Food Chemistry 135 (2012) 17081717Contents lists available at SciVerse ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchemReview

Hydrolyzable tannin analysis in food

Panagiotis Arapitsas Food Quality and Nutrition Department, Research and Innovation Centre, Fondazione Edmund Mach, via E. Mach, 1, 38010 San Michele allAdige, Italy

a r t i c l e i n f o a b s t r a c tArticle history:Received 1 February 2012Accepted 24 May 2012Available online 1 June 2012

Keywords:TanninsFoodGallotanninsEllagitanninsAnalytical chemistry0308-8146/$ - see front matter 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.05.096

Tel.: +39 0461 615 564; fax: +39 0461 615 200.E-mail addresses: panagiotis.arapitsas@fmach.it,

comThe discovery of plant polyphenols in food is perhaps one of the biggest breakthroughs in modern foodscience. Plant polyphenols are known for their role in food quality and safety, since they contribute sig-nificantly to taste, flavour, colour, stability etc., while they are increasingly recognised as important fac-tors in long-term health, contributing towards reducing the risk of chronic disease. Almost 200 years ago,hydrolyzable tannins (HTs) were the first group of plant polyphenols subjected to analytical chemicalresearch. Despite the lack of commercially available standards, food analysis research offers a wealthof papers dealing with extraction optimisation, identification and quantification of HTs. The object of thisreview is to summarise analytical chemistry applications and the tools currently used for the analysis ofHTs in food.

2012 Elsevier Ltd. All rights reserved.Contents1. Historical background introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17082. Classification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17103. Importance of food HTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17104. Analytical chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17114.1. Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17114.2. Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17114.3. Chromatography separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17144.4. Detection Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17144.5. Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17155. Concluding remarks and future aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1715References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17161. Historical background introduction

With the start of the modern scientific revolution and the devel-opment of chemistry from alchemy, plant polyphenols became oneof the first subjects of scientific research. Many of the fathers ofchemistry worked in this field and provided the first results in rela-tion to the quantitative and qualitative analysis of HTs (Fischer,1914; Haslam, 1989; Russell, 1935).

Around 200 years ago, in the leather industry chemists wereasked to improve leather processing techniques, due to the de-mand for greater quantities of leather and better quality. In thisera, traditional techniques were the result of the application ofll rights reserved.

panagiotis.arapitsas@gmail.empirical methods as well as reasoning, and most products weretanned with an infusion of oak bark for a period of three to sixmonths. Probably for this reason, the first term used for theseproducts, which is still adopted today, was vegetable tannins (tannin Celtic means oak tree). Over the centuries and around the world,other rawmaterials in addition to oak bark, all from the plant king-dom, were also used to tan hides, such as myrtle leaf extract in17th century Rome. The quality of the final product was not alwayssimilar, even when exactly the same method was used. The firstproblem was finding the most efficient source of vegetable tannin,while the second step was standardisation. Since the most well-known and effective tanning material was oak bark extract, calledtannic acid, numerous chemists tried to analyse it, discover its com-position and develop quantitative analytical methods, in order toproduce a standard raw material (Darton, 1882a, 1882b; Fischer,1914; Rau, 1887; Russell, 1935; Wilson, 1934).

http://dx.doi.org/10.1016/j.foodchem.2012.05.096mailto:panagiotis.arapitsas@fmach.itmailto:panagiotis.arapitsas@gmail. commailto:panagiotis.arapitsas@gmail. comhttp://dx.doi.org/10.1016/j.foodchem.2012.05.096http://www.sciencedirect.com/science/journal/03088146http://www.elsevier.com/locate/foodchem

OO

OO

O

O

G

GG

G

O

O

OH

OH

OO

OO

O

O

GGO

OHO

HO

HO OH

HO

HO

G

O

HO

HOHO

HO

HO

OH

O

OO

OO

OH

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GG

O

O OO

OHO

GO

OHO

HOOH

HO

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GO

O

HO

HO

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O

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OHHO

OH

OH

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O OO

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G

GG

GG

penta-galloyl-glucose

sanguiin H-6

oenothein B

HOOH

OH

O

OHOHHO

OHO

OHO

HO OHO

HO

O

OH

OH

OH

G=

OO

O OO

O

G

GG

GG

OG

OG

OG O

G OG

OO

O OO

O

GO

G

OH

OHO

G

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OHOH

HO

G

glucogallin

m-depsidic link

penta-(digalloyl)-glucose

O

OOH

OH

O

O

O

HOHO

HO

HO

HOHO

HO

HO

O

OO

OO

OH

O

GG pedunculagin II

punicalagin A

Fig. 1. Structures of Hydrolyzable Tannins.

P. Arapitsas / Food Chemistry 135 (2012) 17081717 1709Carl Wilhelm Scheele, a German-Swedish pharmaceuticalchemist born in 1742, is famous for discovering many chemicalsubstances, such as oxygen and chlorine. Scheele also isolatedand characterised various phenolic natural compounds, includinggallic acid from tannic acid (Fischer, 1914; Russell, 1935). The termtannin was probably introduced by A. Seguin around the end of the17th century to describe the extractable matter used to converthide to leather, while he also demonstrated the ability of tanninsto precipitate gelatin from solution (Haslam, 1989). In the middleof the 18th century, Adolph Strecker seems to have been the firstto point out that tannic acid yields gallic acid and glucose followinghydrolysis and that it has the formula C27H22O17, corresponding toa combination of 3 moles of gallic acid and one mol of glucose(Fischer, 1914). Probably due to this observation, today the chem-ical dictionary The Merck Indexwrongly cites the term tannic acid asa tri-galloyl-glucose derivative (The Merk Index, 2001). R.J. Man-ning isolated a pentaethyl ester of pentagalloyl glucoside from tannicacid. K. Feist arrived at the view that tannic acid from Turkish nutg-alls was a combination of glucogallic acidwith two ester-like boundmolecules of gallic acid (Fischer, 1914).The German chemist Hermann Emil Fisher (18521919), Nobellaureate in 1902, famous for his studies on sugars and purines, alsodevoted his genius to the analysis and synthesis of active principlesfrom the plant kingdom. More than 100 years ago, Fischer, togetherwith Karl Jonnan Freudenberg, carried out the most complete stud-ies in relation to tannic acid identification. They called the ester-like anhydride compounds between gallic acids, such as UgoSchiffs digallic acid, depsides. The term derives from the Greekdewim (to tan), because many of these substances resemble tan-nins. Initially, they suggested that tannic acid is the penta-(digal-loy)-glucose (Fig. 1), whereas ultimately they were the first tosuggest the possibility that tannic acid could be a mixture of verysimilar substances, for example a penta-(digalloyl)-glucose with atetra-(digalloyl)-glucose or tri-(digalloyl)-di-(galloyl)-glucose, etc.(Fischer, 1914).

