Food Research International 63 (2014) 275–289

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Food Research International

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

Review

Untargeted and targeted methodologies in the study of tea

(Camellia sinensis L.)
Maria Daglia a, Riccarda Antiochia b, Anatoly P. Sobolev c, Luisa Mannina b,c,⁎
a
Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section, Pavia University, Via Taramelli 12, 27100 Pavia, Italy
b
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Rome, Italy
c
Istituto di Metodologie Chimiche, Laboratorio di Risonanza Magnetica “Annalaura Segre”, CNR, I-00015 Monterotondo, Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The chemical composition of tea beverage is very complex and not yet completely elucidated. Many factors
Received 5 January 2014 contribute to the chemical complexity of tea, from plant growth conditions (soil, climate, growth altitude and
Received in revised form 14 March 2014 agricultural practices) and manufacturing processes (drying, steaming, fermentation and storage), to preparation
Accepted 29 March 2014
methods (quality of water, infusion time and, not least, the time between tea preparation and consumption).
Available online 16 April 2014
Besides primary metabolites, tea leaves contain a number of secondary metabolites, belonging to many different
Keywords:
classes of compounds (such as polyphenols, xanthines, proteic and nonproteic amino acids, sugars, volatile
Tea composition compounds) that are extracted during the infusion and transferred into the beverage. Epidemiological studies
Targeted analytical methodologies have suggested that tea consumption is inversely associated with the risk of developing chronic diseases such
Untargeted analytical methodologies as cardiovascular diseases, stroke, some forms of cancer and diabetes. Thus, increasing interest in the health
Liquid chromatography properties of tea resulted in a significant rise in scientific investigation on tea chemical composition. This review
Mass spectrometry article highlights the recent results obtained in the tea beverage characterization using different analytical
Nuclear magnetic resonance spectroscopy methodologies. The analytical approaches have been subdivided into two groups: targeted chromatographic
and NMR techniques and untargeted NMR analytical approaches. Several examples of untargeted chromato-
graphic techniques coupled with mass spectrometry methodologies are also reported. Advantages, drawbacks
and significant applications of the different analytical approaches are discussed.
© 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
2. Targeted analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
2.1. Chromatographic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
2.1.1. Determination of polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
2.1.2. Determination of proteic and nonproteic amino acids (theanine and GABA) . . . . . . . . . . . . . . . . . . . . . . . . . 280
2.1.3. Determination of xanthines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
2.1.4. Determination of polyamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
2.2. NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

Abbreviations: CE, capillary electrophoresis; CG, catechin gallate; DART, direct analysis in real time; ESI, electrospray ionization; EGCG, epigallocatechin gallate; EC, epicatechin; ECG,
epicatechin gallate; EGC, epigallocatechin; FTICR-MS, Fourier transform ion cyclotron resonance mass spectrometry; GCG, gallocatechin gallate; Gc, gallocatechin; GABA, gamma-
aminobutyric acid; GC, gas chromatography; HPLC, high performance liquid chromatography; HPLC–MSn, HPLC coupled with tandem mass spectrometry; HSCCC, high-speed
countercurrent chromatography; ITMS, ion trap mass spectrometers; LODs, limits of detection; LOQs, limits of quantification; MS, mass spectrometry; MALDI, matrix-
assisted laser desorption ionization; MEKC-LIF, micellar electrokinetic chromatography with laser-induced fluorescence detection; NAC, N-acetyl- L-cysteine; NMR, nuclear
magnetic resonance; PAD, photodiode array detection; OPA, phthaladehyde; RP, reverse-phase; SEC, size exclusion chromatography; TFs, theaflavins; TRs, thearubigins; TLC,
thin layer chromatography; TOF, time of flight; TSQ, triple quadrupole; UPLC, ultra performance liquid chromatography; UV–vis, ultraviolet–visible spectrophotometry.
⁎ Corresponding author at: Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Rome, Italy. Tel.: +39 06 49913735; fax: +39
06 90672476.
E-mail address: luisa.mannina@uniroma1.it (L. Mannina).

http://dx.doi.org/10.1016/j.foodres.2014.03.070
0963-9969/© 2014 Elsevier Ltd. All rights reserved.
276 M. Daglia et al. / Food Research International 63 (2014) 275–289

3. Untargeted methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

3.1. Chemometric analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
3.2. NMR methodologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
3.2.1. Liquid state NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
3.2.2. Solid state NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
3.3. LC–MS and GC–MS methodologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

1. Introduction amount of retained polyphenols. On the other hand, in the case of

Tea is one of the most popular beverages in the world, next to water.
The total consumption of tea is about 3,000,000 tons a year, with a
world average per capita consumption of 0.3 kg in 2009. The increase
in tea consumption is mainly driven by sales of black specialty tea
bags, followed by green tea and other types (Basu Majumder, Bera, &
Rajan, 2010).
There are approximately 1500 different varieties of tea, all offering
interesting and varied style, taste and color. The quality of tea beverage
is mainly assessed through sensorial properties (aroma, flavor and
appearance) that, like in coffee and wine, depend on many different
factors such as climatic conditions, soil, growth altitude and horticultural
practices, plucking season, sorting of leaves, processing and storage.
Depending on the manufacturing process, teas are classified into four
major types: 1) non-fermented green and white teas, produced by
drying and steaming fresh tea leaves, 2) semi-fermented oolong tea,
obtained by a partial fermentation stage of the fresh leaves before drying,
3) full-fermented black and red teas, produced by a post-harvest fresh
leaf fermentation stage before drying and steaming and 4) post-
fermented tea (Pu-erh tea) that undergoes secondary fermentation and
oxidation in open air.
Black tea represents the majority of tea traded internationally and
takes about 80% of the global tea market (Tanui, Fang, Feng, Zhuang, &
Li, 2012) whereas green tea, originating in China, is traditionally con-
sumed by Asian cultures, although recently it has become widespread
also in Western countries where black tea is usually consumed (Basu
Majumder et al., 2010; Belitz, Grosch, & Schieberle, 2009). The constant
increase in the consumption of green tea is mainly due to its beneficial
effects on human health (Suzuki, Miyoshi, & Isemura, 2012). Epidemio-
logical studies have suggested that tea consumption, especially green
tea, is inversely associated with the risk of developing cardiovascular
diseases, stroke, some forms of cancer and diabetes (Bøhn, Ward,
Hodgson, & Croft, 2012; Geybels, Neuhouser, & Stanford, 2013; Silva
et al., 2013).
Green tea leaves contain proteic and nonproteic amino acids such as
L-theanine, responsible for the enhancement of umami taste and the in-
crease of the production of gamma-aminobutyric acid (GABA), which
shows several physiological functions, such as anxiety reduction and
stress relief (Zhao et al., 2011). Moreover, tea leaves contain minerals
such as Ca, Cu, Fe, Mg, Mn, Zn, and above all F, since the tea plant is a
fluorine accumulator (Lu, Guo, & Yang, 2004), and xanthine compounds
such as caffeine, theobromine and theophylline that are very stable and
remain unaltered in the semi-fermented and fermented teas and are
transferred to the beverage during the infusion process. Other minor
compounds such as vitamin C, terpenoids, fatty acids, and essential
oils (Belitz et al., 2009) are also present in tea leaves.
Green tea leaves also contain about 30% of polyphenols. The main
polyphenols present in green tea are flavonoids that include flavan-3-
ols (catechins), flavonols, flavones, proanthocyanidins and their
derivatives. Besides flavonoids, phenolic acids and their derivatives are
also present (Belitz et al., 2009). The high green tea polyphenol content
is due to the method of production, which inactivates polyphenol
oxidase enzymes and so avoids oxidation reactions, maximizing the
black tea, the fermentation process promotes oxidation and subsequent
condensation reactions of fresh tea leave polyphenols, reducing the
amount of catechins, which are converted to theaflavins (TFs) and
thearubigins (TRs) responsible for the characteristic black tea taste
and bright red-orange color (Cheynier, 2005).
To have an exhaustive characterization of the different commercially
available teas, different analytical approaches both targeted and
untargeted have been used.
Targeted analysis is used when the aim of the research is to deter-
mine a specific metabolite or a class of metabolites. In this case, selective
extractions and/or separations can be performed before the analysis to
isolate and concentrate the selected metabolites and to avoid interfer-
ence from other compounds. The traditional targeted approach allows
the reliable and sensitive identification and precise and accurate quan-
tification of specific compounds. Nevertheless, targeted methods do
not provide a comprehensive picture of the chemical composition of
the investigated sample, having the drawback of missing compounds
not selected at the start of the analysis. In literature, many analytical
techniques such as thin layer chromatography (TLC), high performance
liquid chromatography (HPLC), ultra performance liquid chromatogra-
phy (UPLC), capillary electrophoresis (CE), gas chromatography (GC)
and nuclear magnetic resonance (NMR) spectroscopy have been ap-
plied to determine tea target metabolites. Typical target compounds
are phenolic compounds, proteic and nonproteic amino acids, xan-
thines, and polyamines. This review will be focused on tea target com-
pounds determined using HPLC and UPLC separation techniques
coupled with different detection techniques, such as ultraviolet–visible
spectrophotometry (UV–vis) and mass spectrometry (MS). Moreover,
the use of NMR for the structural elucidation of partially or completely
unknown tea substances is reported.
Untargeted analysis in foodstuff characterization has been carried
out using different approaches such as metabolic fingerprinting, metab-
olite profiling and metabolomics. Metabolic fingerprinting is used when
classification of samples often without identification of individual me-
tabolites is required, whereas metabolite profiling requires the identifi-
cation and quantification of metabolites, belonging to various classes of
compounds. Finally, the metabolomic approach requires the identifica-
tion of all metabolites present in the samples, within the detection
limits of the specific analytical technique. NMR spectroscopy is one of
the main analytical techniques employed for untargeted metabolic
studies (Mannina, Sobolev, & Viel, 2012). This is because it can bring
“high-throughput” spectroscopic/structural information on a wide
range of metabolites simultaneously with high analytical precision.
This methodology also avoids biases against certain classes of com-
pounds and is helpful for the identification of secondary metabolites.
Molecular identification is quite easy and straightforward and can be
obtained from the NMR spectrum of the mixture itself or, after purifica-
tion, by means of 1D and 2D experiments, standard additions and by
comparison with a database of standard compounds. Using 1H NMR-
based metabolomic approach, many foodstuffs have been analyzed
obtaining information on geographical characterization, varieties,
quality and adulteration (Mannina et al., 2012). In this review, the
results obtained using untargeted NMR methodologies in the analysis
 M. Daglia et al. / Food Research International 63 (2014) 275–289 277

