Introduction

The emerging concept of green analytical chemistry (GAC) emphasizes strategies that produce accurate results while minimizing chemical consumption, hazardous waste, and exposure risks, without compromising analytical accuracy1. Several tools have been developed to evaluate the greenness of analytical methods. The Analytical Eco-Scale provides a simple scoring system by deducting penalty points from an ideal value of 1002, while the The Modified Green Analytical Procedure Index (MoGAPI) is an advanced extension of GAPI that combines the visual pentagram assessment with a quantitative total score, enabling easier comparison between analytical methods3. More recently, the Analytical GREEnness metric (AGREE)4 was introduced, integrating the 12 principles of GAC into a single numerical score. In parallel, the whiteness concept of analytical methods, rooted in White Analytical Chemistry (WAC), has been assessed using the RGB-12 algorithm, which balances analytical performance (red), environmental impact (green), and cost/time efficiency (blue)5.

In addition to these holistic assessment tools, we also employed a recently developed model known as D-CHEMS-16. While GAPI and AGREE provide broad, qualitative assessments based on procedure-wide principles, D-CHEMS-1 is specifically tailored for chromatographic methods, quantifying the environmental burden of mobile phases. It integrates the inherent toxicity of each solvent (total analytical hazard value, taHV) with its proportion in the mobile phase, the flow rate, and the chromatographic run time.

On the other hand, the Carbon Footprint Reduction Index (CaFRI) was also applied to assess the carbon footprint of the proposed method7.

In addition to greenness assessment, the practicality of the method was assessed using two indices: Click Analytical Chemistry Index (CACI) and the Blue Applicability Grade Index (BAGI)8,9, respectively.

This offers a more precise estimate of solvent-related hazards, complementing the broader qualitative insights of GAPI and AGREE.

Bambuterol HCl (BBL) Fig. 1a is described chemically as (3-[2-(tert-butylamino)-1-hydroxyethyl]-5-[(dimethylcarbamoyl)oxy]phenyl N, N-dimethyl carbamate hydrochloride). It acts as a long-acting beta-adrenoceptor agonist in asthma treatment, serving as a prodrug for terbutaline. Its roles include being an anti-asthmatic drug, a bronchodilator agent, a prodrug, a beta-adrenergic agonist, a sympathomimetic agent, and an acetylcholinesterase inhibitor10.

Fig. 1
Fig. 1
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Chemical structure of: (a) Bambuterol (b) Montelukast sodium.

Montelukast sodium (MTK) Fig. 1b is chemically designated as 1-[({(R)-m-[(E)-2-(7-chloro-2-quinolyl) vinyl]-α-[o-(1-hydroxyl-1-methylethyl) phenethyl] benzyl}thio) methyl] cyclopropane acetate sodium, functioning as a Leukotriene Receptor Antagonist with anti-inflammatory and bronchodilating activities11.

A literature survey revealed various methods for determining BBL and MTK in biological fluids or in pharmaceutical dosage forms alone or in combination, including RP-HPLC/UPLC12,13,14,15,16,17,18,19, TLC/HPTLC18,20, UV–Vis spectrophotometry (several dual-wavelength/ratio derivative variants)21,22,23,24, and spectrofluorimetry25. While these studies collectively demonstrate that simultaneous determination is feasible, they also reveal important trade-offs, solvent hazard and sustainability. Most chromatographic methods rely on acetonitrile and/or methanol often with acidic modifiers or even trichloroacetic acid, increasing toxicological burden and waste management needs12,13,14,15,17,25. Only a few reports explicitly appraise greenness with recognized metrics; among these, greener profiles are still constrained by solvent choice and total run waste16.

UV spectrophotometric approaches offer simplicity and low cost but can struggle with specificity in multi-component tablets and in dissolution media, where excipient/background absorption and surfactants (e.g., SLS) complicate deconvolution21,22,23,24. Fluorimetric methods increase sensitivity but may require stricter control of experimental conditions and are typically developed for simple matrices25.

Some HPLC/UPLC methods achieve short runtimes, but at the expense of using higher-hazard organic fractions or gradient programs that elevate solvent consumption per sample13,14,15. TLC/HPTLC can be rapid and economical, yet plate-to-plate reproducibility and quantitative robustness for routine QC may be limiting compared with column chromatography17,19.

Several reports provide partial validation (e.g., linearity and accuracy) in limited ranges or do not extend to dissolution testing, a high-throughput task in QC that benefits from a single, selective assay working across media and formulation matrices12,13,14,15,21,22,23,24.

Even when “green” is claimed, comprehensive, multi-tool evaluation (Eco-Scale + GAPI + AGREE + RGB-12) is seldom presented side-by-side with a conventional baseline, and solvent-hazard quantification tailored to actual LC conditions (proportions, flow, runtime) is rarely performed. This makes it difficult to rank methods beyond qualitative statements16.