At the same time that European chemists were focusing on theidentification of vegetable tannins, on the other side of the Atlantic,in the United States, several scientists were turning their attentionto the development of a method for quantification and standardi-sation. Nelson H. Darton, Loewenthal, Hammels, Allen and Carpenk

1710 P. Arapitsas / Food Chemistry 135 (2012) 17081717are some of the scientists who tried to create methods to quantifytanning materials. Most of these methods aimed to facilitate thedetermination of vegetable tannins and yielded very discordant re-sults when compared to the actual weight of the leather formed bythem. In the end, tanners lost all faith in organic chemists generallyand started to use inorganic chemical products for tanning (Darton,1882a, 1882b; Russell, 1935).

According to A.G. Perkin and A.E. Everests polyphenol classifi-cation in 1915, tannins were divided into three groups: depsides,diphenyldimethylodil tannins and phlobatannins. Freudenbergpreferred to call the first two groups hydrolyzable tannins. E.Fischer and Stracker called the first group gallotannins, since theirhydrolysis yields gallic acid. Nierenstein was probably the first tocall the second group ellagitannins, since their hydrolysis yields el-lagic acid. The third group is the class of compound now known asflavonoids (Fischer, 1914; Russell, 1935).2. Classification

In the last few decades, the secondary metabolites widespreadthroughout the plant kingdom and characterised by water solubil-ity and molecular masses between 500 and 5000 Dalton have beencalled HTs; they give the usual phenolic reaction and precipitatealkaloids and proteins. As regards their chemical structure, HTsare multiple esters of gallic acid with glucose and products of theiroxidative reactions. Consequently, they are clearly distinguishedfrom other tannins such as condensed tannins (syn. proanthocy-anidins) which are derivatives of catechin and tara tannins, estersof gallic acid with quinic acid (Harborne &Williams, 2000; Haslam,1996, 2007).

As far as biosynthesis is concerned, 1-galloyl-b-D-glucose (syn.glucogallin) has been proposed as the first intermediate and akey-metabolite in the biosynthetic pathway of tannins. A specificglucosyltranferase catalyses the esterification between gallic acidand UDP-glucose to give glucogallin (Fig. 1). In a second step,glucogallin plays a dual role, functioning as an acyl acceptor andacyl donor, in order to form di-, tri-galloylglucoses, etc. This firstbiosynthetic pathway is completed by the formation of pentagal-loyl-glucose (Fig. 1), and the products are simple galloylglucosederivatives. In the second biosynthetic route, the galloylation ofpentagalloyl-glucose continues to hexa-, hepta-, octa-, etc. gal-loylglucose derivatives (Fig. 1). These compounds are called gallo-tannins or depsidic metabolites, and the esteric-link between twogalloy moieties is known as the m-depsidic link (Fig. 1), accordingto Fischer. The third pathway yields ellagitannins, which are typi-cal constituents of many plant families, in contrast to gallotannins,which are not so widespread in nature. Ellagitannins are the prod-ucts of oxidation, leading to C-C linkages between suitably orien-tated galloyl residues of glucogalloyl molecules that formhexahydroxydiphenoyl (HHDP) units (Buzzini et al., 2008; Gross,2008; Okuda & Ito, 2011; Okuda, Yoshida, & Hatano, 1989a; Qui-deau & Feldman, 1996). The oak C-glycosidic ellagitanins (castala-gin, vescalagin) and their flavano-ellagitannin derivatives(acutissimin B and epiacutissimin B), detected in aged wine andwhisky, and gallagyl-glucosides, whose structure has an ellagicacid moiety (punicalagin, peduncalagin), also belong to the samegroup. Okuda et al. have proposed two more biosynthetic transfor-mations, which deliver HTs with the dehydrohexahydroxydiphe-noyl (DHHDP) group and the transformed DHHDP group (Okuda& Ito, 2011).

While the distribution of gallotannins is rather limited in nat-ure, ellagitannins are widespread in many plant families (Gross,2008; Okuda & Ito, 2011). In total more than 1000 HTs have beenidentified, from the simple glucogallin (MW 332) to pentamericellagitannins with MWs over 5000 Dalton. However, to date thereis not even one commercially available standard, making the worldof analytical chemistry more difficult and of course morefascinating.

3. Importance of food HTs

From the discovery of HTs up to the present day, interest in HTsin various scientific and commercial areas has increased con-stantly. In the food sector, HTs affect food quality in different ways.The most well-known and researched property of these com-pounds, which has major importance in food quality, is theirastringency. Salivary proline-rich proteins form complexes withdietary HTs and precipitate. The result of this action is an astrin-gent sensation, which is perceived as a diffuse feeling of extremedryness and roughness and is not confined to a particular regionof the mouth or tongue (Glabasnia & Hofmann, 2006; Haslam,1989, 1996; Quideau & Feldman, 1996).

Numerous scientific studies and widespread epidemiologicalevidence has correlated fruit and vegetable consumption with alower risk of cancer, degenerative and cardiovascular diseases(Halliwell, 2007; Harborne & Williams, 2000; Haslam, 1989; Ha-slam, 1996; Havsteen, 2002; Landete, 2011; Larrosa, Garca-Cone-sa, Espn, & Toms-Barbern, 2010; Le Marchand, 2002; Steinmetz& Potter, 1991, 1996). Food components playing an important rolein this context include plant polyphenols such as HTs. However,the metabolism of plant polyphenols in the human organism andmechanisms relating to how they work and their bioavailabilityare still not clear. The mechanism which has been most closelystudied in order to explore the biological activities of HTs relatesto their antioxidant properties (Borges, Mullen, & Crozier, 2010;Cai, Luo, Sun, & Corke, 2004; Cai, Sun, Xing, Luo, & Corke, 2006; Ha-slam, 1996; Labieniec, & Gabryelak, 2007; Larrosa et al., 2010; Oku-da & Ito, 2011; Okuda, Yoshida, & Hatano, 1989b; Tzulker et al.,2007; Yoshida, Amakura, & Yoshimura, 2010; Zhao et al., 2005).Different isolated HTs from edible and/or non-edible plants haveshown strong biological activity in the form of anti-tumour, anti-mutagenic, anti-diabetic, anti-proliferative, anti-bacteric andanti-mycotic properties (Buzzini et al., 2008; Formentini et al.,2008; Halberstein, 2005; Halliwell, 2007; Haslam et al., 1989; Ha-slam, 1996; Havsteen, 2002; Landete, 2011; Larrosa et al., 2010;Okuda et al., 1989; Romani et al., 2005; Salminen, Karonen, & Sink-konen, 2011; Steinmetz & Potter, 1991, 1996). Unfortunately, dueto the lack of a commercial standard, there is a lack of HT struc-tureactivity relationship studies in enzymatic and cell lines assaysin the literature.

