Development and Validation of a Rapid RP-HPLC Method for the Analysis of Afatinib in Tablet Dosage Form Using AQBD Approach

Afatinib, a second-generation tyrosine kinase inhibitor, is widely used for the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. Its potent and irreversible inhibition of EGFR, along with other members of the ERBB family, provides significant clinical advantages over first-generation inhibitors. The accurate quantification of Afatinib in pharmaceutical dosage forms is essential for quality control, ensuring therapeutic efficacy, and maintaining regulatory compliance.  Reverse-phase high-performance liquid chromatography (RP-HPLC) is a widely employed analytical technique due to its robustness, sensitivity, and precision in pharmaceutical analysis. Recent advancements in analytical quality by design (AQbD) have revolutionized method development by providing a systematic, risk-based approach that ensures reliability and reproducibility. The AQbD framework emphasizes a science-driven understanding of method parameters and their impact on critical quality attributes (CQAs), enabling the development of a robust analytical method with minimal variability. The current study focuses on the development and validation of a rapid RP-HPLC method for the quantitative analysis of Afatinib in tablet dosage forms using the AQbD approach. The method was designed to achieve optimal separation, sensitivity, and reproducibility within a minimal runtime, ensuring efficiency for routine quality control applications. Method validation was performed in accordance with International Council for Harmonisation (ICH) guidelines, assessing parameters such as specificity, linearity, accuracy, precision, and robustness. This work aims to bridge the gap in existing literature by providing a comprehensive and efficient analytical solution for Afatinib quantification. By employing the AQbD approach, the developed method ensures enhanced analytical performance while aligning with regulatory expectations for method robustness and quality assurance. This study highlights the critical role of AQBD in modern pharmaceutical analysis, paving the way for future advancements in method development.

MATERIALS AND METHOD:

1. Selection and Procurement of Drug:  In method development and validation of preservatives following chemicals and reagents were used.

Table 1: List of Reagents and Chemicals used

2. Selection of formulation: From the literature survey and market survey we selected Maxide formulation for work.

 Marketed Preparation: The marketed preparation was obtained from local market.

Table 2: List of brand names of combined formulations of Afatinib

3. Selection of Analytical Technique

HPLC was selected as analytical technique for estimation of Afatinib. The analysis of the drug was carried out on Agilent Tech. Gradient System with Auto injector, (DAD) &Gradient Detector. Equipped with Reverse Phase (Agilent) C₁₈ column (4.6mm x 100mm; 2.5µm), a 20µl and UV730D Absorbance detector and running chemstation 10.1 software.

Stock preparations:

Stock I: Standard Sample Preparation: Std. AFATINIB 10 mg in 10 ml Methanol = 1000 µgm/ml.

Stock II: Tab solution Preparation: Take 15.5 mgs in 10 ml water i.e. =   1000 µgm/ml

For Accuracy Solution Preparations: Take 10 µgm/ml tab solution for accuracy,

80 % = 0.1 ml tab solution and add 8µgm/ml std. and make up vol 10 ml with mobile phase

100 % =0.1 ml tab solution and add 10 µgm/ml std. and make up vol 10 ml with mobile phase

120 %= 0.1 ml tab solution and add12 µgm/ml std. and make up vol 10 ml with mobile phase

Chromatographic conditions: The  following  chromatographic  conditions  were  established  by  trial  and  error  and  were  kept  constant throughout the experimentation.

RESULT AND DISCUSSION:

Preliminary studies on Afatinib

1. Melting point: The procured reference standard of Afatinib was found to melt in the range of 183-186 ⁰C respectively.

2. Solubility: The drug was found to be freely soluble in Methanol.

3. UV Spectroscopy: UV absorption of 10 µg/mL solution of Afatinib in Methanol was generated and absorbance was taken in the range of 200-400 nm. ? max of Afatinib in Methanol was found to be 256 nm.

Table 3: Optimized Method

       
           
       
Fig No. 2: Chromatogram of standard Combination of Afatinib

5. Std. Calibration curve: The data obtained in the calibration experiments when subjected to linear regression analysis showed a linear relationship between peak areas and concentrations in the range 10-50 µg/mL for Afatinib (Table 5) depict the calibration data of Afatinib The respective linear equation for Afatinib was y = 8.7621x – 0.564 equation where x is the concentration and y is area of peak. The correlation coefficient was 0.999. The calibration curve of Afatinib is depicted in (Fig No. 3).

Table 5: Result of Linearity data for Afatinib

       
           
       
Fig.No.3:  Std. Calibration curve of Afatinib.

Result of Analytical of Method Validation:

Linearity: From Afatinib standard stock solution, different working standard solutions (10-50?g/ml) were prepared in mobile phase were prepared in mobile phase 20 ?L of sample solution was injected into the chromatographic system using fixed volume loop injector. Chromatograms were recorded.  The area for   each Conc. was recorded (Table 6). The Calibration curves are shown in Fig. No. 4)

Table 6: Linearity of AFATINIB

       
           
       
Fig. No. 4: Calibration curve for HPLC method.

