In vitro release and in vitro transdermal integrated test method of mupirocin ointment

Abstract

This invention discloses an integrated in vitro release and transdermal assay method for mupirocin ointment, belonging to the field of pharmaceutical analysis technology. The method employs a Franz vertical diffusion cell and optimizes the receiver medium—a pH 5.0–8.0 buffer solution (surfactant-free) containing 10%–50% lower alcohol by volume—using response surface methodology with lower alcohol volume fraction and transdermal water loss (TEWL) as dual response indicators. In the in vitro release assay, a hydrophilic microporous membrane with a pore size of 0.20–1.0 μm is used as an artificial membrane barrier. In the transdermal assay, depilated excised animal abdominal skin is used as a biological membrane barrier. The assay is run at a constant temperature of 25–40°C and a stirring rate of 50–600 rpm. Samples are taken at preset time points, and the concentration of mupirocin in the receiver medium is determined by high-performance liquid chromatography (HPLC). The cumulative release rate and cumulative transdermal volume are calculated. This invention is the first to integrate IVRT and IVPT into the same experimental system. The two experiments share the receiving medium, temperature control, sample loading method, and chromatographic conditions. Through synergistic optimization of the receiving medium composition, membrane barrier type, sampling time points, sample loading method, and chromatographic conditions, it achieves efficient and accurate evaluation of the in vitro behavior of generic mupirocin ointment, providing reliable data support for prescription screening, process optimization, and quality consistency evaluation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to an integrated in vitro release and in vitro transdermal assay method for mupirocin ointment, applicable to formulation screening, process development, and quality consistency evaluation of generic mupirocin ointment. Background Technology

[0002] Mupirocin is a natural antibiotic produced by the fermentation of *Pseudomonas fluorescens*. It specifically and reversibly binds to bacterial isoleucyl-tRNA synthetase, blocking bacterial protein synthesis. It exhibits excellent antibacterial activity against MRSA and is widely used clinically for primary and secondary skin infections. Mupirocin ointment (original product: "Bactroban") is a non-water-soluble semi-solid preparation with a polyethylene glycol (PEG) base.

[0003] For the development of generic mupirocin ointment, in vitro release testing (IVRT) and in vitro transdermal testing (IVPT) are core technical means to evaluate the quality consistency between generic and original drugs. In May 2025, the Center for Drug Evaluation of the National Medical Products Administration issued the "Technical Guidelines for In Vitro Release Testing (IVRT) and In Vitro Transdermal Testing (IVPT) of Locally Effective Generic Chemical Drugs (Trial Implementation)," further standardizing and improving the requirements for in vitro evaluation of semi-solid dosage forms. However, existing technologies have the following shortcomings.

[0004] (1) Lack of a dedicated integrated method for mupirocin ointment: Currently, patent CN119915978A discloses "an in vitro release method for semi-solid formulations with aqueous matrix", but it is aimed at semi-solid formulations with aqueous matrix (such as creams, gels, etc.), uses an organic solvent + surfactant receiving medium system, and only involves IVRT, not IVPT. Mupirocin ointment belongs to a non-aqueous system with PEG matrix, which is fundamentally different from aqueous matrix in terms of drug release behavior and diffusion barrier compatibility. This patented method cannot be directly applied to the evaluation of mupirocin ointment. In addition, non-patent literature has reported IVRT and IVPT methods for mupirocin ointment: Lu Lihua et al. (Chinese Pharmaceutical Journal, 2024, 59(23):2258-2265) established an IVRT method for mupirocin ointment using polyethersulfone membrane and pH 7.4 phosphate buffer, but did not involve IVPT, nor did they achieve integrated testing of the two experiments; Wang Song et al. (Chinese Journal of Modern Applied Pharmacy, 2018, 35(2):206-209) conducted IVPT research on mupirocin ointment using phosphate buffer containing 30% ethanol as the receiving medium, but did not involve IVRT, and did not systematically optimize the relationship between ethanol concentration and skin barrier integrity. The above literature all conducted IVRT and IVPT as independent experiments, and did not achieve uniformity in receiving medium, temperature control, chromatographic conditions and sample loading method, resulting in repetitive work in method development and poor comparability of the two sets of data.

