Simultaneous determination of monodesethylchloroquine, chloroquine, cycloguanil and proguanil on dried blood spots by reverse-phase liquid chromatography

A method for simultaneous analysis of chloroquine, proguanil and their metabolites from a whole blood sample (80 μL) dried on a filter paper was developed. Sample preparation included a liquid extraction from the filter paper, followed by a solid-phase extraction (C18 Bond Elut® cartridge). Separation was obtained by reverse-phase liquid chromatography (HPLC) using a gradient elution on an X-Terra® column; UV detection was made at 254 nm. This assay was linear between 150 and 2500 ng mL−1 for chloroquine (and metabolite) and 300 and 2500 ng mL−1 for proguanil and cycloguanil. The lower limit of quantification was close to 50 ng mL−1 for chloroquine (and its metabolite) and 100 ng mL−1 for proguanil (and its metabolite). No chromatographic interference from endogenous compounds or other tested anti-malarial drugs was evidenced. Chromatographic separation takes about 40 min with a coefficient of variation below 10.3% for within- and between-batch precision. The paper sampling method was validated in 10 healthy subjects treated by Savarine®. The stability of compounds and metabolites on the filter paper was evaluated at four temperatures (−20, +4, 20 and 50 °C) and for 1, 5 and 20 days. Cycloguanil concentrations were not influenced by storage conditions, whereas, high temperatures and prolonged storage decreased chloroquine and proguanil levels. The proposed HPLC assay is accurate, precise and cost-effective; it can be used for pharmacokinetic and epidemiological studies on anti-malarial treatments.