UPLC-MS/MS法分析新型多柔比星前药在肿瘤细胞中的浓度  

A UPLC-MS/MS method for quantification of a novel doxorubicin conjugation prodrug in tumor cells

作　　者：郑楠[1] 王兴 王瑶琪 徐国兵[1] 张华[2] 代文兵[2] 何冰[2] 张强[2] 王学清[2] 

机构地区：[1]北京大学肿瘤医院暨北京市肿瘤防治研究所国家药物临床试验机构,恶性肿瘤发病机制及转化研究教育部重点实验室,北京100142 [2]北京大学药学院药剂学系,北京100191

出　　处：《药学学报》2018年第2期278-283,共6页Acta Pharmaceutica Sinica

基　　金：北京市自然科学基金资助项目(7162108,7164244);国家自然科学基金青年基金资助项目(81603038).#共同贡献.

摘　　要：本研究采用超高效液相色谱-三重四级杆串联质谱技术,建立在肿瘤细胞中测定单硫键连接的多柔比星前药(DOX-S-DOX)浓度的方法学,并应用于人乳腺癌细胞(MCF-7)的体外药代动力学研究。细胞样品前处理采用乙腈沉淀蛋白的方法。以柔红霉素为内标,采用反相C18色谱柱(Agilent Eclipse plus C18 RRHD 1.8μm,2.1 mm×50 mm),流动相为甲醇-0.1%甲酸水溶液,以0.4 m L·min^(-1)的流速进行梯度洗脱,每个样品的分析时间为6.0 min,样品经电喷雾离子源正离子化后,在多反应监测模式下测定DOX-S-DOX(m/z 1251.3→459.4)的浓度。线性范围为20.0~400 ng·m L^(-1)(r^2≥0.99)。高、中、低三个浓度质控样本的准确度(RE%)在-2.04%~2.62%之间,批内、批间精密度(RSD%)均在3.77%~8.35%以内。基质效应在104.8%~113.9%之间,提取回收率在97.67%~104.2%之间。该分析方法较为简便,其线性范围和灵敏度能够满足DOX-S-DOX(10μg·m L^(-1))在MCF-7细胞中的药代动力学研究。In this study, we developed a rapid and sensitive ultra high-performance liquid chromatographytandem mass spectrometry （UPLC-MS/MS） method to detect a sulfide bond doxorubicin conjugation prodrug （DOX-S-DOX） in human breast cancer tumor cells （MCF-7）. The samples were prepared by acetonitrile precipitation using daunorubicin as internal standard （IS）. A reversed phase C18 analytical column （Agilent Eclipse plus C18 RRHD 1.8 μm, 2.1 mm×50 mm） was utilized to separate the samples under gradient elution conditions. Mobile phase was a mixture of 0.1% formic acid in water and methanol at a flow rate of 0.4 mL ·min^-1. The analysis was conducted on the mass spectrometer using an electrospray interface （ESI） in the positive ionization model. The calibration range was 20.0-400 ng·mL^-1 with the correlation coefficients （r^2） ≥ 0.99. The inter-and intra-assay precision （relative standard deviation, RSD%） of quality control samples was within 3.77%-8.35% and relative error （RE%） for accuracy was between -2.04% and 2.62%. Recovery （97.67%-104.2%） and matrix effect （104.8%-113.9%） were consistent, precise, and reproducible at different quality control levels in accordance with FDA guidance. The assay was successfully used in the cellular pharmacokinetics study of DOX-S-DOX, which may provide a clue to explore analytical methods of other prodrug forms of DOX.

关 键 词：多柔比星前药 超高效液相色谱串联质谱 细胞药代动力学 

分 类 号：R917[医药卫生—药物分析学]