Research article
Open Access
Development of an Improved Isocratic HPLC Method
for the Determination of Gallic Acid, Caffeine and
Catechins in Tea
SM Kingori1*, PO Ongoma2 and SO Ochanda1
1Kalro- Tea Research Institute, P.o Box 820-20200, Kericho, Kenya
2Department of Chemistry, Egerton University, P.o Box 536-20115 Egerton, Kenya
*Corresponding author: Kalro- Tea Research Institute, P.o Box 820-20200, Kericho, Kenya, Tel: +254721900104 E-Mail:
@
Received: April 04, 2018; Accepted: June 06, 2018; Published: June 25, 2018
Citation: Kingori SM, Ongoma PO, Ochanda SO (2018) Development of an Improved Isocratic HPLC Method for the Determination of Gallic Acid, Caffeine and Catechins in Tea. J Nutrition Health Food Sci 6(4):1-9 DOI:
10.15226/jnhfs.2018.001135
Abstract
A simple and sensitive reversed phase high performance liquid
chromatographic (HPLC) method was developed for the determination
of biomolecules in different types of tea. Most of the High Performance
Liquid Chromatography (HPLC) methods used for the determination
of tea biochemicals include gradient elution systems which involve
expensive instrumentation. The aim of this study was to develop
an improved sensitive, fast, cost effective and accurate isocratic
HPLC method with photo diode array (PDA) detection for analysis
of Gallic acid, caffeine and catechins in tea, using a suitable internal
standard. The developed HPLC analytical method consisted of a C6-
phenyl column and an isocratic elution system of Water: acetonitrile:
methanol: Ortho phosphoric acid: ethyl acetate (77.5:18:2.0:0.5:2.0
v/v/v/v/v) at a flow rate of 1.0 mL/min. The detection wavelength
was chosen at 278 nm with guaiacol (2-methoxyphenol) used as an
internal standard as it did not co-elute with the analytes of interest.
Statistical comparison of the analytical result obtained for gallic acid,
caffeine and catechins in four tea types - green CTC (cut, tear and curl),
black CTC, green orthodox and black orthodox using the developed
method and ISO 1405-2:2005(E) method did not show significant
difference. The method was validated and has showed consistency to
qualitative and quantitative determination of the tea biomolecules of
interest.
Introduction
Tea (Camellia sinensis) is a beverage consumed for
refreshment and health benefits since ancient times [27]. Tea
has been consumed for various reasons including its antioxidant
properties sensory properties and potential health benefits [2,
5, 16, 18, 26]. It is the second most consumed beverage in the
world after water and is commonly served hot or iced [20]. Tea
is produced mostly from the two tender leaves and a bud of
the plant. Studies have shown that tea provides several health
benefits, such as reduction of cholesterol, obesity, protection
against cardiovascular disease and cancer [7, 21]. Tea is a unique
beverage with biomolecules whose chemical compositions can
be used as indicators of the quality of tea [9, 31]. Levels of these
chemicals are directly proportional to quality indicators both
in aerated and non-aerated tea products [25]. Therefore, there
is need to explore easy scientific techniques of determining
quality parameters with an aim of complimenting the subjective
organoleptic evaluation techniques commonly used in the tea
industry.
Catechins are the primary polyphenols in tea and account for
75-80% of the soluble ingredients [24, 28]. They are powerful
antioxidants that provide several benefits [23]. Gallic acid and
caffeine are also found in both aerated and nonaerated CTC and
orthodox teas [15, 19]. Figure 1 shows biomolecules of interest
in this study found in tea.
High performance liquid chromatography (HPLC) methods
have been developed to separate, identify and quantify gallic
acid, caffeine and catechins present in tea. The methods are
mostly gradient elution systems [12, 22, 30]. However, gradient
elution compared to isocratic systems, requires expensive
instrumentation and computerized processors [11]. It is
also difficult to optimize the operating conditions and obtain
reproducible results [32]. Moreover, some isocratic methods
studied using C18 (ODS stationary phase) column are also
irreproducible, have poor resolution and low chromatographic
efficiency especially under methanol based mobile phases [6, 29].
