Food Chemistry 135 (2012) 17081717

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Review

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

Article history:
Received 1 February 2012
Accepted 24 May 2012
Available online 1 June 2012
Keywords:
Tannins
Food
Gallotannins
Ellagitannins
Analytical chemistry

a b s t r a c t
The discovery of plant polyphenols in food is perhaps one of the biggest breakthroughs in modern food
science. Plant polyphenols are known for their role in food quality and safety, since they contribute signicantly to taste, avour, colour, stability etc., while they are increasingly recognised as important factors in long-term health, contributing towards reducing the risk of chronic disease. Almost 200 years ago,
hydrolyzable tannins (HTs) were the rst group of plant polyphenols subjected to analytical chemical
research. Despite the lack of commercially available standards, food analysis research offers a wealth
of papers dealing with extraction optimisation, identication and quantication of HTs. The object of this
review is to summarise analytical chemistry applications and the tools currently used for the analysis of
HTs in food.
2012 Elsevier Ltd. All rights reserved.

Contents
1.
2.
3.
4.

5.

Historical background introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Classification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Importance of food HTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Analytical chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Chromatography separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Detection Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.
Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks and future aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Historical background introduction

With the start of the modern scientic revolution and the development of chemistry from alchemy, plant polyphenols became one
of the rst subjects of scientic research. Many of the fathers of
chemistry worked in this eld and provided the rst results in relation to the quantitative and qualitative analysis of HTs (Fischer,
1914; Haslam, 1989; Russell, 1935).
Around 200 years ago, in the leather industry chemists were
asked to improve leather processing techniques, due to the demand for greater quantities of leather and better quality. In this
era, traditional techniques were the result of the application of
Tel.: +39 0461 615 564; fax: +39 0461 615 200.
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com
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.05.096

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empirical methods as well as reasoning, and most products were

tanned with an infusion of oak bark for a period of three to six
months. Probably for this reason, the rst term used for these
products, which is still adopted today, was vegetable tannins (tann
in Celtic means oak tree). Over the centuries and around the world,
other raw materials in addition to oak bark, all from the plant kingdom, were also used to tan hides, such as myrtle leaf extract in
17th century Rome. The quality of the nal product was not always
similar, even when exactly the same method was used. The rst
problem was nding the most efcient source of vegetable tannin,
while the second step was standardisation. Since the most wellknown and effective tanning material was oak bark extract, called
tannic acid, numerous chemists tried to analyse it, discover its composition and develop quantitative analytical methods, in order to
produce a standard raw material (Darton, 1882a, 1882b; Fischer,
1914; Rau, 1887; Russell, 1935; Wilson, 1934).

1709

P. Arapitsas / Food Chemistry 135 (2012) 17081717

HO

HO

HO

OH

HO
HO

OH
OH

OH

OH

HO

G=

O
O

HO

HO

OH

HO
HO

OH

HO
G

OH

glucogallin

O
G

O
O
G

sanguiin H-6

OH

OH

OH

HO

HO
HO

G
O

HO
O

OH

OH

HO

HO

penta-(digalloyl)-glucose

HO

HO

HO

HO
OH

O
O

O
O

OH

HO

OH

HO

HO

OH
HO

HO

HO
O

G
O
G

OH

oenothein B

HO

OH

OH

HO

OH

G O
G O
O
G G
O O
O O
O
G
G
O
G
G

HO

m-depsidic
link

HO

OH
O

HO

OH
O

OO
G
O

HO
O

O
G

OH

O
O

OH

penta-galloyl-glucose

O
O
G

OH

O
O

G O
O

O
G

OH

O
OH

G O
G
O punicalagin A

O
O
G

O
OH
G

pedunculagin II

HO
Fig. 1. Structures of Hydrolyzable Tannins.

Carl Wilhelm Scheele, a German-Swedish pharmaceutical

chemist born in 1742, is famous for discovering many chemical
substances, such as oxygen and chlorine. Scheele also isolated
and characterised various phenolic natural compounds, including
gallic acid from tannic acid (Fischer, 1914; Russell, 1935). The term
tannin was probably introduced by A. Seguin around the end of the
17th century to describe the extractable matter used to convert
hide to leather, while he also demonstrated the ability of tannins
to precipitate gelatin from solution (Haslam, 1989). In the middle
of the 18th century, Adolph Strecker seems to have been the rst
to point out that tannic acid yields gallic acid and glucose following
hydrolysis and that it has the formula C27H22O17, corresponding to
a combination of 3 moles of gallic acid and one mol of glucose
(Fischer, 1914). Probably due to this observation, today the chemical dictionary The Merck Index wrongly cites the term tannic acid as
a tri-galloyl-glucose derivative (The Merk Index, 2001). R.J. Manning isolated a pentaethyl ester of pentagalloyl glucoside from tannic
acid. K. Feist arrived at the view that tannic acid from Turkish nutgalls was a combination of glucogallic acid with two ester-like bound
molecules of gallic acid (Fischer, 1914).

