INTRODUCTION:

Food and Drug Administration approved Afatinib as a kinase inhibitor indicated for first -line treatment of patients with metallic non-small cell lung cancer (NSCLC) in 2013. Afatinib diamaleate (AFT) is an anilinoquinazoline derivative with chemical name as ([N-[4-[(3-chloro-4-fluorophenyl) amino]-7-[[(3S)-tetrahydro-3-furanyl] oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide]). Lung cancer is one of the most aggressive and challenging malignancies worldwide. Non-small cell lung cancer (NSCLC) often involves tumors with specific genetic mutations, such as the epidermal growth factor receptor (EGFR) exon 19 deletions or the exon 21 (L858R) substitution mutations. Afatinib, a tyrosine kinase inhibitor (TKI), exhibits potential anti-neoplastic activity by specifically targeting these EGFR mutations.¹

Afatinib is a second-generation tyrosine kinase inhibitor (TKI) that demonstrates significantly improved clinical outcomes in patients with EGFR-sensitizing mutations compared to first-generation TKIs. These advancements have been supported by studies such as those by Paz-Ares et al., and Mok et al.^2,3

Ibrutinib is an inhibitor of Bruton's tyrosine kinase (BTK) with significant antineoplastic potential. It belongs to the acrylamide class of compounds and is chemically represented as (3R)-3-[4-amino-3-(4-phenoxyphenyl) pyrazolo [3, 4-d] pyrimidin-1-yl] piperidine.

Ibrutinib establishes a covalent bond with the cysteine residue C481 within the active site of Bruton's tyrosine kinase (BTK). This interaction results in prolonged inhibition of BTK enzymatic activity. BTK plays a critical role as a signaling molecule in the B-cell antigen receptor pathway, which is implicated in the pathogenesis of various B-cell malignancies. Ibrutinib is approved for the treatment of multiple conditions, including mantle cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström's macroglobulinemia, marginal zone lymphoma, and chronic graft-versus-host disease.⁴ The chemical structures of Afatinib dimaleate and Ibrutinib are depicted in figure 1.

Dissolution profiling is used as an in vitro release test to evaluate the performance of a dosage form. It is a critical chemical test in pharmaceutical drug product development. Dissolution testing is designed to provide a predetermined assessment of the predicted in vivo release behavior of the drug product.⁵

The US FDA dissolution database has given the dissolution method parameters for Afatinib tablets and Ibrutinib Capsules. The dissolution method parameters for Afatinib Tablets are given as to refer to FDA’s dissolution guidance 2018. As per the FDA’s dissolution Guidance, 2018 guidance the following dissolution parameters were recommended as Paddle Method (USP apparatus 2), stirring rate of 50 RPM. Dissolution media employed is 500mL of 0.1N HCl in aqueous medium maintained at 37±0.5°C.⁶

The dissolution method parameters for Ibrutinib for capsules include USP apparatus II, paddle maintained at 75rpm. Dissolution media includes 900mL of 3% polysorbate 20 in 50mM phosphate buffers, pH 6.8. Sampling upto 45min. with adequate sampling points.

