Development of the RP-HPLC Method for Simultaneous Determination and Quantification of Artemether and Lumefantrine in Fixed-Dose Combination Pharmaceutical Dosage Forms

Developing countries face enormous challenges with substandard and falsified antimalarial drugs. One specific issue is the lack of a simple, cost-effective, and robust HPLC method to simultaneously determine and quantify the active pharmaceutical ingredients (APIs) in fixed-dose artemether-lumefantrine pharmaceutical dosage forms. The current study developed a novel, simple, sensitive, precise, accurate, and cost-effective RP-HPLC method for the simultaneous determination and quantification of artemether and lumefantrine in pharmaceutical dosage forms. The HPLC analysis was carried out on an Agilent 1260 Infinity Series HPLC system equipped with an ODS Intersil-C8 (150 × 4.6 mm) 5.0 µm column, by isocratic elution. The mobile phase composition consisted of acetonitrile and 0.05% orthophosphoric acid buffer of pH 3.5 in the ratio of 70 : 30 v/v. The analysis was performed at a 1 mL/min flow rate and a column temperature of 25°C. The total run time was 6 minutes. The detection was done with a variable wavelength detector (VWD) at an isosbestic point wavelength (λ) of 210 nm. The developed method was validated according to the ICH guidelines concerning system suitability, specificity, linearity, accuracy, precision, and robustness. The system suitability of the developed method revealed satisfactory theoretical plates and symmetry factors. The method proved to be specific, with no interference of mobile phase or excipients. The calibration plot exhibited linearity over the concentration range of 275–1925 μg/mL with R2 = 0.9992 for artemether and a range of 150–1050 μg/mL with R2 = 0.9985 for lumefantrine. The accuracy of the method, determined by the recovery study, was 99.79–100.16% for artemether and 99.04–99.50% for lumefantrine. The % RSD values for intraday precision were 0.175 and 0.203, while interday precision values were 0.340 and 0.554 for artemether and lumefantrine, respectively. The method demonstrated robustness when subjected to slight modifications in the flow rate, column temperature, and mobile phase composition. The developed analytical method proved satisfactory as per ICH guidelines and hence can be used for the determination and quantification of artemether and lumefantrine in bulk drug and pharmaceutical dosage forms.


