Department of Pharmacy & Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands; Division of Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
Department of Pharmacy & Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Sep 1;1125:121723. doi: 10.1016/j.jchromb.2019.121723. Epub 2019 Jul 18.
The first bioanalytical assay for tivozanib in human and mouse plasma, mouse tissue homogenates and culture medium was developed and validated over a linear dynamic range from 0.5 to 5000 ng/mL. The extended concentration range will cover the quantification of tivozanib in the majority of study samples, reducing the need for reanalysis which is often not possible due to limited amount of sample in preclinical studies. A simple and fast pretreatment method was used consisting of protein precipitation with acetonitrile followed by dilution of the supernatant. The final extract was injected onto an Ultra-Performance Liquid Chromatography (UPLC) BEH C18 column with gradient elution of formic acid in water and formic acid in acetonitrile mobile phase. Chromatographic separation was followed by detection with a triple-quadrupole mass spectrometer operating in the positive ion-mode. By simultaneously monitoring the sensitive conventional [M + H] isotopologue- product transition for quantification of low concentrations and a less abundant [M + H]+1 isotopologue- product transition to reduce the sensitivity for quantification of high concentrations, we were able to extend the overall linear dynamic range up to 0.5-5000 ng/mL. A full validation was performed in human plasma and a partial validation was executed for the other matrices. All results were within the acceptance criteria of the European Medicines Agency (EMA) guidelines and the US Food and Drug Administration (FDA) guidance, except for the carry-over. This was solved by the analysis of extra matrix blanks and by grouping study samples containing a high tivozanib concentration in the sample sequence. In this way carry-over did not impact the data integrity. We demonstrated that by measuring two multiple reaction monitoring (MRM) transitions for tivozanib, the linear dynamic range could be extended from two to four decades. The assay was successfully applied in pharmacokinetic studies in mice and a transport assay.
开发并验证了一种用于人血浆、鼠组织匀浆和培养基中替沃扎尼的首个生物分析测定法，其线性动态范围为 0.5 至 5000ng/mL。扩展的浓度范围将涵盖大多数研究样本中替沃扎尼的定量，减少因临床前研究中样本量有限而经常无法重新分析的需要。采用简单快速的预处理方法，包括用乙腈进行蛋白沉淀，然后稀释上清液。最终提取物在 Ultra-Performance Liquid Chromatography (UPLC) BEH C18 柱上进行分析，采用水相中的甲酸和乙腈中的甲酸进行梯度洗脱。色谱分离后，采用正离子模式的三重四极杆质谱仪进行检测。通过同时监测低浓度定量的灵敏常规 [M+H] 同位素产物转化和减少高浓度定量的较少丰度 [M+H]+1 同位素产物转化，我们能够将整体线性动态范围扩展至 0.5-5000ng/mL。在人血浆中进行了全面验证，并对其他基质进行了部分验证。除了交叉污染外，所有结果均符合欧洲药品管理局 (EMA) 指南和美国食品和药物管理局 (FDA) 指南的验收标准。通过分析额外的基质空白和将含有高浓度替沃扎尼的研究样本分组在样品序列中，解决了交叉污染问题。这样，交叉污染不会影响数据完整性。我们证明，通过测量替沃扎尼的两个多重反应监测 (MRM) 转化，可以将线性动态范围从两个扩展到四个数量级。该测定法成功应用于小鼠的药代动力学研究和转运研究。
