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Analytical Methods

Rapid tea catechins and caffeine determination by HPLC using

microwave-assisted extraction and silica monolithic column

A.A. Rahim a,⇑, S. Nofrizal a, Bahruddin Saad a,b

a School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysiab 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 form11 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 flowrate of 1.4 mL min1, thecatechins and caffeine were iso-

cratically separated in about 7 min. The limits of detection and quantification were in the range of 0.11–

0.29 and 0.33–0.87 mg L 1, respectively. Satisfactory recoveries were obtained (94.2–105.2 ± 1.8%) for all

samples when spiked at three concentrations (5, 40 and 70 mg L 1). In combination with microwave-

assisted 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 beneficial effects of tea, e.g., anti-oxidant (Vin-

son & Dabbagh, 1998; Yen & Chen, 1995), anti-carcinoma (Sadzuka,

Sugiyama, & Sonobe, 2000) and arteriosclerosis prevention (Kritz &

Sinzinger, 1997). The major nutraceuticals in teas are the cate-

chins. There is already growing evidence that tea polyphenols re-

duce 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, & Maeda-

Yamamoto, 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 gal-

late (EGCG) inhibits glucuronidation and sulfation of orally admin-

istered 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, oesoph-

agus and lungs (Hakim, Harris, Chow, Dean, Brown & Ali, 2004).

Therefore, tea appears to be an effective chemopreventive agent

for toxic chemicals and carcinogens (Karori, Wachira, Wanyoko,& Ngure, 2010).

The weight percentage of the soluble ingredients account for as

much as 30% of tea, which differs according to variety, production

area, climate, and processing conditions (Bronner & Beecher, 1998;

Han, Tian, & Chen, 1997; Lu, Chu, Yan, & Chen, 2009). The content

of soluble ingredients in green tea is generally greater than those in

black tea as the latter is completely fermented. Polyphenols consti-

tute the major portion of the soluble ingredients and are also the

essential components of tea which have physiological functions.

Catechins are the primary polyphenols in the tea, and accounts

for 75–80% of the soluble ingredients (Lu et al., 2009).

Caffeine is the major alkaloid of tea, present in the range of 

3.0–4.0% (Fernandez, Martin, Gonzalez, & Pablos, 2000; Naik &

Nagalakshmi, 1997; Zhang, Lin, He, & Petteruti, 2002). In humans,caffeine stimulates the heart (Ammon, 1991; Ashihara, Sano, &

Crozier, 2008), central nervous system (Davis, Zhao, Stock, Mehl,

Buggy & Hand, 2003; Nehlig, Daval, & Debry, 1992), and the respi-

ratory system (Doherty & Smith, 2005; Richmond, 1949). It is a

diuretic and has the effect of delaying fatigue (Grandhi, Donnelly,

& Rogers, 2007; Haskell, 1926). The structures of the various

catechin monomers and caffeine are shown in Table 1.

High performance liquid chromatography (HPLC) is by far the

most popular method for the analysis of tea catechins, gallic acid,

purine alkaloids, theanine, etc. (Peng, Song, Shi, Li, & Ye, 2008). In

these methods, the stationary phase used is based on particulate

packing materials (mainly C18 with 5 lm particle size). Particulate

0308-8146/$ - see front matter    2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.09.131

⇑ Corresponding author. Tel.: +60 4 653388; fax: +60 4 6574854.

E-mail address:  afidah@usm.my (A.A. Rahim).

Food Chemistry 147 (2014) 262–268

Contents lists available at  ScienceDirect

Food Chemistry

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packing materials, however, are beset by problems of backpres-

sures when higher flow 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 anal-

yses to be performed in a fraction of the time previously required

(Neue et al., 2007).