The German chemist Hermann Emil Fisher (18521919), Nobel

laureate in 1902, famous for his studies on sugars and purines, also
devoted his genius to the analysis and synthesis of active principles
from the plant kingdom. More than 100 years ago, Fischer, together
with Karl Jonnan Freudenberg, carried out the most complete studies in relation to tannic acid identication. They called the esterlike anhydride compounds between gallic acids, such as Ugo
Schiffs digallic acid, depsides. The term derives from the Greek
de wim (to tan), because many of these substances resemble tannins. Initially, they suggested that tannic acid is the penta-(digalloy)-glucose (Fig. 1), whereas ultimately they were the rst to
suggest the possibility that tannic acid could be a mixture of very
similar substances, for example a penta-(digalloyl)-glucose with a
tetra-(digalloyl)-glucose or tri-(digalloyl)-di-(galloyl)-glucose, etc.
(Fischer, 1914).
At the same time that European chemists were focusing on the
identication of vegetable tannins, on the other side of the Atlantic,
in the United States, several scientists were turning their attention
to the development of a method for quantication and standardisation. Nelson H. Darton, Loewenthal, Hammels, Allen and Carpenk

1710

P. Arapitsas / Food Chemistry 135 (2012) 17081717

are some of the scientists who tried to create methods to quantify

tanning materials. Most of these methods aimed to facilitate the
determination of vegetable tannins and yielded very discordant results when compared to the actual weight of the leather formed by
them. In the end, tanners lost all faith in organic chemists generally
and started to use inorganic chemical products for tanning (Darton,
1882a, 1882b; Russell, 1935).
According to A.G. Perkin and A.E. Everests polyphenol classication in 1915, tannins were divided into three groups: depsides,
diphenyldimethylodil tannins and phlobatannins. Freudenberg
preferred to call the rst two groups hydrolyzable tannins. E.
Fischer and Stracker called the rst group gallotannins, since their
hydrolysis yields gallic acid. Nierenstein was probably the rst to
call the second group ellagitannins, since their hydrolysis yields ellagic acid. The third group is the class of compound now known as
avonoids (Fischer, 1914; Russell, 1935).

2. Classication
In the last few decades, the secondary metabolites widespread
throughout the plant kingdom and characterised by water solubility and molecular masses between 500 and 5000 Dalton have been
called HTs; they give the usual phenolic reaction and precipitate
alkaloids and proteins. As regards their chemical structure, HTs
are multiple esters of gallic acid with glucose and products of their
oxidative reactions. Consequently, they are clearly distinguished
from other tannins such as condensed tannins (syn. proanthocyanidins) which are derivatives of catechin and tara tannins, esters
of 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 rst intermediate and a
key-metabolite in the biosynthetic pathway of tannins. A specic
glucosyltranferase catalyses the esterication between gallic acid
and UDP-glucose to give glucogallin (Fig. 1). In a second step,
glucogallin plays a dual role, functioning as an acyl acceptor and
acyl donor, in order to form di-, tri-galloylglucoses, etc. This rst
biosynthetic pathway is completed by the formation of pentagalloyl-glucose (Fig. 1), and the products are simple galloylglucose
derivatives. In the second biosynthetic route, the galloylation of
pentagalloyl-glucose continues to hexa-, hepta-, octa-, etc. galloylglucose derivatives (Fig. 1). These compounds are called gallotannins or depsidic metabolites, and the esteric-link between two
galloy moieties is known as the m-depsidic link (Fig. 1), according
to Fischer. The third pathway yields ellagitannins, which are typical constituents of many plant families, in contrast to gallotannins,
which are not so widespread in nature. Ellagitannins are the products of oxidation, leading to C-C linkages between suitably orientated galloyl residues of glucogalloyl molecules that form
hexahydroxydiphenoyl (HHDP) units (Buzzini et al., 2008; Gross,
2008; Okuda & Ito, 2011; Okuda, Yoshida, & Hatano, 1989a; Quideau & Feldman, 1996). The oak C-glycosidic ellagitanins (castalagin, vescalagin) and their avano-ellagitannin derivatives
(acutissimin B and epiacutissimin B), detected in aged wine and
whisky, and gallagyl-glucosides, whose structure has an ellagic
acid moiety (punicalagin, peduncalagin), also belong to the same
group. Okuda et al. have proposed two more biosynthetic transformations, which deliver HTs with the dehydrohexahydroxydiphenoyl (DHHDP) group and the transformed DHHDP group (Okuda
& Ito, 2011).
While the distribution of gallotannins is rather limited in nature, ellagitannins are widespread in many plant families (Gross,
2008; Okuda & Ito, 2011). In total more than 1000 HTs have been
identied, from the simple glucogallin (MW 332) to pentameric
ellagitannins with MWs over 5000 Dalton. However, to date there

is not even one commercially available standard, making the world

of analytical chemistry more difcult and of course more
fascinating.
3. Importance of food HTs
From the discovery of HTs up to the present day, interest in HTs
in various scientic and commercial areas has increased constantly. In the food sector, HTs affect food quality in different ways.
The most well-known and researched property of these compounds, which has major importance in food quality, is their
astringency. Salivary proline-rich proteins form complexes with
dietary HTs and precipitate. The result of this action is an astringent sensation, which is perceived as a diffuse feeling of extreme
dryness and roughness and is not conned to a particular region
of the mouth or tongue (Glabasnia & Hofmann, 2006; Haslam,
1989, 1996; Quideau & Feldman, 1996).
Numerous scientic studies and widespread epidemiological
evidence has correlated fruit and vegetable consumption with a
lower risk of cancer, degenerative and cardiovascular diseases
(Halliwell, 2007; Harborne & Williams, 2000; Haslam, 1989; Haslam, 1996; Havsteen, 2002; Landete, 2011; Larrosa, Garca-Conesa, Espn, & Toms-Barbern, 2010; Le Marchand, 2002; Steinmetz
& Potter, 1991, 1996). Food components playing an important role
in this context include plant polyphenols such as HTs. However,
the metabolism of plant polyphenols in the human organism and
mechanisms relating to how they work and their bioavailability
are still not clear. The mechanism which has been most closely
studied in order to explore the biological activities of HTs relates
to their antioxidant properties (Borges, Mullen, & Crozier, 2010;
Cai, Luo, Sun, & Corke, 2004; Cai, Sun, Xing, Luo, & Corke, 2006; Haslam, 1996; Labieniec, & Gabryelak, 2007; Larrosa et al., 2010; Okuda & 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 have
shown strong biological activity in the form of anti-tumour, antimutagenic, anti-diabetic, anti-proliferative, anti-bacteric and
anti-mycotic properties (Buzzini et al., 2008; Formentini et al.,
2008; Halberstein, 2005; Halliwell, 2007; Haslam et al., 1989; Haslam, 1996; Havsteen, 2002; Landete, 2011; Larrosa et al., 2010;
Okuda et al., 1989; Romani et al., 2005; Salminen, Karonen, & Sinkkonen, 2011; Steinmetz & Potter, 1991, 1996). Unfortunately, due
to the lack of a commercial standard, there is a lack of HT structureactivity relationship studies in enzymatic and cell lines assays
in the literature.
Ellagitannins and gallotannins may also affect the life of foodstuff due to their antioxidant properties and/or antimicrobial activity (Cao, Soc, & 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 Rosidae
but only in a small number of Dilleniidae and Hamamelidae plants
(baby berries etc.), while they are absent in Caryophyllales and
Magnolidae. Rosales, in the Rosidae order, mostly produce simple
glucogalloyl and HHDP tannins (raspberries, blackberries, cloudberries, arctic raspberries, strawberries, etc.). Other orders of Rosidae rich in HTs are Fragales (oak, carob, nuts, tonoak, etc.),
Brassicales (capparis), Myrtales (myrtle, pomegranate, etc.) and
Sapindales (Chinese olives, mango, etc.). HTs have also been detected in the Vitaceae family (muscadine grapes), in processed food
products such as aged wines and spirits or food to which HTs have
been added, such as meat, sh or wine, in order to take in advance
their antioxidant activity. Tannic acid is indeed a food avouring
according to European Union regulation (EC) No. 2232/96. The to-

