Bioanalytics, Metabolomics, and Pharmacokinetics Shared Resource, Roswell Park Comprehensive Cancer Center, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Department of Medicine, Roswell Park Comprehensive Cancer Center, Elm and Carlton Streets, Buffalo, NY 14263, USA.
J Pharm Biomed Anal. 2023 Sep 20;234:115594. doi: 10.1016/j.jpba.2023.115594. Epub 2023 Jul 17.
This article describes the development and validation of a liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantitation of the BRAF inhibitors dabrafenib and encorafenib, and semi-quantitation of their major metabolites (i.e., carboxy-dabrafenib, desmethyl-dabrafenib, hydroxy-dabrafenib, M42.5A) in human plasma. Analytes were extracted from human plasma by protein precipitation, followed by reversed phase high-performance liquid chromatography. Analyte detection was performed using tandem mass spectrometry with heated electrospray ionization operating in positive ion mode. The assay was validated in accordance with the current U.S. Food and Drug Administration Guidance on Bioanalytical Method Validation. Results showed that measurements were both accurate (94.6-112.0 %) and precise (within-run: 1.9-3.4 %; between-run: 1.7-12.0 %) spanning a concentration range of 5 to 2000 ng/mL for dabrafenib and 10 to 4000 ng/mL for encorafenib. Recoveries for these analytes were consistent with mean values ranging from 85.6 % to 90.9 %. The mean internal standard-normalized matrix factors for each drug ranged between 0.87 and 0.98 and were found to be precise (% RSD <6.4 %). Dabrafenib and encorafenib were stable in the final extract and in human plasma held under various storage conditions. The metabolites also passed the validation criteria for precision and selectivity. Finally, the clinical applicability of the assay was confirmed by (semi-)quantitation of all six analytes in plasma samples from cancer patients receiving standard-of-care treatment with dabrafenib and encorafenib. Reproducibility of the measured analyte concentrations in study samples was confirmed successfully by incurred sample reanalysis. In conclusion, this sensitive LC-MS/MS assay has been validated successfully and is suitable for therapeutic drug monitoring of dabrafenib and encorafenib and clinical pharmacokinetic studies with these BRAF inhibitors.
本文描述了一种液相色谱-串联质谱（LC-MS/MS）法的建立与验证，该方法可用于同时定量检测人血浆中的 BRAF 抑制剂达布拉非尼和恩考芬尼，以及半定量检测其主要代谢物（即羧基达布拉非尼、去甲基达布拉非尼、羟基达布拉非尼、M42.5A）。分析物经蛋白沉淀后从人血浆中提取，然后进行反相高效液相色谱分离。采用加热电喷雾电离源正离子模式进行串联质谱检测。该方法的验证符合美国食品和药物管理局关于生物分析方法验证的指南。结果表明，该方法在 5 至 2000ng/mL 范围内测定达布拉非尼，在 10 至 4000ng/mL 范围内测定恩考芬尼，其准确度（94.6%-112.0%）和精密度（批内：1.9%-3.4%；批间：1.7%-12.0%）均良好。这些分析物的回收率与 85.6%-90.9%的平均值一致。每种药物的内标归一化基质因子的平均值在 0.87 至 0.98 之间，且精密度良好（%RSD<6.4%）。达布拉非尼和恩考芬尼在最终提取物中和在各种储存条件下的人血浆中均稳定。代谢物的精密度和选择性也符合验证标准。最后，通过对接受达布拉非尼和恩考芬尼标准治疗的癌症患者血浆样品中所有 6 种分析物的（半）定量分析，证实了该方法的临床适用性。通过对研究样品中测定的分析物浓度进行重复分析，成功确认了其重现性。总之，该灵敏的 LC-MS/MS 分析方法已成功验证，适用于达布拉非尼和恩考芬尼的治疗药物监测以及这些 BRAF 抑制剂的临床药代动力学研究。
