Flurbiprofen cataplasms: Development and validation of in-vitro dissolution methods and evaluation of multimedia dissolution profiles

Abstract

The investigation of the systemic release performance of dosage forms using in vitro tools is a crucial objective in the realm of pharmaceutical development. The dissolution methodology is an effective tool for monitoring batch-to-batch variabilities in quality control and gaining insight into the release mechanisms of pharmaceutical drugs. The majority of the dissolution techniques that have been approved are primarily intended for solid oral dosage forms. Nevertheless, these techniques have also been applied to various other dosage forms, including transdermal drug delivery systems. The administration of medication through cataplasm, a transdermal application, poses challenges in understanding the characteristics of drug release. Flurbiprofen is classified as a class II drug according to the Biopharmaceutics Classification System and is commonly administered in the form of a cataplasm for its analgesic properties. A dissolution method was developed to assess the in vitro release profile of the flurbiprofen transdermal delivery system. This method utilized a United States Pharmacopeia dissolution apparatus V, with a disc assembled over the paddle. Additionally, a method for quantification was developed using liquid chromatography. The discriminatory aspect of the developed method has faced criticism due to substantial alterations in excipient composition. Furthermore, we have developed clearly defined multimedia release profiles within the physiological pH range. The validation of the dissolution and chromatography systems was conducted.

Highlights

• •In-vitro and in-vivo are crucial goals in pharmaceutical development.
• •Cataplasm is a transdermal application in which comprehension of drug release characteristics is difficult.
• •The discriminatory nature of the developed method has been challenged with significant excipient composition changes.

Abbreviations

TDDS

Transdermal drug delivery Systems

GIT

Gastrointestinal Track

USP

United States pharmacopeia

ICH

The international council for harmonization of technical requirements for Pharmaceuticals for human use

HPLC

High-performance Chromatography

1. Introduction

A transdermal delivery system (TDDS) is a painless way to administer medicine systemically by simply placing the drug formulation on the skin. The medication enters the skin through the stratum corneum, and then travels across the epidermis and dermis without building up in the dermis. The skin's microcirculation is able to transport drugs throughout the body once they have made it to the dermal layer. TDDS provides several benefits over more traditional medication delivery methods. It can be used as a non-invasive substitute for parenteral methods, helping those who suffer from needle phobia and similar conditions. There are a variety of potential sites for transdermal absorption on the skin due to its enormous surface area and convenience of access [[1], [2], [3], [4], [5], [6]]. Ointments, creams, large adhesive patches, plasters, poultices, and cataplasms are all classic topical formulations [7]. Simple, low-dose molecules and active chemicals are what really make up first-generation TDDS, which are released locally or topically. The TDDS of the second generation is the one that allows you to control the rate at which the dose is released. The third-generation TDDS is an extended-release pharmaceutical active delivery system that utilizes multiple skin-layer adsorptions. In the quest to find effective treatments for hormone diseases, rate-limiting, and long-acting TDDS have been shown to be invaluable [8].

The very low solubility of pharmaceuticals in the Biopharmaceutical Classification System (BCS), classes II and IV has led to a wide range of literature discussing the difficulties of drug product development for these compounds. The pharmaceutical compounds belonging to class II and IV exhibit inferior solubility and permeability in comparison to those in class I and III. Consequently, the primary challenge lies in developing appropriate dosage forms that can be administered orally or intravenously [[9], [10], [11]]. In order to mitigate the negative impact on the gastrointestinal tract (GIT) in terms of changes in absorption and distribution that can result in damage or necrosis of GIT epithelial cells, transdermal dosage forms have emerged as a promising solution to address GIT disruptions. The establishment of the quality, quantity, and efficacy of drug products necessitates the implementation of impeccable analytical procedures. The dissolution test is considered a crucial parameter for evaluating the performance of a product within the realm of quality testing protocols. A bio-simulation comparison tool, also known as an in-vitro correlation tool, is a valuable resource for predicting the behavior of a product in vivo [[12], [13], [14], [15], [16], [17], [18], [19]].

Flurbiprofen exhibits analgesic, anti-inflammatory, and antipyretic characteristics akin to those observed in non-steroidal anti-inflammatory drugs (NSAIDs). The compound in question is a derivative of propionic acid and falls within the category of phenyl alkanoic acid non-steroidal anti-inflammatory drugs (NSAIDs). This intervention is employed for the purpose of mitigating moderate pain and managing symptoms associated with chronic arthritis [[20], [21], [22]]. In the context of treating knee osteoarthritis, a transdermal therapeutic system (TDDS) formulation of flurbiprofen is offered as a plaster or cataplasm. This particular form is designed to address gastrointestinal complications associated with alternative dosage forms. Based on the available literature, it has been established that flurbiprofen falls under the BCS Class II category, exhibiting limited solubility and significant permeability [[23], [24], [25]]. Fig. 1 displays the chemical structure of flurbiprofen. A cataplasm refers to a malleable substance composed of powders or absorbent materials that are moistened with either oily or aqueous fluids. It is often medicated and applied to the skin while in a heated state. Cataplasms are known to have emollient, relaxing, stimulant, or counterirritant properties on the skin and underlying tissues. Hydrogel patches, also referred to as cataplasms, are alternative designations for this particular medical application. Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently employed as topical applications to alleviate localized pain, particularly within the context of Japan [26].