P. Arapitsas / Food Chemistry 135 (2012) 17081717

tal concentration of HTs in food varies from 1 mg/kg (in chestnuts)

to 2 g/kg (in blackberries), with berries representing the richest
dietary source (Larrosa et al., 2010).
4. Analytical chemistry
While biochemistry has revealed many valuable properties of
HTs and a high level of knowledge has been reached as regards
their organic synthesis, analytical chemistry on the other hand still
has many elementary problems to resolve, apart from isolation and
NMR characterisation. Extraction processes have still not been fully
optimised, separation methods are not suitably efcient and identication patterns are not particularly clear. While HTs are more
stable during food processing and storage as compared to other
polyphenolic compounds, foods and supplements rich in HTs lack
authentication (Aaby, Wrolstad, Ekeberg, & Skrede, 2007; Hager
et al., 2010; Kim, Lounds-Singleton, & Talcott, 2009; Koponen
et al., 2007; Madrigal-Carballo, Rodriguez, Krueger, Dreher, & Reed,
2009; Mullen et al., 2002; Zhang et al., 2009). Knowledge about
condensed tannins, which have a similar complexity, is instead
more advanced. These problems are probably due to the lack of
commercially available standard, which leads to further misunderstanding between scientists. There are still scientists who consider
tannic acid to be a single compound, principally because large
chemical industries are selling tannic acid as pentagalloyl glucose.
The high molecular weight, the large number found in nature and
the considerable structural complexity of HTs represent other
complicating factors.
4.1. Extraction
Up to few years ago most of the information about HTs extraction derived from the isolation and NMR identication studies.
Aqueous solutions of methanol, ethanol or acetone, as well as ethyl
acetate have been used to extract HTs from natural tissues (Arapitsas, Menichetti, Vincieri, & Romani, 2007; Garca-Estvez, Escribano-Bailn, Rivas-Gonzalo, & Alcalde-Eon, 2010; Gironi &
Piemonte, 2011; Harborne & Williams, 2000; Hatano, Yoshida, &
Okuda, 1988; Markom, Hasan, Daud, Singh, & Jahim, 2007; Mueller-Harvey, 2001; Okuda & Ito, 2011; Okuda, Yoshida, & Hatano,
1989a; Quideau & Feldman, 1996). Non-polar organic solvents
(i.e. n-hexane, petroleum ether), chloroform and dichloromethane
have a low extraction strength for HTs and are usually used in sample treatment to remove lipids and chlorophyll and/or to prevent
enzymatic reactions (Mueller-Harvey, 2001; Okuda et al., 1989).
Extraction methods with the use of small percentage of ascorbic
acid or under Argon atmosphere have been used for the protection
of analytes by oxidation (Aaby, Ekeberg, & Skrede, 2007; Glabasnia
& Hofmann, 2006; Okuda et al., 1989). Methanol tends to be better
for low molecular weight tannins or in the case of matrices containing large amounts of enzymes (i.e. bark or fruit), while acetone
is preferred for high molecular weight tannins and because it is less
liable to react with them (Mueller-Harvey, 2001; Okuda et al.,
1989). High temperatures for extended times may cause hydrolysis
of the galloyl moiety attached to the glucose anomeric C-1 position
and can also release ellagic acid from ellagitannins (Okuda et al.,
1989). Extraction with ethanol and/or methanol may produce ethyl
or methyl ester of gallic acid, respectively (Arapitsas et al., 2007;
Mueller-Harvey, 2001; Okuda et al., 1989b). Unfortunately, the literature provides little information on extraction techniques comparing HTs, in food products so most of the information in this
eld comes from HTs from non-edible products. Soxhlet apparatus
has been used with very good results for the extraction of HTs from
carob bres and traditional medicinal herbs, with an initial cleanup step with an unpolar solvent (haxene, petroleum ether or chloroform) applied to remove lipids. Tian, Li, Ji, Zhang, and Luo (2009)