Accordingly, there remains a need for a single, routine-ready method that uses low-hazard solvents in isocratic RP-HPLC, delivers robust selectivity for simultaneous BBL/MTK assay in tablets and in-vitro dissolution samples, and undergoes transparent, multi-metric greenness evaluation, ideally including a chromatographic-specific index that ties solvent hazard to real operating conditions.

Objective of the present study

We report an ethanol–phosphate buffer, isocratic RP-HPLC method for the simultaneous determination of BBL and MTK that is validated per ICH, applicable to finished product assay and dissolution profiling, and benchmarked with Eco-Scale, MoGAPI), AGREE, RGB-12, CaFRI, CACI, BAGI and a dedicated D-CHEMS-1 model that quantifies solvent-related hazards based on composition, flow rate, and runtime. This integrated evaluation evidences a substantially greener profile versus conventional ACN/MeOH-based methods while maintaining analytical performance suitable for routine QC.

Experimental

Instruments

  • For the RP-HPLC method: an Agilent HPLC 1200 infinity II series (USA) was utilized, equipped with a quaternary gradient pump (model: Agilent 1200), an auto-sampler (featuring a 100 µL sample loop and a capacity of 132 vials), and a UV detector. Separation occurred on an Inertsil C18 column (250 × 4.6 mm, 5 μm), with data analysis performed using ChemStation software.

  • For dissolution testing: an Agilent dissolution tester employed with standard USP paddles (USA).

Samples

Standard samples

Bambuterol HCl (purity 99.80% ± 0.61) and Montelukast sodium (purity 101.60% ± 0.76) were obtained from Western Medical Company, El-Obour, Cairo, Egypt, and their purities were determined against certified reference standards using reported methods16.

Pharmaceutical formulation

Montair plus® tablets (Batch No. SA20898), bought from India and designated to contain 10 mg of Bambuterol HCl and 10 mg of Montelukast sodium per tablet.

Chemicals

Analytical grade chemicals, including ethanol (HPLC grade), potassium dihydrogen phosphate, O-phosphoric acid and sodium lauryl sulfate were used. Purified water was collected using a Milli-Q water purification system.

Standard solutions

Stock standard solutions of Bambuterol HCL and Montelukast sodium (200 µg/mL in ethanol) were prepared by dissolving 10 mg of each drug in a 50 mL volumetric flask with 30 mL ethanol, followed by sonication for 5 min and then the volume was completed with ethanol.

Chromatographic conditions

Optimal separation conditions include a mobile phase of ethanol and 0.025 M potassium dihydrogen phosphate buffer (pH 3.0) in a 70:30 (v/v) ratio. Isocratic separation was obtained by Inertsil C18 column (4.6 × 250 mm) at a flow rate of 1 mL/min. UV detector was adjusted at 220 nm, and all steps were conducted at ambient temperature.

Method validation

Validation followed ICH guidelines26.

Linearity

Aliquots ranging from 0.06 to 5.00 mL for BBL and 0.25 to 5.00 mL for MTK were accurately transferred from their respective 200 µg/mL stock standard solutions into two groups of 10-mL volumetric flasks. The mobile phase was used to dissolve and complete the volume to the mark. Injection of 20 µL of each concentration onto the analytical column achieved separation under the specified chromatographic conditions. Peaks were detected using a UV detector at 220 nm. Linearity was assessed by constructing calibration curves of BBL and MTK over the specified ranges using six non-zero concentrations. Each concentration was injected in triplicate, and calibration plots of mean peak area ratio versus concentration were obtained and regression equations were then computed.

Accuracy

Pure samples of BBL and MTK were analysed in triplicates at varying concentrations (10.00, 20.00, 30.00 µg/mL) to ensure accuracy. Regression equations were then applied to determine the concentrations of each drug. To validate the method’s accuracy at different stages of standard additions, recovery percentages from tablet dosage forms were obtained. The percentage recoveries of added standards were calculated using the established regression equations.

Precision

Repeatability and Intermediate check were obtained by analysing the same three concentrations of BBL and MTK three times within a day and three times in three different days, respectively, to check accuracy of the developed method, then the RSD% were calculated for each sample.

Robustness

Effects of deliberate changes in certain parameters were observed to evaluate the robustness, such as mobile phase flow rate, column temperature, pH values, and ethanol percentage.

Specificity

Specificity was assessed using the standard addition technique to evaluate potential matrix/excipient interference. Aliquots of tablet extract were spiked with known amounts of BBL and MTK at four concentration levels within the calibration range. Each spiked level was analysed in triplicate and percent recoveries were calculated by comparing measured concentrations to the added amounts.

