Simultaneous Estimation of Artemether and Lumefantrine by RP-HPLC Method Development in Pharmaceutical Tablet Dosage Form

Introduction

Malaria is a life-threatening but preventable and curable disease caused by the Plasmodium parasites, which are transmitted primarily through the bites of infected female Anopheles mosquitoes. The most dangerous species, Plasmodium falciparum, can cause severe illness and death within 24 hours if left untreated. Infants, young children, pregnant women, travellers, and people with HIV/AIDS are especially vulnerable to severe malaria infections.¹

The development and spread of parasite resistance to certain anti-malarial agents has presented a major barrier to successful disease management in malaria-endemic areas.² Artemether is a fast-acting drug that reduces the number of malaria parasites in the blood, while Lumefantrine works to eliminate the remaining parasites. Both these drugs are used to treat uncomplicated malaria caused by Plasmodium falciparum by clearing the parasites from the bloodstream.³

Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) is considered a highly reliable and efficient approach for simultaneously determining Artemether and Lumefantrine in pharmaceutical dosage forms. It operates using a hydrophobic stationary phase typically a C₁₈ column such as Hypersil-and a hydrophilic mobile phase, usually a phosphate buffer combined with acetonitrile in a 45:55 volume ratio, adjusted to around pH 3.5. This configuration ensures optimal retention and clear separation of the lipid-soluble drugs. Detection is commonly carried out within the 210 to 254 nm wavelength range, with 218 nm being frequently used for best results. This method offers notable advantages including high sensitivity, quick run times (often less than 4 minutes), and excellent consistency. Furthermore, it meets International Conference on Harmonisation (ICH) guidelines for validation parameters like accuracy, precision, linearity, and robustness. When compared to UV-visible spectrophotometric methods, RP-HPLC provides greater selectivity and sensitivity, effectively resolving both active ingredients even in the presence of other formulation components or degradation products. Consequently, RP-HPLC is widely adopted in both research and pharmaceutical quality control environments for the accurate quantification of these antimalarial agents.^4,5,6

Materials and Methods

Materials

Unadulterated Artemether (ART) and Lumefantrine (LUM) were used as working standards, sourced from Balaji drugs, Pontasahib (HP), India. Tablets containing 20 mg of ART and 120 mg of LUM were purchased from the market, manufactured by Themis Pharma, India, and were used within their labeled shelf life. Acetonitrile and water (HPLC-grade) were procured from Merck, India. All other substances and reagents used were of analytical grade, and purchased from Merck and Ranbaxy, India.

Solubility studies of Artemether and Lumefantrine

Artemether (ART) and Lumefantrine (LUM) are freely soluble in methanol and ethanol. They are highly soluble in acetonitrile, soluble in chloroform and water.

Instrumentation

A Shimadzu HPLC framework comprising of a LC-2010 CHT parallel inclination siphon, an inbuilt auto sampler, a section stove and double frequency absorbance indicator (DAD) was used for the investigation. The information was accessed through the Empower-2 programming. The segment utilized was Hypersil BDS balance C18 (250×4.6 mm, 5 μm). A Bandline Sonerex sonicator was utilized for upgrading the disintegration of the mixes.

Preparation of stock and standard solutions

A stock solution of ART and LUM (500 μg/mL) was prepared by accurately weighing 50 mg each of ART and LUM reference standards and transferring them into a 100 mL volumetric flask containing 50 mL of acetonitrile. Following this, the volume was made up to the mark with acetonitrile. The stock solution was protected from exposure to light using aluminum foil. Aliquots of the standard stock solutions of ART and LUM were transferred using an A-grade bulb pipette into 10 mL volumetric flasks, and the volumes were made up to the mark, thus obtaining final concentrations of 4-24 μg/mL and 24-144 μg/mL of ART and LUM, respectively.

