An aqueous containing emodin glucuronide and emodin was extracted three times with dichloromethane to get rid of emodin. The extracted aqueous sample was therefore split into two equal parts, one portion was incubated with water and then analyzed by UPLC and another one by hydrolysis with glucuronidase at 37 C for 30 min and then analyzed by UPLC. The natural compound library difference in peak areas of metabolite and emodin obtained from the samples before and following the hydrolysis, that have been represented as Peak areaM and Peak areaE, was determined to be the ratio K Peak areaM Peak areaE e T. Therefore, the concentration of metabolite might be estimated using emodin standard curve. The typical SD conversion factor was 1. 0054 0. 023 at a wavelength of 254 nm, determined independently at three different concentrations. LC and uplc MS/MS Analysis of Emodin and its Glucuronides The conditions used to evaluate emodin and its metabolites were as follows: system, Waters Acquity UPLC with photodiode array detector and Empower pc software, column, BEH C18, 1. 85%A, wavelength, 254 nm for emodin and its glucuronide and testosterone, and treatment volume, 10 L. The check linear Immune system response range was 0. 625 C100 M for emodin. The mass spectrometer boundaries were set as follows: capillary voltage, 4. 5KV, ion resource temperature, 350 C, desolvation temperature, 108 C, nebulizer gas, nitrogen, 40 psi, turbo gas, argon gas, 20 psi. Identification of Emodin and its Glucuronide Metabolite by LC MS/MS and NMR A mixture of reaction products in aqueous solution was extracted with dichloromethane 3 times. The aqueous fraction was loaded onto an ODS column and cleaned using pure water. The mono glucuronide emodin was eluted utilizing a solvent of H2O/MeOH. The structure of mono glucuronide emodin was identified by UPLC ESI Q TOF MS and 1H NMR. The mass spectrometer guidelines were set as follows: capillary voltage, 4. 5KV, ion supply temperature, 350 C, desolvation E3 ligase inhibitor temperature, 108 C, nebulizer gas, nitrogen, 40 psi, turbo gas, argon gas, 20 psi. Kinetic Analysis Permeability of emodin was represented by G eff, which was acquired as described previously. Amounts of emodin absorbed, amounts of glucuronidated emodin excreted into the intestinal lumen, and the percentage absorbed and percentage digested values were calculated as described previously. Briefly, Mab and Mgut were expressed as Eqs. 1 and 2: Mab Qt CAin CAout e T e1T Mgut QtCMout e2T where Q could be the flow rate of perfusion, is the interval time of testing, CAin and CAout are the inlet and outlet concentrations of emodin, and CMout is the concentration of emodin 3 O glucuronide. 1% Metabolized and %absorbed were calculated as: 1% Absorbed in the intestine Mab Mtotal e3T 1% Metabolites excreted within the intestine Mgut Mtotal e4T where Mtotal is the total level of element perfused over the first 30 min period.