ISO 10727:2002

INTERNATIONAL ISO
STANDARD 10727
Second edition
2002-07-15
Tea and instant tea in solid form —
Determination of caffeine content — Method
using high-performance liquid
chromatography
Thé et thé soluble sous forme solide — Détermination de la teneur en
caféine — Méthode par chromatographie liquide à haute performance

Reference number
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not be
edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file,
parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters
were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event
that a problem relating to it is found, please inform the Central Secretariat at the address given below.
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's
member body in the country of the requester.
ISO copyright office
Case postale 56  CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.ch
Web www.iso.ch
Printed in Switzerland
©
ii ISO 2002 – All rights reserved

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 3.
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 International Standard may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 10727 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee
SC 8, Tea.
This second edition cancels and replaces the first edition (ISO 10727:1995), which has been technically revised.
Annex A of this International Standard is for information only.
©
ISO 2002 – All rights reserved iii

INTERNATIONAL STANDARD ISO 10727:2002(E)
Tea and instant tea in solid form — Determination of caffeine
content — Method using high-performance liquid chromatography
1 Scope
This International Standard specifies a method for the determination by high-performance liquid chromatography
(HPLC) of the caffeine content of teas and instant teas. It is applicable to green tea, black tea and decaffeinated tea
products.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 1572:1980, Tea — Preparation of ground sample of known dry matter content
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 7513:1990, Instant tea in solid form — Determination of moisture content (loss in mass at 103 °C)
3 Principle
The caffeine from a test portion is extracted by reflux with water in the presence of magnesium oxide. After filtration,
the caffeine content is quantified using high-performance liquid chromatography with ultraviolet detection.
4Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
4.1 Water, in accordance with grade 1 of ISO 3696.
4.2 Methanol, HPLC grade.
4.3 Mobile phase, methanol/water mixture.
Add 600 ml of the methanol (4.2) to a 2 litre one-mark volumetric flask. Dilute to the mark with water and mix. Filter
the mixture through a filter of 0,45µm pore size (5.3).
By adjusting the methanol concentration, the retention time of the caffeine can be modified to optimize the HPLC
◦
separation. This can also be done by increasing the column temperature, but a temperature of 60 C should not be
exceeded.
4.4 Ethanol/water mixture, 1/4 (volume fraction).
©
ISO 2002 – All rights reserved 1

4.5 Magnesium oxide (so-called “dense magnesium oxide”).
NOTE Light magnesium oxide may result in inaccurate caffeine content values.
4.6 Caffeine stock standard solution, corresponding to 500µg/ml.
Weigh, to the nearest 0,001 g, 0,125 g of caffeine into a 250 ml one-mark volumetric flask. Add sufficient
ethanol/water mixture (4.4) to half fill the flask. Swirl to dissolve the caffeine and then dilute to the mark with the
ethanol/water mixture.
This solution will remain stable for up to 1 month if stored in a refrigerator.
4.7 Caffeine standard solutions.
Standard solutions A, B, C and D shall be prepared on the day of use.
4.7.1 Caffeine standard solution A, corresponding to 15µg/ml.
Allow the caffeine stock standard solution (4.6) to warm up to room temperature. Using a pipette, transfer 3,0 ml of
this solution to a 100 ml one-mark volumetric flask. Dilute to the mark with water and mix.
4.7.2 Caffeine standard solution B, corresponding to 10µg/ml.
Follow the procedure given in 4.7.1 but take, using a pipette, 2,0 ml of the stock standard solution (4.6).
4.7.3 Caffeine standard solution C, corresponding to 5µg/ml.
Follow the procedure given in 4.7.1 but take, using a pipette, 1,0 ml of the stock standard solution (4.6).
4.7.4 Caffeine standard solution D, corresponding to 2µg/ml.
Using a pipette, transfer 20 ml of the standard solution B (4.7.2) to a 100 ml one-mark volumetric flask. Dilute to the
mark with water and mix.
5 Apparatus
Usual laboratory apparatus, and, in particular, the following.
5.1 High-performance liquid chromatograph, equipped with an ultraviolet detector, allowing measurements to
be made at a wavelength between 254 nm and 280 nm, and a data collection/integration system or chart recorder.
NOTE A wavelength close to 280 nm is preferred since the maximum UV absorption of caffeine is at 272 nm.
Where diurnal variations in temperature are wide, a means of ensuring a constant column temperature should be
provided, for example a column oven or water jacket.
5.2 Chromatographic column for HPLC, a reversed-phase C18 type, preferably with spherical particles and
1)
having an efficiency of at least 5 000 theoretical plates . The theoretical plate number N of a column can be
calculated as follows, from the shape of the peak obtained by injection of one of the caffeine standard solutions (4.7):
� �
t
N= 5,54
W
0,5
1) Spherisorb 5 ODS, Spherisorb 10 ODS, Nucleosil 5 C18, Nucleosil 7 C18, Nucleosil 10 C18, Zorbax BP C18, Hypersil ODS,
CP-Sper C18, Bondapak C18, Supelcosil L C18 and Partisphere C18 are examples of suitable products available commercially.
This information is given for the convenience of users of this International Standard and does not constitute an endorsement by
ISO of these products.
©
2 ISO 2002 – All rights reserved

where
t is the retention time of the peak;
W is the peak width at half peak height.
0,5
NOTE In this International Standard, the chromatographic conditions and the composition of the mobile phase (4.3) specified are
suitable for a Partisphere C18 cartridge column of dimensions 110 mm× 4,6 mm, fitted in a Whatman
...