High-Throughput Fractionation Coupled to Mass Spectrometry for Improved Quantitation in Metabolomics

Metabolomics
is emerging as an important field in life sciences.
However, a weakness of current mass spectrometry (MS) based metabolomics
platforms is the time-consuming analysis and the occurrence of severe
matrix effects in complex mixtures. To overcome this problem, we have
developed an automated and fast fractionation module coupled online
to MS. The fractionation is realized by the implementation of three
consecutive high performance solid-phase extraction columns consisting
of a reversed phase, mixed-mode anion exchange, and mixed-mode cation
exchange sorbent chemistry. The different chemistries resulted in
an efficient interaction with a wide range of metabolites based on
polarity, charge, and allocation of important matrix interferences
like salts and phospholipids. The use of short columns and direct
solvent switches allowed for fast screening (3 min per polarity).
In total, 50 commonly reported diagnostic or explorative biomarkers
were validated with a limit of quantification that was comparable
with conventional LC–MS­(/MS). In comparison with a flow injection
analysis without fractionation, ion suppression decreased from 89%
to 25%, and the sensitivity was 21 times higher. The validated method
was used to investigate the effects of circadian rhythm and food intake
on several metabolite classes. The significant diurnal changes that
were observed stress the importance of standardized sampling times
and fasting states when metabolite biomarkers are used. Our method
demonstrates a fast approach for global profiling of the metabolome.
This brings metabolomics one step closer to implementation into the
clinic.

代谢组学（Metabolomics）正逐步成长为生命科学领域的重要研究方向。然而，当前基于质谱（MS）的代谢组学分析平台存在两大核心缺陷：分析流程耗时冗长，且在复杂基质样本中易产生严重的基质效应。为攻克这一难题，本研究开发了一套可与质谱在线联用的自动化快速分级模块。该分级流程通过串联三支高效固相萃取柱实现，三支色谱柱分别采用反相、混合模式阴离子交换及混合模式阳离子交换吸附剂化学体系。不同的吸附剂化学特性可基于极性、电荷特性与多种代谢物实现高效相互作用，同时可有效截留盐类、磷脂等常见基质干扰物。采用短柱搭配直接溶剂切换的策略，可实现快速筛查（每个极性模式仅需3分钟）。本研究共验证了50种常见的诊断或探索性生物标志物，其定量限性能与常规液相色谱-质谱（/串联质谱，LC–MS(/MS)）技术相当。与未进行分级处理的流动注射分析相比，该方法的离子抑制率从89%降至25%，灵敏度提升21倍。借助该验证后的分析方法，本研究探究了昼夜节律与进食状态对多种代谢物类别的影响。实验观测到的显著昼夜变化提示，在使用代谢物生物标志物时，标准化采样时间与禁食状态至关重要。本方法为代谢组全局谱分析提供了一种快速可行的技术路径，推动代谢组学向临床落地应用迈出了关键一步。