Fig. 1.

Chemical structure of flurbiprofen.

The classification of drug substances as BCS Class 2 poses a challenge in the development of an appropriate dissolution method that can effectively discern changes in formulations. In order to develop a generic drug, having a control release dissolution method and the establishment of multimedia dissolution correlations are major aspects of simulating the test product with a reference-listed drug. With the aim of drug formulation development, this study seeks to establish a quality control laboratory dissolution method and optimize the in vitro multi-media and quality control dissolution release profiles across the physiological pH range from 1.2 to 7.4.

The primary objective of this research is to develop a methodology that enables quality control laboratories to assess the dissolution profile and dissolution profiles in multimedia of flurbiprofen in cataplasms. This will be achieved through the utilization of a highly accurate High Performance Liquid Chromatography (HPLC) technique, which allows for the precise quantification of the released drug. The topic of discussion in the United States Pharmacopeia (USP) general chapter <724> Drug Release pertains to the dissolution equipment specifically designed for transdermal systems [27]. While the USP recommends the use of a conventional paddle over disc method for transdermal applications, the suitability of any conventional instrument is contingent upon various method-set parameters. These parameters include the type of dissolution media, rotations per minute, sample operation temperature, sample concentrations, and quantification method, among others. The comprehensive investigation of specific products, taking into account their physico-chemical properties, is essential for the development of general monographed methods. In addition, it should be noted that USP monograph methods were available for the dissolution testing of flurbiprofen tablets and flurbiprofen sodium ophthalmic solutions, but no similar methods were provided for transdermal drug products [28,29]. Multiple testing protocols were recorded to assess the effectiveness of flurbiprofen. However, a dearth of publicly accessible literature exists pertaining to the dissolution profiles and characteristics of flurbiprofen cataplasm in physiological solutions. This represents a novel area of study that has not yet been documented in published works [[30], [31], [32], [33], [34]]. The results of these studies demonstrate that they provide a basis for the development of a drug product that exhibits in vitro equivalence to the reference listed drug.

2. Materials and methods

2.1. Chemicals and reagents

The research study utilized a glass apparatus of Grade-A quality, specifically manufactured by Make-Borosil in China. The apparatus was classified as Grade-Class A. The chemicals and solvents used in this study were as follows: Glacial acetic acid (Analytical Reagent Grade, Guangzhou Chemicals, China), sodium hydroxide (NaOH) and hydrochloric acid (HCl) (Analytical Reagent Grade, Sigma Aldrich, USA), sodium acetate trihydrate (Reagent Grade, Aladdin, China), potassium dihydrogen phosphate (Analytical Reagent Grade, Aladdin, China), di sodium hydrogen phosphate (Analytical Reagent grade, Aladdin, China), ultra-pure grade water produced from a Milli-Q water purification system, methanol (HPLC grade, Merck, China), Tetrahydrofuran (HLE Grade, Merck, China) and Acetonitrile (HPLC grade, Merck, China). The flurbiprofen reference standard was procured from Supplier-USP, located in the United States. Additionally, Cataplasm Zepolas was obtained from Jinan Jianfeng Chemical Co., Ltd., a company based in Japan.

2.2. Equipment and software

The equipment includes an analytical balance (Manufacturer-Mettler Toledo, Switzerland; Model: ME303), pH meter (Manufacturer-Mettler Toledo, Switzerland; Model: FE20), and HPLC (Manufacturer- Shimadzu, Japan; Model: 20 A Series-l201343) with PDA (photo Diode Array) and UV (Ultra Violet) detectors. The HPLC LC solution software (Manufacturer- Shimadzu, Japan, Type- LC version-2.0) made controller used. Mechanical shaker (Manufacturer- Ai Si Li test Equipment co-Limited, China), centrifuge (Wincom Company Limited, China), and hot air oven (Manufacturer- Shanghai Atec Laboratory Equipment Limited, China), were also used for studies. The 0.45μm PVDF (polyvinylidene difluoride), Nylon, and PTFE (Polytetrafluoroethylene) filters made by Merck are employed for sample filtration. Dissolution tests were conducted using a dissolution apparatus with an autosampler (Make- Electrolab, India; Model: TrustE-08).

2.3. Chromatographic method conditions

All solubility and dissolution study samples were analyzed with high accuracy using a reverse phase HPLC and an Agilent Zorbax SB (Stable Bond) C18 stationary phase HPLC column (250 ​× ​4.6 ​mm, 5 ​μm). The temperature of the column was controlled to be 40​°C, while the temperature of the sample vials was kept at room temperature. A mobile phase consisting of 70% v/v methanol, 30% v/v water, and 0.1% v/v acetic acid was utilized at a flow rate of 1.0 ​mL/min for peak elution. After injecting 20 ​μL of sample solutions into the chromatography system and monitoring it for 14 ​min at a wavelength of 245 ​nm, to reach our conclusions.