Due to variability in the compositions of tea catechins,
caffeine and gallic acid and their potential health benefits, it is
important to establish a simple and reliable analytical method
for the determination of these compounds. The current method
is simple as it involves a less complex isocratic system and can
be used in common laboratories having low cost HPLC machines.
The method can be used to determine quality parameters of tea,
levels of tea biomolecules in tea value added products especially
tea based supplements and estimation of levels of adulteration of
tea in the local market.
Material and Methods
Tea Samples for Analysis
Non - aerated green, aerated black CTC teas and orthodox non
- aerated, orthodox aerated black teas were sourced in triplicate
from Kangaita tea factory of Kirinyaga County in Kenya.
Figure 1: Chemical Structures of the major tea catechins, gallic acid and caffeine: (a) (+) Catechin, (b) (-) Epicatechin, (c) (-) Epigallocatechin, (d)(-) Epigallocatechingallate, (e) Gallic acid, (f) Caffeine, (g)(-) Epicatechingallate
Reagent and Chemicals
All standards viz. gallic acid (GA), epigallocatechin (EGC),
(+)-catechin (+C), ( ̶ )- epigallocatechin (EC), ( ̶ )-epigallocatechin
gallate (EGCG), ( ̶ )- epicatechin gallate (ECG), caffeine,
3-fluorocatechol, guaiacol (2-methoxyphenol), 4-methylcatecol
and EDTA were purchased from Sigma Chemical Co., US.
Acetonitrile, methanol, glacial acetic acid (both HPLC grade),
ethyl acetate, methanoic acid, ortho-phosphoric acid and
acetone were purchased from Finar India Ltd. All solvents were
degassed and filtered through a 0.45 μm filter (Millipore filter No.
HAWP04700).
HPLC Instrumentation and Conditions
The HPLC system Shimadzu LC 20A series consisted of binary
pump with vacuum degasser (DGU-20A5R), thermostated column
compartment (CTO ̶ 10AS vp), auto sampler (SIL 20 AT HT), diode
array detector (SPD-20 MA) all from Shimadzu Corporation,
Japan. C6-phenyl reversed-phase column (4.6 x 250mm, 5μ) was
used and the column temperature was maintained at 35°C. A
suitable mobile phase was investigated from the following matrix
combinations.
1. Water: methanol: acetic acid: EDTA
2. Water: methanol: Ortho- phosphoric acid: EDTA
3. Water: acetonitrile: methanol: acetic acid: ethyl acetate
4. Water: acetonitrile: methanol: Ortho- phosphoric acid: ethyl
acetate
5. Methanol: water:methanoic acid
6. Acetonitrile: water: methanoic acid
7. Acetonitrile: water:methanol:acetic acid
8. Water:methanol: ethyl acetate
9. Methanol:EDTA: water
10. Acetic acid: acetone:water
11. Water: acetonitrile: acetic acid: EDTA; starting with
(0:100:0.1% v/v/v) and increasing stepwise by 10%; e.g.
Methanol:water:methanoic acid (100:0:0.1% v/v/v).
Preparation of Stabilizing Solution
A solution of 25 ml of EDTA (10mg/ml), 25 ml ascorbic
(10mg/ml) acid solution and 50ml acetonitrile (HPLC grade) was
transferred to a 500 ml one-mark volumetric flask, diluted to the
mark with distilled water and mixed.
Preparation of Standard Solution
Stock solutions of the standards -GA, EGC, +C, EC, EGCG, ECG,
caffeine and guaiacol (2-methoxyphenol) were prepared at 1000
μg/ml by dissolving in the stabilizing solution, gently warming
if necessary (max. 40°C) and then cooling to room temperature
(20 - 25°C). Five different concentration mixtures, 2- 1000 μg/
ml of each of the standards were diluted from the standard stock
solutions and passed through 0.45μm Millipore filter before
injecting into HPLC. Standard curves for all the standards were
plotted. Peak area responses were obtained for each of the
standards relative to the peak area of guaiacol (2-methoxyphenol).