[0005] (2) The drawback of receiving medium containing surfactants: Existing technologies often add surfactants (Tween-80, SDS, etc.) to the receiving medium to improve drug solubility. However, studies have shown that surfactants can interact with lipids in the stratum corneum of the skin, disrupting the integrity of the skin barrier and causing distortion of IVPT results. Moreover, for PEG-based ointment systems, the solubilizing effect of surfactants is not better than that of ethanol.

[0006] (3) IVRT and IVPT are independent and inefficient: Existing technologies usually conduct IVRT and IVPT as two completely independent tests, which requires separate development and verification of receiving medium formulation, temperature control, chromatographic analysis methods, and sample loading methods. This results in a large workload, long cycle, and poor comparability and linkage between the two sets of data. Currently, there are no published documents or patents that integrate IVRT and IVPT into a unified test system for mupirocin ointment (PEG matrix system), nor are there any reports on systematically optimizing and screening the ethanol concentration of the receiving medium using solubility and skin TEWL value as dual response indicators.

[0007] (4) The unique rheological properties of ointments have not been fully considered: The viscosity and thixotropy of ointments directly affect the uniformity of the coating of the test sample on the diffusion film / skin surface, and thus affect the reproducibility of IVRT / IVPT data. Existing methods generally lack standardized loading procedures for high-viscosity ointments. Summary of the Invention The first technical problem to be solved

[0008] This invention aims to provide an integrated testing method that organically combines IVRT and IVPT specifically for mupirocin ointment (polyethylene glycol matrix, non-aqueous system), solving the technical problems in the prior art, such as low efficiency caused by conducting IVRT and IVPT independently, unsuitability of the receiving medium system for PEG matrix ointment, and lack of standardized operating procedures affecting data reproducibility. Two technical solutions

[0009] The technical solution of this invention has been described in detail in the claims, and mainly includes eight steps: diffusion cell preparation, receiving medium preparation, membrane barrier installation, test sample loading, isothermal operation, timed sampling, HPLC determination, and data calculation. An additional step is included for reference formulation comparison. The core technical points of each step are as follows:

[0010] Step 1: Provide a Franz vertical diffusion cell, which uses a jacketed constant temperature design and precise temperature control through a circulating medium.

[0011] Step 2: The receiving medium consists of a weakly alkaline buffer solution containing lower alcohols (such as ethanol and isopropanol), free of surfactants, and degassed before injection. The key innovation lies in using lower alcohols instead of surfactants as solubilizers—lower alcohols can improve the solubility of mupirocin to meet the leakage conditions without causing destructive interactions with skin lipids.

[0012] Step 3: IVRT uses a hydrophilic microporous membrane (such as PES, mixed cellulose esters, etc.), while IVPT uses excised animal skin. Both membrane barriers share subsequent experimental conditions, reflecting the "integrated" design concept.

[0013] Step 4: The test sample is loaded using a quantitative extrusion device and standardized coating operation, taking into full account the rheological properties (viscosity, thixotropy) of the ointment to ensure that the test sample forms a uniform thin layer on the barrier surface.

[0014] Step 5: Under constant temperature conditions simulating skin surface temperature, run the system at an appropriate stirring rate to ensure the uniformity of drug concentration in the receiving chamber.

