Alkaline Degradation of Clavulanic Acid and High Performance Liquid Chromatographic Determination by Post-Column Alkaline Degradation

Abstract

Alkaline degradation of clavulanic acid in methanol and in aqueous solution has been investigated. Potassium clavulanate was degraded in methanol and in NaOH-saturated methanol to yield methyl 8-hydroxy-6-oxo-4-aza-2-octenoate (I). which showed ultraviolet (UV) absorption with λ_max 268nm (methanol). The UV absorption of I almost disappeared in acidic conditions, and reappeared on subsequent realkalization, suggesting interconversion between I and its protonated form. It was suggested that potassium clavulanate might be rapidly hydrolyzed in alkaline aqueous solutions to 8-hydroxy-6-oxo-4-aza-2-octenoic acid (II), which has strong UV absorption around 260 nm. The acid-base interconversion was also observed between II and its protonated form, the latter exhibiting almost no UV absorption around 260 nm. An ion-pair reversed-phase high performance liquid chromatography method with the alkaline degradation reaction incorporated into post-column has been developed for the determination of clavulanic acid in plasma and urine. After separation from regular components of plasma and urine, clavulanic acid is degraded in NaOH solution in a reaction coil followed by detection of the UV absorbance of the degradation product at 270 nm. The procedure was quantitative over a wide range of clavulanic acid concentrations in plasma and urine down to 0.1 μg/ml.