ISO/DIS 18447

Tea -- Determination of theaflavins in black tea -- Method using high performance liquid chromatography

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DRAFT INTERNATIONAL STANDARD
ISO/DIS 18447
ISO/TC 34/SC 8 Secretariat: BSI
Voting begins on: Voting terminates on:
2020-03-06 2020-05-29
Tea — Determination of theaflavins in black tea — Method
using high performance liquid chromatography
ICS: 67.140.10
THIS DOCUMENT IS A DRAFT CIRCULATED
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ISO/DIS 18447:2020(E)
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PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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ISO/DIS 18447:2020(E)
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ISO/DIS 18447:2020(E)
Contents  Page

Foreword ........................................................................................................................................................................................................................................iv

1  Scope ................................................................................................................................................................................................................................. 1

2  Normative references ...................................................................................................................................................................................... 1

3  Principle ........................................................................................................................................................................................................................ 1

4  Reagents ........................................................................................................................................................................................................................ 1

5  Apparatus ..................................................................................................................................................................................................................... 4

6  Sampling ........................................................................................................................................................................................................................ 5

7  Preparation of test samples ...................................................................................................................................................................... 5

8  Procedure..................................................................................................................................................................................................................... 5

8.1 General ........................................................................................................................................................................................................... 5

8.2 Determination of dry matter content .................................................................................................................................. 5

8.3 Test portion ................................................................................................................................................................................................ 5

8.3.1 Instant tea .............................................................................................................................................................................. 5

8.3.2 Leaf tea ..................................................................................................................................................................................... 5

8.3.3 Brews ......................................................................................................................................................................................... 6

8.4 Extraction of theaflavins ................................................................................................................................................................. 6

8.4.1 Instant tea .............................................................................................................................................................................. 6

8.4.2 Leaf tea ..................................................................................................................................................................................... 6

8.5 Dilution .......................................................................................................................................................................................................... 6

8.6 Determination ......................................................................................................................................................................................... 7

8.6.1 General...................................................................................................................................................................................... 7

8.6.2 Adjustment of the apparatus ................................................................................................................................. 7

8.6.3 HPLC analysis...................................................................................................................................................................... 7

8.6.4 Identification ....................................................................................................................................................................... 7

9  Calculation .................................................................................................................................................................................................................. 8

9.1 General ........................................................................................................................................................................................................... 8

9.1.1 Quantitation using theaflavin standards ..................................................................................................... 8

9.1.2 Quantitation using a caffeine standard and theaflavin 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 the purity of standards used in the RRF work ...................................12

Annex C (informative) Typical HPLC chromatograms .....................................................................................................................13

Bibliography .............................................................................................................................................................................................................................18

© ISO 2020 – All rights reserved iii
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ISO/DIS 18447:2020(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

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electrotechnical standardization.

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described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

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This document was prepared by Technical Committee ISO/TC 34 Food products, Subcommittee SC 8, Tea .

iv © ISO 2020 – All rights reserved
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DRAFT INTERNATIONAL STANDARD ISO/DIS 18447:2020(E)
Tea — Determination of theaflavins in black tea — Method
using high performance liquid chromatography
1  Scope

This International Standard specifies a high performance liquid chromatographic (HPLC) or ultra-high

performance liquid chromatographic (UHPLC) method for the determination of content of the four

major theaflavins of tea, and is applicable to both leaf and instant black and oolong teas. The method is

currently not validated for ready to drink beverages.
2  Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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, 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 degrees C)

3  Principle

Extraction of the theaflavins from a test portion of finely ground leaf tea is achieved with 70 %

methanol at 70 °C. Instant teas are dissolved in hot water with a volume fraction of 10 % acetonitrile

added to stabilise the extract. The individual theaflavins in the extract are determined by HPLC on

a reversed-phase column using isocratic elution with UV detection at 274 nm, optional at 375 nm (as

an alternative detection wavelength not used in the method validation). External standards are used

for quantitation. External theaflavin standards of defined purity and moisture content may be used

directly. Alternatively, caffeine may be used as a standard in conjunction with individual theaflavins

Relative Response Factors (RRFs) established by an ISO international interlaboratory test (see 9.1.2,

Table 3).
4  Reagents
Use only reagents of recognised analytical grade, unless otherwise specified.

SAFETY PRECAUTIONS — Wear gloves, eye protection and dispense reagents in a fume cupboard.

4.1  Water, grade 1 according to ISO 3696.
4.2  Acetonitrile, HPLC grade.
4.3  Methanol, HPLC grade.
4.4  Acetic acid, glacial HPLC grade.
4.5  Ascorbic acid, analytical grade

4.6  Ethylendiaminetetraacetic acid disodium salt, dihydrate (EDTA), analytical grade

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ISO/DIS 18447:2020(E)
4.7  Methanol/water extraction mixture, a volume fraction of 70 % methanol.

