK-24-II model manual transdermal diffusion tester is used.

[0011] Preferably, a suitable inert and commercially available artificial membrane is installed in the Franz diffusion cell system; more preferably, the artificial membrane is 0.45 μm PTFE.

[0012] Preferably, the receiving medium used in the in vitro release test is tetrahydrofuran-water, and the volume ratio is (15:85) to (5:95); more preferably, the volume ratio of tetrahydrofuran-water is (10:90).

[0013] Preferably, the temperature of the receiving medium in the in vitro release test is maintained at 32℃±1℃.

[0014] Preferably, the rotation speed of the in vitro release test is 600 rpm.

[0015] Preferably, the sampling or replenishment volume for the in vitro release test is 5 ml.

[0016] Preferably, in the in vitro release test, the sample loading amount and sampling method are as follows: a quantitative loop is preferred for loading the sample to make the sample as flat as possible, so that the sample is less subjected to shearing action and the matrix structure is less disturbed, which can improve the repeatability of the loading operation. At the same time, the consistency of the loading amount (±5%) should be ensured, and the loss of sample moisture during the test should be avoided as much as possible.

[0017] More preferably, when using a transdermal diffusion tester (manual), a 2 mm quantitative loop is used for sample loading, with a sample loading amount of approximately 550 mg (±5%); when using a fully automated transdermal diffusion tester, a 1 mm quantitative loop is used for sample loading, with a sample loading amount of approximately 150 mg (±5%).

[0018] Preferably, the sampling time points for the in vitro release test are 1h, 2h, 3h, 4h, 5h, and 6h.

[0019] Specifically, the chromatographic conditions for step (2) above are as follows:

[0020] chromatographic column Yuexu Xtimate C18 250mm×4.6mm,5μm Column temperature 30℃ Detection wavelength 245nm Injection volume 80μl Runtime 6min mobile phase Water-acetonitrile (50:50) Flow rate 1.2 ml / min Sample inlet temperature room temperature Washing method Isocratic elution

[0021] Specifically, the calculation formula for step (3) above.

[0022] 1) Linear fitting equation of the standard curve

[0023] Y=kX+b

[0024] in:

[0025] X: Concentration value of the analyte, μg / ml.

[0026] Y: The response area value of the analyte.

[0027] k: slope

[0028] b: Intercept

[0029] 2) Cumulative release per unit area

[0030]

[0031] in:

[0032] A SPLn : Peak area of ​​the analyte in the test solution at the nth sampling point

[0033] b: Intercept value in the linear fitting equation

[0034] k: The slope value in the linear fitting equation

[0035] n: Number of current sampling points

[0036] Vspl: Sample volume, 5ml

[0037] Vc: Volume of diffusion cell, 18ml

[0038] S: Effective diffusion area of ​​the filter membrane, 3.14 cm² 2

[0039] 3) Release rate:

[0040] Using the square root of time as the x-axis and the cumulative release per unit area as the y-axis, a linear regression was performed, and the slope represents the release rate of the formulation.

[0041] 4) Acceptable standards:

[0042] If the 90% confidence interval of the ratio of the median (8th to 29th) in vitro release rate of the self-developed formulation and the reference formulation is within the range of 75% to 133.33%, it indicates that the in vitro release behavior of the self-developed formulation and the reference formulation is consistent.

[0043] The present invention provides a method for rapidly evaluating the in vitro release of topical formulations, which has the following advantages:

[0044] (1) The in vitro test method is simple and easy to perform, does not require animal dissection, and eliminates individual differences in animal skin.

[0045] (2) Liquid chromatography is used for detection. It is a commonly used instrument in laboratories and is highly accessible.

[0046] (3) High in vitro release enhances the "discrimination power" of the method, making it easier to optimize prescriptions and control quality.

[0047] (4) The method has good day-to-day repeatability, ensuring the reliability of the method and the credibility of the data, meeting the compliance requirements of method validation, and facilitating data comparison between laboratories and industrialization transformation.

[0048] (4) The experimental results are highly correlated with the formulation process, which can evaluate the quality difference between the self-developed formulation and the reference formulation. They are also more correlated with the in vivo bioequivalence results, which can better assess the quality of the sample.