2.4. Solutions preparations

2.4.1. Buffer solution preparations

2.4.1.1. 0.2 ​M hydrochloric acid solution

A volume of 16.6 ​mL of concentrated hydrochloric acid was diluted with purified water to a final volume of 1000 ​mL. The resulting solution was thoroughly mixed. It is important to note that the preparations were provided for a 1000 ​mL solution, but the solutions were prepared according to specific requirements.

2.4.1.2. 0.2 ​M sodium hydroxide solution

Dissolved 8.0 ​g of Sodium Hydroxide pellets into 1000 ​mL of purified water and missed well.

2.4.1.3. Potassium phosphate, monobasic 0.2 ​M

Dissolved 27.22 ​g of pot assium dihydrogen phosphate in 1000 ​mL of purified water and mixed well.

2.4.1.4. 0.2 ​M acetic acid solution

A volume of 11.9 ​mL of acetic acid was diluted with purified water to a final volume of 1000 ​mL, and the resulting solution was thoroughly mixed.

2.4.1.5. Preparation of 0.1 ​M hydrochloric acid solution

A volume of 8.3 ​mL of concentrated hydrochloric acid was diluted with purified water to a total volume of 1000 ​mL, and the resulting mixture was thoroughly mixed.

2.4.2. Preparation of acetate buffer solution pH 4.5

Transferred 2.99 ​g of sodium acetate trihydrate into a glass container and added 14 ​mL of 0.2 ​M acetic acid solution, made up to volume with water and mixed well. Verified the pH of the solution; the observed pH is about 4.5 (if the required pH is not attained, adjust pH with 0.2 ​M acetic acid solution).

2.4.3. Preparation of acetate buffer solution pH 5.5

Transferred 5.98 ​g of sodium acetate trihydrate into a glass container and added 3 ​mL of 0.2 ​M acetic acid solution, made up to volume with water and mixed well. Verified the pH of the solution; the observed pH is about 5.5 (if the required pH is not attained, adjust pH with 0.2 ​M acetic acid solution).

2.4.4. Preparation of phosphate buffer solution pH 6.80

Transferred 250 ​mL of 0.2 ​M potassium phosphate monobasic solution into a container, added 112 ​mL of 0.2 ​M NaOH solution, diluted volume to 1000 ​mL with purified water, and mixed well. Verified the pH of the solution and, on a need-based basis, adjusted the pH to 6.8 with either a 0.2 ​M NaOH or 0.2 ​M HCl solution.

2.4.5. Preparation of phosphate buffer solution pH 7.40

Transferred 250 ​mL of 0.2 ​M potassium phosphate monobasic solution into a container, added 195.5 ​mL of 0.2 ​M NaOH solution, diluted the volume up to 1000 ​mL with purified water, and mixed well. Verified the pH of the solution and, on a need-based basis, adjusted the pH to 7.4 with either a 0.2 ​M NaOH or 0.2 ​M HCl solution.

2.4.6. Solubility study

In order to determine the solubility, a saturation shake flask method was used, as described in the USP general chapter <1236> Solubility Measurements of the USP [35]. To test the solubility of flurbiprofen in different buffer solutions, a known amount was added to a 250 ​mL volumetric flask with about 100 ​mL of each solution (0.1 ​N hydrochloric acid, pH 4.5 acetate buffer, pH 6.8 phosphate buffer, pH 67.4 phosphate buffer, and water) prepared according to the method described in USP buffer solutions [36]. The flasks were maintained in a 37​°C incubator with a shaking speed of 200 RPM (Revolutions Per Minute). After 24 ​h, samples were taken out and filtered via 0.45 ​μm nylon syringe filters. The samples were first injected into a chromatography system. Subsequently, the data obtained from the system was analyzed to determine if the sample exhibited chromatographic peak saturation. If saturation was observed, the sample was subsequently diluted and adjusted to a standard concentration of approximately 3 ​ppm using the appropriate buffer solution.

2.4.7. Reference substance solution

By adding about 12.0 ​mg of flurbiprofen standard to a 100 ​mL volumetric flask, a standard stock solution with a concentration of 120 ​ppm was prepared. The material was first dissolved in 5 ​mL of methanol (as a co-solvent, mix well), and then up to 100 ​mL of the solution was made using dissolution media. After that, 5 ​mL of the stock solution was diluted to 100 ​mL with the same dissolution medium to prepare a secondary standard stock with a concentration of 30 ​ppm. The standard solution, which had a concentration of 3 ​ppm, was created by dissolving 5 ​mL of standard stock-2 into 100 ​mL of dissolution media.

2.5. Dissolution

2.5.1. The process of disc assembly and sample preparation

The unit dose weight of a single flurbiprofen cataplasm is about 12,000 ​mg, and it contains 40 ​mg of drug substance. It has a surface area of 13.6 ​cm by 10.0 ​cm, and the surface is covered with a film that contains 40 ​mg of flurbiprofen. To get the cataplasm sample ready for the dissolution release test, we measured the USP disc assembly with a precision protractor, marked 6 circles on the unit surface of the cataplasms, carefully cut the cataplasm into 6-unit circular pieces with a surgical blade, wrote down the weight, and attached it to 6 units of the dissolution disc assembly; the approximate weight observed for the defined range of the cut sample is about 0.8 ​g. The amount of drug present in it will be approximately 2.67 ​mg as the drug uniformity was controlled and confirmed by an in-process assay and the uniformity of dosage units with a specification of 95.0​%–105.0​% levels in the manufacturing process. The complete formula composition of the inhouse test product is detailed in Table 1.

