ISO 18447:2021

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

This document specifies a high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC) method for the determination of content of the four major theaflavins of tea. It is applicable to both leaf and instant black and oolong teas. The method is currently not validated for ready-to-drink (RTD) beverages.

Thé — Détermination des théaflavines dans le thé noir — Méthode par chromatographie liquide à haute performance

General Information

• Status: Published
• Publication Date: 09-Jun-2021
• ICS: 67.140.10 - Tea
• Technical Committee: ISO/TC 34/SC 8 - Tea
• Drafting Committee: ISO/TC 34/SC 8 - Tea
• Current Stage: 6060 - International Standard published
• Start Date: 10-Jun-2021
• Due Date: 09-Feb-2022
• Completion Date: 10-Jun-2021

Overview

ISO 18447:2021 - Tea - Determination of theaflavins in black tea - Method using high performance liquid chromatography defines a validated HPLC/UHPLC procedure for quantifying the four major theaflavins in tea. The standard applies to leaf and instant black and oolong teas (not validated for ready-to-drink/RTD beverages) and specifies extraction, chromatographic conditions, calibration approaches and reporting requirements for reproducible theaflavin analysis.

Keywords: ISO 18447:2021, theaflavins, black tea, HPLC, UHPLC, tea analysis, tea quality, chromatographic method

Key topics and technical requirements

• Analytes covered: the four major theaflavins - TF, TF‑3‑gallate, TF‑3′‑gallate and TF‑3,3′‑digallate.
• Method type: Reversed‑phase HPLC or UHPLC with isocratic elution and UV detection (primary wavelength 274 nm; 375 nm optional).
• Sample scope: Finely ground leaf tea and instant tea (leaf extraction and instant tea dissolution procedures described). Not validated for RTD beverages.
• Extraction and stabilization: Leaf extraction with 70% methanol at 70 °C; instant tea dissolved in hot water with acetonitrile stabilizer. Antioxidants/chelators (ascorbic acid, EDTA) used to stabilize extracts.
• Calibration: External theaflavin standards of known purity and moisture content are preferred. Alternatively, a caffeine external standard may be used together with consensus relative response factors (RRFs) established through ISO interlaboratory testing.
• Reagents and mobile phases: HPLC-grade methanol, acetonitrile, acetic acid, water (ISO 3696 grade) and stabilizer solutions specified. Mobile phases include acidified aqueous and acetonitrile phases (2% acetic acid).
• Quality and precision: The standard includes interlaboratory test results, repeatability and reproducibility guidance, and requirements for test reporting.

Practical applications

• Quality control and specification testing for black and oolong tea producers and packers.
• Product development and process optimization to monitor fermentation and oxidation (theaflavin formation).
• Research laboratories studying tea chemistry, health-related components or processing effects.
• Regulatory and certification bodies requiring traceable, standardized quantitation of theaflavins for labeling, trade or conformity assessment.

Who should use this standard

• Analytical laboratories performing tea composition testing
• Tea manufacturers and ingredient suppliers for QC and shelf‑life studies
• Food scientists and academic researchers focusing on polyphenol analyses

Related standards

• ISO 1572 - Preparation of ground sample of known dry matter content (tea)
• ISO 3696 - Water for analytical laboratory use - Specification and test methods
• ISO 7513 - Instant tea - Determination of moisture content

This ISO method provides a standardized, reproducible HPLC/UHPLC workflow for accurate theaflavin quantitation - a key metric in assessing black/oolong tea quality and processing outcomes.

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 18447
First edition
2021-06
Tea — Determination of theaflavins
in black tea — Method using high
performance liquid chromatography
Thé — Détermination des théaflavines dans le thé noir — Méthode
par chromatographie liquide à haute performance
Reference number
©
ISO 2021
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: [phone]
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Published in Switzerland
ii © ISO 2021 – All rights reserved

Contents  Page
Foreword .iv
1  Scope . 1
2  Normative references . 1
3  Terms and definitions . 1
4  Principle . 1
5  Reagents . 1
5.1 General . 1
5.2 HPLC mobile phases . 2
5.3 Stock standard solutions . 2
5.3.1 General. 2
5.3.2 Preparation of individual theaflavin stock standard solutions . 3
5.4 Mixed standard solutions. 3
5.5 Caffeine standard — Preparation of caffeine stock solution, corresponding to
2,00 mg/ml . 3
6  Apparatus . 4
7  Sampling . 5
8  Preparation of test samples . 5
9  Procedure. 5
9.1 General . 5
9.2 Determination of dry matter content . 5
9.3 Test portion . 6
9.3.1 Instant tea . 6
9.3.2 Leaf tea . 6
9.4 Extraction of theaflavins . 6
9.4.1 Instant tea . 6
9.4.2 Leaf tea . 6
9.5 Dilution . 6
9.6 Determination . 7
9.6.1 General. 7
9.6.2 Adjustment of the apparatus . 7
9.6.3 HPLC analysis. 7
9.6.4 Identification . 7
10  Calculation . 8
10.1 General . 8
10.2 Quantitation using theaflavin standards . 8
10.3 Quantitation using a caffeine standard and theaflavin RRFs . 9
11  Precision .10
11.1 Interlaboratory test.10
11.2 Repeatability .10
11.3 Reproducibility .10
12  Test report .10
Annex A (informative) Results of interlaboratory tests.12
Annex B (informative) Assessment of the purity of standards used in the RRF work .13
Annex C (informative) Typical HPLC chromatograms .14
Bibliography .19
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.
The procedures used to develop this document and those intended for its further maintenance are
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).
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. Details of
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on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
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expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 8,
Tea.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2021 – All rights reserved

