Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100 Toruń, Poland.
Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100 Toruń, Poland.
Talanta. 2019 May 1;196:54-63. doi: 10.1016/j.talanta.2018.12.023. Epub 2018 Dec 13.
The aim of the present research was to study the impact of six various ion-pairing reagents in ion pair chromatography mode on both retention and ionization of seven antisense oligonucleotides which have the same sequence but different types of modifications and length. Furthermore, retention and selectivity studies were performed with the use of four different stationary phases, including octadecyl, octyl, and pentafluorophenyl groups as well as ligands with embedded polar groups. The results of the research showed that the main factors influencing the retention of the studied compounds are amine hydrophobicity and chain branching. The greatest retention was obtained for hexylamine, while the lowest for propylamine. Octadecyl and pentafluorophenyl columns were characterized by the best selectivity and the former was selected for further investigation. What is more, tandem mass spectrometry parameters were optimized and the sensitivity of oligonucleotide mass spectrometry determination was assessed. The greatest sensitivity was obtained for 5 mM N,N-dimethylbutylamine combined with 1,1,1,3,3,3-hexafluoroisopropanol and methanol. The developed method was successfully applied for the determination and separation of the mixture of seven different oligonucleotides in fortified serum, which was completed in 8 min. The LOQ values of the developed method were in the range of 0.15-0.56 µM (0.91-3.56 ng on column). Moreover, it has to be highlighted that it was the first time that such comprehensive investigations were carried out for various types of oligonucleotide modifications and for different stationary phases as well as ion pair reagents.
本研究的目的是研究六种不同的离子对试剂在离子对色谱模式下对 7 种具有相同序列但不同修饰类型和长度的反义寡核苷酸的保留和离解的影响。此外，还使用四种不同的固定相进行了保留和选择性研究，包括十八烷基、辛基和五氟苯基基团以及带有嵌入式极性基团的配体。研究结果表明，影响研究化合物保留的主要因素是胺的疏水性和支链。对于己胺，获得了最大的保留，而对于丙胺，获得了最小的保留。十八烷基和五氟苯基柱的选择性最好，选择前者进行进一步研究。此外，优化了串联质谱参数，并评估了寡核苷酸质谱测定的灵敏度。使用 5 mM N,N-二甲基丁胺与 1,1,1,3,3,3-六氟异丙醇和甲醇组合，获得了最大的灵敏度。开发的方法成功应用于加标血清中 7 种不同寡核苷酸混合物的测定和分离，在 8 分钟内完成。所开发方法的 LOQ 值在 0.15-0.56 µM 范围内（柱上 0.91-3.56 ng）。此外，值得强调的是，这是首次对各种类型的寡核苷酸修饰以及不同的固定相和离子对试剂进行如此全面的研究。
