A Stability-Indicating RP-HPLC Method For The Development And Validation Of APD Quantification In Cream Formulation

Figure 1: Systematic Diagram of HPLC

Method validation is an important part of pharmaceutical analytical research since it is a documentation of proof that the analytical procedure gives accurate results for its intended use. Analytical methods need to be validated prior to being used in quality control laboratories according to international regulatory authorities such as ICH, USP, and FDA. Parameters for validation may include selectivity, linearity, accuracy, precision, robustness, limit of detection, limit of quantitation, and system suitability. Validation ensures the accuracy of data generated from analysis. A stability indicating analytical method is specifically designed to accurately measure the active pharmaceutical ingredient in the presence of its degradation products, process impurities, and formulation excipients. Such methods are crucial for evaluating the stability profile of pharmaceutical formulations and predicting product shelf life. According to ICH guidelines, stability studies are essential for determining the impact of environmental factors such as temperature, humidity, and light exposure on drug products. A validated stability indicating method facilitates the detection of degradation products and ensures the maintenance of product quality throughout its storage period [8].

Several analytical methods have been reported for the estimation of anti psoriasis drugs including Tapinarof, Calcipotriol, Betamethasone Dipropionate, Halobetasol, Tazarotene, Methoxsalen, and Roflumilast using chromatographic and spectrophotometric techniques. Although these methods have demonstrated satisfactory analytical performance, limited information is available regarding a validated stability indicating RP HPLC method for the quantitative estimation of APD in cream formulation. Therefore, the present investigation was undertaken to develop and validate a simple, precise, accurate, and stability indicating RP HPLC method for APD in cream formulation according to ICH Q2(R1) guidelines and to evaluate its applicability for routine quality control and stability assessment.[9,15].

2. MATERIALS AND METHODS

2.1 Materials

APD working standard was obtained from a certified pharmaceutical manufacturer and was used as received without further purification. The marketed cream formulation containing 0.3% APD was procured from the local market and utilized for analytical investigations. HPLC grade methanol, acetonitrile, orthophosphoric acid, and water were employed throughout the study. All chemicals and reagents used during the experimental work were of analytical reagent grade and complied with pharmaceutical analytical standards.

Table 1: Chemicals and Reagents Used in the Study

2.2 Instrumentation

Chromatographic analysis was performed using a High Performance Liquid Chromatography system equipped with a quaternary solvent delivery pump, manual injector, ultraviolet visible detector, and computerized data acquisition software. Chromatographic separation was accomplished by means of the reverse phase C18 analytical column at optimal operational conditions. The analytical weightings were carried out by the calibrated digital analytical balance with sensitivity up to 0.1 mg. The ultrasonic sonication was used as the method of degassing of the mobile phase and for the purpose of the dissolution of the drug substance. All the equipment utilized within the course of the study was calibrated and qualified according to the laboratory quality assurance protocols.

The HPLC system included a solvent reservoir, a high pressure pump, an injector, a chromatographic column, a detector, and a data collecting module.

Figure 2: Systematic Diagram of HPLC

2.3 Method Development Strategy

The creation of a technique involves the systematic optimization of conditions of the chromatography in order to provide good separation, reasonable retention time, symmetrical peaks, and adequate sensitivity. Several experiments were performed using different compositions of mobile phase, columns, and different wavelengths of detection. System suitability indicators such as the number of theoretical plates, tailing factor, resolution, and retention time were used for the optimization.

Figure 3: Steps in HPLC Method Development

Development procedure was based on choosing a suitable stationary phase and then optimizing the mobile phase composition. Wavelength for detection was chosen according to UV absorption properties of APD. Different parameters of the method were studied to achieve good symmetry and sharpness of the peak.

2.4 Selection of Detection Wavelength

The UV Spectra of APD was measured using the UV Visible Spectrophotometer in the wavelength range of 200 to 400 nm. A standard sample of APD was taken and scanned to find the wavelength which gave maximum absorbance. The wavelength which had the maximum absorbance was chosen as the detection wavelength in the HPLC method.

2.5 Selection of Chromatographic Conditions

Various combination systems made up of methanol, acetonitrile, water, and buffers have been tested during initial trials. The purpose here was to obtain suitable peak symmetry, proper retention, and baseline separation from excipients of the formulation.

1. These systems have been optimized using the following factors:
2. Stationary phase characteristics.
3. Mobile phase composition.
4. Flow rate.
5. Detection wavelength.
6. Injection volume.
7. Column temperature.
8. Run time.