[0049] The method of this invention is universal and can be used for in vitro release testing of desonide ointment prepared by various methods. This is because: firstly, the selection of a tetrahydrofuran aqueous solution with strong dissolving power ensures that the main component meets the leakage conditions within a dosage range of 50% to 150%. Secondly, current generic drugs are all studied according to the reference formulation, and this product's excipients only contain white petrolatum. This method has been tested on samples from the formulation process research, and is applicable to samples from different manufacturers or models of white petrolatum and samples from different processes. Referring to the "Technical Guidelines for In Vitro Release (IVRT) and In Vitro Transdermal (IVPT) Research of Locally Acting Generic Chemical Drugs (Trial Implementation)," this method has been validated, and key indicators such as specificity, precision, discriminant power, and supplementary selectivity all meet the requirements. Therefore, this method can be confirmed as a universal detection method for the in vitro release of desonide ointment, applicable to desonide ointment prepared by various processes.

[0050] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below.

[0051] Attached Figure

[0052] Appendix Figure 1 Schematic diagram of alternating sampling of self-developed desonide ointment (0.05%) and desonide ointment reference formulation.

[0053] Appendix Figure 2 3.2.2 Specificity curve Specific Implementation

[0054] The experimental instruments and reagents involved in this invention are as follows:

[0055] (1) Instruments and equipment

[0056] name Manufacturer (Model) Analytical balance (1) METTLER TOLEDO (XSR105); (2) Sartorius Scientific Instruments Ltd. (BSA224S) High Performance Liquid Chromatography Thermo Fisher Scientific (Mltimate 3000) Transdermal diffusion tester (manual) Shanghai Kaikai Technology Trading Co., Ltd. (TK-24Ⅱ) Fully automated transdermal diffusion device (1) Shenzhen Huarong Analytical Instruments Co., Ltd. (DSC-800); (2) LOGAN (TD-12AT) pH meter METTLER TOLEDO (FE28)

[0057] (2) Reagents and materials

[0058] name Grade / Specification Source (manufacturer) Propylene glycol pharmaceutical excipients Hubei Gedian Renfu Pharmaceutical Excipients Co., Ltd. Tetrahydrofuran HPLC Shanghai Aladdin Biochemical Technology Co., Ltd. Acetonitrile HPLC Wuhan Fulton Holdings Co., Ltd. ethanol HPLC Wuhan Fulton Holdings Co., Ltd. Filter membrane PTFE, 0.45μm Tianjin Jinteng Test Equipment Co., Ltd. Filter membrane Nylon 6, 0.45μm Tianjin Jinteng Test Equipment Co., Ltd. Filter membrane 0.4μm polycarbonate Whatman

[0059] (3) Chromatographic column

[0060] Specifications and Models Source (manufacturer) Yuexu Xtimate C18 250mm×4.6mm,5μm Moonrise

[0061] (4) Raw materials

[0062] name content Source (manufacturer) Desonide raw materials 99.7% Hubei Gedian Renfu Pharmaceutical Co., Ltd.

[0063] (5) Reference preparation

[0064] name batch number Specification Source (manufacturer) Desonide ointment reference preparation 4KT0516 0.05% (15g:7.5mg) Padagis

[0065] (6) Self-made preparations

[0066] Name (Batch Number) API strength The main prescription specifications examined Homemade Desonide Ointment (0.025% strength) 50% 0.025% Homemade Desonide Ointment (0.05% strength) 100% 0.05% Homemade Desonide Ointment (0.075% strength) 150% 0.075%

[0067] The preparation method is as follows:

[0068] 1) Matrix preparation: Weigh the prescribed amount of white petrolatum and add it to a container. Heat and stir until completely melted and clear. Cool down to 50~60℃ and set aside.

[0069] 2) Total mixing: Weigh the prescribed amount of desonide, add it to the matrix, and stir under vacuum until homogenized.

[0070] 3) Cooling: Vacuum stir to room temperature, then collect the material.

[0071] Example 1

[0072] 1.1 Chromatographic conditions

[0073] chromatographic column Yuexu Xtimate C18 250mm×4.6mm,5μm Column temperature 30℃ Detection wavelength 245nm Injection volume 80μl Runtime 6min mobile phase Water-acetonitrile (50:50) Flow rate 1.2 ml / min Sample inlet temperature room temperature Washing method Isocratic elution

[0074] 1.2 In vitro release conditions

[0075] Transdermal diffusion tester TK-24-II Filter membrane 0.45μmPTFE Receiving liquid Tetrahydrofuran-water (10:90) temperature 32℃ rotational speed 600rpm Sampling / Replenishment Volume 5ml Sample loading amount (sample loading method) 550mg (2mm metering ring) Sampling time point 1h, 2h, 3h, 4h, 5h, 6h

[0076] 1.3 Solution Preparation

[0077] 1.3.1 Receiving Medium

[0078] Take 3600ml of purified water, add 400ml of tetrahydrofuran, shake well, and degas by sonication.