A comparative study between the HPLC and capillary electro-

phoresis (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 repro-

ducibility of the CE method were much more difficult to be

achieved compared to the HPLC method (Arce, Rios, & Valcarcel,

1998). Ultra high performance liquid chromatography (UPLC) rep-

resents another interesting development in separation science

(Klejdus, Vacek, Lojková, Benesova, & Kuban, 2008). The higher

separation efficiency made possible by using sub-2lm particle

stationary phase allows faster separation without compromising

the resolution (Guillarme, Nguyen, Rudaz, & Veuthey, 2007). UPLC

methods for the determination of flavonoids and phenolic acids

have been developed (Gruz, Novak, & Strnad, 2008). The UPLC sep-

aration of 29 phenolic compounds (including eight catechins) was

feasible in about 20 min (Nováková, Spácil, Seifrtová, Opletal, & So-

lich, 2010). However, the UPLC unit is costly and requires higher

maintenance costs.

Generally, before analytes are ready for the analytical determi-

nation, they need to be isolated from the sample. Liquid–liquid

extraction, reflux and ultrasonic treatment for the extraction of 

catechins and caffeine in tea samples were reported (Buqing, Huil-

ing, Qingling, Buchang, & Shengfang, 1996; Dawidowicz & Wia-

nowska, 2005). While effective, these conventional sample

preparation techniques are laborious, time-consuming, and require

large amounts of solvents. Microwave-assisted extraction (MAE)

has received considerable attention as an alternative extraction

technique (Spigno & De Faveri, 2009). The technique is viewed as

‘‘green’’ as the extraction is carried out in vessels in a closed envi-

ronment, and has been demonstrated to be time- and energy-sav-

ing and highly efficient. It has been widely used for the extraction

of analytes from plants and herbs (Li, Huang, Tang, & Deng, 2010).

In the present studies, a new HPLC method using a monolithic col-

umn was developed for the simultaneous determination of eight

catechins and caffeine in tea. In conjunction with the MAE, the

method was applied to the determination of catechins and caffeine

in several green, black and oolong teas. The requirement of rapid

methods for determination of catechins in the standardization of 

nutraceuticals, clinical, industrial processing of tea beverages and

evaluation of stability of catechin in bakery products are some

other justifications for the development of a rapid analytical meth-

od (Nishitani & Sagesaka, 2004).

2. Materials and methods

 2.1. Materials

Caffeine, methanol and acetonitrile of HPLC grade were pur-

chased from Merck (Darmstadt, Germany). () catechin (C), ()

epicathecin (EC), () epigallocatechin gallate (EGCG), () gallo-

catechin gallate (GCG), () epicathechin gallate (ECG), ()- cate-

chin gallate (CG), ()-gallocatechin (GC) and ()-epigallo

catechin (EGC) standards were purchased from Sigma Aldrich

(Steinheim, Germany). Tea samples were purchased from super-

markets in Penang, Malaysia and were produced from Malaysia,

United Kingdom, Japan, Taiwan, Indonesia and China.

 2.2. Instrumentation and chromatographic conditions

The HPLC system consisted of two LC-10 AD VP pumps and

SPD-10Avp UV detector (Shimadzu, Japan). The mobile phase

composition that comprises of water:acetonitrile:methanol with

compositions of 85:6:9, 84:6:10, 83:6:11 were studied at different

flow rates (1.0, 1.2 and 1.4 mL min1). Monolithic Rp-18 e 100–

4.6 mm (Merck KGaA, Germany) and BDS Hypersil gold C-18

(4.6 mm I.D. 250 mm) columns were used. Signals were moni-

tored at 280 nm. Standard solutions and tea samples were filtered

through a 0.45 lm acrylic polymer filter before being injected into

the HPLC unit. Peak areas versus concentrations were plotted usinglinear least-squares analysis. 20lL of samples were injected.

 Table 1

Structure of catechin monomers and caffeine.