Table 1.

Inhouse Test formulation composition details.

No.	Component	mg/Unit Dose
1	Flurbiprofen	40.0
2	Glycerin	3360.0
3	Titanium Dioxide	30.0
4	Sodium poly acrylate	720.0
5	Sodium CMC	360.0
6	Polysorbate 80	60.0
7	SPAN 80	6.0
8	L-Menthol	24.0
9	Tartaric Acid	120.0
10	White Clay	120.0
11	Crodomol	60.0
12	Gelatin	360.0
13	Disodium EDTA	26.2
14	Polyvinyl Alcohol	240.0
15	Peppermint Oil	24.0
16	Magnesium Aluminum Silicate	18.0
17	Acrypol	120.0
18	Sodium Tartrate dihydrate	7.2
19	Water	6304.6
Total		12,000

The dissolution medium underwent degassing and was subsequently preheated to a temperature of 32 (±0.5) degrees before being transferred into the dissolution vessels. In each vessel a volume of 900 ​mL was transferred using a measuring cylinder. The paddles were affixed to the instrument and the operator awaited the instrument's indication of readiness. The necessary program requirements, including sampling time intervals and input data for the RPM method, were accurately inputted into the dissolution load position. The attached disc sample was gradually immersed into dissolution vessels, with 6 units being dipped. The level of the paddles was then adjusted using a level adjuster, and the dissolution process was promptly initiated. After the dissolution process, the cataplasm cloth (consisting of 6 units) was extracted from the disc, followed by a thorough rinse using tetrahydrofuran. The total drug quantity in a single cataplasm piece used in a dissolution vessel is approximately 2.67 ​mg. With a dissolution volume of 900 ​mL, assuming complete solubility of the drug in the volume, the concentration of the resulting solution would be 2.97 ​ppm, which closely resembles the concentration of the final standard solution.

2.5.2. Dissolution procedure

To evaluate the regular dissolution testing in quality control labs, the dissolution methodology employed with 900 ​mL of pH 7.4 buffer as the media volume, a paddle apparatus over the disc (USP apparatus -V) as illustrated in Fig. 2 with a rotating at a speed of 50 RPM was employed. The dissolution bowl temperature at 32​°C, and bath temperature at 32.5​°C was maintained. As it is a controlled-release drug formulation, long time intervals of 1 ​h, 2 ​h, 4 ​h, 6 ​h, 8 ​h, 12 ​h, 16 ​h, 20 ​h, 22 ​h, and 24 ​h are operated for sample collection. At each time point, 10 ​mL of solution is withdrawn and filtered (filter information: PTFE, 25 ​mm, 0.45 ​μm), and a timely addition of medium with the same temperature and volume is added automatically. The filtered sample was transferred into the HPLC vial and run the chromatography detection as mentioned above analytical method recorded the detection and calculate the percent drug release.

Fig. 2.

Pictorial form of USP APPARATUS 5.

To check the dissolution profile in multi dissolution buffer solutions, the dissolution profile was evaluated using the above-mentioned dissolution conditions by changing the dissolution media to a multibuffered solution of 0.1 ​M HCl, pH 4.5 acetate buffer, pH 6.8 phosphate buffer, and water for both the reference drug product and the in-house formulated drug product.

3. Result and discussion

3.1. Chromatographic method

To ensure the well robust chromatography, a 1 ​mg/mL Flurbiprofen standard was prepared in different dissolution mediums pH range of 1.2–7.4. Flurbiprofen exhibits a spectral maximum of 247 ​nm based on its UV spectral data and chromophore characteristics. Since 245 ​nm is frequently used in the literature as a wavelength for quantification [37], and experimental data also demonstrate that there is no significant difference in response between 245 and 247 ​nm, therefore, 245 is considered as the method wavelength. Since the dissolution study was performed throughout a wide pH range (from 1.2 to 7.4), the chosen column must be both robust and reliable across this range. Columns typically have a pH range of 2–8 that is useable, but the lowest pH in the present study sample is about 1.2. At low pH of 1.2 the general columns will be not having longer life with respect to number of injections. Considering the fact of broad pH range, A Zorbax Stable Bond C18, 250 ​× ​4.6 ​mm, 5μL column has opted for method optimization because Zorbax SB columns are defined based on the stability at low pH, these columns can be used with in a pH range of 0.8–8.0. When applied consistently, it achieves high efficiency and near-perfect with a peak symmetry. To simplify the method in terms of the mobile phase, the common solvents, such as Methanol, Acetonitrile, and Water were chosen as preliminary solvents. Furthermore, flurbiprofen has the highest acidic pKa, around 4.50 [38], implying that acidic mobile phase conditions are suitable to control peak retentions, given that acetic acid is used as a buffering system for the mobile phase. As a results of multiple experiments, a combination of methanol, water, and acetic acid at 70:30:0.1 v/v/v is finalized as a suitable composition with a flow rate of 1.0 ​mL/min to have proper retention and a shorter runtime to save cost and time. We established 40​°C as the method's temperature after studying the temperatures and finding that it provided the best peak symmetry characteristics. The defined temperature of 40​°C is also within the range of the column operable temperature, which is not to exceed 70​°C when used at low pH [39]. Based on the results obtained with a 20 ​μL injection volume, the aliquot sample quantities are 20 ​μL. Refer to Fig. 3 for representative chromatograms.

