Xyloketal B, a marine compound, acts on a network of molecular proteins and regulates the activity and expression of rat cytochrome P450 3a: a bioinformatic and animal study

Su, Junhui; Chang, Cui; Xiang, Qi; Zhou, Zhi-Wei; Luo, Rong; Yang, Lun; He, Zhi-Xu; Yang, Hongtu; Li, Jianan; Bei, Yu; Xu, Jinmei; Zhang, Minjing; Zhang, Qihao; Su, Zhijian; Huang, Yadong; Pang, Jiyan; Zhou, Shu-Feng

The plasma concentrations of midazolam and its metabolites 1′-OH-MDZ were measured by HPLC as previously described, 30 with slight modifications. The HPLC method was validated prior to determination of the plasma PKs of midazolam as per the International Conference on Harmonization guidelines on validation of analytical procedures, including the specificity (ability to unequivocally determine the analyte), linearity (response curve of the analyte), lower limit of quantification (concentration of the analyte which has a signal to noise ratio of 3:1), precision and accuracy (results of repeatability), recovery (satisfactory accuracy and precision), and stability (degradation of analyte). 2, 4 A 100 μL aliquot of plasma was diluted with 400 μL of 0.1 M NaOH, and diazepam 8 μg/mL was added as an internal standard. The mixture was extracted with 2.5 mL of dichloromethane and pentane (at a ratio of 3:7), then vortexed for 20 seconds. The upper organic phase was transferred into a clean glass tube and the residual liquid was extracted again. The upper organic phase mixture was evaporated to dryness. The dried residue was dissolved in 100 μL of mobile phase and centrifuged at 12,000× g for 10 minutes, and the supernatant was subjected to HPLC analysis and detected using an ultraviolet detector at 254 nm. An HPLC system with a Shim-Pack 250 mm ×4.6 mm reverse phase column packed with 5 μm VP-ODS C 18 (Shimadzu Corporation, Tokyo, Japan) was used. The column temperature was set at 40°C. The mobile phase was 10 mM ammonium acetate buffer (pH 4.5) and acetonitrile (63:37, v/v) at a flow rate of 1.0 mL per minute. All samples were analyzed within the validated stability timeframe.