Fast and Scalable Sample Preparation for Integrated Metabolome and Exposome-Wide Association Studies (MWAS/ExWAS)

Broad coverage exposome- and metabolome-wide association studies rely on advanced instrumentation, typically anchored in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to investigate exposure-effect associations. Sample preparation for biological fluids is a delicate matter, as it is essential to strike a pragmatic balance between sensitivity, chemical coverage, robustness, and time efficiency to meet the requirements of large-scale epidemiological studies. Here, we established a protein precipitation workflow in a scalable, high-throughput format for human plasma and urine. Different protocols were evaluated utilizing a xenobiotic mixture containing >200 exposure compounds based on extraction recovery, repeatability, and applicability for nontargeted analysis (NTA) and suspect screening. The tested high-throughput options included a protein precipitation workflow in 96-well plates and a phospholipid removal plate. For urine, a dilute-and-shoot approach was additionally tested. The plate-based protein precipitation resulted in superior performance with acceptable extraction recoveries (RE, 60-140%) for >70% of the highly diverse analyte panel in plasma and >80% in urine, and acceptable repeatability with relative standard deviations of <30% for more than 95% of analytes in plasma and 80% in urine. NTA results exhibited only a small number of altered annotated features compared to a tube-based protocol used for benchmarking, while increasing the overall time efficiency by a factor of five. To further test the protocol, it was applied to the NIST plasma standard reference material SRM1950. A total of 22 toxicants were identified and (semi-)quantified, with most concentrations comparable to available reference values. The results demonstrate the suitability of the established protocol for combined MWAS/ExWAS.