Lindegårdh N, Blessborn D, Bergqvist Y
Dalarna University College, 781 88 Borlänge, Sweden.
J Chromatogr Sci. 2005 May-Jun;43(5):259-66. doi: 10.1093/chromsci/43.5.259.
A bioanalytical method is described for the simultaneous quantitative analysis of the highly lipophilic atovaquone and the strong basic proguanil with metabolites in plasma. The drugs are extracted from protein precipitated plasma samples on a novel mixed-mode solid-phase extraction (SPE) column containing carboxypropyl and octyl silica as functional groups. The analytes are further separated and quantitated using a steep-gradient liquid chromatographic method on a Zorbax SB-CN column with UV detection at 245 nm. Two different internal standards (IS) are used in the method to compensate for both types of analytes. A structurally similar IS to atovaquone is added with acetonitrile to precipitate proteins from plasma. A structurally similar IS to proguanil and its metabolites is added with phosphate buffer before samples are loaded onto the SPE columns. A single elution step is sufficient to elute all analytes. The method is validated according to published guidelines and shows excellent performance. The within-day precisions, expressed as relative standard deviation, are lower than 5% for all analytes at three tested concentrations within the calibration range. The between-day precisions are lower than 13% for all analytes at the same tested concentrations. The limit of quantitation is 25 nM for the basic substances and 50 nM for atovaquone. Several considerations regarding development and optimization of a method for determination of analytes with such a difference in physiochemical properties are discussed.
本文描述了一种生物分析方法，用于同时定量分析血浆中亲脂性很强的阿托伐醌和强碱性的氯胍及其代谢物。药物从经蛋白沉淀的血浆样品中，在一种新型的混合模式固相萃取（SPE）柱上进行萃取，该柱含有羧丙基和辛基硅胶作为官能团。使用Zorbax SB-CN柱上的陡梯度液相色谱法，在245 nm处进行紫外检测，对分析物进行进一步分离和定量。该方法使用两种不同的内标（IS）来补偿两种类型的分析物。一种与阿托伐醌结构相似的内标与乙腈一起加入，以从血浆中沉淀蛋白质。一种与氯胍及其代谢物结构相似的内标在样品加载到SPE柱之前与磷酸盐缓冲液一起加入。单个洗脱步骤足以洗脱所有分析物。该方法根据已发表的指南进行了验证，表现出优异的性能。在校准范围内的三个测试浓度下，所有分析物的日内精密度（以相对标准偏差表示）均低于5%。在相同测试浓度下，所有分析物的日间精密度均低于13%。碱性物质的定量限为25 nM，阿托伐醌的定量限为50 nM。讨论了关于开发和优化一种用于测定具有如此大物理化学性质差异的分析物的方法的若干注意事项。
