Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100, Toruń, Poland.
Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100, Toruń, Poland.
J Chromatogr A. 2018 Jun 15;1554:71-80. doi: 10.1016/j.chroma.2018.04.037. Epub 2018 Apr 17.
The present study highlights the application of ultra high performance liquid chromatography coupled with mass spectrometry for the selective separation and sensitive quantification of microRNAs and modified phosphorothioate oligonucleotide. The Central Composite Design was used for comprehensive optimization of mass spectrometer parameters (for tandem mass spectrometer and quadrupole-time-of-flight mass spectrometer). Ion pair chromatography was used in order to separate the studied compounds. Furthermore, the optimization of concentration of ion pair reagents in the mobile phase was done with respect to mass spectrometry sensitivity and liquid chromatography separation. The greatest sensitivity for studied compounds was determined for the mixture of 100 mM hexafluoroisopropanol, 5 mM N,N-dimethylbutylamine and methanol. This mobile phase also provided the best separation results in the shortest time for two of the four columns used in the study. Finally, the Hypersil GOLD aQ was selected for routine analysis of microRNA and modified phosphorothioate oligonucleotide in serum samples. These compounds were extracted from the sample with the use of combined liquid-liquid and solid phase extraction. The method developed during the study was then applied for the qualitative and quantitative analysis with limits od quantification equal to 49-63 nM.
本研究重点介绍了超高效液相色谱与质谱联用技术在 microRNA 和修饰的硫代磷酸寡核苷酸的选择性分离和灵敏定量分析中的应用。采用中心复合设计对质谱仪参数（串联质谱仪和四极杆飞行时间质谱仪）进行了全面优化。采用离子对色谱法分离研究化合物。此外，还针对质谱灵敏度和液相色谱分离对流动相中离子对试剂浓度进行了优化。研究发现，对于 100mM 六氟异丙醇、5mM N,N-二甲基丁胺和甲醇的混合物，研究化合物的灵敏度最高。该流动相还为研究中使用的四列柱中的两列提供了最佳的分离效果，且用时最短。最后，Hypersil GOLD aQ 被选为血清样本中 microRNA 和修饰的硫代磷酸寡核苷酸的常规分析柱。这些化合物是使用液液和固相萃取相结合的方法从样品中提取出来的。研究过程中建立的方法可用于定性和定量分析，其定量下限为 49-63nM。