Introduction
At present, artemisinin-based combination therapy (ACT) is regarded as the most rapid and efective antimalarial treatment available.Malaria is a common fever disease that frequently necessitates hospitalization after travelling to areas where it is endemic [1].Following the WHO recommendations, Ghana moved from chloroquine to artemisinin-based combination therapy (ACT) as the frst-line treatment for uncomplicated malaria in 2005.Since 2014, Ghana has made extensive use of three ACTs, including dihydroartemisinin-piperaquine (DHAP), artemether-lumefantrine (AL), and artesunate-amodiaquine (AA).Fixed-dose combination (FDC) artemether-lumefantrine (AL) drug is authorized for the treatment of malaria caused by Plasmodium falciparum [2].AL is an ACT made of two active ingredients, namely, artemether, which is derived from the plant Artemisia annua, and lumefantrine, which is synthesized chemically [3].Together, these two compounds work to inhibit the growth and replication of the malaria parasite, thus curing the infection.Although the preferred treatments for severe malaria are intravenous/intramuscular (IV/IM) artesunate, IV/IM quinine, and IM artemether [4]; tablets and suspensions are considered the most widely used dosage forms of fxed-dose combination artemether-lumefantrine drug available in the market.
At the moment, poor-quality medicines present tremendous hurdles for poor nations [5] due to the lack of a simple, less expensive, robust, and stable HPLC method that could simultaneously determine the content of the APIs in artemether-lumefantrine tablets and suspensions in resource-limited settings.Tere is, therefore, an urgent need for methods that are accurate, cost-efective, easy to use, and rapid and require the use of uncomplicated equipment to facilitate easy identifcation and quantifcation of the active components in fxed-dose combinations of artemether and lumefantrine dosage forms for in-process quality control checks or routine pharmacovigilance [6].
High-pressure liquid chromatography (HPLC) is a widely used analytical technique for the separation, identifcation, and quantifcation of drugs in pharmaceutical formulations.It is a highly sensitive and selective method that can detect and quantify trace amounts of drugs [7].However, developing an HPLC method for the simultaneous determination of artemether and lumefantrine in FDC has proven to be difcult due to the physicochemical properties of the drugs [8].
Artemether is a form of artemisinin in which the lactone has undergone conversion to the associated lactol methyl ether [9].It is insoluble in water but soluble in oil.Lumefantrine belongs to the class of fuorenes and is composed of the compound 9-(p-chlorobenzylidene)-9H-fuorene, which has chlorine substituents at positions 2 and 7 and a 2-(dibutylamino)-1-hydroxyethyl group at position 4 [10].Figures 1 and 2 illustrate the chemical structure of artemether and lumefantrine, respectively.
Many studies have developed methods to quantify the API content in drugs, with the majority being used for tablet formulations.Most of these methods are not able to determine simultaneously the active ingredients present in fxed-dose combination medicines [11].Due to their distinct chemical properties, including factors such as polarity, solubility, and UV absorption, the APIs artemether and lumefantrine (classifed as BCS class II or IV drugs) exhibit poor water solubility and limited permeability [12].Tese diferences make it challenging to develop an HPLC method that can separate and quantify both drugs simultaneously in the same sample.In addition, artemether and lumefantrine have diferent retention times in HPLC columns, which further complicate the simultaneous determination of both drugs.Numerous endeavors have been undertaken to establish high-performance liquid chromatography (HPLC) methodologies for the concurrent quantifcation of artemether and lumefantrine in fxed-dose combination (FDC) drugs.Regrettably, many of these methods exhibit defciencies in sensitivity, selectivity, precision, and accuracy.Notably, the prevalent issue persists where, even within a fxed-dose combination drug, the analysis of each active pharmaceutical ingredient (API) in pharmaceutical and biological matrices necessitates separate procedures.Other challenging issues are the cost of analytical reagents used and the long run time.A study by Shamshad et al. [13] utilized a high injection volume of 20 µL, making the method less economical.Another study by Debrah and colleagues [14] was done at a temperature of 40 °C.A high column temperature makes the method unfavorable for commercial use.
Tis study aimed to develop and validate a novel reversephase high-performance liquid chromatography (RP-HPLC) method capable of concurrently detecting artemether and lumefantrine within a single solvent system within a single short run.Tis has the potential to signifcantly reduce analysis time for fxed-dose combination artemether and lumefantrine pharmaceutical dosage forms, consequently minimizing overall costs associated with the analytical process and thus underscoring the necessity of the study.

Chemicals and Analytical Reference Standards.
Primary analytical reference standards of purity ≥98% artemether and lumefantrine (Sigma-Aldrich, USA), deionized water (gifted by Tradewinds Chemist Limited,  of pore size 8 μm and used at a fow rate of 1.0 mL/min.Te separation of artemether and lumefantrine was evaluated in diferent proportions of these solvents and, for each condition, the retention factor (k) and resolution (R) were determined.
2.2.9.Wavelength and the Flow Rate.Employing a highperformance liquid chromatography (HPLC) instrument, a comprehensive scan was conducted over a wavelength range spanning from 200 nm to 400 nm, ensuring a thorough examination of absorption characteristics.Te subsequent narrowing down of this spectrum to the selection of 210 nm yielded an isosbestic point, which proved advantageous for both APIs.
In tandem with the spectral optimization, adjustments were made to the pump system to regulate the delivery of the mobile phase at varying fow rates, namely, 0.5, 0.6, 0.8, 0.9, 1.0, 1.10, 1.5, and 2.0 ml/min.Subsequent analysis of all experimental runs led to the determination that a fow rate of 1 ml/min was the optimal choice for method development.Tis particular fow rate was chosen for its demonstrated ability to facilitate efcient separations, maintain reasonable retention times, and enhance the resolution of chromatographic peaks when compared to the other tested fow rates.