System suitability parameters

Parameters including retention time (Rt), capacity factor (K), selectivity (α), resolution factor (Rs), tailing factor (T), theoretical plates number (N), and height equivalent to theoretical plate (HETP) (mm) were calculated, to ascertain that the reproducibility and resolution were adequate for the analysis performed. Reference values followed USP27.

Application to pharmaceutical preparation

Ten tablets of Montair plus® drug were weighed and grinded into fine powder, the average weight of one tablet (labelled to contain 10 mg of each drug) was calculated and transferred into a 50-mLvolumetric flask, dissolved with 30 mL mobile phase and ultrasonicated for 10 min. The mobile phase was used to complete the volume to the mark. Then the obtained solution was filtered. Final concentration of 20 µg/mL of BBL and MTK were prepared by transferring 2 mL of the filtered solution into a 20-mL volumetric flask, and the mobile phase was used to complete the volume to the mark.

Application to dissolution testing

Dissolution testing followed US FDA28 using 900 mL of 0.5% w/v sodium dodecyl sulfate in distilled water as a dissolution medium. Apparatus II (paddle) was used at speed of 50 revolutions per minute and the temperature of vessel was adjusted at 37 ± 0.5 °C for 60 min. One tablet was used for monitoring the dissolution of BBL and MTK. Standard solution of 12.00 µg/mL of both BBL and MTK was obtained by transferring 3.00 mL of stock standard solution (200 µg/ml) into a 50-mL volumetric flask, and the dissolution medium was used to complete the volume to the mark. Samples of 5 mL were withdrawn each 5 min from the dissolution medium with replacement by 5 mL of dissolution medium immediately. By filtering the withdrawn samples by 0.45 μm syringe filter and injecting them directly alongside with the standard solution into the chromatographic system to test the dissolution pattern of the dosage form.

Evaluation tools of greenness and sustainability of analytical methods

Green principles signify a transformative shift toward continuity in analytical chemistry. These principles, encapsulated as the word “SIGNIFICANCE” guide analysts in applying key concepts associated with Green Analytical Chemistry (GAC) in their laboratory practices29,30. Emerging green evaluation tools, such as Eco-Scale points, GAPI, AGREE, and D-CHEMS-1, ensure analytical procedures align with GAC principles.

Different green evaluation tools have emerged to ensure that analytical procedures align with GAC principles. One such tool is Eco-Scale points, which assigns penalty points to parameters deviating from ideal green analysis. In 2018, GAPI was introduced to assess the eco-friendly assigns of the complete analytical procedure, starting from sample collection to final determination. In 2020, AGREE was developed to provide an overall evaluation of the analytical method in concert with the 12 GAC principles. D-CHEMS-1, a specialized tool, focuses on evaluating the hazards of mobile phases utilized in liquid chromatography methods by the environmental impact. The RGB12 algorithm, based on four green principles, serves as a complete tool for assessing the sustainability of analytical methods, aligning with White Analytical Chemistry (WAC) principles. These tools together contribute to enhancing and facilitating the green characteristics of the analytical process, offering unique insights into different aspects of environmental impact.

The combination of WAC and GAC principles enables the development of environmentally safe and efficient resource utilization in analytical methods while preserving high validity and efficiency31.

Analytical eco-scale evaluation

The Analytical Eco-Scale is a semiquantitative tool used to evaluate the greenness of an analytical procedure. It assigns penalty points to various method components (type and amount of reagents, energy consumption, occupational hazards, and waste generation) and deducts them from an ideal score of 100. A final score ≥ 75 indicates an excellent green method, 50–74 an acceptable method, and < 50 a non-green method2.

Modified green analytical procedure index (MoGAPI)

The Modified Green Analytical Procedure Index (MoGAPI) was applied to obtain a cumulative greenness score. The MoGAPI tool evaluates analytical methods across multiple parameters such as sample preparation, solvents/reagents, instrumentation, energy consumption, waste, and occupational hazards. Each criterion is scored, and the total value ranges from 0 to 100, with higher scores indicating greener procedures. The MoGAPI assessment was carried out based on the experimental details of the developed RP-HPLC method, including solvent type, run time, waste volume, energy use, and sample preparation steps3.

AGREE assessment

The Analytical GREEnness (AGREE) metric is a software-based tool that evaluates compliance of an analytical procedure with the 12 principles of Green Analytical Chemistry. Each principle is individually scored, and the final output is a circular diagram with a numerical score from 0 (least green) to 1 (most green). This provides a simple yet comprehensive overview of method sustainability4.

RGB 12-model assessment

The RGB-12 model, developed as part of the White Analytical Chemistry (WAC) framework, evaluates sustainability by integrating analytical efficiency, ecological impact, and economic feasibility5. The model uses the three primary colors to represent different criteria:

  • Red: analytical performance (accuracy, precision, LOQ, robustness).

