Optimisation of Heated Electrospray Ionisation Parameters to Minimise In-Source Generated Impurities in the Analysis of Oligonucleotide Therapeutics

IF 1.8 3区 化学 Q4 BIOCHEMICAL RESEARCH METHODS
Mollie A. Glenister, Ulrik Mistarz, Ken Cook, James L. Stephenson, Mark J. Dickman
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引用次数: 0

Abstract

Rationale

Oligonucleotides have emerged as an important new class of therapeutic. Due to their structural complexity, this presents significant challenges for the development of analytical methods to characterise and determine their impurity profile. In this study, we introduce a sensitive ion-pair reverse phase method interfaced with mass spectrometry for analysis of antisense oligonucleotides and small interfering RNAs.

Methods

Liquid chromatography–mass spectrometry analysis of antisense oligonucleotides and small interfering RNAs was performed using hexylamine: hexafluoro-2-propanol mobiles phases. LC-MS analysis was performed in both negative and positive ion mode. Electrospray ionisation source conditions including collision energy and temperature were optimised to minimise in-source generated impurities and alkylamine adducts in the analysis of oligonucleotide therapeutics.

Results

The results show that under low or no in-source collision energy the presence of hexylamine adducts are observed and are predominantly on the lowest charge states present. As the in-source collision energy is increased, a reduction of hexylamine adducts is observed in conjunction with an increase in nucleobase loss in the gas phase, therefore generating in-source impurities. In comparison to tributylammonium acetate, increased MS sensitivity, higher charge states and effective removal of hexylamine adducts using mild source conditions was achieved.

Conclusions

Optimisation of the mild source conditions in conjunction with high pH mobile phases was combined with high-resolution accurate mass spectrometry analysis and automated deconvolution workflows to develop a simplified and streamlined approach for characterising oligonucleotide therapeutics and their related impurities.

Abstract Image

优化加热电喷雾电离参数,以尽量减少源内产生的杂质在寡核苷酸治疗分析
寡核苷酸已成为一类重要的新型治疗药物。由于其结构的复杂性,这对表征和确定其杂质谱的分析方法的发展提出了重大挑战。在这项研究中,我们引入了一种敏感的离子对反相方法与质谱相结合,用于分析反义寡核苷酸和小干扰rna。方法采用六胺-六氟-2-丙醇流动相进行反义寡核苷酸和小干扰rna的液相色谱-质谱分析。LC-MS分析在负离子和正离子模式下进行。优化了电喷雾电离源条件,包括碰撞能量和温度,以最大限度地减少源内产生的杂质和烷基胺加合物在寡核苷酸治疗分析中。结果表明,在低源碰撞能或无源碰撞能的情况下,已胺加合物主要存在于最低电荷态。随着源内碰撞能量的增加,观察到己胺加合物的减少与气相核碱损失的增加一起,因此产生源内杂质。与醋酸三丁基铵相比,在温和的源条件下,质谱灵敏度提高,电荷态更高,并能有效去除己胺加合物。结论温和源条件的优化与高pH流动相结合,结合高分辨率准确的质谱分析和自动反褶积工作流程,开发了一种简化和流线化的方法来表征寡核苷酸治疗药物及其相关杂质。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
4.10
自引率
5.00%
发文量
219
审稿时长
2.6 months
期刊介绍: Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.
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