Quantitation of locked nucleic acid antisense oligonucleotides in mouse tissue using a liquid-liquid extraction LC-MS/MS analytical approach
Quantitation of locked nucleic acid antisense oligonucleotides in mouse tissue using a liquid-liquid extraction LC-MS/MS analytical approach
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DOI：
10.4155/bio.11.100
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发表时间：
2011-09-01
期刊：
影响因子：
1.8
通讯作者：
Brady, Kevin
中科院分区：
文献类型：
--
作者：
Turnpenny, Paul;Rawal, Jaiessh;Brady, Kevin
背景资料：寡核苷酸生物分析的一个重大挑战是从复杂组织样品中选择性提取，其中分布到细胞内空间中的分子广泛地与蛋白质结合，并且位于高浓度的内源性核酸材料之间。已发表的分析方法目前声称有广泛的样品制备要求，包括细胞裂解步骤、均质化和进样前液-液萃取和固相萃取的双重净化。结果如下：我们开发了一种简单的液-液萃取方法，以高回收率从生物组织中快速分离反义寡核苷酸，并将这些制备步骤与强大的整体柱LC-MS/MS设置相结合。与标准反相柱相比，该平台显示出更高的色谱分辨率和检测灵敏度，并且需要较低的样品体积。结论：高通量方法足以准确定量小鼠组织和血浆中的多种反义寡核苷酸，分别低至ng/g和ng/ml水平，用于药代动力学测定，并表现出高度的特异性。
Background: A significant challenge of oligonucleotide bioanalysis is the selective extraction from complex tissue samples, where the molecules that distribute into the intracellular space are extensively protein bound and sit amongst a high concentration of endogenous nucleic acid material. Published analytical methodology currently purports extensive sample preparation requirements that include cell lysis steps, homogenization and dual cleanup with liquid-liquid extraction and solid-phase extraction, prior to injection. Results: We have developed a simple liquid-liquid extraction approach to rapidly isolate antisense oligonucleotides from biological tissues with high recovery and combined these preparative steps with a robust monolithic column LC-MS/MS setup. The platform showed improved chromatographic resolution and detection sensitivity over standard reversed-phase columns and required a low sample volume. Conclusion: The high-throughput method was sufficient to accurately quantify multiple antisense oligonucleotides in mouse tissue and plasma down to low ng/g and ng/ml levels, respectively, for pharmacokinetic determination, and exhibited a high degree of specificity.