1711

showed that for the soxhlet technique, ethyl acetate was better for
extraction of high MW gallotannins, water for small gallatannins
(with 14 galloyl moieties), while ethanol provided medium results but was able to extract all gallotannins tested. Markom
et al. (2007) published a study showing that corilagin was best extracted from the herbal plant P. niruri using aqueous ethanol, while
pressurized water extraction (PWE) gave a higher yield in a shorter
time as compared to Soxhlet and supercritical uid extraction
(SFE). In the same study, as far as the choice of solvent was concerned, aqueous ethanol gave better results when a Soxhlet extractor was used, but aqueous acetone was the best solvent mixture for
liquidsolid extraction. PWE also gave better results according to a
study by Papagiannopoulos, Wallseifen, Mellenthin, Haber, and Galensa (2004), but the best solvent was a wateracetone mixture. On
the other hand, the results published by Cam et al. based on the total polyphenolic amount in pomegranate peel (HTs are the predominant polyphenolic metabolites according to the paper),
showed no signicant difference between PWE and simple liquidsolid extraction (am & HIsIl, 2010). Moreover, extractability
of HTs may depend on the seasonal maturity of tissues and on the
type of plants or their structural physicochemical properties (Okuda et al., 1989).
Consequently, according to the literature, there is no optimal
extraction procedure which can be universally employed for all
HTs and all types of samples. However, as shown in Table 1, simple
and time-consuming techniques are mainly used, acetone being
more frequently used as the extraction solvent, with acidic methanol as the second choice. Final PWE would seem to be a very
promising technique, which could also give very effective, fast
and environmentally friendly results in the eld of HTs.

4.2. Isolation
There are plenty of studies regarding the isolation of HTs. As
most HTs are strongly absorbed on Silica gel, this is not a suitable
material for normal phase preparative chromatographic separation, although it has been used for analytical HPLC when developed
with an acid-containing eluent (Mueller-Harvey, 2001; Okuda
et al., 1989b). The separation of tannins on columns of hydroxypropylated dextran gel (i.e. Sephadex LH-20), is induced by the different adsorption of each compound on the gel, rather by gel
ltration, and tannins of high molecular weight are often not
recovered from the column. Usually tannins are initially selectively
absorbed on Sephadex LH-20, then equilibrated with alcohols
(methanol and ethanol) and nally 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 gels
such 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 lately
for HT isolation, adopted for the typical stationary phase for analytical columns (Supercosil, Phenomenex acqua, Chromatonex ODS
Descovery HS, ODS-C18 silica gel, Purospher, Hichrom, Synergy
Hydro RP, Latex C-18, etc.). For the fractionation of extracts rich
in gallotannin, Engels, Ganzle, and Schieber (2009) optimised and
used a High Speed Counter-Current Chromatography (HSCCC)
column, with a preparative run solvent system of hexane/ethyl
acetate/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 of
ascorbic acid are added to the mobile phase for the prevention of
oxidation, but the main drawback of this practice is the presence
of the antioxidant in all the eluted analytes (Mueller-Harvey,
2001; Okuda et al., 1989b).

1712

Table 1
HTs analysis in Food and Beverages.
Sample

HTs detected

Extraction

Strawberry

Ellagitannins

(CH3)2CO

Muscadine grape

CH3COOH:(CH3)2CO:H2O
(0.3:70:29.7)
Rasberry, tannic acid
Sanguiin H-6, lambertianin C, gallotannins of tannin CH3CH2OH:H2O (70:30)
acid
14 Raspberry cultivars Lambertianin C, sanguiin H-6
CH3COOH:CH3OH:H2O
(0.1:62.5:37.4)
CH3OH
36 Euripean fruit juice Punicalagin-like, punicalagin, A, punicalagin B,
punicalin A, punicalin B, Granatin A, Granatin B,
containing
punicalagin isomer
pomegranate
15 Strawberry cultivars Sanguiin H-6, lambertianin C, galloyl-bis-HHDPCH3COOH:(CH3)2CO:H2O
(0.5:70:29.5); sonication
glucose
Mango kernel
From tetra- to dodeca-galloyl glucose
(CH3)2CO:H2O (80:20)

Whisky

Boysenberry seeds
and juice

Gallotannins and ellagitannins

HCl:CH3OH:H2O
Pedunculagin I, pedunculagin II, casuarinin,
lagerstannin B, granatin B, castalagin derivative,
(80:19.9:0.1)
lagerstannin B derivative, digalloyl glucose,
monogalloyl glucose, galloyl-HHDP-glucose,
lagerstannin C, punigluconin and others ellagitannins
and gallotannins
Whisky tannin A, whisky tannin B, gallic acid, ellagic
acid, castalin, 6-galloyl glucose, 3-galloyl glucose,
2,3-digalloyl glucose, 2,3-HHDP glucose, castacrenin
B, castalagin and other ellagitannins
CH3CH2OH:H2O (80:20)
Peducilagin, peduculagin isomer, sanguiin H-6,
sanguiin H-10, lambertianin A, bis-HHDP-glucose,
sanguiin H.2, lambertianin C,

Oak-aged wine

Grandinin, vescalagin, castalagin

11 Rubus berries
cultivars

Three sanguiin H-10 isomers, sanguiin H-2, sanguiin (CH3)2CO:H2O (70:30).

Anthocyanins clean up
H-6, lambertianin C and other ellagitannins
with shephadex LH-20
From tri- to deca- galloyl glucose
(CH3)2CO:H2O (70:30);
liquidliquid extraction
with CH3COOCH3
CH3COOH:(CH3)2CO:H2O
Two isomers of pedunculagin, two isomers of
castalagin/vescalagin, two isomers of lambertianin C, (0.5:70:29.5)
two isomers of sanguiin H-6/lambertianin A,
lambertianin D, galloylbisHHDPglucose
Gallic acid, methyl gallate, ethyl gallate, corilagin,
(CH3)2CO:H2O (70:30)
hyperin
CH3CH2OH:H2O (70:30)
Glansreginin A, glansreginin B, glansrin D, 1,2,3,6
tetragalloyl glucose, 1,2,4,6 tetragalloyl glucose,
pentagalloyl glucose, pterocarinin A, platycaryanin A,
euprostin A, rugosin F, 1-desgalloylrugosin F,
heterophylliin D
1,3,6 Trigalloyl-2-chebuloyl-glucose, 1,3,6 trigalloyl- CH3OH:H2O (50:50)
2-(4-methyl)-chebuloyl-glucose

