Determination of unbound cephalothin in rat blood by on-line microdialysis and microbore liquid chromatography

A method of analysis for the determination of unbound cephalothin in rat blood has been developed. The method was fully automated using an on-line microdialysis procedure. A microdialysis probe was inserted into the jugular vein/right atrium of male Sprague-Dawley rats to examine the unbound cephalothin level in the rat blood after cephalothin administration (50 mg/kg, i.v.). Dialysates were directly input to a liquid chromatographic system using an on-line injector. Samples were eluted with a mobile phase containing methanol-acetonitrile-100 mM monosodium phosphate (pH 5.0) (20:20:60, v/v). The UV wavelength was set at 254 nm for monitoring the analyte. Using the retrograde method, at infusion concentrations of 1 and 5 microg/ml of cephalothin, the in vivo microdialysis recoveries were 48.4+/-4.5% and 52.9+/-4.7% for the rat blood (n=6). Intra- and inter-assay accuracy and precision of the analyses were < or = 10% in the range of 0.01 through 10 microg/ml. Pharmacokinetic parameters were calculated from the recovery corrected dialysate concentrations of cephalothin versus time data. The results suggest that the pharmacokinetics of unbound cephalothin in blood fitted best to the two-compartmental model following cephalothin administration (50 mg/kg, i.v.).