Liquid chromatographic assay for the simultaneous determination of indole-3-carbinol and its acid condensation products in plasma

A high-performance liquid chromatographic method was developed for the simultaneous determination of indole-3-carbinol (I3C), 3,3′-diindolylmethane (DIM), [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr1), and indolo[3,2b]carbazole (ICZ). Compounds were extracted from mouse plasma using tert.-butyl methyl ether, incorporating 4-methoxy-indole as internal standard. Chromatographic separation utilized a Waters Symmetry RP18 in tandem with a Thermoquest BDS C18 column, an acetonitrile–water gradient and UV (280 nm) in series with fluorescence (ex. 335 nm; em. 415 nm) detection. Calibration curves were linear (r2>0.99) between 50 and 15,000 ng/ml for I3C; 150 and 15,000 ng/ml for LTr1; and 0.15 and 37.5 ng/ml for ICZ and the method was reproducible and precise (within-day and between-day coefficients of variation below 9.7 and 13%, respectively). The method described is suitable for comprehensive pharmacokinetic studies with indole-3-carbinol.