Ellagitannins and gallotannins may also affect the life of food-stuff due to their antioxidant properties and/or antimicrobial activ-ity (Cao, Sofic, & Prior, 1997; Hager, Howard, & Prior, 2010; Haslam,1989; Koponen, Happonen, Mattila, & Trrnen, 2007; Lau, King, &Waterhouse, 2003; Michel et al., 2011; Zhang, Wang, Lee, Henning,& Heber, 2009).

Finally, HTs are widespread in fruit, vegetables and other food.In particular, they are frequently found in large amounts in Rosidaebut only in a small number of Dilleniidae and Hamamelidae plants(baby berries etc.), while they are absent in Caryophyllales andMagnolidae. Rosales, in the Rosidae order, mostly produce simpleglucogalloyl and HHDP tannins (raspberries, blackberries, cloud-berries, arctic raspberries, strawberries, etc.). Other orders of Rosi-dae rich in HTs are Fragales (oak, carob, nuts, tonoak, etc.),Brassicales (capparis), Myrtales (myrtle, pomegranate, etc.) andSapindales (Chinese olives, mango, etc.). HTs have also been de-tected in the Vitaceae family (muscadine grapes), in processed foodproducts such as aged wines and spirits or food to which HTs havebeen added, such as meat, fish or wine, in order to take in advancetheir antioxidant activity. Tannic acid is indeed a food flavouringaccording to European Union regulation (EC) No. 2232/96. The to-

P. Arapitsas / Food Chemistry 135 (2012) 17081717 1711tal concentration of HTs in food varies from 1 mg/kg (in chestnuts)to 2 g/kg (in blackberries), with berries representing the richestdietary source (Larrosa et al., 2010).

4. Analytical chemistry

While biochemistry has revealed many valuable properties ofHTs and a high level of knowledge has been reached as regardstheir organic synthesis, analytical chemistry on the other hand stillhas many elementary problems to resolve, apart from isolation andNMR characterisation. Extraction processes have still not been fullyoptimised, separation methods are not suitably efficient and iden-tification patterns are not particularly clear. While HTs are morestable during food processing and storage as compared to otherpolyphenolic compounds, foods and supplements rich in HTs lackauthentication (Aaby, Wrolstad, Ekeberg, & Skrede, 2007; Hageret al., 2010; Kim, Lounds-Singleton, & Talcott, 2009; Koponenet al., 2007; Madrigal-Carballo, Rodriguez, Krueger, Dreher, & Reed,2009; Mullen et al., 2002; Zhang et al., 2009). Knowledge aboutcondensed tannins, which have a similar complexity, is insteadmore advanced. These problems are probably due to the lack ofcommercially available standard, which leads to further misunder-standing between scientists. There are still scientists who considertannic acid to be a single compound, principally because largechemical industries are selling tannic acid as pentagalloyl glucose.The high molecular weight, the large number found in nature andthe considerable structural complexity of HTs represent othercomplicating factors.

4.1. Extraction

Up to few years ago most of the information about HTs extrac-tion derived from the isolation and NMR identification studies.Aqueous solutions of methanol, ethanol or acetone, as well as ethylacetate have been used to extract HTs from natural tissues (Arapit-sas, Menichetti, Vincieri, & Romani, 2007; Garca-Estvez, Escrib-ano-Bailn, Rivas-Gonzalo, & Alcalde-Eon, 2010; Gironi &Piemonte, 2011; Harborne & Williams, 2000; Hatano, Yoshida, &Okuda, 1988; Markom, Hasan, Daud, Singh, & Jahim, 2007; Muel-ler-Harvey, 2001; Okuda & Ito, 2011; Okuda, Yoshida, & Hatano,1989a; Quideau & Feldman, 1996). Non-polar organic solvents(i.e. n-hexane, petroleum ether), chloroform and dichloromethanehave a low extraction strength for HTs and are usually used in sam-ple treatment to remove lipids and chlorophyll and/or to preventenzymatic reactions (Mueller-Harvey, 2001; Okuda et al., 1989).Extraction methods with the use of small percentage of ascorbicacid or under Argon atmosphere have been used for the protectionof analytes by oxidation (Aaby, Ekeberg, & Skrede, 2007; Glabasnia& Hofmann, 2006; Okuda et al., 1989). Methanol tends to be betterfor low molecular weight tannins or in the case of matrices con-taining large amounts of enzymes (i.e. bark or fruit), while acetoneis preferred for high molecular weight tannins and because it is lessliable to react with them (Mueller-Harvey, 2001; Okuda et al.,1989). High temperatures for extended times may cause hydrolysisof the galloyl moiety attached to the glucose anomeric C-1 positionand can also release ellagic acid from ellagitannins (Okuda et al.,1989). Extraction with ethanol and/or methanol may produce ethylor methyl ester of gallic acid, respectively (Arapitsas et al., 2007;Mueller-Harvey, 2001; Okuda et al., 1989b). Unfortunately, the lit-erature provides little information on extraction techniques com-paring HTs, in food products so most of the information in thisfield comes from HTs from non-edible products. Soxhlet apparatushas been used with very good results for the extraction of HTs fromcarob fibres and traditional medicinal herbs, with an initial clean-up step with an unpolar solvent (haxene, petroleum ether or chlo-roform) applied to remove lipids. Tian, Li, Ji, Zhang, and Luo (2009)showed that for the soxhlet technique, ethyl acetate was better forextraction of high MW gallotannins, water for small gallatannins(with 14 galloyl moieties), while ethanol provided medium re-sults but was able to extract all gallotannins tested. Markomet al. (2007) published a study showing that corilagin was best ex-tracted from the herbal plant P. niruri using aqueous ethanol, whilepressurized water extraction (PWE) gave a higher yield in a shortertime as compared to Soxhlet and supercritical fluid extraction(SFE). In the same study, as far as the choice of solvent was con-cerned, aqueous ethanol gave better results when a Soxhlet extrac-tor was used, but aqueous acetone was the best solvent mixture forliquidsolid extraction. PWE also gave better results according to astudy by Papagiannopoulos, Wallseifen, Mellenthin, Haber, and Ga-lensa (2004), but the best solvent was a wateracetonemixture. Onthe other hand, the results published by Cam et al. based on the to-tal polyphenolic amount in pomegranate peel (HTs are the pre-dominant polyphenolic metabolites according to the paper),showed no significant difference between PWE and simple li-quidsolid extraction (am & HIsIl, 2010). Moreover, extractabilityof HTs may depend on the seasonal maturity of tissues and on thetype of plants or their structural physicochemical properties (Oku-da et al., 1989).

