ISO 18447:2021

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