Turnpenny Paul, Rawal Jaiessh, Schardt Torsten, Lamoratta Sandro, Mueller Heiko, Weber Markus, Brady Kevin
Pharmacokinetics, Dynamics & Metabolism Department, Pfizer Global Research & Development, Sandwich, UK.
Bioanalysis. 2011 Sep;3(17):1911-21. doi: 10.4155/bio.11.100.
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.
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.
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.
寡核苷酸生物分析面临的一个重大挑战是从复杂的组织样本中进行选择性提取，其中分布到细胞内空间的分子与蛋白质广泛结合，并且处于高浓度的内源性核酸物质之中。目前已发表的分析方法声称需要大量的样品制备步骤，包括细胞裂解步骤、匀浆以及在进样前通过液液萃取和固相萃取进行双重净化。
我们开发了一种简单的液液萃取方法，能够从生物组织中快速分离反义寡核苷酸，回收率高，并将这些制备步骤与强大的整体柱液相色谱 - 串联质谱装置相结合。该平台相对于标准反相柱显示出更高的色谱分辨率和检测灵敏度，并且所需样品体积较小。
该高通量方法足以分别准确地将小鼠组织和血浆中的多种反义寡核苷酸定量至低至纳克/克和纳克/毫升水平，用于药代动力学测定，并且具有高度的特异性。