Consequently, according to the literature, there is no optimalextraction procedure which can be universally employed for allHTs and all types of samples. However, as shown in Table 1, simpleand time-consuming techniques are mainly used, acetone beingmore frequently used as the extraction solvent, with acidic meth-anol as the second choice. Final PWE would seem to be a verypromising technique, which could also give very effective, fastand environmentally friendly results in the field of HTs.4.2. Isolation

There are plenty of studies regarding the isolation of HTs. Asmost HTs are strongly absorbed on Silica gel, this is not a suitablematerial for normal phase preparative chromatographic separa-tion, although it has been used for analytical HPLC when developedwith an acid-containing eluent (Mueller-Harvey, 2001; Okudaet al., 1989b). The separation of tannins on columns of hydroxypro-pylated dextran gel (i.e. Sephadex LH-20), is induced by the differ-ent adsorption of each compound on the gel, rather by gelfiltration, and tannins of high molecular weight are often notrecovered from the column. Usually tannins are initially selectivelyabsorbed on Sephadex LH-20, then equilibrated with alcohols(methanol and ethanol) and finally released with aqueous acetone(Arapitsas et al., 2007; Engels et al., 2009; Fujieda, Tanaka, Suwa,Koshimizu, & Kouno, 2008; Mueller-Harvey, 2001; Okuda et al.,1989). Better results have been obtained by vinyl polymer gelssuch as Toyopearl HW-40 and Diaion HP-20 (Fujieda et al., 2008;He, Xia, & Chen, 2007; Mueller-Harvey, 2001; Okuda et al., 1989).As shown in Table 1, other materials have also been used latelyfor HT isolation, adopted for the typical stationary phase for analyt-ical columns (Supercosil, Phenomenex acqua, Chromatonex ODSDescovery HS, ODS-C18 silica gel, Purospher, Hichrom, SynergyHydro RP, Latex C-18, etc.). For the fractionation of extracts richin gallotannin, Engels, Ganzle, and Schieber (2009) optimised andused a High Speed Counter-Current Chromatography (HSCCC)column, with a preparative run solvent system of hexane/ethylacetate/methanol/water with initial ratios of 0.5/5/1/5 (v/v/v/v),followed by a 0.75/5/1/5 ratio. Sometimes small quantities ofascorbic acid are added to the mobile phase for the prevention ofoxidation, but the main drawback of this practice is the presenceof the antioxidant in all the eluted analytes (Mueller-Harvey,2001; Okuda et al., 1989b).

Table 1HTs analysis in Food and Beverages.

Sample HTs detected Extraction Isolation fractionation LC parameters Detection Reference

Strawberry Ellagitannins (CH3)2CO Betasil C18; 25 C; CH3COOH:H2O (2:98),CH3COOH:CH3CN:H2O (2:50:48); 74 min

LC-DAD-MSn Aaby, Ekeberg, andSkrede (2007a)

Muscadine grape Gallotannins and ellagitannins CH3COOH:(CH3)2CO:H2O(0.3:70:29.7)

Zorbax stablebond SB-C-18;CH3COOH:H2O (0.5:99.5), CH3OH; 70 min

LC-DAD-MSn Amandeep andLiwei (2010)

Rasberry, tannic acid Sanguiin H-6, lambertianin C, gallotannins of tanninacid

CH3CH2OH:H2O (70:30) Phenomenex Luna C18; 27 C;CH3COOH:H2O (0.1:99.9), CH3CN; 30 min

LC-DAD-MS; NMR (1H,13C; CD3OD)

Arapitsas et al.(2007)

14 Raspberry cultivars Lambertianin C, sanguiin H-6 CH3COOH:CH3OH:H2O(0.1:62.5:37.4)

Synergi C-18; CH3COOH:H2O (0.5:99.5),CH3COOH:CH3CN (0.5:99.5); 60 min

HPLC-QTOF-MS Beekwilder et al.(2005)

36 Euripean fruit juicecontainingpomegranate

Punicalagin-like, punicalagin, A, punicalagin B,punicalin A, punicalin B, Granatin A, Granatin B,punicalagin isomer

CH3OH Gemini C6 phenyl; 40 C; CH3COOH:H2O(0.1:99.9), CH3OH

LC-DAD-MS/MS Borges et al.(2010)

15 Strawberry cultivars Sanguiin H-6, lambertianin C, galloyl-bis-HHDP-glucose

CH3COOH:(CH3)2CO:H2O(0.5:70:29.5); sonication

LiCrocart C18; CH3COOH:H2O (5:95),CH3OH; 70 min

LC-DAD & LC-MS/MS Buendia et al.(2010)

Mango kernel From tetra- to dodeca-galloyl glucose (CH3)2CO:H2O (80:20) Shephadex LH-20,Supercosil LC-18-DB(isolation); HSCCC(fractionation)

Supercosil LC-18-DB and Synergi Hydro-RP; 21 oC; HCOOH:H2O (2:98),HCOOH:CH3OH:H20 (0.5:49.5:50);72 min

LC-MS/MS Engles et al.(2009), Engleset al. (2010)

Pomegranate peel,mesocarp and arils

Pedunculagin I, pedunculagin II, casuarinin,lagerstannin B, granatin B, castalagin derivative,lagerstannin B derivative, digalloyl glucose,monogalloyl glucose, galloyl-HHDP-glucose,lagerstannin C, punigluconin and others ellagitanninsand gallotannins

