ISO 14502-2:2005

INTERNATIONAL ISO
STANDARD 14502-2
First edition
2005-03-01


Determination of substances
characteristic of green and black tea —
Part 2:
Content of catechins in green tea —
Method using high-performance liquid
chromatography
Détermination des substances caractéristiques du thé vert et du thé
noir —
Partie 2: Dosage des catechins dans le thé vert — Méthode par
chromatographie en phase liquide à haute performance




Reference number
ISO 14502-2:2005(E)
©
ISO 2005

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ISO 14502-2:2005(E)
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ISO 14502-2:2005(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references. 1
3 Principle. 1
4 Reagents. 1
5 Apparatus. 5
6 Sampling. 5
7 Preparation of test samples. 6
8 Procedure. 6
8.1 General. 6
8.2 Determination of dry matter content. 6
8.3 Test portion. 6
8.4 Extraction of polyphenols. 6
8.5 Dilution. 7
8.6 Determination. 7
9 Calculation. 8
9.1 General. 8
9.2 Quantitation using catechin standards . 8
9.3 Quantitation using a caffeine standard and catechin Relative Response Factors (RRFs) . 9
10 Precision. 10
10.1 Interlaboratory test. 10
10.2 Repeatability. 10
10.3 Reproducibility. 10
11 Test report. 10
Annex A (informative) Results of interlaboratory tests . 11
Annex B (informative) Assessment of purity of standards . 13
Annex C (informative) Typical HPLC chromatograms. 14
Annex D (informative) The effect of ferric ions on catechin RRFs. 18
Annex E (informative) Quantitative comparison — Use of catechin standards or a caffeine
standard in conjunction with catechin RRFs. 21
Bibliography . 23

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ISO 14502-2:2005(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 14502-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 8, Tea.
ISO 14502 consists of the following parts, under the general title Determination of substances characteristic of
green and black tea:
 Part 1: Content of total polyphenols in tea — Colorimetric method using Folin-Ciocalteu reagent
 Part 2: Content of catechins in green tea — Method using high-performance liquid chromatography

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INTERNATIONAL STANDARD ISO 14502-2:2005(E)

