Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, GA, 30602-2352, USA.
Rapid Commun Mass Spectrom. 2013 Dec 15;27(23):2655-64. doi: 10.1002/rcm.6733.
New methods to enhance the electrospray ionization (ESI) signals are essential for low-level analysis of oligonucleotides. We report a systematic evaluation comparing 13 ion-pairing agents with and without hexafluoroisopropanol to understand their effect on the ion abundance of hetero-oligonucleotides.
A Waters Synapt G2 HDMS quadrupole time-of-flight instrument was used to compare oligonucleotide signal intensity with 13 alkylamine ion-pairing agents at varying concentrations. The alkylamines that yielded the highest signal intensity were further evaluated with hexafluoroisopropanol at concentrations between 5 and 100 mM. The chemical properties of the solution components and analytes were evaluated to identify key factors in predicting optimal mobile phase conditions for different classes of oligonucleotides.
We identified a series of optimized mobile phase systems using diisopropylamine, tripropylamine, dimethylbutylamine, methyldibutylamine, and dimethylhexylamine along with 25 to 50 mM hexafluoroisopropanol that yielded significantly higher MS signal intensity for both siRNA and DNA compared with the traditionally used triethlyamine/hexafluoroisopropanol system. We explored charge state reduction, adduct formation and ESI mechanisms and identify the Henry's Law constant k aq/g as a key chemical property in predicting alkylamines that will increase oligonucleotide ion intensity. We also find that the hydrophobicity of the oligonucleotide plays a major role in choosing ion-pairing agents that will increase ion abundance.
This comprehensive and systematic optimization finds that the hydrophobicity of the oligonucleotide was a key factor in choosing alkylamine ion-pairing agents to increase ESI abundance. We identified that diisopropylamine and tripropylamine combined with lower concentrations of hexafluoroisopropanol yielded the highest signal intensity for these oligonucleotides.
新方法来增强电喷雾电离（ESI）信号对于寡核苷酸的低水平分析至关重要。我们报告了一个系统的评估，比较了 13 种带有和不带有六氟异丙醇的离子对试剂，以了解它们对异源寡核苷酸离子丰度的影响。
使用沃特世 Synapt G2 HDMS 四极杆飞行时间仪器，在不同浓度下比较了 13 种烷基胺离子对试剂对寡核苷酸信号强度的影响。在 5 至 100mM 的浓度下，进一步评估了产生最高信号强度的烷基胺与六氟异丙醇的结合情况。评估了溶液成分和分析物的化学性质，以确定不同类别寡核苷酸最佳流动相条件的关键因素。
我们使用二异丙基胺、三丙基胺、二甲基丁基胺、甲基二丁基胺和二甲基己基胺以及 25 至 50mM 的六氟异丙醇，确定了一系列优化的流动相体系，与传统的三乙胺/六氟异丙醇体系相比，这些体系显著提高了 siRNA 和 DNA 的 MS 信号强度。我们探讨了电荷状态还原、加合物形成和 ESI 机制，并确定亨利定律常数 k aq/g 为预测增加寡核苷酸离子强度的烷基胺的关键化学性质。我们还发现，寡核苷酸的疏水性在选择增加离子丰度的离子对试剂方面起着主要作用。
这项全面而系统的优化发现，寡核苷酸的疏水性是选择烷基胺离子对试剂来增加 ESI 丰度的关键因素。我们发现，二异丙基胺和三丙基胺与较低浓度的六氟异丙醇结合，为这些寡核苷酸产生了最高的信号强度。