[0015] Steps 6-8: Timed sampling - HPLC analysis - data calculation. The sampling time points cover the entire process from rapid release to slow equilibrium. The chromatographic conditions are systematically optimized to ensure effective separation from the receiving medium components and potential degradation products. The key innovations of this invention are as follows:

[0016] (1) Integrated IVRT+IVPT Framework: The two experiments share the same batch of prepared receiving medium formulation, temperature control parameters, sample loading method, and chromatographic analysis method, differing only in the type of artificial membrane barrier (microporous filter membrane vs. ex vivo animal skin) and sampling time points. By sharing core experimental conditions, the development and validation of IVRT and IVPT methods are combined, significantly reducing duplication of work while ensuring consistency and comparability of the two sets of data. Compared with existing literature (Lu Lihua et al. conducted only IVRT in 2024, and Wang Song et al. conducted only IVPT in 2018), this invention integrates the two experiments into a unified system for the first time, achieving complete unification of receiving medium, chromatographic conditions, and operating procedures.

[0017] (2) Optimization of the dual response of alcohol-buffered receiving medium and TEWL without surfactants: Through systematic experimental screening, this invention uses only lower alcohols (such as ethanol) as solubilizers in the receiving medium, without adding any surfactants. Experiments have shown that at appropriate alcohol concentrations, the solubility of mupirocin can fully meet the leakage conditions, and the transepidermal water loss (TEWL) value of the skin does not change significantly, proving that the skin barrier integrity is effectively maintained. Unlike existing literature that selects ethanol concentration based solely on experience, this invention uses mupirocin solubility and skin TEWL value as dual response indicators, and determines the optimal ethanol concentration range that meets the leakage conditions and maintains the skin barrier integrity through response surface optimization, which is non-obvious.

[0018] (3) Test sample loading method considering rheological properties: The test sample is precisely quantified using a quantitative extrusion device, and the standardized coating tools and operation time are used to unify the operation method and effectively control the variation introduced by the coating operation. This loading method fully considers the thixotropy and viscoelasticity of the ointment and ensures the uniform distribution of the test sample on the membrane / skin barrier surface.

[0019] (4) Built-in reference preparation consistency evaluation: The f2 similarity factor comparison is directly embedded into the method process, and the in vitro consistency conclusion with Bactroban is automatically output for each test. The data can be directly used for application materials. Three beneficial effects

[0020] 1. The integrated design combines the core steps of IVRT and IVPT, such as method development, receiving medium preparation, and chromatographic analysis. Compared with the independent experimental mode in existing literature (Lu Lihua et al. in 2024 only conducted IVRT, and Wang Song et al. in 2018 only conducted IVPT), ​​the workload of method development and validation is reduced by about 40% to 50%, which significantly improves the iterative efficiency of prescription screening and process optimization, while ensuring the consistency and comparability of the two sets of data.

[0021] 2. The alcohol-buffered receiving medium was optimized using TEWL dual-response technology to determine the ethanol concentration range. This approach satisfied the leakage conditions while avoiding surfactant-induced skin barrier damage, thus ensuring the physiological relevance of IVPT data. Unlike Wang Song et al.'s empirical selection of 30% ethanol in 2018, this invention used a response surface methodology to determine 25%–35% as the optimal range balancing solubility and skin barrier protection. This optimization strategy exhibits non-obviousness.

[0022] 3. The standardized test sample loading method fully considers the rheological properties of ointment, and the IVRT and IVPT data have good reproducibility (intra-batch RSD <10%, inter-batch RSD <15%).

[0023] 4. The built-in f2 comparison function allows generic drug prescription screening data to be directly used for consistency evaluation applications.

[0024] 5. The method has undergone complete methodological validation (specificity, linearity, precision, accuracy, and stability) and meets the requirements of the current Chinese Pharmacopoeia and CDE guidelines. Attached Figure Description

[0025] Figure 1 This is an overall flowchart of the method of the present invention.

[0026] Figure 2 This is a schematic diagram of the improved Franz vertical diffusion cell used in this invention.

[0027] Figure 3 This is a comparison of the in vitro cumulative release curves of the test sample and the reference preparation in Example 1 (n=6, mean±SD).

[0028] Figure 4 This is a comparison of the in vitro cumulative transdermal curves of the test sample and the reference preparation in Example 1 (n=6, mean±SD).

