Development and validation of an HPLC method for the simultaneous quantification of indole-3-carbinol acetate, indole-3-carbinol, and 3,3′-diindolylmethane in mouse plasma, liver, and kidney tissues

Abstract

A novel Indole-3-carbinol derivative (I3C) prodrug, indole-3-carbinol acetate (I3CA), was synthesized and a rapid high-performance liquid chromatography (HPLC) method for the quantification of I3CA, I3C, and the major metabolite of I3C, diindolylmethane (DIM), in mouse plasma, liver and kidney tissues was developed and validated. 4-Methoxy-1-methylindole was used as the internal standard. Chromatographic separation was achieved on a Symmetry(®) C18 column (75mm×4.6mm, 3.5μm) and the detection was made at 280nm. A gradient elution was programmed with the mobile phases of water (A) and acetonitrile (B) and a flow rate of 1ml/min. The total run time was 15min. The calibration curves were linear over the range of 0.06-1.6μg/ml for both I3C and DIM with a correlation coefficient (r(2)) higher than 0.997 and the lower limit of quantitation (LLOQ) of 0.06μg/ml. The calibration curve of I3CA was linear over the range of 0.15-4.0μg/ml, with a r(2)>0.995 and LLOQ of 0.15μg/ml. I3CA, I3C, and DIM intra-day accuracy values of plasma, liver and kidney samples ranged from 90.0 to 101.3%, while the inter-day ones were between 93.3 and 101.9%. Precision evaluated by the relative standard deviation was ranged from 2.0 to 14.8% for intra-day and 1.9 to 14.4% for inter-day variability. I3CA, I3C, and DIM were stable in mouse plasma, liver and kidney samples containing an esterase inhibitor dichlorvos. This method was successfully applied to a pharmacokinetic study in mice following oral and intravenous administration of I3C and I3CA.