[0079] 1.3.2 Blank solution

[0080] Same receiving medium.

[0081] 1.3.3 Mobile Phase

[0082] Take 2000ml of purified water, add 2000ml of acetonitrile, shake well, and degas by sonication.

[0083] 1.3.4 Standard Curve Solution

[0084] Stock solution: Weigh about 20 mg of desonide raw material into a 100 ml volumetric flask, add tetrahydrofuran-water (10:90) to dissolve and dilute to the mark, shake well to obtain the stock solution, take 1 ml into a 100 ml volumetric flask, add tetrahydrofuran-water (10:90) to the mark, shake well to obtain the stock solution.

[0085] Transfer the above-mentioned stock solution to prepare the calibration standard solution according to the table below, with the diluent as the receiving medium.

[0086]

[0087] 1.3.5 Quality Control Solution

[0088] LQC solution: Prepared in the same way as "L-1 calibration standard solution". (Prepared independently of the standard curve solution)

[0089] LMQC solution: Prepared in the same way as the "L-2 calibration standard solution". (Prepared independently of the standard curve solution)

[0090] HMQC solution: Prepared in the same way as the "L-6 calibration standard solution". (Prepared independently of the standard curve solution)

[0091] HQC solution: Prepared in the same way as "L-7 calibration standard solution". (Prepared independently of the standard curve solution)

[0092] The calibration standard and the quality control stock solution should be prepared by weighing them separately twice to obtain the standard curve and the quality control sample stock solution.

[0093] 1.3.6 System Applicability

[0094] Prepare the same solution as "L-2 calibration standard solution".

[0095] 1.3.7 Test solution

[0096] Take the filter membrane, cut it into a circular piece with a diameter of 28 mm, soak it in the medium for 30 min and wipe it dry. Use a quantitative loop (2 mm) (about 550 mg) to weigh the reference preparation of desonide ointment and the self-made desonide ointment (0.05% specification) as samples and spread them evenly on the filter membrane. Install it on the diffusion cell and take samples and replenish the solution according to the in vitro release conditions.

[0097] 1.3.8 Calculation Formula

[0098] 1) Linear fitting equation of the standard curve

[0099] Y=kX+b

[0100] in:

[0101] X: Concentration value of the analyte, μg / ml.

[0102] Y: The response area value of the analyte.

[0103] k: slope

[0104] b: Intercept

[0105] 2) Cumulative release per unit area

[0106]

[0107] in:

[0108] A SPLn : Peak area of ​​the analyte in the test solution at the nth sampling point

[0109] b: Intercept value in the linear fitting equation

[0110] k: The slope value in the linear fitting equation

[0111] n: Number of current sampling points

[0112] Vspl: Sample volume, 5ml

[0113] Vc: Volume of diffusion cell, 18ml

[0114] S: Effective diffusion area of ​​the filter membrane, 3.14 cm² 2

[0115] 3) Release rate:

[0116] Using the square root of time as the x-axis and the cumulative release per unit area as the y-axis, a linear regression was performed, and the slope represents the release rate of the formulation.

[0117] 4) Acceptable standards:

[0118] If the 90% confidence interval of the ratio of the median (8th to 29th) in vitro release rate of the self-developed formulation and the reference formulation is within the range of 75% to 133.33%, it indicates that the in vitro release behavior of the self-developed formulation and the reference formulation is consistent.

[0119] 1.3.9 Recommended Sequence

[0120] name Number of injection needles Blank solution (receiving medium) 1~Multiple needles SST 6 L-0 1 L -1 1 L -2 1 L -3 1 L -4 1 L -5 1 L -6 1 L -7 1 L -8 1 blank solution 1 LQC 1 Test solution 1 … … LMQC 1 Test solution 1 … … HMQC 1 Test solution 1 … … HQC 1

[0121] 1.3.10 System Applicability

[0122] 1) For the suitability of the 6-needle system, the coefficient of variation (%CV) of the retention time of the analyte in the solution should not exceed 5%, and the coefficient of variation (%CV) of the peak area of ​​the analyte should not exceed 15%.

