Comparison of Tacrolimus (FK506) Levels Determined by Three Different Methods in the Rat Blood.

Abstract

Blood levels of tacrolimus (FK506) determined by three different methods, were com pared after intravenous (i.v.) and oral administration of 14C-FK506 to rats. The methods used were high performance liquid chromatography-mass spectrometry (LC/MS) with electrospray ionization mode, enzyme immunoassay (EIA) using anti-FK506 monoclonal antibody, and the counting of radioactivity. Also, the composition of the radioactivity in the whole blood and plasma of rats after i.v. and oral administration was analyzed by the high performance liquid chromatography (HPLC). 1. FK506 concentrations determined by EIA were almost equal to those determined by LC/MS in the whole blood and plasma of rats after ix. injection. The ratio of FK506 levels measured by EIA to those by LC/MS ranged from 1.1 to 1.3 and from 1.4 to 2.0 in the whole blood and plasma, respectively, after oral administration. 2. Levels of radioactivity in all blood samples were higher than those determined by LC/MS and EIA. The ratio of the concentrations of the unchanged FK506 to total radioac tivity decreased gradually after administration of FK506 and the respective values at 0.25 and 8 hours after dosing were 0.8 and 0.6 in the whole blood and 0.7 and 0.2 in the plasma af ter i.v. injection, and 0.4 and 0.1 in the whole blood and 0.08 and 0.04 in the plasma after oral administration. 3. At 0.5 and 8 hours after i.v. injection, the unchanged FK 506 in the whole blood and plasma accounted for more than 84 and 59%, respectively, of total radioactivity eluted on HPLC. After oral administration, the unchanged FK506 accounted for 62% in the whole blood and 35% in the plasma at 0.25 hour. The metabolite, 13-0-mono-desmethyl FK506 was detected in all blood samples, and ranged from 2 to 8 % of the eluted radioactivity in the whole blood and from 3 to 20% of that in the plasma. 4. The results indicate that FK506 levels measured by EIA reflect the levels of the un changed FK506 in the whole blood and plasma after i.v. injection of FK506 to rats and those in the whole blood after oral administration, and part of metabolites in the blood in crease with time after administration.