Comparative in vitro Release Test Using Franz Diffusion Cell for Luliconazole % 1 Cream with Reference Drug Product

Sep 10 2021

Author: Didem Cetin, Turkan Gunes Turker, Nihal Yilmaz, Basak Acar Karakoy and Muhammad Safadı on behalf of Bilim Pharmaceuticals

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Skin is a valuable route for administration of drugs. However, the challenge for topical applications is to deliver a given dose to the patient while the skin prevents the absorption of foreign matter. Therefore, drug effectiveness varies according to the active ingredient release from the vehicle, and the percutaneous absorption by the skin after administration. To this end, analysing the rate of active pharmaceutical ingredient release from transdermal drug administration has become a critical performance data for semisolid preparations. Despite being in the literature over the years, Franz Cell Diffusion testing for cream products recently evolved into a mostly used tool for in vitro release testing (IVRT). In this paper, IVRT was performed on Bilconazol 1% cream (Bilim Pharmaceuticals, Turkey) that contains the active ingredient Luliconazole, using the Teledyne Hanson’s Phoenix manual diffusion testing system. The drug release rate was observed over 6 hours with PBS:Ethanol receptor medium at 32ºC with 600 rpm stir bar speed. Following the IVRT, a HPLC method was used for the analysis of samples. Statistical comparisons were made between the reference product according to the EMA guidelines. The results were confirmed that there is no statistically significant difference between the test and the reference product. Validation studies also performed to evaluate the influence of the different parameters of the Franz diffusion cells.

The global market for topical products is estimated to reach $129.8 billion by 2025, and in 2019, it had the highest market share of the application routes [1]. Although overlooked in favour of oral and injectable routes, semi-solid dosage forms such as ointments, creams, gels, pastes and emulsions have unique advantages such as avoiding metabolic transition, patient compliance, ease of use and reduction of side effects caused by external administration [2, 3]. Due to the challenges in the controlled release and dosage accuracy of the active compounds, achieving their unique composition and requirements in the formulation of semisolid dosage forms can be complicated. Thus making pharmaceutical companies focus more on transdermal delivery systems.
Semi-solid dosage forms require comprehensive demonstration of their extended release in controlled clinical trials.  However, the duration and cost of these trials make them unsuitable as routine control methods [4, 5]. Therefore, in vitro testing is often used to assure performance in the laboratory and check the feasibility of formulation.
In vitro release testing (IVRT) enables obtaining consistent delivery data of the active component from semisolid products. The European Medicines Agency (EMA) guideline on quality and equivalence of topical products [6] and United States Pharmacopeia (USP) Semisolid Drug Products - Performance Tests [7] provide procedures for determining drug release from semisolid dosage forms. However, the complex nature of skin, changes in formulation and the application process significantly affect the release rate, and guides cannot present a single procedure to address such diversity. The general guidelines do not provide a product-specific protocol to demonstrate equivalence.
This article aims to present IVRT research conducted for Bilconazol 1% cream by using Teledyne Hanson’s Phoenix Manual diffusion testing system (Figure 1). Adequate drug transport from the sample through a membrane was achieved with no rate limiting effect on release. Critical parameters were evaluated, and the developed IVRT method was validated with high-performance liquid chromatography (HPLC). A successful IVRT comparison was made between the test product and the reference product through statistical analysis to justify the parameters discussed in the EMA guideline [6]. Although such validation studies already exist, this study could shed light on various new applications.

Figure 1. Teledyne Hanson’s Phoenix Manual diffusion testing system.

