Cross platform solutions to improve the zebrafish polar metabolome coverage using LC-QTOF MS: Optimization of separation mechanisms, solvent additives, and resuspension solvents

Untargeted metabolomics has been widely used for studies with zebrafish embryos. Until now, the number of analytical approaches to determine metabolites in zebrafish is limited, and there is a lack of consensus on the best platforms for comprehensive metabolomics analysis of zebrafish embryos. In addition, the capacity of these methods to detect metabolites is unsatisfactory and the confidence level for identifying compounds is relatively low. To improve the metabolome coverage, we mainly focused on the optimization of separation mechanisms, mobile phase additives, and resuspension solvents based on liquid chromatography (LC) coupling to high-resolution mass spectrometry (HRMS) techniques. Moreover, the procedures for optimizing methods were assessed when taking metabolite profiles in both positive and negative ionization modes into account. Four LC columns were studied: C18, T3, PFP, and HILIC. In positive ionization mode, it was strongly recommended to employ the HILIC approach operated at the neutral condition, which led to the presence of more than 4700 features and the annotation of 151 metabolites, mainly zwitterionic and basic compounds, in comparison to reverse phase (RP)-based methods with less than 1000 features. In negative ionization mode, the PFP column operated at 0.02% acetic acid showed the best performance in terms of metabolite coverage: 3100 metabolic features were detected and 218 metabolites were annotated in zebrafish embryos. Metabolite profiles mainly contained acidic and zwitterionic compounds. HILIC-based platforms were complementary to RP columns when analyzing highly polar metabolites. Additionally, it was preferable to reconstitute zebrafish extracts in 100% water for analysis of metabolites on RP columns, with a 20-30% increase in the number of identified metabolites compared to a 50% water in methanol solution. However, water/methanol (1:9, v/v), as resuspension solution, was advantageous over water/methanol (1:1, v/v) for HILIC analysis showing an 8-15% increase in detected metabolites. In total 336 polar metabolites were annotated by the combination of the optimized HILIC (positive) and PFP (negative) approaches. The largest metabolome coverage of polar metabolites in zebrafish embryos was obtained when three approaches were combined (negative PFP and HILIC, and HILIC positive) resulting in more than 420 annotated compounds.