A newly validated high-performance liquid chromatography method with diode array ultraviolet detection for analysis of the antimalarial drug primaquine in the blood plasma

Introduction: Primaquine (PQ) diphosphate is an 8-aminoquinoline antimalarial drug with unique therapeutic properties. It is the only drug that prevents relapses of Plasmodium vivax or Plasmodium ovale infections. In this study, a fast, sensitive, cost-effective, and robust method for the extraction and high-performance liquid chromatography with diode array ultraviolet detection (HPLC-DAD-UV) analysis of PQ in the blood plasma was developed and validated. Methods: After plasma protein precipitation, PQ was obtained by liquid-liquid extraction and analyzed by HPLC-DAD-UV with a modified-silica cyanopropyl column (250mm x 4.6mm i.d. x 5 mu m) as the stationary phase and a mixture of acetonitrile and 10mM ammonium acetate buffer (pH = 3.80) (45: 55) as the mobile phase. The flow rate was 1.0mL center dot min(-1), the oven temperature was 50 degrees C, and absorbance was measured at 264nm. The method was validated for linearity, intra-day and inter-day precision, accuracy, recovery, and robustness. The detection (LOD) and quantification (LOQ) limits were 1.0 and 3.5ng mL-1, respectively. The method was used to analyze the plasma of female DBA-2 mice treated with 20mg center dot kg(-1) (oral) PQ diphosphate. Results: By combining a simple, lowcost extraction procedure with a sensitive, precise, accurate, and robust method, it was possible to analyze PQ in small volumes of plasma. The new method presents lower LOD and LOQ limits and requires a shorter analysis time and smaller plasma volumes than those of previously reported HPLC methods with DAD-UV detection. Conclusions: The new validated method is suitable for kinetic studies of PQ in small rodents, including mouse models for the study of malaria.