Green chromatography includes the green chemistry principles. Implication of green chromatography reduces the usage of hazardous chemicals.⁷ Father of Green chemistry is Paul T. Anastas. Green chemistry involves 12 principles which lead to minimization on environmental and health impacts⁸ 3R rules of green chromatography are reducing, replace and recycle. These rules help in developing of chromatographic methods to minimize harmful affect on environment. Reduce refers to reduction of solvent usage, energy consumption, dimensions of columns, etc. Replace refers to substitution of hazardous solvents with green solvents, replacement of traditional chromatographic methods with micellar or ultra performance techniques. Recycle refers to recyclization of outlet solvents, which leads to reduction of harmful vapors to air.⁹ Green liquid chromatographic methods can be evaluated for its greenness by various greenness assessment tools. Overall parameters such as waste output, energy consumption, safety of chemicals, etc. are considered. Certain greenness assessment tools are National Environmental Methods Index Label (NEMI), Modified NEMI, Analytical Eco-Scale Score calculation, Green Analytical Procedure Index (GAPI), Analytical GREEnness (AGREE), AGREE prep, HPLC-EAT, etc. NEMI is the earliest qualitative tool to assess the greenness of method by considering four environmental criteria ie. persistence, bioaccumulation, toxicity, and hazardous waste generation. Modified NEMI is an extension of NEMI, which some additional criteria’s. Analytical eco-score is a quantitative tool that considered the hazard and amount penalty points. The total penalty points are subtracted from 100.^10,11 GAPI pictogram is a tool which contains five pentagonal pictograms overall which includes consideration of sample preparation, sample analysis, and waste generation. The results are represented in a color coded (red, yellow, green) radial diagram for interpretation of ecological impact on environment.¹² Complex Green Analytical Procedure Index (Complex GAPI) is an additional version of GAPI which contains hexagonal glyph.¹³ AGREE is a software-based tool which considers 12 principles of green analytical chemistry.¹⁴AGREE prep is an additional version of AGREE specifically considers evaluation of the sample preparation techniques.¹⁵

A comprehensive literature review revealed that only a few reports are available on the liquid chromatographic tandem mass spectrometric (LC-MS/MS) methods for the determination of afatinib (AFT) in animals¹⁶ and humans¹⁷ Additionally, the liquid chromatographic determination of several tyrosine kinase inhibitors, including afatinib, has been reported using diode array detection^18,19 Pharmacokinetic²⁰ and metabolism studies²¹ of afatinib in humans have also been documented. However, there are very few reports on the determination of the drug in bulk and dosage forms.²² Eco-friendly green liquid chromatographic method was developed for the assay of Afatinib tablets.²³

Based on the literature survey conducted on chromatographic methods for Ibrutinib, the disadvantages of current chromatographic methods, particularly their reliance on toxic solvents (e.g., acetonitrile, methanol) for mobile phases and diluents and the usage of long lengths of column leads to longer run time increases the time analysis and effluent waste.^24-27 Eco-friendly green liquid chromatographic method was developed for the assay of Ibrutinib capsules.²⁸ Literatures are documented and works are done related to green chemistry.^29-36

Given the lack of a green analytical method, particularly for the estimation of the dissolution profiles of Afatinib and Ibrutinib in different drug products, this research aims to develop multi-analyte quantification in a single method which is one of the principles of green chemistry to get the sustainable and eco-friendly liquid chromatographic method.^37,38 This method will be designed for the dissolution profiling of Afatinib in Afatinib tablets and Ibrutinib in Ibrutinib capsules.

Figure 1. Chemical Structures

MATERIALS AND METHODS:

Chemicals, standards and materials:

Potassium dihydrogen orthophosphate (Merck), 85% Ortho-phosphoric acid (Labchem UK), Ethanol (JT baker), Hydrochloric acid (85%) (Merck), Sodium hydroxide (Merck), hydrogen peroxide (Merck) and Polysorbate 20 (SRL) are the chemicals involved. Milli-Q water is employed throughout the experiment. Afatinib dimaleate tablets AFANAT-40mg manufactured by Natco is utilized. The Ibrutinib capsules, specifically IMBRUVICA-140mg (Pharmacyclics [Janssen]), were purchased from the market. Afatinib dimaleate and Ibrutinib standard were obtained from MSN Laboratories.

Instrumentation and Software:

Shimadzu HPLC model equipped with binary gradient pumps and photodiode array detector was utilized for the separation. Data acquisition and evaluation done by Lab solutions software. Other softwares employed are design expert version 11.0, GAPI and complex GAPI 02 beta version; AGREE 0.5 beta version and AGREEprep 1 betaversion.

