Overview of the seven extraction protocols used as well as the model organisms and sample types investigated. The protocols increase in handling effort and complexity from left to right. The color code indicates similarities amongst the protocols either through solvents or chemicals used. All extraction products were stored at −80°C until further processing. A list of abbreviations can be found above.

Metabolite coverage per extraction protocol across all sample types and model organisms. Indicated by color are the different metabolite classes measured. A metabolite was counted as detectable when at least 2 out of 3 replicates were >LOD within a given extraction protocol. The legend is categorized between compound classes measured via LC-MS/MS or FIA-MS/MS. Ordering from left to right follows the level of complexity and required time per extraction.

Number of metabolites per class with the highest concentration per extraction protocol across all sample types and model organisms. Metabolites that appear in the bar chart are only counted when they produce the highest or a non-significantly lower concentration (p > 0.05, see Material and Methods) than another tested extraction protocol. The dotted line shows the number of detectable metabolites for each sample type. Indicated by color are the different metabolite classes measured. The legend is categorized between the LC-MS/MS and FIA-MS/MS measurements. A list of the metabolites per extraction protocol can be viewed online in “MetaboExtract”. Of note, the metabolite classes that were best suited for a single extraction protocol are depicted in Supplementary Figure S4.

Variability of extraction procedures across all sample types and model organisms. Indicated by color are the different extraction protocols used. The CV% was generated based on the triplicate analyses. For each of the CV% categories the percentage of the total 630 metabolites. Note that 2xMeOH/MTBE/MeOH was only used for mice sample types. Alternative visualizations of the CV% and MAD are shown in the (Supplementary Figure S3, S4).

Venn diagram showing number of metabolites that are common or unique across all sample types and model organisms within the 75EtOH/MTBE extraction protocol. A comparison of the remaining extraction protocols can be found in the supplement (see Supplementary Figure S5). A list of the metabolites that are unique and overlapping can be found in the supplementary data file (see supplementary Data File S1).