Unveiling Metabolite Interference for Reliable LC–MS Targeted Metabolomics Analysis

Targeted metabolomics has revolutionized the field of metabolite measurement, offering a straightforward annotation workflow and excellent quantitative linearity. However, a recent study conducted by Li Chen and colleagues at Fudan University in Shanghai, China, sheds light on an essential factor that demands attention in LC–MS targeted metabolomics analysis: metabolite interference. Their research, published in Analytical Chemistry, emphasizes the significance of identifying and addressing metabolite interference to ensure accurate metabolite annotation and quantification (1).

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Metabolite interference occurs when one metabolite produces a peak within another metabolite's MRM setting (Q1/Q3) due to closely aligned retention times (RT). While isomeric metabolites with identical precursor and product ions are well-known sources of interference, the study uncovers additional interferences resulting from inadequate mass resolution in triple-quadrupole mass spectrometry and in-source fragmentation of metabolite ions.

Inadequate mass resolution in triple-quadrupole mass spectrometry refers to the instrument's limited ability to differentiate ions with similar mass-to-charge ratios (m/z) accurately. Triple-quadrupole mass spectrometers employ three sets of quadrupole analyzers to selectively filter ions based on their m/z values. However, when the mass resolution is inadequate, ions with slightly different m/z values can overlap, leading to spectral interference. This interference can result in erroneous identification or quantification of metabolites, particularly when metabolites with similar masses co-elute during chromatographic separation. On the other hand, in-source fragmentation of metabolite ions occurs within the ion source of the mass spectrometer. The high-energy environment during ionization can cause metabolite ions to undergo fragmentation, producing multiple product ions from a single precursor ion. In targeted metabolomics, where specific precursor-product ion transitions are monitored, in-source fragmentation can introduce interference by generating product ions that coincide with those of other metabolites, leading to false peaks and misinterpretation of the targeted metabolomics data.

To investigate the extent of metabolite interference, the researchers characterized targeted metabolomics data using 334 metabolite standards. Surprisingly, their analysis revealed that approximately 75% of the metabolites exhibited measurable signals in at least one other metabolite's MRM setting. However, employing different chromatography techniques allowed for the resolution of 65–85% of these interfering signals among the standards.

The team further delved into the implications of metabolite interference by combining interference analysis with manual inspection of cell lysate and serum data. Their findings indicated that approximately 10% of the approximately 180 annotated metabolites were mis-annotated or mis-quantified. These results underscore the critical importance of a comprehensive investigation into metabolite interference for ensuring accurate and reliable metabolite measurement in targeted metabolomics studies.

The research highlights the necessity of identifying and addressing metabolite interference in LC–MS targeted metabolomics analysis. By enhancing our understanding of this phenomenon, scientists can refine their methodologies and protocols, leading to more precise and trustworthy results in metabolomics research.

Reference

(1) Jia, Z.; Qiu, Q.; He, R.; Zhou, T.; Chen, L. Identification of Metabolite Interference Is Necessary for Accurate LC-MS Targeted Metabolomics Analysis. Anal. Chem. 2023, 95 (20), 7985–7992. DOI: https://doi.org/10.1021/acs.analchem.3c00804