[0123] 2) The L-0 solution does not interfere with the analyte. If there is interference, the response area at the analyte should be less than 20% of the area in the L-1 solution.

[0124] 3) Using the concentration values ​​of the analyte in solutions L-1 to L-8 as the x-axis (X) and the peak area values ​​as the y-axis (Y), perform linear regression and report the linear fit equation and correlation coefficient r. The correlation coefficient r should not be less than 0.9900.

[0125] 4) The calculated concentration of L-1 should be within ±20% of the labeled concentration, and the calculated concentrations of the remaining standards should be within ±15% of the labeled concentration. At least six calibration standards must meet the above requirements. If any calibration standard fails to meet the standard, it should be rejected, the calibration curve excluding that point should be recalculated, and a new regression analysis should be performed.

[0126] At least two-thirds of the quality control samples, and at least 50% of each concentration level, should be within ±15% of the labeled value.

[0127] 1.3.11 Experimental Procedure

[0128] Take a 0.45μm PTFE filter membrane, cut it into 28mm diameter discs, soak it in the medium for 30 minutes, and then wipe it dry. Place a 2mm metering loop on the filter membrane in a manual transdermal diffusion apparatus. Weigh approximately 550mg of the self-made desonide ointment (0.05%) and desonide ointment reference preparation, and place them in the metering loop. Perform parallel operations for both ointments, with 6 VDCs (vertical diffusion cells) per group. Weigh the two ointments alternately during sample weighing. Place the filter membrane on the diffusion cell (manual) of the transdermal apparatus, and alternately place the two ointments during sample placement, as shown in the attached diagram. Figure 1 As shown. Add receiving medium to the receiving cell, ensuring there are no air bubbles between it and the artificial membrane, filling to the mark. The temperature is 32℃, the rotation speed is 600 rpm, and sampling points are 1h, 2h, 3h, 4h, 5h, and 6h. At each specified time point, take 5ml of the receiving solution as the test solution. Add receiving medium at the same temperature and volume.

[0129] Following the sequence in 1.3.9, the test solution was injected into the liquid chromatograph, the peak area was recorded, the release amount was calculated using the formula in 1.3.8, the release curve was established, the release rate was obtained, and then the 90% confidence interval was obtained.

[0130] 1.1.2 Experimental Results

[0131] (1) List of release rates of self-developed formulation and reference formulation:

[0132] (2) Ratio of release rate of self-developed desonide ointment (0.05% specification) to that of the reference desonide ointment:

[0133]

[0134] (3) 90% confidence interval between the self-developed desonide ointment (0.05% specification) and the reference desonide ointment:

[0135] limit Serial Number ratio 1 93.25% 2 94.99% 3 96.64% 4 97.41% 5 97.89% 6 98.43% 7 99.12% 75% 8 100.28% 9 100.98% 10 101.20% 11 101.23% 12 102.01% 13 102.73% 14 102.82% 15 103.10% 16 103.13% 17 103.33% 18 103.55% 19 103.60% 20 104.06% 21 104.88% 22 104.92% 23 105.72% 24 105.75% 25 106.24% 26 106.28% 27 108.65% 28 109.36% 133.33% 29 110.13% 30 110.68% 31 112.44% 32 112.48% 33 112.60% 34 113.49% 35 114.06% 36 120.71%

[0136] Results: The 90% confidence interval for the in vitro release of the self-developed desonide ointment (0.05%) and the reference desonide ointment was 100.28%–110.13%. Within the range of 75%–133.33%, the in vitro release rates of the self-developed and reference formulations were consistent.

[0137] Example 2: Investigation of the receiving medium and filter membrane in (1) in vitro release test

[0138] 2.1 Preliminary exploration of filter membranes and media

[0139] To further investigate the effects of different receiving media and filter membranes on the in vitro release of desonide ointment, an automated transdermal diffusion instrument was used. The in vitro release curves of the reference formulation of desonide ointment were investigated in the laboratory under the following conditions: sample diameter 15 mm, sample volume 150 mg (±5%), 600 rpm, 32℃, replenishment volume 12 ml, sampling time points of 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and filter membranes of 0.45 μm PTFE and 0.45 μm nylon 6. The media used were ethanol-water (30:70), ethanol-water (50:50), 0.9% sodium chloride solution, 2.7% sodium chloride solution, 0.9% sodium chloride solution-ethanol (50:50), and 2.7% sodium chloride solution-ethanol (50:50). The analytical results are shown in Table 1.