INTERNATIONAL STANDARD ISO 18447:2021(E)
Tea — Determination of theaflavins in black tea — Method
using high performance liquid chromatography
1  Scope
This document specifies a high performance liquid chromatography (HPLC) or ultra-high performance
liquid chromatography (UHPLC) method for the determination of content of the four major theaflavins
of tea.
It is applicable to both leaf and instant black and oolong teas. The method is currently not validated for
ready-to-drink (RTD) 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 °C)
3  Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
4  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 stabilize 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 Table 3).
5  Reagents
5.1  General
Use only reagents of recognized analytical grade, unless otherwise specified.
SAFETY PRECAUTIONS — Wear gloves and eye protection, and dispense reagents in a fume
cupboard.
5.1.1 Water, grade 1 in accordance with ISO 3696.
5.1.2 Acetonitrile, HPLC grade.
5.1.3 Methanol, HPLC grade.
5.1.4 Acetic acid, glacial, HPLC grade.
5.1.5 Ascorbic acid, analytical grade.
5.1.6 Ethylenediaminetetraacetic acid disodium salt, dihydrate (EDTA), analytical grade.
5.1.7 Methanol/water extraction mixture, a volume fraction of 70 % methanol.
Add 700 ml of the methanol (5.1.3) to a 1 l one-mark volumetric flask. Dilute to the mark with water
(5.1.1) and mix.
5.1.8 20 % acetonitrile stabilizer solution.
Fill a 500 ml volumetric flask half with water (5.1.1). Add 10 ml of glacial acetic acid (5.1.4) and 100 ml
of acetonitrile (5.1.2). Add 125 mg each of ascorbic acid (5.1.5) and EDTA (5.1.6) and fill up to the mark
with water (5.1.1). This solution may be stored in the fridge for up to a week.
5.1.9 Leaf/infusion stabilizing solution.
Weigh 0,062 5 g EDTA (5.1.6) and 0,062 5 g ascorbic acid (5.1.5) into a 25 ml volumetric flask and fill
up to the mark with water (5.1.1), 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.
5.2  HPLC mobile phases
5.2.1  Mobile phase A, 2 % (volume fraction) acetic acid in water.
Transfer 40 ml of acetic acid (5.1.4) into a 2 l one-mark volumetric flask. Add sufficient water (5.1.1) to
fill half of the flask and mix well. Dilute to the mark with water, mix and filter (6.10).
5.2.2  Mobile phase B, 2 % (volume fraction) acetic acid in acetonitrile.
Transfer 20 ml of acetic acid (5.1.4) into a 1 l one-mark volumetric flask. Add approximately 400 ml
acetonitrile (5.1.2), mix well and dilute to the mark with acetonitrile, mix again and filter.
5.3  Stock standard solutions
5.3.1  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 Table 3).
NOTE The statistical data in Annex A have been generated using the calibration against caffeine (see 5.5).
2 © ISO 2021 – All rights reserved

5.3.2  Preparation of individual theaflavin stock standard solutions
Weigh approximately 20 mg (exact weight recorded) of the following individual theaflavins into
separate 10 ml volumetric flasks: theaflavin (TF), theaflavin-3-gallate (TF-3-g), theaflavin-3′-gallate
(TF-3′-g) and theaflavin-3,3′-gallate (TF-3,3′-dig). Fill up to the mark with 20 % acetonitrile stabilizing
solution (5.1.8) 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.
5.4  Mixed standard solutions
To prepare the mixed standards, pipette the aliquots of the individual standards into 20 ml volumetric
flasks in accordance with Table 1 and fill up to the mark with 20 % acetonitrile stabilizing solution
(5.1.8).
Table 1 — Preparation of mixed TF working strength standards
Standard name Individual stock theafla- Nominal concentration  Nominal concentration
vin standards diluted to  of individual theaflavins of injection volume
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 TFs given in Table 1 is based on 100 % purity of the standards.
The purity of the standard TFs should be determined by high resolution nuclear magnetic resonance
(NMR) spectroscopy and the nominal concentrations of the standards accordingly should give the exact
concentration of the individual TFs in each standard.
With theaflavins of unknown purity, it is strongly recommended that an individual HPLC assessment is
first carried out to check for other potentially interfering components.
The nominal concentrations of the mixed standard solutions A to 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.
The mixed working standard solutions A to F will remain stable for at least two 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.
5.5  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 stabilizing solution (5.1.8).
...