HCl:CH3OH:H2O(80:19.9:0.1)

Phenomenex Aqua C18(isolation)

Synergi Hydro-RP; 30 C, HCOOH:H2O(2:98), HCOOH:CH3OH:H2O (0.5:90:9.5);71 min

LC-DAD-MS Fischer et al.(2011)

Whisky Whisky tannin A, whisky tannin B, gallic acid, ellagicacid, castalin, 6-galloyl glucose, 3-galloyl glucose,2,3-digalloyl glucose, 2,3-HHDP glucose, castacreninB, castalagin and other ellagitannins

Chromatorex ODS column,MCl gel CHP20P column,Sephadex LH-20, ToyopearlHW40F column (isolation)

NMR (1H, 13C, COSY,NOESY, HSQC, HMBC;(CD3)2CO, CD3OD,DMSO, (CD3)2CO-D2O)

Fujieda et al.(2008)

Boysenberry seedsand juice

Peducilagin, peduculagin isomer, sanguiin H-6,sanguiin H-10, lambertianin A, bis-HHDP-glucose,sanguiin H.2, lambertianin C,

CH3CH2OH:H2O (80:20) Amberlite XAD7 HP resin,Shephadex HL-20(fractionation)

RP L-column; 40 C; CH3COOH:H2O(0.1:99.9), CH3COOH:CH3OH (0.1:99.9);65 min & Develosil 100 Diol-S; 35 C;HCOOH:CH3CN (2:98),HCOOH:CH3CN:H2O (2:95:3); 80 min.

LC-DAD-MS/MS; LC-NP-Fluorescence;Maldi-Tof-MS.

Furuuchi et al.(2011)

Oak-aged wine Grandinin, vescalagin, castalagin SPE Water C-18 Sep-Pakand Sephadex LH-20(fractionation)

Aqua C-18; 35 C; HCOOH:H2O (2.5:97.5),(CH3)2CHOH, CH(CH3)2COOH; 65 min

LC-DAD; MS Ion Trap Garca-Estvezet al. (2010)

11 Rubus berriescultivars

Three sanguiin H-10 isomers, sanguiin H-2, sanguiinH-6, lambertianin C and other ellagitannins

(CH3)2CO:H2O (70:30).Anthocyanins clean upwith shephadex LH-20

ENV + resin, Descovery HSC18 (isolation)

Phenomenex Luna C18; 40 C;CH3COOH:H2O (1:99), CH3CN; 52 min

LC-DAD-QTOF Gasperotti et al.(2010)

Witch hazel From tri- to deca- galloyl glucose (CH3)2CO:H2O (70:30);liquidliquid extractionwith CH3COOCH3

Waters C-18 Symmetry; 25 C;HCOOH:H2O (0.05:99.95), CH3CN; 52 min

LC-DAD-MS/MS Gonzalez, Torres,and Medina (2010)

Blackberry products Two isomers of pedunculagin, two isomers ofcastalagin/vescalagin, two isomers of lambertianin C,two isomers of sanguiin H-6/lambertianin A,lambertianin D, galloylbisHHDPglucose

CH3COOH:(CH3)2CO:H2O(0.5:70:29.5)

Phenomenex Aqua; CH3COOH:H2O (2:98),CH3COOH:CH3CN:H2O (0.5:50:49.5);65 min,

Hager et al. (2010)

Chinese olive Gallic acid, methyl gallate, ethyl gallate, corilagin,hyperin

(CH3)2CO:H2O (70:30) Polyamine, Toyopearl Hw-40 (isolation)

Purospher STAR C-18; 30 C; HCOOH:H2O(0.5:99.5), CH3OH; 30 min

LC-DAD-MS; NMR(CD3OD; 1H, 13C)

He et al. (2007)

Walnuts Glansreginin A, glansreginin B, glansrin D, 1,2,3,6tetragalloyl glucose, 1,2,4,6 tetragalloyl glucose,pentagalloyl glucose, pterocarinin A, platycaryanin A,euprostin A, rugosin F, 1-desgalloylrugosin F,heterophylliin D

CH3CH2OH:H2O (70:30) Diaian HP-20 CC, Toyopearl,MCl gel CHP-20P CC, YMCGEL ODS-AQ 120-S50 CC,Mega Bond Elut C18 CC(isolation)

NMR ((CD3)2CO; 1H,13C, COSY, NOESY,HSQS, HMBC)

Ito et al. (2008)

Large caper fruit 1,3,6 Trigalloyl-2-chebuloyl-glucose, 1,3,6 trigalloyl-2-(4-methyl)-chebuloyl-glucose

CH3OH:H2O (50:50) Partition with chloroformand n-butanol, ODS-C-18Dilica gel, Purospher,Hichrom (isolation)

NMR (CD3OD, DMSO;1H, 13C, COSY, HSQC,HMBC, DEPT-90 & 135)

Kanaujia et al.(2010)

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Mango Gallotannins Waters Spherisorb ODS 2; HCOOH:H2O(2:98), HCOOH:CH3CN:H2O (2:68:30);60 min

LC-DAD Kim et al. (2009)

Boysenberry Sanguiin H-2, sanguiin H-6, sanguiin H-10, (galloyl-bis-HHDP-glucose)-2-gallate

CH3COOH:CH3CH2OH:H2O(1:80:20)

Synergi Hydro-RP(isolation)

Zorbax Rapid Resolution SB-18;CH3COOH:H2O (5:95), CH3CN; 17 min &Prodigy ODS(3) 100 A; 35 C;CH3COOH:H2O (0.1:99.9), CH3CN; 48 min

LS-MS/MS; Maldi-Tof;NMR ((CD3)2CO-H2O;1H, 13C)

Kool et al. (2010)

Various berries Ellagitannins and gallotannins CH3COOCH3 and CH3OH LiChroCART Purospher RP-18e;CH3COOH:H2O (1:99), CH3CN; 20 min

LC-DAD-MS Mtt-Riihinenet al. (2004)

Pomegranate Punicalin, punicalagin A, punicalagon B Spherisorb ODS2 C-18; TFA:H2O (1:99),CH3OH; 50 min

LC-DAD Madrigal-Carballoet al. (2009)

Tanoak acorns Gallotannins CH3OH:H2O (80:20) Prodigy ODS column; CH3COOH:H2O(1:99), CH3COOH:CH3OH (1:99); 45 min

LC-DAD-MS/MS Meyers, Swiecki,and Mitchell(2006)

Oak-aged wine Castalagin, vesclagin, grandinin, robunin E, robuninA, robunin D, robunin B, robunin C.