  • Green: environmental and operator safety (toxicity, hazards, waste).

  • Blue: cost, time-effectiveness, and simplicity.

The RGB model then combines these into a “whiteness” score, which symbolizes overall sustainability. The method brilliance parameter merges the three colors according to user priorities. Evaluation is performed using standard Excel spreadsheets, freely available from the developers.

RGB-12 scoring was computed with the authors’ Excel tool using equal weights (R = G=B). Input parameters were:

  • Red (analytical performance): accuracy (mean recovery), precision (RSD), LOQ, robustness (per Tables I–III). Green (environment & safety): solvent hazard and waste per run (ethanol/buffer vs. MeOH/ACN/TCA), operator safety; cross-checked with Eco-Scale, AGREE, and D-CHEMS-1 outcomes. Blue (cost & time): total run time, sample prep complexity, and per-run solvent volume at the stated flow rate.

  • Normalized 0–100 scores for R, G, and B were exported along with the overall ‘whiteness’.

D-CHEMS-1 model

D-CHEMS-1 model scores solvents frequently utilized in analytical chemistry6. The results obtained by the CHEMS-1 model aid analysts in selecting greener solvents for environmentally friendly analytical methods. It calculates the environmental impact (E1) for a single run in the HPLC method using the total analytical hazard value for the employed solvent (taHV), the percentage volume of solvent used (S%), the flow rate (FR), and the retention time (Rt) for the last analyte.

The algorithm is as follows:

$$E1=\left( \begin{gathered} \left( {S1 \times taHV \times S1\% } \right) + \left( {S2 \times taHV \times S2\% } \right) \hfill \\ + \left( {S3 \times taHV \times S3\% } \right) + \ldots \hfill \\ \end{gathered} \right) \times FR \times \left( {Rt\, + \,0.5} \right)$$

Whereas S1, S2 and S3 represent the reagents used to constitute the mobile phase, taHV is obtained for each chemical [34], and 0.5 is added to the last retention time.

The total analytical hazard value was calculated for the mobile phases used in the HPLC methods, based on the type and amount of the used solvent. In the reported RP-HPLC method, methanol, acetonitrile, and trichloroacetic acid were used as solvents, while the developed method used ethanol and buffer. Ethanol is considered the least hazardous solvent used as a mobile phase compared to methanol, acetonitrile, or trichloroacetic acid. The taHVs were 15.7 and 26.8 for methanol and acetonitrile, respectively and 76 for trichloroacetic acid. The taHV for ethanol and buffer is defaulted to zero.

GEAR

The Green Environmental Assessment and Rating for Solvents (GEARS) is a solvent-focused greenness evaluation approach developed to rank organic solvents based on their intrinsic environmental, health, and safety (EHS) characteristics. The GEARS concept relies on literature-reported physicochemical, toxicological, and regulatory data to assess solvent-related hazards such as human toxicity, environmental impact, volatility, and occupational exposure risks. Unlike holistic greenness assessment tools that evaluate the entire analytical procedure, GEARS specifically targets solvent selection, allowing direct comparison between commonly used organic solvents. Consequently, GEARS is particularly useful as a complementary tool to chromatography-dependent models, providing additional insight into the inherent safety and sustainability of solvents employed in analytical methods33.

CaFRI

The Carbon Footprint Reduction Index (CaFRI) was also applied to assess the carbon footprint of the proposed method. CaFRI is a dedicated metric that estimates CO₂-equivalent emissions from solvent usage, energy consumption, and waste generation, and compares them with a conventional baseline method. The resulting index ranges from 0 to 1, with values closer to 1 indicating a higher reduction in carbon footprint7.

CACI and BAGI

CACI is a recent multi-criteria metric for quickly rating analytical methods—designed to be as simple and decisive as “click chemistry,” hence the name. CACI focuses on practicality and feasibility as well as greenness. In its framework, you score a method across core aspects such as sample size, sample preparation, feasibility/applicability, portability, sensitivity, and automation, then the tool aggregates these into an overall index and a pictogram to compare methods side-by-side. It’s proposed as a lightweight complement to tools like GAPI/AGREE and RGB/ “white” assessments, helping avoid overly complex checklists while still capturing safety/simplicity/throughput considerations. BAGI is a metric tool for evaluating the practicability of a method in analytical chemistry and scoring them from 25 to 100 (the higher the score, the more practical the method). It can be used to quickly find the strong and weak points of a method in terms of its applicability and to compare the performance of different analytical methods. Both indices were calculated using the respective online tools provided by their developers8,9.

