Department of Pharmaceutical and Biomedical Sciences, The University of Georgia College of Pharmacy, 250 W. Green Street, Athens, GA, 30602-2352, USA.
J Am Soc Mass Spectrom. 2017 Aug;28(8):1647-1656. doi: 10.1007/s13361-017-1671-6. Epub 2017 Apr 12.
Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions into the gas phase, cause charge state reduction, and decrease cation adduction. However, the choice of such ion-pairing agents has considerable influence on the MS signal intensity of oligonucleotides as they can also cause significant ion suppression. Interestingly, optimal ion-pairing agents should be selected on a case by case basis as their choice is strongly influenced by the sequence of the oligonucleotide under investigation. Despite imposing major practical difficulties to analytical method development, such a highly variable system that responds very strongly to the nuances of the electrospray composition provides an excellent opportunity for a fundamental study of the electrospray ionization process. Our investigations using this system quantitatively revealed the major factors that influenced the ESI ionization efficiency of oligonucleotides. Parameters such as boiling point, proton affinity, partition coefficient, water solubility, and Henry's law constants for the ion-pairing reagents and the hydrophobic thymine content of the oligonucleotides were found to be the most significant contributors. Identification of these parameters also allowed for the development of a statistical predictive algorithm that can assist with the choice of an optimum IP agent for each particular oligonucleotide sequence. We believe that research in the field of oligonucleotide bioanalysis will significantly benefit from this algorithm (included in Supplementary Material) as it advocates for the use of lesser-known but more suitable ion-pair alternatives to TEA for many oligonucleotide sequences. Graphical Abstract ᅟ.
烷基胺在寡核苷酸的 LC-MS 分析中被广泛用作离子对试剂。除了更好的色谱分离外，它们还有助于将寡核苷酸离子解吸到气相中，降低离子的荷质比，并减少阳离子加合物的形成。然而，这种离子对试剂的选择对寡核苷酸的 MS 信号强度有相当大的影响，因为它们也会导致显著的离子抑制。有趣的是，最优的离子对试剂应该根据具体情况进行选择，因为它们的选择受到研究中寡核苷酸序列的强烈影响。尽管这种高度可变的系统对分析方法的开发造成了重大的实际困难，但它对电喷雾组成的细微变化反应非常强烈，为电喷雾电离过程的基础研究提供了极好的机会。我们使用该系统进行的研究定量揭示了影响寡核苷酸 ESI 电离效率的主要因素。沸点、质子亲和力、分配系数、离子对试剂的水溶性和亨利常数以及寡核苷酸的疏水性胸腺嘧啶含量等参数被发现是最重要的影响因素。这些参数的确定也允许开发一个统计预测算法，该算法可以帮助为每个特定的寡核苷酸序列选择最佳的 IP 试剂。我们相信，该算法（包含在补充材料中）将使寡核苷酸生物分析领域的研究受益匪浅，因为它提倡在许多寡核苷酸序列中使用不太知名但更合适的离子对替代物 TEA。