Lichrospher 100RP 18; H3PO4:H2O(0.1:99.1), H3PO4:CH3OH (0.1:99.9); &CH3COOH:H2O (0.4:99.6), HCOOH:CH3OH(0.4:99.6); 40 min

LC-DAD; LC-MSn Michel et al.(2011)

Red rasberry Lambertianin C, sanguiin H-6 HCl:CH3OH (0.1:99.9) RP-MAX C12; 40 oC; TFA:H2O (0.5:99.5),CH3CN; 30 min

LC-DAD Mullen et al.(2002)

Rasberry Sanguiin H-10, lambertianin C, sanguiin H-6, ellagicacid pentose conjugate, nobotanin A, and othersellagitanins

HCl:CH3OH (0.1:99.9) Synergi RP Max; 40 C; CH3COOH:H2O(1:99), CH3CN; 60 min.

LC-MSn Mullen et al.(2003)

Carob 1,6 Digalloyl glucose, 1,2,6 trigalloyl glucose, 1,2,3,6tetragalloyl glucose,

Soxhlet extractor, CH3OH Silica gel 60, Latex C-18(isolation)

Latex C-18; CH3COOH:H2O (2:98),CH3OH; 45 min. GCMS after acididhydrolysis

GCMS, LC-MS, nano-ESI-MS; NMR (CD3OD;1H, 13C, DEPT-135,COSY, 2D CH COLOCand COLOC-S)

Owen et al. (2003)

Carob Gallotannins Pressurised liquidextraction optimization;(CH3)2CO:H2O (50:50)

Aqua C18, 25 C, CH3COOH:H2O (1:99),CH3COOH:CH3CN (1:99); 77 min

LC-DAD-MS/MS Papagiannopouloset al. (2004)

9 Longan fruit cultivars Corilagin CH3OH:H2O (70:30) LiChrospher RP-18; 25 C; CH3COOH:H2O(0.4:99.6), CH3OH; 20 min & PhenomenexLuna C18; THF:TFA:H2O (2:0.1:98),CH3CN, 45 min

LC-DAD and LC-DAD-MS/MS

Rangkadilok et al.(2005)

Myrthle berries Gallotannins and ellagitannins CH3CH2OH:H2O (70:30) Phenomenex luna C-18; 25 C;CH3COOH:H2O (0.1:99.9), CH3CN; 25 min

LC-DAD-MS Romani et al.(2006)

Oak aged wine Vescalagin, castalagin, acutissimin, epiacutissimin A,epiacutissimin B, ethylvescalagin

Amberlite XAD7 HP resin,TSK HW 40F gelchromatography(fractionation)

CH3COOH:H2O (1:99), CH3COOH:CH3OH(1:99); 50 min

LC-DAD-MS/MS Saucier et al.(2006)

Strawberry Gallotannins and ellagitannins HCl:CH3OH (0.1:99.9) and(CH3)2CO:H20 (70:30)

Symmetry C-18; CH3COOH:H2O (1:99),CH3CN; 70 min

LC-DAD-MS/MS Seeram et al.(2006)

White strawberry Three bis-HHDP-glucose, tetragalloyl glucose,potentilin/casaurictin

HCOOH:CH3OH (1:99) C-18 Luna; 25 C, CH3COOH:H2O (1:99),CH3CN; 50 min

LC-DAD-MS/MS Simirgiotis andSchmeda-Hirschmann(2010)

Longan seeds Gallotannins and ellagitannins CH3CH2OH:H2O (50:50);70 C.

Shim-Pack VP-ODS; HCOOH:H2O (3:97),CH3OH, 50 min & Shephadex LH-20;CH3OH

LC-DAD-MS/MS Soong and Barlow(2005)

Rubus berry Sanguiin H-10, sanguiin H-6, lambertianin A, B, C & Dand other ellagitannins

(CH3)2CO:H2O (70:30) Purospher Star; 40 C; CH3COOH:H2O(1:99), CH3CN, 47 min

LC-DAD-MS Vrhovsek et al.(2006)

Pomegranate basesuplements

Punicalagins A and B, punicalin DMSO Agilemt Zorbax SB C-18, H3PO4:H2O(0.4:99.6), CH3CN; 30 min

LC-DAD Zhang et al. (2009)

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1714 P. Arapitsas / Food Chemistry 135 (2012) 170817174.3. Chromatography separation

The analytical separation of HTs is another issue which has notbeen studied in depth. Due to the physicochemical properties ofthese analytes (e.g. high molecular weight and high polarity), sep-aration techniques such as gas chromatography, electrophoresiscapillary and superfluid chromatography cannot be applied, ortheir results are not yet adequate. Gas Chromatography is usedonly after acidic hydrolysis, so HT identification is not possible,allowing only consideration of the total amounts (Owen et al.,2003). Liquid chromatography appears to be the only method usedboth in reversed and normal phase mode (Table 1). The most com-mon technique used in HT food analysis is RP C-18 chromatogra-phy, with acidic methanol or acetonitrile used as the strongsolvent eluent and acidic water as the weak eluent (Table 1). Foracidification, 0.053% formic or acetic acid are usually used (Ta-ble 1). Acetic acid is principally used due to its higher volatilityand its lower ion suppression in LC/MS, while formic acid is mainlyused because of its lower pK as compared to acetic acid, which alsohelps HT solubility in water. Of course a higher percentage of acid(up to 5%) was also used when measurement of anthocyanins wasalso required within the same chromatographic run (Buendia et al.,2010). For LC-DAD analysis, non-volatile acids such as phosphoricacid or TFA are often used as eluents.

Alternatively, Soong and Barlow (2005) used a Shephadex LH-20 column, Borges et al. a C-6 phenyl column, and Furuuchi, Yokoy-ama, Watanabe, and Hirayama (2011) a Diol column for their LC-DAD, LC-DAD-MS/MS and LC-fluorescence analyses. The chromato-graphic runs varied from 20 min to 80 min, depending mainly fromthe column diameter, and 40 C was the maximum temperatureapplied also for UPLC systems (Table 1).