of tea beverages are reported together with some examples of
chromatographic-based untargeted analyses coupled with MS spec-
trometry (LC–MS, and HPLC/MS).
2. Targeted analysis
2.1. Chromatographic methods
Chromatography is an analytical method widely used for the separa-
tion and identification of the components occurring in complex mix-
tures, such as foods and beverages. The most frequently used analytical
techniques for the separation of the compounds occurring in tea are
reverse-phase (RP)-HPLC, with photodiode array detection (PAD),
fluorescence, electrochemical (ECD) and MS detection. HPLC-PAD,
HPLC–MS and HPLC coupled with tandem mass spectrometry (HPLC–
MSn) are often used in combination because these types of detection
provide different types of information for structural characterization.
UV–vis spectra allow preliminary identification and quantification
using characteristic absorption maxima, whereas mass spectra provide
information about molecular mass. A wide variety of ionization
techniques are used in MS. The most common example of ionization is
electrospray ionization (ESI) that may be performed both in the positive
and/or negative ion mode. Electrospray ionization is often coupled with
triple quadrupole (TSQ) and ion trap mass spectrometers (ITMS). ESI,
coupled with tandem mass spectrometry (ESI–MSn), through the
multistage fragmentations starting from the parent ion to the fragments
derived from it, enables the partial chemical characterization and the
identification of the metabolite, also in the absence of fragmentation
pattern of reference substances. Another ionization technique, which
has been first used for protein and nucleic acid analysis and then has
been applied for the study of complex molecules, is matrix-assisted
laser desorption ionization (MALDI). The technology of MALDI is based
on the excitation of the molecules of a sample (dissolved in an excess
of matrix, sinapinic acid or alpha-cyano-4-hydroxycinnamic), with a
laser beam to produce the sample vaporization and ionization. MALDI
is often coupled with a time of flight (TOF) analyzer. In a MALDI–TOF
instrument, the mass of the ions generated in the source is measured
by means of a time of flight analyzer (time of flight, TOF). The mass-to-
charge ratio (m/z) of the sample is derived from the time taken by the
ion, which, accelerated by an electric field in the ionization source,
reaches a detector at a known distance. More recently, Kuhnert,
Drynan, Obuchowicz, Clifford, and Witt (2010) have used Fourier trans-
form ion cyclotron resonance mass spectrometry (FTICR-MS) for the
analysis of black tea polymeric polyphenols. In this technique, the m/z
of an ion is experimentally determined through the measurement of
the frequency at which the ion processes in a magnetic field. FT-ICR MS
has the highest mass accuracy and mass resolution of any mass analyzer,
thus FT-ICR MS has proven to be a powerful platform across a large
variety of metabolomic studies.
water–acetonitrile–phosphoric acid (or formic acid, or acetic acid) mo-
bile phase. Since then, dozens of articles have been published reporting
different sample preparation, polyphenol identification and quantifica-
tion protocols. Different sample preparation procedures have been
suggested that aimed to avoid possible alterations, especially for tea
catechins that have a high tendency to be epimerized, oxidized and
polymerized. The sample preparation protocol involves first the prepa-
ration of the tea extract by the infusion method and thereafter the ex-
traction of polyphenols by liquid–liquid extraction, solid phase
extraction (SPE) and solid phase microextraction. The infusion method
has been widely used since the composition of the obtained extract is
expected to be similar to that of consumed tea beverage. Nkhili et al.
(2009) have reported an alternative method for the preparation of tea
extract using microwave extraction. The results of this study have
shown that at the same water-to-tea ratio of 20:1 (mL/g) and time of
extraction, the concentration of flavan-3-ols, especially epigallocatechin
gallate (EGCG), is higher in the microwave-assisted extract than in the
conventional extract. As regards the identification of polyphenols,
peak identity and purity have been based on 1) chromatographic be-
havior (retention time and elution order), 2) UV–vis and MS spectra,
and 3) comparisons with the literature data. Quantitative determination
of each identified polyphenol has been generally performed through the
response of the diode array detector (Bronner & Beecher, 1998; Chiu &
Lin, 2005; Kuhr & Engelhardt, 1991; Goto, Yoshida, Kiso, & Nagashima,
1996; Lee, Prabhu, Meng, Li, & Yang, 2000; Lin, Wu & Lin, 2003; Yagi,
Goto, & Nanjo, 2009).
The main polyphenols identified in green tea are flavan-3-ols, which
are the most abundant green tea polyphenols, flavonols, flavones and
their different glycosides. Among flavan-3-ols (Fig. 1), the more
representative catechins are epicatechin (EC, approximately 1–3%), epi-
catechin gallate (ECG, approximately 3–6%), epigallocatechin (EGC, ap-
proximately 3–6%) and EGCG, with the last one being the most