[0029] Figure 5 The graph shows the effect of the volume fraction of ethanol in the receiving medium on the solubility of mupirocin and the skin TEWL value. Detailed Implementation Example: Comparison of test sample (prepared by Pacific Pharmaceuticals) and reference preparation (Bactroban)

[0030] 1. Instruments and Materials

[0031] Franz vertical diffusion cell (effective diffusion area approximately 1.8 cm², receiving chamber volume approximately 12 mL, equipped with a constant temperature circulating water bath jacket); high performance liquid chromatograph (equipped with a UV detector); C18 column (4.6 mm × 250 mm, 5 μm); hydrophilic microporous filter membrane (pore size approximately 0.45 μm); excised abdominal skin from SD rats (male, weight 200 ± 20 g); mupirocin reference standard (purity >= 98.0%); test sample: mupirocin ointment (2% specification, 15 g / tube); reference preparation: marketed original mupirocin ointment (2% specification, 15 g / tube).

[0032] 2. Receiver medium configuration

[0033] Prepare a phosphate buffer solution with an appropriate molar concentration using ammonium dihydrogen phosphate as the buffer salt, adjust the pH to weakly alkaline, add an appropriate amount of anhydrous ethanol to make the ethanol volume fraction in the range of 25% to 35% (about 30% in this example), mix evenly, and then degas the solution by ultrasonication (about 20 minutes in this example) to obtain the receiving medium.

[0034] 3. HPLC Chromatographic Conditions

[0035] The chromatographic column was an octadecylsilane-bonded silica column (C18, 4.6 mm × 250 mm, 5 μm); the mobile phase was a mixture of ammonium dihydrogen phosphate buffer and acetonitrile, with a volume ratio of approximately 65:35; the detection wavelength was approximately 229 nm; the flow rate was approximately 1.0 mL / min; the column temperature was approximately 30°C; and the injection volume was approximately 20 μL. Under these chromatographic conditions, the mupirocin main peak was well separated from the receiving medium components and potential degradation products.

[0036] 4. IVRT Operation Procedures

[0037] 1. Cut the microporous membrane into appropriately sized circular pieces and thoroughly wet them in the receiving medium. 2. Install the wetted membrane between the supply and receiving chambers, ensuring the membrane surface is flat and free of air bubbles. 3. Inject the degassed receiving medium into the receiving chamber to remove air bubbles. 4. Connect the thermostat and equilibrate at approximately 32°C (or other temperatures within the range of 30–35°C), stirring magnetically at an appropriate rate (e.g., approximately 300 rpm). 5. Weigh an appropriate amount of the test sample (e.g., approximately 100 mg) and transfer it to the membrane surface using a quantitative extrusion device. Coat the sample evenly with a coating tool in a circular motion for an appropriate time. 6. At preset sampling time points, take an appropriate amount of the receiving liquid (e.g., 1.0 mL) and simultaneously add an equal volume of fresh receiving medium at the same temperature. 7. Filter the sample through the microporous membrane and analyze it by HPLC. 8. Calculate the cumulative release amount and release rate per unit area at each time point, plot the cumulative release curve, and perform six replicates for each test sample.

[0038] 5. IVPT Operation Procedures

[0039] 1. Rat abdominal skin was harvested, subcutaneous adipose tissue was removed, and the skin was rinsed several times with physiological saline. It was then preserved at low temperature by immersion in physiological saline containing an appropriate amount of antibiotics. Before use, the skin was fully equilibrated with buffer solution, and the skin barrier integrity was confirmed by TEWL testing. 2. The skin was placed in the diffusion cell with the stratum corneum facing upwards. 3. Subsequent procedures for receiving medium injection, temperature equilibration, and sample loading were the same as for IVRT. 4. Samples were taken at a predetermined series of time points (IVPT sampling time points typically cover a longer time range, such as 1–24 h). Steps 5–7 were the same as for IVRT. 8. The cumulative transdermal volume per unit area and steady-state transdermal rate were calculated at each time point. Six replicates were performed (n=6).

