1、BS ISO 19563:2017Determination of theanine intea and instant tea in solidform using high-performanceliquid chromatographyBSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS ISO 19563:2017 BRITISH STANDARDNational forewordThis British Standard is the UK implementation
2、of ISO 19563:2017.The UK participation in its preparation was entrusted to Technical Committee AW/8, Tea.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users ar
3、e responsible for its correct application. The British Standards Institution 2017.Published by BSI Standards Limited 2017ISBN 978 0 580 86547 3 ICS 67.140.10 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of th
4、e Standards Policy and Strategy Committee on 31 January 2017.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 19563:2017 ISO 2017Determination of theanine in tea and instant tea in solid form using high-performance liquid chromatographyDtermination de la thanine dans
5、le th et le th instantan sous forme solide en utilisant la chromatographie en phase liquide haute performanceINTERNATIONAL STANDARDISO19563First edition2017-01Reference numberISO 19563:2017(E)BS ISO 19563:2017ISO 19563:2017(E)ii ISO 2017 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2017, Publ
6、ished in SwitzerlandAll rights reserved. Unless otherwise specified, 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. Permiss
7、ion can be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 19563:2017ISO 19563:2017(E)Forewo
8、rd ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 15 Reagents 26 Apparatus . 37 Sampling 38 Preparation of test samples 39 Procedure. 39.1 General . 39.2 Determination of dry matter content 49.3 Test portion 49.3.1 Sample preparations 49.3.2 Sample clean-up
9、 (using a polyamide column) Optional . 49.4 Determination . 59.4.1 Adjustment of the apparatus . 59.4.2 HPLC analysis 59.4.3 Identification . 510 Calculation 611 Precision . 611.1 Interlaboratory test. 611.2 Repeatability . 611.3 Reproducibility . 612 Test report . 7Annex A (informative) Typical the
10、anine calibration graph 8Annex B (informative) Typical chromatograms 9Annex C (informative) Results of interlaboratory tests .12Bibliography .13 ISO 2017 All rights reserved iiiContents PageBS ISO 19563:2017ISO 19563:2017(E)ForewordISO (the International Organization for Standardization) is a worldw
11、ide 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 th
12、at 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 docu
13、ment 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 Directiv
14、es, 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 any patent rights identified during the develop
15、ment of the document will be in the Introduction and/or 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.For an explanation on the meaning of ISO
16、specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www .iso .org/ iso/ foreword .html.The committee responsible for this document
17、 is ISO/TC 34, Food products, Subcommittee SC 8, Tea.iv ISO 2017 All rights reservedBS ISO 19563:2017ISO 19563:2017(E)IntroductionTheanine (N-ethyl-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
18、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-th
19、eanine by HPLC is not achieved by this method; however, L-theanine is the major form (more than 95 %) in all teas23. 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 asso
20、ciated 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. ISO 2017 All rights reserved vBS ISO 19563:2017BS ISO 19563:2017Determination of theanine in tea and insta
21、nt tea in solid form using high-performance liquid chromatography1 ScopeThis 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-t
22、heanine is not possible using this method; however, the L-enantiomer is the major form in tea.2 Normative referencesThe 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 ci
23、ted 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 contentISO 1573, Tea Determination of loss in mass at 103 CISO 1839, Tea SamplingISO 3696, Water for analytical laborat
24、ory use Specification and test methodsISO 7513, Instant tea in solid form Determination of moisture content (loss in mass at 103 C)ISO 7516, Instant tea in solid form Sampling3 Terms and definitionsNo terms and definitions are listed in this document.ISO and IEC maintain terminological databases for
25、 use in standardization at the following addresses: IEC Electropedia: available at h t t p :/ www .electropedia .org/ ISO Online browsing platform: available at h t t p :/ www .iso .org/ obp4 PrincipleA reversed-phase (RP)-C18 column is used which is capable of separating more polar compounds using
26、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
27、 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.INTERNATIONAL STANDARD ISO 19563:2017(E) ISO 2017 All rights reserved 1BS
28、ISO 19563:2017ISO 19563:2017(E)5 ReagentsUse 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.5.4 Polyamide (Polyami
29、de CC 61), 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
30、 % 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 volumetric flask dissolve with purified water using sonication to aid dissolution and make up to the volume with purified water.5.7.2 Stan
31、dard working solution.The stock solution is diluted with purified water to prepare the standard solutions in the concentration range of 5 g/ml to 200 g/ml as detailed in Table 1.Table 1 Stock solution dilutionsStandard solutionWorking solution (g/ml)Volume taken from the stock solution (ml)Final vol
32、ume (ml)A 5 0,5 100B 10 1,0 100C 20 1,0 50D 50 2,5 50E 100 5,0 50F 150 7, 5 50G 200 10,0 501) Polyamide CC 6 is the required/minimum specification required for the separation which is available commercially. This information is given for the convenience of users of this document and does not constit
33、ute an endorsement by ISO of this product.2 ISO 2017 All rights reservedBS ISO 19563:2017ISO 19563:2017(E)6 ApparatusUsual laboratory apparatus and, in particular, the following:6.1 Analytical balances, capable of weighing to an accuracy of 0,000 1 g.6.2 Magnetic stirrer, capable of 500 r/min.6.3 Fi
34、lters, membrane filter units of 0,45 m pore size for filtration of mobile phase and diluted sample extracts. All membranes used should be checked using a simple calibration solution to ensure that theanine retention does not occur.6.4 Centrifuge, capable of 12 300 relative centrifugal force (R.C.F.)