2.1.1. Determination of polyphenols

Over the course of the last 20 years, polyphenols have been studied
for their potential involvement in the prevention of chronic diseases,
such as cardiovascular diseases, cancer, osteoporosis, diabetes mellitus
and neurodegenerative diseases. Their protective activity has been
attributed initially to their antioxidant, free radical scavenging and
metal chelating properties, then to their capability of inhibiting or
reducing different enzymes such as telomerase, cyclooxygenase, or
lipoxygenase and, in more recent years, to their interaction with signal
transduction pathways and cell receptors (Daglia, 2011).
Tea polyphenols were first studied in the 1950s using paper chroma-
tography (Roberts & Wood, 1951). In the 1990s, a number of polyphe-
nols have been identified both in fresh tea leaves and different
commercially available types of tea beverages, using HPLC–UV–vis and
HPLC–MS methods. These methodologies have allowed simultaneous
determination of polyphenols using C18 reverse-phase LC column and Fig. 1. Chemical structures of the main flavan-3-ols occurring in green tea.
278 M. Daglia et al. / Food Research International 63 (2014) 275–289
abundant (approximately 9–13%). Besides these major catechins, in
green tea obtained from Camellia sinensis var. assamica, other flavan-
3-ols such as gallocatechin gallate (GCG), gallocatechin (Gc), catechin
gallate (CG), catechin, epigallocatechin digallates, epicatechin digallate,
3-O-methyl EC, 3-O-methyl EGC, and afzelechin (Zhu, Li, Liu, & Chen,
2013) have been found in small amounts. Thirteen compounds have
been isolated by means of semi-preparative HPLC and their structure
has been determined by PAD and MS detection. Among these com-
pounds, a new natural product namely (+)-afzelechin-3-O-gallate has
been identified, whereas four compounds (theobromine, ampelopsin,
(+)-catechin-3-O-gallate, and (−)-epicatechin-3-O-p-hydroxybenzoate)
have been detected in tea plant for the first time.
Moreover, Chiu and Lin (2005) have identified three methylated
EGCGs, namely (−)-epigallocatechin-3-O-(3-O-methyl)gallate, (−)-
epigallocatechin-3-O-(4-O-methyl)gallate, and (−)-4′-methyl
epigallocatechin-3-O-(4-O-methyl)gallate, in different Chinese tea
samples cultivated in the mountain area of Sansia, Taipei County,
Taiwan by using a RP-HPLC–ECD method in isocratic conditions. The
content of O-methylated EGCG derivatives in tea leaves was found to
be dependent on season, age of leaf (young leaves or old leaves) and
above all manufacturing process. In particular, the O-methylated EGCG
contents of green and oolong teas are higher than that of fresh tea
leaves, suggesting that the manufacturing process, converting the
fresh leaves to green tea and oolong tea, increases the level of these
EGCG derivatives, or that the mechanical operation in the withering
process may induce a rupture of the outer membrane of the tea leaves,
thus liberating the intracellular O-methylated EGCG derivatives and
making them more available for extraction.
As far as flavonols are concerned, kaempferol, myricetin and quercetin
and their glycosides (Fig. 2) have been detected in 41 green tea samples
of different geographical origins, using an RP-HPLC coupled with PAD and
TSQ.
In their research, Lin, Chen, and Harnly (2008) have identified in
different green teas O- and C- mono-, di-, and triglycoside flavonols, in
which the sugar moiety is represented by pentose (xylose) or hexose
(glucose, galattose and rhamnose) units. Furthermore, acylated
glycosylated flavonols, such as kaempferol and quercetin 3-O-acetyl-
or 3-O-p-coumaroyl glycosides have been isolated and identified.
Fig. 2. Chemical structures of other polyphenols occurring in tea.
Moreover, apigenin (Fig. 2), the only flavone occurring in tea, has
been determined together with its C-glycosylated derivatives (such as
6,8-C-diglucosylapigenin, apigenin 6-C glucosyl-8-C-arabinoside,
apigenin 6-C-arabinosyl-8-C-glucoside, apigenin 6,8-C-dipentoside
and other derivatives). In the same investigation, Lin et al. (2008)
have also identified six tea proanthocyanidins, called theasinensins in
fermented black tea of different geographical origins.
More recently, using a comprehensive two dimensional liquid
chromatography (2D-LC) technique employing size exclusion chroma-
tography (SEC) × reverse phase (C18), Scoparo et al. (2012) have
determined tetraglycoside flavonols not previously described. In the
same investigation, other green tea nonflavonoid polyphenols such as
derivatives of benzoic acid (gallic acid, 3-, 4- and 5-galloylquinic
acid) and hydroxycinnamate quinic esters (caffeoylquinic acid and
p-coumaroylquinic acid isomers) have been detected (Fig. 2).
Besides the polyphenol compounds reported above, semi-fermented
and fermented teas contain a heterogeneous fraction of polyphenolic
fermentation compounds, represented by TFs (orange or orange-red
in color) and TRs (red-brown or dark-brown in color) formed by the
oxidation and polymerization of catechins (Fig. 3). TFs, accounting for
2–6% of black tea dry matter, have a benzotropolone skeleton (a bicyclic
ring containing the 2-hydroxy-2,4,6-heptatrienone moiety) that derives
from co-oxidation of catechins under catalysis of polyphenol oxidase
and peroxidase. The four main TFs occurring in fermented tea are
theaflavin, theaflavin 3-gallate, theaflavin 3′-gallate, and theaflavin
3-3′digallate. Del Rio et al. (2004) have developed a RP-HPLC–
PAD–MSn method useful to separate and identify 14 flavonols from
the four major TFs, using 10–30% acetonitrile gradient, in a 60 min
run. Since then, many RP-HPLC methods have been developed for
the determination of major TFs, minor TFs (e.g. epitheaflavic acid
and theaflagallins) and methylated TFs, both in isocratic and
gradient conditions with UV–vis (λ = 278, 375, and 450 nm), PAD
or MS detection. These methods employ a complex sample preparation
as the TFs are extracted with organic solvents or separated by SPE
(Neilson, Green, Wood, & Ferruzzi, 2006, Nishimura et al., 2007). In
2008, two preparative high-speed countercurrent chromatography
(HSCCC) protocols, with a solvent system composed of n-hexane-
ethyl acetate–methanol–water or water acidified with acetic acid,
 M. Daglia et al. / Food Research International 63 (2014) 275–289 279

Fig. 3. Chemical structures of theaflavins and thearubigins occurring in fermented teas.

