Innovative Research for a Sustainable Future www.epa.gov/research m/z 455.2897, 6.70 min 0.1 1 Verapamil (Parent Drug) Normalized Abundance Tyler S. Larson l larson.tyler@epa.gov l 434-942-5470 Untargeted LC-MS/MS Analysis of Drug Metabolites in Human Hepatocyte Suspensions for Comparison to Liver S9 Fractions Tyler S. Larson1, Krishna Ravindra2, Barbara A. Wetmore3, Denise K. MacMillan3 1Oak Ridge Institute for Science and Education (ORISE), 2Oak Ridge Associated Universities (ORAU) 3U.S. Environmental Protection Agency, Office of Research and Development Introduction Experimental Coumarin Verapamil Conclusions Rosiglitazone The U.S. EPA, in collaboration with Unilever, is developing new approach methods (NAMs) to quickly evaluate chemicals for human health hazards. Incorporating metabolism in NAMs is important to evaluate both parent compound and metabolite exposures during risk evaluations. Here, untargeted LC- MS/MS analysis is used to characterize metabolism of Coumarin, Rosiglitazone, and Verapamil in human hepatocyte suspensions and S9 protein fractions, and rat S9 protein fractions. Human hepatocytes are the gold standard for evaluating metabolism, but S9 fractions are easier to use and are not limited by toxicity. O O Rosiglitazone DTXSID7037131 Verapamil DTXSID9041152 Coumarin DTXSID7020348 O O Top: (left) Abundance of the coumarin parent drug in each system. The Human S9 fractions showed the highest clearance. (right) Abundances of the detected coumarin metabolites Metabolite ID: (left) Each metabolite m/z value matched to two possible isomers. For C-Met-1a-b two features were detected while only one was detected for C-Met-2a-b. (right) Example of an MS/MS spectral match using MetFrag. MetFrag does not match abundances, only m/z values. All identifications were Level 2: Probable Structure on the Schymanski scale. Label m/z (M+H)+ RT (min) Matched Structure C-Met-1a-b 163.0389 5.60 and 6.25 C-Met-2a-b 339.0690 5.60 Label m/z (M+H)+ RT (min) Matched Structure R-Met-1 344.1072 5.69 R-Met- 2a-d 374.11 5.61, 5.80, 6.60 O S NH O O N + N CH3 O – O S NH O O N N CH3 OH Label m/z (M+H)+ RT (min) Matched Structure V-Met-1 196.1325 5.22 V-Met-2 291.2048 6.08 V-Met-3a-c 441.2739 (total) 6.50 and 6.70 V-Met-4a-c 471.285 6.15, 6.41, and 6.59 V-Met-5a-b 427.2568 6.48 V-Met-6a-b 457.2678 6.55 NH O O O O CH3 C H3 C H3 CH3 C H3 N CH3 N O O O O H CH3 CH3 C H3 CH3 C H3 N CH3 N OH O O O CH3 C H3 C H3 CH3 C H3 N CH3 N O O O O CH3 CH3 C H3 C H3 CH3 C H3 CH CH3 OH N O O O O CH3 CH3 CH3 C H3 CH3 C H3 N CH3 OH N O O O O CH3 CH3 C H3 C H3 CH3 C H3 N CH3 OH NH O O O O CH3 C H3 C H3 CH3 C H3 N CH3 OH NH O O O O CH3 C H3 C H3 CH3 CH3 N C H3 OH NH O H O O O C H3 C H3 CH3 C H3 N CH3 C H3 NH O O O O H C H3 CH3 C H3 N CH3 A B A B A B C D A B C A B C A B A B Top: (left) Abundance of the verapamil parent drug in each metabolic system. The human S9s showed the highest clearance. (right) Abundances of the detected verapamil metabolites in each system. Metabolite ID: All metabolites were annotated as Level 2 except for V-Met-1 for which no MS/MS was acquired (Level 5: Accurate mass only). Many metabolite MS/MS spectra matched to two or more isomers. Two metabolites were detected that were not predicted. They were annotated the same way as the others. N O O O O CH3 CH3 C H3 C H3 CH3 C H3 N CH3 Top: (left) Abundance of the rosiglitazone precursor. The rat S9s showed the highest clearance. (right) Abundance of the detected rosiglitazone metabolites. Metabolite ID: All matched structures were considered Level 2: Probable Structure on the Schymanski scale. R- Met 2a-d matched to four possible isomers despite three m/z values being detected. Level 2: Probable Structure 50 100 150 200 m/z Experimental MetFrag Not Predicted Not Predicted • Metabolism was compared between three different systems: human hepatocytes, human S9 fractions, and rat S9 fractions which all differed slightly in the metabolites observed. • Human liver S9s contain the native enzymes present