Abstract:
We have successfully developed and validated a bioanalytical assay using liquid chromatography tandem mass spectrometry to simultaneously quantify the first approved KRASG12C inhibitor sotorasib and its major circulating metabolite (M24) in various mouse matrices. M24 was synthesized in-house via low-pH hydrolysis. We utilized a fast and efficient protein precipitation method in a 96-well plate format to extract both analytes from biological matrices. Erlotinib was selected as the internal standard in this assay. Gradient elution using methanol and 0.1 % formic acid in water (v/v) was applied on an Acquity UPLC BEH C18 column to separate all analytes. Sotorasib, M24, and erlotinib were detected with a triple quadrupole mass spectrometer in positive electrospray ionization in multiple reaction monitoring mode. During the validation and sample quantification, a linear calibration range was observed for both sotorasib and M24 in a range of 4 - 4000 nM and 1 - 1000 nM, respectively. The %bias and %CV (both intra- and inter-day) for all tested levels in all investigated matrices were lower than 15 % as required by the guidelines. Sotorasib had a rather short room temperature stability in mouse plasma for up to 8 h compared to M24 which was stable up to 16 h at room temperature. This method has been successfully applied to measure sotorasib and M24 in several mouse matrices from three different mouse strains. We can conclude that the plasma exposure of sotorasib in mice is limited via human CYP3A4- and mouse Cyp3a-mediated metabolism of sotorasib into M24.
摘要:
我们已经成功开发并验证了使用液相色谱串联质谱法的生物分析测定,以同时定量各种小鼠基质中第一个批准的KRASG12C抑制剂sotorasib及其主要循环代谢物(M24)。M24通过低pH水解在内部合成。我们在96孔板格式中利用快速有效的蛋白沉淀方法从生物基质中提取两种分析物。选择厄洛替尼作为该测定中的内标。将使用甲醇和0.1%甲酸水溶液(v/v)的梯度洗脱应用于AcquityUPLCPEHC18柱以分离所有分析物。Sotorasib,M24和厄洛替尼在多反应监测模式下用三重四极杆质谱仪在正电喷雾电离中检测。在验证和样品定量过程中,在4-4000nM和1-1000nM的范围内观察到sotorasib和M24的线性校准范围,分别。在所有研究的基质中,所有测试水平的%偏倚和%CV(在一天内和在一天间)低于指南要求的15%。与在室温下稳定长达16小时的M24相比,Sotorasib在小鼠血浆中具有相当短的室温稳定性长达8小时。该方法已成功应用于测量来自三种不同小鼠品系的几种小鼠基质中的sotorasib和M24。我们可以得出结论,通过人CYP3A4-和小鼠Cyp3a介导的索托拉西布向M24的代谢,索托拉西布在小鼠中的血浆暴露受到限制。