Lumefantrine quantification on pre-treated filter paper dried blood spots for clinical field trials

Artemether-lumefantrine is the most widely used antimalarial treatment for uncomplicated Plasmodium falciparum malaria. Malaria predominantly affects populations in rural areas of tropical and subtropical regions. Drug resistance is a growing concern, and monitoring antimalarial efficacy remains crucial for detecting changes in treatment performance and tracking potential resistance trends. In this study, we present an efficient method for collecting blood samples in resource-limited settings, using dried blood spots on pre-treated filter paper, which enhances the stability of lumefantrine. This approach builds on previous methods by incorporating rapid sample processing through semi-automated punching robots for 96-well plate formats, quick sample cleanup via filtration through a phospholipid removal plate, and expedited analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a turnaround time of just 2 min per sample. The method also achieves a significantly improved the lower limit of quantification of 15 ng of lumefantrine per ml of blood (i.e. LLOQ of 15 ng/ml). A Phenomenex Luna-CN 50 × 2 mm, 3 µm LC column was used, with a flow rate of 400 µl/min and a mobile phase consisting of acetonitrile and ammonium formate buffer (10 mM, 0.5% formic acid) in a 67:33 (v/v) ratio. The linear range for quantification spanned from 15 to 15,000 ng/ml, based on three 3.2 mm punched discs from a 50 µl blood spot. Accuracy was within ± 15% of the validation criteria, and precision was demonstrated by intra-assay variability of less than 7.2% and inter-assay variability of less than 12% across all concentration levels. The stability of lumefantrine in dried blood spots on pre-treated filter paper was demonstrated using a sample set obtained from another research group, which had been stored in the dark under low-humidity conditions for up to 16 years. The method showed no endogenous interference or interference from the concomitant drug artemether and is suitable for use in clinical trials.