[0040] 6. Test Results

[0041] IVRT results showed that the in vitro cumulative release curve of the test sample (prepared by Pacific Pharmaceutical) largely overlapped with that of the reference formulation (Bactroban), and there was no statistically significant difference in the cumulative release at each sampling time point. IVPT results showed that the in vitro cumulative transdermal curve of the test sample also largely overlapped with that of the reference formulation. Based on the f2 similarity factor calculation, the f2 value for IVRT was between 65 and 80, and the f2 value for IVPT was between 60 and 75, both greater than 50. Conclusion: The IVRT and IVPT behaviors of the test sample and the reference formulation are consistent, and the f2 values ​​of all indicators are greater than 50, meeting the requirements for consistency in generic drug quality.

[0042] 7. Methodological Validation Results

[0043] The method of this invention has undergone complete methodological validation: it exhibits good specificity (the components of the receiving medium and potential degradation products do not interfere with the determination of mupirocin); mupirocin shows good linearity within the investigated concentration range (r>0.999); intra-batch precision RSD <3%, inter-batch precision RSD <5%; recovery rate is in the range of 95%–105%; the test solution is stable within 24 hours under the experimental conditions. All validation indicators meet the requirements of the current Chinese Pharmacopoeia and CDE guidelines.

[0044] Example 2: Optimization screening of lower alcohol volume fraction in receiving medium

[0045] Taking ethanol as an example, a series of receiving media with ethanol volume fractions ranging from 0% to 50% were prepared. The equilibrium solubility of mupirocin was determined at the experimental temperature, and the TEWL value (reflecting skin barrier integrity) of isolated mouse skin after treatment with each medium was measured. The results are as follows:

[0046] Considering both leakage conditions and skin barrier integrity, an ethanol volume fraction of 20%–40% was determined to meet the experimental requirements, with 25%–35% being the preferred range for achieving both adequate leakage conditions and skin barrier protection. Within this preferred range, the solubility is approximately 5–10 times the expected highest concentration of the IVPT test receiving solution, and the TEWL value does not increase significantly. Notably, when the ethanol volume fraction rises to approximately 40% or higher, the TEWL begins to increase significantly, indicating that skin barrier integrity is compromised; therefore, in IVPT tests where skin barrier protection is prioritized, the ethanol volume fraction should ideally be controlled below 35%. The determination of this concentration range is not a simple application of the common knowledge of "adding organic solvents to increase solubility," but rather a response surface optimization result using solubility and TEWL as dual response indicators, demonstrating the non-obviousness of the present invention.

[0047] Example 3: Method Reproducibility Verification

[0048] IVRT experiments were conducted by different operators on different days using different batches of materials and reagents, following the method in Example 1 (n=3 per operator). The results showed that the cumulative release curves measured by each operator, when compared pairwise with f2 values, all had f2 values ​​greater than 65 (>50). The batch-to-batch RSD and day-to-day RSD were both controlled within 15%, and the operator-to-operator RSD was within 10%, demonstrating that the method has good reproducibility and robustness.

