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Analytical Methods
School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
Doping Control Center, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
a r t i c l e
i n f o
Article history:
Received 11 October 2011
Received in revised form 11 September 2013
Accepted 25 September 2013
Available online 3 October 2013
Keywords:
Catechin
Monolithic column
High performance liquid chromatography
Green tea
Oolong tea
Black tea
a b s t r a c t
A rapid reversed-phase high performance liquid chromatographic method using a monolithic column for
the determination of eight catechin monomers and caffeine was developed. Using a mobile phase of
water:acetonitrile:methanol (83:6:11) at a ow rate of 1.4 mL min1, the catechins and caffeine were isocratically separated in about 7 min. The limits of detection and quantication were in the range of 0.11
0.29 and 0.330.87 mg L1, respectively. Satisfactory recoveries were obtained (94.2105.2 1.8%) for all
samples when spiked at three concentrations (5, 40 and 70 mg L1). In combination with microwaveassisted extraction (MAE), the method was applied to the determination of the catechins and caffeine
in eleven tea samples (6 green, 3 black and 2 oolong teas). Relatively high levels of caffeine were found
in black tea, but higher levels of the catechins, especially epigallocatechin gallate (EGCG) were found in
green teas.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Tea is one of the most frequently consumed beverages in the
world, dating back to more than 5000 years ago. Numerous studies
have recorded the benecial effects of tea, e.g., anti-oxidant (Vinson & Dabbagh, 1998; Yen & Chen, 1995), anti-carcinoma (Sadzuka,
Sugiyama, & Sonobe, 2000) and arteriosclerosis prevention (Kritz &
Sinzinger, 1997). The major nutraceuticals in teas are the catechins. There is already growing evidence that tea polyphenols reduce the risk of heart diseases and cancer in humans (Crespy &
Williamson, 2004). In some studies, tea has been associated with
antiallergic action (Sano, Suzuki, Miyase, Yoshino, & MaedaYamamoto, 1999) and antimicrobial properties (Greenwalt,
Ledford, & Steinkraus, 1998; Vaquero, Alberto & Nandra, 2007).
Further studies have demonstrated that the co-administration of
drugs with catechins (C), epicatechin (EC) and epigallocatechin gallate (EGCG) inhibits glucuronidation and sulfation of orally administered drugs thereby increasing the bioavailability of such drugs
(Prasain & Barnes, 2007). Moreover some epidemiological studies
have linked the consumption of tea with a lower risk of several
types of cancer including those of the stomach, oral cavity, oesophagus and lungs (Hakim, Harris, Chow, Dean, Brown & Ali, 2004).
Therefore, tea appears to be an effective chemopreventive agent
R, R conformation
OH
OH
OH
OH
HO
O
A
HO
O
A
C
OH
OH
OH
OH
Epicatechin (EC)
Catechin (C)
OH
OH
OH
HO
A
OH
HO
OH
O
A
OH
OH
C
OH
OH
OH
Gallocatechin (GC)
Epigallocatechin (EGC)
OH
OH
OH
HO
A
O
C
HO
A
O
C
O
OH
OH
B
O
OH
OH
OH
OH
OH
Catechin gallate
(CG)
OH
Epicatechin gallate
(ECG)
OH
OH
OH
OH
HO
O
A
OH
C
O
Gallocatechin gallate
(GCG)
O
C
OH
OH
D
OH
OH
OH
OH
D
OH
N
O
OH
Epigallocatechin gallate
(EGCG)
CH3
N
N
O
H3C
HO
B
O
O
OH
OH
263
N
CH3
Caffeine
packing materials, however, are beset by problems of backpressures when higher ow rates are attempted. Separation times of
20 min or more is often required. Monolithic columns represent
an innovative type of column for rapid chromatographic analysis.
In contrast to the conventional HPLC columns, monolith columns
are formed from a single piece of porous silica gel, thus giving them
greater porosity and permeability, allowing chromatographic analyses to be performed in a fraction of the time previously required
(Neue et al., 2007).
A comparative study between the HPLC and capillary electrophoresis (CE) technique for the separation of catechins in tea has
been reported (Bonoli, Pelillo, Toschi, & Lercker, 2003; Lee & Ong,
2000). The CE is in certain ways advantageous due to the shorter
analysis time and reduced consumption of solvents and sample;
but is nevertheless less sensitive due to the short sample path
length of the CE capillary. In addition, the consistency and reproducibility of the CE method were much more difcult to be
264
Quantication of the analytes was based on the peak area using the
external calibration method.
2.3. Analytical characteristics
2.3.1. Linearity
Linearity of the curves was established by injecting standard
mixtures of catechins and caffeine (0.1100 mg L1). Linear least
squares regression was used to calculate the coefcient of determination (R2).
2.3.2. Limit of detection (LOD) and limit of quantication (LOQ)
The LOD and LOQ were determined as follows:
LOD 3 SD=slope
LOQ 10 SD=slope
where SD is the standard deviation.