have been suggested for the separation of TFs in black tea and catechins
in green tea. The results indicate that pure theaflavin, theaflavin-3-
gallate, theaflavin-3′-gallate and theaflavin-3,3′-digallate can be
obtained from crude TF samples and black tea (Wang et al., 2008,
Yang, Li, & Wan, 2008).
More recently, Chen, Shurlknight, Leung, and Sang (2012) have
reported the presence of two new TF derivatives, theaflavin trigallate
and tetragallate in black tea using HPLC–ESI-MSn.
The TF concentration was found to depend on the degree of fermen-
tation, with fully fermented black tea containing more TFs than semi-
fermented tea (oolong and Pu-erh teas). However, over-fermented
black tea contains only trace amounts of TFs that probably undergo
further polymerization.
The other most represented pigments of black tea are TRs, accounting
for an estimated 30–60% of the dry matter. TRs are complex mixtures
of polymeric polyphenols (about 10,000 different molecules) with
280 M. Daglia et al. / Food Research International 63 (2014) 275–289
molecular weights ranging from 1000 to 2100 Da, deriving from the
oxidation and polymerization of EGC and EGCG. Even if they were
discovered by Roberts and Myers (1959), their chemical structure re-
mains largely to be elucidated. The first information on the structure of
TRs has been reported by Menet, Sang, Yang, Ho, and Rosen (2004).
Through the MALDI–TOF mass spectra analysis, Menet et al. have
hypothesized that the structure of TRs is similar to that of TFs, having
similar fragmentation patterns. Due to their structural complexity and
composition, some indications on the chemical structure of TRs have
been recently reported by Kuhnert et al. (2010) using a combination
of complementary MS techniques, such as MALDI–TOF-MS, FTICR-MS,
LC/TOF-MS and LC/MSn. They suggest that TFs are oxidized to ortho-
quinones that add water to yield polyhydroxylated dimers. These
reactions, which involved catechins, water and oxygen, continue until
polymers (TRs) of about 2000 Da are formed.
2.1.2. Determination of proteic and nonproteic amino acids (theanine and
GABA)
Tea beverage contains both proteic and nonproteic free amino acids
(Fig. 4). Among nonproteic amino acids, theanine (2-amino-5-
(ethylamino)-5-oxopentanoic acid), which occurs almost exclusively
in tea leaves, shows positive effects on human health, as it has been
found to increase serotonin, dopamine and GABA levels in the brain
(promoting concentration and learning ability), prevent some types of
cancers and cardiovascular diseases, induce weight loss and enhance
the immune system (Vuong, Bowyer, & Roach, 2011). Theanine is the
most abundant amino acid in tea, accounting for more than 37.2% of
the amino acids. Another nonproteic amino acid is GABA, an inhibitory
neurotransmitter in the mammalian forebrain, with a number of posi-
tive effects in humans such as hypotensive, diuretic, and tranquilizing
effects (Jiang, Fu, & Zhang, 2010). More than 20 years ago, a new type
of tea, named GABA tea, with a high level of GABA (Ntagsecquiz50 mg
of GABA/100 g of tea) has been produced in Japan, submitting tea leaves
to anaerobic fermentation under nitrogen atmosphere (Tsushida,
Murai, Omori, & Okamoto, 1987).
Due to their physiological properties, the determination of both
proteic and nonproteic (theanine and GABA) amino acids in different
types of tea is an important topic and many research articles are fo-
cused on the identification of the main factors that affect their con-
tent, such as manufacturing process, production season and growth
altitude. Xu, Song, Li, and Wan (2012) used the AccQ·Tag method
(Paramas, Barez, Marcos, Garcia-Villanova, & Sanchez, 2006) for the
determination of free amino acids in 160 Chinese green tea samples.
Free amino acids have been submitted to pre-column derivatization
with 6-aminoquinolyl-N-hydroxysuccinimidyl carbammate (ACQ)
and then analyzed by HPLC with fluorescence detection (λ ex =
theanine
GABA
Fig. 4. Chemical structures of nonproteic amino acids of tea.
250 nm and λ em = 395 nm). Using individual calibration curves,
free amino acids have been quantified. The authors demonstrated
that the content of certain amino acids varied according to the
season of production. For example, the mean level of theanine in
spring tea was 16.3 mg/g, 1.5 times higher than that in summer tea
(10.7 mg/g). In the same investigation the content of catechins
(catechin, EC, EGC, ECG, GCG, and EGCG) and caffeine has been deter-
mined by a RP-HPLC–UV–vis method. Also in this case, the content of
catechins and caffeine differs on the basis of the season of produc-
tion. Finally, the content of these compounds was elaborated by
means of multivariate statistical analysis, namely K-nearest neigh-
bor (KNN) algorithm (see Chemometric analysis), which has been
suggested as a useful tool for green tea sample classification.
One of the most common analytical approaches to theanine and
GABA determination consists in the RP-HPLC–UV determination after
pre-column derivatization. In 2008, two easy methods for the simulta-
neous determination of theanine and GABA have been developed,
requiring dabsyl-chloride as a derivatizing reagent and a HPLC system
coupled with a UV–vis detector at λ = 425 nm absorbance with a RP
column (Hypersil GOLD and Zorbax ODS) (Syu, Lin, Huang, & Lin,
2008; Zhao et al., 2011). In particular, Syu et al. have focused their atten-
tion on theanine and GABA content in different kinds of Chinese tea.
Their results show that green and oolong tea samples have a theanine
concentration ranging from 686.53 to 3029.98 μg/g and 170.06 to
2831.40 μg/g, respectively, and very low concentrations of GABA.
Fermented tea samples (black tea, Pu-erh tea, and GABA tea) have
theanine concentrations ranging from 470.77 to 1070.19 μg/g, and
their GABA contents are lower than 55.45 μg/g, whereas high-
mountain teas usually contain higher levels of theanine.
Zhao et al. (2011) have studied 98 Chinese tea samples showing for
the first time that Chinese white tea (45.7 ± 7.7 mg/100 g) and GABA
tea samples (Ntagsecquiz50 mg/100 g) also have mean GABA concen-
trations significantly higher than Pu-erh (1.6 ± 1.0 mg/100 g), green
(18.2 ± 11.0 mg/100 g), black (22.9 ± 14.7 mg/100 g), and oolong
(15.9 ± 7.9 mg/100 g) teas, respectively.
More recently, Tu, Yang, Zhang, and Zhu (2012) have developed
and validated a very sensitive HPLC method in which the derivatiza-
tion is carried out with phthaladehyde (OPA) and N-acetyl- L -
cysteine (NAC). The limits of detection (LODs) for GABA and
theanine are 3.01 × 10− 5 mmol/L and 7.98 × 10 − 5 mmol/L, and
the limits of quantification (LOQs) are 9.99 × 10 − 5 mmol/L and
2.658 × 10− 4 mmol/L, respectively. The influence of different contents
of polyphenols, proline and vitamin C on the derivatization yield has
been investigated. The results have shown that vitamin C increases
the derivatization yield of GABA, but does not affect the derivatization
yield of theanine. Moreover, data coming from the validation procedure
have showed that the method is accurate and precise and is suitable for
a simple and efficient determination of theanine and GABA in different
types of tea.
Micellar electrokinetic chromatography with laser-induced fluores-
cence detection (MEKC-LIF) is a CE technique that shows all the advan-
tages of CE (short analysis time, high separation efficiency, and minimal
solvent consumption) and moreover, is one of the most sensitive
methods available for detection in CE, for both neutral and charged
analytes. Yan, Cai, Wang, Lin, and Li (2014) for the first time have devel-
oped a rapid (11 min) and effective method of micellar electrokinetic
chromatography with laser-induced fluorescence detection (MEKC-
LIF) for the simultaneous determination of 15 amino acids in different
types of tea samples. For the pre-column derivatization of the amino
acids, 4-chloro-7-nitrobenzofurazan (NDB-Cl) has been used. The re-
sults of the validation procedure showed good linearity for each analyte
and low LOD and LOQ. Moreover, repeatability (intra-day and inter-
day) of the migration time and peak area for each amino acid is more
than satisfactory. Finally the accuracy and reliability of the method in
tea sample analysis are good. This method was applied to different
types of tea (black, jasmine, green, biluochun, and maofeng tea leaves).
 M. Daglia et al. / Food Research International 63 (2014) 275–289
Only the contents of Lys, Phe, Leu, Val, Thea, GABA, Gly, Glu, and Asp
were determined because Cys and His occurred in low concentrations
and Thr, Ala, Ser, and Met were not determined due to the interference
of other tea components.
Overall the results of the above reported investigations have identi-
fied the manufacturing process followed by growth altitude as the main
factors that affect theanine and GABA content.
2.1.3. Determination of xanthines
Tea beverage contains caffeine, plus, in minor amounts, theophylline
and theobromine (Fig. 5). Among xanthines, caffeine is the most widely
used psychoactive substance in the world and about 80% of the world's
