Simultaneous Estimation of Adapalene from Marketed Gel Formulation along with the Preservative Phenoxyethanol by UV- Visible Spectroscopy

 

Tejasvini Neve*, Vijaya Vichare, Manasi Rokade, S.N. Dhole

PES Modern College of Pharmacy (For Ladies) Moshi, Savitribai Phule University, Pune, Maharashtra, India.

 

ABSTRACT:

A simple, rapid and eco-friendly UV spectroscopic method was developed for the estimation of Adapalene along with Phenoxyethanol for laboratory purpose. Bulk drug and marketed formulation both are analyzed and validated by this method. Tetrahydrofuran was selected for the solubility of Adapalene and Phenoxyethanol as per solubility profile. Wavelength selected for Adapalene and Phenoxyethanol was 325nm and 269nm respectively. In the proposed method, adapalene and phenoxyethanol follows linearity in the concentration range 1-5µg/mL and 2.5-12.5µg/mL with a correlation coefficient 0.999 and 0.998 respectively. Proposed method was statistically validated as per ICH guidelines and by recovery studies. The standard deviation was found to be less than 2, which showed excellent precision and accuracy.

 

 

 

INTRODUCTION:

Adapalene (AD) is a topical retinoid-like compound, chemically similar to vitamin A. It is a derivative of naphthoic acid having chemical name 6-[3-(1-adamantyl)-4-methoxy-phenyl] naphthalene-2- carboxylic acid. Molecular formula of AD is C₂₈H₂₈O₃ with molecular weight 412.52 g/mol¹. It is a widely used in acne vulgaris, keratosis pilaris, warts, callus and variety of skin diseases^2-3. AD is a subject of monograph in European Pharmacopoeia⁴.

 

AD modulates cellular keratinisation, stimulate humoral and cellular immunity and also modulates inflammatory process. This anti inflammatory effect is due to inhibition of the lipooxygenase activity and also to oxidative metabolism of arachidonic acid. These mechanism may be the reason for decreased risk of irritation with adapalene⁵.

 

Topical formulations must include a preservative component to prevent microbial growth during storage. Preservatives are an important part of the product composition because of their antibacterial and antifungal activities⁶. PE is a common preservative in aqueous-based topical formulations, monitoring of these preservatives is essential in pharmaceutical products to ensure their effectiveness throughout their shelf life⁷.

 

Fig 1: Structure of Adapalene     Fig 2: Structure of Phenoxyethanol

 

Literature survey revealed that there are numerous analytical methods such as flourimetry⁸, Derivative spectroscopic⁹, HPTLC¹⁰, LC-MS/MS¹¹and HPLC^12-15 which are reported for AD as alone or in combination with other drugs^16-17. PE is the most commonly used preservatives in AD topical gel formulation. But, there is not single method reported for estimation of AD along with preservative (PE). Hence, there is a need for simple UV spectroscopic method, which is more accurate and rapid. As a result, the current goal of this analysis is to develop and validate a UV-Visible spectroscopic method for estimating AD along with preservative PE in pharmaceutical topical formulation simultaneously^18-19.

 

MATERIALS AND METHODS:

Instrument:

UV-Visible Spectrophotometer (Shimadzu-1800, Japan), Sonicator, and electronic balance (Shimadzu AUX 220).

 

Reagents and Chemicals:

Tetrahydrofuran (AR), Methanol AR grade, Distilled water.

The following are the details of the formulation

 

Brand Name:

Adapen Gel

 

Manufacturer:

INTAS Pharmaceuticals.

 

Composition:

0.1% AD + 0.25 % PE²⁰.

 

Method Development:

Selection of solvent: As per solubility criteria of AD, THF was selected for analysis (stock) and further dilutions from stock solution were made with methanol and distilled water.

 

Preparation of Standard Stock and Working Standard Stock:

10mg of AD was weighed and dissolved in 10ml of tetrahydrofuran to make 1000µg/ml of stock solution. To make 100µg/ml solutions, 1ml of each drug's stock solution was diluted to 10ml using methanol. The solutions were further diluted with distilled water to achieve the required concentrations of wavelength ranges (1-5)µg/ml.

 

0.1ml of PE was pipetted out and dissolved in 10ml THF to make 10,000µg/ml of stock solution. From this stock 1ml is diluted with methanol upto 10ml to get 1000 µg/ml. From this dilution 1ml was pipetted out to make 100µg/ml solution. Further dilutions were made from 100µg/ml of stock solution and diluted with water up to mark 10ml to achieve concentrations in the range of (2.5-12.5)µg/ml .

 

 

FIG. 3

Selection of Area and Method of Analysis:

Using distilled water as a blank, a solution of 2µg/ml of AD and 5µg/ml of PE was scanned in the region of 200-400 nm individually. From the overlain spectra, 325nm wavelength was selected for AD and 269nm for PE. (Fig 3).

 

 

From the overlay of the spectrum simultaneous equation method is selected for Analysis^21.

 

Simultaneous Equation Method²²:

The absorbance of each resulting solution was measured at 269nm and 325nm. The Absorptivity (µg/ml) values (ax1, ax2, ay1 and ay2) were determined from five different concentrations of 2 and 5µg/ml of AD and PE using following equation. Linearity graph was plotted as absorbance vs. concentration.

