ISO 19563:2017

INTERNATIONAL ISO
STANDARD 19563
First edition
2017-01
Determination of theanine in tea and
instant tea in solid form using high-
performance liquid chromatography
Détermination de la théanine dans le thé et le thé instantané sous
forme solide en utilisant la chromatographie en phase liquide à haute
performance
Reference number
©
ISO 2017
© ISO 2017, Published in Switzerland
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ii © ISO 2017 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus . 3
7 Sampling . 3
8 Preparation of test samples . 3
9 Procedure. 3
9.1 General . 3
9.2 Determination of dry matter content . 4
9.3 Test portion . 4
9.3.1 Sample preparations . 4
9.3.2 Sample clean-up (using a polyamide column) — Optional . 4
9.4 Determination . 5
9.4.1 Adjustment of the apparatus . 5
9.4.2 HPLC analysis. 5
9.4.3 Identification . 5
10 Calculation . 6
11 Precision . 6
11.1 Interlaboratory test. 6
11.2 Repeatability . 6
11.3 Reproducibility . 6
12 Test report . 7
Annex A (informative) Typical theanine calibration graph . 8
Annex B (informative) Typical chromatograms . 9
Annex C (informative) Results of interlaboratory tests .12
Bibliography .13
Foreword
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The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 8, Tea.
iv © ISO 2017 – All rights reserved

Introduction
Theanine (N-ethyl-γ-L-glutamine) is a non-protein derived (free) amino acid. In the plant world, it is a
unique amino acid found in Camellia genus and the mushroom Boletus badius and Ilex guayusa. The most
common and the most prominent occurrence is in Camellia sinensis (L.) O. Kuntze species.
Theanine constitutes circa 0,1 % to 2 % w/w of the dry weight in tea leaves (Camellia sinensis) and
is approximately 50 % of the total free amino acids. Separation of L- and D-theanine by HPLC is not
[2][3]
achieved by this method; however, L-theanine is the major form (more than 95 %) in all teas .
Theanine has been associated with the typical tea umami taste and is also being studied to evaluate
potential beneficial effects to health.
The umami taste characteristic has been associated with theanine which is also being evaluated for its
potential benefits to health. This is also creating interest in theanine as an important novel food and
dietary supplement ingredient.
INTERNATIONAL STANDARD ISO 19563:2017(E)
Determination of theanine in tea and instant tea in solid
form using high-performance liquid chromatography
1 Scope
This document specifies a high-performance liquid chromatographic (HPLC) method for the
determination of theanine content in tea (Camellia sinensis). It is applicable to both tea and instant tea
samples. Separation of L- and D-theanine is not possible using this method; however, the L-enantiomer
is the major form in tea.
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 1573, Tea — Determination of loss in mass at 103 °C
ISO 1839, Tea — Sampling
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)
ISO 7516, Instant tea in solid form — Sampling
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:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
4 Principle
A reversed-phase (RP)-C18 column is used which is capable of separating more polar compounds using
water as an eluent and UV detection at 210 nm. Theanine is separated from the other amino acids;
however, the potential co-elution of theanine with methionine is not relevant due to the very small
concentration of methionine in tea. It is recommended that polyamide column extract clean-up is used
to remove interfering polyphenols for column protection when separation of theanine from other
constituents in the extract sample is difficult. Alternatively, the column should be washed after each tea
sample using acetonitrile.
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
5.1 Water, conforming to grade 1 of ISO 3696.
5.2 Acetonitrile, HPLC grade (however, super grade is preferable).
5.3 Methanol, regular laboratory distilled grade.
1)
5.4 Polyamide (Polyamide CC 6 , Particle size: 0,05 mm to 0,16 mm for column chromatography).
5.5 L-theanine reference standard, >99 %, anhydrous.
5.6 HPLC mobile phases.
WARNING — Wear gloves and eye protection and dispense reagents in a fume cupboard.
5.6.1 Mobile phase A, 100 % purified water (5.1).
5.6.2 Mobile phase B, 100 % acetonitrile (5.2).
5.7 Standard solution.
5.7.1 Standard stock solution, corresponding to 1 mg/ml.
Weigh (0,050 0 ± 0,000 1) g of pure L-theanine (5.5) in a 50 ml
...