Session: Session 4
Optimization of a high-throughput HRMS-based non-targeted analysis workflow for steroids characterization and endocrine disruptors screening
Introduction: 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. Here, we developed a complete and robust non-targeted analysis (NTA) workflow based on high-resolution mass spectrometry (HRMS), i.e. running from sample preparation up to compound quantification or annotation, to screen media from the NCI-H295R cell line exposed to known endocrine disruptors (EDs).
Methodology: On the cell culture medium as well as in human plasma and serum samples, we compared absolute quantifications of 13 steroids, determined from HRMS at the MS1 level using the NTA workflow with those from targeted analyses (TA) using a multiple reaction monitoring (MRM) method performed on a triple quadrupole mass spectrometer.
Results: This comparison showed that HRMS-based steroid measurements were nearly as sensitive and as reproducible as those of MRM in blood samples, even though the MRM method performed better on cell media. We obtained similar results comparing absolute concentrations of steroids obtained using NTA, TA and ELISA kits (testosterone only) on cell culture media from NCI-H295R cells exposed to bisphenol A, prochloraz, ketoconazole, atrazine and forskolin in a dose-response experiment.
Conclusion: Our NTA workflow was optimized to detect, identify and quantify steroids as well as other compounds using the XCMS R package and the suspect screening tool Scannotation. The results demonstrate that our NTA workflow can accurately measure absolute steroid concentrations, abundances of EDs and those of their metabolites and abundances of endogenous molecules. Future developments should make it possible to include ion mobility values and MS2 measurements in the Scannotation tool's scoring system to improve the annotations of the detected compounds.
Methodology: On the cell culture medium as well as in human plasma and serum samples, we compared absolute quantifications of 13 steroids, determined from HRMS at the MS1 level using the NTA workflow with those from targeted analyses (TA) using a multiple reaction monitoring (MRM) method performed on a triple quadrupole mass spectrometer.
Results: This comparison showed that HRMS-based steroid measurements were nearly as sensitive and as reproducible as those of MRM in blood samples, even though the MRM method performed better on cell media. We obtained similar results comparing absolute concentrations of steroids obtained using NTA, TA and ELISA kits (testosterone only) on cell culture media from NCI-H295R cells exposed to bisphenol A, prochloraz, ketoconazole, atrazine and forskolin in a dose-response experiment.
Conclusion: Our NTA workflow was optimized to detect, identify and quantify steroids as well as other compounds using the XCMS R package and the suspect screening tool Scannotation. The results demonstrate that our NTA workflow can accurately measure absolute steroid concentrations, abundances of EDs and those of their metabolites and abundances of endogenous molecules. Future developments should make it possible to include ion mobility values and MS2 measurements in the Scannotation tool's scoring system to improve the annotations of the detected compounds.