Journl of Chromtogrphy A, 922 (2001) 359 363 www.elsevier.com/ locte/ chrom Short communiction Anlysis of phenolic constituents of biologicl interest in red wines q by high-performnce liquid chromtogrphy M. Lopez *, F. Mrtınez, C. Del Vlle, C. Orte, M. Miro, b Deprtment of Physicl Chemistry, Fculty of Phrmcy, University of Grnd, 18071 Grnd, Spin b Deprtment of Phrmcology, Fculty of Phrmcy, University of Grnd, 18071 Grnd, Spin Received 17 Jnury 2001; received in revised form 17 April 2001; ccepted 24 April 2001 Abstrct We describe reversed-phse HPLC method tht uses grdient elution nd diode rry detection to determine four biologiclly ctive phenolic constituents of red wines: gllic cid, trns-resvertrol, quercetin nd rutin. The method permits direct injection without smple pre-tretment. ODS Hypersil served s the sttionry phse; the grdient ws formed by cetic cid, methnol, nd wter. Ech nlysis required n equilibrtion period of 10 min nd run time of 50 min for completion. Previously, totl phenols were nlysed ccording to the Folin Cioclteu method, using gllic cid s the stndrd, nd the results re given s gllic cid equivlent. 2001 Elsevier Science B.V. All rights reserved. Keywords: Wine; Food nlysis; Phenolic compounds; Gllic cid; Resvertrol; Quercetin; Rutin 1. Introduction Grpes nd wines contin lrge mounts of phenolic compounds, mostly flvonoids t high concentrtions It is of gret interest to evlute the phenolic of 1000 1800 mg/ l [4]. A lrge prt of the phenolics constituents of red wines becuse flvonoids nd in wines my ct s ntioxidnts [5]. Some of these phenolic cids re widely distributed in higher plnts compounds my ct selectively t very low connd form prt of the humn diet. Common foods of centrtions to inhibit ex vivo low-density lipoproplnt origin contin vriety of hydroxylted flvo- teins (LDLs) oxidtion in vitro [6,7]. noids nd other phenolics in mounts rnging from Reduced mortlity from coronry hert disese trces to severl grms per kilogrm [1]. Flvonoids (CHD) mong moderte consumers of lcohol is nd other plnt phenolics hve been reported to hve well-estblished epidemiologic phenomenon [8]. multiple biologicl effects such s ntioxidnt ctivi- There is some evidence tht those who regulrly ty, nti-inflmmtory ction, inhibition of pltelet drink wine my hve lower coronry hert disese ggregtion, nd ntimicrobil ctivities [2,3]. mortlity thn those whose preference lies with other lcoholic beverges. This ltter possibility hs creted gret interest in constituents found in wine Presented t the 29th Scientific Meeting of the Spnish Group tht my be responsible for these puttive effects. It of Chromtogrphy nd Relted Techniques, Alcl de Henres (Mdrid), 12 14 July 2000. hs been suggested tht the moderte intke of wine *Corresponding uthor. provides protection ginst CHD becuse the nti- 0021-9673/ 01/ $ see front mtter 2001 Elsevier Science B.V. All rights reserved. PII: S0021-9673(01)00913-X
360 M. Lopez et l. / J. Chromtogr. A 922 (2001) 359 363 oxidnt properties of the phenolic compounds of 2.2. Chemicls nd stndrds wine dely the onset of therogenesis nd regulte thrombotic tendencies. The French prdox (p- Stndrd substnces of trns-resvertrol, quercetin prent comptibility of high ft diet with low nd rutin were purchsed from Sigm (St. Louis, incidence of CHD) hs been ttributed to the regulr MO, USA). Gllic cid ws obtined from Merck consumption of red wine [9]. The constituents of red (Drmstdt, Germny). Solvents used for chromtogwine hve lwys been of interest due to the French rphy were cetic cid nd methnol of high-perprdox nd the potentil contributory role of the formnce liquid chromtogrphy (HPLC) ultr grflvonoid constituents to decresing coronry hert dient grde supplied by Merck nd deionised wter. disese [10]. Recent interest in these phenolic con- Methnol of Uvsol grde from Merck ws used for stituents of red wine, including gllic cid, trns- prepring stndrd solutions. 