Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education & Research, IDPL R&D Campus, Balanagar, Hyderabad- 500 037. India.
National Center for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India.
J Pharm Biomed Anal. 2019 Mar 20;166:139-146. doi: 10.1016/j.jpba.2019.01.004. Epub 2019 Jan 7.
Afatinib is an irreversible tyrosine kinase receptor inhibitor which was approved lately by USFDA for the treatment of metastatic non-small cell lung cancer (NSCLC). AFT was subjected to stress degradation studies under hydrolytic (acid, base and neutral), oxidative, thermal and photolytic conditions to investigate the inherent stability of the drug. The present study describes the simple and rapid HPLC method development for the selective separation of the AFT and its degradation products. The drug and degradation products were separated on Agilent Eclipse plus C18 (150 × 4.6 mm, 5 μ) column with ammonium acetate buffer (10 mM, pH 6.7) in gradient elution mode. The drug was found to be unstable in all the conditions studied. The developed chromatographic method was extended to tandem mass spectrometry (QTOF-MS) for the characterization of the degradation products. A total of 11 unknown degradation products were characterized using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS/MS). Two major degradation products (DP2 and DP3) were isolated using preparative HPLC and their structures were confirmed by conducting H and C NMR experiments. The isolated DPs were evaluated for their anticancer potential using non small cell lung cancer cell line A549. The IC values for AFT, DP2 and DP3 were found to be 15.02 ± 1.49, 25.00 ± 1.26 and 32.56 ± 0.11 respectively. The in silico toxicity studies were performed employing ProTox-II software for the assessment of toxicity potential of drug and its degradation products. Finally, the developed chromatographic method was validated as per the International Conference on Harmonization guideline Q2 (R1).
阿法替尼是一种不可逆的酪氨酸激酶受体抑制剂，最近被美国食品和药物管理局批准用于治疗转移性非小细胞肺癌（NSCLC）。AFT 在水解（酸、碱和中性）、氧化、热和光解条件下进行了强制降解研究，以研究药物的固有稳定性。本研究描述了一种简单、快速的 HPLC 方法开发，用于选择性分离 AFT 及其降解产物。药物和降解产物在 Agilent Eclipse plus C18（150×4.6mm，5μm）柱上，以梯度洗脱模式，用乙酸铵缓冲液（10mM，pH6.7）分离。研究发现，在所有研究条件下，该药物均不稳定。所开发的色谱方法已扩展到串联质谱（QTOF-MS），用于降解产物的表征。使用液相色谱-四极杆飞行时间质谱（LC-Q-TOF/MS/MS）对 11 种未知降解产物进行了表征。使用制备型 HPLC 分离出两种主要的降解产物（DP2 和 DP3），并通过进行 H 和 C NMR 实验确认其结构。对这些 DP 进行了非小细胞肺癌细胞系 A549 的抗癌潜力评估。AFT、DP2 和 DP3 的 IC 值分别为 15.02±1.49、25.00±1.26 和 32.56±0.11。采用 ProTox-II 软件进行了计算机毒性研究，以评估药物及其降解产物的毒性潜力。最后，按照国际人用药品注册技术协调会（ICH）指导原则 Q2（R1）对所开发的色谱方法进行了验证。