Fig. 3.

Chromatograms of blank, standard, and sample.

3.2. Dissolution method

The drug substance exhibits a pH-dependent solubility in buffer solutions, as seen by the solubility data; the solubility values in mg/mL are pH 1.2–0.10, pH 4.5–0.11, pH 5.5–0.14, water-0.45, pH 6.8–2.46, and pH 7.4–5.31. The results were tabulated in Table 2. The highest solubility was observed at pH 7.4, moderate solubility at pH 6.8, and extremely poor solubility at pH 1.2 while using 0.1 ​M HCl. While taking sink condition into account, a single dose of the medication (40 mg)should be dissolved three times at the designated volumes. The buffer solutions with pH 1.2, 4.5, 5.5 and water are obviously not going to satisfy the sink condition criterion; hence the remaining two buffer solutions with pH 6.8 and 7.4 were selected for analysis. The ultimate concentrations at sink conditions for testing with 500 ​mL and 900 ​mL volume medium are 0.24 ​mg/mL and 0.13 ​mg/mL, respectively. Both pH buffers, at 6.8 and 7.4, satisfied the requirement for sink conditions. Flurbiprofen, when administered orally, has maximal absorption in the buccal cavity [40]. Despite the solubility of the drug substance showing a higher solubility in both 6.8 and 7.4, even though the suggested formulation is a TDDS, the buccal pH of 7.4 is taken into account as the final dissolution medium to perform the studies. Regarding buffer volume, the solubility and test product dissolution data have recommended using both 500 ​mL and 900 ​mL as a method final dissolution volume, but taking into account the type of formulation, i.e., the controlled-release dose and its dissolution have to be tested for 24 ​h time, a 900 ​mL volume is chosen. Table 3 refers to the results of dissolution data in different volumes. The reason for opting for the 900 ​mL volume is that if the dissolution is employed for 24 ​h with a low volume of 500 ​mL, volume evaporations are anticipated as the empty space in the dissolution bowl will be greater and the chances are high to form water vapors. In order to support the same investigations, volume optimization at 500 ​mL, 750 ​mL, and 900 ​mL volumes and degassed mediums of water, pH 6.8, and 7.4 buffers were undertaken. The results showed that the acquired release data for 500 ​mL and 750 ​mL volumes had a high RSD fluctuation at initial time intervals. As discussed in 2.5.1, a small portion of the cataplasm, which weighs about 0.8 ​g and contains 2.67 ​mg of the drug substance, was carefully taken for analysis.

Table 2.

Solubility data.

No.	Buffer Type	Solubility in mg/mL
1	0.1 ​N Hydrochloride acid	0.10
2	pH 4.5 Acetate Buffer	0.11
3	pH 5.5 Acetate Buffer	0.14
4	pH 6.8 Phosphate Buffer	2.46
5	pH 7.4 Phosphate Buffer	5.31
6	Water	0.45

Table 3.

Dissolution method optimization data.

No.	Condition	% Drug Release
		Time	1 h	2 h	4 h	6 h	8 h	12 h	16 h	20 h	22 h	24 h
1	pH 6.8, 500 ​mL volume	Average	11	15	31	43	52	61	72	81	92	96
		% RSD	8.0	6.8	9.7	9.9	8.6	6.8	6.8	5.76	4.7	4.9
2	pH 6.8, 900 ​mL volume	Average	11	17	32	45	53	63	72	82	94	98
		% RSD	5.1	4.2	3.9	3.6	2.1	2.1	1.9	1.9	1.8	0.9
3	pH 7.4, 500 ​mL volume	Average	13	23	37	49	66	65	70	81	94	95
		% RSD	7.9	6.0	6.5	6.1	5.9	5.8	5.0	4.3	2.9	1.9
4	pH 7.4, 900 ​mL volume	Average	10	20	35	47	56	70	83	90	95	97
		% RSD	4.3	3.2	3.1	2.1	1.9	1.9	1.8	0.9	0.9	1.0

h-Hours; Min- Minutes.