Preparation of Standards.
HPLC grade reference standards equivalent to 30 mg and 55 mg of lumefantrine and artemether, respectively, were transferred into a 50 mL clean dry volumetric fask.Acetonitrile was added frst to the powdered mixture and sonicated for 10 minutes and then topped up with 0.05% orthophosphoric acid to make up the volume of 50 mL in an equivalent ratio of 70 : 30 (v/v).Te resulting mixture was fnally sonicated for better dissolution and fltered via grade 2 qualitative flter paper (Whatman ® ) of pore size 8 μm into vials.
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Preparation of Tablet and Suspension Samples.
In this study, six tablets of artemether-lumefantrine (AL) were analyzed for their average weight.Te tablets were then crushed into a fne powder and a specifc amount of this powder, equivalent to 30 mg of lumefantrine and 55 mg of artemether, was placed in a 50 mL fask.To prepare the analytical solution, acetonitrile was added to the powder and sonicated for 10 minutes.Tis mixture was then combined with a 0.05% orthophosphoric acid solution in a ratio of 70 : 30 v/v, resulting in a fnal volume of 50 mL.Te mixture was further sonicated for better dissolution and was fltered through 8 μm pore size grade 2 qualitative flter paper (Whatman ® ).Te vials used for fltration were thoroughly cleaned, rinsed, and dried beforehand.
Te suspensions were reconstituted according to the specifcations given by the manufacturer.A quantity of the suspension sample equivalent to 30 mg of lumefantrine and 55 mg of artemether was accurately measured in a 50 ml volumetric fask.Acetonitrile was added frst to the powdered mixture and sonicated for 10 minutes and then topped up with the bufer (0.05% orthophosphoric acid) to make up the volume of 50 ml in an equivalent ratio of 70 : 30 (v/v).Te resulting mixture was fnally sonicated for better dissolution and fltered via grade 2 qualitative flter paper (Whatman ® ) of pore size 8 μm into vials that have been washed, rinsed, and dried completely.

Validation of the Method.
Multiple metrics, including linearity, precision, accuracy, robustness, limit of detection, and limit of quantitation, were evaluated as part of the validation process [15].To ensure conformity to globally accepted standards, the chromatographic method's validation was carried out following a research validation protocol inspired by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2b) [16][17][18].

System Suitability.
System suitability parameters, such as the theoretical plate, the tailing factor, the peak-to-noise ratio, the similarity factor, and the retention time, were carefully examined to make sure they complied with the acceptable ranges outlined in the guidelines provided by the European Pharmacopoeia and the Center for Drug Evaluation and Research (CDER).Te peak area, retention time, and symmetry factor were calculated and analyzed for sextuplicate injections, and the symmetry factor was carefully scrutinized.Furthermore, theoretical plates that indicate column efciency were analyzed.

Specifcity.
High-performance liquid chromatography (HPLC) relies heavily on specifcity since it allows the analytical technique to identify and separate the analyte from other components in a mixture.A series of injections were carried out using several solutions that each contained 5 µL of the standard solutions of each API, a mixture of the APIs, an AL drug sample, and the blank (diluent) to evaluate the method's specifcity.Te subsequent analysis entailed scrutinizing the chromatographic profle for the presence of any interfering peaks that may compromise the accurate determination of the analyte of interest.

