title: Integrated liquid chromatography method in enantioselective studies: Biodegradation of ofloxacin by an activated sludge consortium.
abstract: Ofloxacin is a chiral fluoroquinolone commercialized as racemate and as its enantiomerically pure form levofloxacin. This work presents an integrated liquid chromatography (LC) method with fluorescence detection (FD) and exact mass spectrometry (EMS) developed to assess the enantiomeric biodegradation of ofloxacin and levofloxacin in laboratory-scale microcosms. The optimized enantioseparation conditions were achieved using a macrocyclic antibiotic ristocetin A-bonded CSP (150×2.1mm i.d.; particle size 5μm) under reversed-phase elution mode. The method was validated using a mineral salts medium as matrix and presented selectivity and linearity over a concentration range from 5μgL(-1) (quantification limit) to 350μgL(-1) for each enantiomer. The method was successfully applied to evaluate biodegradation of ofloxacin enantiomers at 250μgL(-1) by an activated sludge inoculum. Ofloxacin (racemic mixture) and (S)-enantiomer (levofloxacin) were degraded up to 58 and 52%, respectively. An additional degradable carbon source, acetate, enhanced biodegradation up to 23%. (S)-enantiomer presented the highest extent of degradation (66.8%) when ofloxacin was supplied along with acetate. Results indicated slightly higher biodegradation extents for the (S)-enantiomer when supplementation was done with ofloxacin. Degradation occurred faster in the first 3days and proceeded slowly until the end of the assays. The chromatographic results from LC-FD suggested the formation of the (R)-enantiomer during levofloxacin biodegradation which was confirmed by LC-MS with a LTQ Orbitrap XL.
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal; Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Rua Arquiteto Lobão Vital, Apartado 2511, 4202-401 Porto, Portugal.
authors.forename: Alexandra S
authors.initials: AS
authors.lastname: Maia
authors.affiliation: Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Rua Arquiteto Lobão Vital, Apartado 2511, 4202-401 Porto, Portugal.
authors.forename: Paula M L
authors.initials: PML
authors.lastname: Castro
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal; Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. Electronic address: elizabeth.tiritan@iscsn.cespu.pt.
authors.forename: Maria Elizabeth
authors.initials: ME
authors.lastname: Tiritan
mesh_terms: D000890:Anti-Infective Agents / Q000302:isolation & purification*; D001673:Biodegradation, Environmental; D002853:Chromatography, Liquid / Q000379:methods; D064704:Levofloxacin / Q000302:isolation & purification*; D057230:Limit of Detection; D012722:Sewage / Q000032:analysis* / Q000382:microbiology*; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods; D014874:Water Pollutants, Chemical / Q000302:isolation & purification*
chemical_list: D000890:Anti-Infective Agents; D012722:Sewage; D014874:Water Pollutants, Chemical; D064704:Levofloxacin
keywords: Activated sludge; Biodegradation; Enantioselectivity; Levofloxacin; Macrocyclic antibiotic-based stationary phases; Ofloxacin