High-throughput screening to identify endocrine disruptors: Contribution of low-resolution tandem MS and high-resolution MS

Background: Considering the large diversity of chemicals present in the environment and the need to study their effects (alone or as mixtures), the development of high-throughput in vitro assays in line with the Replacement, Reduction, Refinement (3R) strategy is essential for chemical risk assessments.

Results: We developed a robust analytical workflow based on both low resolution tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry (HRMS) to quantify 13 steroids in NCI-H295R cell culture medium, human plasma and serum. The workflow was validated by screening media from the NCI-H295R cell line exposed in dose-response experiments to 5 endocrine disruptors (EDs) such as bisphenol A, prochloraz, ketoconazole, atrazine and forskolin. Absolute quantifications of the 13 steroids performed on a triple quadrupole (QqQ) MS/MS demonstrated that the performances obtained were in line with OECD recommendations. HRMS (MS1-HRMS) provided measurements nearly as sensitive and as reproducible as those obtained using multiple reaction monitoring (MRM) and ELISA. A bioinformatics workflow, using HRMS, was implemented to detect and annotate disrupted metabolites. HRMS allowed to detect disruptions in pathways associated to fatty acids, purines and amino acids metabolisms after exposure to the EDs tested, in addition to that linked to steroidogenesis.

Significance: We developed a robust MS1-HRMS workflow, from sample preparation to compound quantification or annotation, compatible with absolute steroid quantification, to screen NCI-H295R cell media exposed to potential EDs. Using only 200 μL of medium, the method integrates MS/MS and HRMS analyses, 96-well plate solid-phase extraction for high throughput, and automated pre-annotation for cost efficiency. This optimized workflow identifies EDs in cell assays by detecting disruptions in steroidogenesis and other biological pathways.