Food Chemistry 81 (2003) 631–638

www.elsevier.com/locate/foodchem

Analytical, Nutritional and Clinical Methods Section

Analysis of green tea catechins: comparative study between

HPLC and HPCE
Matteo Bonoli*, Marco Pelillo, Tullia Gallina Toschi, Giovanni Lercker
Dipartimento di Scienze degli Alimenti, Università di Bologna, Via Ravennate 1020, Cesena (FC), 47023, Italy

Received 11 July 2002; received in revised form 13 November 2002; accepted 13 November 2002

Abstract
A comparison between a borate–phosphate–SDS based MEKC and an RP-HPLC method for the separation of seven tea cate-
chins and gallic acid in a green tea extract is here proposed. Under optimised conditions, HPCE offered several advantages respect
to time of analysis (compounds were separated within 4.5 min), sensitivity (HPCE LODs were about 20–100 times lower than
HPLC ones) and solvent consumption. HPCE displayed excellent migration time repeatability (RSD% on MT < 2%, and RSD%
on RMT < 1%), whereas HPLC showed slightly more quantification ruggedness (total amount catechins RSD% was < 2% for
HPLC and <6% for HPCE). This study highlights the effective possibilities of application of HPCE in the food chemistry field for
routine analysis.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: MEKC; HPLC-DAD; Catechins; Green tea; Antioxidants

