Determination of intracellular anlotinib, osimertinib, afatinib and gefitinib accumulations in human brain microvascular endothelial cells by liquid chromatography/tandem mass spectrometry
Determination of intracellular anlotinib, osimertinib, afatinib and gefitinib accumulations in human brain microvascular endothelial cells by liquid chromatography/tandem mass spectrometry
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液相色谱/串联质谱法测定人脑微血管内皮细胞内安罗替尼、奥希替尼、阿法替尼和吉非替尼的蓄积
DOI：
10.1002/rcm.8955
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发表时间：
2021-01-15
影响因子：
2
通讯作者：
Lin, Nengming
中科院分区：
文献类型：
--
作者：
Ma, Zhiyuan;Lu, Shuanghui;Lin, Nengming
脑转移是非小细胞肺癌（NSCLC）患者的常见并发症。盐酸安洛替尼是一种新型多靶点酪氨酸激酶抑制剂（TKI），对NSCLC脑转移瘤具有上级总体缓解率。方法采用液相色谱-串联质谱法（LC/MS/MS），建立了4种表皮生长因子受体（EGFR）TKI对脑微血管内皮细胞（HBMECs）的定量分析方法。直接蛋白沉淀后，在ACQUITY BEH C18色谱柱上分离安洛替尼和三种EGFR TKI，然后使用正离子模式下的电喷雾电离进行分析。结果4种分析物均能在3.8min内快速定量，各验证参数均符合生物分析方法学标准。校准范围为0.2-200 ng/mL（安洛替尼和吉非替尼）、1-500 ng/mL（奥希替尼）和1-200 ng/mL（阿法替尼）。阿法替尼和吉非替尼在HBMEC中的渗透性与阿法替尼和吉非替尼相当，但低于osimertinib.Conclusions一种灵敏的LC/MS/MS方法，同时测量细胞提取物中的阿法替尼，osimertinib，阿法替尼和吉非替尼，成功地验证和应用于确定HBMEC内的摄取，这可能为将来研究阿法替尼在NSCLC脑转移中的作用铺平道路。
Rationale Brain metastases are a common complication in patients with non-small-cell lung cancer (NSCLC). Anlotinib hydrochloride is a novel multi-target tyrosine kinase inhibitor (TKI) exhibiting a superior overall response rate for brain metastases from NSCLC. The penetrability of anlotinib and three generations of epidermal growth factor receptor (EGFR) TKIs (osimertinib, afatinib and gefitinib) into brain microvascular endothelial cells (HBMECs) was compared.Methods A sensitive quantification method for the four TKIs was developed using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). Anlotinib and the three EGFR TKIs were separated on an ACQUITY BEH C18 column after a direct protein precipitation, and then analyzed using electrospray ionization in positive ion mode. The linearity, accuracy, precision, limit of quantification, specificity and stability were assessed.Results The four analytes could be efficiently quantified in a single run of 3.8 min. The validation parameters of all analytes satisfy the acceptance criteria of bioanalytical method guidelines. The calibration range was 0.2-200 ng mL(-1) for anlotinib and gefitinib, 1-500 ng mL(-1) for osimertinib and 1-200 ng mL(-1) for afatinib. The penetration of anlotinib across HBMECs was comparable with that of afatinib and gefitinib but less than that of osimertinib.Conclusions A sensitive LC/MS/MS method to simultaneously measure anlotinib, osimertinib, afatinib and gefitinib in cell extracts was successfully validated and applied to determine their uptake inside HBMECs, which could pave the way for future research on the role of anlotinib in NSCLC brain metastases.