Preparation of Samples
Finely milled tea test samples were weighed (0.200 ± 0.001)
g into extraction tubes. The extraction tubes containing the
sample were placed in a water bath set at 70°C and 5.0 ml of hot
methanol/water (7:3 v/v) extraction mixture was dispensed. The
extraction tubes were stoppered and mixing done with the help
of a vortex mixer. The heating of the extraction tubes in the water
bath continued for 10 min, mixing on the vortex mixer after 5
min and 10 min. The extraction tubes from the water bath were
removed and allowed to cool to room temperature. The stoppers
were removed and the tubes placed in a centrifuge at 3500
rpm for 10 min. The supernatant was carefully decanted into a
graduated tube. The extraction steps were repeated resulting in
two extracts. The two extracts were combined and made to 10
ml with cold methanol/water extraction mixture. On mixing,
the extract was allowed to attain room temperature (20 - 25°C)
before carrying out the assay.
Method Validation
Method for quantitative and qualitative analysis of GA,
( ̶) -EGC, caffeine, (+)-C, ( ̶) -EC, ( ̶) –EGCG and ( ̶) – ECG was
validated for its specificity, linearity, accuracy, level of detection
(LOD), level of quantitation (LOQ) and precision by utilization the
Food and Drug Administration (FDA) guidelines [10].
Results and Discussion
Optimization of Chromatographic Conditions
A simple, sensitive, fast, cost effective and accurate isocratic
HPLC method with diode array detection for analysis of gallic
acid, caffeine and catechins in tea, using a suitable internal
standard was developed. The method consisted of the use of a
C6-phenyl column which combines low adsorption ability of the
hexyl spacer for less polar groups and strong π-π interactions
between the phenyl group and the substrate (polyphenols) via
dipole- dipole and dipole-induced dipole forces to enhance its
performance [4].
A comprehensive study was systematically carried out to
determine the best mobile phase combination for the method as
shown in table 1.
Table 1: Isocratic elution systems investigated for separation of gallic acid, caffeine and catechins in tea at 35 °C
System |
Matrix composition |
Matrix ratio (v/v) |
Runtime (min) |
A |
Water: methanol: acetic acid: EDTA (20µg/ml) |
80:19.3:0.5:0.2 |
60 |
B |
Water: methanol: Ortho phosphoric acid (50%): EDTA |
79.3:20:0.5:0.2 (20µg/ml) |
60 |
C |
Water: acetonitrile: methanol: acetic acid: ethyl acetate |
77.5:18:2.0:0.5:2.0 |
12.5 |
D |
Water: acetonitrile: methanol :Ortho phosphoric acid ethyl acetate |
77.5:18:2.0:0.5:2.0 |
12.5 |
E |
Water: methanol: methanoic acid |
79.5:20:2.0:0.5 |
60 |
F |
Water: acetonitrile: methanoic acid |
79.5:20:0.5 |
50 |
G |
Water: acetonitrile: methanol: acetic acid |
79.5 :18:2.0:0.5 |
17 |
H |
Water: methanol: ethyl acetate |
None |
None |
I |
Water: methanol: EDTA (20µg/ml) |
79:20:01 |
60 |
J |
Water: acetic acid: acetone |
None |
None |
K |
Water: acetonitrile: acetic acid: EDTA (20µg/ml) |
86.3:13:0.5:0.5:0.2 |
45 |
Finally, a mobile phase containing water: acetonitrile:
methanol: Orth-phosphoric acid: ethyl acetate (77.5:18:2.0:0.5:2.0)
was adapted for use in the method (figure2). The mobile phase
flow rate was 1.0 mL/minute and the injection volume 20 μL.