Chromatographic Method:

HPLC instrument employed was a Shimadzu instrument equipped with a binary pump, and a photodiode array detector. Mobile phase- A comprised of pH 3.0 Potassium dihydrogen phosphate (25mM) buffer. Mobile phase- B consists of ethanol and HPLC water in the ratio of 90:10% V/V respectively. The final mobile phase composition is Mobile phase-A and Mobile phase-B in the ratio of 20:80% V/V. The flow rate was set at 0.80 mL/min. The separation was done on Shimadzu Shim-pack solar C₁₈(150mm x 4.6mm, 5µm) column with injection volume of 10µL. Afatinib and Ibrutinib were detected at a wavelength of 254nm. Diluent employed for the standard preparations composed of ethanol and HPLC water in the ratio of 90:10% V/V respectively.

Preparation of Dissolution media:

For Afatinib Tablets: 0.1N Hydrochloric acid solution was prepared by transferring 85mL of concentrated HCl in 1litre of purified water.

For Ibrutinib Capsules (3% polysorbate 20 in 50mM phosphate buffer, pH 6.8) Dissolved 68.0g of Potassium dihydrogen phosphate and 9.0g of sodium hydroxide into 1litre of purified water with continuous stirring and sonication. Adjusted pH with 5N Sodium hydroxide solution to 6.80±0.05. To this solution, added 30.0gm Polysorbate 20 and sonicated to dissolve completely.

Preparation of Afatinib+Ibrutinib standard solution:

Weighed and transferred about 30.0mg of Afatinib dimaleate (equivalent to about 20.0mg of Afatinib) standard and 20 mg of Ibrutinib standard into a 100mL volumetric flask and added about 70mL of diluent and sonicated to dissolve. Make up to volume with diluent and mixed well. Transferred 4.0mL of Afatinib+ Ibrutinib Standard Stock solution into a 100mL volumetric flask and diluted to volume with diluent and mixed well, to obtain a concentration of about 8ppm.

Preparation of Sample Solution for the Dissolution of Afatinib Tablets:

Prepared the dissolution bath as per dissolution parameters. Filled six dissolution vessels with a specified volume of dissolution media and allowed to attain the temperature of each vessel to 37.0°C±0.5°C. Weighed and dropped a tablet in each of the dissolution vessels and started the dissolution. At the specified time point, withdrawn about 10mL of the aliquot from each dissolution vessel and replaced an equal volume of dissolution media into the vessel. Pipette out 1.0mL of the above solution into a 10mL volumetric flask, diluted to volume with dissolution media, and mixed well.

Preparation of Sample Solution for the Dissolution of Ibrutinib Capsule:

Prepared the dissolution bath as per dissolution parameters. Filled six dissolution vessels with a specified volume of dissolution media and allowed to attain the temperature of each vessel to 37.0°C±0.5°C. Weighed and dropped a Capsule in each of the dissolution vessels and started the dissolution. At the specified time point, withdrawn about 10mL of the aliquot from each dissolution vessel and replaced an equal volume of dissolution media into the vessel. Pipette out 1.0mL of the above solution into a 20mL volumetric flask, diluted to volume with dissolution media, and mixed well.

Greenness Assessment:

The common optimized liquid chromatographic method for the evaluation of dissolution profile of Afatinib in tablets and Ibrutinib in capsules was subjected to greenness assessment. Various greenness parameters such as NEMI (National Environmental Methods Index), Modified NEMI pictograms, Analytical eco-score value, GAPI (Green Analytical Procedure Index), Complex GAPI, AGREE (Analytical greenness) and AGREE prep were utilized to show the method greenness

RESULTS AND DISCUSSION:

Optimization of Chromatographic Conditions:

The research work focused on development of a single eco-friendly liquid chromatographic method for the dissolution of Afatinib tablets and Ibrutinib Capsules. Afatinib dimaleate and ibrutinib are basic molecules with pKa value of 2.6 and 3.74 respectively. These small molecules are characterized by their molecular weights. Initial trials utilized C8 and C18 columns, which are linear alkyl silane non-polar stationary phases commonly employed for small molecules in reverse-phase HPLC.

The optimization trials were conducted in isocratic mode, using combinations of potassium dihydrogen phosphate (pH 3.0) and 90% ethanol in water as mobile phases, with varying flow rates. Using the specified mobile phase in a 20:80 (v/v) ratio and a flow rate of 0.8 mL/min, optimized separation of the two analytes was achieved within a 10-minute run time, with satisfactory chromatographic system suitability parameters.