Determination of substances characteristic of green and black
tea —
Part 2:
Content of catechins in green tea — Method using
high-performance liquid chromatography
1 Scope
This part of ISO 14502 specifies a high-performance liquid chromatographic (HPLC) method for the
determination of the total catechin content of tea from the summation of the individual catechins. It is
applicable to both leaf and instant green tea, and with precision limitations to black tea (see Annex A).
Gallic acid and caffeine can also be determined by this method, as can theogallin and theaflavins.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 1572, Tea — Preparation of ground sample of known dry matter content
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 7513, Instant tea in solid form — Determination of moisture content (loss in mass at 103 °C)
3 Principle
The total catechin content from a test portion of finely ground leaf tea is extracted with 70 % methanol at 70 °C.
Instant teas are dissolved in hot water with 10 % (volume fraction) acetonitrile added to stabilize the extract.
The individual catechins in the extract are determined by HPLC on a phenyl-bonded column using gradient
elution with UV detection at 278 nm. External standards are used for quantitation. External catechin standards
of defined purity and moisture content may be used directly. Alternatively, caffeine may be used as a standard
in conjunction with individual catechin Relative Response Factors (RRFs) established by an ISO international
interlaboratory test.
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
4.1 Water, conforming to grade 1 of ISO 3696:1987.
4.2 Acetonitrile, HPLC grade.
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ISO 14502-2:2005(E)
4.3 Methanol.
4.4 Acetic acid, glacial HPLC grade.
4.5 Methanol/water extraction mixture, 70 % methanol (volume fraction).
Add 700 ml of the methanol (4.3) to a 1 litre one-mark volumetric flask. Dilute to the mark with water (4.1) and
mix.
4.6 EDTA solution, 10 mg/ml.
Weigh (1,00 ± 0,01) g of EDTA (ethylenediaminetetraacetic acid disodium salt, dihydrate) into a 100 ml one-
mark volumetric flask. Add sufficient warm water to half-fill the flask. Swirl to dissolve the EDTA, cool to room
temperature, dilute to the mark with water and mix.
Prepare a fresh solution daily.
4.7 Ascorbic acid solution, 10 mg/ml.
Weigh (1,00 ± 0,01) g of L-ascorbic acid into a 100 ml one-mark volumetric flask. Dissolve in water, dilute to
the mark and mix.
Prepare a fresh solution daily.
4.8 Stabilizing solution, 10 % (volume fraction) acetonitrile with 500 µg/ml of EDTA and ascorbic acid.
Using a pipette, transfer 25 ml of EDTA solution (4.6), 25 ml ascorbic acid solution (4.7) and 50 ml of
acetonitrile (4.2) to a 500 ml one-mark volumetric flask. Dilute to the mark with water and mix.
Prepare a fresh solution daily.
4.9 HPLC mobile phases.
SAFETY PRECAUTIONS — Wear gloves, eye protection and dispense reagents in a fume cupboard.
4.9.1 Mobile phase A, 9 % (volume fraction) acetonitrile, 2 % (volume fraction) acetic acid with 20 µg/ml
EDTA.
Transfer 180 ml of acetonitrile (4.2) and 40 ml of acetic acid (4.4) to a 2 litre one-mark volumetric flask. Add
sufficient water to half-fill the flask and add 4,0 ml of EDTA solution (4.6). Dilute to the mark with water, mix
and filter through a filter of 0,45 µm pore size (5.10).
4.9.2 Mobile phase B, 80 % (volume fraction) acetonitrile, 2 % (volume fraction) acetic acid with 20 µg/ml
EDTA.
Transfer 800 ml of acetonitrile (4.2) and 20 ml of acetic acid (4.4) into a 1 litre one-mark volumetric flask. Add
approximately 100 ml water and 2,0 ml of EDTA solution (4.6). Dilute to the mark with water, mix and filter
through a filter of 0,45 µm pore size (5.10).
4.10 Stock standard solutions.
4.10.1 General.
If catechins of known purity are available, they may be used directly as external standards. In addition to the
normally quoted HPLC purity, it is important that their moisture contents be also known, as high levels of water
of crystallization will not be accounted for in the HPLC assessment. The purity and moisture content data on
standards used in the interlaboratory test are given in Annex B. If comprehensive purity data are unavailable
or cannot be determined, catechin materials should only be used as marker compounds to aid identification. In
these circumstances, quantitation may be achieved using a caffeine external standard in conjunction with
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ISO 14502-2:2005(E)
consensus individual catechin RRF values (with respect to caffeine) obtained from interlaboratory tests (see
9.2 and Reference [3]).
4.10.2 Caffeine stock standard solution, corresponding to 2,00 mg/ml.
Weigh (0,200 ± 0,001) g of anhydrous caffeine into a 100 ml one-mark volumetric flask. Add sufficient warm
water to half-fill the flask. Swirl to dissolve the caffeine then cool to room temperature. Dilute to the mark with
water and mix.
4.10.3 Gallic acid stock standard solution, corresponding to approximately 1,00 mg/ml of anhydrous gallic
acid.
Weigh (0,110 ± 0,001) g of gallic acid monohydrate (M.W. 188,14) into a 100 ml one-mark volumetric flask.
Dissolve in water, dilute to the mark and mix.
Prepare fresh standard solution daily.
Gallic acid monohydrate is preferred over anhydrous, due to its greater solubility, reduced hygroscopic
properties and availability of certified reagent grades, e.g. A.C.S., which is used to denote chemicals that meet
specifications set by the American Chemical Society. If not known, the moisture content (loss in mass at
103 °C) should be determined on a portion of the standard material.
4.10.4 Preparation of individual catechin stock standard solutions
Accurately weigh the amounts of standards given in Table 1 into separate one-mark volumetric flasks.
Dissolve in stabilizing solution (4.8), gently warming if necessary (max. 40 °C). Cool to room temperature,
dilute to the mark with stabilizing solution and mix.
Table 1 — Catechin stock standard solutions
Mass of standard Volume of stabilizing Nominal concentration
solution of stock standard
Standard component
g ml mg/ml
(+)-Catechin 20 1,0
0,020 ± 0,001
(–)-Epicatechin 20 1,0
0,020 ± 0,001
(–)-Epigallocatechin 0,040 ± 0,001 20 2,0
(–)-Epigallocatechin gallate 0,040 ± 0,001 20 2,0
(–)-Epicatechin gallate 20 2,0
0,040 ± 0,001

Where sufficient quantities (i.e. > 20 mg) are available, an analytical balance capable of weighing to an
accuracy of at least 0,1 mg is required for the preparation of the individual stock standard solutions. For
limited quantities (i.e. < 20 mg), an analytical balance capable of weighing to 0,01 mg is required.
4.11 Dilute standard solutions.
4.11.1 Dilute gallic acid standard solution, corresponding to approximately 200 µg/ml.
Using a pipette, transfer 20 ml of the gallic acid stock standard solution (4.10.3) to a 100 ml one-mark
volumetric flask. Dilute to the mark with stabilizing solution (4.8) and mix.
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ISO 14502-2:2005(E)
4.11.2 Mixed standard solutions.
Prepare the three mixed standard solutions A, B and C containing caffeine, gallic acid and the catechins being
used for external standardization or as marker compounds. Carefully pipette the volumes given in Table 2 of
caffeine stock standard solution (4.10.2), dilute gallic acid standard solution (4.11.1) and any available
individual catechin stock standard solutions (4.10.4) into three separate 20 ml one-mark volumetric flasks.
Dilute to the mark with stabilizing solution (4.8) and mix. Pipette 1,0 ml aliquots of each mixed standard
solution into labelled small amber glass vials. Gently flush with nitrogen prior to sealing and store frozen at
−20 °C. The nominal concentrations of components of standard solutions A, B and C are given in Table 3.
With catechins of unknown purity it is essential that an individual HPLC assessment be first carried out to
check for other potentially interfering components.
NOTE The nominal concentrations of the mixed standard solutions A, B and C are given in Table 2 and have been
selected to cover the range typically found in tea.
Calculate the actual anhydrous concentrations from the masses used for preparation of the stock standard
solutions along with the standard moisture contents.
The mixed working standard solutions A, B and C will remain stable for at least 2 months when stored frozen
at −20 °C. Only thaw sufficient mixed working standard solution vials for each batch of analysis. Discard any
remaining solution, and do not refreeze.
Table 2 — Preparation of mixed standard solutions A, B and C
Aliquots required for the preparation of
20 ml of mixed standard solution
Component Solution
ml
A B C
Caffeine 2,00 mg/ml caffeine stock standard solution (4.10.2) 0,5 1,0 1,5
Gallic acid 200 µg/ml dilute gallic acid standard solution (4.11.1) 0,5 1,0 2,5
+C 1,00 mg/ml +C stock standard solution (4.10.4) 1,0 2,0 3,0
EC 1,00 mg/ml EC stock standard solution (4.10.4) 1,0 2,0 3,0
EGC 2,00 mg/ml EGC stock standard solution (4.10.4) 1,0 2,0 3,0
EGCG 2,00 mg/ml EGCG stock standard solution (4.10.4) 1,0 2,0 4,0
ECG 2,00 mg/ml ECG stock standard solution (4.10.4) 0,5 1,0 2,0

