[Direct determination of clavulanic acid in biological fluids using HPLC]

The so far described HPLC methods for clavulanic acid (CA) monitoring needed post-column derivatization with imidazole, resulting in poorly practicable methods. We propose here a direct determination of CA in human biological fluids with a ion-pairing technology using the bathochromic shift of tetrabutylammonium bromide (TBAB). The separation is performed on a reversed phase analytical column (250 X 4.6 mm) with the following mobile phase: 10% acetonitrile in 1 mM TAB and 20 mM ammonium acetate (pH = 5). The U.V. detection is at 214 nm. Serum and bile are prepared with acetonitrile and methylene chloride, and urines are diluted 1/10 prior the analysis. Retention time of CA is 8.4 min. Detection limit for bile and serum is 0.1 mg/l and 5 mg/l for urines. Within and between-day reproducibility is respectively 5.4% and 7.2% for serum and bile, and 4.7% and 6.8% for urine. This method may be suitable for clinical routine analysis and pharmacokinetic studies.