Date of Award
College of Pharmacy and Health Sciences (COPHS)
Ph.D. in Administration of Justice
• LC-MS/MS • Metabolism • Pharmacokinetics • UPLC
Upon oral administration, polydatin undergoes hydrolysis by intestinal microbial b- glucuronidase, releasing its active metabolite, resveratrol. Resveratrol is easily absorbed by the gut lumen and is further metabolized in the liver, producing a glucuronide conjugated metabolite. Following conjugation, the metabolite is actively secreted through the bile duct back into the small intestine. There, the microbiota may again hydrolyze the compound, releasing resveratrol back into the gastrointestinal tract for absorption, a process known as enterohepatic recycling. A sensitive, specific, and reproducible ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated. This method was applied for the determination of both resveratrol and polydatin following pharmacokinetic studies in rats, permitting a comprehensive evaluation of total drug concentrations within the system. A Shimadzu Ultra performance liquid chromatography (UPLC) system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for analysis. Separation was achieved using an Aquity UPLC BEH C18 column (2.1 x 50 mm) in acetonitrile and 0.1% formic acid v/v in water. Analysis was performed under negative ionization mode using the multiple reaction monitoring (MRM) approach, with the Transitions of m/z 389àm/z 227 for polydatin, m/z 227àm/z 185 for resveratrol, and m/z 283à162 for wogonin (as internal standard). Rat plasma samples were extracted by protein precipitation in acetonitrile for quantification. The method was linear in the range of 9.77–1250 nM for both resveratrol and polydatin with correlation coefficient values >0.99. The method has been shown to be reproducible, with intra- and inter-day accuracy and precision ±10.4% of nominal values, for both analytes. Extraction recovery, matrix effect, and stability tests were performed at low, medium, and high concentration levels of 20 ng/mL, 400 ng/mL, and 800 ng/mL, respectively. The average extraction recovery rates were 81.78–98.3% for polydatin and 86.4–103.2% for resveratrol. Matrix effect was in the acceptable range (<15%) and the analytes in plasma were found to be stable under bench-top, freeze- thaw, and storage (21 days, − 20 oC) conditions. Polydatin hydrolysis by intestinal microflora was determined using fecal S9 fraction following the protocol published by us previously. To determine hydrolysis rates, different concentrations of polydatin were incubated with fecal S9 fractions for 1 hour, after which the reaction was terminated using 6% acetic acid in acetonitrile. Samples were prepared and the concentration of polydatin and resveratrol in the reaction system was determined using UPLC quantification. Multiple assays were carried out to evaluate differences in the rate of hydrolysis based on various animal conditions (age, gender, sex, Dss induced colitis, and PRIC rats). A significant difference in the kinetic parameters of hydrolysis was found only between male and female wild-type rats. The previously developed LC-MS/MS method was applied to multiple pharmacokinetic studies in male and female rats (n = 4/group). Polydatin (50 and100 mg/kg) was administered through oral gavage. Additionally, polydatin (10 mg/Kg) was administered intravenously through the tail vein to male and female rats. Blood samples (50 ~ 80 μL) were collected into heparinized microcentrifuge tubes at predetermined time points for 24 hours following administration. The oral bioavailability of polydatin was evaluated using a crossover study design. The pharmacokinetic parameters were calculated using Phoenix WinNonlin® 8.0 software.
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Sunsong, Robin, "An In Depth In Vitro and In Vivo Analysis of Metabolism and Pharmacokinetics of Polydatin and Its Active Metabolite, Resveratrol: In Rats." (2022). Dissertations (2016-Present). 52.