After optimization, the chromatographic conditions providing acceptable system suitability parameters were selected for further validation studies.

2.6 Preparation of Mobile Phase

The selected mobile phase was prepared by accurately measuring the required quantities of organic solvent and aqueous phase according to the optimized ratio. The prepared mobile phase was filtered through a 0.45 μm membrane filter to remove particulate matter and subsequently degassed using an ultrasonic bath for approximately fifteen minutes prior to chromatographic analysis.

2.7 Preparation of Standard Stock Solution

An accurately weighed quantity of APD working standard equivalent to 10 mg was transferred into a 100 mL volumetric flask. Approximately 70 mL of diluent was added and the solution was sonicated until complete dissolution was achieved. The volume was then adjusted to the mark with the same diluent to obtain a stock solution containing 100 μg/mL of APD.

2.8 Preparation of Working Standard Solution

Aliquots of the standard stock solution were transferred into a series of volumetric flasks and diluted appropriately with the selected diluent to obtain working standard solutions covering the concentration range required for method validation studies. These solutions were used for linearity, precision, accuracy, and robustness investigations.

2.9 Sample Preparation

A measured amount of the cream sample equivalent to 10 mg APD was added to the volumetric flask containing the appropriate amount of diluent. The mixture was sonicated to aid in extracting the drug from the semisolid base. This solution was then filtered using a membrane filter to separate the insoluble excipients. The necessary dilutions were done to get the required concentration.

2.10 System Suitability Evaluation

Before analyzing the samples, system suitability test was done to check for the acceptability of the HPLC system. The HPLC system suitability test was done by making six replicates of the standard and recording the system suitability parameters. The acceptance criteria were determined based on pharmaceutical regulation guidelines.

Table 2: System Suitability Parameters

2.11 Method Validation

The established RP-HPLC method was validated based on the ICH guideline for analytical procedure validation, Q2(R1). The parameters validated were specificity, linearity, precision, accuracy, limit of detection, limit of quantitation, robustness, and system suitability.

2.11.1 Specificity

Specificity was assessed by the comparison of the chromatograms from blank solution, placebo solution, standard solution, and sample solution. The capability of the method to quantify APD free from interference due to any excipient or degradation product was studied.

2.11.2 Linearity

Linearity was assessed by preparing a series of APD standard solutions covering the anticipated analytical concentration range. Each concentration level was injected into the HPLC system and the corresponding peak areas were recorded. A calibration curve was constructed by plotting concentration versus peak area, and linear regression analysis was performed.

2.11.3 Precision

Precision was evaluated through repeatability and intermediate precision studies. Repeatability was determined by analyzing six replicate preparations of the same concentration under identical operating conditions. Intermediate precision was assessed by performing the analysis on different days and by different analysts.

2.11.4 Accuracy

Accuracy was determined using recovery studies at three concentration levels corresponding to eighty percent, one hundred percent, and one hundred twenty percent of the target concentration. Known quantities of APD standard were added to the pre analyzed sample and percentage recovery values were calculated.

Table 3: Accuracy Study Levels

2.11.5 Limit of Detection and Limit of Quantification

The sensitivity of the method was evaluated by calculating the limit of detection and limit of quantification using the standard deviation of response and slope of the calibration curve. The calculations were performed according to ICH recommendations.

LOD = 3.3 × (σ/S)

LOQ = 10 × (σ/S)

Where σ represents the standard deviation of response and S represents the slope of the calibration curve.

2.11.6 Robustness

Robustness was examined by deliberately introducing minor variations in chromatographic parameters including flow rate, mobile phase composition, and detection wavelength. The influence of these changes on chromatographic performance was assessed.

2.11.7 Ruggedness

Ruggedness studies were performed by conducting analyses using different analysts, instruments, and experimental conditions. The obtained results were compared to evaluate the reproducibility of the analytical procedure.

2.12 Stability Indicating Assay

Stability indicating ability of the method developed was tested by storing the cream formulation in different storage conditions. The samples were stored at controlled temperatures for three months. At specific intervals, the samples were analyzed using the developed RP HPLC method and percentage assays were calculated. The chromatograms of stressed samples were evaluated for the appearance of any degradation product and change in retention behavior.

The studies of stability were conducted in order to prove the suitability of the method developed for stability testing of APD cream formulation.