Materials and Method

2.1 Drug Products
Bilconazol cream 1%, placebo and differently formulated creams containing 0.5% and 2% concentrations manufactured in Bilim Pharmaceuticals (Gebze, Turkey) Luzu Cream 1% (8116357, Valeant, USA) used as a reference product for the method validation.
    Figure 2. Luliconazole molecule [8].
2.2. IVRT system
IVRT studies employed the Phoenix DB - 6 dry heat diffusion system (59-201-101, Hanson Research Cooperation, USA), which meets USP specifications [7]. Commercially available cellulose ester membrane (0.45µM, 10401606, A29437704, GE Healthcare) were studied as a modal synthetic membrane for drug diffusion. The receptor medium chosen was phosphate buffer (pH 7.4) containing 50% (v/v) ethanol. The system was maintained at 32 ± 0.5°C with a rotational stirring speed of 600 rpm throughout the experiment. Approximately 400 mg of semi-solid product was placed on the membranes and the test was started. Samples were then taken from the receptor compartment of the cell using a syringe at one-hour intervals over 6 hours. The withdrawn volume was adjusted to the fill mark on the sampling arm with the warmed stock receptor medium. A set of 6 cells were operated together in a single run and for each run, two products compared to the ABABAB form with an alternative pattern for unbiased comparison [9].
2.3 IVRT Calculation Method
For each cell, the withdrawn Luliconazole samples were analysed by HPLC-UV (e2695, Alliance, Waters Corporation) at 277 nm wavelength. Mobile phases were used in isocratic conditions at 25°C sample tray temperature and 30°C column temperature at a ratio of 10:90 with an injection volume of 10 µL. Mobile Phase A was prepared with 0.79 g Ammonium Acetate in 1000 mL distilled water and was filtered through a 0.45µM filter and was degassed. Acetonitrile was degassed and used as Mobile Phase B. Luliconazole was eluted from a Symmetry C18 column (250x4.6 mm, 5 µm column, Waters, USA) at a flow rate of 1.5 mL/min. Concentration values obtained as mg/mL were converted to mg/cm2, taking into account the cell volume, correction factor and effective diffusion area. For each cell, the amount of drug released at each sampling time was determined and the cumulative amount released was plotted against √t according to Higuchi Eq (1) (Higuchi, 1961).
Q=k×√t  (1)
In Eq. (1), Q (mg/cm2) is the amount of drug released per unit area, t (hour) is the time and k is the slope, which is a measure of the rate of drug release. The average of the 6 slopes for each run is a measure of the drug release rate for the dosage form.
2.4 Statistical analysis
IVRT method was qualified based on the approach from EMA guideline [6] in 6 runs. For the product similarity tests, statistical analyses were carried out using ANOVA and student t-test with the significance level of p<0.10. Twelve samples of test and reference product were used to demonstrate equivalence as specified [6]. Another parameter for acceptance was the linearity of the cumulative amount of active substance released versus the square root of the time plot.
2.5 Validation parameters
The HPLC-UV method validation parameters for Luliconazole 1% cream included specificity, selectivity, linearity, range, accuracy, precision limit of qualification, robustness, stability and system suitability. System suitability tests were also performed when the relative standard deviation of the 100% standard solution exceeded 2.0% or the European Pharmacopoeia (EP) plate count was below 2000.
2.5.1. Specificity
Possible interfering peaks that may be caused by the receptor medium, membrane, or placebo was investigated by conducting an IVRT study with blank receptor medium and placebo cream. In each run, medium and placebo must be free of interference at the retention times of the luliconazole.
2.5.2. Selectivity
To determine the luliconazole release rates, different concentration formulations were evaluated according to their release rates. The release rate as a function of drug concentration was plotted to accurately observe the proportionality of changes and was reported as the linearity (R2 ≥0.90).
2.5.3. Linearity
Drug assay standard solutions with six different concentrations between 150% of working concentration and LOQ were prepared. The calculated R2 value for the linear regression analysis was checked to be greater than 0.995.
2.5.4. Accuracy
For the accuracy of the IVRT samples, placebo cream was spiked with luliconazole concentrations of LOQ, 50%, 100% and 200% in triplicate. The recovery should be within 95% and 105%. For the LOQ, results should not deviate more than ±20%. Additionally, RSD should be below 5%.
To test the precision of the method, the variation among slopes were calculated and checked whether the RSD across inter six runs was less than 2%. Then repeatability was studied with test and the reference product’s release rates and compared within 90% confidence level. Finally, for intermediate precision, the RSD of the release rates of two different runs from the same batch was carried out by different operators on various days, ensuring that it did not exceed 10.0%.
2.5.6. Robustness
For both the chromatography system and the IVRT system, different parameters were tested by running an IVRT test with minor perturbations in the method parameters. Two column temperature variation (27ºC and 33ºC) and two mobile phase ratios (Mobile Phase A: B ratio as 5:95 and 15:85) were tested for the chromatography system changes. Also mixing rate changes (540 rpm and 660 rpm) and receptor temperature variations (30ºC and 34ºC) were performed for the IVRT system alterations. The results were expected not to deviate more than 15%.

Results & Discussion

Two formulations were investigated for their release characteristics and differences in between. As the first step, the analytical HPLC-UV method for luliconazole was established. The membrane selected was mixed cellulose; for commercial availability and its inertness and for enabling high permeability with the least possible diffusional resistance, since it is critical that the membrane has no physical or chemical interaction with the formulation that could cause affect the IVRT results [10].
One of the key parameters was ensuring that no bubbles were introduced into the cell. Therefore, it was essential to degas the medium before starting the experiment. Also, the semi-solid product was spread onto the membrane throughout the room to eliminate diffusion-based variations, since the membrane was assigned to keep the product separate from the receptor medium while enabling a diffusive communication between them.
System suitability parameters were checked for the EP plate count and the relative standard deviation of %100 standard solutions. The results were found as 7128 with 0.28%RSD.
3.1 Validation Parameters
3.1.1. Specificity
Possible interfering peaks were investigated by conducting two separate IVRT study with a blank membrane and with the placebo. The peak purity was examined at the detected retention time region and passed the test.
3.1.2. Selectivity
Three different concentration of Luliconazole cream’s IVRT results were evaluated, and their release rates was plotted against drug concentration. The linearity found 0.918, which meets the EMA specifications [6] (Figure 4).