Results

Development and optimization of chromatographic conditions

Mobile phase pH and buffer strength were optimized to resolve BBL and MTK with symmetric peak shapes and robust retention in an ethanol–aqueous system. Screening across pH 2.5–4.5 and 0.010–0.050 M phosphate showed that pH 3.0 minimized silanol-related tailing of the basic BBL (protonated at pH 3) while maintaining stronger hydrophobic retention for MTK (carboxyl group largely protonated), thereby enhancing selectivity. A 0.025 M phosphate buffer afforded adequate buffering capacity and baseline stability without unnecessary increases in viscosity or backpressure and avoided precipitation risks at higher organic content. Accordingly, 0.025 M phosphate (pH 3.0) in ethanol–buffer was adopted as the optimum condition for simultaneous assay and dissolution applications. Efficient separation of BBL and MTK was achieved after several attempts to resolve the two peaks in the chromatogram. Initially, varying ratios of water and ethanol were tested, but these trials did not provide satisfactory separation. The use of 0.025 M phosphate buffer with ethanol, instead of water, resulted in improved separation. The buffer’s pH was adjusted to 3.0 using phosphoric acid, further enhancing peak shape and separation.

Multiple ethanol-to-buffer ratios (30:70, 40:60, 50:50, 60:40, 70:30, and 80:20 v/v) were evaluated, with the optimal separation and good peak symmetry for BBL and MTK obtained using a 70:30 v/v ratio of ethanol to 0.025 M buffer. This chromatographic condition improved system suitability parameters, enhancing resolution between the two peaks and ensuring selectivity.

Different analytical columns and flow rates were also tested. The final method employed an Inertsil C18 column (250 × 4.6 mm, 5 μm) with a 1.0 mL/min flow rate at ambient temperature, providing optimal resolution and separation efficiency (Fig. 2).

Fig. 2
Fig. 2
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HPLC chromatogram of bambuterol HCl (BBL) (Rt: 2.258 min) and montelukast sodium (MTK) (Rt: 15.646 min) using mobile phase composed of ethanol and 0.025 M phosphate buffer pH = 3 (70: 30, v/v), with flow rate 1 mL/min.

Method validation

Linearity

Calibration curves demonstrated a linear relationship between the peak areas and concentrations and the linearity was demonstrated up to 100 µg/mL. To maintain ≤ 5% relative intercept contribution to signal, the validated working ranges are 1.2–100 µg/mL for BBL and 5.0–100 µg/mL for MTK. The regression equations for both compounds are presented in Table 1. Representative calibration curves are provided in Supplementary Figures S1 and S2, confirming the linear relationship within the validated ranges.

Table 1 Regression and analytical parameters of the proposed RP-HPLC method for the determination of BBL and MTK.
Full size table

Limits of detection and quantification

The limits of detection (LOD) and quantification (LOQ) were determined based on the standard deviation of the response and the slope of the calibration curve. The LOD and LOQ values, calculated using (3.3σ/S) and (10σ/S), respectively, are detailed in Table 1, indicating the high sensitivity of the developed method.

Accuracy

The method’s accuracy was confirmed by analysing nine samples of BBL and MTK at three concentration levels (10, 20, and 30 µg/mL), each in triplicate. The mean percent recoveries and standard deviations (SD) for both drugs are summarized in Table 1.

Precision

Repeatability and intermediate precision results for BBL and MTK were consistent, with relative standard deviation (RSD%) values below 2%, as shown in Table 1. This highlights the precision and reliability of the developed method.

System suitability parameters

System suitability parameters, such as resolution, tailing factor, and theoretical plates, met the criteria recommended by the FDA. The detailed parameters are listed in Table 2.

Table 2 System suitability parameters for BBL and MTK by the proposed RP-HPLC method.
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Robustness

Robustness was assessed by introducing minor variations in the mobile phase pH, column temperature, flow rate, and ethanol percentage in the mobile phase. The method remained unaffected by these changes, demonstrating its robustness. The results are presented in Table 3.

Table 3 Robustness of the proposed RP-HPLC method.
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Specificity

Results of the standard addition experiments, as shown in Table 4 demonstrated accurate recoveries for both analytes in the presence of tablet excipients and dissolution media. Mean recoveries for BBL and MTK were 100.40 and 100.86, respectively. The percent relative standard deviation (RSD) for all levels did not exceed 1.377 confirming that the matrix did not significantly affect quantitation. These findings confirm method specificity for the intended matrices. Although placebo or blank matrix samples were not available for experimental analysis, the absence of co-eluting peaks, together with stable retention times and acceptable peak symmetry, supports the specificity of the developed method for the intended analytical application.

Table 4 Determination of BBL and MTK in pharmaceutical formulation by the proposed HPLC method and application of standard addition technique.
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Application of the method for Montair plus® tablet analysis

The developed method was successfully applied to quantify BBL and MTK in Montair Plus® tablets. The standard addition technique further confirmed accuracy, with results for mean percentage recovery and RSD% within acceptable limits, as shown in Table 4.