The separations were performed at 35°C, absorption measured
at 278 nm, with the compounds of interest effectively detected
and separated. Saito, et al. 2006 worked with a similar mobile
phase matrix composition using acetic acid and incorporating
both isocratic and time gradient conditions in their method.
The total run time was 37 minutes, when flow rate gradient was
introduced, all the components were eluted within 27 minutes.
Additionally, only caffeine and three catechins (EGCG, EC and
+C) were reported to have been determined [29]. The systems
of the developed method can elute the components of interest
in less than 10 minutes and both qualitative and quantitative
determinations have yielded success. With the inclusion of the
internal standard of interest and an allowance of a short wash
time total analysis time can be achieved in 12.5 min as shown in
figure 2. The mobile phase composition of the developed method
uses low pH (2– 3), these is necessary as catechins are unstable in
basic solutions and can bind to various metals especially calcium,
magnesium, iron, zinc as well as trace levels other minerals that
could react with the catechins [3, 14]. Addition of very small
amount of ethyl acetate increased resolution efficiency especially
for the closely eluting peaks of caffeine and EGC [13]. Also, being
a chelating agent helped to prevent the decomposition of the
catechins by binding with trace ions in the chromatographic
system [1].
Figure 2: HPLC chromatogram of mixed standards at 278 nm. Peaks:
1-gallic acid (GA); 2- (-) epigallocatechin (EGC); 3-caffeine (CA), 4- (+)
catechin (+C), 5- (-) epicatechin (EC); 6-(-) epigallocatechin gallate
(EGCG); 7-(-) epicatechin gallate (ECG), 8-internal standard guaiacol (2-
methoxyphenol)
The choice of a suitable internal standard was investigated
amongst 3-fluorocatechol, guaiacol (2-methoxyphenol), and
4-methylcatecol as they are phenols. Guaiacol (2-methoxyphenol)
was a good internal standard for the method as it showed no coelution
with the analytes (figure 2) and gave good quantitation
levels for gallic acid, caffeine and the major catechins. Figure
3 and figure 4 shows an HPLC chromatogram of nonaerated
green CTC tea before and after being spiked with the internal
standard guaiacol (2- methoxyphenol) respectively and further
demonstrates that the internal standard does not coelute with
components in sample matrix. HPLC chromatogram for aerated
black CTC also showed similar result figure 5 and figure 6.
Figure 3: HPLC chromatogram of unaerated green CTC tea at 278 nm.
Peaks: 1- gallic acid (GA); 2- (-) epigallocatechin (EGC); 3 - caffeine (CA),
4 - (+) catechin (+C), 5 - (-) epicatechin (EC); 6 ̶ (-) epigallocatechin gallate
(EGCG); 7- (-) epicatechin gallate (ECG)
Figure 4: HPLC chromatogram of unaerated green CTC tea at 278 nm
spiked internal standard guaiacol (2- methoxyphenol). Peaks: 1- gallic
acid (GA); 2- (-) epigallocatechin (EGC); 3 - caffeine (CA), 4- (+) catechin
(+C), 5- (-) epicatechin (EC); 6- (-) epigallocatechin gallate (EGCG); 7- (-)
epicatechin gallate (ECG), 8 - guaiacol (2- methoxyphenol)
Figure 5: HPLC chromatogram of aerated black CTC at 278 nm. Peaks:
1- gallic acid (GA); 2- (-) epigallocatechin (EGC); 3- caffeine (CA), 4- (+)
catechin (+C), 5- (-) epicatechin (EC); 6- (-) epigallocatechin gallate
(EGCG); 7- (-) epicatechin gallate (ECG)
Figure 6: HPLC chromatogram of aerated black CTC tea at 278 nm
spiked with internal standard guaiacol (2- methoxyphenol). Mixed
standards at 278 nm. Peaks: 1- gallic acid (GA); 2- (-) epigallocatechin
(EGC); 3- caffeine (CA), 4 -(+) catechin (+C), 5- (-) epicatechin (EC); 6-
(-) epigallocatechin gallate (EGCG); 7- (-) epicatechin gallate (ECG), 8-
(2- methoxyphenol)
Development of Internal Standard Relative Response
Factors (RRFS)
A volume of 20 μL of the mixed standard of GA, EGC,
Caffeine, (+) C, EC, EGCG and ECG internal standard (2- 1000
μg/ml) was injected into the isocratic HPLC system. The result
obtained was used to determine relative response factors, (table
2) for GA, caffeine and the catechins in relation to the internal
standard using equation 1. These factors were consequently used
for Quantitation purposes and gave satisfactory levels of the
biomolecules. The RRF can hence be reliably used in Quantitation
of GA, EGC, caffeine, (+) C, EC, EGCG and ECG in different types of
tea and tea based products.
