Chemical exposomics in human plasma wasenhanced by an optimizedphospholipid removal step that increased targeted method sensitivitywhile also revealing >13,000 new molecular features by LC-HRMSnon-targetedacquisition. The challenge of chemical exposomics in human plasmais the 1000-foldconcentration gap between endogenous substances and environmentalpollutants. Phospholipids are the major endogenous small moleculesin plasma, thus we validated a chemical exposomics protocol with anoptimized phospholipid-removal step prior to targeted and non-targetedliquid chromatography high-resolution mass spectrometry. Increasedinjection volume with negligible matrix effect permitted sensitivemulticlass targeted analysis of 77 priority analytes; median MLOQ= 0.05 ng/mL for 200 & mu;L plasma.In non-targeted acquisition, mean total signal intensities of non-phospholipidswere enhanced 6-fold in positive (max 28-fold) and 4-fold in negativemode (max 58-fold) compared to a control method without phospholipidremoval. Moreover, 109 and 28% more non-phospholipid molecular featureswere detected by exposomics in positive and negative mode, respectively,allowing new substances to be annotated that were non-detectable withoutphospholipid removal. In individual adult plasma (100 & mu;L, n = 34), 28 analytes were detected and quantified among10 chemical classes, and quantitation of per- and polyfluoroalkylsubstances (PFAS) was externally validated by independent targetedanalysis. Retrospective discovery and semi-quantification of PFAS-precursorswas demonstrated, and widespread fenuron exposure is reported in plasmafor the first time. The new exposomics method is complementary tometabolomics protocols, relies on open science resources, and canbe scaled to support large studies of the exposome.

Chemical exposomes can now be comprehensively measured in human blood, but knowledge of their variability and longitudinal stability is required for robust application in cohort studies. Here, we applied high-resolution chemical exposomics to plasma of 46 adults, each sampled 6 times over 2 years in a multiomic cohort, resulting in 276 individual exposomes. In addition to quantitative analysis of 83 priority target analytes, we discovered and semiquantified substances that have rarely or never been reported in humans, including personal care products, pesticide transformation products, and polymer additives. Hierarchical cluster analysis for 519 confidently annotated substances revealed unique and distinctive coexposures, including clustered pesticides, poly(ethylene glycols), chlorinated phenols, or natural substances from tea and coffee; interactive heatmaps were publicly deposited to support open exploration of the complex (meta)data. Intraclass correlation coefficients (ICC) for all annotated substances demonstrated the relatively low stability of the exposome compared to that of proteome, microbiome, and endogenous small molecules. Implications are that the chemical exposome must be measured more frequently than other omics in longitudinal studies and four longitudinal exposure types are defined that can be considered in study design. In this small cohort, mixed-effect models nevertheless revealed significant associations between testosterone and perfluoroalkyl substances, demonstrating great potential for longitudinal exposomics in precision health research.