Mixtures Biotransformation: Multilayer Molecular Networking of Kratom Liver Metabolites

Metabolites generated from therapeutic drugs can exhibit diverse pharmacological effects, making knowledge of metabolites important to understand the overall mechanisms of therapeutic action. Methods to study the metabolism of single or simple mixtures of bioactive agents are often insufficient to address the chemical diversity of natural product mixtures. Molecular network analysis has emerged as a powerful approach to gain an understanding of complex chemical mixtures. In the present study, we used <i>in vitro</i> biotransformation and molecular network analysis of single compounds and extracts of the medicinal plant, kratom (<i>Mitragyna speciosa</i> Korth., Rubiaceae), to test the utility for characterizing natural product mixtures. We analyzed biotransformation products from single compounds and multiple chemotypes of kratom leaf extracts using human liver S9 and liquid chromatography-high-resolution mass spectrometry. Multilayer molecular networking of the polymetabolism of the entire natural product mixture provided predicted metabolites from precursors in a semitargeted manner. Both phase I and phase II metabolites of precursors were detected, with the metabolic profiles of individual components being substantially different in the context of total extracts. Application of molecular networking to mixture metabolism will enhance metabolite identification and the understanding of the therapeutic and toxicological mode(s) of action of pharmacologically active plants.