Linearity.
Te exact weights of each sample were accurately measured and then diluted to a fxed volume of 50 mL using the mobile phase as the diluent, resulting in the creation of nine (9) distinct concentration levels.Standard solutions encompassing concentrations ranging from 275 μg/mL to 1925 μg/mL of artemether and from 150 μg/ mL to 1050 μg/mL of lumefantrine were prepared and injected.For each concentration level, a consistent injection volume of fve microliters (5.0 µL) was used.Te resulting chromatographic data were graphically plotted against their corresponding concentrations.Te obtained R 2 values demonstrated a linear correlation between peak areas and the concentrations of the reference solutions, afrming the method's linearity.Furthermore, rigorous statistical analyses were conducted to validate the linearity of this novel method.

Calibration Curve for Artemether and Lumefantrine.
To establish the relationship between the concentrations of the solutions and the corresponding peak areas, the mean of the peak areas was calculated and plotted against their respective concentrations.By plotting these data points, distinct calibration curves for artemether and lumefantrine were generated.Tese calibration curves serve as references to estimate the amount of the respective APIs present in the samples.

Precision. Repeatability and intermediate precisions
were identifed and statistically evaluated to establish the precision of the approach proposed.
(1) Repeatability Precision.Te intraday precision was analyzed using sextuplicate of the standard solutions containing 0.800 mg/mL (80%), 1.100 mg/mL (100%), and 1.320 mg/mL (120%) of the stock solution.Te mean chromatogram peak areas of each standard solution of various concentrations were collected, and the relative standard deviation (RSD) was calculated.
(2) Intermediate Precision.Te interday precision is useful in the validation process as it gives the precision of the results of the HPLC when run on diferent days or by diferent analysts using the same developed method.Sextuplicate of the standard solutions containing 0.800 mg/mL (80%), 1.100 mg/mL (100%), and 1.320 mg/mL (120%) of the stock solution was run on three diferent days.Te mean chromatogram peak areas of each standard solution of various concentrations were collected, and the relative standard deviation (RSD) was calculated.
2.3.6.Sensitivity.Te limit of detection (LOD) and limit of quantifcation (LOQ) of the developed method were, respectively, calculated from the standard deviation and slope 4 Advances in Pharmacological and Pharmaceutical Sciences obtained from the calibration curve and the linearity test based on the signal-to-noise (S/N) ratio of 3 : 1 and 10 : 1 in the following formula: where SD � the standard deviation.S � slope of the calibration curve.

2.3.7.
Accuracy.In this study, to determine the accuracy of the proposed analytical method and detect any interference matrix from excipients used in the dosage forms, recovery experiments were conducted using the standard addition method (spiking).Tis method involved adding a known concentration of a standard to a fxed concentration of the preanalyzed sample solution.Te percent recovery was then calculated by comparing the area before and after the addition of the working standard.Recovery of the method was carried out in three sets at diferent concentration levels corresponding to 80%, 100%, and 120% of the expected concentration.By comparing the amount claimed and the amount recovered after the addition of the standard, the percentage recovery was determined and % RSD was calculated.

Robustness.
A few chromatographic parameters were intentionally altered to test the robustness of the established approach.Specifcally, the fow rate, column temperature, and mobile phase composition ratio were altered.Te analysis was done by generating six sample solutions at 100% concentration.Sample solutions were injected, and the chromatographic parameters indicated above were modifed in accordance to test the robustness of the devised method.

Method Development.
To optimize the separation of analytes of interest, extensive investigations were conducted on various chromatographic conditions.Parameters such as the mobile phase composition, stationary phase, column temperature, fow rate, wavelength, and injection volume were carefully studied.Trough a series of rigorous trials, a reverse-phase, ODS Intersil-C8 column (Phenomenex) measuring 150 × 4.6 mm with 5.0 μm particles was determined to be the most suitable stationary phase for the analysis.Te mobile phase composition of acetonitrile and 0.05% orthophosphoric acid in a ratio of 70 : 30 (v/v) was found to provide optimal separation.Moreover, a detection wavelength of 210 nm was selected as the isosbestic wavelength, allowing for maximum sensitivity and selectivity.To maintain consistent and reproducible results, a column temperature of 25 °C was deemed appropriate for the analysis.Te fow rate of 1.0 mL/min was identifed as the optimal rate, striking a balance between separation efciency and analysis time.
A minimal injection volume of 5.0 μL was determined to be sufcient for achieving well-defned peaks and maintaining sensitivity.
Utilizing an isocratic elution method, the developed chromatographic conditions were further refned to yield well-distinguished peaks with consistent retention times.Te entire analysis process was completed within a run time of 6 minutes.

