title: Automated evaluation of protein binding affinity of anti-inflammatory choline based ionic liquids.
abstract: In this work, an automated system for the study of the interaction of drugs with human serum albumin (HSA) was developed. The methodology was based on the quenching of the intrinsic fluorescence of HSA by binding of the drug to one of its binding sites. The fluorescence quenching assay was implemented in a sequential injection analysis (SIA) system and the optimized assay was applied to ionic liquids based on the association of non-steroidal anti-inflammatory drugs with choline (IL-API). In each cycle, 100 µL of HSA and 100 µL of IL-API (variable concentration) were aspirated at a flow rate of 1 mL min(-1) and then sent through the reaction coil to the detector where the fluorescence intensity was measured. In the optimized conditions the effect of increasing concentrations of choline ketoprofenate and choline naproxenate (and respective starting materials: ketoprofen and naproxen) on the intrinsic fluorescence of HSA was studied and the dissociation constants (Kd) were calculated by means of models of drug-protein binding in the equilibrium. The calculated Kd showed that all the compounds bind strongly to HSA (Kd<100 µmol L(-1)) and that the use of the drugs in the IL format does not affect or can even improve their HSA binding. The obtained results were compared with those provided by a conventional batch assay and the relative errors were lower than 4.5%. The developed SIA methodology showed to be robust and exhibited good repeatability in all the assay conditions (rsd<6.5%).
authors.affiliation: REQUIMTE, Chemical Science Department, Faculty of Pharmacy, University of Oporto, Rua Jorge Viterbo Ferreira, 4050-313 OPorto, Portugal.
authors.forename: Rosa
authors.initials: R
authors.lastname: Ribeiro
authors.affiliation: REQUIMTE, Chemical Science Department, Faculty of Pharmacy, University of Oporto, Rua Jorge Viterbo Ferreira, 4050-313 OPorto, Portugal. Electronic address: ppinto@ff.up.pt.
authors.forename: Paula C A G
authors.initials: PC
authors.lastname: Pinto
authors.affiliation: REQUIMTE, Chemical Science Department, Faculty of Pharmacy, University of Oporto, Rua Jorge Viterbo Ferreira, 4050-313 OPorto, Portugal.
authors.forename: Ana M O
authors.initials: AM
authors.lastname: Azevedo
authors.affiliation: Vienna University of Technology, Institute of Applied and Synthetic Chemistry, Getreidemarkt 9/163, A-1060 Vienna, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Bica
authors.affiliation: Vienna University of Technology, Institute of Applied and Synthetic Chemistry, Getreidemarkt 9/163, A-1060 Vienna, Austria.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Ressmann
authors.affiliation: REQUIMTE, Chemical Science Department, Faculty of Pharmacy, University of Oporto, Rua Jorge Viterbo Ferreira, 4050-313 OPorto, Portugal.
authors.forename: Salette
authors.initials: S
authors.lastname: Reis
authors.affiliation: REQUIMTE, Chemical Science Department, Faculty of Pharmacy, University of Oporto, Rua Jorge Viterbo Ferreira, 4050-313 OPorto, Portugal. Electronic address: lsaraiva@ff.up.pt.
authors.forename: M Lúcia M F S
authors.initials: ML
authors.lastname: Saraiva
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry*; D001331:Automation; D002794:Choline / Q000737:chemistry*; D006801:Humans; D052578:Ionic Liquids / Q000737:chemistry*; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D052578:Ionic Liquids; D012709:Serum Albumin; D002794:Choline
keywords: Automation; Human serum albumin; Ionic liquids with active pharmaceutical ingredient (IL-API); Quenching