title: LC-MS-MS determination of ibuprofen, 2-hydroxyibuprofen enantiomers, and carboxyibuprofen stereoisomers for application in biotransformation studies employing endophytic fungi.
abstract: The purpose of this study was the development and validation of an LC-MS-MS method for simultaneous analysis of ibuprofen (IBP), 2-hydroxyibuprofen (2-OH-IBP) enantiomers, and carboxyibuprofen (COOH-IBP) stereoisomers in fungi culture medium, to investigate the ability of some endophytic fungi to biotransform the chiral drug IBP into its metabolites. Resolution of IBP and the stereoisomers of its main metabolites was achieved by use of a Chiralpak AS-H column (150 × 4.6 mm, 5 μm particle size), column temperature 8 °C, and the mobile phase hexane-isopropanol-trifluoroacetic acid (95: 5: 0.1, v/v) at a flow rate of 1.2 mL min(-1). Post-column infusion with 10 mmol L(-1) ammonium acetate in methanol at a flow rate of 0.3 mL min(-1) was performed to enhance MS detection (positive electrospray ionization). Liquid-liquid extraction was used for sample preparation with hexane-ethyl acetate (1:1, v/v) as extraction solvent. Linearity was obtained in the range 0.1-20 μg mL(-1) for IBP, 0.05-7.5 μg mL(-1) for each 2-OH-IBP enantiomer, and 0.025-5.0 μg mL(-1) for each COOH-IBP stereoisomer (r ≥ 0.99). The coefficients of variation and relative errors obtained in precision and accuracy studies (within-day and between-day) were below 15%. The stability studies showed that the samples were stable (p > 0.05) during freeze and thaw cycles, short-term exposure to room temperature, storage at -20 °C, and biotransformation conditions. Among the six fungi studied, only the strains Nigrospora sphaerica (SS67) and Chaetomium globosum (VR10) biotransformed IBP enantioselectively, with greater formation of the metabolite (+)-(S)-2-OH-IBP. Formation of the COOH-IBP stereoisomers, which involves hydroxylation at C3 and further oxidation to form the carboxyl group, was not observed.
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, 14040-903, Brazil.
authors.forename: Keyller Bastos
authors.initials: KB
authors.lastname: Borges
authors.forename: Anderson Rodrigo Moraes
authors.initials: AR
authors.lastname: de Oliveira
authors.forename: Thiago
authors.initials: T
authors.lastname: Barth
authors.forename: Valquíria Aparecida Polizel
authors.initials: VA
authors.lastname: Jabor
authors.forename: Mônica Tallarico
authors.initials: MT
authors.lastname: Pupo
authors.forename: Pierina Sueli
authors.initials: PS
authors.lastname: Bonato
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000032:analysis* / Q000378:metabolism; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005658:Fungi / Q000378:metabolism*; D007052:Ibuprofen / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; C109994:hydroxyibuprofen; C109993:carboxyibuprofen; D007052:Ibuprofen