Improved sensitivity of the nano ultra-high performance liquid chromatography-tandem mass spectrometric analysis of low-concentrated neuropeptides by reducing aspecific adsorption and optimizing the injection solvent
Improved sensitivity of the nano ultra-high performance liquid chromatography-tandem mass spectrometric analysis of low-concentrated neuropeptides by reducing aspecific adsorption and optimizing the injection solvent
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DOI：
10.1016/j.chroma.2014.07.086
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发表时间：
2014-09-19
影响因子：
4.1
通讯作者：
Van Eeckhauta, Ann
中科院分区：
文献类型：
--
作者：
Maes, Katrien;Van Liefferinge, Joeri;Van Eeckhauta, Ann
获得纳米 UHPLC-MS/MS 方法的最大灵敏度是量化微透析样品中神经肽皮摩尔浓度的首要条件。由于肽对 Eppendorf 管、移液器吸头和 UHPLC 小瓶的非特异性吸附不利于方法灵敏度，因此提出了一种策略来减少标准品制备过程中这些肽的吸附。在这方面，研究了从冻干粉末溶解到将样品注入系统的所有程序步骤。对神经调节肽家族的两种肽，即神经调节肽 B 和神经调节肽 N，以及神经调节肽 N 相关的神经肽神经降压素进行了评估。本研究的第一部分概述了一些已知影响肽溶解度的参数。第二部分的主要重点涉及通过实验设计优化 UHPLC 小瓶中的样品成分。关于乙腈、盐和基质成分的影响，观察到了相互矛盾的结果。人们发现它们对于将肽注入系统很重要，但至关重要的是需要将其排除在稀释溶剂之外。此外，表面材料的类型、温度和移液方案对吸附现象有很大影响。对中心复合设计结果的统计分析表明，使用由 13.1% V/V ACN 和 4.4% V/V FA 组成的注射溶剂获得了最高的肽响应。优化策略的这一方面可以被认为是提高方法灵敏度的主要因素。由于肽吸附的减少和进样溶剂的优化导致了三种肽的清晰且可定量的信号，因此在方法开发的早期阶段应考虑这两个问题的优化，特别是在设想分析低浓度肽溶液时。 (C) 2014 Elsevier B.V. 保留所有权利。
Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N, and a neuromedin N-related neuropeptide, neurotensin, are evaluated.The first part of this study outlines a number of parameters which are known to affect peptide solubility. The main focus of the second part involves the optimization of the sample composition in the UHPLC vial by using design of experiments.Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified as the main contributor to the gain in method sensitivity.Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged. (C) 2014 Elsevier B.V. All rights reserved.