A straightforward choice was chosen for the USP dissolution apparatus because it is a TDDS system and is in the form of a cataplasm. The USP V paddle over the disc is the specified apparatus and is chosen for the evaluation of the dissolution. The evaluation of the in vitro drug diffusion process by USP apparatus V paddle across the disc is suggested, as is the transdermal system drug release through epidermal cells. According to USP, 50 RPM is the preferred RPM for paddles, so 50 RPM has been used as the final RPM, and its applicability was assessed. In order to support it, a study on paddle agitation speed at 50, 75, and 100 ​revolutions per minute was conducted, with the higher agitation patches losing connectivity from the disc assembly at 75 and 100 RPM after 10 ​h. The results at 50 RPM are favorable, and the same was decided upon. Even though there is a chance to conduct dissolution studies at lower speeds than 50 RPM because doing so is only advised for formulation development investigational studies and not for routine quality control studies, the studies weren't carried out. Since it is a TDDS, the external temperature of 32°C was assumed to be the operating temperature for the dissolution media. The profile monitoring time points were agreed upon as 1 ​h, 2 ​h, 4 ​h, 6 ​h, 8 ​h, 12 ​h, 16 ​h, 20 ​h, 22 ​h, and 24 ​h given that it is an extended-release formulation. The three filters—PTFE, Nylon, and PVDF verified and PTFE was determined to be the final one for examination based on the filter verification results. Finally, it was shown that the dissolution method, which is optimized for quality control testing, has a discriminatory nature and that there is a significant difference in the results when key excipients of the drug product, such as glycerin, sodium polyacrylate, sodium CMC, polysorbate 80, SPAN 80, and disodium EDTA, are modified by 10 %. The summary of results is represented in Fig. 4.

Fig. 4.

Discriminatory evaluation summary.

3.3. Multimedia dissolutions

The specific goal of the multimedia dissolution is to give important information on how the generic drug product behaves when compared to the referenced drug product. Better chances of achieving bioequivalence between test and reference products are provided by providing similarity between two drug products in a multimedia dissolution, in acidic pH, neutral pH, and the highest pH conditions of the philological pH range of the human body. The multimedia dissolution profiles were assessed in light of this. In study, the dissolution profiles were performed using 0.1 ​M HCl, pH 4.5 Acetate buffer, pH 6.8 phosphate buffer, pH 7.4 phosphate buffer, and water to determine how closely the internal test product behaves to the reference listed drug product. Results indicated a closer relationship between the two formulations under test. The significant finding of the multimedia study is the observed similarity factor values of test vs reference drug product are all above 50 (50 is a significant value for f2 correlations), which is generally a recommended value to show a significant correlation between two products [41]. A minimum of 71 and a maximum of 82 values were found as an f2. The results were tabulated in Table 4, and graphs were imaged in Fig. 5.

Table 4.

Dissolution Profiles Data in multimedia.

No.	Condition	% Drug Release											F2
		Time	1 h	2 h	4 h	6 h	8 h	12 h	16 h	20 h	22 h	24 h
1	RLD - 0.1 ​N HCl	Average	9	21	32	37	43	52	60	70	75	77	81
		% RSD	8.7	9.1	8.6	7.6	5.6	4.9	3.0	2.7	2.7	2.6
2	INHOUSE - 0.1 ​N HCl	Average	8	20	28	35	42	50	58	68	72	75
		% RSD	9.0	8.7	8.5	7.9	7.4	5.6	4.8	3.2	3.1	2.9
3	RLD - pH 4.5	Average	11	18	29	38	45	54	62	68	73	76	79
		% RSD	5.6	5.7	5.1	3.8	3.2	3.1	2.7	2.6	1.9	1.9
4	INHOUSE - pH 4.5	Average	9	17	26	35	42	52	60	66	70	74
		% RSD	9.1	8.9	9.1	8.7	8.9	7.8	7.5	6.9	4.3	3.2
5	RLD - pH 5.5	% RSD	12	19	28	37	46	55	61	67	74	78	82
		Average	8.1	7.2	7.1	7.0	4.5	3.8	3.8	2.9	2.9	2.8
6	INHOUSE - pH 5.5	% RSD	11	17	27	35	44	53	59	64	72	75
		Average	7.3	6.7	6.8	6.7	4.5	3.9	3.8	2.8	2.8	2.7
7	RLD - pH 6.80	Average	11	19	35	47	55	66	75	85	94	98	81
		% RSD	4.3	4.1	3.9	3.8	3.2	3.0	2.9	2.0	2.0	0.4
8	INHOUSE - pH 6.80	Average	11	17	32	45	53	63	72	82	94	98
		% RSD	4.5	5.0	3.8	4.2	3.9	3.7	3.2	2.8	2.1	1.0
9	RLD - Water	Average	12	20	38	50	57	66	73	85	92	98	71
		% RSD	6.0	5.9	4.8	4.2	4.2	3.9	2.8	2.1	1.5	0.7
10	INHOUSE - Water	Average	10	21	36	48	55	63	70	79	90	95
		% RSD	5.1	4.8	4.2	3.8	3.9	3.7	2.8	2.2	1.8	1.2
11	RLD - pH 7.40	Average	12	22	38	51	61	74	87	95	98	98	76
		% RSD	2.9	2.1	2.1	1.9	1.8	1.8	1.2	1.2	0.9	0.9
12	INHOUSE - pH 7.40	Average	10	20	35	47	56	70	83	90	95	97
		% RSD	4.3	3.2	3.1	2.1	1.9	1.9	1.8	0.9	0.9	1.0

RLD – Reference Listed Drug.

Fig. 5.

Profiles of RLD Vs INHOUSE in multimedia (s).

