Core Facilities-Clinical Proteomics and Metabolomics, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy.
Department of Chemistry and Industrial Chemistry, University of Genoa, 16146 Genoa, Italy.
Molecules. 2021 Jul 13;26(14):4256. doi: 10.3390/molecules26144256.
Liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is currently the method of choice for untargeted metabolomic analysis. The availability of established protocols to achieve a high confidence identification of metabolites is crucial. The aim of this work is to describe the workflow that we have applied to build an Accurate Mass Retention Time (AMRT) database using a commercial metabolite library of standards. LC-HRMS analysis was carried out using a Vanquish Horizon UHPLC system coupled to a Q-Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Milan, Italy). The fragmentation spectra, obtained with 12 collision energies, were acquired for each metabolite, in both polarities, through flow injection analysis. Several chromatographic conditions were tested to obtain a protocol that yielded stable retention times. The adopted chromatographic protocol included a gradient separation using a reversed phase (Waters Acquity BEH C18) and a HILIC (Waters Acquity BEH Amide) column. An AMRT database of 518 compounds was obtained and tested on real plasma and urine samples analyzed in data-dependent acquisition mode. Our AMRT library allowed a level 1 identification, according to the Metabolomics Standards Initiative, of 132 and 124 metabolites in human pediatric plasma and urine samples, respectively. This library represents a starting point for future metabolomic studies in pediatric settings.
液相色谱-高分辨质谱联用（LC-HRMS）目前是用于非靶向代谢组学分析的首选方法。建立具有高置信度代谢物鉴定能力的既定方案至关重要。本工作旨在描述我们应用于使用商业标准代谢物库构建精确质量保留时间（AMRT）数据库的工作流程。LC-HRMS 分析使用 Vanquish Horizon UHPLC 系统与 Q-Exactive Plus 混合四极杆-轨道阱质谱联用仪（Thermo Fisher Scientific，意大利米兰）进行。通过流注射分析，对每个代谢物在两种极性下，使用 12 种碰撞能获得碎片光谱。测试了几种色谱条件，以获得产生稳定保留时间的方案。所采用的色谱方案包括使用反相（Waters Acquity BEH C18）和亲水作用色谱（Waters Acquity BEH Amide）柱的梯度分离。获得了 518 种化合物的 AMRT 数据库，并在数据依赖采集模式下对真实血浆和尿液样本进行了测试。根据代谢组学标准倡议，我们的 AMRT 库分别对人儿科血浆和尿液样本中的 132 种和 124 种代谢物进行了 1 级鉴定。该库代表了儿科环境中未来代谢组学研究的起点。
