A rapid stability-indicating, fused-core HPLC method for simultaneous determination of β-artemether and lumefantrine in anti-malarial fixed dose combination products

Abstract

BackgroundArtemisinin-based fixed dose combination (FDC) products are recommended by World Health Organization (WHO) as a first-line treatment. However, the current artemisinin FDC products, such as β-artemether and lumefantrine, are inherently unstable and require controlled distribution and storage conditions, which are not always available in resource-limited settings. Moreover, quality control is hampered by lack of suitable analytical methods. Thus, there is a need for a rapid and simple, but stability-indicating method for the simultaneous assay of β-artemether and lumefantrine FDC products.MethodsThree reversed-phase fused-core HPLC columns (Halo RP-Amide, Halo C18 and Halo Phenyl-hexyl), all thermostated at 30°C, were evaluated. β-artemether and lumefantrine (unstressed and stressed), and reference-related impurities were injected and chromatographic parameters were assessed. Optimal chromatographic parameters were obtained using Halo RP-Amide column and an isocratic mobile phase composed of acetonitrile and 1mM phosphate buffer pH 3.0 (52:48; V/V) at a flow of 1.0 ml/min and 3 μl injection volume. Quantification was performed at 210 nm and 335 nm for β-artemether and for lumefantrine, respectively. In-silico toxicological evaluation of the related impurities was made using Derek Nexus v2.0®.ResultsBoth β-artemether and lumefantrine were separated from each other as well as from the specified and unspecified related impurities including degradants. A complete chromatographic run only took four minutes. Evaluation of the method, including a Plackett-Burman robustness verification within analytical QbD-principles, and real-life samples showed the method is suitable for quantitative assay purposes of both active pharmaceutical ingredients, with a mean recovery relative standard deviation (± RSD) of 99.7 % (± 0.7%) for β-artemether and 99.7 % (± 0.6%) for lumefantrine. All identified β-artemether-related impurities were predicted in Derek Nexus v2.0® to have toxicity risks similar to β-artemether active pharmaceutical ingredient (API) itself.ConclusionsA rapid, robust, precise and accurate stability-indicating, quantitative fused-core isocratic HPLC method was developed for simultaneous assay of β-artemether and lumefantrine. This method can be applied in the routine regulatory quality control of FDC products. The in-silico toxicological investigation using Derek Nexus® indicated that the overall toxicity risk for β-artemether-related impurities is comparable to that of β-artemether API.

Determination of artemether and lumefantrine in anti-malarial fixed-dose combination tablets by microemulsion electrokinetic chromatography with short-end injection procedure

“…For the assay of AS and AQ in FDCs, high performance liquid chromatography (HPLC) [4-6] and capillary electrophoresis (CE) methods have been proposed [7]. For AL, only HPLC methods [8-12] have been reported. César et al [8] were the first authors to propose a HPLC method allowing a separation of the two analytes (within 5 min.)…”

“…Very recently Suleman et al . [12] have developed and validated a stability indicating assay for the simultaneous determination of AL tablets. After extraction by tetrahydrofuran, artemether and lumefantrine are separated in isocratic conditions using a fused-core amide stationary phase and detection at 210 nm (β- artemether) and 335 nm (lumefantrine).…”

Development of a dissolution method for lumefantrine and artemether in immediate release fixed dose artemether/lumefantrine tablets

“…The amount of ART and LUM in samples of FDC ART/ LUM tablets was determined based on the previously published HPLC method [26]. In brief, the analysis of ART and LUM was conducted using Agilent 1260 Infinity Series HPLC system (Agilent Technologies, Santa Clara, California, USA) equipped with a Halo-RP-Amide column (50 × 4.6 mm, 2.7 µm) coupled to a diode-array detector (DAD).…”

“…The flasks were incubated at 37 °C for 24 h whilst shaking at 100 rpm. The samples were rapidly filtered using 0.45 µm PVDF (polyvinylidene fluoride) syringe filter, suitably diluted (2.5 ± 0.5 times) with acetonitrile and analysed at the auto-sampler temperature of 37 °C using HPLC system (Waters Alliance 2695 Separations Module Milford, MA, USA) equipped with a Halo-RP-Amide column (50 × 4.6 mm, 2.7 µm) as described in previous HPLC method [26].…”

Validated Reverse Phase High Performance Liquid Chromatographic Method for the Simultaneous Determination of Artemether and Lumefantrine in Fixed Combined Dosage Form

“…Very few high performance liquid chromatographic (HPLC) methods were reported for the determination of artemether and lumefantrine in combination forms [6][7][8][9][10][11]. Basing on this accord, it made essential to develop a new reverse phase HPLC method (RP-HPLC) for routine analysis of the above said drugs in combined formulations, and in this accord, attempts were made by the author to develop simple, precise, and accurate RP-HPLC method for the simultaneous assay of the titled drugs and extended it for their determination in formulations.…”