R, S conformation R, R conformation

OH

OH

HO O

OH

OH

Catechin (C)

 A C

B

OH

OH

HO O

OH

OH

Epicatechin (EC)

 A C

B

OH

OH

HO O

OH

OH

OH

Gallocatechin (GC)

 A  C

B

OH

OH

HO O

OH

OH

OH

Epigallocatechin (EGC)

 A   C

B

OH

HO O

OH

OH

OH

OH

OH

O

O

Catechin gallate

(CG)

 A C

B

 D

OH

HO O

OH

OH

OH

OH

OH

O

O

Epicatechin gallate

(ECG)

 A C

B

D

OH

HO O

OH

OH

OH

OH

OH

O

O

OH

Gallocatechin gallate

(GCG)

 A C

B

D

OH

HO O

OH

OH

OH

OH

OH

O

O

OH

Epigallocatechin gallate

(EGCG)

 A C

B

D

O   N

CH3

N

CH3

N

NH3C

O

Caffeine

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Quantificationof the analytes was based on the peak area usingthe

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 (0.1–100 mg L 

1

). Linear leastsquares regression was used to calculate the coefficient of determi-

nation (R2).

 2.3.2. Limit of detection (LOD) and limit of quantification (LOQ)

The LOD and LOQ were determined as follows:

LOD ¼ 3 SD=slope

LOQ   ¼ 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, 40and 70 mg L 1) on the same day (intra-day) and over five days

(inter-day), respectively.

 2.3.4. Recovery studies

Recovery studies were carried out by spiking three concentra-

tions (5, 40 and 70 mg L 1) 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 mLof either (i)acetonitrile:water (1:1), (ii) metha-

nol:water (1:1) or (iii) water:acetonitrile:methanol, (83:6:11)) sol-

vent was added. The extraction tube was put into a microwaveunit (Mars 5, CEM Corporation, USA). The power was set at 300 or

600 W. Thesuspensions were irradiatedwith microwaveas follows:

2–10 min (heated to about 80 C at250, 300, 350 or400Psi). It was

allowed to cool for about 15 min. The extract was transferred to a

10 mL volumetric flask (10 mL) and made up to the mark using the

appropriate solvent. The mixture was filtered using nylon syringe

filter (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:acetoni-trile:methanol (85:6:9); (ii) 84:6:10 and (iii) 83:6:11 were studied

at a fixed flow rate (1.0 mL min1). When the first mobile phase

was used, catechin and caffeine were not well resolved. All the cat-

echins 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 > caf-

feine > EGCG > EC > GCG > ECG> and >CG. As compared to the mo-

bile 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 acetoni-trile:water:ortho phosphoric acid (10:89.9:0.1) for the isocratic

separation and determination of catechin monomers and caffeine.

A reasonable separation with distinct multiple peaks was achieved

for gallic acid, GC, caffeine, EGCG, EC, GCG and ECG with this sol-

vent system. However, EGC and C monomers were eluted as a sin-

gle peak and furthermore required longer analysis time (50 min).

 3.1.2. Effect of flow rates

The effect of flow rates (1.0, 1.2 and 1.4 mL min1) on the sep-

aration of the analytes were studied. It was noted that retention

time decreased with increase in flow rate and all the peaks were

well resolved. Therefore, 1.4 mL min1 was chosen.

The adopted HPLC conditions were: isocratic elution using mo-

bile phase composition of water:acetonitrile:methanol (83:6:11);

flow rate, 1.4 mL min1; monitored at 280 nm. Under these condi-

tions, all the analytes were separated in less than 7 min. As com-

parison, the analytes were also separated using a particulate DBS

Hypersil C-18 column (Fig. 1a) with the same HPLC conditions.

The use of this column resulted in good separations, but the sepa-

ration time was longer (about 30 min). Similar order of elution to

that of the monolithic column was found (Fig. 1b), suggesting sim-

ilar separation mechanism between the columns.

 3.2. Analytical characteristics

The results of developed HPLC method were validated in terms

of linearity, LOD, LOQ, precision and recovery.

 3.2.1. Linearity

Linear calibration curves were obtained by plotting the peak

area against the concentration of the respective standards and

were found to be linear over the range 0.1–80 mg L 1 (Table 2).

All the analytes showed good linearity with coefficient of determi-

nation (R2) ranging from 0.9989 to 0.9998 for the eight catechins

standards and caffeine.