Linearity of of Afatinib was observed in the range of 10-50?g/ml. Detection wavelength used was 256nm (Table 7). The plot should be linear passing through the origin, Correlation Coefficient should not be less than 0.999.that concluded..

Accuracy: Recovery studies were performed to validate the accuracy of developed method. To pre analyzed tablet solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analyzed (Table 8).

Table 8:  Result of Recovery data for Afatinib

Accuracy of RP-HPLC method is ascertained by recovery studies performed at different levels of concentrations (80%, 100% and 120%). The % recovery was found to be within 99-101% (Table 8).

 System suitability parameters :( Repeatability): To ascertain the resolution and reproducibility of the proposed chromatographic system for estimation of Afatinib system suitability parameters were studied the result shown in below (Table 9, and 10).                   

Table 10: Repeatability studies on RP-HPLC for Afatinib:

Repeatability studies on RP-HPLC for Afatinib  was found to be ,The  %RSD was less than 2%, which shows high percentage amount found  in between 100.14%  indicates the analytical method that concluded (Table 10).

Precision: The method was established by analyzing various replicates standards of Afatinib. All the solution was analyzed thrice in order to record any intra-day & inter-day variation in the result that concluded. The result obtained for intraday is shown in (Table 11) respectively.

Table 11:  Result of Intraday and Inter day Precision studies on RP-HPLC

*Mean of each 3 reading for RP-HPLC method

Intraday and Inter day Precision studies on RP-HPLC method for Afatinib  which shows the high precision %amount in between 98% to 102% indicates to analytical method that concluded.

Robustness: The Robustness of a method is its ability to remain unaffected by small deliberate changes in parameters. To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done. The effect of changes in mobile phase composition and flow rate, wavelength on retention time and tailing factor of drug peak was studied. The mobile phase composition was changed in (±1 ml/min^-1) proportion and the flow rate was varied by(±1ml/min^-1), and wavelength change(±1 ml/min^-1)of optimized chromatographic condition. The results of robustness studies are shown in (Table 12, 13, and 14).Robustness parameters were also found satisfactory; hence the analytical method would be concluded.

Table 12: Chromatogram of flow change 0.9 ml

Table 13: Chromatogram of flow change 1.1 ml

Table 14: Chromatogram of Mobile phase composition

Mobile phase change (66% MEOH, 34% 0.1% TEA) :-

Table 15: Chromatogram of Mobile phase composition (66 MEOH+34% 0.1 %TEA):

2)  Wavelength changes 255 nm:

Table 16: Chromatogram change wavelength change 255 nm:

3) Wavelength change 257 nm:

Table 17: Chromatogram of comp change wavelength change 257nm:

Table 18: Result of Robustness Study of Afatinib:

Robustness Study of Afatinib: The changes were did flow rate (±1 ml/ min^-1), PH of mobile phase composition (±1 ml/ min^-1), and Wavelength (±1 ml/ min^-1). %RSD for peak area was calculated which should be less than 2%.the result shown in analytical method that concluded.

Result of Limit of detection =   3.3 X 0.24/8.762= 0.5174 (?g/mL). The LOD of Afatinib was found to be 0.5174 (?g/mL) analytical methods that concluded

Result of Limit of Quantitation =   10 X0.24/ 8.762=0.2739 (?g/mL).  The LOQ of Afatinib was found to be 0.2739 (?g/mL), analytical method that concluded.

Result of Analysis tablet formulation:  The simple chromatogram of test Afatinib Shown in (Fig No. 8) the amounts of Afatinib per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated five times with tablet formulation. Tablet Assay for % Label claim for %RSD Calculated, Result was shown in (Table No. 19)

Table 19: Chromatogram of Marketed Formulation:

Table 20: Analysis of marketed formulation:

Analysis of marketed formulation were also %Lable Claim was found to be 99-101% Satisfactory are concluded (Table No.20).

Result of Specificity and Selectivity: The analytes should have no interference from the extraneous components and be well resolved from them. Specificity is the procedure to detect quantitatively the analyte in presence of component that may be expected to be present in the sample matrix, while selectivity is the procedure to detect qualitatively the analyte in presence of components that may be expected to be present in the sample matrix.

Result of Force Degradation: Forced degradation studies of both the drugs namely Afatinib were carried out individually and in combination under different stress conditions like acid hydrolysis, alkaline hydrolysis, hydrogen peroxide oxidation and photolysis. The results are shown in (Table 22).

Table 22: Degradation of different stress condition:

1. Alkaline hydrolysis: The alkaline hydrolysis condition was performed using 0.1N NaOH for 1hr Afatinib Degradation of Afatinib. The major degradation products for Afatinib were observed at 2 hr ?gradation 100 % for 2 hrs.