0.45-mm pore size resvertrol, quercetin nd rutin hs been stimulted membrne filters from Millipore were used for by the potentil beneficil effects on helth. filtrtion of the mobile phse nd the smples. Some recent studies hve determined polyphenolic compounds in wines principlly using chromto- 2.3. Wine smples grphic techniques, with detection being crried out by spectrophotometry [11 13] or fluorimetry A group of commercilly vilble red wines from [14,15]. different Spnish regions ws nlysed. Smples In the present study, seprtion of the phenolic were opened, protected ginst sunlight nd stored t components, gllic cid, trns-resvertrol, quercetin 48C. Anlyses were crried out within few dys. nd its glucoside rutin, ws determined using re- The smples were filtered through 0.45-mm memversed-phse liquid chromtogrphy. Detection ws brne Millipore chromtogrphic filter. performed using photodiode rry detection. The method permitted the identifiction of the phenols in 2.4. Procedure different types of wines by direct injection, without ny prior purifiction of the smple. Previously, totl 2.4.1. Colorimetric determintion of polyphenols phenols were nlysed ccording to the Folin content Cioclteu method, using gllic cid s the stndrd, The concentrtion of wine phenolics ws estind the results re given s gllic cid equivlents mted by nlysing for totl phenol by the Folin (GAEs). Cioclteu procedure nd expressing results in microgrms per milliliter or molr equivlents of gllic cid, nturlly occurring polyphenol. A clibrtion curve ws prepred using concentrtions of gllic 2. Experimentl cid rnging from 0 to 500 mg/l. 2.4.2. Chromtogrphic conditions 2.1. Apprtus The seprtion ws performed using n ODS Hypersil 5 mm column, 250 mm34 mm I.D. s Absorption spectr were recorded on Perkin- sttionry phse preceded by gurd column of Elmer (Beconsfield, UK) Lmbd 16 UV Vis LiChrospher 100 RP-18, 5 mm, 4 mm34 mm. spectrophotometer. The liquid chromtogrph ws Smples of 20 ml of wine or clibrtion stndrd from Merck Hitchi (Drmstdt, Germny) nd ws were directly injected onto the column nd eluted equipped with diode-rry detector (L-4500, with grdient comprising cetic cid (A), methnol Merck), biocomptible intelligent pump (Merck (B) nd wter (C). Zero-time conditions were A B L-6220), n eluent mixing chmber, mnul injec- C (5:15:80) t flow-rte of 0.4 ml/min. After 5 tor with 20-ml loop nd chromtogrphic dt min, the pumps were djusted to A B C (5:20:75) processing softwre (Model D-6500, Hitchi). The t flow-rte of 0.5 ml/ min, nd t 30 min to operting conditions were t room temperture. A B C (5:45:50) t 0.5 ml/ min until termintion of
M. Lopez et l. / J. Chromtogr. A 922 (2001) 359 363 361 the run t 50 min. The system ws equilibrted using the originl concentrtion by distilled wter t room the strting conditions for 10 min prior to injection temperture. of the next smple. Detection ws routinely crried Recently much ttention hs been focused on the out by monitoring the bsorbnce signls t 280, 306 protective biochemicl function of nturlly occurnd 360 nm. At the end of ech dy, the column ws ring ntioxidnts in biologicl systems nd on the wshed with the zero-time solvent mixture. The wine mechnisms of their ction. Most phenolic comsmples, stndrd solutions nd mobile phses were pounds, which occur widely in plnts, re present in filtered by 0.45-mm pore size membrne filter, nd red wines. Our results confirm vrition in phenolic degssed before their use. content mong severl red wines, from 1800 to 2300 mg/l phenolics. These results re in greement with 2.4.3. Clibrtion grphs those published by others uthors [4]. Stndrd clibrtion curves were estblished by plotting the re of peks ginst different con- 3.2. Spectroscopic identifiction nd mximum centrtions of phenolic compounds (vrying from 0.5 wvelength of stndrd phenols to 25 mg/ml for gllic cid, quercetin nd rutin, nd from 0.1 to 15 mg/ ml for trns-resvertrol). The It is well known tht most flvonols (quercetin nd optimum bsorbnce found for trns-resvertrol is in its glycoside rutin) exhibit two mjor bsorption ccordnce with some recent ccurte studies bnds in the ultrviolet visible region, Bnd I in the [16,17]. Stndrd solutions of trns-resvertrol were 320 385 nm rnge, nd bnd II in the 250 285 nm. prepred in obscurity. Gllic cid nd trns-resvertrol present mximum of bsorbnce t 280 nd 306 nm, respectively. The 2.4.4. Evlution of the pek purity nd linerity lmx nd