In line with green chromatographic principles, the objective was to develop a method for the estimation of drug release of analytes in the respective dissolution mediums using columns with a short length. This approach was chosen to achieve a shorter run time, thereby minimizing effluent generation from HPLC. Unlike conventional HPLC methods that commonly use hazardous solvents as mobile phases; this study employs ethanol as a greener and safer alternative.

Optimization of Similarity factor:

Similarity factor was evaluated as method development was done parallely for two analytes. The mixed standard solution of Afatinib and Ibrutinib was prepared in the diluent, while individual standards of Afatinib and Ibrutinib were prepared in their respective dissolution media. The similarity factor, with criteria ranging from 95% to 105%, was successfully achieved between the mixed standard of Afatinib and Ibrutinib in the diluent and individual standard preparation of Afatinib and Ibrutinib in their respective dissolution media. Consequently, the mixed standard solution of Afatinib and Ibrutinib was finalized for further dissolution testing of Afatinib tablets and Ibrutinib capsules, regardless of their FDA-suggested different dissolution media. The data associated with similarity factor is tabulated under Table 1.

Table 1. Determination of Similarity factor for Afatinib and Ibrutinib in different solution

Name of drug	Solution	Weight of standard	Average peak of Afatinib	Similarity factor
Afatinib	Diluent	20.31	527538	0.99
Afatinib	Afatinib tablets Dissolution media	20.62	531363	0.99
Ibrutinib	Diluent	20.31	406488	0.99
Ibrutinib	Ibrutinib capsule dissolution medium	20.62	406712	0.99

Validation:

System suitability and Specificity: The blank (Diluent) and mixed standard solution of Afatinib and Ibrutinib in diluent (N=6) was injected into HPLC. Specificity was assessed by injecting the blank (diluent), dissolution medium for Afatinib, dissolution medium for Ibrutinib, and the Afatinib + Ibrutinib standard solution into the HPLC. As a result, no interference was observed from the blank solution, dissolution medium for Afatinib, or dissolution medium for Ibrutinib at the retention times of Afatinib and Ibrutinib. The Afatinib peak was well resolved from the Ibrutinib peak, with retention times of approximately 4.3 and 7.1 minutes, respectively. The HPLC chromatograms of the blank (diluent), dissolution medium for Afatinib, dissolution medium for Ibrutinib and Afatinib + Ibrutinib standard solution are shown in Figure 2. The system suitability parameters are presented in Table 2.

Figure 2. HPLC Chromatograms

Linearity:

The standard and sample concentration for Afatinib and Ibrutinib is ~8ppm. The linearity is performed at 50% level to 150% level of standard concentration of Afatinib and Ibrutinib. The proposed method demonstrated linearity within the concentration range of approximately 4.0–12μg/mL. The corresponding statistical values are provided in Table 2.

Precision:

Precision of Afatinib tablets: Performed dissolution on six units of Afatinib Tablets, 40 mg as per dissolution parameter. Collect the samples at 5, 10, 20, 30 and 45 minutes time point. Reported the precision at each time point for the %release of Afatinib at 5, 10, 20, 30, and 45 minutes. The %RSD results of precision for Afatinib tablets are provided in Table 2. %Drug release for Afatinib tablets is displayed under Figure 3a.

Precision of Ibrutinib Capsules: Performed dissolution on six units of Ibrutinib Capsules 140mg as per dissolution parameter. Collect the samples at 5, 10, 20, 30 and 45 minutes time point. Reported the precision at each time point for the % release of Afatinib at 5, 10, 20, 30, and 45 minutes. The %RSD results of precision for Ibrutinib Capsules are provided in Table 2.% Drug release for Ibrutinib capsules is displayed under Figure 3b.