Table 2: Relative response factors (RRFS) for GA, caffeine and the catechins in relation to guaiacol (2- methoxyphenol) internal standard
Component |
Rep 1 |
Rep 2 |
Rep 3 |
Rep 4 |
Rep 5 |
Mean |
GA |
0.29 |
0.28 |
0.29 |
0.28 |
0.28 |
0.29 |
EGC |
3.72 |
3.49 |
3.53 |
3.41 |
3.49 |
3.56 |
(+) C |
1.85 |
1.82 |
1.85 |
1.81 |
1.82 |
1.83 |
Caffeine |
0.35 |
0.36 |
0.37 |
0.35 |
0.36 |
0.36 |
EC |
1.55 |
1.55 |
1.56 |
1.52 |
1.54 |
1.55 |
EGCG |
0.82 |
0.81 |
0.82 |
0.8 |
0.8 |
0.81 |
ECG |
0.64 |
0.61 |
0.61 |
0.6 |
0.61 |
0.62 |
Validation of the Method
Method validation is an important requirement for any
package of information submitted to international regulatory
agencies in support of new product marketing. Analytical
methods should be validated, including methods published in
relevant standard references. The suitability of all test methods
used should always be verified under the actual conditions of use
and should be well documented.
Specificity of the Method
The specificity of the method was investigated by injecting
extracted placebo to demonstrate the absence coelution of
analyte. The conditions for the method gave good specificity as
there was no coelution in the mixed standard sample matrix,
(figure 2) and both aerated (black) and non-aerated (green) tea
extracts on being analyzed gave peaks that were specific with
respect to each other. The trends did not change with repeated
introduction of sample material (figures 3-6).
Linearity of the Method
The linearity of the method was checked with standard
solutions of GA, ( ̶ )- EGC, caffeine, (+) C, ( ̶ )- EC, ( ̶ )- EGCG, and ( ̶)-
ECG prepared at six concentrations in the concentration range
of 2.5 ̶ 500 μg/mL. Three individually prepared replicates at
each concentration were analyzed. The mean peak area of three
injections and corresponding signal levels were used to generate
equations for the regression line and correlation coefficients (r2)
for each of the standards. Limit of Detection (LOD) and limit of
Quantitation (LOQ) were obtained from the standard deviation
(σ) of the blank response (n=6) and slope (S) of calibration curves
using the formula 3.3 σ/S and 10 σ/S, respectively. All calibration
curves yielded straight lines over a wide range and correlation
coefficient greater (˃) than 0.99. The results are as shown in table
3. The concentration ranges taken were above the LOD and LOQ of
the method and furthermore all types of teas under consideration
here are not known to have their concentration outside the set
limits [17]. The linearity data shows that the method meets the
requirements of validation based on this parameter and hence fit
for this kind of analysis.
