A validated analytical method for the determination of lumefantrine in selected time modulated dried blood spot samples from malaria patients in Botswana

Abstract

A simple liquid chromatography- diode array detector (LC-DAD) method for the determination of lumefantrine in whole blood collected in dried blood spot (DBS) filters was developed and validated. The validation was done using the United States Food and Drug Administration (USFDA) guidelines. Sample preparation was done using solid liquid extraction (SLE) followed by separation using high performance liquid chromatography (HPLC) and diode array detection (DAD). Separation was done at 25ºC using an XTerra C18 column with dimensions of 50 mm x 4.6 mm x 5μm (length x internal diameter x particle size) and a binary solvent system of acetonitrile and water adjusted to pH of 2.3 with formic acid as the mobile phase. The mobile phase was pumped at a flow rate of 0.570 mL/min using a gradient elution program. The analysis time was 2 minutes and the calibration curve obtained was linear over the concentration range of 1-8 μg/mL with a correlation coefficient (R2) of 0.9980. The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 0.4 and 0.8 μg/mL respectively. The extraction efficiency estimated as percent recovery was greater than 60 %. Both the intra and inter-day precision of this method were ˂ ±15% as prescribed by the USFDA guidelines. The method was successfully applied for the quantification of lumefantrine in time modulated dried blood spot samples, previously collected from patients on malarial treatment with the artemisinin/ lumefantrine combination therapy.