Add 700 ml of the methanol (4.3) to a 1 l one-mark volumetric flask. Dilute to the mark with water (4.1)

and mix.
4.8  20 %-acetonitrile stabiliser solution

Fill a 500 ml volumetric flask half with water (4.1). Add 10 ml of glacial acetic acid (4.4) and 100 ml of

acetonitrile (4.2). Add 125 mg each of ascorbic acid (4.5) and EDTA (4.6) and fill up to the mark with

water (4.1). This solution may be stored in the fridge for up to a week.
4.9  Leaf/Infusion stabilising solution

Weigh 0,0625 g EDTA (4.6) and 0,0625 g ascorbic acid (4.5) into a 25 ml volumetric flask and fill up to

the mark with water (4.1) giving a solution containing 2,5 mg/ml EDTA and 2,5 mg/ml ascorbic acid.

This solution may be stored in the fridge for up to a week.
4.10 HPLC mobile phases
4.10.1 Mobile phase A, 2% (volume fraction) acetic acid in water.

Transfer 40 ml of acetic acid (4.4) into a 2 l one-mark volumetric flask. Add sufficient water (4.1) to half

fill the flask and mix well. Dilute to the mark with water, mix and filter (5.10).

4.10.2 Mobile phase B, 2 % (volume fraction) acetic acid in acetonitrile.

Transfer 20 ml of acetic acid (4.4) into a 1 l one-mark volumetric flask. Add approximately 400 ml

acetonitrile (4.2), mix well and dilute to the mark with acetonitrile, mix again and filter.

4.11  Stock standard solutions
4.11.1 General

If theaflavins of known and guaranteed 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

are also known, as high levels of water of crystallization will not be accounted for in the HPLC

assessment. Purity and moisture content data on standards used in ISO interlaboratory testing are

given in Annex A. If comprehensive purity data are unavailable or cannot be determined, theaflavins

should only be used as marker compounds to aid identification. In these circumstances, quantification

may be achieved using a caffeine external standard in conjunction with consensus individual theaflavin

RRF values (with respect to caffeine) obtained from ISO interlaboratory testing (see 9.1.2, Table 3).

4.11.2  Preparation of individual theaflavin stock standard solutions

Weigh approximately 20 mg (exact weight recorded) of individual theaflavins (theaflavin: TF,

theaflavin-3-gallate: TF-3-g, theaflavin-3’-gallate: TF-3’-g and theaflavin-3,3’-gallate: TF-3,3’-dig)) into

separate 10 ml volumetric flasks. Fill up to mark with 20 % acetonitrile stabilising solution (4.6) to

give stock standards with a concentration of approximately 2 mg/ml.

NOTE 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, whereas

for limited quantities (i.e. < 20 mg) an analytical balance capable of weighing to 0,01 mg is required.

1) Statistical data in the annex have been generated using the calibration against caffeine (see 4.11)

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ISO/DIS 18447:2020(E)
4.12 Mixed standard solutions

To prepare the mixed standards pipette the following aliquots of the individual standards into 20 ml

volumetric flasks and fill up to the mark with 20 % acetonitrile stabilising solution (4.6).

Table 1 — Preparation of mixed TF working strength standards
Standard name Individual stock   Nominal concentration    Nominal concentration
theaflavin standards  of individual theaflavins of injection volume
diluted to 20 ml
ml mg/l μg/10 μl
A 4,0 µg std 4 400 4,0
B 3,0 µg std 3 300 3,0
C 2,0 µg std 2 200 2,0
D 1,0 µg std 1 100 1,0
E 0,5 µg std 0,5 50 0,5
F 0,25 µg std 0,25 25 0,25

The concentration of the individual TF’s given in Table 1 is based on 100 % purity of the standards.

The purity of the standard TF’s should be determined by high resolution NMR spectroscopy and the

nominal concentrations of the standards accordingly to give the exact concentration of the individual

TFs in each standard.

NOTE 1 With theaflavins of unknown purity it is essential that an individual HPLC assessment is first carried

out to check for other potentially interfering components.

NOTE 2 The nominal concentrations of the mixed standard solutions A-F are given in Table 1 and have been

selected to cover the range typically found in tea. Calculate actual anhydrous concentrations from the weights

used for preparation of the stock standard solutions along with the standard moisture contents.

NOTE 3 The mixed working standard solutions A-F 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.

4.13  Caffeine standard - Preparation of caffeine stock 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 to give a stock standard with a concentration of 2 mg/ml.

Prepare aliquots of the caffeine stock standard into 20 ml volumetric flasks as detailed in Table 2 and

make up to volume with 20% acetonitrile stabilising solution (4.6).
Table 2 — Preparation of caffeine working strength standards

Standard name Individual stock caf-  Nominal concentration    Nominal concentration

feine standards diluted  of caffeine of injection volume
to 20 ml
ml mg/l μg/10 μl
A 1,0 µg std 1 100 1,0
B 0,5 µg std 0,5 50 0,5
C 0,25 µg std 0,25 25 0,25
D 0,1 µg std 0,1 10 0,1
E 0,05 µg std 0,05 5 0,05
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