35、 (typically 13 500 r/min).6.5 Glass column, 17 cm 2,2 cm in diameter, (for polyamide clean-up only).6.6 One mark volumetric flasks, of capacities 50 ml and 100 ml.6.7 High performance liquid chromatograph, equipped to perform binary gradient elution, with a thermostatically controlled column compart
36、ment and an ultraviolet detector set at 210 nm.6.8 Chromatographic column for HPLC, Phenomenex AquaTM2)250 4,6 in diameter or 2 mm in diameter or equivalent.NOTE This method has been developed with the Aqua which has the minimum specification required for this analysis.7 SamplingThe sampling methods
37、 that shall be used are detailed in ISO 1839 for tea or ISO 7516 for instant tea.A representative sample(s) shall be sent to the laboratory. It shall not be damaged or changed during transport or storage.8 Preparation of test samplesTo ensure homogeneity, grind the sample of leaf tea in accordance w
38、ith ISO 1572 and store samples in well-sealed containers protected from light.Grinding of instant tea samples is only required with samples having a coarse granular structure (e.g. freeze-dried instant tea).9 Procedure9.1 GeneralIf it is required to check whether the results are within the acceptabl
39、e repeatability limit (see 11.2), carry out two single determinations in accordance with 9.2 to 9.4 under repeatability conditions.2) Phenomenex AquaTMis the required/minimum specification required for the separation which is available commercially. This information is given for the convenience of u
40、sers of this document and does not constitute an endorsement by ISO of this product. ISO 2017 All rights reserved 3BS ISO 19563:2017ISO 19563:2017(E)9.2 Determination of dry matter contentCalculate the dry matter content from the moisture content (loss in mass at 103 C) determined on a portion of th
41、e test sample (Clause 8) in accordance with ISO 1573 for tea or ISO 7513 for instant tea.9.3 Test portion9.3.1 Sample preparations9.3.1.1 Leaf tea9.3.1.1.1 Weigh (1,00 0,01) g of a finely ground sample into a 200 ml beaker and add 100 ml of boiling water.9.3.1.1.2 Allow brewing for 5 min using a mag
42、netic stirrer and then filter through a filter paper (Grade 4 or equivalent) into a 100 ml volumetric flask.9.3.1.1.3 Allow the tea brew to cool down to ambient temperature, make up to volume of 100 ml with purified water and filter using a 0,45 m membrane before injection. Alternatively, approximat
43、ely 1 ml of the sample solution can be centrifuged at 13 000 r/min for 10 min prior to HPLC analysis.9.3.1.2 Tea extract or instant tea9.3.1.2.1 Weigh (0,100 0,001) g sample into a 50 ml volumetric flask, dissolve and make up with heated purified water (approximately 50 C).9.3.1.2.2 Sonicate for 2 m
44、in to 5 min for complete dissolution and filter using a 0,45 m membrane before injection. Alternatively, approximately 1 ml of the sample solution can be centrifuged at 13 000 r/min for 10 min prior to HPLC analysis.9.3.2 Sample clean-up (using a polyamide column) OptionalCertain tea samples will no
45、t elute with clean chromatograms. When this occurs in the position where theanine elutes in the chromatogram, the interference will prevent an accurate determination of the peak area. On these occasions, it is necessary to carry out the clean-up procedures as described below in order to improve sepa
46、ration and to protect the column.9.3.2.1 Suspend 1,2 kg polyamide in a mixture of 8 l of water and 1,5 l of methanol, mix well and allow settling overnight.9.3.2.2 Remove supernatant and wash with 1,5 l methanol twice. Store in methanol and mix well before use.9.3.2.3 Column chromatography:9.3.2.3.1
47、 Fill a glass column (17 cm 2,2 cm in diameter) equipped with a thin layer of glass wool and sea sand with swollen polyamide to a height of 8 cm. Condition with 250 ml of water.9.3.2.3.2 Apply 50 ml of the tea solution to the column and immediately collect the eluate into a 100 ml volumetric flask.4
48、 ISO 2017 All rights reservedBS ISO 19563:2017ISO 19563:2017(E)9.3.2.3.3 Wash the column with purified water until the mark of 100 ml is reached. Mix well and filter using a 0,45 m membrane before injection. Alternatively, approximately 1 ml of the sample solution can be centrifuged at 13 000 r/min
49、for 10 min prior to HPLC analysis.The polyphenols will be retained on the polyamide while the theanine elutes. This step corresponds to a twofold dilution.9.4 Determination9.4.1 Adjustment of the apparatusSet up the chromatograph (6.7) in accordance with the manufacturers instructions and adjust it as follows.a) Flow rate of the mobile phase (5.6.1 and 5.6.2): 1,0 ml/min for 4,6 mm columns and 0,25 ml/min for 2 mm columns.b) Binary gradient conditions: according to the elution programme as det
