Development and validation of a liquid chromatography–mass spectrometry assay for the determination of pyronaridine in human blood for application to clinical pharmacokinetic studies

A reliable and sensitive method for the determination of pyronaridine in human blood was developed and validated. A 0.3 ml aliquot of whole blood was extracted using liquid–liquid extraction after addition of amodiaquine as an internal standard. Analysis was performed on a Shimadzu LCMS-2010A in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. The extracted ion for pyronaridine was m/z 518.20 and for amodiaquine was m/z 356.10. Separation was achieved on a Gemini 5 μm C18 3.0 × 150 mm column using a mobile phase composed of 2 mM perflurooctanoic acid–acetonitrile mixture delivered at a flow rate of 0.5 mL/min. The mobile phase was delivered in gradient mode. The retention times of pyronaridine and amodiaquine were 9.2 and 8.2 min, respectively, with a total run time of 14 min. The estimated calibration range of the method was 5.7–855 ng/mL. The analysis of quality control samples for pyronaridine at 11.4, 285, and 760 ng/mL demonstrated excellent precision with relative standard deviation of 11.1, 4.8 and 2.2%, respectively (n = 5). Recoveries at concentrations of 11.4, 285 and 760 ng/mL were all greater than 75%. No interference peaks or matrix effects were observed. This LC-MS method for the determination of pyronaridine in human blood has excellent specifications for sensitivity, reproducibility and accuracy. This LC–MS technique was found to improve the quantitation of pyronaridine in whole blood allowing its use in pharmacokinetic studies with clinically relevant doses.