[0140] It can be seen that the linear correlation coefficient R under the media filter membrane combination 2All values ​​were greater than 0.97, the release conformed to the Higuchi equation, and no backflow was observed in the samples. The RSD of the release rate was less than 15%, but the cumulative release rate was low. Comparatively, the release rate was higher with a higher proportion of ethanol. The release rate of the PTFE membrane was higher than that of the nylon 6 membrane. The release behavior of the medium with added sodium chloride did not significantly change compared to the medium without added sodium chloride, indicating that adding sodium chloride did not help the experimental results. Therefore, future experiments will use a higher proportion of ethanol and PTFE membrane, with partial sampling as a priority, and will not consider adding sodium chloride.

[0141] Table 1. Results of the exploration of receiving media and filter membranes

[0142]

[0143] 2.2 Further Exploration of Filter Membranes and Media

[0144] To further investigate the effect of different receiving media on the in vitro release of desonide ointment, an automated transdermal diffusion instrument was used. The conditions were: sample diameter 15 mm, sample volume 150 mg (±5%), 600 rpm, 32℃, replenishment volume 3 ml, diffusion cell volume 12.5 ml, sampling time points of 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, and a 0.45 μm PTFE filter. The in vitro release curves of the desonide ointment reference formulation were investigated using 1% Tween 60 and ethanol-water (90:10) media. The results showed that the peak surface area of ​​the main component was extremely low; therefore, the above media were not used.

[0145] Under the above conditions, the in vitro release curves of desonide ointment using tetrahydrofuran-water (30:70)-0.45μmPTFE, dimethyl sulfoxide-water (50:50)-0.45μmPTFE, ethanol-water (50:50)-0.4μm polycarbonate, and propylene glycol-water (50:50) filter membranes were further investigated. The results and analysis are shown in Table 2.

[0146] As can be seen, no main components were detected in the dimethyl sulfoxide-water (50:50) and propylene glycol-water (50:50) media, so they were not used; for the other media filter membrane combinations, the obtained linear correlation coefficient R... 2 All samples met the requirement of being no less than 0.97, conformed to the Higuchi equation, exhibited no backflow, and had an RSD of less than 15%. Among these, the cumulative release rate was relatively low with the ethanol-water (50:50)-0.4μm polycarbonate combination. While the release rate and percentage doubled compared to the previous method (3.3.6.1), the release rate remained low. The tetrahydrofuran-water (30:70)-0.45μm PTFE combination achieved a release rate of 82.4%, but its RSD was also low. 2The ratio is relatively low, so we will prioritize trying a combination of tetrahydrofuran media and PTFE filter membrane with an even lower ratio.

[0147] Table 2 Results of the exploration of receiving media and filter membranes

[0148]

[0149] 2.3 Final confirmation of filter membrane and media

[0150] To further investigate the effect of different receiving media on the in vitro release of desonide ointment, a transdermal diffusion assay was performed using a manual instrument. The experiment was conducted at a sample diameter of 20 mm, a sample volume of 550 mg (±5%), 600 rpm, 32℃, a replenishment volume of 5 ml, a diffusion cell volume of 18 ml, sampling time points of 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, and a 0.45 μm PTFE filter membrane. The in vitro release curves of the desonide ointment reference formulation were investigated under different volume ratios of tetrahydrofuran-water medium. The results are shown in Table 3.

[0151] It can be seen that the linear correlation coefficient R of the curves obtained in the experiment is... 2 All values ​​were greater than 0.97, the release conformed to the Higμchi equation, and no backflow was observed in the samples. The RSD of the release rate was less than 15%. Among them, the cumulative release rate was higher in the tetrahydrofuran-water (30:70) medium, but RSD was lower. 2 Poor.

[0152] According to FDA guidelines, the cumulative release rate in in vitro release tests should be below 30%. Preferably, the release rate meets the in vitro release requirements when the tetrahydrofuran-water volume ratio is (15:85) to (5:95); most preferably, the release rate is around 13% in a tetrahydrofuran-water (10:90) medium, which best meets the in vitro release requirements. Therefore, a combination of tetrahydrofuran-water (10:90) medium and a 0.45μm PTFE filter membrane will be used for subsequent trials.