Figure 3. Dissolution profile of Afatinib tablets and Ibrutinib capules

Accuracy:

Established accuracy, spiked samples of Afatinib and Ibrutinib Active Pharmaceutical Ingredients (APIs) are prepared at different concentrations (50%, 100%, and 150% of the expected dissolution concentration). After performing the dissolution test, the accuracy is calculated by comparing the recovered amount of analyte to the known spiked amount. The recovery results obtained for Afatinib and Ibrutinib accuracy are mentioned under table 2.

Robustness:

Robustness was done for the optimized chromatographic method for the parameters of flow rate, % organic composition and pH of mobile phase. Chromatographic results of Rt, peak area, USP plate count and tailing was evaluated. The results are displayed under table 3.

Greenness Assessment of the Optimized Method:

NEMI (National Environmental Methods Index label):

The NEMI pictogram is shown in figure 4a. Among 4 quadrants, 3 are coded as green and one colorless. The reagents employed are non-toxic, hence 1^st quadrant coded as green. As Orthophosphoric acid is hazardous, hence 2^nd quadrant is colorless. 3^rd and 4^th quadrant are green as pH is between 2-12 and total waste generated is less than 50mL respectively. Based, on obtained NEMI pictogram, the proposed method can be coded as green method.

Modified NEMI:

The modified NEMI pictogram consists of 5 quadrants/portions. Among 5 quadrants 2 are green and 3 are yellow. Modified NEMI pictogram coded based on overall utilized chemical’s NFPA health hazard value, flammability value waste generated and environmental risk. Based on the obtained modified NEMI pictogram, as depicted under figure 4b, it can be said that the proposed method is green.

Analytical Eco-Scale Score Value:

Analytical eco-scale score is calculated based on the penalty points. To obtain total penalty points hazard penalty points is multiplied with amount penalty points. Hazard penalty points calculated based on multiplying number of pictograms multiplied with signal of each respective chemical. Based on each respective amount of chemical, amount penalty points considered. Penalty points taken for waste generated and instrument energy. The obtained sub penalty points are summed up to get total penalty points. The total penalty points are subtracted from 100 to get score. Ethanol is major solvent employed here in this method. Ethanol is a green and biodegradable solvent. Based on the green chemistry institute pharmaceutical meetings, AstraZeneca’s GlaxoSmithKline’s employed guidelines stating ethanol is coded as green due to its eco-friendly nature, low toxic, easily biodegradable and disposable. Hence ethanol opted as major mobile phase in compassion to regular acetonitrile and methanol which are toxic and hazardous in nature. The total penalty points obtained for the proposed method is 6. The analytical eco-scale score value obtained for the proposed method is 94, as tabulated under table 4. Based on this score of 94, it can be affirmed that the proposed method for dissolution testing of Afatinib tablets and Ibrutinib capsules is found to be an excellent green method.

Table 4. Analytical eco-scale score value for optimized chromatographic dissolution method for Afatinib tablets and Ibrutinib capsules

Parameter	Hazard Penalty Points	Amount = Amount Penalty Points	Penalty Points
KH₂PO₄	0 × 1 = 0	<10 mL (g) = 1	0 × 1 = 0
Dil. OPA	1 × 2 = 2	<10 mL (g) = 1	2 × 1 = 2
Ethanol	0 × 0 = 0	<10 mL (g) = 1	0 × 1 = 0
Instrument energy	-	<1.5 kWh = 1	=1
Waste	-	8 mL/run (g) = 3	=3
Total penalty points	-	-	Σ = 6
Final Eco-scale score	-	-	=100-6 = 94

GAPI (Green Analytical Procedure Index) pictogram:

GAPI pictogram for the reported method is displayed under figure 4c. GAPI pictogram generated through the software. Each parameter input is given and the color for each portion is generated automatically. criteria are selected. Among 15 quadrants, 10 quadrants are yellow, 5 quadrants are green and 0 quadrants are red. Based on obtained GAPI pictogram it can be said that the proposed method is green method.

Complex GAPI (Complex Green Analytical Procedure Index) pictogram:

Complex GAPI pictogram is displayed in figure 4d. The extension of GAPI, it contains an additional hexagonal glyph. Likewise, GAPI hexagonal glyph also generated. Complex GAPI reveals that the proposed method is green.