Witch hazel

Blackberry products

Chinese olive
Walnuts

Large caper fruit

Shephadex LH-20,
Supercosil LC-18-DB
(isolation); HSCCC
(fractionation)
Phenomenex Aqua C18
(isolation)

LC parameters

Detection

Betasil C18; 25 C; CH3COOH:H2O (2:98),

CH3COOH:CH3CN:H2O (2:50:48); 74 min
Zorbax stablebond SB-C-18;
CH3COOH:H2O (0.5:99.5), CH3OH; 70 min
Phenomenex Luna C18; 27 C;
CH3COOH:H2O (0.1:99.9), CH3CN; 30 min
Synergi C-18; CH3COOH:H2O (0.5:99.5),
CH3COOH:CH3CN (0.5:99.5); 60 min
Gemini C6 phenyl; 40 C; CH3COOH:H2O
(0.1:99.9), CH3OH

LC-DAD-MSn

Reference

Aaby, Ekeberg, and

Skrede (2007a)
Amandeep and
LC-DAD-MS
Liwei (2010)
LC-DAD-MS; NMR (1H, Arapitsas et al.
13
C; CD3OD)
(2007)
HPLC-QTOF-MS
Beekwilder et al.
(2005)
LC-DAD-MS/MS
Borges et al.
(2010)
n

LiCrocart C18; CH3COOH:H2O (5:95),

LC-DAD & LC-MS/MS
CH3OH; 70 min
Supercosil LC-18-DB and Synergi Hydro- LC-MS/MS
RP; 21 oC; HCOOH:H2O (2:98),
HCOOH:CH3OH:H20 (0.5:49.5:50);
72 min
LC-DAD-MS
Synergi Hydro-RP; 30 C, HCOOH:H2O
(2:98), HCOOH:CH3OH:H2O (0.5:90:9.5);
71 min

Chromatorex ODS column,

MCl gel CHP20P column,
Sephadex LH-20, Toyopearl
HW40F column (isolation)
Amberlite XAD7 HP resin, RP L-column; 40 C; CH3COOH:H2O
Shephadex HL-20
(0.1:99.9), CH3COOH:CH3OH (0.1:99.9);
(fractionation)
65 min & Develosil 100 Diol-S; 35 C;
HCOOH:CH3CN (2:98),
HCOOH:CH3CN:H2O (2:95:3); 80 min.
Aqua C-18; 35 C; HCOOH:H2O (2.5:97.5),
SPE Water C-18 Sep-Pak
and Sephadex LH-20
(CH3)2CHOH, CH(CH3)2COOH; 65 min
(fractionation)
ENV + resin, Descovery HS Phenomenex Luna C18; 40 C;
C18 (isolation)
CH3COOH:H2O (1:99), CH3CN; 52 min

NMR (1H, 13C, COSY,

NOESY, HSQC, HMBC;
(CD3)2CO, CD3OD,
DMSO, (CD3)2CO-D2O)
LC-DAD-MS/MS; LCNP-Fluorescence;
Maldi-Tof-MS.

Buendia et al.
(2010)
Engles et al.
(2009), Engles
et al. (2010)
Fischer et al.
(2011)

Fujieda et al.
(2008)

Furuuchi et al.
(2011)

LC-DAD; MS Ion Trap

Garca-Estvez
et al. (2010)

LC-DAD-QTOF

Gasperotti et al.
(2010)

Waters C-18 Symmetry; 25 C;

LC-DAD-MS/MS
HCOOH:H2O (0.05:99.95), CH3CN; 52 min

Gonzalez, Torres,
and Medina (2010)

Phenomenex Aqua; CH3COOH:H2O (2:98),

CH3COOH:CH3CN:H2O (0.5:50:49.5);
65 min,

Hager et al. (2010)

Polyamine, Toyopearl Hw- Purospher STAR C-18; 30 C; HCOOH:H2O

40 (isolation)
(0.5:99.5), CH3OH; 30 min
Diaian HP-20 CC, Toyopearl,
MCl gel CHP-20P CC, YMC
GEL ODS-AQ 120-S50 CC,
Mega Bond Elut C18 CC
(isolation)
Partition with chloroform
and n-butanol, ODS-C-18
Dilica gel, Purospher,
Hichrom (isolation)

LC-DAD-MS; NMR
(CD3OD; 1H, 13C)
NMR ((CD3)2CO; 1H,
13
C, COSY, NOESY,
HSQS, HMBC)

He et al. (2007)
Ito et al. (2008)

NMR (CD3OD, DMSO; Kanaujia et al.

1
(2010)
H, 13C, COSY, HSQC,
HMBC, DEPT-90 & 135)

P. Arapitsas / Food Chemistry 135 (2012) 17081717

Pomegranate peel,
mesocarp and arils

Isolation fractionation

Gallotannins

Boysenberry

Sanguiin H-2, sanguiin H-6, sanguiin H-10, (galloyl- CH3COOH:CH3CH2OH:H2O Synergi Hydro-RP
bis-HHDP-glucose)-2-gallate
(1:80:20)
(isolation)

Various berries

Ellagitannins and gallotannins

Pomegranate

Punicalin, punicalagin A, punicalagon B

Tanoak acorns

Gallotannins

Oak-aged wine

Castalagin, vesclagin, grandinin, robunin E, robunin

A, robunin D, robunin B, robunin C.

