Electrospray ionization and heterogeneous matrix effects in liquid chromatography/mass spectrometry based meta‐metabolomics: A biomarker or a suppressed ion?

Abstract

Rationale Correct biomarker determination in metabolomics is crucial for unbiased conclusions and reliable applications. However, this determination is subject to several drifts, e.g. matrix effects and ion suppression in Liquid Chromatography/Mass Spectrometry (LC/MS)‐based approaches. This phenomenon provokes critical issues for biomarker determination, particularly during comparative studies dealing with samples exhibiting heterogeneous complexities. Methods Occurrence of the issue was coincidentally noticed when studying the environmental impact of a complex bioinsecticide: Bacillus thuringiensis israelensis. The studied samples comprised insecticide‐spiked sediments and untreated control sediments. QuEChERS extractions followed by LC/ESI‐Q/ToF analyses were performed on sediments after 15 days of incubation. Meta‐metabolomes containing pesticide xenometabolites and sediment endometabolites were analyzed in depth using XCMS‐based computational data preprocessing. Multivariate statistical analyses (PCA, OPLS‐DA) and raw data crosschecks were performed to search for environmental biomarkers. Results Multivariate analyses and raw data crosschecks led to the selection of nine metabolites as biomarker candidates. However, when exploring the mass spectra, co‐elutions were noticed between seven of these metabolites and multi‐charged macromolecules originating from the pesticide. Provoked false positives were thus suspected due to a potential ion suppression exclusively occurring in the spiked samples. A dilution‐based approach was then applied. It confirmed five metabolites as suppressed ions. Conclusions Ion suppression should be considered as a critical issue for biomarker determination when comparing heterogeneous metabolic profiles. Raw chromatograms and mass spectra crosschecks are mandatory to reveal potential ion suppressions in such cases. Dilution is a suitable approach to filter reliable biomarker candidates before their identification and absolute quantification.

Chemotaxonomic patterns in intracellular metabolites of marine microbial plankton

“…After removal of metabolites added to culture media and those not detected in any of the 42 organisms (see Methods), there were 111 metabolites present in at least one culture with 99 found in more than one organism and 91 in more than two organisms. We did not determine absolute quantities of metabolites because accurate cell volumes were not measured for all organisms, and further, the differing biological matrices across samples cause varying ion suppression that make direct comparison of metabolite peak areas problematic (Chamberlain et al, 2019;Ghosson et al, 2021). In previous work, we quantified a subset of polar metabolites in 21 of the cultures based on measured or estimated cell volume in order to relate these data to field-based community observations (Heal et al, 2021).…”

Characteristic response of formulation ingredients revealed by ultra high performance liquid chromatography‐electrospray ionization‐high resolution mass spectrometry‐based untargeted screening of pesticides in soil

“…After manual preprocessing of MS/MS data was achieved, putative annotations were addressed. The annotation methodology using SIRIUS 4.4.29 22 is the same described in a previous work 26 . Nonetheless, as the initial/terminal groups of a given series are the same, it could be sufficient to identify with high confidence only one compound belonging to the series and the identity of all the other compounds belonging to it can be concluded via the addition/subtraction of one monomer.…”

“…The annotation methodology using SIR-IUS 4.4.29 22 is the same described in a previous work. 26 Nonetheless, as the initial/terminal groups of a given series are the same, it could be sufficient to identify with high confidence only one compound belonging to the series and the identity of all the other compounds belonging to it can be concluded via the addition/subtraction of one monomer. This fact renders practical the DDA-based approaches for untargeted screening of FIs in complex environmental matrices like soil, as it increases the chances to acquire reliable MS/MS data that allow annotating these compounds.…”

LC–HRMS-Driven Computational Toolbox to Assess Extraction Protocols Dedicated to Untargeted Analysis: How to Ease Analyzing Pesticide-Contaminated Soils?

“…software-based methodology described in a h i b i t e d . previous work18 (using exact m/z, isotope patterns, and MS/MS spectra, if available). Out of these 861 traces, 2 traces (0.23%) were annotated at level 5 of identification confidence according to Schymanski et al19 − derived from Sumner et al20 − (i.e., no elemental composition could be concluded; only m/z and RT were available), 269 traces (31.24%) were annotated at level 4 (only with a suggested elemental composition), 133 traces (15.45%) at level 3 (elemental composition + chemical family); 451 traces (52.38%) at level 2 (putatively identified metabolites); and 6 traces (0.70%) at level 1 (traces of nonanoic acid, leucine, isoleucine, and methionine; confirmed by their reference standard…”