Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016, China; Department of Pharmaceutical and Biomedical Sciences, The University of Georgia College of Pharmacy, 250 W. Green Street, Athens, GA, 30602-2352, USA.
Department of Pharmaceutical and Biomedical Sciences, The University of Georgia College of Pharmacy, 250 W. Green Street, Athens, GA, 30602-2352, USA; Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Ain Shams University, Organization of African Unity Street, Abassia, Cairo, 11566, Egypt.
J Chromatogr A. 2018 Dec 14;1580:110-119. doi: 10.1016/j.chroma.2018.10.040. Epub 2018 Oct 25.
Alkylamines are commonly used to improve both chromatographic and mass spectral performance of electrospray ionization liquid chromatography mass spectrometry based methods for the analysis of oligonucleotides. Recently several new alkylamines have been introduced to enhance the electrospray mass spectral response for oligonucleotides; however, the chromatographic properties of these new alkylamines have not been rigorously assessed. We have investigated the retention, peak width, resolution and general chromatographic performance of fifteen different alkylamines for the separation of a model DNA, RNA and an antisense therapeutic oligonucleotide. Eleven of the fifteen alkylamines were shown to provide similar chromatographic performance across all three classes of oligonucleotides. Based on these findings, a model for the mechanism of retention of oligonucleotides using alkylamines and hexafluoroisopropanol mobile phases is proposed. Depending on the concentrations of alkylamines and pH adjustment, oligonucleotides can be retained by micellar chromatography and not the generally held ion-pairing mechanism. This conclusion is supported by light scattering, transmission electron microscopy and ion mobility experiments detecting three micron aggregates in the mobile phase at concentrations that are routinely used for LC-MS analysis of oligonucleotides. These aggregates are not detected at lower alkylamine concentrations where the retention mechanism follows an ion-pairing mechanism. The formation of these aggregates appears to be dependent on the pH of the mobile phase.
烷基胺常用于改善基于电喷雾电离液相色谱质谱法的寡核苷酸分析方法的色谱和质谱性能。最近，已经引入了几种新的烷基胺来增强寡核苷酸的电喷雾质谱响应；然而，这些新的烷基胺的色谱性质尚未得到严格评估。我们研究了十五种不同的烷基胺对模型 DNA、RNA 和反义治疗性寡核苷酸分离的保留、峰宽、分辨率和一般色谱性能。十五种烷基胺中有十一种在所有三种类型的寡核苷酸中都表现出相似的色谱性能。基于这些发现，提出了一种使用烷基胺和六氟异丙醇流动相的寡核苷酸保留机制模型。根据烷基胺和 pH 值的浓度调整，寡核苷酸可以通过胶束色谱而不是通常的离子对机制保留。这一结论得到了光散射、透射电子显微镜和离子淌度实验的支持，这些实验在常规用于寡核苷酸 LC-MS 分析的浓度下在流动相中检测到了三个微米的聚集体。在较低的烷基胺浓度下，不会检测到这些聚集体，此时保留机制遵循离子对机制。这些聚集体的形成似乎取决于流动相的 pH 值。