1. Introduction varieties, climate and cultivation. Furthermore, for

Green tea, a non-fermented product obtained just by
leaves desiccation, is an excellent source of polyphenolic
antioxidants, which are mainly represented by a group
of compounds having a flavan-3-olic structure and gen-
erally defined as green tea catechins, GTCs. The main
types of catechins present in green tea are seven: (
)-
gallocatechin (GC), (
)-epigallocatechin (EGC), (+)-
catechin (C), (
)-epigallocatechin-3-gallate (EGCG),
(
)-epigallocatechin (EC), (
)-gallocatechingallate
(GCG), (
)-epicatechingallate (ECG). These substances
show a strong antioxidant activity, and are known also
for their effects on human body, such as the anti-
bacterial, antiviral and antiallergenic properties (Dal-
luge & Nelson, 2000; Hertog, Freskens, Hollman,
Katan, & Kromhout, 1993; Hollman, Hertog, & Katan,
1996; Miura et al., 2001; Sesso, Gaziano, Buring, &
Hennekens, 1999).
The amount of green tea catechins varies according to
the type of raw material, in particular to the kind of
* Corresponding author. Tel.: +39-0547-636121; fax: +39-0547-
382348.
E-mail address: mbonoli@foodsci.unibo.it (M. Bonoli).
0308-8146/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0308-8146(02)00565-4
green tea extracts, quantity of GTCs depends mainly on
the technologies applied during extraction, concen-
tration and preservation processes.
The quality control of these extracts must compre-
hend the determination of the total amount of catechins
and the quantification of every single isomer by a rapid
and reproducible method. In literature, as far as the
analysis of GTCs is concerned, there are many applica-
tions by high performance liquid chromatography
(HPLC; Dalluge & Nelson, 2000; Gallina Toschi, Bor-
doni, Hrelia, Bendini, Lercker, & Biagi, 2000; Horie &
Kohata, 2000; Pelillo, Biguzzi, Bendini, Gallina Toschi,
Vanzini, & Lercker, 2002; Sano, Tabata, Suzuki,
Degawa, Miyase, & Maeda-Yamamoto, 2001; Wang,
Helliwell, & You, 2000; Zuo, Chen, & Deng, 2002), but
there are only few examples of analysis by high-perfor-
mance capillary electrophoresis (HPCE) (Aucamp,
Hara, & Apostolides, 2000; Barroso & Van de Werken,
1999; Horie & Kohata, 2000; Larger, Jones, &
Dacombe, 1998; Lee & Ong, 2000; Stach & Schmitz,
2001; Worth, Wielcfsler, & Schmitz, 2000). This pro-
mising analytical technique is still at a developing stage,
especially if compared it to other hyphenated and
already consolidated techniques, such as gas capillary
632 M. Bonoli et al. / Food Chemistry 81 (2003) 631–638
chromatography and liquid chromatography (Robards
& Antolovich, 1997). HPCE is characterised by a nota-
ble separation efficiency (normally ranging from 105 to
106 theoretical plates) and sensitivity, short analysis
times, small sample volumes, low running costs and the
possibility of working mainly with water mobile phases.
This allows the lowering of laboratory costs for the sol-
vents purchase and dismantlement, and it has a positive
impact on the environment. For these reasons, in the last
years, the number of publications regarding the applica-
tion of CE in food analysis has been considerably
increased (Corradini & Cavazza, 1998; Issaq, 1999; Lin-
deberg, 1996a, 1966). In addition, the flexibility of this
technique, which was primarily used for charged mole-
cules of big dimensions, is now increased by various
operative modes, having a wide range of applications.
This work proposes a comparison between a MEKC
and an HPLC separation and quantitation of seven
catechins and gallic acid, in a purified green tea extract.
The MEKC and the HPLC methods cited were previously
optimised in our laboratory (Bonoli, Gallina Toschi, &
Lercker, 2002; Bonoli, Colabufalo, Pelillo, Gallina
Toschi, & Lercker, submitted for publication; Pelillo et al.,
2002), and this study highlights the effective possibilities of
application of CE in the food chemistry field.
2. Materials and methods
2.1. Reagents and chemicals
(
)-Gallocatechin (GC, 98%), (+)-catechin (C,
98%), (
)-epigallocatechin (EGC, 98%), (
)-epigallo-
catechin-3-gallate (EGCG, 95%), (
)-epicatechin (EC,
purity not specified), (
)-epicatechingallate (ECG,
98%), (
)-gallocatechingallate (GCG, purity not speci-
fied) and Gallic acid (GA, 99%) were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Sodium
dodecyl sulfate for HPCE, water for HPCE, 0.1 M
sodium hydroxide, 1 M sodium hydroxide and 0.1 M
hydrochloric acid were from Fluka (Neu-Ulm, Switzer-
land). Potassium dihydrogen phosphate, HPLC-grade
methanol and formic acid were purchased from Carlo
Erba Reagents (Milan, Italy). HPLC-grade water,
HPLC-grade acetone and 50 mM sodium tetraborate
(pH 9.3) for HPCE were from Merck (Darmstadt, Ger-
many). HPLC-grade acetonitrile was supplied by Pro-