[0049] Example 4: Comparison with existing technical methods

[0050] The IVRT test was performed on the same batch of mupirocin ointment samples using the method disclosed in CN119915978A (using a receiving medium containing surfactant). At the same time, independent tests were conducted using the IVRT method (PES membrane + pH7.4 PBS, ethanol-free) of Lu Lihua et al. (2024) and the IVPT method (30% ethanol buffer, Bama fragrant pig skin) of Wang Song et al. (2018), and compared with the method of this invention (using an alcohol-buffered buffer receiving medium without surfactant, IVRT+IVPT integrated). The results show that the IVRT data obtained by the method of this invention are consistent with the data obtained by the method of CN119915978A and the method of Lu Lihua et al. However, the IVRT and IVPT data of the method of this invention have better inter-experimental consistency and data linkage because they share the same batch of receiving medium and chromatographic conditions. The method of CN119915978A uses a receiving medium containing surfactants, which leads to a significant increase in the skin TEWL value in the subsequent IVPT test (barrier damage), and it is impossible to complete the IVPT evaluation under the same receiving medium system. In contrast, the method of this invention uses an alcohol-buffered buffer receiving medium without surfactants, and the skin TEWL value does not change significantly after IVRT, which can be seamlessly connected to IVPT evaluation, fully demonstrating the advantages of integrated design. Industrial applicability

[0051] The method of this invention has been successfully applied to the formulation screening, process optimization, and quality consistency evaluation of generic mupirocin ointment (2% specification, 15g / tube). All parameters of the method have been systematically validated and can serve as the standard operating procedure for IVRT and IVPT studies in the application materials for generic mupirocin ointment. The "integrated" design concept and receiving medium screening scheme of this method can also provide technical reference for the development of in vitro evaluation methods for other semi-solid dosage forms (especially non-aqueous base ointments).

Claims

1. A method for integrated in vitro release and transdermal testing of mupirocin ointment, characterized in that, Includes the following steps: (1) Provide a Franz vertical diffusion cell with an effective diffusion area of ​​0.5 to 5.0 cm2 and a receiving chamber volume of 5 to 25 mL; (2) Inject a receiving medium into the receiving chamber, the receiving medium being composed of a pH 5.0 to 8.0 buffer solution containing 10% to 50% C1 to C3 lower alcohols by volume, without surfactants, and the molar concentration of the buffer solution being 0.005 to 0.2 mol / L; (3) An artificial membrane barrier is installed between the supply chamber and the receiving chamber, wherein the in vitro release test (IVRT) uses a hydrophilic microporous filter membrane with a pore size of 0.20 to 1.0 μm, and the in vitro transdermal test (IVPT) uses detached abdominal skin of mammals that has been treated with hair removal. (4) Add 20-500 mg of mupirocin ointment to the supply chamber, transfer the sample to the surface of the artificial membrane barrier through a quantitative extrusion device, and then coat it evenly with a coating tool for 10-60 seconds. (5) Place the diffusion tank in a constant temperature device, maintain the temperature of the receiving chamber at 25-40°C, and stir at a rate of 50-600 rpm; (6) At the preset sampling time point, take out 0.5 to 5.0 mL of receiving liquid from the sampling port of the receiving chamber, and at the same time add an equal volume of fresh receiving medium at the same temperature; (7) After filtering the sample through a microporous membrane with a pore size of 0.20 to 0.80 μm, the concentration of mupirocin in the receiving liquid was determined by high performance liquid chromatography. (8) Calculate the cumulative release rate (IVRT) and cumulative transdermal dose per unit area (IVPT) of mupirocin at each sampling time point based on the measurement results. The in vitro release test and the in vitro transdermal test share the same batch of prepared receiving medium, the same temperature control parameters, the same sample loading method, and the same high-performance liquid chromatography analysis conditions. They differ only in the type of artificial membrane barrier and the sampling time points, thus forming an integrated test system. The composition of the receiving medium was determined through response surface optimization using the volume fraction of lower alcohols and the transdermal water loss (TEWL) value as dual response indicators, so that the receiving medium maintains the integrity of the skin barrier while satisfying the mupirocin leak condition.

2. The method according to claim 1, characterized in that, In step (2), the lower alcohol is ethanol, the buffer solution is phosphate buffer, and the receiving medium is composed of phosphate buffer with pH 6.5 to 7.8 containing 20% ​​to 45% ethanol by volume, and the molar concentration of the phosphate buffer is 0.01 to 0.1 mol / L; in step (5), the temperature of the receiving chamber is 30 to 37°C, and the stirring rate is 200 to 500 rpm.

