Carmo Ana Paula Barbosa do, Borborema Manoella, Ribeiro Stephan, De-Oliveira Ana Cecilia Xavier, Paumgartten Francisco Jose Roma, Moreira Davyson de Lima
Laboratório de Toxicologia Ambiental, Departamento de Ciências Biológicas, Escola Nacional de Saúde Pública, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil.
Departamento de Produtos Naturais, Instituto de Tecnologia em Fármacos - Farmanguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil.
Rev Soc Bras Med Trop. 2017 Jul-Aug;50(4):499-505. doi: 10.1590/0037-8682-0023-2017.
: 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.
: After plasma protein precipitation, PQ was obtained by liquid-liquid extraction and analyzed by HPLC-DAD-UV with a modified-silica cyanopropyl column (250mm × 4.6mm i.d. × 5μm) 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·min-1, the oven temperature was 50OC, 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.kg-1 (oral) PQ diphosphate.
: By combining a simple, low-cost 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.
: The new validated method is suitable for kinetic studies of PQ in small rodents, including mouse models for the study of malaria.
磷酸伯氨喹（PQ）是一种具有独特治疗特性的8-氨基喹啉类抗疟药物。它是唯一能预防间日疟原虫或卵形疟原虫感染复发的药物。在本研究中，开发并验证了一种快速、灵敏、经济高效且稳健的血浆中PQ提取及高效液相色谱-二极管阵列紫外检测（HPLC-DAD-UV）分析方法。
血浆蛋白沉淀后，通过液-液萃取获得PQ，并采用以改性硅胶氰丙基柱（250mm×4.6mm内径×5μm）为固定相、乙腈和10mM醋酸铵缓冲液（pH = 3.80）（45:55）的混合物为流动相的HPLC-DAD-UV进行分析。流速为1.0mL·min-1，柱温为50℃，在264nm处测定吸光度。该方法针对线性、日内和日间精密度、准确度、回收率及稳健性进行了验证。检测限（LOD）和定量限（LOQ）分别为1.0和3.5ng·mL-1。该方法用于分析经20mg.kg-1（口服）磷酸伯氨喹处理的雌性DBA-2小鼠的血浆。
通过将简单、低成本的提取程序与灵敏、精确、准确且稳健的方法相结合，能够对少量血浆中的PQ进行分析。与先前报道的采用DAD-UV检测的HPLC方法相比，新方法具有更低的LOD和LOQ限，且分析时间更短、所需血浆量更小。
新验证的方法适用于小型啮齿动物中PQ的动力学研究，包括用于疟疾研究的小鼠模型。