 3.2.2. Limit of detection (LOD) and limit of quantification (LOQ)

A blank sample that was spiked with 1 mg L 1 of each standardwas used for the measurement of LOD and LOQ. Calibration curve

was constructed, the standard deviation and slope were used to

calculate the LOD and LOQ. The LOD and LOQ for these catechins

and caffeine (Table 2) were comparable to that reported by Yang,

Ye, Xu, and Jiang (2007) and Wang, Provan, and Helliwell (2003).

 3.2.3. Repeatability and reproducibility studies

The repeatability of the peak area was assessed by injecting

mixtures of each standard (n = 6). The relative standard deviation

(RSD) for the peak area was found to range between 0.76% and

2.53% (Table 2). The reproducibility over different days was carried

out by injecting the same standard solution over 5 days. Good

reproducibility of the peak area (RSD6 2.55%) was found for all

experiments (Table 2).

 3.2.4. Recovery studies

Recovery studies were carried out by spiking three concentra-

tions of a mixture of catechins and caffeine standards to each type

of tea samples. Good recoveries of 94.2–105.2 ± 1.8% were found

for all the spiked samples (Table 3).

 3.3. Comparison with other HPLC methods

Table 4 summarises the main analytical characteristics of some

recent HPLC methods for the determination of catechins and re-

lated compounds. The list is by no means exhaustive, but rather

serves to illustrate the important features of the reported HPLC

techniques. As mentioned earlier, all the previous reports usedparticle-type stationary phase and gradient elution was preferred

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as shorter analysis times and improved resolution can be realised.

The superiority of our proposed method based on monolithic col-

umn due to the short separation time (7 min) is evident. Separation

times rivaling to those of UPLC systems can be realised by employ-

ing suitable gradient programmes.

 3.4. Optimisation of microwave assisted extraction (MAE)

Several parameters viz., type of solvent, pressure, power and

time for the microwave irradiation were studied and optimisedin order to obtain suitable extraction conditions of the interested

tea components. Supporting information is given as a  Supplemen-

tary file.

 3.4.1. Effect of extracting solvents

The effect of (i) acetonitrile:water (1:1), (ii) methanol:water

(1:1) and (iii) water:acetonitrile:methanol (83:6:11) as extracting

solvents were studied. It is clear that the third extracting solvents

(i.e., the HPLC mobile phase) resulted in the highest extraction of 

the components of interest.   Hu et al. (2009)   also obtained high

extraction efficiency for catechin monomers and caffeine whenmethanol:acetonitrile (1:1) were used. On the other hand, our

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); flow 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).

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results were found to be better than the one reported by  Nishitani

and Sagesaka (2004)   who studied acetonitrile:water (1:1) as the

extracting solvent.

 3.4.2. Effect of microwave parameters

The extraction efficiency of catechins and caffeine in tea sam-

ples that were extracted using different pressures (250, 300, 350and 400 Psi) were studied. The compounds were optimally

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 efficiency for the catechins and caffeine increased

with irradiation time until 6 min and remained stable after8–10 min. This value was lower than that reported by   Pan, Niu,

 Table 2

Analytical characteristics of the developed HPLC method.

Analyte Linear range (mg L 1)   R2 LOD (mg L 1) LOQ (mg L  1) Intra-day (%RSD) n  = 6 Inter-day (%RSD) n  = 5

Concentration spike, mg L 1

5 40 80 5 40 80

GC 0.5–80 0.9997 0.21 0.63 2.27 1.02 1.88 1.93 1.98 1.47

EGC 0.5–80 0.9994 0.24 0.72 2.00 2.14 1.78 1.96 2.22 2.28

Catechin 0.5–80 0.9998 0.29 0.87 2.26 1.59 2.53 2.39 1.78 2.21Caffeine 0.5–80 0.9991 0.17 0.51 2.11 1.78 1.97 1.75 1.89 2.27

EGCG 0.5–80 0.9998 0.22 0.66 1.76 1.84 2.44 1.81 0.89 1.49

EC 0.5–80 0.9992 0.13 0.39 1.92 1.61 1.89 2.17 1.95 1.88

GCG 0.5–80 0.9997 0.22 0.66 1.66 1.77 2.17 2.32 1.62 2.53

ECG 0.5–80 0.9995 0.27 0.81 1.89 0.76 2.53 1.23 1.77 2.22

CG 0.5–80 0.9994 0.11 0.33 2.05 1.00 2.35 2.49 1.09 1.98

R2, square of regression coefficient; RSD, %relative standard deviation.