Statistical analysis

A statistical comparison between the results obtained using the proposed method and a reported RP-HPLC method16 was performed using t- and F-tests. No significant differences were observed, as shown in Table 5.

Table 5 Statistical analysis of the results obtained by the proposed method and the reported method for determination of BBL and MTK.
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In-vitro dissolution testing of Montair plus® tablets

The dissolution profile of BBL and MTK in Montair Plus® tablets was monitored using the developed HPLC method. Following United State Food and Drug Administration specifications28, the dissolution medium enabled the calculation of drug release percentages over time. The dissolution profile (Fig. 3) indicated that Montair Plus® tablets exhibit immediate-release characteristics, with over 85% of both drugs released within 20 min. Dissolution results were evaluated descriptively to assess the release behaviour of the studied product. Calculation of the similarity factor (f₂) was not performed, as a suitable reference product was not available for direct comparison. Despite this limitation, the obtained dissolution profiles demonstrated consistent release characteristics and acceptable reproducibility across sampling time points.

Fig. 3
Fig. 3
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Dissolution profiles for BBL and MTL in Montair plus® tablet obtained by the developed RP-HPLC method.

Evaluation tools of greenness and sustainability of analytical methods

The greenness of the proposed ethanol-based RP-HPLC method was evaluated in comparison with the reported method16 using different assessment techniques.

Eco-scale points for evaluation of environmental impact

According to the Analytical Eco-scale, values of 50–75 indicate acceptable green analysis. Both methods thus fall into this category, with the developed method (72) closer to the excellent threshold (> 75) than the reference method (65). As shown in Table S1, the proposed method received significantly fewer penalty points than the reported method due to the use of ethanol instead of methanol, acetonitrile, and trichloroacetic acid. The higher Eco-Scale score confirms the superior environmental performance of the developed method.

The 12 principles of red green blue algorithm

The RGB-12 pictogram (Fig. S3) shows that the developed method scores higher in Green (ethanol/buffer; lower solvent hazard) and comparable in Red (accuracy/precision/robustness), while it scores lower in Blue because the run time and per-run solvent volume are higher than the reported method. With equal weights (R = G=B), the overall ‘whiteness’ of the developed method is comparable to the reported method.

Modified green analytical procedure index model (MoGAPI)

While the GAPI pictogram provides a visual overview, it does not offer a single score to rank method’s greenness. Therefore, MoGAPI was employed as a complementary tool. Both the developed RP-HPLC and the reported methods achieved MoGAPI scores of 77/100, although the overall greenness indices of both methods are similar when expressed as a single aggregated score, their environmental profiles differ substantially. The proposed method substantially reduces solvent toxicity and operator exposure through substitution of ethanol and buffer, whereas the reported method scores better for throughput (shorter run time and lower per-sample runtime). Thus, comparable global scores mask complementary trade-offs — the proposed method improves safety and solvent-related environmental burden while incurring longer analysis time and higher per-run solvent volume. (Fig. 4).

Fig. 4
Fig. 4
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Modified Green Analytical Procedure Index MoGAPI scores for the proposed RP-HPLC method versus a reported method.

Analytical greenness model (AGREE)

AGREE analysis (Fig. 5) yielded an overall score of 0.65 for the proposed method and 0.51 for the reported method. Although both methods exhibited limitations in offline sampling and high energy consumption, the developed method benefited from ethanol substitution, producing greener results in solvent-related sectors.

Fig. 5
Fig. 5
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The AGREE symbols results for the developed and reported HPLC methods.

D-CHEMS-1 model

Application of the D-CHEMS-1 model revealed a dramatic reduction in solvent-related hazards. The reported method produced an E1 value of 5622.5 due to methanol, acetonitrile, and trichloroacetic acid, whereas the proposed ethanol-based method scored only 16.14 (Fig. S4). This striking difference underscores the greenness advantage of ethanol substitution and highlights the unique value of D-CHEMS-1 in quantifying solvent hazards under real chromatographic conditions.

GEAR

To address the intrinsic environmental, health, and safety (EHS) properties of the organic solvents independently of chromatographic operating conditions, the Green Environmental Assessment and Rating for Solvents (GEARS) was applied as a complementary evaluation tool. GEARS focuses specifically on solvent hazards and therefore provides information that is not captured by chromatography-dependent models such as D-CHEMS-1.

As shown in Fig. 6 and Table S2, ethanol used as the organic modifier in the proposed RP-HPLC method exhibited markedly higher GEARS scores compared with methanol and acetonitrile, which were employed in the reported method. Ethanol demonstrated superior performance across all evaluated criteria, including human toxicity, environmental toxicity, volatility/exposure risk, and regulatory concern. In contrast, methanol and acetonitrile showed lower GEARS scores, reflecting their higher toxicity, increased occupational exposure risks, and stricter regulatory classifications. These results clearly indicate that the proposed method benefits from a safer and more environmentally benign solvent system compared with the reported RP-HPLC method.