Table 3: Statistical data for regression plots, LOD and LOQ
Component |
Linear range (µg/mL) |
Equation for regression line |
Correlation coefficient(r2) |
LOD |
LOQ |
EGC |
25 – 400 |
y=0.00033x – 0.0015 |
0.9995 |
0.06 |
0.18 |
+C |
25 - 250 |
y=0.011x – 0.0162 |
0.9969 |
0.09 |
0.27 |
EC |
25 – 400 |
y=0.01361x – 0.027 |
0.997 |
0.02 |
0.18 |
EGCG |
25 - 250 |
y=0.0225x – 0.00098 |
0.9999 |
0.16 |
0.48 |
ECG |
25 - 250 |
y=0.0031x + 0.042 |
0.9969 |
0.11 |
0.33 |
GA |
2.5 – 30 |
y=0.048x – 0.0183 |
0.9998 |
0.04 |
0.12 |
Caffeine |
2.5 - 30 |
y=0.0441x – 0.0058 |
0.999 |
0.07 |
0.21 |
Precision of the Method
The precision of the method was investigated by preparing
one sample solution containing the target level of analyte.
Ten replicates of this sample solution were analyzed with the
retention time and peak area being recorded. The mean, standard
deviation and relative standard deviation (RSD %) were finally
determined (table 4). Both Peak Areas (PA) and Retention Times
(RT) for all the components gave low standard deviation from
the respective means. In terms of relative standard deviation
percentage (RSD %) for the components the range was 0.241 –
2.175 % demonstrating that the method is fairly precise and can
be used to get reproducible results.
Accuracy of the Method
Accuracy of the analytical method was determined by
preparing quality control (QC) materials of GA, ( ̶ )- ECG, (+)-C,
caffeine, ( ̶ )- EC, ( ̶ )- EGCG, and ( ̶ )- EGC in a similar way to
unknown samples at four predetermined concentration levels.
The QC materials were repeatedly measured to determine the
accuracy of the determinations. The mean recovery for the
catechins ranged from 99.2 – 105.5% while the recovery for
gallic acid was 99.9% and that for caffeine was 105.3% (table 5).
This demonstrated that the result found is satisfactory for the
intended purpose and is adequate for routine analysis.
Application of the Method
Factory processed CTC non aerated green tea, CTC aerated
black tea, orthodox non aerated green tea and orthodox aerated
black tea from Kangaita tea factory of Kirinyaga County in Kenya
were extraction for gallic acid, caffeine and catechins according
to ISO 1405-2:2005(E) procedures. All samples were extracted in
triplicate and analyzed using the developed improved isocratic
method and the ISO 1405-2:2005(E) method for comparison
purposes. Statistical analysis was carried out using SAS® V 9.1
for windows statistical software.
ANOVA was used to determine the means, coefficient of
variation and Least Significance Difference (LSD) was used
to separate means. The probability limit was set at P ≤ 0.05
significant level and the Standard Deviation (SD) done using
the student t-test. Results of the parameter determined were
expressed as a mean of the triplicate determination. Table 6
shows the statistics for method comparison for green CTC, green
Table 4: Precision test for GA, ( ̶ )- EGC, Caffeine,(+) C, , ( ̶ )- EC, ( ̶ )- EGCG, and ( ̶ )- ECG
