INTRODUCTION LC-MS-based untargeted metabolomics has become increasingly popular due to the vast amount of information gained in a single analysis. Many studies utilize metabolomics to profile metabolic changes in various representative biofluids, tissues, or other sample types. Most analyses are performed measuring changes in the metabolic pool of a single biological matrix due to an altered phenotype, such as disease versus normal. Measurements in such experiments are typically highly reproducible with little variation due to analytical and technological advancements in mass spectrometry. With the expanded application of metabolomics into various non-analytical scientific disciplines, the emergence of studies comparing the signal intensities of specific analytes across different biological matrices (e.g. plasma vs. urine) is becoming more common, but the matrix effect between sample types is often neglected. Additionally, the practice of comparing the signal intensities of different analytes and correlating to relative abundance is also increasingly prevalent, but the response ratio between analytes due to differences in ionization efficiency is not always accounted for in data analysis. This report serves to communicate and raise awareness of these two well-recognized issues to prevent improper data interpretation in the field of metabolomics. OBJECTIVES We demonstrate the impact of matrix effects and ionization efficiency with labeled analytical standards in human plasma, serum, and urine and describe how the direct comparison of non-quantitative signal intensities between biofluids, as well as between different analytes in the same biofluid, in untargeted metabolomics is inaccurate without proper response corrections. METHODS Human plasma, serum, and urine (n = 4 technical replicates per biofluid) were spiked with a panel of labeled internal standards all at identical concentrations, simultaneously extracted, and analyzed by UHPLC-HRMS. Signal intensities were compared for demonstration of the impact of matrix effects in untargeted metabolomics. A neat mixture of two co-eluting, structurally-similar labeled standards at the same concentration was also analyzed to demonstrate the effect of ionization efficiency on signal intensity. RESULTS Despite being spiked at identical concentrations, labeled standards we examined in this study showed significant differences in their signal intensities between biofluids, as well as from each other in the same biofluid, due to matrix effects. Co-eluting standards were also found to yield significantly different signal intensities at identical concentrations due to differences in ionization efficiency. CONCLUSIONS Due to the presence of matrix effects in untargeted, non-quantitative metabolomics, the signal intensity of any single analyte cannot be directly compared to the signal intensity of that same analyte (or any other analyte) between any two different matrices. Due to differences in ionization efficiency, the signal intensity of any single analyte cannot be directly compared to the signal intensity of any other analyte, even in the same matrix.

中文翻译：

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非定量非靶向代谢组学中基质效应和电离效率的影响。

引言由于基于一次分析的大量信息，基于LC-MS的非靶向代谢组学已变得越来越流行。许多研究利用代谢组学来描述各种代表性生物流体，组织或其他样品类型中的代谢变化。大多数分析都是通过测量表型改变（例如疾病与正常）引起的单个生物基质代谢库的变化来进行的。由于质谱分析和技术的进步，此类实验中的测量值通常具有很高的可重复性，几乎没有变化。随着代谢组学在各种非分析科学领域中的广泛应用，比较不同生物基质（例如血浆与尿液）中特定分析物信号强度的研究的出现变得越来越普遍，但是样本类型之间的矩阵效应常常被忽略。另外，比较不同分析物的信号强度并与相对丰度相关的做法也越来越普遍，但是由于电离效率的差异而导致的分析物之间的响应率并非总是在数据分析中考虑的。该报告旨在交流和提高对这两个公认问题的认识，以防止在代谢组学领域进行不正确的数据解释。目的我们用标记的分析标准品在人血浆，血清和尿液中证明基质效应和电离效率的影响，并描述如何直接比较生物流体之间以及同一生物流体中不同分析物之间的非定量信号强度，没有适当的反应更正，在非目标代谢组学中的应用是不准确的。方法将人血浆，血清和尿液（每个生物流体n = 4次技术重复）与一组标记的内标物加标，浓度均相同，同时萃取并通过UHPLC-HRMS进行分析。比较信号强度以证明基质效应在非靶向代谢组学中的影响。还分析了两种浓度相同的共洗脱，结构相似的标记标准品的纯净混合物，以证明电离效率对信号强度的影响。结果尽管掺入了相同的浓度，但由于基质效应，我们在这项研究中检测的标记标准品在生物流体之间以及在同一生物流体中彼此之间的信号强度存在显着差异。由于电离效率的差异，在相同浓度下，共洗脱标准液也会产生明显不同的信号强度。结论由于在非靶向，非定量代谢组学中存在基质效应，因此无法将任何单个分析物的信号强度直接与任何两个不同基质之间相同分析物（或任何其他分析物）的信号强度进行比较。由于电离效率的差异，即使在同一基质中，也无法将任何一种分析物的信号强度直接与任何其他分析物的信号强度进行比较。在非定量代谢组学中，任何单一分析物的信号强度都不能直接与任何两种不同基质之间相同分析物（或任何其他分析物）的信号强度进行比较。由于电离效率的差异，即使在同一基质中，也无法将任何一种分析物的信号强度直接与任何其他分析物的信号强度进行比较。在非定量代谢组学中，任何单一分析物的信号强度都不能直接与任何两种不同基质之间相同分析物（或任何其他分析物）的信号强度进行比较。由于电离效率的差异，即使在同一基质中，也无法将任何一种分析物的信号强度直接与任何其他分析物的信号强度进行比较。