DUGi

Aerobic activated sludge transformation of methotrexate: Identification of biotransformation products

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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

The financial support of the European Community’s Seventh Framework Programme (FP7/2007–2013), Grant agreement no.265264: CytoThreat (Fate and effects of cytostatic pharmaceuticals in the environment and the identification of biomarkers for improved risk assessment on environmental exposure), the Slovenian Ministry of Higher Education, Science and Technology, programme P1-0143 (Cycling of substances in the environment, mass balances, modelling of environmental processes and risk assessment) and Project BI-HR/12-13-034 (Determination of toxicity and physico-chemical properties of pharmaceuticals), the Spanish Ministry of Economy and Competitiveness through the Project SCARCE (Consolider-Ingenio 2010 CSD2009-00065) and the Generalitat de Catalunya (Consolidated Research Group: Water and Soil Quality Unit 2009-SGR-965) are acknowledged. Merck is acknowledged for the gift of LC columns. TK wishes to thank Dr. Ester Heath for her support in conducting this research and preparation of the manuscript

Elsevier