Fig. 6
Fig. 6
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GEARS-based comparison of organic solvents used in the proposed and reported RP-HPLC methods.

CaFRI

The environmental impact of the proposed HPLC method was evaluated using the Carbon Footprint Reduction Index (CaFRI) framework. The calculated total CaFRI score was 69. The detailed profile is presented in (Fig. 7), which visualizes the performance across eight key criteria: Energy, CO₂ Emissions, Reagents/Solvents, Waste, Storage, Transportation, Personnel, and Recycling. As illustrated, the method performs well in terms of sample storage, transportation, and personnel efficiency, all indicated by green zones, due to the lack of required storage, on-site analysis, and full automation. However, the assessment highlights several areas with medium environmental impact (yellow zones), namely energy consumption, associated CO₂ emissions, solvent usage, and waste generation. Most critically, the recycling of reagents or solvents was identified as a major weakness (red zone), presenting a significant opportunity for reducing the method’s overall environmental burden.

Fig. 7
Fig. 7
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CaFRI profile of the proposed HPLC method showing its environmental performance across eight impact criteria (total score = 69).

Consequently, future improvements should prioritize the implementation of a solvent recycling system and investigate pathways to reduce energy consumption and organic solvent volumes, which would directly enhance the method’s greenness profile.

CACI and BAGI

To ensure that the developed RP-HPLC method is not only green but also applicable, the CACI and BAGI tools were applied. The method achieved a CACI score of 69/80 for the proposed method versus 70/80 for the reported method, suggesting low methodological complexity and strong suitability for routine analysis (Fig. 8). In parallel, the BAGI comparison (Fig. 9) classified both procedures as practical (≥ 60). The reported method scored 75.0/100 versus 72.5/100 for the developed ethanol/buffer method. The small edge for the reported method derives from the samples-per-hour attribute (≈ 3.2-min vs. ≈ 16-min runs), while other attributes (instrumentation, sample prep, automation, reagent availability, preconcentration) were comparable. When considered alongside the other greenness tools (Eco-Scale, AGREE, GAPI/MoGAPI, RGB 12, D-CHEMS-1, and CaFRI), the CACI and BAGI outcomes provide a more comprehensive evaluation of the method’s sustainability and practicality.

Fig. 8
Fig. 8
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Click Analytical Chemistry Index (CACI) scores for the proposed RP-HPLC method versus a reported method.

Fig. 9
Fig. 9
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Blue Applicability Grade Index (BAGI) metric for the developed and reported HPLC methods.

Discussion

Optimization of chromatographic conditions

The method development process demonstrated the critical role of buffer composition and pH in achieving optimal separation of BBL and MTK. Initial trials with water and ethanol mixtures proved insufficient in resolving the two peaks, necessitating the introduction of a phosphate buffer. The selection of 0.025 M phosphate buffer, combined with ethanol, significantly improved peak resolution and system suitability.

Moreover, the pH adjustment to 3.00 contributed to better peak shapes, highlighting the importance of pH in controlling the ionization of the analytes and, consequently, their retention behaviour. The ratio of ethanol to buffer (70:30 v/v) proved optimal, balancing separation efficiency and peak symmetry. This condition, combined with the use of the Inertsil C18 column and an optimized flow rate, resulted in a robust method with high selectivity for both compounds.

Method validation

The method validation confirmed its reliability, accuracy, precision, and sensitivity. The linearity range for both BBL and MTK covers the relevant concentrations typically encountered in tablet dosage forms, making the method highly applicable in pharmaceutical analysis. The low LOD and LOQ values further emphasize the method’s ability to detect trace amounts of both drugs, enhancing its utility for quality control purposes.

The accuracy and precision assessments, demonstrated through mean recovery percentages and low RSD values, ensure that the method consistently provides reproducible results. The robustness evaluation, which tested the method’s performance under minor variations in experimental conditions, highlights its suitability for routine application without compromising accuracy or precision. The unavailability of placebo samples represents a limitation of the present study; however, the chromatographic separation achieved ensures reliable quantification of the analytes without observable interference under the applied conditions.

Application to tablet dosage forms

The successful application of the method in the assay of Montair Plus® tablets underscores its practical utility. The standard addition technique confirmed the method’s accuracy, making it reliable for routine analysis of BBL and MTK in pharmaceutical formulations. Additionally, the statistical comparison with a reported RP-HPLC method demonstrated no significant differences, confirming the proposed method’s equivalency and efficiency.

Dissolution profile analysis

The dissolution study revealed that Montair Plus® tablets possess immediate-release properties, with over 85% of both BBL and MTK dissolved within 20 min. This rapid dissolution profile is essential for ensuring timely therapeutic effects. The HPLC method’s capability to monitor the dissolution behaviour of both drugs further enhances its value in quality control and formulation development, where understanding dissolution characteristics is critical for optimizing drug release.

