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A Facile and Sensitive HPLC-MS/MS Method for Quantification of 3,3'-Diindolylmethane in Plasma Samples.

Indole-3-carbinol is the subject of ongoing biomedical research due to its potential antiatherogenic, anticarcinogenic, and antioxidant effects. The antitumor properties are mainly associated with its major metabolite, i.e. 3,3'-diindolylmethane (DIM). Typically, the biological activity of the chemical compound is manifested in the ng/mL concentration range. Consequently, the development of highly sensitive analytical methods to determine the DIM in various biological samples is an urgent issue. In this study, an HPLC-MS/MS method was established for the DIM quantification in human plasma. The developed method was validated according to the EMA guidelines. Sensitivity, selectivity, accuracy, and precision were found good allowing the DIM quantification in the concentration range of 5-500 ng/mL. The limit of detection and the lower limit of quantification were 1 and 5 ng/mL, respectively. 4-Methoxy-1-methylindole was used as an internal standard (IS). The analytes were taken from the human plasma by the acetonitrile-induced protein precipitation method with the addition of 3 mol/L ammonium sulfate as a salting-out agent, which is a facile and efficient approach for high-throughput bioanalysis. The chromatographic separation was performed on the Synergi Fusion-RP C18 column (50 × 2.0 mm, 4 μm, 80 Å) under isocratic elution at 40°C. The mobile phase consisting of acetonitrile and water (0.1% formic acid) (85:15, v/v) was delivered at a flow rate of 0.20 mL/min. The DIM and IS were eluted at 2.36 ± 0.04 and 2.43 ± 0.03 min, respectively. The total analysis time was 3.20 min. Atmospheric pressure chemical ionization was carried out using multiple reaction monitoring in the positive polarity mode. The ion transitions were set to m/z 247.1 → 130.1 (DIM) and 162.1 → 147.1 (IS). The method was successfully applied to the analysis of plasma samples after a single oral administration of the Indinol® Forto drug (200 mg) to Russian female healthy volunteers. Also, the developed method was used for the analysis of rabbit plasma samples after a single oral dose of DIM (20 mg/kg).