 Table 4

Some reported HPLC methods for the analysis of tea flavonoids and phenolic acids.

No. Analytes Detector Column Dimension Program LOD LOQ SeparationTime

(min)

References

1. Catechins PDA Wakosil-II 5 C-18 3 mm150 mm, 5 lm Gradient 0.5–1.2 ng – 40   Nishitani and Sagesaka

(2004)

2. Phenolic acid, purine,

theanine

UV RP-Amine C-16 4.6 m m 150 mm, 5 lm Gradient 0.2–2.8 n g 0.7–6.3 n g 45   Peng et al. (2008)

3. Catechins, purine,

gallic acid

DAD ODS-100Z C-18 3.9mm 100 mm, 5 lm Gradient 0.04–0.89 ng – 11   Hu et al. (2009)

4. Catechins UV C18 packing 3.9 mm 150 mm, 5 lm Isocratic – – 40   Row and Jin (2006)

5. Catechins,

theaflavines

UV Partispere C-18 4.6 m m 110 mm, 5 lm Gradient 0.05 lg mL 1 – 25   Lee and Ong, (2000)

6. Catechins, caffeine,

gallic acid

UV Kingsorb/Nucleosil

C-18

4.6mm 150 mm, 5 lm Isocratic – – 50   Wang et al. (2000)

7. Catechins DAD C-18 packing 4.6 mm 150 mm, 5 lm Isocratic 0.19–

1.16mg L 1

0.64–3.86

mg L 1

25   Wang et al. (2003)

8 Purine alkaloide,

catechins

DAD Mightysil Rp-18 4.6 mm 150 mm, 5 lm Gradient 0.84–2.98

mg L 1

2.2–8.5 mg

L 1

20   Yang et al. (2007)

9 Catechins and

caffeine

UV Monolithic C-18 4.6mm 100 mm Isocratic 0.11–0.29

mg L 1

0.33–

0.87mg L 1

7 Our method

PDA, photo diode array; UV, ultraviolet visible spectrometry; DAD, diode array detector.

 Table 3

Recoveries of catechins and caffeine in green, black and oolong tea samples.

Concentration

spike, mg L 1

Type Analytes (%)

GC EGC Catechin Caffeine EGCG EC GCG ECG CG

5 Green tea (#1) 97.4 ± 1.2 96.6 ± 1.9 98.8 ± 1 .4 100.2 ± 0 .8 102.5 ± 2.0 99.9 ± 2.1 101.5 ± 0 .9 99.4 ± 2.2 100.4 ± 1.6

Black tea (#7) 98.1 ± 1.8 94.4 ± 2.9 94.9 ± 1.4 96.6 ± 2.3 99.2 ± 0.8 95.7 ± 1.7 96.1 ± 2.4 100.7 ± 2.0 102.2 ± 2.7

Oolong tea (#10) 97.1 ± 2.9 100.8 ± 2.0 105.2 ± 2.5 102.1 ± 1 .8 100.2 ± 0.7 99.4 ± 1.8 95.7 ± 2.2 98.3 ± 1.9 101.1 ± 0.6

40 Green tea (#1) 102.3 ± 2.3 103.2 ± 1.9 96.3 ± 2 .6 96.9 ± 1 .1 97.3 ± 2.5 104.2 ± 0 .8 96.1 ± 1.5 99.1 ± 1.1 95.8 ± 2.1

