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The totality of environmental exposures and lifestyle factors, commonly referred to as the exposome, is poorly understood. Measuring the myriad of chemicals that humans are exposed to is immensely challenging, and identifying disrupted metabolic pathways is even more complex. Here, we present a novel technological approach for the comprehensive, rapid, and integrated analysis of the endogenous human metabolome and the chemical exposome. By combining reverse-phase and hydrophilic interaction liquid chromatography (HILIC) and fast polarityswitching, molecules with highly diverse chemical structures can be analyzed in 15 min with a single analytical run as both column's effluents are combined before analysis. Standard reference materials and authentic standards were evaluated to critically benchmark performance. Highly sensitive median limits of detection (LODs) with 0.04 μM for >140 quantitatively assessed endogenous metabolites and 0.08 ng/mL for the >100 model xenobiotics and human estrogens in solvent were obtained. In matrix, the median LOD values were higher with 0.7 ng/mL (urine) and 0.5 ng/mL (plasma) for exogenous chemicals. To prove the dualcolumn approach's applicability, real-life urine samples from sub-Saharan Africa (high-exposure scenario) and Europe (low-exposure scenario) were assessed in a targeted and nontargeted manner. Our liquid chromatography high-resolution mass spectrometry (LC-HRMS) approach demonstrates the feasibility of quantitatively and simultaneously assessing the endogenous metabolome and the chemical exposome for the high-throughput measurement of environmental drivers of diseases.

We introduce a new concept of yeast-derived biological matrix reference material for metabolomics research relying on in vivo synthesis of a defined biomass, standardized extraction followed by absolute quantification with isotope dilution. The yeast Pichia pastoris was grown using full control- and online monitoring fed-batch fermentations followed by fast cold methanol quenching and boiling ethanol extraction. Dried extracts served for the quantification campaign. A metabolite panel of the evolutionarily conserved primary metabolome (amino acids, nucleotides, organic acids, and metabolites of the central carbon metabolism) was absolutely quantified by isotope dilution utilizing uniformly labeled 13C-yeast-based internal standards. The study involved two independent laboratories employing complementary mass spectrometry platforms, namely hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) and gas chromatography-tandem mass spectrometry (GC–MS/MS). Homogeneity, stability tests (on a panel of >70 metabolites over a period of 6 months), and excellent biological repeatability of independent fermentations over a period of 2 years showed the feasibility of producing biological reference materials on demand. The obtained control ranges proved to be fit for purpose as they were either superior or comparable to the established reference materials in the field.

Covering a wide spectrum of molecules is essential for global metabolome assessment. While metabolomics assays are most frequently carried out in microbore LC-MS analysis, reducing the size of the analytical platform has proven its ability to boost sensitivity for specific -omics applications. In this study, we elaborate the impact of LC miniaturization on exploratory small-molecule LC-MS analysis, focusing on chromatographic properties with critical impact on peak picking and statistical analysis. We have assessed a panel of small molecules comprising endogenous metabolites and environmental contaminants covering three flow regimes—analytical, micro-, and nano-flow. Miniaturization to the micro-flow regime yields moderately increased sensitivity as compared to the nano setup, where median sensitivity gains around 80-fold are observed in protein-precipitated blood plasma extract. This gain resulting in higher coverage at low µg/L concentrations is compound dependent. At the same time, the nano-LC-high-resolution mass spectrometry (HRMS) approach reduces the investigated chemical space as a consequence of the trap-and-elute nano-LC platform. Finally, while all three setups show excellent retention time stabilities, rapid gradients jeopardize the peak area repeatability of the nano-LC setup. Micro-LC offers the best compromise between improving signal intensity and metabolome coverage, despite the fact that only incremental gains can be achieved. Hence, we recommend using micro-LC for wide-target small-molecule trace bioanalysis and global metabolomics of abundant samples.