In conclusion, the developed RP-HPLC method is efficient, accurate, precise, and eco-friendly, making it an ideal choice for the simultaneous determination of BBL and MTK in both dosage forms and dissolution studies. The absence of a reference product represents a limitation for statistical profile comparison; however, the primary objective of the dissolution study was to confirm adequate release behaviour rather than establish equivalence.

Evaluation tools of greenness and sustainability of analytical methods

The comprehensive evaluation of both the developed and reported RP-HPLC methods using multiple green analytical tools clearly demonstrates the environmental superiority of the proposed method.

The Eco-Scale assessment reflected the reduced environmental impact of the developed method, primarily due to the substitution of ethanol for more hazardous solvents such as methanol and acetonitrile.

The RGB 12 algorithm further underscored these advantages, with the developed method achieving higher scores in both eco-friendliness and cost-effectiveness. These gains were attributed to lower energy consumption and the use of safer chemicals, while analytical performance remained comparable between the two methods.

The MoGAPI provided a detailed step-by-step evaluation of the environmental footprint. The developed method received more green zones, particularly in parameters related to reagent hazards and sample handling, highlighting the benefits of avoiding highly toxic solvents like trichloroacetic acid, which were employed in the reported method.

The AGREE model, based on the 12 principles of Green Analytical Chemistry, offered additional confirmation. The proposed method obtained a higher overall score, reflecting improvements in hazardous waste reduction and reagent safety. Nevertheless, both methods showed limitations related to high energy consumption, a common challenge for HPLC techniques.

Finally, the D-CHEMS-1 model delivered a focused assessment of the mobile phase. Here, the developed method dramatically outperformed the reported one, with a striking reduction in the E1 score. This improvement stemmed from the replacement of methanol, acetonitrile, and trichloroacetic acid with ethanol and buffer. Unlike other tools, D-CHEMS-1 uniquely integrates solvent hazard indices with actual solvent volumes and chromatographic conditions, providing a more precise evaluation of greenness in LC methods.

The combined use of GEARS and D-CHEMS-1 offers a more complete evaluation of solvent greenness. GEARS confirms the intrinsic safety advantage of ethanol relative to methanol and acetonitrile, whereas D-CHEMS-1 demonstrates how these solvent choices impact the overall environmental burden under real chromatographic conditions.

Compared to conventional RP-HPLC methods for simultaneous determination of BBL and MTK, our developed RP-HPLC method offers a more environmentally safe alternative. It exhibits enhanced peak efficiency and resolution for both drugs, utilizing an eco-friendly mobile phase in a straightforward isocratic elution. The method’s uniqueness lies in its safe applicability for daily analysis of BBL and MTK in their pharmaceutical dosage form, addressing both chemical and physical aspects.

Furthermore, the evaluation of whiteness and greenness confirms the environmental safety of the developed method, minimizing harmful effects on human health and the environment. However, a limitation of the current method is the relatively long retention gap between the first and last peaks, which extends the run time. While gradient elution or further optimization might shorten the analysis, repeating the separation was beyond the scope of the present study.

Additionally, the CaFRI value of 69 highlights a notable reduction in the method’s carbon footprint, further supporting its classification as a sustainable and eco-friendly analytical procedure. Consistent with the RGB-12 Blue dimension and the reviewer’s point on throughput, BAGI shows that our method trades speed for solvent safety: practicality remains high (72.5) but is slightly lower than the reported procedure (75.0) due to the longer run time, whereas analytical performance and operational simplicity are preserved.

Future work may therefore focus on optimizing chromatographic conditions to reduce analysis time while preserving the method’s eco-friendly profile.

Conclusion

In this work, a simple, isocratic, and eco-friendly RP-HPLC method was successfully developed and validated for the simultaneous determination of BBL and MTK in pharmaceutical dosage forms. The method, based on ethanol and phosphate buffer, avoids the use of hazardous solvents such as acetonitrile and methanol, offering a greener and safer alternative. In addition to assay determination, the method was effectively applied to dissolution profile testing, demonstrating its practical utility for routine quality control. The greenness and sustainability of the proposed method were comprehensively evaluated using multiple assessment tools, including the Analytical Eco-Scale, MoGAPI, AGREE, RGB-12, D-CHEMS-1, GEAR, CaFRI, CACI, and BAGI, providing a holistic and reliable environmental profile. While the method demonstrates strong environmental and analytical performance, it is acknowledged that the relatively longer run time, compared with some conventional HPLC methods, may limit its suitability for high-throughput analysis. Nevertheless, this trade-off is justified by the substantial reduction in environmental impact and solvent toxicity.