Black tea (#7) 96.7 ± 0.8 98.1 ± 1.2 97.2 ± 2.3 104.2 ± 1.3 101.5 ± 0.8 100.9 ± 1.5 102.3 ± 2.5 96.4 ± 2.7 95.4 ± 1.9

Oolong tea (#10) 95.7 ± 1.9 98.5 ± 1.1 100.3 ± 2.1 101.6 ± 2.9 105.3 ± 1.8 97.8 ± 0.8 99.1 ± 1.7 102.4 ± 2.1 98.7 ± 0.7

70 Green tea (#1) 103.9 ± 2.1 97.3 ± 1.6 100.1 ± 2 .7 96.3 ± 1 .9 95.8 ± 2.0 99.5 ± 2.2 94.6 ± 2.8 94.2 ± 1.1 95.1 ± 2.0

Black tea (#7) 97.6 ± 1.1 105.2 ± 0.9 100.2 ± 2.1 102.6 ± 0.4 103.3 ± 2.1 101.7 ± 1.6 96.3 ± 0.3 99.0 ± 0.8 99.1 ± 2.6

Oolong tea (#10) 101.5 ± 1.9 102.9 ± 2.1 99.7 ± 2.1 98.6 ± 2.2 94.5 ± 1.8 96.8 ± 2.7 97.9 ± 2.9 99.2 ± 1.1 95.7 ± 0.7

# = sample number (Table 5).

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and Liu (2003) and Li et al. (2010), when 15 min and 20 min of irra-

diation time was used, respectively at 400 W.

The adopted microwave extraction conditions were: pressure,

350 Psi; irradiation power, 600 W; irradiation time, 6 min.

 3.5. Analysis of tea samples

In the analysis of tea samples, peak identification was based on

the comparison between the retention times of standard com-

pounds and was confirmed by spiking standards to the samples

(Fig. 1c–e). Quantification was based on the external standard

method using calibration curves fitted by linear regression analy-

sis. The analysis was performed in triplicate.

The MAE and HPLC method were applied for the determination

of several commercial tea samples. The levels of the catechin

monomers and caffeine found are shown in Table 5.

Green tea (samples 1–6) is a type of tea obtained exclusively

from the leaves of   Camellia sinensis  that has undergone minimaloxidation during its production. It was observed that EGCG was

the major catechin monomer found in green tea (a non fermented

tea) in the range of 24.52–36.08 mg g1, followed by EGC, ECG, EC,

GC, GCG, catechin and CG. The identified epicatechins (EGCG, EGC,

ECG and EC) are in cis  structure. They can be reversibly converted

to their corresponding epimers that are non-epicatechins (GCG, GC,

CG and C). Thus, the chemical structures of epicatechins and non-

epicatechins differ only between the 2R, 3R (2,3-cis, epi-form) and

2S, 3R (2,3-trans, non-epi form) (Table 1). The accumulation of 

EGCG in green tea is due to inactivation of the polyphenol oxidase

by drying and steaming the fresh leaves until non oxidation pro-

cess occurs (McKay & Blumberg, 2002). The results shown in  Ta-

ble 5  are generally in agreement with those previously reported

by Yang et al. (2007) and Wang et al. (2003).Oolong tea (samples 7–8) is a semi-fermented tea that is pro-

ducedwhenthe fresh leaves are subjected to a partial fermentation

stage before drying (McKay & Blumberg, 2002). It can be thought of 

as being half-way between green and black tea (Sano et al., 1999;

Wang, Lu, Miao, Xie, & Yang, 2008). The results shown in  Table 5

are in good agreement with the finding of   Zuo, Cheng and Deng

(2002)   and  Horzic Komes, Belscak, Ganic, Ivekovic and Karlovic

(2009)  where the level of catechin monomers in oolong tea was

higher than black tea but relatively lower compared to green tea.

Oolong tea that is rich in antioxidants known as polyphenols

showed high levels of the EGC, ECG, EGCG and EC (R, R conforma-

tion) followed by GC, catechin, GCG and CG (R, S conformation).