System Suitability.
System suitability testing was performed to assess the suitability of the developed chromatographic system for analysis.Six replicate solutions of artemether and lumefantrine standards were utilized to evaluate retention times, symmetry factors, and theoretical plates.Te obtained results indicate favorable system performance.
Te peak symmetry exhibited satisfactory values, with an average of 0.91 for artemether and 1.32 for lumefantrine.Te average retention times were determined as 5.38 minutes for artemether and 1.42 minutes for lumefantrine.In addition, the resolution was 2.03, and average theoretical plates were found to be 2297 for artemether and 5035 for lumefantrine, signifying a sufcient level of chromatographic resolution.Te assessed parameters, including % RSD (≤2%) of peak areas and retention time, tailing factor (≤2), theoretical plates (≥2000), and peak resolution (R) (≥2), remained within the acceptable limits outlined by the British Pharmacopoeia (BP) requirements.Tus, the system suitability data, presented in Table 1, afrm that the developed chromatographic system meets the necessary criteria for the intended analysis.

Specifcity.
Te developed method exhibited excellent specifcity for the resolution of all active pharmaceutical ingredients (APIs) used in the study.No interference peaks were observed during the elution periods of the APIs, even in the presence of excipients.Te chromatograms in Figure 3 illustrate the method's specifcity.Tese chromatograms provide visual evidence of the distinct separation of the APIs, without any overlapping or interfering peaks.Te absence of extraneous signals within the elution regions of interest confrms the high specifcity of the method.

Linearity.
A graphical representation of the analytical curve displaying the peak area against concentration was generated, and the result was subjected to regression analysis.From the regression analysis, the linear equation was obtained as follows: y � 0.2296x − 1.529 for artemether and y � 15.609x + 167.14 for lumefantrine, and their coefcient of determination (R 2 ) was 0.9992 and 0.9985, respectively.Te calibration curve of artemether and lumefantrine at 210 nm is shown in Figures I and II (supplementary materials), respectively, and the linearity results of artemether and lumefantrine are shown in Table 2.  Te LOD signifes the lowest concentration at which the analyte can be reliably detected, while LOQ represents the lowest concentration at which the analyte can be quantifed with a satisfactory level of accuracy and precision.In this study, the signal-to-noise ratios of 3 : 1 and 10 : 1 were obtained for the LOD and LOQ, respectively.Te calculated LOD for artemether was found to be 7.95 μg/mL and that of lumefantrine was 0.064 μg/mL.Te LOQ was measured to be 24.10 μg/mL for artemether and 0.193 μg/mL for lumefantrine.Table 3 shows the results of the LOD and LOQ of artemether and lumefantrine.

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3.2.5.Precision.Repeatability (intraday) studies showed a % RSD of 0.175 and 0.203 for artemether and lumefantrine, respectively.It was also observed that the intermediate studies (interday) showed a % RSD of 0.340 and 0.554 for artemether and lumefantrine, respectively.Tis implies that a change in days did not have a change in the precision of the developed method by more than 2% for both APIs.Te results of the precision study for artemether and lumefantrine are presented in Table 4.

Accuracy.
Te accuracy of the developed method is shown in Table 5.Based on the standard deviation (SD), relative standard deviation (% RSD) values, and the % drug recovery, the developed method is accurate at 3 levels of concentrations with % RSD in the range 0.004-0.072for artemether and 0.092-0.511for lumefantrine.

Robustness.
A slight modifcation of certain chromatographic conditions, including adjustments to the fow rate, column temperature, and mobile phase composition, resulted in a negligible variance of no more than 2% in the assay values, as illustrated in Table 6.

