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A modified method for determination of lumefantrine in human plasma by HPLC-UV and combination of protein precipitation and solid-phase extraction: application to a pharmacokinetic study.

ABSTRACT: An HPLC-UV method was developed and validated for the determination of lumefantrine in human plasma. Lumefantrine and its internal standard halofantrine were extracted from plasma samples using protein precipitation with acetonitrile (0.2% perchloric acid) followed by solid-phase extraction with Hypersep C(8) cartridges. Chromatographic separation was performed on a Zorbax SB-CN HPLC column (3.0 x 150 mm, 3.5 microm) with water/methanol (0.1% TFA) as the mobile phases in a gradient elution mode. Detection was performed using UV/vis detector at lambda = 335 nm. The method showed to be linear over a range of 50-10,000 ng/mL with acceptable intra- and inter-day precision and accuracy. The mean recoveries were 88.2% for lumefatrine and 84.5% for the I.S. The internal standard halofantrine is readily available from commercial sources. This method was successfully applied to a pharmacokinetic interaction study between a first-line antimalarial combination (artemether-lumefantrine) and antiretroviral therapy.

SUBMITTER: Huang L

PROVIDER: S-EPMC2865164 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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A modified method for determination of lumefantrine in human plasma by HPLC-UV and combination of protein precipitation and solid-phase extraction: application to a pharmacokinetic study.

Huang Liusheng L Lizak Patricia S PS Jayewardene Anura L AL Marzan Florence F Lee Ming-Na Tina MN Aweeka Francesca T FT

Analytical chemistry insights 20100329

An HPLC-UV method was developed and validated for the determination of lumefantrine in human plasma. Lumefantrine and its internal standard halofantrine were extracted from plasma samples using protein precipitation with acetonitrile (0.2% perchloric acid) followed by solid-phase extraction with Hypersep C(8) cartridges. Chromatographic separation was performed on a Zorbax SB-CN HPLC column (3.0 x 150 mm, 3.5 microm) with water/methanol (0.1% TFA) as the mobile phases in a gradient elution mode. ...[more]

PMID: 20448843

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Project description: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 ?-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 ?-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. ?-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 1 mM phosphate buffer pH 3.0 (52:48; V/V) at a flow of 1.0 ml/min and 3 ?l injection volume. Quantification was performed at 210 nm and 335 nm for ?-artemether and for lumefantrine, respectively. In-silico toxicological evaluation of the related impurities was made using Derek Nexus v2.0®.ResultsBoth ?-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 ?-artemether and 99.7 % (± 0.6%) for lumefantrine. All identified ?-artemether-related impurities were predicted in Derek Nexus v2.0® to have toxicity risks similar to ?-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 ?-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 ?-artemether-related impurities is comparable to that of ?-artemether API.

Project description: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.

Dataset Information

A modified method for determination of lumefantrine in human plasma by HPLC-UV and combination of protein precipitation and solid-phase extraction: application to a pharmacokinetic study.

Publications

A modified method for determination of lumefantrine in human plasma by HPLC-UV and combination of protein precipitation and solid-phase extraction: application to a pharmacokinetic study.

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