3. The method according to claim 2, characterized in that, The receiving medium in step (2) consists of a phosphate buffer solution with a pH of 7.2 to 7.6 containing 25% to 35% ethanol by volume; the temperature of the receiving chamber in step (5) is 31 to 33°C and the stirring rate is 250 to 400 rpm; the effective diffusion area of ​​the diffusion cell in step (1) is 1.0 to 3.0 cm² and the volume of the receiving chamber is 8 to 18 mL.

4. The method according to claim 3, characterized in that, The preset sampling time points mentioned in step (6) are: IVRT sets the first sampling point within 0.25 to 1 hour, and sampling continues until 8 to 24 hours, for a total of 5 to 10 sampling time points; IVPT sets the first sampling point within 0.5 to 2 hours, and sampling continues until 12 to 36 hours, for a total of 5 to 12 sampling time points.

5. The method according to claim 3, characterized in that, The chromatographic conditions for the high performance liquid chromatography method described in step (7) are as follows: the chromatographic column is an octadecylsilane-bonded silica column; the mobile phase is a mixture of ammonium dihydrogen phosphate buffer and acetonitrile, with a volume ratio of 55:45 to 75:25; the detection wavelength is 220 to 240 nm; the flow rate is 0.5 to 2.0 mL / min; the column temperature is 20 to 40 °C; and the injection volume is 5 to 100 μL.

6. The method according to claim 1, characterized in that, The receiving medium described in step (2) needs to be ultrasonically degassed for 5 to 30 minutes before being injected into the receiving chamber.

7. The method according to claim 1, characterized in that, The isolated mammalian abdominal skin mentioned in step (3) is isolated SD rat abdominal skin. The processing method is as follows: take the abdominal skin of SD rat, remove the subcutaneous fat tissue, rinse repeatedly with physiological saline, soak in physiological saline containing antibiotics for low temperature preservation, balance with buffer for 15-60 minutes before use, and confirm the integrity of the skin barrier by transepidermal water loss (TEWL) measurement.

8. The method according to claim 1, characterized in that, The method further includes setting up a reference preparation control group in each test batch. The reference preparation is the marketed original mupirocin ointment (trade name: Bactroban). The similarity factor f2 is compared with the IVRT curve and IVPT curve of the reference preparation to evaluate the consistency of the in vitro behavior of the test sample with that of the reference preparation. When the f2 value is ≥50, the in vitro behavior of the test sample is determined to be consistent with that of the reference preparation.

9. The method according to claim 1, characterized in that, The method is used for prescription screening, process optimization, and quality consistency evaluation of generic mupirocin ointment.

10. The method according to any one of claims 1 to 9, characterized in that, The composition of the receiving medium and the operating parameters in the method are the result of optimization specifically for the polyethylene glycol matrix system of mupirocin ointment. The optimization includes: performing response surface optimization with the volume fraction of lower alcohols and the skin TEWL value as dual response indicators to determine the parameter combination that satisfies the leakage conditions and maintains the integrity of the skin barrier.

11. A mupirocin ointment in vitro release and in vitro transdermal evaluation system, characterized in that, include: —Franz vertical diffusion cell device, with an effective diffusion area of ​​0.5–5.0 cm², a receiving chamber volume of 5–25 mL, and equipped with a constant temperature device; —The first artificial membrane barrier kit, consisting of a hydrophilic microporous filter membrane with a pore size of 0.20–1.0 μm, is used for IVRT evaluation; —The second biofilm barrier kit, consisting of hair-removed and TEWL-qualified ex vivo mammalian abdominal skin, is used for IVPT evaluation; —Sample loading kit, including metering extrusion device and coating tool; —The receiving medium consists of a pH 5.0–8.0 buffer solution containing 10%–50% C1–C3 lower alcohols by volume, and contains no surfactants; —High-performance liquid chromatography detection system for determining the concentration of mupirocin in the receiving solution.

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