Component |
Parameter |
Mean |
SD |
RSD % |
GA - (2.5µg/ml) |
RT |
3.776 |
0.029 |
0.768 |
PA |
113606 |
2471 |
2.175 |
EGC - (50µg/ml) |
RT |
4.084 |
0.031 |
0.759 |
PA |
197057 |
474 |
0.241 |
Caff - (2.5 µg/ml) |
RT |
4.516 |
0.054 |
1.196 |
PA |
133484 |
785 |
0.588 |
+ C - (25 µg/ml) |
RT |
4.9 |
0.055 |
1.122 |
PA |
326029 |
75975 |
1.833 |
EC - (25 µg/ml) |
RT |
5.192 |
0.05 |
0.963 |
PA |
430567 |
2541 |
0.59 |
EGCG - (25 µg/ml) |
RT |
6.011 |
0.054 |
0.898 |
PA |
627129 |
5796 |
0.924 |
ECG - (25 µg/ml) |
RT |
9.121 |
0.087 |
0.954 |
PA |
114368 |
574 |
0.502 |
Table 5: Accuracy test for gallic acid, ( ̶ )- ECG, (+)-catechin, caffeine, ( ̶ )- EC, ( ̶ )- EGCG, and ( ̶ )- EGC
Tea Component |
Concentration (µg/ml) |
Accuracy (µg/ml) |
Recovery (%) |
Gallic acid |
2 |
2.10 ± 0.50 |
105.0 |
5 |
4.84 ± 0.50 |
96.8 |
10 |
9.79 ± 0.50 |
97.9 |
15 |
14.66 ± 0.50 |
97.7 |
Epigallocatechin |
25 |
24.32 ± 0.90 |
97.3 |
50 |
50.89 ± 0.20 |
101.8 |
100 |
98.70 ± 0.60 |
98.7 |
150 |
148.79 ± 0.80 |
99.2 |
+ Catechin |
25 |
26.81 ± 0.50 |
107.2 |
50 |
50.15 ± 0.50 |
100.2 |
100 |
99.39 ± 0.50 |
99.4 |
150 |
149.02 ± 0.50 |
99.3 |
Caffeine |
2 |
2.04 ± 0.90 |
102.0 |
5 |
5.55 ± 0.70 |
111.0 |
10 |
10.35 ± 0.50 |
103.5 |
15 |
14.95 ± 0.90 |
99.7 |
Epicatechin |
25 |
26.53 ± 0.90 |
106.1 |
50 |
48.98 ± 0.80 |
98 |
100 |
102.35 ± 0.70 |
102.4 |
150 |
148.83 ± 0.90 |
99.2 |
Epigallocatechin gallate |
25 |
25.23 ± 0.40 |
100.9 |
50 |
50.10 ± 0.90 |
100.2 |
100 |
103.71 ± 0.82 |
103.7 |
150 |
151.52 ± 0.60 |
101.0 |
Epicatechin gallate |
25 |
25.32 ± 0.50 |
101.3 |
50 |
49.37 ± 0.90 |
98.7 |
100 |
100.97 ± 0.70 |
101.0 |
150 |
152.39 ± 0.70 |
101.6 |
Table 6: Method comparison for levels of GA, EGC, Caffeine, +C, EC, EGCG, ECG and total catechin in the different types of tea
Tea type |
Component |
SD |
ANOVA |
|
|
|
Method 1 |
Method 2 |
CV |
LSD |
Green CTC |
GA |
0.61a ± 0.03 |
0.63a ± 0.20 |
12.8 |
0.28 |
EGC |
5.76a ± 0.61 |
5.86a ± 0.50 |
5.8 |
1.10 |
Caffeine |
3.52a ± 0.24 |
3.73a ± 0.21 |
3.8 |
0.49 |
+C |
0.56a ± 0.03 |
0.53a ± 0.03 |
7.3 |
0.14 |
EC |
1.97a ± 0.24 |
1.36a ± 0.19 |
14.8 |
0.86 |
EGCG |
8.86a ± 1.29 |
8.71a ± 0.43 |
13.8 |
4.24 |
ECG |
2.31a ± 0.37 |
2.29a ± 0.19 |
8.7 |
0.70 |
Total catechin |
19.46a ± 1.27 |
18.74a ± 1.02 |
8.4 |
5.63 |
Green orthodox |
GA |
0.72a ± 0.05 |
0.68a ± 0.04 |
7.9 |
0.19 |
EGC |
6.20a ± 0.21 |
5.92a ± 0.06 |
2.9 |
0.62 |
Caffeine |
3.28a ± 0.46 |
3.82a ± 0.20 |
7.5 |
0.94 |
+C |
0.5a ± 0.03 |
0.5a ± 0.02 |
3.4 |
0.06 |
EC |
1.80a ± 0.09 |
1.55a ± 0.13 |
5.9 |
0.35 |
EGCG |
9.09a ± 0.56 |
9.37a ± 0.53 |
8.2 |
2.67 |
ECG |
2.30a ± 0.08 |
2.65a ± 0.21 |
6.2 |
0.54 |
Total catechin |
19.88a ± 0.50 |
20.03a ± 0.36 |
2.9 |
2 |
Black CTC |
GA |
0.41a ± 0.04 |
0.48a ± 0.06 |
8.8 |
0.14 |
EGC |
2.45a ± 0.08 |
2.49a ± 0.06 |
3.7 |
0.32 |
Caffeine |
3.52a ± 0.42 |
3.70a ± 0.37 |
6.9 |
0.87 |
+C |
0.19a ± 0.02 |
0.20a ± 0.02 |
12.6 |
0.09 |
EC |
0.16a ± 0.04 |
