High performance liquid chromatographic determination of clavulanic acid in human urine

An ion pair reversed phase HPLC method for the determination of clavulanic acid has been developed. Since clavulanic acid had poor absorption (lambda max 201 nm in water) in a UV-region suitable for HPLC detection, the detectability was enhanced by bathochromic shift of lambda max due to solvent effect. The shifts of lambda max were measured with the solutions containing clavulanic acid, tetrabutylammonium bromide, and phosphate buffer salts in aqueous methanol. The magnitudes of the observed shifts were investigated with respect to pH, ionic strength, methanol content, and TBAB concentration. The results indicated that TBAB concentration was the predominant factor responsible for the bathochromic shifts. Taking account of the results together with solvent effects on the retention of clavulanic acid on hydrophobic stationary phase, HPLC condition suitable for detection and separation of clavulanic acid in urine was established as follows; mobile phase: 10 mM TBAB + 0.6 mM NaH2PO4 + 0.4 mM Na2HPO4 in H2O - MeOH, 10:1 (v/v)(pH 7.02), flow rate: 1.5 ml/minute, stationary phase: LiChrosorb RP-18 (25 cm x 4.6 mm i.d.), detection: UV 220 nm. The applicability of the present method is demonstrated by determining the time course of urinary excretion of clavulanic acid after oral administration of a conjugated tablet of clavulanic acid and amoxicillin to human subject.