4.4. Detection Identification

The lack of a detector with high specificity for HTs has led to theuse of different techniques and the combining of data for identifi-cation of HTs.

DAD detectors provide important information for the identifica-tion of HTs and are widely used for routine identification andquantification analysis (Aaby, Ekeberg et al., 2007; Aaby, Wrolstadet al., 2007; Arapitsas et al., 2007; Engels et al., 2010; Fischer, Carle,& Kammerer, 2011; Furuuchi et al., 2011; Harborne & Williams,2000; Koponen et al., 2007; Mueller-Harvey, 2001; Mullen et al.,2002; Mtt-Riihinen, Kamal-Eldin, & Trrnen, 2004; Okudaet al., 1989; Romani et al., 2004). As shown in Fig. 2, gallotannins(simple glucogalloyl esters i.e. pentagalloylglucose (Fig. 2c)) havea characteristic UVVis spectra with a maximum at 280 nm. Allglucogalloyl derivatives have a very similar UVVis spectra, witha bathochromic shift of 1012 nm when compared to gallic acidspectrum (a). Ellagitannins also have a characteristic UV spectra(i.e. Fig. 2df) with maximum absorbance bellow 270 nm. Whenonly HHDP moieties are present in the molecule, the UVVis spec-trum of Fig. 2d is the most probable case. When the ellagitanninalso has simple galloyl esters the UVVis spectrum of Fig. 2f ismore likely, and finally when ellagic acid is part of the moleculestructure (gallagyl-glucosides) the Fig. 2i UV spectrum is morelikely (Arapitsas et al., 2007; Mtt-Riihinen et al., 2004). C-glyco-sidic ellagitannins have UVVis spectra with a maximum of around245 nm (Fernandes, Sousa, Mateus, Cabral, & de Freitas, 2011). Forcompounds such as 2,3-(HHDP)-glucose, without a simple glucog-alloyl ester, DAD have very low selectivity (Fig. 2d). Gallotanninswith m-depsidic links have spectra similar to the (b) UVVis spec-tra of Fig. 2b (Arapitsas et al., 2007).

Mass spectrometry detectors also offer valuable information forthe identification of HTs, the negative ion mode of electrosprayionisation generally being employed (Arapitsas et al., 2007; Buen-dia et al., 2010; Fischer et al., 2011; Franceschi, Vrhovsek, & Guella,2011; Garca-Estvez et al., 2010; Gasperotti, Masuero, Vrhovsek,Guella, & Mattivi, 2010; Harborne & Williams, 2000; Hatanoet al., 1988; Kool, Comeskey, Cooney, & McGhie, 2010; Mueller-Harvey, 2001; Mullen, Yokota, Lean, & Crozier, 2003; Mtt, Ka-mal-Eldin, & Trrnen, 2003; Mtt-Riihinen et al., 2004; Reed,Krueger, & Vestling, 2005; Soong & Barlow, 2005). When positiveion mode is used, the metal adducts of Na and K are usually iden-tified (Franceschi et al., 2011; Furuuchi et al., 2011). Almost all MSdetectors have been used for the identification of HTs, from singleand triple quadruple, to ion trap and MALDI-TOF-TOF MS (Table 1).In addition to the deprotonated molecule [M-H], ellagitannins of-ten also provide [M2H]2 or [2MH] ions, depending on themass of the compound. Typical losses during fragmentation aregalloyl (152 amu), HHDP (302 amu), galloylglucose (332 amu),HHDP-glucose (482 amu), and galloyl-HHDP-glucose (634 amu).When the m/z 169 ion is present in the fragmentation pattern thisindicates the presence of a simple galloyl ester in the molecule ofHTs, and the m/z 301 ion indicates the presence of a HHDP moiety(Arapitsas et al., 2007; Mtt-Riihinen et al., 2004; Seeram, Lee,Scheuller, & Heber, 2006). The diagnostic fragment ion for chebulicellagitannin is the m/z 337 [chebulic H H2O], which furtherbreaks into m/z 319 [chebulic H2H2O], 293 [chebulic HH2OCO2] and 275 [chebulic H2H2OCO2] or the m/z 351[methyl neochebuloyl HH2O] (Pfundstein et al., 2010). Ellagit-annins of the gallagyl-glucoside group have m/z 601 as the charac-teristic fragment ion, indicating the loss of a gallagic acid group(Fischer et al., 2011; Pfundstein et al., 2010). Both quercetin (themost common flavonoid in plants) and ellagitannins with HHDPmoiety produce identical molecular ions on fragmentation (m/z301), but when tandem MS is used, quercetin m/z 301 further frag-ments to form characteristic m/z 179 and 151 ions whereas theequivalent EA m/z 301 ion yields ions at m/z 257 and 229 (Mullenet al., 2003; Seeram et al., 2006). The simultaneous tandem use ofDAD and ESI-MS is the most common technique applied for theidentification of HTs. For the analysis of high MWHTs, the soft ion-isation of the MALDI technique is more suitable than ESI, as it doesnot form multiply charged ions and gives high-resolution results.In contrast to ESI, MALDI frequently provides better detection inpositive ionisation, with ellagitannins being typically detected asadducts. Using MALDI-TOF MS, Afag et al. identified up to penta-meric forms of ellagitanins in pomegranates (Afag, Seleem, Krue-ger, Reed, & Mukhtar, 2005); Kool et al. published the MALDI-TOF MS spectra of sanguiin H-2, lambertianin C, sanguiin H-10(Kool et al., 2010); and Franceschi et al. used Ion Mobility Separa-tion with a MALDI-TOF MS instrument and identified four chargedmolecular species (single, double, triple and quadruple) of sanguiinH-6 (Franceschi et al., 2011).

Aaby, Ekeberg et al., 2007; Aaby, Wrolstad et al., 2007 used acoulometric array for the analysis of HTs, a method based on thecharacteristics of phenols with adjacent OH groups (e.g. catecholand pyrogallol) to stabilise the phenoxy radical and lead to loweroxidation potential. So ellagitannins which have several pyrogallolgroups are oxidised at the lowest oxidation potential.