Drug Assay of Market Samples (Quantitative Estimation of the API Content) Using the Validated Method.
Assay were performed on some selected brands of marketed drug products to investigate the content and concentration of the API present.Te quantity of the two APIs was estimated using the calibration curve equation.Table 7 presents the results of the analyzed market samples using the developed RP-HPLC method.

Greenness Study.
Te method development and validation processes were meticulously conducted in compliance with the principles of green chemistry and environmental friendliness (Table 8).Tis was achieved through the careful selection of chemicals for the mobile phase and the subsequent application of the developed method to analyze marketed samples.

Discussion
Numerous studies have endeavored to develop methodologies for quantifying active pharmaceutical ingredients (APIs) in fxed-dose combination drugs.However, challenges emerge in simultaneously determining artemether and lumefantrine in fxed-dose combination medicines.Given the diference in chemical properties of artemether and lumefantrine, classifed as BCS class II or IV drugs with characteristics such as poor water solubility and limited permeability, a surge in research has been witnessed, aiming to establish methods for their distinct analysis in pharmaceutical and biological matrices.Tis approach persists due to the complexities of achieving optimal separation when both drugs coexist in a solution.Te growing need for swift and cost-efective high-performance liquid chromatography (HPLC) methods is particularly pronounced in the resourcelimited setting.Te investigation revealed the signifcance of the stationary phase selection, particularly concerning the separation efciency of the drug.While the prevalent use of C18 (octadecyl) columns with acetonitrile as the main solvent is common, our study found that employing a C18 column resulted in prolonged analysis times and indistinctly separated peaks.Subsequently, utilizing an ODS Intersil-C8 column (Phenomenex) (150 × 4.6 mm with 5.0 μm particles) as the stationary phase improved the peak shape and signifcantly reduced analysis time.Tis improvement is attributed to the shorter alkyl length of the C8 (octyl) column.Tese fndings align with a method previously developed by Abd-AlGhafar et al. in 2022 [19].Tey discovered that using the C8 column gave a shorter elution time and nicely separated peaks.Also, in a study where the Phenomenex Luna C18 column (150 × 4.6 mm with 5.0 μm particles) was used as the stationary phase in the development and validation of an RP-HPLC method for simultaneous quantifcation of geftinib and resveratrol, it resulted in short retention times of 2.56 minutes and 1.80 minutes, respectively [20].
Te study employed a mobile phase composed of a 70 : 30 (v/v) mixture of acetonitrile and 0.05% orthophosphoric 8 Advances in Pharmacological and Pharmaceutical Sciences acid.Acetonitrile, chosen for its versatile ability to dissolve both polar and nonpolar analytes, is widely utilized in highliquid chromatography (HPLC) due to its high chromatographic performance [21] and availability in the high-purity HPLC grade [22].In addition, it is compatible with various stationary phases.Orthophosphoric acid was      Advances in Pharmacological and Pharmaceutical Sciences selected for its UV transparency and efcacy in optimizing the separation of artemether and lumefantrine when the pH is adjusted approximately 3-4.Similar to a study conducted by Patel et al., a fltrate compatibility study was conducted to ensure the selection of an optimal flter for obtaining a refned fltrate for subsequent analysis [23].
Te validation of the RP-HPLC method for artemether and lumefantrine assay was meticulously performed, adhering to the stringent guidelines inspired by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).Tis encompassed a thorough examination of specifcity, linearity, accuracy, precision, sensitivity (LOD and LOQ), and robustness by the regulatory framework set forth by the European Medicines Agency (1994) and ICH (Q2B, 1996).System suitability testing, involving six replicate analyses at concentrations of 1100 μg/mL for artemether and 600 μg/mL for lumefantrine, elucidated a notably favorable system performance.Te obtained results indicate favorable system performance.Under ideal circumstances, a chromatographic peak is expected to exhibit a Gaussian shape.However, if the migration and distribution of the drug within the HPLC column are nonuniform, an asymmetric peak is obtained [24].When the symmetry factor approaches 1, it suggests that the peak shape is approaching a desirable Gaussian distribution [20].Tailing or fronting can occur when there is an uneven fow in the column.To mitigate tailing, various strategies can be employed, such as adjusting the pH or ionic strength of the mobile phase, optimizing the column temperature, using diferent stationary phases or additives in the mobile phase, or modifying the sample preparation techniques [24].
Te method is specifc, highlighting the method's ability to accurately identify artemether and lumefantrine, even in the presence of potential interferences.Te linear regression analysis revealed exceptional linearity (R 2 ≥ 0.99), a key indicator of the strong correlation between the signals obtained and the various levels of concentrations prepared.Determining LOD and LOQ values, using the signal-to-noise ratio (S/N) method, demonstrated the method's sensitivity in reliably detecting and quantifying artemether and lumefantrine irrespective of other impurities or excipients that might be present.Te precision analysis, both intraday and interday, reveals a relative standard deviation (RSD) below 2%.Tis indicates that the method consistently yielded closely aligned results when the HPLC analysis was conducted within the same day or on diferent days under identical conditions.A method incapable of providing consistent results over multiple days suggests the potential for disparate values, rendering it unsuitable.Intentionally varying certain chromatographic conditions and parameters, the researchers assessed the method's robustness, and the % RSD value confrmed that the observed change was less than 2%, afrming its resilience and reliability in delivering expected results.
Comparing the proposed method with that developed by Habib et al. [25], the present method revealed better economic efciency and shorter retention time, enhancing its practical utility in pharmaceutical analyses.Furthermore, the method's alignment with green chemistry principles, encompassing considerations of reagent toxicity, compatibility, energy consumption, and biodegradability, attested to its environmental conscientiousness.
In ensuring environmental friendliness, green chemistry plays a crucial role in method development.A greenness study was conducted, considering factors such as reagent toxicity, reagent compatibility, the energy consumption of electronic devices, biodegradability of reagents and other disposable materials, and the duration of the analysis.Te results strongly indicate that the method development and validation process align very well with the principles of green chemistry.

