Aerobic activated sludge transformation of methotrexate: identification of biotransformation products

This study describes the biotransformation of cytostatic and immunosuppressive pharmaceutical methotrexate. Its susceptibility to microbiological breakdown was studied in a batch biotransformation system, in presence or absence of carbon source and at two activated sludge concentrations. The primary focus of the present study are methotrexate biotransformation products, which were tentatively identified by the ultra-high performance liquid chromatography-quadrupole--Orbitrap-MS. Data-dependent experiments, combining full-scan MS data with product ion spectra were acquired, in order to identify the molecular ions of methotrexate transformation products, to propose the molecular formulae and to elucidate their chemical structures. Among the identified transformation products 2,4-diamino-N10-methyl-pteroic acid is most abundant and persistent. Other biotransformation reactions involve demethylation, oxidative cleavage of amine, cleavage of C-N bond, aldehyde to carboxylate transformation and hydroxylation. Finally, a breakdown pathway is proposed, which shows that most of methotrexate breakdown products retain the diaminopteridine structural segment. In total we propose nine transformation products, among them eight are described as methotrexate transformation products for the first time.