Red rasberry

Lambertianin C, sanguiin H-6

Rasberry

Sanguiin H-10, lambertianin C, sanguiin H-6, ellagic HCl:CH3OH (0.1:99.9)

acid pentose conjugate, nobotanin A, and others
ellagitanins
1,6 Digalloyl glucose, 1,2,6 trigalloyl glucose, 1,2,3,6 Soxhlet extractor, CH3OH
tetragalloyl glucose,

Carob

Carob

CH3OH:H2O (80:20)

9 Longan fruit cultivars Corilagin

Myrthle berries

Gallotannins and ellagitannins

CH3CH2OH:H2O (70:30)

Oak aged wine

Vescalagin, castalagin, acutissimin, epiacutissimin A,

epiacutissimin B, ethylvescalagin

Strawberry

Gallotannins and ellagitannins

White strawberry

Three bis-HHDP-glucose, tetragalloyl glucose,

potentilin/casaurictin

Longan seeds

Gallotannins and ellagitannins

Rubus berry

Sanguiin H-10, sanguiin H-6, lambertianin A, B, C & D (CH3)2CO:H2O (70:30)

and other ellagitannins
Punicalagins A and B, punicalin
DMSO

LC-DAD

Kim et al. (2009)

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

Kool et al. (2010)

LC-DAD-MS

Mtt-Riihinen
et al. (2004)
Madrigal-Carballo
et al. (2009)
Meyers, Swiecki,
and Mitchell
(2006)
Michel et al.
(2011)

LC-DAD
LC-DAD-MS/MS

Lichrospher 100RP 18; H3PO4:H2O

LC-DAD; LC-MSn
(0.1:99.1), H3PO4:CH3OH (0.1:99.9); &
CH3COOH:H2O (0.4:99.6), HCOOH:CH3OH
(0.4:99.6); 40 min
RP-MAX C12; 40 oC; TFA:H2O (0.5:99.5), LC-DAD
CH3CN; 30 min
Synergi RP Max; 40 C; CH3COOH:H2O
LC-MSn
(1:99), CH3CN; 60 min.

HCl:CH3OH (0.1:99.9)

Pressurised liquid
extraction optimization;
(CH3)2CO:H2O (50:50)
CH3OH:H2O (70:30)

Pomegranate base
suplements

Gallotannins

CH3COOCH3 and CH3OH

Waters Spherisorb ODS 2; HCOOH:H2O

(2:98), HCOOH:CH3CN:H2O (2:68:30);
60 min
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
LiChroCART Purospher RP-18e;
CH3COOH:H2O (1:99), CH3CN; 20 min
Spherisorb ODS2 C-18; TFA:H2O (1:99),
CH3OH; 50 min
Prodigy ODS column; CH3COOH:H2O
(1:99), CH3COOH:CH3OH (1:99); 45 min

Silica gel 60, Latex C-18

(isolation)

Latex C-18; CH3COOH:H2O (2:98),

CH3OH; 45 min. GCMS after acidid
hydrolysis

Aqua C18, 25 C, CH3COOH:H2O (1:99),

CH3COOH:CH3CN (1:99); 77 min

Mullen et al.
(2002)
Mullen et al.
(2003)

GCMS, LC-MS, nano- Owen et al. (2003)

ESI-MS; NMR (CD3OD;
1
H, 13C, DEPT-135,
COSY, 2D CH COLOC
and COLOC-S)
LC-DAD-MS/MS
Papagiannopoulos
et al. (2004)

LiChrospher RP-18; 25 C; CH3COOH:H2O

(0.4:99.6), CH3OH; 20 min & Phenomenex
Luna C18; THF:TFA:H2O (2:0.1:98),
CH3CN, 45 min
Phenomenex luna C-18; 25 C;
CH3COOH:H2O (0.1:99.9), CH3CN; 25 min
CH3COOH:H2O (1:99), CH3COOH:CH3OH
(1:99); 50 min

LC-DAD and LC-DADMS/MS

Rangkadilok et al.
(2005)

LC-DAD-MS

Romani et al.
(2006)
Saucier et al.
(2006)

HCl:CH3OH (0.1:99.9) and

(CH3)2CO:H20 (70:30)
HCOOH:CH3OH (1:99)

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

CH3CN; 70 min
C-18 Luna; 25 C, CH3COOH:H2O (1:99),
CH3CN; 50 min

LC-DAD-MS/MS

CH3CH2OH:H2O (50:50);
70 C.

Shim-Pack VP-ODS; HCOOH:H2O (3:97),

CH3OH, 50 min & Shephadex LH-20;
CH3OH
Purospher Star; 40 C; CH3COOH:H2O
(1:99), CH3CN, 47 min
Agilemt Zorbax SB C-18, H3PO4:H2O
(0.4:99.6), CH3CN; 30 min

LC-DAD-MS/MS

Amberlite XAD7 HP resin,

TSK HW 40F gel
chromatography
(fractionation)

LC-DAD-MS/MS

LC-DAD-MS/MS

LC-DAD-MS
LC-DAD

P. Arapitsas / Food Chemistry 135 (2012) 17081717

Mango

Seeram et al.
(2006)
Simirgiotis and
SchmedaHirschmann
(2010)
Soong and Barlow
(2005)
Vrhovsek et al.
(2006)
Zhang et al. (2009)