labo (Paris, France). All the other chemicals and solvents
were high-analytical grade. Double distilled water was
prepared in our laboratory from deionized water.
2.2. Sample preparation
Indena (Milan, Italy) kindly donated green tea extract
(GTE, GreenselectTM). This aqueous spray-dried
extract contains less than 0.1% of caffeine (w/w, HPLC
determination), 60% of polyphenols (expressed as
EGCG; w/w, HPLC determination), and not less than
40% of EGCG. A 1000 mg/ml GTE solution was pre-
pared by dissolving 100 mg of GTE in 100 ml of an
HPLC-grade water/formic acid solution (99.7/0.3, v/v).
Samples to be analysed by MEKC were added with
acetone (GTE/acetone, 99/1, v/v) as a marker, since it
does not interact with the micelles.
2.3. Standard preparation and method evaluation
The stock solutions were prepared dissolving 0.5–1
mg of each standard compound in 1 ml of HPLC-grade
water/formic acid solution (99.7/0.3, v/v). Each stock
solution was then used for the preparation of the diluted
solutions, for the calibration curves (from 0.005 to 1000
mg/ml for ECG, EC, EGC, GCG, EGCG and GA; from
0.005 to 500 for C; from 0.005 and 200 mg/ml for GC).
For both analytical methods, each point of the cali-
bration curves corresponded to the mean value obtained
from three injections. Detection limits (LODs) were
estimated from the calibration curves (signal/noise
ratio, S/N, equal to 3).
The intra-assay precision was calculated from ten
consecutive determinations of the same GTE sample;
the inter-assay precision was calculated from five differ-
ent samples. Both statistical parameters were re-assessed
1 week later, using the same samples.
2.4. Micellar electrokinetic capillary chromatography:
instrument and conditions
Electrophoretic analyses were performed using a
Beckman P/ACE 5000 model equipped with an UV–vis
detector (Beckman Instruments, Inc., Fullerton, CA). A
PC, equipped with Beckman P/ACE Station software,
accomplished data acquisition and processing. The
capillary cartridge contained undeactivated fused silica
tubing (50 mm i.d.375 mm o.d.), supplied from Beck-
man. Total capillary length was 47 cm, whereas effective
length was 40 cm.
The capillary cartridge was conditioned by flushing
with 1 M sodium hydroxide for 3 min, 0.1 M sodium
hydroxide for 3 min, HPCE-grade water for 3 min and
finally with the running buffer for 5 min; when not in
use, it was stored in water to prevent buffer crystal-
lization. The running buffer was daily prepared by mix-
ing three parts of 20 mM potassium dihydrogen
phosphate (solid salt diluted in HPLC-grade water), one
part of 50 mM sodium tetraborate and two parts of 200
mM sodium dodecyl sulfate (solid salt diluted in HPLC-
grade water). It was optimized to pH=7 by adding 0.1
M hydrochloric acid; it was then filtered through a cel-
lulose acetate 0.2 m syringe filter (Orange Scientific,
Waterloo, Belgium) and sonicated in a ultrasonic bath
for 10 min.
 M. Bonoli et al. / Food Chemistry 81 (2003) 631–638
Samples were injected hydrodynamically in the anodic
end at low pressure mode (0.5 psi) for 1 s. Electro-
phoretic run was carried out at 30 kV for 5 min, main-
taining the capillary temperature at 29  C, resulting in a
current of approximately 85 mA. Between each run the
capillary was rinsed at high pressure (20 psi) with
NaOH 0.1 M for 2 min, HPCE-grade water for 2 min
and then re-equilibrated with the running buffer for 2
min. At the end of each run, the capillary cartridge was
also rinsed with HPCE-grade water for 2 min. All
washing steps were performed at 29  C. The running
buffer was changed every three runs.
UV detection was performed at 200 nm; rise time was
set at 0.17 s and data rate was 10 Hz. Peak identification
was carried out by spiking the GTE sample with stan-
dard compounds.
2.5. High-performance liquid chromatography:
instrument and conditions
HPLC analyses were performed on a HP Series 1100
(Hewlett Packard, Wilmington, DE, USA), equipped
with a binary pump delivery system, a degaser (model
G1322A), an autosampler (Automatic Liquid Sampler,
ALS, model G1312A), a HP diode-array UV–vis detec-
tor (DAD, model G1315A); integration and data ela-
boration were performed by ChemStation software
(Hewlett Packard). A column RP LunaTM 5 mm C18, 25
cm3.0 mm i.d. (Phenomenex, Torrance, CA, USA)
with Rheodyne precolumn filter 7335 model, was used.
All solvents were filtered with 0.45mm Millipore filter
disk. The following gradient elution was carried out:
mobile phase A, double distilled water/methanol/formic
acid (74.7/25/0.3; v/v/v); mobile phase B, acetonitrile/
formic acid (99.7/0.3; v/v). The linear gradient elution
system was: 100% A for the first 8 min, and then
reached the 100% B in 24 min, standing at 100% B for 6
min. The gradient returned to 100% A in 4 min, stand-
