Development and validation of a LC-MS/MS method for the quantitation of lumefantrine in mouse whole blood and plasma.

Abstract

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of the antimalarial drug, lumefantrine (LF), in mouse whole blood and plasma. The analyte was extracted using a protein precipitation method followed by chromatographic separation on a Phenomenex Luna, PFP (50mm×2.0mm, 5μm) analytical column with a mobile phase consisting of acetonitrile and 0.1% formic acid (formic acid:water, 1:1000, v/v) at a ratio of 3:7 (v/v), delivered at a constant flow rate of 0.5ml/min. Stable isotope labeled lumefantrine (D9-LF) was used as the internal standard. Multiple reaction monitoring was performed using the transitions m/z 530.1→m/z 347.9 and m/z 539.1→m/z 347.9 for the quantification of LF and D9-LF, respectively. Calibration curves were constructed over the concentration range 15.6-4000ng/ml. The mean intra- and inter-assay accuracy values for the analysis of LF in WB was 103% (%CV=5.5) and 99.5% (%CV=5.5), respectively. The mean intra- and inter-assay accuracy values for the analysis of LF in plasma was 93.7% (%CV=3.5) and 93.9% (%CV=5.5), respectively. No significant matrix effect was observed during the method validation. The validated method was applied to an absorption study in mice, to determine and compare LF concentrations in whole blood and plasma samples. Results of the statistical analysis using a linear mixed effects growth curve model concluded that there was no significant difference (p-value=0.668) between WB and plasma LF concentrations. This method utilizes a small sample volume of 20μl, facilitating low blood collection volumes and a short chromatographic run time of 3min which allows for high sample throughput analysis.