3. RESULTS AND DISCUSSION

3.1 UV Visible Spectrophotometric Analysis

The development of the UV Visible spectrophotometric technique began with the identification of the wavelength of maximum absorption (λmax) of APD before proceeding to the development of the RP HPLC method. Choice of methanol as the solvent was based on the solvent's capacity to dissolve the analyte. The standard solution of APD was scanned across a range of 200 – 400 nm. The absorption spectrum revealed an absorption peak at 245 nm, which indicated the presence of high absorbency of the analyte at this wavelength. Hence, 245 nm was chosen as the wavelength for analysis through UV spectrophotometry and RP HPLC techniques.

Choice of λmax ensures high analytical sensitivity by generating maximum detector response without any background interference. This wavelength was also ideal for the quantitative estimation of APD.

Figure 4: UV-Spectrum of APD

After selecting the wavelength for measurement, the linearity of the UV Spectrophotometry was determined using Beer Lambert’s law. This was done by preparing standard solutions that had varying concentrations from 10 µg/mL to 50 µg/mL, and their respective absorbance values were recorded at 245nm. It was noted that the absorbance increased linearly with increasing concentration of the drug, thus showing excellent compliance with Beer Lambert’s law.

Table 4: Linearity study of APD on UV- Visible Spectometry

Figure 5: Linearity curve of APD on UV- Visible Spectometry

It can be seen from the linear regression analysis that a very good correlation exists between concentration and absorbance. The calibration graph shows a slope of 0.307 and intercept value of 0.015, and R2 = 0.9999 validates a very good linear correlation. It is seen that the value of the intercept is negligibly low, suggesting a very low level of background absorption, and the high regression coefficient implies that almost all variations in the absorbance are due to the variation in analyte concentration.

3.2 RP HPLC Method Development

RP HPLC method development was aimed at creating a fast, selective, and stability-indicating chromatographic method for accurate quantitation of APD in cream formulation. Method development was based on the use of various chromatographic columns with the same composition of the mobile phase and constant flow rate. Chromatographic properties were evaluated through the consideration of peak symmetry, peak sharpness, retention characteristics, and resolution.

At first, Kromasil C8 column was studied; however, poor peak definition and poor chromatographic characteristics were provided. Inertsil ODS column showed too long retention time of the analyte peaks. Agilent Eclipse XDB C18 column gave poor peak definition with poor peak characteristics. YMC Pack Pro C18 column significantly increased peak resolution, but additional modifications were necessary. The Prontosil Prontol C18 column provided sharp, symmetrical, and well-resolved peaks with high reproducibility and was chosen for further method validation.

Table 5: Trial for selection of Chromatographic column

1. Kromasil C8 Column

2. Inertsil ODS Column

3. Agilent Eclipse XDB C18

4. YMC Pack Pro C18

5. Prontosil Prontol C18 Column (APD)

6. Prontosil Prontol C18 Column (AP Cream)

Figure 6: Chromatograms of selected HPLC columns used during the optimization of chromatography method: (A) Kromasil C8 column, (B) Inertsil ODS column, (C) Agilent Eclipse XDB C18 column, (D) YMC Pack Pro C18 column, (E) Prontosil ProntoSIL C18 column for APD formula, and (F) Prontosil ProntoSIL C18

It can be seen from the above optimization study that the Prontosil Prontol C18 column is better than the other columns that were studied in terms of providing good chromatographic efficiency. It has produced an ideal symmetrical peak, theoretical number of plates, adequate retention property and separation of APD from the other formulation components.

3.3 Optimized Chromatographic Conditions

After optimizing the conditions, the optimized condition for the RP-HPLC analysis for APD was determined. The optimized technique included the use of the Prontosil Prontol C18 analytical column with a mobile phase consisting of acetate buffer, acetonitrile, and methanol. The detection was done using wavelength of 245 nm and flow rate of 1.0 mL/min. With the optimized conditions, APD was eluted as a sharp and symmetrical peak at retention time of 9.87 minutes.

Table 6: Final Chromatographic Conditions

Figure 7: HPLC Graph of APD

The optimal conditions gave rise to well-resolved and highly symmetric chromatographic peaks with stable baseline. There were no interferences from formulation excipients or the solvent peak at the retention time of APD. The developed method thus met all the necessary chromatographic criteria for its validation according to ICH Q2(R1).

3.4 System Suitability Studies

Prior to the method validation, the system suitability tests were carried out to confirm the adequacy and suitability of the chromatographic system. The freshly prepared APD standard solution was injected using optimal chromatographic conditions and the system suitability criteria such as retention time, theoretical plates, tailing factor, capacity factor, and asymmetry factor were determined.

Table 7: System Suitability Parameters