population takes caffeine daily with an average intake of 200 mg
(Ogawa & Ueki, 2007). In the last five years, more than 400 papers
have been published on the effects of caffeine on human health. Overall,
the results indicate that in healthy subjects, the daily intake of caffeine
can positively stimulate neuromuscular, metabolic and cognitive
activities without having considerable adverse effects on the organism.
However, under special pathological or physiological conditions, the
theobromine
theophylline
caffeine
Fig. 5. Chemical structures of xanthines.
281
intake of caffeine can have undesirable effects. The intake of caffeine
produces reversible increases in systolic and diastolic blood pressure,
and, in pregnancy, the intake of more than 200 mg/day increases the
risk of miscarriage whereas, at quantities of over 300 mg/day, caffeine
increases the risk of reduced fetal growth (Kuczkowski, 2009). There-
fore, it is important to have accurate and precise analytical methods to
determine the content of xanthines in common beverages such as tea.
Even if the earliest methods for the determination of caffeine (based
on GC methodology) date back to the late 70s (Gilbert, Marshman,
Schwieder, & Berg, 1976), over the last years, new methods have been
developed and validated. Rahim, Nofrizal, and Saad (2014) have devel-
oped a rapid RP-HPLC method to determine caffeine (and catechins)
using a monolithic column, a mobile phase of water:acetonitrile:meth-
anol (83:6:11) at a flow rate of 1.4 mL/min. Catechins and caffeine are
separated in about 7 min. The method turned out to be sensitive and
to have good accuracy and precision. In fact the limits of detection and
quantification are in the range of 0.11–0.29 and 0.33–0.87 mg/L, respec-
tively and satisfactory recoveries are obtained (94.2–105.2 ± 1.8%) for
all samples, when spiked at three concentrations. Sample preparation
involves the use of microwave-assisted extraction that allows a very
efficient extraction of the analytes.
Moreover, a validated RP-UHPLC with UV detection method has
been developed for the fast quantitation of caffeine and catechins,
separated in a 3 min run. The limits of detection are in the range 0.1–
0.4 μg mL −1 and the selectivity is confirmed by comparison with
UHPLC-MSn analysis (Naldi et al., 2014).
In another innovative method published in the last year, caffeine has
been determined using an untargeted method, without chromato-
graphic separation, that requires ambient ionization with a direct anal-
ysis in real time (DART) ion source and MS spectrometry (Fraser et al.,
2013). This investigation suggests the potential to utilise DART-MS to
monitor tea fermentation process. Correlation analyses on the MS spec-
tra also revealed the presence of two volatile compounds, tentatively
identified as indole and geranic acid. The tentative identifications have
been carried out using a combination of DART-ion-trap MSn and
DART-accurate mass MS1 and MS2 on tea samples and standard
compounds.
2.1.4. Determination of polyamines
Polyamines are small aliphatic molecules naturally occurring in
plants, animals and microorganisms. Most of the polyamines' roles are
due to their positive charge that allows interaction with negatively
charged molecules. Thus, they are found to be involved in many pro-
cesses such as control of cell cycle (growth, survival and proliferation)
and DNA, RNA, protein, and phospholipid binding. Polyamines play a
crucial role in plant development and growth even if little is known
about their molecular mechanism. In humans, polyamines, which are
derived from cellular synthesis, food intake, and production by the gut
microflora, have been associated with both positive and negative effects
on health. Growing evidence suggests that a high polyamine food intake
is correlated to a prolonged life expectancy. In vitro and in vivo studies
have showed that polyamines are important for eukaryotic cell growth
and viability, promoting positively vital functions (such as proliferation,
cell reprogramming, autophagy, migration and differentiation).
Otherwise, other investigations have reported a negative effect on
health due to their involvement in cell death (apoptosis) and their
high concentration in tumor cells in comparison with normal cells.
Some authors have suggested to consider the content of polyamines in
urine and plasma as a diagnostic marker for cancer (Ghisalberti,
Morisetti, Bestetti, & Cairo, 2013; Park & Igarashi, 2013).
Due to the controversial roles of polyamines, in recent years many
attempts have been carried out to determine polyamine food content
(Hanhineva et al., 2008; La Torre, Saitta, Potortì, Di Bella, & Dugo,
2010; Latorre-Moratalla et al., 2009; Magwamba, Matsheka, Mpuchane,
& Gashe, 2010; Sobolev, Sy, & Gloer, 2008).
282 M. Daglia et al. / Food Research International 63 (2014) 275–289
spermine
spermidine
putrescine
Fig. 6. Chemical structure of polyamines.
The main polyamines occurring in tea are putrescine, spermidine
and spermine (Fig. 6). They are determined by HPLC with UV or fluores-
cence detectors after reaction with chromophoric and fluorescent
derivatizing reagents, such as 2-naphthyloxycarbonyl chloride, 2-
naphthyloxycarbonyl chloride, and 9-fluorenylmethoxycarbonyl
chloride. The derivatizing reagents are necessary since polyamines do
not show sufficient absorption in the ultraviolet and visible wavelength
range (from 190 to 800 nm) and fluorescence.
Palavan-Unsal, Arisan, and Terzioglu (2007) developed a RP-HPLC–
UV method for the determination of polyamines in tea leaves in the
different manufacturing phases of tea production. The pre-column
derivatization is carried out with benzoyl chloride and the
benzoylamines, extracted with diethyl ether, after the evaporation
of organic solvent, are suspended in the HPLC mobile phase and
submitted to a chromatographic analysis with UV detection at λ =
254 nm. The highest content of spermidine has been found in fresh
tea leaves, whereas a relevant spermidine decrease (higher than
80%) has been observed with the manufacturing processes,
especially fermentation and drying. On the contrary, the content of
putrescine and spermine increases with the manufacturing
treatment. More recently, 9-fluorenylmethoxycarbonyl chloride
has been used for the pre-column derivatization of putrescine,
spermidine, spermine and other biogenic amines in different tea
beverages (5 green, 1 oolong, 14 black tea and 1 instant beverage
samples) (Brückner, Flassig, & Kirschbaum, 2012). The fluorescent
polyamine derivatives, obtained with an automated derivatization
procedure, have been directly resolved by RP-HPLC with fluorescence
detection (λex = 263 nm and λem = 313 nm). This method allowed
the determination of polyamines and also tyramine, a biogenic amine
derived from the decarboxylation reaction of tyrosine. The content of
the other biogenic amines has been found to be very low in all samples
(Brückner et al., 2012). Besides free polyamines, spermidine–phenolic
acid conjugates have been determined in tea flowers by UPLC/TOFMS
(Facchini, Hagel, & Zulak, 2002). These secondary metabolites, which
are widely distributed in the floral part of plants, are involved in many
different plant functions (such as defense against wounding, pathogens
and insects, floral induction, flower formation, sexual differentiation,
tuberization, and cell division). In this study, the methanol extract
obtained from tea flowers has been submitted to chromatographic
analysis, with four spermidine derivatives identified by MS2 and MS3
data (tricoumaroyl-, feruoyl dicoumaroyl-, coumaroyl diferuoyl-, and
triferuoyl-spermidine). The main spermidine derivative in flowers
was found to be the tricoumaroyl-derivative, whose content varied
depending on the stage of floral development (so that the higher
content has been found in flower buds) and the localization in the
flower (the higher content has been found in anthers) and therefore
in pollen (Yang et al., 2012).
2.2. NMR
The unambiguous structural elucidation of molecules present in tea
extract as a general rule is the result of multitechnique approaches. MS
spectral data usually provide information on elemental formulas and
MS fragmentation can add valuable structural information but without
reference compounds MS alone cannot give unequivocal identification.
For structural elucidation of partially or completely unknown
metabolites NMR spectroscopy remains essential. The NMR based ap-
proach leans upon intimate connections between molecular structures
and parameters measured by NMR such as chemical shifts, fine
structure of observed NMR signals, and NOE due to space proximity of
specific molecular fragments, among others (Sanders & Hunter, 1993).
A simple 1D 1H NMR spectrum provides a lot of useful structural
information, moreover, specific 2D NMR editing correlation experi-
ments have been developed for the unambiguous assignment of 1H
NMR spectra (Braun, Kalinowski, & Berger, 1998; Sanders & Hunter,
1993). The most important 2D homonuclear NMR experiments that
can be used for structural elucidation are as follows:
• 2D J-resolved experiment. Information on the multiplicity and