ing at 100% A for others 6 min as post-time. The flow
rate was 0.5 ml/min. The quantification of the catechins
by DAD was performed at 270 nm. Of each sample 1 ml
was injected, after filtration through a 0.45-mm filter
disk.
Identification of compounds was carried out by com-
paring retention times and UV spectra of the unknown
peaks to those of the standards.
3. Results and discussion
3.1. HPLC and MEKC analyses
As previously cited, the optimization of both methods
was previously performed in our laboratory (Bonoli et
al., 2002, submitted for publication; Pelillo et al., 2002),
with a standard mixture constituted by the seven cate-
633
chins cited in this work, gallic acid and caffeine. To
optimize the capillary electrophoresis method, theo-
bromine and theophylline were added to the standard
mixture too. At the optimized conditions, the HPCE
method permitted whole separation of those 11 com-
pounds, while two peaks, corresponding to GA and
GC, have not been resolved by the HPLC method. In
fact, the separation of GA from GC is not easy to
obtain, and most of works in literature does not report
their separation (Dalluge & Nelson, 2000; Horie &
Kohata, 2000), but using long elution gradients (Wang
et al., 2000). Applying both optimized methods caffeine
was completely separated to the others compounds,
even if it was not detected in here-analyzed extract. It is
important to separate caffeine to others components,
since it is one of the main substances in tea infusions.
Both optimised methods were employed to perform
analyses of real tea infusions, but only the here-reported
GTE extract was utilized to compare these two analy-
tical methods (Bonoli et al., 2003 in press; Pelillo et al.,
2002). To date, HPLC determination of tea catechins
have been prevalently carried out by using RP octadecyl
silyl columns (25 cm4.6 mm i.d) (Horie & Kohata,
2000). From a preliminary work, we have demonstrated
that using a 3.0 mm i.d. column, the resulting improve-
ment in LODs (limits of detection) was higher than one
order of magnitude, and the retention times of the tes-
ted compounds, in the two types of columns, were
similar (paper to be published).
As shown in Fig. 1, the seven catechins were sepa-
rated within 18 min; the total analysis time was about 40
min, including the 6 min for column conditioning after
each run. The elution order was: GC (coeluting with
GA), EGC, C, EGCG, EC, GCG and ECG. Fig. 1A
shows the separation of a standard mixture (all stan-
dard solution were 100 mg/ml), while Fig. 1B shows the
typical chromatogram of a GTE sample (concentration:
1000 mg/ml; injection volume: 1 ml). Detection limits
ranged from 0.385 to 0.025 mg/ml, which correspond to
EGC and GA, respectively.
In literature, HPCE catechin separation methods are
very heterogeneous, in terms of application modes
(CZE, MEKC), kind of buffer (background electrolytes
composition, micellar agents and their concentration)
and run conditions (applied voltage, capillary tempera-
ture). This fact may be due to the relatively recent
introduction of this technique in food analysis.
Fig. 2 shows the HPCE-electropherograms of the
same samples (standard mixture and GTE solution,
respectively). The main advantage of here developed
capillary electrophoresis method, respect to the HPLC
method, was the whole separation of the eight com-
pounds each others, in a very short time (within 4.5
min); as earlier cited, the separation of GA from other
polyphenolic species is not easy to perform, especially
from GC. Furthermore, the HPLC GA–LOD is about
 634
M. Bonoli et al. / Food Chemistry 81 (2003) 631–638
Fig. 1. (A) HPLC-chromatogram of pure standards (all standard solutions were 100 mg/ml); (B) HPLC-chromatogram of GTE sample (concentration: 1000 mg/ml; injection volume: 1 ml). UV
detection was performed at 270 nm; the other analytical conditions are described in the methodology section. Peak identification: GC, (
)-gallocatechin; EGC, (
)-epigallocatechin; C, (+)-catechin;
EGCG, (
)-epigallocatechin-3-gallate; EC, (
)-epigallocatechin; GCG, (
)-gallocatechingallate; ECG, (
)-epicatechingallate; GA, gallic acid.
 M. Bonoli et al. / Food Chemistry 81 (2003) 631–638
Fig. 2. (A) HPCE-electropherogram of the standard mixture (C, 500 mg/ml; GC, 200 mg/ml; EGC, 1000 mg/ml; EGCG, 1000 mg/ml; GCG, 1000 mg/ml; ECG, 1000 mg/ml; EC 1000; GA, 1000 mg/ml);
(B) HPCE-electropherogram of GTE sample. UV detection was performed at 200 nm; the other analytical conditions are described in the methodology section. Peak identification: GC, (
)-gallo-
catechin; EGC, (
)-epigallocatechin; C, (+)-catechin; EGCG, (
)-epigallocatechin-3-gallate; EC, (
)-epigallocatechin; GCG, (
)-gallocatechingallate; ECG, (
)-epicatechingallate; GA, gallic acid;
Ac, acetone (EOF marker).