Comparable results were also reported by Fernández-Cáceres, Mar-

tín, Pablos, and González (2001) where the EGCG and EGC were themajor catechins found in semi fermented oolong teas.

‘Fermented’ black tea (samples 9–11) undergoes a post-harvest

fermentation stage before drying and steaming processes (McKay

& Blumberg, 2002). The fermentation of black tea is due to an oxi-

dation process that is catalysed by polyphenol oxidase, and also at-

tained by using microorganisms. The level of catechin monomersin black tea is lower than green tea and oolong tea. This is due to

the alteration of catechin to form theaflavin, thearubigins during

fermentation process (Wang et al., 2000).Naturally, all the fresh

tea leaves contain certain levels of caffeine. The levels of caffeine

vary depending on the variety of tea origin, structure of the tea leaf 

and the differences in the tea processing itself (Song, Lin, Qu, &

Huie, 2003).

From Table 5, the level of caffeine in fully fermented black tea

was in the range 61.1–69.2 mg g1, whereas semi fermented oo-

long tea and non fermented green tea give values in the range of 

41.0–43.3 and 20.4–38.7 mg g1, respectively.

4. Conclusion

A rapid HPLC method using a monolithic column was devel-

oped, validated and applied to the simultaneous determination of 

eight catechin monomers and caffeine in tea samples. The use of 

the monolithic column enables these analytes to be separated al-

most four times faster than that of a conventional particulate col-

umn (7 min versus   27 min). The attractiveness of the MAE

technique is further demonstrated where significant improve-

ments in the extraction efficiency of the catechins and caffeine

were obtained. In conjunction with MAE, the HPLC method readily

lends itself as a useful analytical technique for the determination of 

catechins and caffeine.

 Acknowledgement

The authors gratefully acknowledge the financial support of this

work by the Short Term Grant of Universiti Sains Malaysia (304/

PKIMIA/6310028).

 Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at   http://dx.doi.org/10.1016/j.foodchem.

2013.09.131.

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 Table 5

Levels of catechins and caffeine in tea samples analysed.

Samples Concentration (mg g1)

GC EGC Catechin Caffeine EGCG EC GCG ECG CG

GT 1 4.24 ± 0.66 17.0 ± 0.23 2.36 ± 0.02 35.7 ± 0.77 33.7 ± 0.65 8.32 ± 0.92 2.38 ± 0.89 9.48 ± 0.95 0.22 ± 0.03

2 5.32 ± 0.11 17.9 ± 0.39 3.56 ± 0.23 38.7 ± 0.89 36.1 ± 0.54 9.08 ± 0.14 2.53 ± 0.06 10.2 ± 0.32 0.28 ± 0.08

3 5.04 ± 0.17 10.9 ± 0.22 2.08 ± 0.08 24.9 ± 0.32 27.1 ± 0.41 6.72 ± 0.18 0.84 ± 0.02 7.26 ± 0.86 0.31 ± 0.05

4 3.76 ± 0.13 18.5 ± 0.85 1.24 ± 0.11 20.4 ± 0.43 24.6 ± 0.52 5.56 ± 0.09 0.96 ± 0.11 5.06 ± 0.34 0.22 ± 0.03

5 4.44 ± 0.23 12.2 ± 0.17 2.28 ± 0.12 30.2 ± 0.88 24.5 ± 0.16 7.08 ± 0.29 0.92 ± 0.13 6.12 ± 0.27 0.17 ± 0.04

6 6.24 ± 0.55 13.8 ± 0.15 2.24 ± 0.05 32.6 ± 0.23 28.8 ± 0.19 7.69 ± 0.05 1.64 ± 0.36 8.26 ± 0.25 0.23 ± 0.01

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BT 9 0.39 ± 0.02 1.14 ± 0.05 0.46 ± 0.08 69.2 ± 0.91 5.7 ± 0.22 1.14 ± 0.06 0.44 ± 0.04 4.08 ± 0.54 0.24 ± 0.05

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GT = green tea; OT = oolong tea; BT = black tea.

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