coupling patterns of the resonances can be obtained. A projection of
such a spectrum onto the chemical shift axis yields a simplified
spectrum with all spin-coupling peak multiplicities removed.
• 2D COSY. It shows correlations between scalar coupled hydrogen
atoms over two to several (up to 4 in favorable cases) chemical bonds.
• 2D TOCSY. It provides 1H–1H spin–spin coupling connectivities for all
hydrogen atoms within a spin system that form an unbroken chain of
couplings, thereby unraveling the number and nature of the molecu-
lar fragments. TOCSY experiment can be used not only for targeted
analysis but is also very useful in untargeted studies of mixture of me-
tabolites as in the case of black tea infusion (Ohno, Oka, Sakuma,
Okuda, & Fukuhara, 2011).
• 2D NOESY. It allows detection of spatial proximities between 1H spins
by taking advantage of the dipolar coupling interaction and can bring
valuable information regarding double-bond configuration and mole-
cule spatial organization which would be otherwise very difficult to
obtain.
Moreover, the following so called heteronuclear 2D NMR experi-
ments allow less sensitive 13C NMR (and other heteronuclei) to be ob-
served through the use of inverse detection. The most common
heteronuclear correlation 2D NMR experiments are:
• 2D HSQC, which shows correlations between hydrogen atoms and the
carbon atom to which they are attached;
• 2D HMBC, which provides correlations between hydrogen and
heteronucleus generally two or three bonds away.
In spite of a broad range of NMR based experiments, identification of
a compound could be a very difficult task, hence, it requires comple-
mentary MS data. Combining spectral data from MS and NMR can con-
siderably decrease the time for structural elucidation of metabolites by
candidate rejection and substructure conformation.
An interesting example of MS and NMR combination in the analysis
of tea extracts has been reported by Van der Hooft et al. (2012). In this
study SPE–NMR has been used for unambiguous identification and
structural elucidation of selected LC–MSn detectable metabolites in
black and green teas. Structural information, such as elemental formulas
and fragmentation patterns from mass LC–MSn, of 23 metabolites from
black tea and 26 metabolites from green tea has been combined with 1D
1
H and 2D COSY NMR spectral information. Using this approach the full
structure identification of 33 out of 49 metabolites has been achieved
including highly complex conjugated phenolic compounds with
m/z N 800 Da. For instance, derivatives of quercetin and kaempferol,
such as kaempferol 3-O-(2G-p-coumaroyl(trans))-3G-O-β-L-arabinosyl-
3R-O-β-D-glucosylrutinoside have been identified. Apart from LC–
TOF-MS coupled with SPE NMR, another type of chromatography,
 M. Daglia et al. / Food Research International 63 (2014) 275–289
LC-LTQ-Orbitrap Fourier transformed-MS has also been used in the
same study (Van der Hooft et al., 2012) giving 177 metabolites, 82
of which were not previously reported.
The classical targeted approach for compound identification requires
HPLC fractionations and purifications, before dissolving the fractions in
suitable deuterated solvents for NMR measurements. For instance, in
the study of astringent taste compounds in black tea infusions
(Scharbert, Holzmann, & Hofmann, 2004), catechins and flavon-3-ol
glycosides have been separated using various HPLC protocols followed
by structural identification using LC–MS and NMR. Among glycosides, the
apigenin-8-C-[α-L-rhamnopyranosyl-(1 → 2)-O-β-D-glucopyranoside]
has been identified for the first time in tea infusions.
The routine use of quantitative HPLC in tea analysis requires validated
standard compounds, therefore an independent standard validation
method has to be applied. The importance of 1H NMR as a rapid, highly
informative, and nondestructive analysis for reference standard
validation has been highlighted by Kelman and Wright (2012) in a
study undertaken on green tea extracts. One of the commercially avail-
able polyphenol standards sold as EGC, in spite of physical appearance
and HPLC characteristics appropriate for EGC, has been found to be a
completely different compound. A simple 1H NMR spectrum of
suspected standard has been an essential proof of this mislabeling. The
authors reasonably questioned the validity of published pieces of
research based on quantitative HPLC without rigorous validation of
employed standards.
The systematic approach to NMR characterization of principal green
tea polyphenols has been undertaken by Davis, Cai, Davies, and Lewis
(1996). In this study green tea polyphenols such as epiafzelechin, cate-
chin, EC, Gc, EGC, CG, ECG, GCG, EGCG and epigallocatechin-3-O-(3′-O-
methyl)-gallate have been extracted and isolated from green tea
infusion using chromatographic techniques. The 1H and 13C NMR
spectra of individual compounds in acetone-d6 have been fully assigned,
giving a strong basis as a reference work for subsequent studies. The
comprehensive NMR characterization of three black tea polyphenols
(theaflavate B, isotheaflavin-3′-O-gallate, and neotheaflavin-3-O-
gallate) undertaken by the same group has been also reported
(Lewis et al., 1998).
Apart from the structural identification of targeted metabolites in
green tea, NMR spectroscopy can also be used as a robust quantitative
analytical technique. Recently a validation of the quantitative 1H NMR
analysis of five green tea components has been reported (Yuan et al.,
2014). The contents of selected compounds, namely caffeine, gallic
acid, theanine, EGC, and (−)-epigallocatechin-3-gallate, determined
simultaneously in green tea infusions, turned out to be in the range
between 10 and 1 mg mL− 1. LODs and LOQs were in the range
29–18 μg mL−1, and 56–37 μg mL−1, respectively, whereas the relative
standard deviation of both precision and repeatability assays was lower
than 4.5%.
3. Untargeted methodology
From a chemical point of view, tea beverage can be considered a
complex mixture containing many metabolites widely ranging in
concentration and chemical properties. Untargeted methodologies
giving a comprehensive view of the tea metabolites based on chemo-
metric statistical analysis have provided relevant results in terms of
quality evaluation, origin authentication, tea category differentiation,
geographical origin, manufacturing process and so on.
3.1. Chemometric analysis
Data coming from different analytical methodologies can be ana-
lyzed using several suitable chemometric tools widely described in
many papers and reviews (Izquierdo-García et al., 2011, McKenzie,
Donarski, Wilson, & Charlton, 2011). The chemometric tools reduce
the dimension of the data obtained from the untargeted analysis
283
identifying possible patterns among samples. In tea analysis, explorative
unsupervised approaches used to discover the natural grouping of sam-
ples and supervised approaches used in the case of classification and
prediction problems have been applied giving relevant results. Here, a
brief description of the statistical approaches used in tea investigations
is reported.
Principal components analysis (PCA) is commonly used as an
explorative technique in multivariate analysis (Brereton, 2003). It al-
lows the transformation of the multidimensional space of experi-
mental variables into a space with reduced dimensions that are
linear combinations of variables, which are mutually orthogonal,
called principal components. Principal components retain the most
important part of information present in the data, their variability.
Using two or three principal components as variables, data can be vi-
sualized in the new system of coordinates and basic features of data
such as similarity between samples, the presence of outliers and
clustering can be revealed.
Tree clustering analysis (TCA) or hierarchical clustering analysis
(HCA) joins together objects, e.g. samples, into successively larger
clusters, using amalgamation rules for clusters and some measure-
ments of the distance between single objects (Brereton, 2003).
Results are reported as a dendrogram and different classifications
in a given number of groups can be obtained by cutting the tree at
a suitable level.
Partial least square (PLS) regression, PLS-discriminant analysis
(PLS-DA), orthogonal projections to latent structures (OPLS), soft
independent modeling of class analogies (SIMCA), and K-nearest
neighbor (KNN) are termed supervised pattern recognition methods,
whereby a training set with known classes is used to build a mathemat-
ical model which is then evaluated by an independent validation data
set. After this external validation procedure, the models are ready for
classification of unknown samples. PLS regression can be chosen to
create a prediction model (Brereton, 2003) in which the features from
PCA and multiple regression are generalized and combined. PLS studies
the relationship between two data matrices, one matrix containing
data obtained from the analytical methodology, whereas the second
one containing extra information on samples obtained using reference
