Simultaneous and enantiospecific quantification of primaquine and carboxyprimaquine in human plasma using liquid chromatography-tandem mass spectrometry.

BACKGROUND: The enantiomers of the 8-aminoquinoline anti-malarial primaquine have different pharmacological properties. Development of an analytical method for simultaneous quantification of the enantiomers of primaquine and its metabolite, carboxyprimaquine, will support clinical pharmacometric assessments. METHODS: A simple and sensitive method consisting of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for simultaneous and enantiospecific determination of primaquine and its metabolite, carboxyprimaquine, in human plasma. Stable isotopes were used as internal standards to compensate for potential interference and matrix effects. Plasma samples (100 µL) were precipitated with 1% formic acid in acetonitrile followed by phospholipid removal solid phase extraction. Primaquine and carboxyprimaquine enantiomers were separated on a Chiralcel OD-3R (150 mm × 4.6 mm; I.D. 3 μm) column using a LC gradient mode. For separation of racemic primaquine and carboxyprimaquine, the LC method was modified and validated using a reverse phase column (Hypersil Gold 100 mm × 4.6 mm; I.D. 3 µm) and a mobile phase composed of 10 mM ammonium acetate buffer, pH 3.5 and acetonitrile in the isocratic mode. Method validation was performed according to regulatory guidelines. RESULTS: The calibration range was set to 0.571-260 ng/mL and 2.44-2,500 ng/mL for primaquine and carboxyprimaquine enantiomers, respectively, resulting in a correlation coefficient (r(2)) ≥ 0.0998 for all calibration curves. The intra- and inter-day assay precisions were < 10% and the accuracy was between 94.7 to 103% for all enantiomers of primaquine and carboxyprimaquine. The enantiospecific method was also modified and validated to quantify racemic primaquine and carboxyprimaquine, reducing the total run time from 30 to 8 min. The inter-, intra-day assay precision of the racemic quantification method was < 15%. The absolute recoveries of primaquine and carboxyprimaquine were between 70 and 80%. Stability was demonstrated for up to 2 years in - 80 °C. Both the enantiomeric and racemic LC-MS/MS methods were successfully implemented in pharmacokinetic studies in healthy volunteers. CONCLUSIONS: Simple, sensitive and accurate LC-MS/MS methods for the quantification of enantiomeric and racemic primaquine and carboxyprimaquine in human plasma were validated successfully and implemented in clinical routine drug analysis.