Department of Pharmacy and Pharmaceutical Science, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Molecules. 2022 Aug 25;27(17):5448. doi: 10.3390/molecules27175448.
IWR-1-endo, a small molecule that potently inhibits the Wnt/β-catenin signaling pathway by stabilizing the AXIN2 destruction complex, can inhibit drug efflux at the blood−brain barrier. To conduct murine cerebral microdialysis research, validated, sensitive, and reliable liquid chromatography−tandem mass spectrometry (LC-MS/MS) methods were used to determine IWR-1-endo concentration in the murine plasma and brain microdialysate. IWR-1-endo and the internal standard (ISTD) dabrafenib were extracted from murine plasma and microdialysate samples by a simple solid-phase extraction protocol performed on an Oasis HLB µElution plate. Chromatographic separation was executed on a Kinetex C18 (100A, 50 × 2.1 mm, 4 µm particle size) column with a binary gradient of water and acetonitrile, each having 0.1% formic acid, pumped at a flow rate of 0.6 mL/min. Detection by mass spectrometry was conducted in the positive selected reaction monitoring ion mode by monitoring mass transitions 410.40 > 344.10 (IWR-1-endo) and 520.40 > 307.20 (ISTD). The validated curve range of IWR-1-endo was 5−1000 ng/mL for the murine plasma method (r2 ≥ 0.99) and 0.5−500 ng/mL for the microdialysate method (r2 ≥ 0.99). The lower limit of quantification (LLOQ) was 5 ng/mL and 0.5 ng/mL for the murine plasma and microdialysate sample analysis method, respectively. Negligible matrix effects were observed in murine plasma and microdialysate samples. IWR-1-endo was extremely unstable in murine plasma. To improve the stability of IWR-1-endo, pH adjustments of 1.5 were introduced to murine plasma and microdialysate samples before sample storage and processing. With pH adjustment of 1.5 to the murine plasma and microdialysate samples, IWR-1-endo was stable across several tested conditions such as benchtop, autosampler, freeze−thaw, and long term at −80 °C. The LC-MS/MS methods were successfully applied to a murine pharmacokinetic and cerebral microdialysis study to characterize the unbound IWR-1-endo exposure in brain extracellular fluid and plasma.
IWR-1-endo 是一种小分子化合物，通过稳定 AXIN2 降解复合物，强力抑制 Wnt/β-连环蛋白信号通路，可抑制血脑屏障中的药物外排。为了进行小鼠脑微透析研究，使用经过验证、灵敏和可靠的液相色谱-串联质谱 (LC-MS/MS) 方法来测定小鼠血浆和脑微透析液中的 IWR-1-endo 浓度。IWR-1-endo 和内标 (ISTD) dabrafenib 通过固相萃取方案从小鼠血浆和微透析液样品中提取，该方案在 Oasis HLB µElution 板上进行。色谱分离在 Kinetex C18 (100A, 50×2.1mm, 4μm 粒径) 柱上进行，采用水和乙腈的二元梯度，均含有 0.1%甲酸，以 0.6mL/min 的流速泵入。通过监测质量转移 410.40 > 344.10 (IWR-1-endo) 和 520.40 > 307.20 (ISTD)，在正选择反应监测离子模式下进行质谱检测。IWR-1-endo 的验证曲线范围为 5-1000ng/mL 的小鼠血浆方法 (r2≥0.99) 和 0.5-500ng/mL 的微透析液方法 (r2≥0.99)。小鼠血浆和微透析液样品分析方法的定量下限 (LLOQ) 分别为 5ng/mL 和 0.5ng/mL。在小鼠血浆和微透析液样品中观察到基质效应可忽略不计。IWR-1-endo 在小鼠血浆中极不稳定。为了提高 IWR-1-endo 的稳定性，在样品储存和处理前，将 pH 值调节至 1.5。通过将 pH 值调节至 1.5，可使 IWR-1-endo 在多种测试条件下保持稳定，例如在台式、自动进样器、冻融和长期在-80°C 下。LC-MS/MS 方法成功应用于小鼠药代动力学和脑微透析研究，以描述脑细胞外液和血浆中未结合的 IWR-1-endo 暴露情况。