0.14a ± 0.05 |
9.2 |
0.05 |
EGCG |
0.48a ± 0.04 |
0.36a ± 0.03 |
12.2 |
0.18 |
ECG |
0.47a ± 0.05 |
0.44a ± 0.04 |
7.1 |
0.11 |
Total catechin |
3.65a ± 0.04 |
3.63a ± 0.08 |
8.81 |
0.21 |
Black orthodox |
GA |
0.52a ± 0.02 |
0.54a ± 0.04 |
6.7 |
0.13 |
EGC |
1.74a ± 0.01 |
1.57a ± 0.51 |
21.4 |
1.25 |
Caffeine |
3.07a ± 0.05 |
3.46a ± 0.18 |
4.9 |
0.56 |
+C |
0.21a ± 0.04 |
0.20a ± 0.05 |
13.9 |
0.1 |
EC |
0.30a ± 0.05 |
0.22a ± 0.02 |
12.2 |
0.11 |
EGCG |
0.43a ± 0.02 |
0.58a ± 0.10 |
14.8 |
0.26 |
ECG |
0.69a ± 0.06 |
0.71a ± 0.04 |
8.4 |
0.21 |
Total catechin |
3.37a ± 0.07 |
3.29a ± 0.56 |
11.2 |
1.31 |
Means with the same letter in the same column are not statistically significant
orthodox, black CTC, and black orthodox teas. All the biomolecules
of interest in this study - GA, EGC, +C, EC, EGCG, ECG and caffeine
were present in all the teas. In general, both green CTC and green
orthodox teas contained significantly higher concentrations of
catechins than the black teas whereas the contents of GA and
caffeine remained constant. The differences can be attributed
to the manufacture process where catechins combine with the
biological enzyme polyphenol oxidase and with the help of the
atmospheric oxygen oxidize to the aflavins and the arubigins.
GA and caffeine are not affected by the oxidative process of
manufacture and hence remain relatively constant during the
oxidative degradation of the catechins and other phenols. All
these facts were well demonstrated by the conditions set in the
newly developed method and the conditions of the ISO 1405-
2:2005(E) method which is widely used in determination of tea
biomolecules. Additionally, all parameters of measurement did
not show significant differences between the two methods and
hence the two methods are in close agreement.
Conclusions
Tea is known for its complex mixture of phytochemicals.
Polyphenols, catechins, caffeine and gallic acid have lately elicited
attention in line with the health benefits associated with these
biomolecules. Tea value added products are increasingly becoming
common in the local market and dietary polyphenols comprise a
wide range of aromatic compounds which can easily be sourced
from tea. The growing importance of the commonly known tea
types, specialty developed types and tea value added products
necessitated the development of a sensitive, fast, cost effective
and accurate HPLC method. A simple isocratic HPLC method that
can perform qualitative and quantitative determination of gallic
acid, (+) - catechin, ( ̶ )- EC, ( ̶ ) - ECG, ( ̶ ) - EGCG, ( ̶ ) – EGC and
caffeine in tea and tea related products has been developed. The
method has proved to be specific and precise and has shown good
performance with the column of choice. The total elution time is
impressively short (10minutes) and with the internal standard
being incorporated analysis can be achieved in 12.5 minutes.
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