e vlues in methnol t vrious wve- To check the pek purity, the elutes were moni- lengths of the phenolic compounds studied in this tored with photodiode rry detector ( l5200 400 work re presented in Tble 1. nm). The three spectr corresponding to the upslope, pex nd downslope of ech pek were computer 3.3. Anlysis of phenolic compounds using normlised nd superimposed. Peks were consid- photodiode rry detection ered pure when there ws exct coincidence between the three spectr (mtch fctor $99.5). Although norml-phse chromtogrphy hs been The linerity of the detector responses for the used for the seprtion of phenolic compounds, it is prepred stndrds ws ssessed by mens of liner now generlly greed tht reversed-phse HPLC is regression nlysis regrding the mounts of ech the method of choice for the seprtion of wide stndrd (mesures in mg) introduced in the loop of vriety of phenolic compounds. Generlly, such the chromtogrphic system nd the re of the corresponding pek on the chromtogrm. seprtions re rpid nd provide high resolution nd sensitivity. Besides, the reversed-phse chromtogrphy ws selected becuse polyphenols re insoluble in wter but soluble in lcohols. The sttionry 3. Results nd discussion phse ws ODS (C ), which permitted greter 18 3.1. Totl polyphenols content Tble 1 Absorbnce dt for stndrd phenolic compounds Red wines contin lrge mounts of phenolic 21 21 Phenolic compound l mx (nm) e l mx (M cm ) compounds. The concentrtion of phenolics, esti- Gllic cid 272 10 210 mted by nlysing totl phenols in wines, is pre- trns-resvertrol 306 29 352 sented in Tble 2. The results show tht the red Quercetin 373 24 373 wines contin high concentrtions of phenolics. 256 23 797 Similrity results were obtined when wines were Rutin 360 17 699 distilled to remove lcohol by vcuum nd diluted to 259 21 153
362 M. Lopez et l. / J. Chromtogr. A 922 (2001) 359 363 the hydroxyl groups. An initil mobile phse contining methnol 5% queous cetic cid ws selected. The chromtogrph obtined using this progrmme is shown in Fig. 1. The elution order nd the retention chrcteristics were: 1, gllic cid (t 56.7 R min); 2, rutin (t R536.5 min); trns-resvertrol (t R5 37.1 min); quercetin (t R541.5 min). 3.4. Anlysis of wines Once the chromtogrphic conditions for the seprtion hd been studied, the procedure ws pplied to the determintion of phenol components in wines. These studies were crried out using photodiode rry detection. The identifiction of the different compounds ws chieved by comprison of both retention time nd the bsorption spectr obtined for ech eluted pek with those obtined for the stndrds. The concentrtions of the components were clculted from the chromtogrm pek res (Tble 2). Five Spnish red wines were nlysed for their Fig. 1. Chromtogrphic profile using grdient elution. The peks concentrtion in phenolic compounds. A typicl correspond to: 1, gllic cid (25 mg/ml) mesured t 280 nm; 2, chromtogrm is shown in Fig. 2 (monitored t 280 rutin (25 mg/ml) mesured t 360 nm; trns-resvertrol (5 mg/ nd 360 nm). As regrds red wines, the concentrtion ml) mesured t 306 nm nd 4, quercetin (25 mg/ml) mesured t 360 nm. of these substnces seem to vry considerbly, since it depends on diverse fctors, such s cultivr, climte nd the vinifiction techniques [2,3]. Gllic cid ws present in ll nlysed wines (Tble 2) nd retention. The orgnic solvent selected for prelimin- the level of this compound in wines ws similr to ry experiments ws methnol (MeOH) due to the those described in globl survey [1]. Smple 2 high solubility of phenols in this solvent. The contined considerbly level of trns-resvertrol. retention behviour of phenols ws studied in the Quercetin nd rutin were mesurble in only one of presence of n cid, which prevented ionistion of five wines. Tble 2 Phenolic compound concentrtions nd phenol detected in severl commercil red wines Wine Phenolics Gllic cid Rutin trns-resvertrol Quercetin (mg / l) (mg/ ml) (mg/ ml) (mg/ ml) (mg/ ml) Smple 1 1857 53.3 ND ND ND Smple 2 2315 46.2 ND 1.34 ND Smple 3 1848 48.1 ND ND ND Smple 4 2050 38.8 4.62 ND ND Smple 5 1950 27.7 ND ND 4.66 ND, Not detected. Totl phenols re expressed s gllic cid equivlents (GAEs).