2.3.3. Repeatability and reproducibility studies
The repeatability and reproducibility of the peak areas were
investigated by injecting each of the standard mixtures (5, 40
and 70 mg L1) on the same day (intra-day) and over ve days
(inter-day), respectively.
2.3.4. Recovery studies
Recovery studies were carried out by spiking three concentrations (5, 40 and 70 mg L1) of a mixture of catechins and caffeine
standards to each type of tea samples studied.
2.4. Optimisation of microwave assisted extraction (MAE)
The tea sample (0.5 g) was placed into an extraction tube
(100 mL) and 25 mL of either (i) acetonitrile:water (1:1), (ii) methanol:water (1:1) or (iii) water:acetonitrile:methanol, (83:6:11)) solvent was added. The extraction tube was put into a microwave
unit (Mars 5, CEM Corporation, USA). The power was set at 300 or
600 W. The suspensions were irradiated with microwave as follows:
210 min (heated to about 80 C at 250, 300, 350 or 400 Psi). It was
allowed to cool for about 15 min. The extract was transferred to a
10 mL volumetric ask (10 mL) and made up to the mark using the
appropriate solvent. The mixture was ltered using nylon syringe
lter (0.45 lm, 13 mm diameter) (Gema Medical, Germany).
3. Results and discussion
3.1. HPLC method development
3.1.1. Effect of mobile phase compositions
The effect of mobile phase compositions viz., (i) water:acetonitrile:methanol (85:6:9); (ii) 84:6:10 and (iii) 83:6:11 were studied
at a xed ow rate (1.0 mL min1). When the rst mobile phase
was used, catechin and caffeine were not well resolved. All the catechins and caffeine were separated when the second composition
was used, however the separation time was relatively long (about
14 min). The third mobile phase provided good separation of the
components in about 12 min, and was thus used for the remaining
studies. The order of elution was: GC > EGC > C > caffeine > EGCG > EC > GCG > ECG> and >CG. As compared to the mobile phases mentioned in the works of Wang, Helliwell, and You
(2000), Fernandez et al. (2000) and Nishitami and Sagesaka
(2004), the present study was found to be the most suitable in
the analysis of catechin monomers and caffeine in tea samples.
For example, Wang et al. (2000) used a mobile phase of acetonitrile:water:ortho phosphoric acid (10:89.9:0.1) for the isocratic
265
Fig. 1. Typical chromatograms for the separation of catechins and caffeine standards using particulate DBS Hypersil gold C-18 (a), monolithic column (b) and green tea (c),
oolong tea (d), black tea (e) samples on monolithic column; isocratic elution with water:acetonitrile:methanol (83:6:11); ow rate,1.4 mL min1. Assignment of peaks: (1)
()-gallocatechin (GC), (2) ()-epigallo catechin (EGC),(3) ()-catechin (C), (4) ()-caffeine, (5) ()-epigallocatechin gallate (EGCG), (6) ()-epicatechin(EC), (7) ()gallocatechin gallate (GCG), (8) ()-epicatechin gallate (ECG), and (9) ()- catechin gallate (CG).
266
Table 2
Analytical characteristics of the developed HPLC method.
Analyte
R2
Intra-day (%RSD) n = 6
Inter-day (%RSD) n = 5
1
Concentration spike, mg L
GC
EGC
Catechin
Caffeine
EGCG
EC
GCG
ECG
CG
0.580
0.580
0.580
0.580
0.580
0.580
0.580
0.580
0.580
0.9997
0.9994
0.9998
0.9991
0.9998
0.9992
0.9997
0.9995
0.9994
0.21
0.24
0.29
0.17
0.22
0.13
0.22
0.27
0.11
0.63
0.72
0.87
0.51
0.66
0.39
0.66
0.81
0.33
40
80
40
80
2.27
2.00
2.26
2.11
1.76
1.92
1.66
1.89
2.05
1.02
2.14
1.59
1.78
1.84
1.61
1.77
0.76
1.00
1.88
1.78
2.53
1.97
2.44
1.89
2.17
2.53
2.35
1.93
1.96
2.39
1.75
1.81
2.17
2.32
1.23
2.49
1.98
2.22
1.78
1.89
0.89
1.95
1.62
1.77
1.09
1.47
2.28
2.21
2.27
1.49
1.88
2.53
2.22
1.98
Table 3
Recoveries of catechins and caffeine in green, black and oolong tea samples.
