The use of liquid chromatography coupled with mass spectrometry (LC-MS) for the characterization of oligonucleotides and nucleic acids is a powerful analytical method. Recently, hydrophilic interaction chromatography (HILIC) has been proposed as a reasonable alternative to ion-pair reversed phase separations of oligonucleotides prior to MS. A wide variety of HILIC stationary phase surface chemistries are currently available. Although their selectivity can be considerably different, few studies have compared these chemistries for LC-MS analysis of oligonucleotides. We evaluated ten different HILIC column chemistries to understand their capabilities for separating a variety of oligonucleotides. In general, we found that most columns were ineffective at separating larger (n > 15-mer) oligonucleotides under the mobile phase and gradient conditions evaluated here. However, several stationary phases were found to be effective for separating smaller oligonucleotides such as endonuclease digestion products. Given that early eluting oligonucleotides were found to be compatible with standard electrospray ionization conditions, several different HILIC stationary phase options are available for LC-MS studies of smaller oligonucleotides including those generated in RNA modification mapping experiments.

中文翻译：

---

寡核苷酸亲水相互作用液相色谱分离中的固定相效应


使用液相色谱-质谱联用 （LC-MS） 表征寡核苷酸和核酸是一种强大的分析方法。最近，亲水作用色谱 （HILIC） 被提议作为 MS 之前寡核苷酸离子对反相分离的合理替代方案。目前有多种 HILIC 固定相表面填料可供选择。尽管它们的选择性可能有很大差异，但很少有研究将这些填料用于寡核苷酸的 LC-MS 分析。我们评估了 10 种不同的 HILIC 色谱柱填料，以了解它们分离各种寡核苷酸的能力。总的来说，我们发现，在本文评估的流动相和梯度条件下，大多数色谱柱在分离较大的 （n > 15-mer） 寡核苷酸方面无效。然而，发现几种固定相可有效分离较小的寡核苷酸，例如核酸内切酶消化产物。鉴于发现早期洗脱的寡核苷酸与标准电喷雾电离条件兼容，因此有几种不同的 HILIC 固定相选项可用于较小寡核苷酸的 LC-MS 研究，包括在 RNA 修饰图谱实验中生成的寡核苷酸。