Birdsall Robert E, Gilar Martin, Shion Henry, Yu Ying Qing, Chen Weibin
Waters Corp., 34 Maple St, Milford, MA, 01757-3604, USA.
Rapid Commun Mass Spectrom. 2016 Jul 30;30(14):1667-1679. doi: 10.1002/rcm.7596.
Electrospray ionization mass spectrometry (ESI-MS)-based techniques commonly used in oligonucleotide analyses are known to be sensitive to alkali metal adduct formation. Adducts directly impact the sensitivity of MS-based analyses as the available charge is distributed across the parent peak and adduct(s). The current study systematically evaluated common liquid chromatography (LC) components in LC/ESI-MS configurations used in oligonucleotide analysis to identify metal adduct contributions from LC instrumentation.
A UPLC liquid chromatography system was configured with a single quadrupole MS detector (ACQUITY QDa, Waters Corp.) to monitor adduct formation in oligonucleotide separations. An ion-pairing mobile phase comprised of 15 mM triethylamine and 400 mM hexafluoro-2-propanol was used in conjunction with an oligonucleotide separation column (Waters OST BEH C18, 2.1 mm × 50 mm) for all separations. A 10-min method was used to provide statistical figures of merit and evaluate adduct formation over time.
Trace alkali metal salts in the mobile phase and reagents were determined to be the main source of metal salt adducts in LC/ESI-MS-based configurations. Non-specific adsorption sites located throughout the fluidic path contribute to adduct formation in oligonucleotide analyses. Ion-pairing mobile phases prepared at neutral or slightly basic pH result in up to a 57% loss of spectral abundance to adduct formation in the current study.
Implementation of a short low pH reconditioning step was observed to effectively displace trace metal salts non-specifically adsorbed to surfaces in the fluidic path and was able to maintain an average MS spectral abundance ≥94% with a high degree of repeatability (relative standard deviation (R.S.D.) 0.8%) over an extended time study. The proposed method offers the ability to rapidly regenerate adsorption sites with minimal impact on productivity while retaining assay sensitivity afforded by MS detection with reduced adduct formation. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.
基于电喷雾电离质谱（ESI-MS）的技术常用于寡核苷酸分析，已知其对碱金属加合物的形成敏感。加合物会直接影响基于质谱分析的灵敏度，因为可用电荷分布在母峰和加合物之间。本研究系统评估了寡核苷酸分析中使用的液相色谱（LC）/ESI-MS配置中的常见液相色谱组件，以确定液相色谱仪器产生的金属加合物的贡献。
将超高效液相色谱（UPLC）系统与单四极杆质谱检测器（ACQUITY QDa，沃特世公司）配置在一起，以监测寡核苷酸分离过程中的加合物形成。所有分离均使用由15 mM三乙胺和400 mM六氟-2-丙醇组成的离子对流动相，并结合寡核苷酸分离柱（沃特世OST BEH C18，2.1 mm×50 mm）。采用10分钟的方法来提供统计性能指标，并评估随时间的加合物形成情况。
流动相和试剂中的痕量碱金属盐被确定为基于LC/ESI-MS配置中金属盐加合物的主要来源。整个流体路径中存在的非特异性吸附位点会导致寡核苷酸分析中的加合物形成。在本研究中，在中性或略碱性pH下制备的离子对流动相会导致高达57%的光谱丰度损失于加合物形成。
观察到实施一个短的低pH再生步骤能够有效地置换非特异性吸附在流体路径表面的痕量金属盐，并且在长时间研究中能够以高度的重复性（相对标准偏差（R.S.D.）0.8%）维持平均质谱光谱丰度≥94%。所提出的方法能够在对生产力影响最小的情况下快速再生吸附位点，同时通过减少加合物形成来保持质谱检测提供的分析灵敏度。© 2016作者。《质谱快报》由约翰·威利父子有限公司出版。
