Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, 7 Gagarin Str., PL-87-100 Toruń, Poland.
Analyst. 2019 Aug 7;144(15):4622-4632. doi: 10.1039/c9an00740g. Epub 2019 Jun 27.
Our research focused on applying microextraction by packed sorbent to extracting antisense oligonucleotides from serum samples. The tested sorbents included poly(styrene-co-divinylbenzene), octyl, octadecyl, and unmodified silica gel. As nonpolar sorbents were used for highly-polar molecules, this required ion-pair mode. Comprehensive optimization of extraction conditions was performed for 20-mer phosphorothioate oligonucleotide. Several parametres - the number of "draw-eject" cycles during the conditioning and load step, the amine type and concentration, and the volume of elution mixture - and the influence they had on recovery were studied for nonpolar sorbents, which made it possible to obtain high (ca. 90%) recovery values. The most influential parameter turned out to be the volume of elution mixture. Similar optimization was performed for silica sorbents; however, despite optimization of various parameters, the recovery values stayed relatively low. The optimized procedures for nonpolar sorbents were applied in extraction of six different oligonucleotides of various length and with different structure modifications. The highest recoveries were obtained for octyl and octadecyl sorbents, ranging between 80-99%. The developed microextraction method was used to extract phosphorothioate and 2'-O-(2-methoxyethyl) oligonucleotides and their two synthetic metabolites from enriched human plasma, with recoveries around 70-80%.
我们的研究集中于应用填充固相微萃取从血清样品中提取反义寡核苷酸。测试的固定相包括聚苯乙烯-二乙烯基苯共聚物、辛基、十八烷基和未改性硅胶。由于非极性固定相用于高极性分子，因此需要采用离子对模式。我们对 20 个碱基的硫代磷酸酯寡核苷酸进行了综合的萃取条件优化。对非极性固定相进行了几个参数（在预处理和加载步骤中“抽取-注入”的循环次数、胺的类型和浓度以及洗脱混合物的体积）及其对回收率影响的研究，从而实现了高（约 90%）的回收率。最具影响力的参数是洗脱混合物的体积。对硅胶固定相也进行了类似的优化；然而，尽管对各种参数进行了优化，回收率仍然相对较低。优化后的非极性固定相程序应用于提取六种不同长度和不同结构修饰的寡核苷酸。辛基和十八烷基固定相的回收率最高，在 80-99%之间。开发的微萃取方法用于从富有人血浆中提取硫代磷酸酯和 2'-O-(2-甲氧基乙基)寡核苷酸及其两种合成代谢物，回收率约为 70-80%。
