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Clinical Chemistry 53: 292-299, 2007. First published December 14, 2006; 10.1373/clinchem.2006.071936
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(Clinical Chemistry. 2007;53:292-299.)
© 2007 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

HPLC–Tandem Mass Spectrometric Method to Characterize Resveratrol Metabolism in Humans

Mireia Urpi-Sarda1, Raul Zamora-Ros1, Rosa Lamuela-Raventos1, Antonio Cherubini2, Olga Jauregui3, Rafael de la Torre4, Maria Isabel Covas5, Ramon Estruch6, Walter Jaeger7 and Cristina Andres-Lacueva1,a

1 Nutrition and Food Science Department, CeRTA, Pharmacy School, University of Barcelona, Barcelona, Spain.
2 Institute of Gerontology and Geriatrics, Department of Clinical and Experimental Medicine, University of Perugia Medical School, Perugia, Italy.
3 Scientific and Technical Services and 6 Department of Internal Medicine, Hospital Clínic, Institut d’Investigació, Biomèdica August Pi i Sunyer, University of Barcelona, Barcelona, Spain.
4 Pharmacology Research Unit and 5 Lipids and Cardiovascular Epidemiology Unit, Institut Municipal d’Investigació Mèdica, Barcelona, Spain.
7 Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria.

aAddress correspondence to this author at: Nutrition and Food Science Department, CeRTA, Pharmacy School, University of Barcelona, 08028 Barcelona, Spain. Fax 34-93-4035931; e-mail candres{at}ub.edu.

Background: Nutritional biomarkers are alternatives to traditional dietary assessment tools. We sought to develop a method for nutritional analysis of resveratrol, a phenolic compound with purported health-promoting properties, and to determine all resveratrol metabolites.

Methods: We obtained LDL and urine samples from 11 healthy male volunteers who had consumed 250 mL of Merlot red wine. We measured resveratrol and its metabolites with 96-well solid-phase extraction plates coupled with HPLC-tandem mass spectrometry. Hexestrol was used as the internal standard. Gradient chromatography in multiple reaction monitoring mode was performed on a Luna C18 column, maintained at 40 °C; m/z transitions were as follows: resveratrol, 227/185; resveratrol glucosides, 389/227; resveratrol glucuronides, 403/227; resveratrol sulfates, 307/227; taxifolin, 303/285; and hexestrol, 269/134.

Results: Standard calibration curves were linear at 4.4–3289.5 nmol/L. Residual analyses were 100% (3.2) for trans-resveratrol and 100% (11.1) for trans-piceid. In both matrices, imprecision (CV) was <10.8% at all concentrations. Detection limits for resveratrol were 0.2 nmol/L (LDL), 0.3 nmol/L (synthetic urine), and 4.0 nmol/L (blank urine). Resveratrol and metabolites were checked for stability, and no degradation was observed.

Conclusions: The HPLC–tandem mass spectrometry method enabled us to identify resveratrol sulfates in human LDL and to characterize the complete profile of resveratrol metabolism in human LDL and urine. This method provides an accurate index of exposure to resveratrol and its metabolites, which can be used as nutritional biomarkers for evaluating the biological effects of moderate wine intake on human health.






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