[0153] Table 3. Results of the exploration of receiving media and filter membranes

[0154]

[0155] Conclusion: When the volume ratio of tetrahydrofuran to water is (15:85) to (5:95), the linear fitting coefficient (R) of the in vitro release curve is [missing information]. 2 Both ) and RSD meet the requirements.

[0156] Example 3: Confirmatory experiment on (1) in vitro release test method

[0157] 3.1 Solution stability study

[0158] To investigate the stability of the drug in the receiving medium, a transdermal diffusion assay was performed using a manual instrument. The conditions were: sample diameter 20 mm, sample volume 550 mg (±5%), 600 rpm, 32℃, replenishment volume 5 ml, diffusion cell volume 18 ml, sampling time points of 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, and a 0.45 μm PTFE filter membrane. Low, medium, and high concentrations of calibration standards and the test solution of desonide were selected and tested for stability at room temperature. A stability was considered achieved when the ratio of the main peak area or concentration at each time point to the peak area or concentration at 0 h was between 0.95 and 1.05.

[0159] The results are shown in Table 4. The ratio of the main peak area or concentration to the peak area at 0h for the low (medium / high) concentration test sample (standard curve) solution at each time point was a minimum of 0.96 and a maximum of 1.02, ranging from 0.95 to 1.05. This indicates that the low (medium / high) concentration test sample (standard curve) solution is stable at room temperature for at least 38 hours.

[0160] Table 4 Solution stability results

[0161]

[0162] 3.2 Differentiation Test

[0163] 3.2.1 Preliminary Examination of Discriminating Ability

[0164] Under the conditions of a sample loading amount of 150 mg (±5%), 600 rpm, 32℃, 0.45 μm PTFE filter membrane, manual transdermal diffusion instrument, sample diameter of 15 mm, replenishment volume of 3 ml, and diffusion cell volume of 12.5 ml, the in vitro release of self-developed desonide ointment with 50%, 100%, and 150% principal component strength was investigated using ethanol-water (50:50) as the receiving medium and sampling time points of 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. The analytical results are shown in Table 5. The release rate did not meet the requirement of V(50%) < V(100%) < V(150%), indicating that the discrimination power of this method does not meet the requirements for in vitro release.

[0165] Table 5. Results of IVRT studies for formulations of different strengths

[0166] Desonide principal component strength 50% 100% 150% Average release rate (pg / cm 2 / h 1 / 2 )]]> 0.131 0.131 0.232

[0167] 3.2.2 Final Examination of Discriminating Ability

[0168] The transdermal diffusion assay was performed using a manual instrument. Under the conditions of a sample loading of 550 mg (±5%), 600 rpm, 32℃, a 0.45 μm PTFE filter membrane, a diffusion cell diameter of 20 mm, a replenishment volume of 5 ml, and a diffusion cell volume of 18 ml, the in vitro release of desonide ointment with a main component strength of 50%, 100%, and 150% was investigated at sampling time points of 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, with tetrahydrofuran-water (10:90) as the receiving medium. The results are shown in Tables 6 and 7. The release rate satisfied V(50%) < V(100%) < V(150%), and the release rate increased with increasing sample strength, indicating good sensitivity of the method.

[0169] A linear regression was performed with principal component intensity on the x-axis and release rate on the y-axis. The linear correlation coefficient R0 was calculated. 2 The value is 0.99, which is greater than 0.95, indicating good specificity of the method.

[0170] The 90% confidence intervals for the ratios of the release rates of the 50%, 150%, and 100% principal components were 64.14%–70.18% and 144.37%–147.37%, respectively, both outside the 75%–133.33% range, indicating good selectivity of the method.