NMR spectroscopy is a powerful technique for the structuralidentification of HTs, and other substances. The 1D (1H- and 13C-NMR), 2D (cosy, roesy) and 2D etero-correlated (HSQC and HMBC)spectra of more than 1000 HTs have been studied in the literature,greatly facilitating structural analysis of new compounds. Never-theless, most of them have been isolated from medicinal plantsand only a few papers have reported the NMR spectra of HTs iso-lated from food products. These papers are limited to a few ellag-itanins isolated from raspberries (sanguiins and lambertianins),pomegranates (gallagyl-glucosides), aged wine and whisky (flava-no-ellagittaninis and C-glucosides of ellagitannins) and carob fibre(depsidic gallotannins) (Arapitsas et al., 2007; Franceschi et al.,

nm250 300 350 400 450 500 550 nm250 300 350 400 450 500 550

nm300 350 400 450 500 550nm250 300 350 400 450 500 550

nm250 300 350 400 450 500

nm250 300 350 400 450 500 550 nm300 350 400 450 500 550

nm250 300 350 400 450 500 550

gallic acid

270 nm

penta(digalloyl)-glucose

270 nm

pentagalloyl-glucose

280 nm

pedunculagin II

sanguiin H-6

oenothein B

275 nm

ellagic acid

254 nm

punicalagin

300 nm

360 nm

254 nm 374 nm

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(i)

Fig. 2. UV Spectra of gallic acid, ellagic acid and different gallotannins and ellagitannins.

P. Arapitsas / Food Chemistry 135 (2012) 17081717 17152011; Fujieda et al., 2008; Gasperotti et al., 2010; He, Shi, Yao, Luo,& Ma, 2001; He et al., 2007; Ito, Iguchi, & Hatano, 2008; Kanaujiaet al., 2010; Kool et al., 2010; Owen et al., 2003; Quideau & Feld-man, 1996). An interesting diagnostic characteristic for the 1H-NMR spectrum, demonstrated by Arapitsas et al. (2007), is thataromatic protons de-shield in the presence of the m-depsidic link(7.197.39 ppm) and shield in the presence of the CC link of theHDDP group (6.586.66 ppm), as compared to the simple glucogal-loyl esters aromatic protons (6.997.04 ppm). As shown in Table 1the most common deuterared solvents used for the registration ofNMR spectra are methanol and acetone, and more rarely DMSO.The temperature and concentration of HTs with high MW, whichtend to aggregate, are also important for clarifying structure(Franceschi et al., 2011; Fujieda et al., 2008; Okuda et al., 1989). Re-cently, Jourdes et al. (2011) produced an interesting review con-taining critical information about signal recognition for HTs NMRspectra.

Chemical degradation, such as complete hydrolysis with acid,partial hydrolysis with hot water or enzyme, or thiol degradation,are often applied for structural elucidation of HTs (Mueller-Harvey,2001; Okuda et al., 1989; Owen et al., 2003; Vrhovsek et al., 2006).

4.5. Quantification

The absence of commercial standards has obliged scientists todevelop and use conventional methods both for total and individ-ual quantitative determination of HTs.

Methods for total determination of HTs include Owades sugges-tion for the use of absorbance at 270 nm in 1958 and the FolinCio-calteau assay of 1927. However both methods measured totalphenols and are not specific for HTs. The KIO3-reagent assay de-scribed by Bate-Smith in 1977 for the quantification of gallotan-nins and ellagitannins is not suitable for complex mixtures oftannins and is not particularly effective, since it depends criticallyon the temperature and duration of the analysis (Harborne & Wil-liams, 2000; Hartzfeld, Forkner, Hunter, & Hagerman, 2002; Muel-ler-Harvey, 2001; Okuda et al., 1989). A flow injection system usingthe KIO3-reagent has also been reported. This is a rhodanine-re-agent assay developed by Inoue and Hagerman in 1988, whichmeasures only gallic acid and not galloyl esters, ellagic acid orellagitannins, while it requires the absence of oxygen (Falco &Arajo, 2011). The NaNO2-reagent method reported by Wilsonand Hagerman in 1990 is only selective for ellagic acid and is alsosensitive to oxygen (Wilson, 1934). Other assays based on theacidic hydrolysis of ellagitannins are also described with the useof different acids, such as anhydrous methanolic HCl or trifluoro-acetic acid, but none of these procedures represent an optimalsolution, as they are time-consuming, require the use of hazardoussolvents or have low sensitivity and selectivity (Harborne & Wil-liams, 2000; Hartzfeld et al., 2002; Mueller-Harvey, 2001; Okudaet al., 1989).

As far as HPLC/DAD quantification analysis is concerned, single,group or total HTs are frequently conventionally measured as gallicacid, ellagic acid or a similar ellagitannin, without evaluating theselectivity of the method (Borges et al., 2010a; Fischer et al.,2011; Michel et al., 2011; Mueller-Harvey, 2001; Mtt-Riihinenet al., 2004; Okuda et al., 1989; Pfundstein et al., 2010; Sandhu &Gu, 2010). Groups working both on isolation/characterisation andquantification analysis have used the isolated compounds as stan-dards for quantitative determination using HPLC/DAD or HPLC/MStechniques, although this is an exception (Fischer et al., 2011;Gasperotti et al., 2010; Michel et al., 2011; Rangkadilok, Worasut-tayangkurn, Bennett, & Satayavivad, 2005; Saucier, Jourdes,Glories, & Quideau, 2006; Vrhovsek et al., 2006).

5. Concluding remarks and future aspects

Since the very birth of chemistry, there has always been consid-erable interest in HTs on the part of scientists, consumers and foodmanufacturers. On the one hand the beneficial effects of HTs onhealth and food quality, and on the other their structural variety,complexity and widespread distribution have represented themain characteristics underlying this continuing interest. The mainproblems, causing serious misunderstanding, are the lack of

1716 P. Arapitsas / Food Chemistry 135 (2012) 17081717commercially available standards and selective methods for theanalysis of HTs. As knowledge and technological achievementshave improved, more and more researchers from different back-grounds have directed their attention towards the implementationof better extraction, purification and detection methodologies forHTs. The establishing of new HT analytical tools for productauthentication and the discovery of sophisticated adulteration willalways be a major field of interest. Furthermore there is still a gen-eral need for analytical techniques able to assist with establishingnew, fast, accurate, effective and environmentally friendly meth-ods for the identification and quantification of HTs.References

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Hydrolyzable tannin analysis in food1 Historical background introduction2 Classification3 Importance of food HTs4 Analytical chemistry4.1 Extraction4.2 Isolation4.3 Chromatography separation4.4 Detection Identification4.5 Quantification

5 Concluding remarks and future aspectsReferences