M. Lopez et l. / J. Chromtogr. A 922 (2001) 359 363 363 lengths were ccurtely determined for phenolic compounds. References [1] J. Burns, P.T. Grdner, J. O Neil, S. Crwford, I. Morecroft, D.B. McPhil, C. Lister, D. Mtthews, M.R. McLen, M.E.J. Len, G.G. Duthie, A. Crozier, J. Agric. Food Chem. 48 (2000) 220. [2] D.M. Goldberg, A. Krumnchiri, G.J. Soles, E. Tsng, Am. J. Enol. Vitic. 50 (1999) 185. [3] K. Kwng-Seok, S. Ghim, Y. Seu, B. Song, J. Microbiol. Biotechnol. 9 (1999) 691. [4] J. Knner, E. Frnkel, R. Grnit, B. Germn, J.E. Kinsell, J. Agric. Food Chem. 42 (1994) 64. [5] S. Mxwell, A. Cruikshnk, G. Thorpe, Lncet 334 (1994) 193. [6] E.N. Frnkel, A.L. Wterhouse, J.E. Kinsell, Lncet 341 (1993) 1103. [7] E.N. Frnkel, A.L. Wterhouse, P.L. Teissedre, J. Agric. Food Chem. 43 (1995) 890. [8] E.B. Rim, E.L. Giovnnucci, W.C. Willet, G.A. Colditz, A. Fig. 2. Chromtogrm for red wine smple using grdient Ascherio, B. Rosner, M. Stmper, Lncet 338 (1991) 464. elution nd photodiode rry detection. The peks correspond to: [9] E.N. Frnkel, J. Knner, J.B. Germn, E. Prks, J.E. Kinsel- 1, gllic cid, mesured t 280 nm, nd 2, rutin, mesured t 360 l, Lncet 341 (1993) 454. nm. [10] S. Renud, M. De Lorgeril, Lncet 339 (1992) 1523. [11] D.M. Goldberg, E. Tsng, A. Krumnchiri, E.P. Dimndis, 4. Conclusions G. Soles, E. Ng, Anl. Chem. 68 (1996) 1688. [12] R.M. Lmuel-Rventos, A.I. Romero-Perez, A.L. Wterhouse, M.C. de l Torre Boront, J. Agric. Food Chem. 43 We hve described method for the nlysis of (1995) 281. four phenolic constituents of wine by HPLC of [13] R.M. Lmuel-Rventos, A.L. Wterhouse, Am. J. Enol. 20-ml smple which is directly introduced without Vitic. 45 (1994) 1. the need for prior preprtory procedures. Further [14] R. Pezet, V. Pont, P. Cuent, J. Chromtogr. A 663 (1994) investigtion is required to determine the effects of 191. [15] P. Jendet, A.C. Breuil, M. Adrin, L.A. Weston, S. Debord, geogrphicl origin, geing, climte nd the vinific- P. Meunier, G. Mume, R. Bessis, Anl. Chem. 69 (1997) tion techniques. We expect this ssy to lso be 5172. suitble for the nlysis of the phenolic compounds [16] B.C. Trel, A.L. Wterhouse, J. Agric. Food Chem. 44 in vrious types of wines nd other beverges such (1996) 1253. s beers nd liqueurs. Furthermore, molr bsorp- [17] V.G. Dourtoglou, D.P. Mkris, F. Bois-Douns, C. Zons, J. Food Compos. Anl. 12 (1999) 227. tivities over rnge of commonly reported wve-