3.4. Analytical method validation

According to ICH Guidelines on Validation of Analytical Procedures Q2 (R1) and USP General Chapter <621>, the method has been validated for its system suitability, specificity, precision, accuracy, linearity, robustness, and solution stability. Also, the de-aeration, dissolution volume change, buffer pH change, and RPM variations of the dissolution method parameters were assessed by favoring the USP general chapters of <711> and <1092> [[42], [43], [44], [45]].

A list of system suitability requirements for the method has been established using the validation data. As compared with the placebo and blank samples, the method has shown excellent specificity with no interference at the active retention time, and the peak purity results of the standard and samples show a pure peak; the values are 999.9 and 999.8 for the standard and samples, respectively. The linearity was determined for a concertation range of 0.3 ​ppm–4.5 ​ppm, or 10​%–150​% of the desired concertation of 3 ​ppm, and the resulting correlation coefficient satisfies the requirement of not being less than 0.995. By adding the known amounts of drug substance and placebo compositions into dissolution baths at 50​% (20 ​mg of active and 11,960 ​mg of placebo) and 150​% (60 ​mg of drug substance and 11,960 ​mg of placebo) at triplicate samples and 100​% (40 ​mg drug substance and 11,960 ​mg of placebo) at six samples, the sample recovery in a range of 50 %–150​% of the test concentrations was assessed. The procedure is accurate, as evidenced by the findings, which fell within the allowed range of 97.0​%–103.0%. By performing the method precision and intermediate precision tests with six samples, the method's precision and adaptability were also demonstrated the results. The experimental conditions were altered deliberately to test the method's robustness; assessment %RSD, tailing factor, and theoretical plates of analyte peaks were all taken into account. The impact of a flow rate variation of ±0.2 ​mL/min on the established flow rate of 1.0 ​mL/min was investigated. The column temperature variation was studied with a variation ±5 ​°C from the actual value of 40​°C. Also, the mobile phase organic solvent composition was altered from its original organic solvent by ±10​% and examined how the method behaves. All the results we showed that the deliberate changes in the method were not impacting the finalized method. In order to obtain an accurate result (without any changes due to the sample degrading over time), it is necessary to establish the solution stability because the type of study is one that involves dissolution and because the HPLC analysis may take longer depending on the number of dissolution sampling time points. An essential component is the establishment of solution stability. Thus, the solutions' stability was established after 72 ​h at room temperature and in the refrigerator. The prepared solutions were injected at 0 and 72 ​h. Less than 2.0 was the ratio of the difference between the 0 ​h and 72 ​h analyte test samples. Various filters were assessed for their efficacy in filtering the solution, and the findings of the study indicate that PTFE and nylon filters are appropriate for this purpose, while PVDF filters are deemed unsuitable. The complete chromatographic method validation data are summarized in Table 5.

Table 5.

Chromatographic method validation data.

No	Parameter	Results	Criteria
1	System suitability (SST)
	Tailing factor	1.09	NMT 2.0
	Plate count	11,952	NLT 2000
	% RSD, 6 injections	0.5	NMT 2.0
2	Specificity
	Blank Interference	No interference	No interference
	Placebo Interference	No interference	No interference
	Peak Purity	Pass	Should pass
3	Linearity and Range
	Range	10 % to 150 ​%	–
	Slope	82,792	Report values
	Intercept	399,956	Report values
	Correlation coefficient	0.9996	NLT 0.997
4	Accuracy
	50 ​% mean ​± ​SD, n ​= ​3	98.1 ​± ​0.7	Accuracy should be between 93.0 and 107.0 ​% and RSD NMT 3.0 %
	100 ​% mean ​± ​SD, n ​= ​6	99.7 ​± ​0.5
	150 ​% mean ​± ​SD, n ​= ​3	100.4 ​± ​0.6
5	Precision
	Precision, % RSD, n ​= ​6	1.2	% RSD should not be more than 5.0
	Intermediate Precision, % RSD, n ​= ​6	1.0
	Ruggedness, % RSD, n ​= ​12	0.8
6	Solution Stability
	Standard Solution	Stable for 72 ​h	Similarity 0.97 to 1.03
	Sample Solution	Stable for 72 ​h	% difference NMT 3.0
	Mobile Phase	Stable for 72 ​h	SST should meet
7	Robustness (Tailing, Plate count, %RSD)
	Actual method	1.09, 11,952, 0.5	The system suitability criteria should meet. Tailing factor - NMT 2.0, Plate count - NLT 2000, %RSD of 6 standard injections - NMT 2.0
	Flow, −10 ​%	1.12,10,657, 0.7
	Flow, +10 ​%	1.01, 12,456, 0.4
	Column temperature, −5 ​°C	1.21, 10,282, 0.7
	Column temperature, +5 ​°C	1.01, 14,662, 0.3
	Organic Solvent, −10 ​%	1.19, 10,076, 0.6
	Organic Solvent, +10 ​%	1.02, 13,456, 0.2
8	Filter Verification
	Centrifuge, % Drug	100.5	Difference should be not more than 3.0 from centrifuged data.
	PTFE Filtered, % Drug, difference	100.1, 0.4
	PVDF Filtered, % Drug, difference	96.4, 4.1
	Nylon Filtered, % Drug, difference	98.0, 2.5

RSD – Relative Standard Deviation; n-number of preparations; NMT-Not more than, NLT – Not less than.

