title: An improved HPLC-DAD method for clavulanic acid quantification in fermentation broths of Streptomyces clavuligerus.
abstract: Clavulanic acid (CA) is an important secondary metabolite commercially produced by cultivation of Streptomyces clavuligerus (Sc). It is a potent inhibitor of bacterial β-lactamases. In this work, a specific and improved high performance liquid chromatography (HPLC) method, using a C-18 reversed phase column, diode array detector and gradient elution for CA quantification in fermentation broths of Sc, was developed and successfully validated. Samples were imidazole-derivatized for the purpose of creating a stable chromophore (clavulanate-imidazole). The calibration curve was linear over a typical range of CA concentration between 0.2 and 400mg/L. The detection and quantification limits were 0.01 and 0.02mg/L, respectively. The precision of the method was evaluated for CA spiked into production media and a recovery of 103.8%, on average, was obtained. The clavulanate-imidazole complex was not stable when the samples were not cooled during the analysis. The recovery rate was 39.3% on average. This assay was successfully tested for CA quantification in samples from Sc fermentation, using both, a chemically defined and a complex medium.
authors.affiliation: Grupo de Bioprocesos, Departamento de Ingeniería Química, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia.
authors.forename: Howard
authors.initials: H
authors.lastname: Ramirez-Malule
authors.affiliation: Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 76, ACK 24, D-13355 Berlin, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Junne
authors.affiliation: Laboratorio de Análisis de Residuos, Instituto de Química, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia.
authors.forename: Carlos
authors.initials: C
authors.lastname: López
authors.affiliation: Laboratorio de Análisis de Residuos, Instituto de Química, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia.
authors.forename: Julian
authors.initials: J
authors.lastname: Zapata
authors.affiliation: Grupo CIBIOP, Escuela de Ciencias, Universidad EAFIT, Carrera 49 No. 7 sur-50, Medellín, Colombia.
authors.forename: Alex
authors.initials: A
authors.lastname: Sáez
authors.affiliation: Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 76, ACK 24, D-13355 Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Neubauer
authors.affiliation: Grupo de Bioprocesos, Departamento de Ingeniería Química, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia. Electronic address: rigoberto.rios@udea.edu.co.
authors.forename: Rigoberto
authors.initials: R
authors.lastname: Rios-Estepa
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019818:Clavulanic Acid / Q000032:analysis*; D003470:Culture Media / Q000032:analysis* / Q000737:chemistry; D005285:Fermentation / Q000502:physiology*; D013302:Streptomyces / Q000378:metabolism*
chemical_list: D003470:Culture Media; D019818:Clavulanic Acid
keywords: Clavulanate–imidazole; Clavulanic acid; Fermentation processes; High performance liquid chromatography method; Streptomyces clavuligerus