635
636 M. Bonoli et al. / Food Chemistry 81 (2003) 631–638

20 times higher than the HPCE GA–LOD. Although
the RSD% for GA quantification, by HPCE, was
somehow high (> 30%), the fact that it was possible to
detect this compound by this technique is quite impor-
tant from the qualitative standpoint.
The slight difference between migration times of
the components of the standard mixture and those
of the GTE samples (see Fig. 2A and B) could be
due to the different analytes concentration
employed.
The elution order is listed as follows: GC, C, EGC,
EGCG, GCG, ECG, EC and GA. These eight com-
pounds were completely separated within 4.5 min; a whole
analysis required about 12 min (rinse steps included) and
no sample clean-up pre-treatment was necessary.
3.2. HPLC and MEKC performances
Table 1 shows performance parameters (calibration
parameters, intra- and inter-assay precision), of GTE
samples of both HPLC and MEKC methods. The
correlation coefficients (r) of the calibration curves (cal-
culated using peak areas) are not significantly different,
while HPCE-LODs are 20–100 times lower than those
obtained by HPLC. Probably, we could justify lower
LODs of the MECK method respect to the HPLC
method mentioning the higher sensitivity in the detec-
tion of catechins at 200 nm and the higher signal-to-
noise ratio at this wavelength (Lee & Ong, 2000).
Unfortunately, it was not possible to use 200 nm as
detection wavelength in HPLC due to the interference
of the mobile phase.
Relative standard deviations of HPLC retention times
ranged from 0.20 to 4.14%, whereas average RSD% of
HPCE migration times varied between 0.17 and 1.78%.
Considering relative migration times (RMT) with ace-
tone as marker, the RSDs% did not reach 1% (max.
value was 0.89%). In terms of quantification, the results
obtained by HPLC method displayed lower variability
than those of HPCE: RSDs% of total amounts of cate-
chins were 0.77–1.72% and 1.01–5.54% for HPLC and
HPCE, respectively (Table 1). Nevertheless, data varia-

Table 1
HPLC and HPCE calibration curves parameters and repeatability study on quantification (values are expressed in mg of catechins for 100 mg of
extract)

Analytes r Areasa LOD (mg/ml)b First quantification (n=5) After 1 week quantification (n=5)

INTRA-assay INTER-assay INTRA-assay INTER-assay

MeanS.D. %RSD MeanS.D. %RSD MeanS.D. %RSD Mean S.D. %RSD

GC-HPLC 0.995 0.166 4.23 0.15 3.58 4.400.13 2.95 4.550.11 2.50 4.580.10 2.10
GC-HPCE 0.997 0.0013 1.21 0.05 4.38 1.160.11 9.62 1.160.08 7.26 1.190.04 3.07

C-HPLC 0.999 0.224 0.83 0.02 2.59 0.800.06 7.06 0.890.08 8.70 0.860.03 3.53
C-HPCE 0.992 0.0012 0.75 0.02 2.38 0.720.04 5.17 0.770.03 3.32 0.760.01 1.39

EGC-HPLC 0.999 0.385 11.82 0.15 1.27 11.920.16 1.38 12.070.12 0.96 12.130.26 2.17
EGC-HPCE 0.999 0.0017 10.34 0.21 2.06 10.740.77 7.13 10.080.89 8.79 10.680.35 3.23

EGCG-HPLC 0.999 0.192 38.78 0.40 1.02 39.070.30 0.77 40.300.50 1.24 40.290.67 1.67
EGCG-HPCE 0.999 0.0018 34.46 0.51 1.47 34.521.95 5.66 36.651.91 5.20 37.161.12 3.03

GCG-HPLC 0.997 0.084 0.69 0.03 4.35 0.720.03 3.94 0.850.04 4.56 0.790.05 6.60
GCG-HPCE 0.994 0.0033 1.10 0.11 10.12 1.190.11 9.36 1.260.15 11.69 1.240.08 6.44