Estimation of Artemether and Lumefantrine in tablets

To determine the content of ART and LUM in tablets (Label guarantee: 20 mg and 120 mg respectively), 20 tablets were selected and their total weight was recorded. An aliquot of powder proportional to the weight of one tablet was precisely gauged and transferred to 50 mL volumetric flask. The tablet was then disintegrated in 25 mL acetonitrile and volume was made up to the mark with acetonitrile. The cup was sonicated for 20 minutes to facilitate complete disintegration. The solution was then filtered through a 0.45 μm Millipore channel. An appropriate aliquot of the tablet extract was transferred into a 100 mL volumetric flask and diluted to volume with the mobile phase to obtain a final concentration of 10 μg/mL for ART and 60 μg/mL for LUM.^5,6,7

The experiments were performed six times under optimized chromatographic conditions as previously described. The peak areas were measured at 215 nm, and the concentration was determined by comparing the area of the sample to the standard.

Results

Linearity Working solutions of Artemether (ART) and Lumefantrine (LUM) were prepared in concentrations ranging from 4 to 24 μg/mL and 24 to 144 μg/mL, respectively. Under optimized chromatographic conditions, triplicate measurements were obtained, and peak areas were plotted against concentrations to generate calibration curves. Both ART and LUM demonstrated good linearity within their respective ranges.

Accuracy

Accuracy was validated through recovery studies using the standard addition method. Known quantities of standard ART and LUM were added to pre-analyzed samples at three levels, 80%, 100%, and 120% of the label claim. The method showed satisfactory recovery, indicating high accuracy.

Precision (Repeatability)

Intra-day and inter-day precision were assessed by analyzing ART and LUM at all concentrations within the linear range. Triplicate measurements were taken on the same day and over three consecutive days. The results demonstrated acceptable repeatability, confirming method precision.

Reproducibility

Reproducibility was evaluated by conducting the analysis on the same instrument but by a different analyst within the same laboratory. Triplicate measurements of ART and LUM at various concentrations confirmed consistent results, supporting the method’s reproducibility.

Robustness

Method robustness was confirmed by introducing deliberate variations in chromatographic parameters. Changes included ±2% in the mobile phase composition and ±0.2 units in buffer pH. These variations did not significantly affect method performance.

System Suitability

System suitability was established using ten injections of a working standard solution containing 10 μg/mL of ART and 60 μg/mL of LUM. Parameters such as retention time, tailing factor, and number of theoretical plates met the acceptance criteria.

Discussion

The method validation results confirm that the developed RP-HPLC method is suitable for the simultaneous estimation of Artemether and Lumefantrine in tablet formulations. The linearity, accuracy, and precision data suggest that the method performs reliably within the specified concentration ranges. Recovery rates near 100% reinforce the method's accuracy, and low RSD values in precision and reproducibility tests affirm its reliability and repeatability.

Furthermore, robustness testing demonstrated that minor deliberate changes in chromatographic conditions did not significantly influence the results, highlighting the method's stability under variable laboratory conditions. The system suitability parameters further support the method's effectiveness for routine quality control.

Overall, the findings suggest that the proposed method is scientifically sound, analytically valid, and appropriate for the intended pharmaceutical applications.

The quality and accuracy of fixed-dose combination formulations of Artemether and Lumefantrine are critical for effective malaria treatment. Nyarko et al., highlighted the concerning presence of falsified and substandard Artemether-Lumefantrine products in parts of Ghana, emphasizing the urgent need for reliable analytical techniques to ensure drug quality.⁸

In this context, the study by Nagvekar et al., demonstrates the utility of UV-visible spectrophotometry using the simultaneous equation method as a simple and costeffective approach for estimating these compounds in tablet form.⁹ While not as selective as chromatographic techniques, this method still offers acceptable precision and accuracy for routine quality checks, especially in resource-limited settings. Together, these studies underscore the importance of validated analytical methods in safeguarding drug quality and public health.^10,11

Conclusion

The proposed RP-HPLC technique is rapid, specific, accurate and precise for the estimation of ART and LUM in tablet dosage forms. Compared to the previously reported methods, this technique offers several advantages, including a wide scope of linearity, utilization of readily available mobile phase, and absence of extraction techniques. Each of these variables make this method highly suitable for evaluation of ART and LUM in tablet dosage forms.

The method can be effectively utilized for routine analysis of ART and LUM in both bulk drugs and pharmaceutical dosage forms, without any interference.

Conflict of Interest

Nil