The Use of Reverse Phase High Performance Liquid Chromatography to Determine 5, 7–dihydroxy-3, 6, 8–trimethoxy Flavone and 3, 5–dihydroxy-6, 7, 8–trimethoxy Flavone Concentrations in Rat Colon Tissue

6, 8–trimethoxy Flavone and 3, 5–dihydroxy-6, 7, 8–trimethoxy Flavone Concentrations in Rat Colon Tissue C. L. Whitted, V. E. Palau, R. D. Torrenegra, O. E. Rodriguez, S. Harirforoosh East Tennessee State University, Universidad de Ciencias Aplicadas y Ambientales, Universidad del Bosque Purpose In vitro antineoplastic activity against colon cancer cell lines has been previously shown [1]for isomeric flavones, 5, 7–dihydroxy-3, 6, 8–trimethoxy flavone (flavone A) and 3, 5–dihydroxy-6, 7, 8–trimethoxy flavone (flavone B). Here, we modified a reverse phase high performance liquid chromatography (HPLC) method developed for the extraction and quantification of flavone A and B developed in rat plasma [2] in order to measure the concentrations of two flavones in rat colon tissue. Methods Flavone A or flavone B was added to homogenized colon samples (1 g tissue: 2 mL water) in concentrations ranging from 250100,000 ng/g and 1,000-25,000 ng/g, respectively. The organic solvent, acetonitrile, was used to extract the flavones from the tissue and the supernatant was filtered with PVDF iso filters before being evaporated using a Labconco centivap concentrator (Kansas City, MO). Residue was reconstituted in mobile phase (60:40 or 70:30 acetonitrile:water with 0.2‰ acetic acid and 0.05‰ trimethylamine for flavone A and B, respectively). Quantification was done using a HPLC system (Shimadzu, United States) with an ACE 5 C18 column. Flow rate (0.4 mL/min), temperature (30 °C), detection wavelength (245 nm), and internal standards (25 μg/mL Celecoxib or Diclofenac, respectively) were the same as previous method [2]. Three trials were performed to determine precision (coefficient of variance-CV) and accuracy. Results The method showed successful separation of HPLC peaks. Representative calibration equations y = 2e-05 + 0.0029 and y = 7e-05 + 0.0531 were linear with r ≥ 0.99 for flavone A and flavone B, respectively. CVs for flavone A or B ranged from 0.91-24.03 and 1.6233.56 with accuracies of 109-116‰ and 98-113‰, respectively. Conclusion The modified method was suitable for extraction and quantification of flavones A and B in rat colon tissue.