A field-adapted sampling and HPLC quantification method for lumefantrine and its desbutyl metabolite in whole blood spotted on filter paper

A quantitative reverse-phase HPLC method with UV detection, for lumefantrine (LF) and desbutyllume-fantrine (DLF) in whole blood spotted on filter paper was developed. The analytes were stabilized on filter paper by treatment of blood with phosphoric acid (1.6 mol/L). Halofantrine was used as internal standard and the analytes were extracted from filter paper using methanol. The methanolic extract was extracted with di-isopropylether after addition of acidic phosphate buffer (pH 2). Chromatographic separation was carried out on a Zorbax Eclipse XDB-phenyl column (4.6 mm x 150 mm, particle size 5 mu m) at a flow rate of 1 mL/min using a mobile phase of acetonitrile-ammonium acetate buffer (0.1 M ammonium acetate and 0.01 M acetic acid, pH 6.5) (10:90). The absorbance of the compounds was monitored at 335 nm. The average extraction recovery from filter paper ranged between 45-51% for LF and 25-33% for DLF for a concentration range between 300 and 3000 nM. Inter- and intra-assay coefficients of variation for LF and DLF were <= 9.2. Limits of quantification for LF and DLF were 300 nM. The method has been applied in malaria patients. In conclusion, a simple procedure for blood sampling and quantitative measurement of lumefantrine and desbutyllumefantrine suitable for field studies in resource-limited laboratories was developed. (C) 2008 Elsevier B.V. All rights reserved.