On the topic of dissolution, the dissolution medium was subjected to a de-aeration experiment by being kept for 5, 10, and 15 ​min. The results reveal that the dissolution outcomes in all conditions were the same, and the %RSD levels are below 1.0​%, which indicates that there is no aeration effect. The dissolution release is not affected by the 10​% dissolution volume variant or 10​% RPM, based on the data. Refer to Table 6 for the validation results summary.

Table 6.

Dissolution method validation data.

No.	Condition	% Drug Release
		Time	1 h	2 h	4 h	6 h	8 h	12 h	16 h	20 h	22 h	24 h
1	Standard Conditions	Average	10	20	35	47	56	70	83	90	95	97
		% RSD	4.3	3.2	3.1	2.1	1.9	1.9	1.8	0.9	0.9	1.0
2	5 Min. Deaeration	Average	11	21	34	47	55	71	83	90	96	98
		% RSD	5.5	4.2	4.2	4.1	3.9	3.8	3.0	1.2	1.1	1.1
3	10 Min. Deaeration	Average	11	22	34	47	56	72	82	90	95	98
		% RSD	4.5	4.1	3.9	3.2	2.9	2.1	2.1	1.6	1.0	1.0
4	15 Min. Deaeration	Average	10	21	34	46	56	70	83	91	95	98
		% RSD	3.8	3.9	3.2	3.2	2.9	2.1	1.0	0.8	0.8	0.8
5	Media Volume 810 ​mL	Average	10	21	33	45	55	70	79	89	94	98
		% RSD	5.1	4.8	3.8	3.2	2.7	2.1	1.6	1.6	1.0	0.1
6	Media Volume 990 ​mL	Average	11	22	37	49	57	72	85	92	96	99
		% RSD	4.1	3.9	3.9	3.2	2.9	2.9	1.8	1.81	0.9	0.2
7	RPM, 48	Average	09	21	35	43	54	69	83	90	94	98
		% RSD	6.9	5.8	5.6	5.4	5.0	4.6	4.2	3.9	0.5	0.5
8	RPM, 52	Average	12	25	37	44	59	71	86	92	96	99
		% RSD	4.2	4.9	6.2	4.2	3.2	3.3	3.1	3.9	2.0	0.8

RPM- Revolutions Per Minute.

4. Conclusion

Based on the primary findings of the comprehensive study, it is recommended that the consistency of the flurbiprofen cataplasm product be maintained through using of the established dissolution method employing USP apparatus V. This apparatus, consisting of a paddle over a disc, allows for the assessment of the dissolution rate of release. Additionally, it is advised to employ a high-performance liquid chromatography (HPLC) method as a means of quantifying the drug content in the cataplasm. This method has been shown to produce accurate results and can be widely used as a quality control tool. The discriminatory nature of the dissolution method has been evaluated by changing the formulation compositions and observing noticeably different releases among finished products. Based on the validation of the method for variables like system suitability, specificity, precision, linearity, accuracy, and robustness, it has been determined that the developed HPLC quantification method is repeatable. Also, the solution stability for the three elements—mobile phase, standard, and sample—has been established for a period of 72 ​h. The multimedia dissolution assessment demonstrates that the comparative release profiles of the in-house test product and reference listed drugs demonstrate equivalent in vitro release over the pH range of 1.2–7.4, including water. In summary, the present study effectively overcame the main obstacles to developing a dissolution method and a quantification method that must be approved by regulatory authorities, such as dissolution sample preparation, optimization of dissolution conditions, and the shorter runtime HPLC method.

Author contributions

All the concepts, techniques, and data were generated by Nathi Rathnakar. In addition, the article was drafted, updated, implemented, and edited. It was confirmed that the research was conducted collaboratively with the assistance of Naga Venkata Durga Prasad Ketha, Leela Prasad Kowtarapu, Siva Krishna Muchakayala, Naresh Konduru, Baby Saroja, Arya Lakshmi Marisetti. All authors have read and approved the final manuscript.

Funding

This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration - ethics approval and consent to participate

There were no studies involved, this subject is not applicable.

Conflicts of interest

The authors Rathnakar Nathi are currently employed at Shenzhen DEC Pharmaceutical Co., Ltd., located in Shenzhen, China. Leela Prasad Kowtarapu holds a position at STA Pharmaceutical Co., Ltd. (Wuxi App Tec Company), situated in Wuxi, China. Siva Krishna Muchakayala is currently affiliated with Catalent Pharma Solutions, located at 1100 Enterprise Drive, Winchester, Kentucky, 40,391, USA. Despite being employed, Dr Naresh Konduru, working as a Sr. Director & HOD of analytical R&D Department, leading the whole Analytical Research works and administration works. Baby Saroja, Arya Lakshmi Marisetti.

The individuals in question did not receive any form of remuneration or non-monetary perks. The authors declare no conflicts of interest.

Data availability

All data are available upon reasonable requests.