Figure 3

Validation experiment: randomised combinatorial metabolite standard mixing. Three metabolite mixtures each contained a set of approximately 30 different non-co-eluting high-purity authentic metabolite standards at known concentrations (typical metabolite concentration: 200 ng/μl). The components of these mixtures were chosen such that no chromatographic co-elution would occur between components of the same mixture but co-elution would occur between components of different mixtures when two or more of the mixtures were combined into a single analysis. An eight point dilution series was prepared from each of the three mixtures, generating three sets of eight solutions. The order of each dilution series was randomised to generate a randomised mixing protocol table and aliquots of solutions were combined accordingly. This randomised mixing process was repeated 5 times to generate 5 sets of 8 solutions (40 solutions). These complex solutions, each containing the same set of 85 metabolite standards, were analysed by a standard GC/MS metabolomics protocol.