Biotransformation of nodakenin and simultaneous quantification of nodakenin and its aglycone in incubated system of human intestinal bacteria by HPLC method

Abstract

When nodakenin (1) was anaerobically incubated with human intestinal bacteria, nodakenetin (2) was found as a main biotransformed product. We developed a simple and selective reversed-phase high-performance liquid chromatographic method for simultaneous quantification of 1 and 2 in incubated system of human intestinal bacteria with 1. Chromatographic separation of 1 and 2 was performed on an analytical C18 column, with a mobile phase of MeOH–H2O (4:6, v/v) at a flow rate of 1.0 ml/min and the UV detection was at 330 nm. The calibration curves were linear over the range of 0.15–24.0 μg/ml for 1 and 0.7–13.2 μg/ml for 2. The lower limits of detection and quantification were 0.01 and 0.1 μg/ml for 1, and 0.005 and 0.05 μg/ml for 2. The recoveries were (87.66 ± 1.66), (79.89 ± 2.53), and (82.96 ± 5.61)% at 1.0, 2.0, and 8.0 μg/ml, respectively, for 1 and (88.32 ± 4.12), (78.15 ± 4.39), and (76.22 ± 3.29)% at 1.0, 4.0, and 16.0 μg/ml, respectively, for 2. The intra- and interday precision and accuracy were validated by relative standard deviation, which were in the ranges of 1.25–4.16 and 2.16–6.12% for 1, and 1.98–6.45 and 2.56–4.57% for 2, respectively. This method has been applied to the simultaneous quantitation of 1 and 2 in incubated system of human intestinal bacteria with 1.