An improved HPLC-DAD method for clavulanic acid quantification in fermentation broths of Streptomyces clavuligerus

• An improved HPLC-DAD method was developed for CA quantification produced in fermentation broths of Streptomyces clavuligerus.
• The HPLC-DAD method was tested using different complex and chemically defined media, and in each case the CA peak was clearly resolved without interferences and/or overlapping peaks.
• The gradient elution configuration avoids overlapping of new peaks, such as β-lactam compounds with 3S, 5S stereochemistry, with the CA peak, a case commonly found in isocratic elution.
• Experimental evidence of the instability of the clavulanate–imidazole complex at room temperature was also found.

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 400 mg/L. The detection and quantification limits were 0.01 and 0.02 mg/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.

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