Table 3 — Nominal concentrations of mixed standard solutions A, B and C
Nominal concentration
Component
µg/ml
A B C
Gallic acid 5 10 25
Caffeine 50 100 150
+C 50 100 150
EC 50 100 150
EGC 100 200 300
EGCG 100 200 400
ECG 50 100 200
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ISO 14502-2:2005(E)
5 Apparatus
Usual laboratory apparatus and, in particular, the following.
5.1 Analytical balances, capable of weighing to an accuracy of ± 0,000 1 g and ± 0,000 01 g (see 4.10.4).
5.2 Water bath, capable of being maintained at (70 ± 1) °C.
5.3 Dispenser, for methanol/water extraction mixture (4.5), and set at 5,0 ml.
5.4 Centrifuge, capable of 2 000 Relative Centrifugal Force (R.C.F.) (typically 3 500 r/min).
5.5 Vortex mixer, for efficient mixing during extraction.
5.6 Extraction tubes, glass, of 10 ml capacity, stoppered and able to withstand being centrifuged.
5.7 Graduated tubes, glass, of 10 ml capacity with 0,1 ml graduations.
5.8 One-mark volumetric flasks, of capacities 5 ml, 10 ml, 20 ml, 100 ml, 500 ml, 1 litre and 2 litres.
5.9 Pipettes, glass or automatic, to cover the volume range for standard and sample extract dilutions.
5.10 Filters, membrane filter units of 0,45 µm pore size for filtration of mobile phases and diluted sample
extracts.
NOTE PTFE and nylon membrane filters have proven to be suitable.
All membranes should be checked to ensure that that catechin retention does not occur.
5.11 High-performance liquid chromatograph, equipped to perform binary gradient elution, with a
thermostatically controlled column compartment and an ultraviolet detector set at 278 nm.
5.12 Data collection /integration system.
5.13 Chromatographic column for HPLC.
NOTE Phenyl-bonded phases give additional selectivity over reversed-phase materials, and result in improved
resolution of the catechins. In this part of ISO 14502 the chromatographic conditions and composition of the mobile phase
1)
specified (4.9) are suitable for a Phenomenex Luna 5 µm Phenyl-Hexyl column of dimensions 250 mm ¥ 4,6 mm, fitted
2)
with a Phenomenex SecurityGuard 4 mm ¥ 3,0 mm Phenyl-Hexyl cartridge. If other types of column are used,
modifications to the mobile phase and chromatographic conditions may be necessary.
6 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this part of ISO 14502. A recommended sampling method is
given in
 ISO 1839 for leaf tea, and
 ISO 7516 for instant tea.

1) Phenyl-Hexyl is an example of a suitable product available commercially. This information is given for the
convenience of users of this part of ISO 14502 and does not constitute an endorsement by ISO of this product.
2) SecurityGuard is an example of a suitable product available commercially. This information is given for the
convenience of users of this part of ISO 14502 and does not constitute an endorsement by ISO of this product.
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ISO 14502-2:2005(E)
7 Preparation of test samples
To ensure homogeniety, grind the sample of leaf tea in accordance with ISO 1572 and store samples in well-
sealed containers protected from light.
Grinding of instant tea is only required on samples of a coarse granular structure (e.g. freeze-dried instant tea).
8 Procedure
8.1 General
If it is required to check whether the repeatability limit (10.2) is met, carry out two single determinations in
accordance with 8.2 to 8.6 under repeatability conditions.
8.2 Determination of dry matter content
Calculate the dry matter content from the moisture content (loss in mass at 103 °C) determined on a portion of
the test sample (Clause 7) in
...