Blood Sample Preparation for Human Chemical Exposomics: Insights from Large-Scale Applications

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) enables profiling of hundreds of externally derived chemicals and their metabolites in human biofluids to capture the internal chemical exposome. Nevertheless, developing sensitive and robust high-throughput methodologies to scale up exposomics remains challenging. We compared two validated sample preparation methods (SPMs) based on protein precipitation (PPT) and PPT combined with phospholipid removal (PLR) in two cohorts using blood plasma (<i>n</i> = 109) and serum (<i>n</i> = 75) to assess their impact on LC-HRMS repeatability and chemical coverage. Additional filtration on PLR plates allowed the injection of extracts twice concentrated without affecting LC-HRMS repeatability. The comparison of chemical coverage between SPMs was then assessed using 184 compounds (including a large variety of exogenous chemicals) annotated with a suspect screening strategy. Most chemicals (78-86%) were detected with both SPMs, but a substantial fraction showed preferential (>2-fold, 37-50%) or exclusive (14-22%) detection with one SPM, reflecting differences in matrix effects and/or recoveries. Low-abundance chemicals were more sensitive to the choice of SPM, though no predictive trends related to physicochemical properties emerged, likely due to higher analytical uncertainty. Overall, both SPMs provided acceptable chemical coverage for large-scale exposomics. Nonetheless, the compound-dependent nature of detection highlights the value of applying complementary SPMs when feasible.