Mass spectrometric characterization of carfentanil metabolism in human, dog, and rat lung microsomes via comparison to chemically synthesized metabolite standards

• Published in: Forensic toxicology 2020-07, Vol.38 (2), p.352-364
• Main Authors: Kong, Li, Walz, Andrew J.
• Format: Article
• Language: English
• Subjects: Forensic Medicine; Forensic Science; Medical Law; Medicinal Chemistry; Medicine; Medicine & Public Health; Original Article; Pharmacology/Toxicology
• ISSN: 1860-8965; 1860-8973
• DOI: 10.1007/s11419-019-00521-x

Purpose The metabolism of carfentanil was assessed using human, dog, and rat pulmonary microsomes. Mass spectrometry based analysis allowed for metabolite identification and species differentiation. Participation of different metabolic enzymes in carfentanil biotransformation was also assessed. Methods Metabolite profiling was accomplished by incubating 10 µM carfentanil in human, dog, and rat lung microsomes. The metabolites were separated and analyzed by ultra-high performance liquid chromatography/high-resolution mass spectrometry. Results In total, 18 metabolites were detected. Nine metabolites were authentically identified through comparison to synthesized reference standards. In human lung microsomes, nine metabolites were identified. In dog lung microsomes, 15 metabolites were identified with three being dog specific. In rat lung microsomes, 15 metabolites were identified and two were rat specific. Proposed metabolic pathways included N -dealkylation, monohydroxylation, dihydroxylation, N -oxidation of piperidine ring nitrogen, and ketone formation. Participation of enzymes CYP2B6, CYP2E1, CYP2J2, and CYP3A4/5 to carfentanil metabolism was suggested by selective enzymatic inhibition. Conclusions The pulmonary clearance in human lung microsomes was lower than the previously reported hepatic metabolism suggesting organ specific metabolic rates. The contribution of multiple cytochrome P450 enzymes to human, dog, and rat pulmonary microsomal carfentanil biotransformation varied between species. The identified metabolites may provide useful markers for possible forensic and clinical determination of the mode of ingestion but the use of dog and rat animal models was not indicated. To our knowledge, this is the first reported use of chemically synthesized reference standards for the unequivocal identification of lung carfentanil metabolites.