Determination and pharmacokinetics study of beta-lactamase in rat plasma by fluorimetric HPLC

For the determination of in vivo beta-lactamase activity, a high-performance liquid chromatographic (HPLC) method was established, and the pharmacokinetics of beta-lactamase after intravenous administration to the rats was analyzed using this standardized HPLC method. The plasma samples containing beta-lactamase were reacted with ampicillin (substrate) and further processed to make them fluorescent. The fluorescent compound of interest was separated using HPLC at room temperature using the excitation and the emission wavelengths of 410 nm and 475 nm, respectively. For the pharmacokinetic studies, 252 mU of beta-lactamase solution was administered to the rats through the tail vein injection (n = 6). The blood samples were withdrawn from the tail vein at different time points and analyzed by HPLC for beta-lactamase activity. For the HPLC method of beta-lactamase in plasma samples, the peak area showed a good correlation within the concentration ranges of 0.126-12.6 mU/mL (10-1000 ng/mL). The coefficients of variations were within 0.56-6.24, and the percentage recovery were within 102-107. After the intravenous injection, plasma concentration at the time zero (C(p0)) was 11.47 +/- 0.48 mU/mL, and no beta-lactamase was detected 24 h after the injection. The volume of distribution (V(d)) was 22 mL. An elimination half-life (t(1/2)) of 4.12 +/- 0.5 h and AUC of 79.4 +/- 12.9 mU.hr/mL were also calculated. The HPLC fluorimetric method was a very sensitive and reproducible method for the detection of beta-lactamase in plasma. The disposition of beta-lactamase after intravenous administration followed one-compartment and first-order kinetics.