Sensitive fluorescence HPLC assay for AQ-13, a candidate aminoquinoline antimalarial, that also detects chloroquine and N-dealkylated metabolites

A sensitive, specific and reproducible fluorescence high performance liquid chromatography (HPLC) assay has been developed for the separate or simultaneous measurement of AQ-13 (a candidate 4-aminoquinoline antimalarial), chloroquine (CQ), and their metabolites in whole blood. After liquid–solid extraction using commercially available extraction cartridges, these two aminoquinolines (AQs) and their metabolites were separated on C18 (Xterra RP18) columns using a mobile phase containing 60% borate buffer (20 mM, pH 9.0) and 40% acetonitrile with isocratic elution at a flow-rate of 1.0 ml/min. The assay uses a biologically inactive 8-chloro-4-aminoquinoline (AQ-18) as its internal standard (IS). There is a linear relationship between the concentrations of these AQs and the peak area ratio (ratio between the peak area of the AQ or metabolite and the peak area of the IS) on the chromatogram. Linear calibration curves with correlation coefficients ≥0.997 (r2 ≥ 0.995, p < 0.001) were obtained for AQ-13, CQ and their N-dealkylated metabolites. Reproducibility of the assay was excellent with coefficients of variation (CVs) ≤3.8% for AQ-13 and its metabolites, and ≤2.5% for CQ and its metabolites. The sensitivity of the assay is 5 nM using 1.0 ml of blood and a 20 μl injection volume, and can be increased by using 5.0 ml of blood with an injection volume of 40 μl.