3.1.3. Linearity
Drug assay standard solutions with 6 different concentrations of 20%, 50%, 70%, 100%,120% of working concentration and LOQ were prepared. The calculated R2 value for the linear regression analysis was found be 0.999 which was greater than 0.995 (Figure 5).

Figure 5. Linear regression curve of 6 different concentrations.
3.1.4. Accuracy
Accuracy analyses were performed in triplicate with volumetric flasks by adding the placebo of the luliconazole cream and diluting it with the medium. Then, 50%, 100%, 200% and LOQ percentage of the working concentration was added into flasks. Except for LOQ concentration, the RSD was observed below 1%. For the LOQ, the average was found 4.9%, which was within the acceptance criteria.
3.1.5. Precision
To observe the variation between HPLC injections, six successive runs were analysed, and the RSD found as 0.23%, which was within acceptance criteria. Repeatability was studied using the test and reference product’s release rates. Student t-test and the ANAVO statistical analyses used for product similarity tests. The results in a 90% confidence interval fell within limits specified in EMA guideline [6], hence passed the test (Table 1).

Table 1. Results of release experiments for reference and test product.
Results    Reference    Test
R2    0.974    0.998
k (µg/cm2/h0.5)    423.67    377.07
t-test (α=0,10)    0.17    
ANOVA (α=0,10)    0.52    

Also, the IVRT results of two different runs that belongs to same batch were statistically analysed according to their release rates found identical, which proves that there is no variation between operators (RSD, 0.0001%).
3.1.6. Robustness
For the chromatography system changes, column temperature variations were tested with 27ºC and 33ºC. It was observed that 33ºC had 377.65 (µg/cm2/h0.5) k value with 0.15% increase and the 27ºC had 376.23 (µg/cm2/h0.5) k value with 0.22% RSD decrease. Mobile phase ratio was another parameter for the chromatography system change. Mobile Phase A: B ratio changed to 5:95 and 15:85. In both situations, the k value dropped 1.6% and 0.39% respectively when compared with the 377.03 (µg/cm2/h0.5) k value.
For the IVRT system changes, mixing rate firstly was tried at 540 rpm and 660 rpm. Variation for these changes were 6.25% and 5.92%, respectively and again were below the 15% deviation ratio. A 48.2% higher release rate was observed in the receptor with a 2ºC higher temperature, and a 52.2% decrease in release rate was observed for the 2ºC cooler receptor (Table 2). These results prove that IVRT mixing rate, column temperature and mobile phase changes do not affect the quantity of the drug, although the IVRT temperature condition has a significant effect on the release rate, and it should be considered as a critical parameter [11].

Table 2. Calculated IVRT results (R2, release rate and % difference) on different parameters and conditions.
System    Parameter    Condition    R2    k    %
HPLC    Column temperature
& Mobile Phase ratios    27ºC, 10:90    0.998    376.23    0.22
        33ºC, 10:90    0.998    377.65    0.15
        30ºC, 85:15    0.998    370.99    1.60
        30ºC, 5:95    0.998    375.58    0.39
IVRT    Receptor Temperature
& Mixing rate    30ºC, 600rpm    0.996    180.34    52.2
        34ºC, 600rpm    0.994    558.77    48.2
        32ºC, 540rpm    0.999    353.51    6.25
        32ºC, 660pm    0.999    399.41    5.92

4. Conclusion

This report illustrates a comprehensive study of the validation parameters of the IVRT method for the Luliconazole 1% cream. IVRT was used as a method for product similarity comparison between reference and the test products. Appropriate inert and commercially available cellulose ester membrane and PBS: Ethanol receptor medium at 32ºC with 600 rpm stir bar speed were used with the diffusion cell system. Diffusion of the luliconazole across the membrane was analysed hourly for drug content by high-pressure liquid chromatography. Possible influences on the IVRT system were determined and validation parameters determined. Limitations and the acceptance criteria were developed according to the EMA guideline [6].
It was found that the critical parameter for IVRT studies is the temperature of the receptor environment. The temperature of the area of use should be taken into account and any possible change should be monitored so that the release rate does not cause a high effect. The method discussed in this paper can be used in other relevant IVRT analyses for further studies.


This work was supported by the R&D Department, Analytical Method Development Laboratory under the Directorate of Scientific Division of Bilim Pharmaceuticals.
Declaration of Competing Interest
There are no conflicts to declare.


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