[0171] Table 6. Results of IVRT studies for formulations of different strengths

[0172]

[0173] Table 7 Results of the Differentiation Test

[0174] Principal component strength 50% 150% Ratio to 100% intensity release rate 0.68 1.46 90% confidence interval (compared to 100%) 64.14%~70.18% 144.37%~147.37% Specificity Appendix Figure 2 —

[0175] 3.3 Media Durability Test

[0176] The transdermal diffusion test instrument (manual) was used to investigate the in vitro release of tetrahydrofuran-water (9:91) and tetrahydrofuran-water (11:89) as receiving media compared with tetrahydrofuran-water (10:90) as receiving media under the following conditions: sample loading amount of 550 mg (±5%), 600 rpm, 32℃, 0.45 μm PTFE filter membrane, diffusion cell diameter of 20 mm, replenishment volume of 5 ml, diffusion cell volume of 18 ml, and sampling time points of 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. The results are shown in Table 7. The 90% confidence intervals for the release rate ratios of tetrahydrofuran-water (9:91) and tetrahydrofuran-water (10:90) media were 77.57%–81.41%, and for tetrahydrofuran-water (11:89) and tetrahydrofuran-water (10:90) media, they were 114.78%–129.36%, both within the range of 75%–133.33%, indicating that the media durability meets the requirements.

[0177] Table 7 Results of Media Durability Test

[0178]

Claims

1. A method for in vitro release assay of desonide ointment, the method comprising: (1) Desonide components in desonide ointment were released using an in vitro transdermal device; (2) the release was determined by high performance liquid chromatography; (3) the release amount of desonide ointment was calculated using the standard curve method. The specific steps of step (1) are as follows: the experimental apparatus is based on the Franz diffusion cell method; appropriate inert and commercially available artificial membranes are installed in the Franz diffusion cell system; in vitro release test parameters are set; and in vitro release test of the model drug is performed. The diffusion cell receiving medium is tetrahydrofuran-water, and its volume ratio is (15:85) to (5:95). The artificial membrane installed in the Franz diffusion cell system is 0.45 μm PTFE.

2. The test method according to claim 1, wherein, The Franz diffusion cell system in step (1) is a diffusion cell system with a standard open-cap frosted glass sample surface, with a system diameter of 15-20 mm and a receiving cell volume of 12-18 ml; preferably, the Franz diffusion cell system is a diffusion cell system with a standard open-cap frosted glass sample surface, with a system diameter of 20 mm and a receiving cell volume of 18 ml.

3. The test method according to claim 2, wherein, Step (1) Select one of the following for the Franz diffusion cell system: a transdermal diffusion tester (manual) or a fully automatic transdermal diffusion tester; more preferably, select a transdermal diffusion tester (manual); most preferably, select a transdermal diffusion tester (manual) of model TK-24-II.

4. The test method according to claim 2, wherein, Step (1) Franz diffusion cell system: When using a transdermal diffusion tester (manual), use a 2 mm quantitative loop for sample loading, with a sample loading amount of approximately 550 mg (±5%); when using a fully automated transdermal diffusion tester, use a 1 mm quantitative loop for sample loading, with a sample loading amount of approximately 150 mg (±5%).

5. The test method according to claim 4, wherein, The diffusion cell receiving medium in step (1) is tetrahydrofuran-water, and its volume ratio is tetrahydrofuran-water volume ratio (10:90).

6. The test method according to claim 5, wherein, The in vitro release test in step (1) is performed at a rotation speed of 600 rpm. According to the test method of claim 6, the temperature of the in vitro release test receiving medium in step (1) is maintained at 32℃±1℃.

7. The test method according to claim 1, wherein, The conditions for the in vitro transdermal device mentioned in step (1) are as follows:

8. In the test method according to claim 1, wherein, The chromatographic conditions for high performance liquid chromatography in step (2) are as follows:

9. In the test method according to claim 1, wherein, Step (3) The formula and standard for calculating the standard curve are: the linear fitting equation of the standard curve. Y=kX+b in: X: Concentration value of the analyte, μg / ml. Y: The response area value of the analyte. k: slope b: Intercept 1) Cumulative release per unit area in: A SPLn : Peak area of ​​the analyte in the test solution at the nth sampling point b: Intercept value in the linear fitting equation k: The slope value in the linear fitting equation n: Number of current sampling points Vspl: Sample volume, 5ml Vc: Volume of diffusion cell, 18ml S: Effective diffusion area of ​​the filter membrane, 3.14 cm² 2 2) Release rate: Using the square root of time as the x-axis and the cumulative release per unit area as the y-axis, a linear regression was performed, and the slope represents the release rate of the formulation. 3) Acceptable standards: If the 90% confidence interval of the ratio of the median (8th to 29th) in vitro release rate of the self-developed formulation and the reference formulation is within the range of 75% to 133.33%, it indicates that the in vitro release behavior of the self-developed formulation and the reference formulation is consistent.

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