methodologies. In this way, the relationship between the analytical
data and the other properties of samples can be used to predict these
properties directly using the analytical methodology.
As regards PLS-DA (Barker & Rayens, 2003) and OPLS (Trygg &
Wold, 2002), taking advantages of both statistical approaches (PLS
and LDA) and avoiding their weaknesses, PLS-DA has found many
applications in the analysis of NMR metabolomic data, especially in
the cases when LDA cannot be used for a low number of samples to
number of variables ratio and high collinearity among the variables.
The utilization of class memberships in PLS-DA allows the algorithm
to better expose separations between classes in score space.
Orthogonal projections to latent structures (OPLS) incorporates an
orthogonal signal correction (OSC) filter into a PLS model (Wold,
Antti, Lindgren, & Öhman, 1998).
As a class modeling technique, SIMCA (Wold & Sjöström, 1977)
acts by defining the category space of one class at a time and is
more focused on the similarities among samples of the same class
than on the differences among the classes as in the case of discrimi-
nant analysis. Consequently, each sample can be assigned to one or
more classes or to none of them. In SIMCA, each class model is
defined on the basis of a principal component model of appropriate
dimensionality. KNN attempts to categorize an unknown sample
based on its proximity to samples from the training set. Specifically,
the predicted class of an unknown sample depends on the class of its
K-nearest neighbors. In a fashion analogous to polling, each of the K
closest training set samples votes once for its class; the unknown is
then assigned to the class mostly voted. An important part of the
process is determining an appropriate value for K, the number of
neighbors polled.
284 M. Daglia et al. / Food Research International 63 (2014) 275–289
3.2. NMR methodologies
The strength points of the 1H NMR untargeted metabolomic
approach are the simplicity of sample preparation and the capacity to
detect and quantify a large number of metabolites of a given sample
using a single experiment.
3.2.1. Liquid state NMR
Beverages have usually been analyzed either without any treatment
(fruit juices, vinegars, and olive oils) or using some pre-treatments
(degassing, lyophilization, dilution, and so on) to improve the results.
In the case of tea, the infusion for the NMR analysis is prepared using
directly deuterated solvents. A given amount of tea, finely ground, is
stirred with a given amount of extraction solvent (methanol-d4 or
D2O) at 60–70 °C, allowed to cool and then centrifuged. Finally, the su-
pernatants are lyophilized and dissolved in buffered solution for NMR
analysis. The tea sample preparations reported in the literature provide
the same procedures only differing in the extraction solvent used, the
infusion temperature and time, and the parameters of centrifugations.
1
H NMR is a quantitative technique owing to the proportionality of
signal integral or intensity to the molar concentration of the corre-
sponding metabolite. Generally, two quantification strategies can be
followed, namely, an absolute quantification which provides the
amount of metabolites in a given sample, and a relative quantification
which provides the percentage of a given metabolite in the mixture.
Data coming from NMR spectra can be analyzed using several suitable
chemometric tools.
Fujiwara, Ando, and Arifuku (2006) reports a 1H NMR-based multi-
variate statistical pattern recognition approach, including PCA and
SIMCA for the classification of green, oolong, black and other teas. A
metabolic fingerprinting approach has been used: each 1H NMR spec-
trum has been considered as a fingerprint of the tea sample and all
the NMR resonances have been measured without any metabolite iden-
tification. PCA has been used to survey the whole profile of the samples
and then SIMCA has been carried out for modeling by effective use of a
priori knowledge of the sample sets and select variables that contribute
to tea categorization. The final PCA performed on selected variables has
shown a clear tea classification reflecting the fermentation and process-
ing of each tea, and revealed marker variables that include catechin and
theanine.
Fig. 7. PCA score plot derived from the 1H NMR spectra of green, partially fermented, and
fully fermented teas, indicating clear changes in tea metabolites during tea fermentation.
Raw and ripened Pu-erh teas were from China and indicated partially and fully fermented
teas, respectively. Japanese green teas were from different producers.
Adapted from Lee, Lee, Chung, et al. (2011).
The metabolic behavior of green tea during tea fermentation has also
been investigated by Lee, Lee, Chung et al. (2011) using a 1H NMR
spectroscopy coupled with multivariate PCA. EC, EGC, ECG, EGCG,
theanine, alanine, acetate, quinate, glutamate, caffeine, sucrose, glucose,
and gallate identified in the 1H NMR spectrum have turned out to be
responsible for metabolic differentiation between green tea and
fermented tea by PCA (Fig. 7). During tea fermentation, levels of EC,
EGC, ECG, EGCG, quinate, caffeine and sucrose decrease, whereas gallate
and glucose levels increase.
Le Gall, Colquhoun, and Defernez (2004) have studied the quality of
different green teas using the 1H NMR-based metabolite profiling ap-
proach. Green teas (191 samples) from China, Japan, Vietnam, India,
Indonesia, and Bangladesh gathered over 2 years have been analyzed.
About 31 compounds have been identified in the 1H NMR spectrum of
green tea extracts. In addition to ubiquitous compounds, such as
amino acids, fatty acids and common sugars (glucose and sucrose), phe-
nols, flavonoids, xanthines, and minor sugars have also been identified.
Tea metabolic profiles have been analyzed by PCA and cluster analysis,
suggesting a potential classification of Chinese teas and teas from
other countries. However, as also underlined by the authors, the num-
ber of non-Chinese tea samples was limited and the interpretation of
the results remained open to debate. On the other hand, a clear distinc-
tion between high quality Longjing teas (which are green teas) and
other Chinese teas has been obtained by PCA. Longjing teas has shown
higher levels of theanine, theogallin, gallic acid, caffeine, EGCG, ECG, 2-
O-(β-L-arabinopyranosyl)-myo-inositol, and other minor sugar com-
pounds, and lower levels of fatty acids, EGC, and sucrose compared to
the other teas.
Some differentiations of green teas coming from three different
growing areas (Seogwang, Dosun and Hanman) of Jeju Island (South
Korea) have been studied by Lee et al. (2010). The 1H NMR-based global
metabolic profile, elaborated by PCA and OPLS-DA statistical tools, has
allowed tea samples to be grouped according to the growing areas
characterized by different climatic conditions such as exposure
time, temperature and precipitation. Green teas grown in an area with
high temperature, long sun exposure time, and high rainfall have
shown higher levels of theanine suggesting the importance of these fac-
tors in stimulation of theanine synthesis in green tea during the spring
season and confirming the results obtained by Xu et al. (2012). On the
other hand, these samples have shown lower levels of isoleucine, leu-
cine, valine, alanine, EC, EGC, EGCG, and caffeine than those grown in
areas with relatively low temperature, short sun exposure time, and
low rainfall.
The tea quality of Japanese green tea samples has been investigated
by Tarachiwin, Kobayashi, and Fukusaki (2007) using a combination of
untargeted 1H NMR based metabolomics and a pattern recognition tech-
nique. The metabolic profile of high and low quality samples has been
analyzed by PCA and PLS giving a good discrimination of tea samples
based on their quality. Moreover, in order to evaluate unknown green
tea samples a quality regression model has been employed using green
tea samples with known ranking judged by professional tea tasters.
The dependence of global green tea metabolome on plucking posi-
tions, from young to old leaves (previously investigated by Chiu & Lin,
2005), has also been studied by Lee, Lee, Hwang, et al. (2011) through
1
H NMR analysis coupled with pattern recognition methods, namely
PCA and OPLS-DA (see Fig. 8). Increase in theanine, caffeine, and gallic
acid levels and reductions in catechins, such as EC, EGC, ECG, and
EGCG, glucose, and sucrose levels have been observed, as the green
tea plant grows up. On the other hand, the younger the green tea leaf
is, the more theanine, caffeine, and gallic acid are present but less cate-
chins are accumulated in the green tea leaf, revealing a reverse associa-
tion between theanine and catechin levels due to incorporation of
theanine into catechins in the growing green tea plant. Moreover, the
observation of markedly higher levels of theanine and lower levels of
catechins in green tea stems compared to the tea leaves indicates a
distinction of tea plant metabolism between the tea leaf and the stem.
 M. Daglia et al. / Food Research International 63 (2014) 275–289 285