Conclusion
A new high-performance liquid chromatography (HPLC) method has been developed and validated for the identifcation and quantifcation of artemether and lumefantrine in pharmaceutical dosage forms.Tis method ofers a reliable and efcient approach to simultaneously analyze these compounds, saving time, eliminating the costs associated with separate analyses, and being very friendly to the environment.Te method was validated for specifcity, precision, accuracy, and robustness according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.Te developed analytical method proved satisfactory as per ICH guidelines and hence can be used for the determination and quantifcation of artemether and lumefantrine in bulk drug and pharmaceutical dosage forms during manufacturing or routine pharmacovigilance [12].
of energy-efcient instrument Low energy consumption (efcient) instruments were used in the study 8 Simple size and resource efciency Minimize sample size A small sample size was utilized 9 Life cycle assessment Assess the entire life cycle impact Ensured a comprehensive understanding of the environmental impact 9 Biodegradability and environmental friendliness Selection of biodegradable and environmentally friendly chemicals All chemicals used were environmentally friendly and fast biodegradation 9 Compliance with green chemistry Tests should comply with green chemistry principles Green chemistry principles ensured 9 Reduction in analysis time Short experimental/reaction time Short run time in HPLC analyses.Use of a sonicator to speed up dissolution 9 * (0): no success, (1-3): low success, (4-6): moderate success, (7-8): high success, and (9-10): very high success.

Table 1 :
System suitability data for artemether and lumefantrine.

Table 4 :
Precision study data for artemether and lumefantrine: repeatability and intermediate precision.

Table 6 :
Results of the robustness study.

Table 7 :
Results of analyses of market samples using the developed RP-HPLC method.

Table 8 :
Results of the greenness study.