1713

1714

P. Arapitsas / Food Chemistry 135 (2012) 17081717

4.3. Chromatography separation

The analytical separation of HTs is another issue which has not
been studied in depth. Due to the physicochemical properties of
these analytes (e.g. high molecular weight and high polarity), separation techniques such as gas chromatography, electrophoresis
capillary and superuid chromatography cannot be applied, or
their results are not yet adequate. Gas Chromatography is used
only after acidic hydrolysis, so HT identication is not possible,
allowing only consideration of the total amounts (Owen et al.,
2003). Liquid chromatography appears to be the only method used
both in reversed and normal phase mode (Table 1). The most common technique used in HT food analysis is RP C-18 chromatography, with acidic methanol or acetonitrile used as the strong
solvent eluent and acidic water as the weak eluent (Table 1). For
acidication, 0.053% formic or acetic acid are usually used (Table 1). Acetic acid is principally used due to its higher volatility
and its lower ion suppression in LC/MS, while formic acid is mainly
used because of its lower pK as compared to acetic acid, which also
helps HT solubility in water. Of course a higher percentage of acid
(up to 5%) was also used when measurement of anthocyanins was
also required within the same chromatographic run (Buendia et al.,
2010). For LC-DAD analysis, non-volatile acids such as phosphoric
acid or TFA are often used as eluents.
Alternatively, Soong and Barlow (2005) used a Shephadex LH20 column, Borges et al. a C-6 phenyl column, and Furuuchi, Yokoyama, Watanabe, and Hirayama (2011) a Diol column for their LCDAD, LC-DAD-MS/MS and LC-uorescence analyses. The chromatographic runs varied from 20 min to 80 min, depending mainly from
the column diameter, and 40 C was the maximum temperature
applied also for UPLC systems (Table 1).
4.4. Detection Identication
The lack of a detector with high specicity for HTs has led to the
use of different techniques and the combining of data for identication of HTs.
DAD detectors provide important information for the identication of HTs and are widely used for routine identication and
quantication analysis (Aaby, Ekeberg et al., 2007; Aaby, Wrolstad
et 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; Okuda
et al., 1989; Romani et al., 2004). As shown in Fig. 2, gallotannins
(simple glucogalloyl esters i.e. pentagalloylglucose (Fig. 2c)) have
a characteristic UVVis spectra with a maximum at 280 nm. All
glucogalloyl derivatives have a very similar UVVis spectra, with
a bathochromic shift of 1012 nm when compared to gallic acid
spectrum (a). Ellagitannins also have a characteristic UV spectra
(i.e. Fig. 2df) with maximum absorbance bellow 270 nm. When
only HHDP moieties are present in the molecule, the UVVis spectrum of Fig. 2d is the most probable case. When the ellagitannin
also has simple galloyl esters the UVVis spectrum of Fig. 2f is
more likely, and nally when ellagic acid is part of the molecule
structure (gallagyl-glucosides) the Fig. 2i UV spectrum is more
likely (Arapitsas et al., 2007; Mtt-Riihinen et al., 2004). C-glycosidic ellagitannins have UVVis spectra with a maximum of around
245 nm (Fernandes, Sousa, Mateus, Cabral, & de Freitas, 2011). For
compounds such as 2,3-(HHDP)-glucose, without a simple glucogalloyl ester, DAD have very low selectivity (Fig. 2d). Gallotannins
with m-depsidic links have spectra similar to the (b) UVVis spectra of Fig. 2b (Arapitsas et al., 2007).
Mass spectrometry detectors also offer valuable information for
the identication of HTs, the negative ion mode of electrospray
ionisation 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; Hatano
et al., 1988; Kool, Comeskey, Cooney, & McGhie, 2010; MuellerHarvey, 2001; Mullen, Yokota, Lean, & Crozier, 2003; Mtt, Kamal-Eldin, & Trrnen, 2003; Mtt-Riihinen et al., 2004; Reed,
Krueger, & Vestling, 2005; Soong & Barlow, 2005). When positive
ion mode is used, the metal adducts of Na and K are usually identied (Franceschi et al., 2011; Furuuchi et al., 2011). Almost all MS
detectors have been used for the identication of HTs, from single
and triple quadruple, to ion trap and MALDI-TOF-TOF MS (Table 1).
In addition to the deprotonated molecule [M-H], ellagitannins often also provide [M 2H]2 or [2M H] ions, depending on the
mass of the compound. Typical losses during fragmentation are
galloyl (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 this
indicates the presence of a simple galloyl ester in the molecule of
HTs, 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 chebulic
ellagitannin is the m/z 337 [chebulic H H2O] , which further
breaks into m/z 319 [chebulic H2H2O] , 293 [chebulic H
H2OCO2] and 275 [chebulic H2H2OCO2] or the m/z 351
[methyl neochebuloyl HH2O] (Pfundstein et al., 2010). Ellagitannins of the gallagyl-glucoside group have m/z 601 as the characteristic fragment ion, indicating the loss of a gallagic acid group
(Fischer et al., 2011; Pfundstein et al., 2010). Both quercetin (the
most common avonoid in plants) and ellagitannins with HHDP
moiety produce identical molecular ions on fragmentation (m/z
301), but when tandem MS is used, quercetin m/z 301 further fragments to form characteristic m/z 179 and 151 ions whereas the
equivalent EA m/z 301 ion yields ions at m/z 257 and 229 (Mullen
et al., 2003; Seeram et al., 2006). The simultaneous tandem use of
DAD and ESI-MS is the most common technique applied for the
identication of HTs. For the analysis of high MW HTs, the soft ionisation of the MALDI technique is more suitable than ESI, as it does
not form multiply charged ions and gives high-resolution results.
In contrast to ESI, MALDI frequently provides better detection in
positive ionisation, with ellagitannins being typically detected as
adducts. Using MALDI-TOF MS, Afag et al. identied up to pentameric forms of ellagitanins in pomegranates (Afag, Seleem, Krueger, Reed, & Mukhtar, 2005); Kool et al. published the MALDITOF MS spectra of sanguiin H-2, lambertianin C, sanguiin H-10
(Kool et al., 2010); and Franceschi et al. used Ion Mobility Separation with a MALDI-TOF MS instrument and identied four charged
molecular species (single, double, triple and quadruple) of sanguiin
H-6 (Franceschi et al., 2011).
Aaby, Ekeberg et al., 2007; Aaby, Wrolstad et al., 2007 used a
coulometric array for the analysis of HTs, a method based on the
characteristics of phenols with adjacent OH groups (e.g. catechol
and pyrogallol) to stabilise the phenoxy radical and lead to lower
oxidation potential. So ellagitannins which have several pyrogallol
groups are oxidised at the lowest oxidation potential.
NMR spectroscopy is a powerful technique for the structural
identication of HTs, and other substances. The 1D (1H- and 13CNMR), 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. Nevertheless, most of them have been isolated from medicinal plants
and only a few papers have reported the NMR spectra of HTs isolated from food products. These papers are limited to a few ellagitanins isolated from raspberries (sanguiins and lambertianins),
pomegranates (gallagyl-glucosides), aged wine and whisky (avano-ellagittaninis and C-glucosides of ellagitannins) and carob bre
(depsidic gallotannins) (Arapitsas et al., 2007; Franceschi et al.,

