The antimalarial drug primaquine (PQ) causes methemoglobinemia and hemolysis in individuals with a genetic deficiency of glucose 6-phosphate dehydrogenase. Reactive oxygen species (ROS) generated by redox cycling of the metabolite primaquine-5,6-orthoquinone (POQ) in erythrocytes has been attributed to be responsible for the toxicity of PQ. Carboxyprimaquine (CPQ), the major human plasma metabolite of PQ, can also form the analogous carboxyprimaquine-5,6-orthoquinone (CPOQ) metabolite, which can also generate ROS in erythrocytes by redox cycling, thus contributing to the hematotoxicity of this drug. In order to study these pathways and characterize such effects in vivo, methods are needed for characterization and quantification of POQ and CPOQ in human erythrocytes. The purpose of this work was to develop a validated method for the quantitative determination of CPOQ and POQ metabolites in human erythrocytes, suitable for clinical studies of PQ metabolism. Several liquid-liquid extraction methods using different organic solvents had been investigated. The solvent mixture of water-methanol-acetonitrile (9:9:5, v/v) was shown to yield the best results for the two analytes. Chromatographic analysis of POQ and CPOQ in human erythrocytes was achieved on a high strength silica (HSS) column and gradient elution (water and acetonitrile, both containing 0.1% formic acid) by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Quantitative estimation of POQ and CPOQ was executed by monitoring ion pairs of m/z 260.23 > 175.03 and m/z 275.19 > 175.04, respectively. The method, which was validated for precision, accuracy, selectivity, and linearity, was successfully applied for the quantitative determination of POQ and CPOQ, the key metabolites of PQ in human erythrocytes in PQ clinical study.

抗疟药伯氨喹（PQ）会导致具有6-磷酸葡萄糖脱氢酶遗传缺陷的个体发生高铁血红蛋白血症和溶血。红细胞中代谢物伯氨喹5,6-邻醌（POQ）的氧化还原循环产生的活性氧（ROS）被认为是造成PQ毒性的原因。羧苄基喹（CPQ）是PQ的主要人类血浆代谢产物，也可以形成类似的羧基伯氨喹5,6-邻醌（CPOQ）代谢产物，它也可以通过氧化还原循环在红细胞中产生ROS，从而导致这种药物的血液毒性。为了研究这些途径并表征体内这种作用，需要用于表征和定量人红细胞中POQ和CPOQ的方法。这项工作的目的是开发一种定量测定人红细胞中CPOQ和POQ代谢产物的有效方法，适用于PQ代谢的临床研究。研究了几种使用不同有机溶剂的液-液萃取方法。水-甲醇-乙腈（9：9：5，v / v）的溶剂混合物显示出对两种分析物均能产生最佳结果。使用高强度硅胶（HSS）色谱柱和超高效液相色谱-串联质谱（UHPLC）进行梯度洗脱（水和乙腈，均含有0.1％甲酸），对人红细胞中的POQ和CPOQ进行色谱分析-MS / MS）。m / z 260.23> 175.03和m / z 275.19> 175.04。该方法经精密度，准确性，选择性和线性度验证，已成功用于PQ临床研究中定量测定POQ和CPOQ，这是人红细胞中PQ的关键代谢产物。