title: Determination of inhibitory potency of argatroban toward thrombin by electrophoretically mediated microanalysis.
abstract: Developing an EMMA method for enzymatic assay remains a challenge, particularly using UV detection. Indeed, it is necessary to optimize the separation conditions while allowing the enzymatic reaction to occur within the capillary respecting kinetic constraints and achieving enough sensitivity. In this work, such EMMA methodology was set up to evaluate the inhibitory potency of drugs toward thrombin, a serine protease implicated in the coagulation cascade. To achieve our goal, the separation buffer, the injection sequence, the internal standard and the chromogenic substrate were investigated. The newly developed system was then assessed determining the kinetic Km constant for the selected substrate and compared with the results obtained with a continuous spectrophotometer cell assay. Secondly, the Ki inhibitory constant of the thrombin inhibitor argatroban was determined and found in agreement with the published value.
authors.affiliation: Namur Medicine & Drug Innovation Center (NaMEDIC - Narilis), University of Namur, Belgium.
authors.forename: Lionel
authors.initials: L
authors.lastname: Pochet
authors.forename: Anne-Catherine
authors.initials: AC
authors.lastname: Servais
authors.forename: Elena
authors.initials: E
authors.lastname: Farcas
authors.forename: Virginie
authors.initials: V
authors.lastname: Bettonville
authors.forename: Charlotte
authors.initials: C
authors.lastname: Bouckaert
authors.forename: Marianne
authors.initials: M
authors.lastname: Fillet
mesh_terms: D000991:Antithrombins / Q000737:chemistry*; D001120:Arginine / Q000031:analogs & derivatives; D001777:Blood Coagulation; D020374:Chromatography, Micellar Electrokinetic Capillary; D002863:Chromogenic Compounds / Q000032:analysis; D004151:Dipeptides / Q000032:analysis; D019075:Electrophoresis, Capillary; D057075:Enzyme Assays; D006801:Humans; D007700:Kinetics; D010875:Pipecolic Acids / Q000737:chemistry*; D012996:Solutions; D013449:Sulfonamides; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D000991:Antithrombins; D002863:Chromogenic Compounds; D004151:Dipeptides; D010875:Pipecolic Acids; D012996:Solutions; D013449:Sulfonamides; C021129:S 2238; D001120:Arginine; D013917:Thrombin; C031942:argatroban
keywords: Argatroban; BGE; CE; EMMA; EOF; Enzymatic assay; IS; MEKC; NF; SDS; THR; Thrombin; background electrolyte; capillary electrophoresis; electro-osmotic flow; electrophoretically mediated microanalysis; internal standard; micellar electrokinetic chromatography; nitrofurantoin; pNA; para-nitroaniline; sodium dodecyl sulfate; thrombin