1715

P. Arapitsas / Food Chemistry 135 (2012) 17081717

270 nm

(a)

(e)

gallic acid
250

300

350

400

450

270 nm

sanguiin H-6
500

550

nm

250

350

300

350 400
280 nm

450

500

300

350

400

550 nm

300

(c)

450

500

350

254 nm

400

450

350

400

450

500

550 nm

(g)

360 nm

550 nm

300

350

254 nm

400

450

374 nm

500

550 nm

(i)
punicalagin

pedunculagin II
300

550 nm

ellagic acid

(d)

250

500

oenothein B

pentagalloyl-glucose
250

450

(f)

penta(digalloyl)-glucose
250

400

275 nm

(b)

300 nm

300

500

nm

250

300

350

400

450

500

550 nm

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

2011; Fujieda et al., 2008; Gasperotti et al., 2010; He, Shi, Yao, Luo,
& Ma, 2001; He et al., 2007; Ito, Iguchi, & Hatano, 2008; Kanaujia
et al., 2010; Kool et al., 2010; Owen et al., 2003; Quideau & Feldman, 1996). An interesting diagnostic characteristic for the 1HNMR spectrum, demonstrated by Arapitsas et al. (2007), is that
aromatic 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 the
HDDP group (6.586.66 ppm), as compared to the simple glucogalloyl esters aromatic protons (6.997.04 ppm). As shown in Table 1
the most common deuterared solvents used for the registration of
NMR spectra are methanol and acetone, and more rarely DMSO.
The temperature and concentration of HTs with high MW, which
tend to aggregate, are also important for clarifying structure
(Franceschi et al., 2011; Fujieda et al., 2008; Okuda et al., 1989). Recently, Jourdes et al. (2011) produced an interesting review containing critical information about signal recognition for HTs NMR
spectra.
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. Quantication
The absence of commercial standards has obliged scientists to
develop and use conventional methods both for total and individual quantitative determination of HTs.
Methods for total determination of HTs include Owades suggestion for the use of absorbance at 270 nm in 1958 and the FolinCiocalteau assay of 1927. However both methods measured total
phenols and are not specic for HTs. The KIO3-reagent assay described by Bate-Smith in 1977 for the quantication of gallotannins and ellagitannins is not suitable for complex mixtures of
tannins and is not particularly effective, since it depends critically
on the temperature and duration of the analysis (Harborne & Williams, 2000; Hartzfeld, Forkner, Hunter, & Hagerman, 2002; Mueller-Harvey, 2001; Okuda et al., 1989). A ow injection system using

the KIO3-reagent has also been reported. This is a rhodanine-reagent assay developed by Inoue and Hagerman in 1988, which
measures only gallic acid and not galloyl esters, ellagic acid or
ellagitannins, while it requires the absence of oxygen (Falco &
Arajo, 2011). The NaNO2-reagent method reported by Wilson
and Hagerman in 1990 is only selective for ellagic acid and is also
sensitive to oxygen (Wilson, 1934). Other assays based on the
acidic hydrolysis of ellagitannins are also described with the use
of different acids, such as anhydrous methanolic HCl or triuoroacetic acid, but none of these procedures represent an optimal
solution, as they are time-consuming, require the use of hazardous
solvents or have low sensitivity and selectivity (Harborne & Williams, 2000; Hartzfeld et al., 2002; Mueller-Harvey, 2001; Okuda
et al., 1989).
As far as HPLC/DAD quantication analysis is concerned, single,
group or total HTs are frequently conventionally measured as gallic
acid, ellagic acid or a similar ellagitannin, without evaluating the
selectivity of the method (Borges et al., 2010a; Fischer et al.,
2011; Michel et al., 2011; Mueller-Harvey, 2001; Mtt-Riihinen
et al., 2004; Okuda et al., 1989; Pfundstein et al., 2010; Sandhu &
Gu, 2010). Groups working both on isolation/characterisation and
quantication analysis have used the isolated compounds as standards for quantitative determination using HPLC/DAD or HPLC/MS
techniques, although this is an exception (Fischer et al., 2011;
Gasperotti et al., 2010; Michel et al., 2011; Rangkadilok, Worasuttayangkurn, 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 considerable interest in HTs on the part of scientists, consumers and food
manufacturers. On the one hand the benecial effects of HTs on
health and food quality, and on the other their structural variety,
complexity and widespread distribution have represented the
main characteristics underlying this continuing interest. The main
problems, causing serious misunderstanding, are the lack of

1716

P. Arapitsas / Food Chemistry 135 (2012) 17081717

commercially available standards and selective methods for the

analysis of HTs. As knowledge and technological achievements
have improved, more and more researchers from different backgrounds have directed their attention towards the implementation
of better extraction, purication and detection methodologies for
HTs. The establishing of new HT analytical tools for product
authentication and the discovery of sophisticated adulteration will
always be a major eld of interest. Furthermore there is still a general need for analytical techniques able to assist with establishing
new, fast, accurate, effective and environmentally friendly methods for the identication and quantication of HTs.

References
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