Concentration
spike, mg L1
Type
Analytes (%)
EGC
Catechin
Caffeine
EGCG
GCG
ECG
CG
97.4 1.2
98.1 1.8
97.1 2.9
96.6 1.9
94.4 2.9
100.8 2.0
98.8 1.4
94.9 1.4
105.2 2.5
100.2 0.8
96.6 2.3
102.1 1.8
102.5 2.0
99.2 0.8
100.2 0.7
99.9 2.1
95.7 1.7
99.4 1.8
101.5 0.9
96.1 2.4
95.7 2.2
99.4 2.2
100.7 2.0
98.3 1.9
100.4 1.6
102.2 2.7
101.1 0.6
40
102.3 2.3
96.7 0.8
95.7 1.9
103.2 1.9
98.1 1.2
98.5 1.1
96.3 2.6
97.2 2.3
100.3 2.1
96.9 1.1
104.2 1.3
101.6 2.9
97.3 2.5
101.5 0.8
105.3 1.8
104.2 0.8
100.9 1.5
97.8 0.8
96.1 1.5
102.3 2.5
99.1 1.7
99.1 1.1
96.4 2.7
102.4 2.1
95.8 2.1
95.4 1.9
98.7 0.7
70
103.9 2.1
97.6 1.1
101.5 1.9
97.3 1.6
105.2 0.9
102.9 2.1
100.1 2.7
100.2 2.1
99.7 2.1
96.3 1.9
102.6 0.4
98.6 2.2
95.8 2.0
103.3 2.1
94.5 1.8
99.5 2.2
101.7 1.6
96.8 2.7
94.6 2.8
96.3 0.3
97.9 2.9
94.2 1.1
99.0 0.8
99.2 1.1
95.1 2.0
99.1 2.6
95.7 0.7
GC
EC
Table 4
Some reported HPLC methods for the analysis of tea avonoids and phenolic acids.
No. Analytes
Detector Column
Dimension
Program LOD
LOQ
Separation References
Time
(min)
1.
Catechins
PDA
Wakosil-II 5 C-18
3 mm 150 mm, 5 lm
Gradient 0.51.2 ng
40
2.
UV
RP-Amine C-16
Gradient 0.22.8 ng
0.76.3 ng
45
DAD
ODS-100Z C-18
Gradient 0.040.89 ng
11
Hu et al. (2009)
UV
UV
C18 packing
Partispere C-18
Isocratic
40
25
UV
Isocratic
50
DAD
DAD
Mightysil Rp-18
20
UV
Monolithic C-18
4.6 mm 100 mm
0.643.86
mg L1
2.28.5 mg
L1
0.33
0.87 mg L1
25
Purine alkaloide,
catechins
Catechins and
caffeine
Isocratic 0.19
1.16 mg L1
Gradient 0.842.98
mg L1
Isocratic 0.110.29
mg L1
3.
4.
5.
6.
7.
8
9
Our method
PDA, photo diode array; UV, ultraviolet visible spectrometry; DAD, diode array detector.
extracted using 350 or 400 Psi, but the lower pressure was used
for the studies.
The tea samples were extracted at different irradiation power
(300 or 600 W) for 6 min. The best extraction was when 600 W
was used. Varying microwave irradiation times (2, 4, 6, 8 and
10 min) using 600 W was then investigated. It was observed that
the extraction efciency for the catechins and caffeine increased
with irradiation time until 6 min and remained stable after
810 min. This value was lower than that reported by Pan, Niu,
267
Samples
GC
EGC
Catechin
Caffeine
EGCG
EC
GCG
ECG
CG
GT
1
2
3
4
5
6
4.24 0.66
5.32 0.11
5.04 0.17
3.76 0.13
4.44 0.23
6.24 0.55
17.0 0.23
17.9 0.39
10.9 0.22
18.5 0.85
12.2 0.17
13.8 0.15
2.36 0.02
3.56 0.23
2.08 0.08
1.24 0.11
2.28 0.12
2.24 0.05
35.7 0.77
38.7 0.89
24.9 0.32
20.4 0.43
30.2 0.88
32.6 0.23
33.7 0.65
36.1 0.54
27.1 0.41
24.6 0.52
24.5 0.16
28.8 0.19
8.32 0.92
9.08 0.14
6.72 0.18
5.56 0.09
7.08 0.29
7.69 0.05
2.38 0.89
2.53 0.06
0.84 0.02
0.96 0.11
0.92 0.13
1.64 0.36
9.48 0.95
10.2 0.32
7.26 0.86
5.06 0.34
6.12 0.27
8.26 0.25
0.22 0.03
0.28 0.08
0.31 0.05
0.22 0.03
0.17 0.04
0.23 0.01
OT
7
8
6.08 0.13
7.69 0.15
26.2 0.62
24.0 0.41
0.64 0.09
0.88 0.11
41.0 0.83
43.3 0.74
13.8 0.22
15.7 0.33
11.1 0.30
12.9 0.30
1.56 0.06
1.44 0.09
12.4 0.36
14.3 0.55
0.22 0.09
0.19 0.07
BT
9
10
11
0.39 0.02
3.32 0.29
7.69 0.15
1.14 0.05
0.84 0.06
0.49 0.06
0.46 0.08
0.42 0.08
0.85 0.10
69.2 0.91
61.1 0.98
63.3 0.88
5.7 0.22
4.6 0.04
5.7 0.33
1.14 0.06
1.46 0.14
1.92 0.30
0.44 0.04
2.72 0.74
1.44 0.09
4.08 0.54
5.72 0.17
4.27 0.55
0.24 0.05
0.19 0.07
0.25 0.07
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