title: Determination of resveratrol and its sulfate and glucuronide metabolites in plasma by LC-MS/MS and their pharmacokinetics in dogs.
abstract: An analytical approach for the determination of trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) and its glucuronide and sulfate conjugates in dog plasma by LC-MS/MS (without enzymatic hydrolysis of the conjugates) was validated to support pre-clinical toxicological and pharmacological studies. The approach required two independent sample extractions and consequent instrument runs. Samples for resveratrol determination were prepared by protein precipitation with acetonitrile; acetonitrile-methanol was used instead for resveratrol metabolites. Chromatographic separation was performed using a C18 column (30 mm × 2.0 mm) at a flow rate of 0.25 mL/min. For resveratrol the mobile phase consisted of A: 5mM ammonium acetate in water-isopropanol (98:2, v/v) and B: methanol-isopropanol (98:2, v/v) and for metabolites the mobile phase was modified as follows: A: 0.1% (v/v) formic acid in water and B: 0.1% (v/v) formic acid in acetonitrile. Total run time was 12 min for each run with retention times of about 4-5 min for all analytes. A turbo ion spray source was used operating in negative mode for resveratrol and resveratrol sulfate and in positive mode for resveratrol glucuronide. Calibration curves were linear from 5 to 1000 ng/mL for resveratrol and its glucuronide, and 10-2000 ng/mL for resveratrol sulfate. Linearity was assessed using the internal standard method for resveratrol and the external standard method for the metabolites. Method accuracy was 90-112% of the true value for all analytes with precision of 9% RSD or less for all validation experiments. The validated method was applied to a preclinical toxicology study in dogs after oral administration (200-1200 mg/kg) of the agent. Peak plasma resveratrol concentration (C(max)) for most animals was observed within 1-5 h of dosing, with group mean values in the 1.7-9.9 μg/mL (7.5-43 μM) range. Area under the plasma concentration-time curve (AUC) mean values for resveratrol ranged from 3.6 to 44 h μg/mL for all study groups and were generally proportional to the dose, with no consistent statistically significant changes observed for gender or number of doses. Mean molecular-weight adjusted ratios of resveratrol metabolites to resveratrol for AUC ranged from 1 to 9 for resveratrol glucuronide and from 2 to 11 for resveratrol sulfate.
authors.affiliation: Life Sciences Group, IIT Research Institute, Chicago, IL 60616, USA. mmuzzio@iitri.org
authors.forename: Miguel
authors.initials: M
authors.lastname: Muzzio
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Huang
authors.forename: Shu-Chieh
authors.initials: SC
authors.lastname: Hu
authors.forename: William D
authors.initials: WD
authors.lastname: Johnson
authors.forename: David L
authors.initials: DL
authors.lastname: McCormick
authors.forename: Izet M
authors.initials: IM
authors.lastname: Kapetanovic
mesh_terms: D000818:Animals; D001835:Body Weight / Q000187:drug effects; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D005260:Female; D020719:Glucuronides / Q000097:blood*; D006801:Humans; D057230:Limit of Detection; D008297:Male; D015394:Molecular Structure; D051381:Rats; D012015:Reference Standards; D015203:Reproducibility of Results; D000077185:Resveratrol; D013045:Species Specificity; D013267:Stilbenes / Q000097:blood* / Q000378:metabolism* / Q000633:toxicity; D013463:Sulfuric Acid Esters / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D020719:Glucuronides; D013267:Stilbenes; D013463:Sulfuric Acid Esters; D000077185:Resveratrol
references.citation: Mol Nutr Food Res. 2005 May;49(5):472-81
references.pmid: 15779070
references.citation: Curr Drug Metab. 2009 Jul;10(6):530-46
references.pmid: 19702538
references.citation: Food Chem Toxicol. 2011 Dec;49(12):3319-27
references.pmid: 21939727
references.citation: J Pharmacol Exp Ther. 2002 Jul;302(1):369-73
references.pmid: 12065739
references.citation: Anal Chem. 2005 May 15;77(10):3149-55
references.pmid: 15889903
references.citation: Free Radic Biol Med. 2002 Aug 1;33(3):387-98
references.pmid: 12126761
references.citation: Cancer Epidemiol Biomarkers Prev. 2007 Jun;16(6):1246-52
references.pmid: 17548692
references.citation: J Med Chem. 2010 Jul 8;53(13):5033-43
references.pmid: 20527891
references.citation: IUBMB Life. 2008 May;60(5):323-32
references.pmid: 18421779
references.citation: Biomed Chromatogr. 2007 Mar;21(3):257-65
references.pmid: 17294509
references.citation: Biochem J. 2010 Jul 15;429(2):273-82
references.pmid: 20450491
references.citation: Cancer Chemother Pharmacol. 2011 Sep;68(3):593-601
references.pmid: 21116625
references.citation: Int J Biochem Cell Biol. 2009 Nov;41(11):2125-8
references.pmid: 19527796
references.citation: Cancer Res. 2010 Oct 1;70(19):7392-9
references.pmid: 20841478
references.citation: Mol Nutr Food Res. 2008 May;52(5):549-57
references.pmid: 18435437
references.citation: Nat Rev Drug Discov. 2006 Jun;5(6):493-506
references.pmid: 16732220
references.citation: Cancer Epidemiol Biomarkers Prev. 2003 Oct;12(10):953-7
references.pmid: 14578128
references.citation: Antioxid Redox Signal. 2009 Nov;11(11):2851-97
references.pmid: 19432534
references.citation: Cancer Chemother Rep. 1966 May;50(4):219-44
references.pmid: 4957125
references.citation: Mol Nutr Food Res. 2010 Jan;54(1):7-16
references.pmid: 20013887
references.citation: Anal Chem. 1999 Feb 1;71(3):747-50
references.pmid: 9989391
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Apr 1;848(2):182-7
references.pmid: 17097357
references.citation: Mol Nutr Food Res. 2005 May;49(5):482-94
references.pmid: 15779067
references.citation: J Agric Food Chem. 2010 Oct 27;58(20):11165-71
references.pmid: 20886884
references.citation: Drug Metab Dispos. 2004 Dec;32(12):1377-82
references.pmid: 15333514
references.citation: Mol Interv. 2006 Feb;6(1):36-47
references.pmid: 16507749
references.citation: Br J Cancer. 2004 Feb 9;90(3):736-44
references.pmid: 14760392
references.citation: Clin Cancer Res. 2010 Dec 15;16(24):5942-8
references.pmid: 21045084
references.citation: J Pharm Biomed Anal. 2010 Jan 20;51(2):391-8
references.pmid: 19406597
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Dec 27;829(1-2):97-106
references.pmid: 16243591
references.citation: Pharm Res. 2002 Dec;19(12):1907-14
references.pmid: 12523673
references.citation: Mol Nutr Food Res. 2009 May;53 Suppl 1:S7-15
references.pmid: 19194969