Fig. 8. Illustration of green tea leaf (left) and representative 1H NMR spectra (right) of green tea leaves plucked at different positions from the youngest or the first (A) to the oldest or the
fourth (D) leaves and stem (E). Asterisks (*) denote unknown compounds; (F) PCA score plot derived from 1H NMR spectra of green tea plucked at different positions from the first (1st, the
youngest or a bud) to the fourth (4th, the oldest) leaves and of stems, showing a strong dependence of green tea metabolome on plucking positions and a clear metabolic difference
between green tea leaves and stems.
Reproduced with permission from Lee, Lee, Hwang, et al. (2011).

The antistaphylococcal activity as well as the metabolic profiling
and polyphenol content of green tea before and after in vitro
simulated gastric, duodenal and gastroduodenal digestion have
been investigated by Marchese et al. (2014). Gastric and duodenal
digested samples have shown antistaphylococcal activity, whereas
gastroduodenal digested samples have not shown any antibacterial
activity. Metabolite analysis, carried out using an explorative
untargeted NMR-based approach and a RP-HPLC–PAD–ESI-MSn
method, showed that green tea polyphenols are stable under gastric
conditions. Duodenal digested samples maintained antibacterial
activity, even if some polyphenols are widely degraded. ECG, under
duodenal digestive conditions, is hydrolyzed to produce EC, whereas
EGCG reacts with digestive enzymes and a galloyl-high molecular
weight derivative is produced. Gastroduodenal digestion results in
degradation of polyphenols, especially Gc, considered as the
polyphenol mainly responsible for the antibacterial activity. These
results explain the loss of activity of gastroduodenal digested sam-
ples and why in vivo green tea has neither protective nor therapeutic
effects against intestinal and systemic bacterial infections.
The 1H NMR based metabolic profiling of black tea cultivated in four
different geographical areas of Sri Lanka has been reported by Ohno
et al. (2011). The cultivation areas were located at different altitudes
from 1800 m down to 300 m above sea level. Using PLS-DA the authors
tried to correlate the differences in metabolic profiles with differences
of the cultivation area. 1H NMR spectra of black tea with the assignment
of major components of metabolic mixture (caffeine, theanine,
theaflavin, theaflavin 3,3′-digallate, thearubigin 3,3′-digallate) have
been reported (see Fig. 9A). For the statistical analysis the entire 1H
NMR spectrum has been binned and the bin integrals have been used
as input variables. The resulting PLS-DA score plot (see Fig. 9B) shows
that the metabolic profile of tea from the highest altitude area (RAN)
is quite different with respect to teas from other locations. Among the
other teas the highest altitude area tea sample is characterized by the
highest content of theanine, caffeine, theaflavin and theaflavin 3,3′-
digallate with the last two components being detected only in this
type of tea. This marked difference has been tentatively explained by
the lower oxygen level and different humidity level that can alter the
fermentation processes during the manufacturing at higher elevations.
286 M. Daglia et al. / Food Research International 63 (2014) 275–289
Fig. 9. (A) Representative 1H NMR spectrum of black tea from RAN. (B) PLS-DA score plot derived from the 1H NMR spectra of black teas from RAN (square), UDA (asterisk), MEDA (di-
amond), and YATA (circle).
Adapted from Ohno et al. (2011).
Reasonably, the difference in the metabolic profile of the raw material –
tea leaves before fermentation – has not been excluded by the authors
as a possible cause of such a difference, and is under further investiga-
tion. Unfortunately, a very small number of samples have been used in
the study to consider the statistical analysis sufficiently robust and the
increasing number of representative samples must be taken into
account in future studies.
Establishing the correlations between sensorial qualities of black tea
and its chemical/metabolic composition could be very important for a
tea quality evaluation method development. Directly measured 1H
NMR spectral profiles of tea are reliable fingerprints of metabolic
composition and can be used as such in the trial to find a correlation
with sensorial qualities (Peňa y Lillo, Sanderson, Wantling, & Pudney,
2006). This study was focused on the contribution of the whole non-
volatile matrix of black tea to its in-mouth sensation, astringency in
particular. A relatively small number of samples, namely eight, repre-
sentative of a wide range of aroma and taste have been used. The aim
of the multivariate statistical analysis (PLS) has been to find an optimal
predictive model for correlation between NMR data and sensory panel
tests with minimum root mean square error. Several regions of 1H
NMR spectrum with the highest contribution to astringency indicate
polyphenolic gallate-like group paired with aliphatic methyl, caffeine,
and a compound with aliphatic methyl and methylene to be responsible
for this correlation. It is clear that a more precise chemical identification
has to be undertaken.
The effects of black and green tea consumption on human metabo-
lism have been compared by Van Dorsten, Daykin, Mudler, and Van
Duynhoven (2006). Urine and blood plasma samples of volunteers
have been analyzed by high-resolution 1H NMR metabolic profiling
combined with multivariate statistics. Green and black tea consump-
tions have resulted in similar increases in urinary excretion of hippuric
acid and 1,3-dihydroxyphenyl-2-O-sulfate, both of which are end prod-
ucts of tea flavonoid degradation by colonic bacteria. Green tea intake
caused a stronger increase in urinary excretion of several citric acid
cycle intermediates, which suggests an effect of green tea flavanols on
human oxidative energy metabolism and/or biosynthetic pathways.
3.2.2. Solid state NMR
The untargeted analysis of tea, carried out mainly using high resolu-
tion 1H NMR in liquid state, has also been performed using solid-state
methodologies directly on tea leaves. A few interesting applications
are reported in the literature. The first application of 13C cross polariza-
tion magic angle spinning (CP-MAS) NMR technique to green and black
tea characterization has been reported by Martínez-Richa and Joseph-
Nathan (2003). NMR spectra have shown complex peak patterns due
to polyphenol, alkaloid, terpene, and carbohydrate components present
in tea leaves. Spectral assignment has been partially obtained using
model compounds (tropolone, (+)-catechin hydrate, gallic acid,
caffeine and flavone derivatives) and literature data. The 13C CP-MAS
spectra of green and black teas have turned out to be quite similar, the
most prominent differences being in the carbonyl peak shape and
position, and in the relative intensities among some peaks. 13 C
CP-MAS spectra of black tea insoluble components after extraction
with hot water have showed peaks due to residual terpenoid and
sesquiterpenoid components and carbohydrates.
Solid-state 1H and 13C CP-MAS NMR methodologies have also been
applied to characterize the theabrownin fraction of Pu-erh tea (Tan,
Peng, Gao, & Gong, 2012). Crude theabrownin extract from tea infusion
 M. Daglia et al. / Food Research International 63 (2014) 275–289
has been fractionated by dialysis and the fraction with molecular
weights between 3.5 and 25 kDa has been analyzed by solid-state
NMR. 1H and 13C CP-MAS NMR experiments have shown the presence
of polymeric compounds such as polysaccharides and polyphenols
with benzene rings and residual protein groups.
3.3. LC–MS and GC–MS methodologies
LC–MS, GC–MS and HPLC–MS are very sensitive and selective
techniques that coupled with suitable chemometric methods can be
very useful tools in metabolomic studies to evaluate the relationships
between the metabolome of vegetable foods/beverages and their
geographical origin, growing treatments, and nutritional and nutraceu-
tical quality.
Ku et al. (2010) have investigated the metabolome changes in green
tea and shade cultured green tea (tencha) by means of LC–MS and GC–
MS coupled with PCA and OPLS-DA. With respect to tencha, green tea
has shown higher levels of galloylquinic acid, EGC, EC, succinic acid, and
fructose, together with lower levels of Gc, strictinin, apigenin glucosyl
arabinoside, quercetin p-coumaroylglucosyl-rhamnosylgalactoside,
kaempferol p-coumaroylglucosylrhamnosylgalactoside, malic acid and
pyroglutamic acid. The effects of some seasonal variations have also
been observed in the primary metabolite concentrations such as
amino acids and organic acids. In addition, green tea has shown
stronger antioxidant activity than tencha in both April and July. The
antioxidant activity of green tea samples has been significantly
correlated with their total phenol and total flavonoid content.
The difference in the nutraceutical properties of green teen coming
from 43 Japanese cultivars has been investigated by Fujimura et al.
(2011) using LC–MS based metabolic approach coupled with PCA and
OPLS-DA. The ability of leaf extracts from different green tea cultivars
to inhibit thrombin-induced phosphorylation of myosin regulatory
light chain (MRLC) in human umbilical vein endothelial cells has been
investigated and among the tested cultivars, Cha Chuukanbohon Nou-
6 and Sunrouge strongly inhibited MRLC phosphorylation. PCA and
OPLS-DA revealed differences among green tea cultivars with respect
to their ability to inhibit MRLC phosphorylation. In the Sunrouge culti-
var, polyphenols have been associated with its unique metabolic profile
and its bioactivity. Finally, using PLS regression analysis, a reliable
bioactivity-prediction model has been built to predict the inhibitory ef-
fect of tea cultivars based on their metabolome.
A comparison of the chemical composition of green tea-based
dietary supplements (GTDSs) and green tea leaves has been carried
out by Sun, Chen, Lin, and Harnly (2011) using an HPLC/MS fingerprint-
ing technique coupled with PCA. ECG, strictinin, trigalloylglucose,
quercetin-3-O-glucosylrhamnosylglucoside, and kaempferol-3-O-
galactosyl-rhamnosylglucoside identified by mass measurements
and MS2 data, turned out to be the components most responsible
for class separation. Flavonol aglycone concentrations are higher in
GTDSs than in tea leaves, indicating the degradation of flavonol
glycosides or the oxidation of catechin during the manufacturing
and storage processes.
An interesting methodology for green tea quality determination has
been developed by Pongsuwan et al. (2007) using a GC-based finger-
printing approach, coupled with time-of-flight mass spectrometry and
PLS analysis. Metabolic fingerprinting of a set of ranked green tea
samples from a Japanese commercial tea contest has been analyzed
and a reliable quality-prediction model has been built.
Fraser et al. (2014) have studied the effect of the fermentation/
oxidation process on the chemical composition of oolong tea produced
in New Zealand, because this topic has not been well understood and
documented. In this investigation 18 sequential samples were collected
during the 36 h oolong production process (from fresh tea leaves to the
final product) belonging to three separate harvest dates, covering
different seasons from late spring to mid-summer and early autumn.
The study was performed using the LC–ESI-MS technique with different
287
types of columns (RP and HILIC) both in positive and negative ESI, to
detect a wide range of compounds with different polarities. The data
obtained from all these analyses were processed with PCA and PLS
multivariate methods, with the first multivariate methods highlighting
the most interesting findings. This approach allowed the identification
of three new metabolic changes occurring throughout the fermentation
process (such as the decrease of flavonoids, the increase of
primeverosides, which could have implications for the flavor and taste
of the beverage, and jasmonic acid metabolites, which have not been
previously reported during the tea manufacturing process) and also
confirmed previously reported biochemical changes, such as the
increase of free amino acids during fermentation, without the need to
perform separate targeted analyses.
4. Conclusion
The growing interest of the scientific community for tea's health
properties has deepened the knowledge about the chemical composi-
tion of this beverage. The development of targeted methods focused
on the different classes of tea compounds and untargeted methods
focused on the metabolite profile of whole beverage allowed an almost
complete picture of the primary and secondary metabolites occurring in
the different tea beverages to be obtained and highlighted the
differences between the different types of commercially available teas.
In recent years, many new derivatives, especially belonging to the poly-
phenol family, have been discovered through the use of MS or NMR or
the combination of both techniques. The only class of compounds, for
which the chemical structure has not been yet fully elucidated to date,
is represented by TRs, as they are very complex polymers with high mo-
lecular weight. Untargeted methods provide a means for the study of
not only tea beverage composition, but also the influence of different
factors such as plant growth conditions, manufacturing processes and
preparation methods on tea composition and the bio-functional
activities, classifying the different types of teas also on the basis of
their nutraceutical properties.
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