ECG-HPLC 0.998 0.040 10.10 0.16 1.56 10.040.09 0.87 10.550.30 2.80 10.310.15 1.47
ECG-HPCE 0.999 0.0023 8.77 0.24 2.77 8.810.30 3.37 9.520.49 5.13 9.740.51 5.28

EC-HPLC 0.999 0.321 5.55 0.14 2.57 5.570.12 2.22 6.210.33 5.37 5.910.24 4.11
EC-HPCE 0.999 0.0023 5.07 0.14 2.70 4.920.29 5.82 5.330.12 2.27 5.470.13 2.29

GA-HPLC 0.999 0.0250 NDc – NDc – NDc – NDc –

GA-HPCE 0.999 0.0051 0.06 0.02 31.49 0.090.03 31.07 0.100.04 37.14 0.110.04 35.92

TOT-HPLC 71.98 0.56 0.77 72.520.64 0.88 75.400.73 0.96 74.881.29 1.72
TOT-HPCE 61.77 0.62 1.01 62.153.45 5.54 64.382.72 4.22 66.341.91 2.88
a
Correlation coefficients of the calibration curves using peak area.
b
For S/N=3
c
Not detectable.
 M. Bonoli et al. / Food Chemistry 81 (2003) 631–638
Table 2
Differences in total amount of catechins and gallic acid (TACs) found
by HPLC and HPCE


a

d
S:D:b t0 Significativityc
Total amount of catechins 10.04 1.06 18.94 S
Pn
a ðTACsHPLC;i
TACsMEKC;i Þ
d ¼ i¼1 ; n ¼ 4:
b
n
Standard deviation (n =4).
c
S=significant; NS=not significant.
bility in HPCE is highly acceptable for a hyphenated
analytical technique. As you can see in Table 1, no GA
data have been reported in HPLC quantification. Only
GC quantification data have been tabulated because,
from further mass spectrometry analyses, GA was not
detected (Pelillo et al., 2002). This evidence confirmed
that the sensitivity of capillary electrophoresis permitted
a better qualitative investigation of this extract than
liquid chromatography.
Regarding elution time, HPCE is much faster than
HPLC, since total analysis time for the eight compo-
nents is 12 min for this MEKC method whereas HPLC
separation is achieved in 40 min. Table 2 shows the sta-
tistical interpretation of results. The following formula
was applied:




d

t0 ¼ pffiffiffi
SD= n



where
d
is defined as the average difference between
total amount of catechins found by HPLC and that
found by MEKC, SD is the standard deviation and n is
the number of replications. Finally, value was compared
with t-Student value (t ¼0:05
n
1¼3 ¼ 2:35); the repeatability
hypothesis was rejected if t0 > t ¼0:05
n
1¼3 . In fact, the sig-
nificant difference between the total amount of catechins
found with the two analytical methods can be attributed
to the different peak area integration software used
(Faller & Engelhardt, 1999; Reijenga & Lee, 2001), and
to the higher efficiency and sensitivity of HPCE than
HPLC. Nevertheless, another factor that could have
influenced these results could be the response variation
related to the different wavelength used for the compo-
nent detection (200 and 270 nm for CE and HPLC,
respectively). Indeed, significant difference between the
quantification of some compounds by the HPCE and
HPLC methods could be due to interferences in the
sample which could have a different weight at the two
selected wavelength. As earlier mentioned, it was not
possible to use 200 nm as detection wavelength for both
methods because the HPLC underwent interference by
the mobile phase.
637
4. Conclusions
Two different analytical techniques (MEKC and
HPLC) for the analysis of green tea catechins, were
compared. This study highlights the effective possibility
of application of HPCE in the food chemistry field. The
MEKC method here suggested show higher sensitivity,
resolution, efficiency and migration times repeatability
than the HPLC method, even if the latter displayed a
slightly better repeatability in the quantification of total
amount of catechins. It should be pointed out that
HPCE LODs were about 20–100 times lower than
HPLC ones, which is extremely advantageous when
analyzing real systems, such as food products. More-
over, HPCE is very convenient in terms of analysis time
(12 min for total analysis against 40 min for the HPLC
one) and solvents consumption for routine analysis of
tea extracts.
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