AGREE (Analytical Greenness):

AGREE pictogram is generated through software and the 12 principles of green chemistry are considered. The 12 parts are color coded with different shades from red, yellow and green. Based on given data, the obtained score is 0.65 from a total score of 1.0 AGREE pictogram is shown under figure 4e. Based on AGREE pictogram, it can be proved that the method is green and eco-friendly in nature.

AGREE prep (Analytical Greenness preparation): AGREE prep pictogram generated through software is displayed in figure 4f. The circular pictogram with 10 parts is subjected to default weights. Then for each parameter input given. The obtained score is 0.69, based on obtained AGREE prep pictogram, it can be affirmed that the method is green.

CONCLUSION:

The development of greener methods that incorporate green chemistry concepts is an emerging trend in both academic and industrial fields. The focus was to optimize a single green liquid chromatographic method for two drugs dissolution profiling i.e., Afatinib and Ibrutinib. A single chromatographic method was validated and is found to be sensitive, specific, linear, accurate, precise and robust. The optimized chromatographic method for dissolution profiling of Afatinib in tablets and Ibrutinib in capsules was subjected to greenness evaluation. Based on obtained analytical eco-score value and pictograms of NEMI, modified NEMI, GAPI, complex GAPI, AGREE and AGREE prep it can be said that the developed method for both tyrosine kinase inhibitor is a green method. Hence, the eco-friendly liquid chromatographic method can be regularly employed for the estimation of % drug release of Afatinib in tablets and Ibrutinib in capsules. Method development time was reduced by developing the multi-analyte in single method. The method can be employed in determine the drug release profiles of Afatinib and Ibrutinib in their dosage forms. Thereby overcoming the hazardous chromatographic methods for dissolution profiling of Afatinib and Ibrutinib in drug product.

DECLARATION OF INTEREST STATEMENT:

No potential conflict of interest was reported by the authors.

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Received on 30.07.2025 Revised on 23.08.2025

Accepted on 16.09.2025 Published on 30.09.2025

Available online from October 07, 2025

Asian J. Research Chem.2025; 18(5):348-356.

DOI: 10.52711/0974-4150.2025.00054

This work is licensed under a Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License. Creative Commons License.

S. No.	Validation Parameter	Observation Values for Afatinib	Observation Values for Ibrutinib
1.	Retention time	4.3 min.	7.1 min.
2.	USP Plate count	4044	5710
3.	USP tailing	1.3	1.1
4.	Linearity Range	4 to 12 ppm	4 to 12 ppm
5.	Regression equation	y = 76684x + 80625	y = 55500x + 35923
6.	Correlation coefficient (r)	0.999	0.998
7.	Precision- Repeatability at 45 min. Samples-Mean % ± RSD	101.0 ± 0.81	103.0 ± 1.80
	-Maximum	102	105
	-Minimum	100	100
8.	Recovery % ± SD
	50 % Level, Mean Recovery % ± SD	98.0 ± 0.20	96.6 ± 0.68
	100% Level, Mean Recovery % ± SD	96.2 ± 1.10	97.5 ± 0.04
	150% Level, Mean Recovery % ± SD	97.5 ± 0.41	99.6 ± 0.07

*Parameter*	Rt (min.)		USP plate count		USP tailing
*Parameter*	AFT*	IBR*	AFT*	IBR*	AFT*	IBR*
Standard (0.8 mL/min.,90% Ethanol pH 3.0)	4.3	7.1	4044	5710	1.3	1.1
Low flow rate (0.7 mL/min.)	5.3	8.9	4530	7105	1.4	1.1
High flow rate (0.9 mL/min.)	3.8	6.8	4578	5502	1.1	1.1
Low Organic (85%)	5.6	9.1	4523	4589	1.3	1.2
High Organic (95%)	4.0	6.5	4387	6268	1.3	1.1
Low pH (pH 2.8)	4.1	7.3	4581	5623	1.2	1.2
High pH (pH 3.2)	3.9	7.4	4326	5375	1.1	1.1