Effect of H/D Isotopomerization in the Assay of Resveratrol by Tandem Mass Spectrometry and Isotope Dilution Method Leonardo Di Donna, † Fabio Mazzotti, † Hicham Benabdelkamel, † Bartolo Gabriele, ‡ Pierluigi Plastina, ‡ and Giovanni Sindona †, * Dipartimento di Chimica, Universita ` della Calabria, Via P. Bucci, Cubo 12/C, I-87030 Arcavacata di Rende (CS)-Italy, and Dipartimento di Scienze Farmaceutiche, Universita ` della Calabria, I-87036 Arcavacata di Rende (CS), Italy Resveratrol is a phytoalexin found in several plant tissues and present in wines, which is supplied as a nutritional supplement. Different studies have revealed its beneficial effects as anticancer, antiviral, neuroprotective, antiaging, and anti-inflammatory natural active principle. The as- saying of resveratrol by mass spectrometry and isotope dilution method, using a stable [ 2 H 4 ] analogue, has required a full elucidation of its gas-phase H/D isoto- pomerization. Either selected ion monitoring (SIM) or multiple reaction monitoring (MRM) methods have been used for the evaluation of the amount of resvera- trol present in wine and plasma samples in the negative ionization mode. In all instances the acquired ac- curacy, limit of quantitation (LOQ), and limit of detec- tion (LOD) are fit for the intended purpose of the assay. Resveratrol (trans-3,5,4′-trihydroxystilbene, 1) is a secondary metabolite bearing powerful antioxidant properties produced by some plants for self-protection against environmental stresses (see Chart 1). 1 It is found mostly in grapes, in particular in the skin, where it acts as shield against fungal diseases and sun damage. The amount of resveratrol in grape skins varies with the cultivar, its geographic origin, and exposure to fungal infection. 2 Wines contain, therefore, appreciable amounts of resveratrol released from the grape skins. 3 The predominant form of resveratrol in grapes and grape juice is trans-resveratrol glucoside (trans-piceid); during fermentation, however, the action of specific glucosidases induces significant amounts of resveratrol aglycon in wines. 4 The latter contains also detectable amounts of cis-resveratrol, which may be formed either during fermentation or released from viniferins (resveratrol polymers). 5 The cis isomer can also be induced by photoisomerization of the trans-form when the wine is exposed to light and oxygen. 6 The potential health benefits of resveratrol, investigated in the early nineties when its presence in red wine was first reported, 7 led to speculation referred as the “French Paradox” and related to the apparently low mortality from coronary heart disease, 8,9 despite the relatively high levels of dietary saturated fat and cigarette smoking. This fact led to the idea that the regular consumption of red wine might provide additional protection from cardiovascular disease. Inhibition of platelet aggregation, in vitro, 10,11 anti-inflammatory and antiestro- genic effects, 12,13 and the prevention of cancer diseases 14 are among those healing effects, recently, associated with the intake of functional foods containing resveratrol. Various analytical methods have been developed for its quantitative determination in wine, most of which are based on * To whom correspondence should be addressed. Giovanni Sindona, Dipar- timento di Chimica, Universita ` della Calabria, via P. Bucci, cubo 12/C, I-87036 Arcavacata di Rende (CS)-Italy. Phone: +39-0984-492046. Fax: +39-0984-493307. E-mail: sindona@unical.it. † Dipartimento di Chimica. ‡ Dipartimento di Scienze Farmaceutiche. (1) Langcake, P.; Pryce, R. J. Physiol. Plant Pathol. 1976, 9, 77–86. (2) Fremont, L. Life Sci. 2000, 66, 663–673. (3) Mattivi, F.; Reniero, F.; Korhammer, S. J. Agric. Food Chem. 1995, 43, 1820–1823. (4) Burns, J.; Yokota, T.; Ashihara, H.; Lean, M. E. J.; Crozier, A. J. Agric. Food Chem. 2002, 50, 3337–3340. (5) Goldberg, D. M.; Karumanchiri, A.; Ng, E.; Yan, J.; Diamandis, E. P.; Soleas, G. J. J. Agric. Food Chem. 1995, 43, 1245–1250. (6) Cantos, E.; Garcia-Viguera, C.; de Pascual-Teresa, S.; Tomas-Berberan, F. A. J. Agric. Food Chem. 2000, 48, 4606–4612. (7) Siemann, E. H.; Creasy, L. L. Am. J. Enol. Viticult. 1992, 43, 49–52. (8) Criqui, M. H.; Ringel, B. L. Lancet 1994, 344, 1719–1723. (9) St. Leger, A. S.; Cochrane, A. L.; Moore, F. Lancet 1979, 314, 1017–1020. (10) Kirk, R. I.; Deitch, J. A.; Wu, J. M.; Lerea, K. M. Blood Cell. Mol. Dis. 2000, 26, 144–150. (11) Paceasciak, C. R.; Hahn, S.; Diamandis, E. P.; Soleas, G.; Goldberg, D. M. Clin. Chim. Acta 1995, 235, 207–219. (12) Pinto, M. C.; Garcia-Barrado, J. A.; Macias, P. J. Agric. Food Chem. 1999, 47, 4842–4846. (13) Gehm, B. D.; McAndrews, J. M.; Chien, P. Y.; Jameson, J. L. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 14138–14143. (14) Jang, M. S.; Cai, E. N.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.; Beecher, C. W. W.; Fong, H. H. S.; Farnsworth, N. R.; Kinghorn, A. D.; Mehta, R. G.; Moon, R. C.; Pezzuto, J. M. Science 1997, 275, 218–220. Chart 1 Anal. Chem. 2009, 81, 8603–8609 10.1021/ac9015243 CCC: $40.75  2009 American Chemical Society 8603 Analytical Chemistry, Vol. 81, No. 20, October 15, 2009 Published on Web 09/21/2009