Y= mx

Where,

Y= Absorbance   

m= Absorbtivity value

x= Concentration (µg/ml) 

The % drug contents were calculated by following formula:                    

Equation 1: simultaneous equation for the estimation of AD

C_x = (A₂ a_y1 - A1a_y2)/ (a_x2 a_y1 - a_x1 a_y2)

Equation 2: simultaneous equation for the estimation of PE

C_y = (A₁ a_x2 - A2a_x1)/ (a_{x2 ay1} - a_{x1 ay2})

Where:

Cx = Conc. of AD (µg/ml) .

Cy = Conc. of PE (µg/ml).

A₁ = Absorbance and Formulation at 269 nm.

A₂ = Absorbance and Formulation at 325 nm.

a_x1 = Absorptivity of AD at 269 nm.

a_x2 = Absorptivity of AD at 325 nm.

a_y1 = Absorptivity of PE at 269 nm.

a_y2 = Absorptivity of PE at 325 nm.

 

METHOD VALIDATION:

Validation is the process of documenting in written that a specific activity will consistently achieve the desired outcome or produce a product that fulfils the specified standard and quality aspects. Validation is becoming an important part of not only quality assurance, but also pharmaceutics and pharmaceutical manufacture^23-25.

 

UV method development was validated using parameters such as linearity, precision, specificity, and robustness. The method has been validated using ICH Q₂ (R₁) criteria²⁶.

 

Linearity and Range:

The linear correlation between drug's absorbance and concentration was tested in three replicates over concentration ranges of 1-5µg/ml AD and 2.5-12.5 µg/ml PE as a combination at specified wavelengths. The calibration curves were plotted, regression equation and R² values were reported.

Accuracy:

The accuracy of the method was determined by the % recovery. To conduct recovery studies, mixtures of drugs containing known quantities of drugs at various levels (50 %, 100 %, and 150 %) spiked in to formulation. The developed method was utilized to analyze the samples that obtained. The percentage recovery and the amount of drug added were calculated.

 

Precision:

The relative standard deviation of the % drug content was used to assess the repeatability and intermediate precision of the developed method. The sample solution, which contained 2 µg/ml AD and 5 µg/ml PE, was tested for repeatability in six repetitions on the same day and intermediate precision in triplicates over three days. The results were expressed as a % RSD.

 

Robustness:

Robustness of the developed method was studies by testing the stability of prepared solutions.

 

Assay of Marketed Gel Formulation:

In a 10 ml volumetric flask, accurately weighed amount of gel, equivalent to 0.1 mg of AD and 0.25 mg of PE was transferred. Small amount of THF was added to the volumetric flask and mixture was sonicated for 15 min. Then volume was made up to the mark with THF to get 0.1 mg (100µg/10ml), from this 1 ml was pipette out and diluted with Methanol upto 10 ml. This solution was filtered through Whatman filter paper no 41. 0.1 ml from this stock was pipette out and volume was made up with distilled water to achieve 2 µg/ml of AD and 5 µg/ml of PE. The solution was then analysed using a newly developed UV Visible spectroscopy method, with the results expressed as a % of drug content.

 

RESULT AND DISCUSSION

1. Assay of gel formulation:

The assay % of AD and PE in marketed gel formulation was found to be in a range of 95-105% as shown in table 1. Therefore, this method is effective in determination of drugs in gel formulation.

 

Table 1: Result of assay of gel formulation.

 

2. Accuracy:

The developed method was used to analyze the recovery samples at (50 %, 100 %, and 150 %) w/w. The proportion of drug added and % recovery was calculated as shown in table 2.

 

Table 2: Accuracy result, (n=3)

 

Table 3: Precision result

 

Table 4: Linearity Data

 

3. Precision:

The % drug contents for precision of studies were found in a range of 100.4 -105.05w/w. The %RSD values of repeatability and intermediate precision was found to be less than 2. Thus, proposed method was found to be precise as shown in table 3.

 

4. Linearity/Range:

Linearity of AD was found to be in a range of 1-5µg/ml and linearity of PE was found in a range of 2.5-12.5 µg/ml. The correlation coefficient was found to be 0.999 and 0.998 for AD and PE respectively as shown in table 4, which indicated good linearity as shown in figure 8 and 9. For both the drugs in the concentration range studied, a well-correlated linear fit graph was obtained.

 

 

Fig 4: Calibration of AD at 325

 

Fig 5: Calibration of PE at 269

 

Robustness:

The prepared standard and sample solutions were found to be stable 8 Hrs.

 

CONCLUSION:

The concentration of adapalene and along with effect phenoxyethanol as a preservative in a gel formulation were estimated using a UV spectrophotometric simeltaneous equation method, developed method was verified in accordance with ICH guidelines. The proposed method for analytical purposes has the following advantages: rapid determination, cost-effectiveness, ease of sample preparation, and a simple, accurate, and precise nature. As a result, the proposed method can be used in regular quality control analysis to determine Adapalene and along with phenoxyethanol in combination formulations.

 

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Received on 14.06.2022         Modified on 01.09.2022

Accepted on 11.11.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2023; 13(3):206-209.