1、BRITISH STANDARD BS ISO 10727:2002 Tea and instant tea in solid form Determination of caffeine content Method using high-performance liquid chromatography ICS 67.140.10 BS ISO 10727:2002 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Poli
2、cy and Strategy Committee, was published under the authority of the Standards Policy and Strategy Committee on 21 August 2002 BSI 21 August 2002 ISBN 0 580 39223 6 National foreword This British Standard reproduces verbatim ISO 10727:2002 and implements it as the UK national standard. It supersedes
3、BS 7820-1:1995 which is withdrawn. The UK participation in its preparation was entrusted to Technical Committee AW/8, Tea, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which
4、implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not p
5、urport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European commi
6、ttee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii an
7、d iii, a blank page, pages 1 to 8, an inside back cover and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsINTERNATIONAL STANDARD ISO 10727 Second edition 2002-07-15 Reference numb
8、er ISO 10727:2002(E) Tea and instant tea in solid form Determination of caffeine content Method using high-performance liquid chromatography Th et th soluble sous forme solide Dtermination de la teneur en cafine Mthode par chromatographie liquide haute performanceBSISO10727:2002ISO :72701(2002)E ii
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13、r SIsO memreb ydob in toc ehtnury fo tr ehtseuqeer. ISO copyrithg fofiec tsop esaCael 65 G 1121-HCeneva 02 Te.l + 22 14 10 947 11 Fax + 14 47 2290 9 74 E-liam copyrightsio.ch Web wwwsi.oc.h Prni detni Sztiwerldna BSISO10727:2002ISO 27012002:7)E( ISO 2002 r llAithgs reservde iii Foreword ISO (the Int
14、ernational 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 ha
15、s 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
16、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 a
17、t 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
18、prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 8, Te a. 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. BSISO10727:20022002:72701OSISBANRETNIITO
19、TS LANDNADRA ISO 27012002:7)E( ISO 2002 r llAithgs reservde 1 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
20、 (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.
21、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.
22、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, Te a Preparation of ground sample of known dry matter content ISO 3696:1987, Water for analytical laboratory
23、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 quantifie
24、d using high-performance liquid chromatography with ultraviolet detection. 4R e a g e n t s 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 of the
25、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 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
26、increasing the column temperature, but a temperature of should not be exceeded. 4.4 Ethanol/water mixture, (volume fraction). 600 ml 0,45 m 60 C 1/4 BSISO10727:2002ISO :72701(2002)E 2 ISO 2002 Ar llithgr seresvde 4.5 Magnesium oxide (so-called “dense magnesium oxide”). NOTE Light magnesium oxide may
27、 result in inaccurate caffeine content values. 4.6 Caffeine stock standard solution, corresponding to . Weigh, to the nearest , of caffeine into a 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
28、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 . Allow the caffeine stock stand
29、ard solution (4.6) to warm up to room temperature. Using a pipette, transfer of this solution to a one-mark volumetric flask. Dilute to the mark with water and mix. 4.7.2 Caffeine standard solution B, corresponding to . Follow the procedure given in 4.7.1 but take, using a pipette, of the stock stan
30、dard solution (4.6). 4.7.3 Caffeine standard solution C, corresponding to . Follow the procedure given in 4.7.1 but take, using a pipette, of the stock standard solution (4.6). 4.7.4 Caffeine standard solution D, corresponding to . Using a pipette, transfer of the standard solution B (4.7.2) to a on
31、e-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 and , and a data colle
32、ction/integration system or chart recorder. NOTE A wavelength close to is preferred since the maximum UV absorption of caffeine is at . 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
33、.2 Chromatographic column for HPLC, a reversed-phase C18 type, preferably with spherical particles and having an efficiency of at least theoretical plates 1) . The theoretical plate number of a column can be calculated as follows, from the shape of the peak obtained by injection of one of the caffei
34、ne standard solutions (4.7): 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 fo
35、r the convenience of users of this International Standard and does not constitute an endorsement by ISO of these products. 500 g/ml 0,001 g 0,125 g 250 ml 15 g/ml 3,0 ml 100 ml 10 g/ml 2,0 ml 5 g/ml 1,0 ml 2 g/ml 20 ml 100 ml 254 nm 280 nm 280 nm 272 nm 5 000 N N= 5,54 t W 0,5 2 BSISO10727:2002ISO 2
36、7012002:7)E( ISO 2002 r llAithgs reservde 3 where is the retention time of the peak; is the peak width at half peak height. 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
37、of dimensions , fitted in a Whatman HPLC cartridge system. If other types of column are used, an alternative mobile phase and alternative chromatographic conditions may be necessary. 5.3 Filter, membrane filter unit of pore size. 5.4 Electric heating mantle, to accommodate a 1 litre round-bottom fla
38、sk. 5.5 Flask, round bottom, of capacity 1 litre, with ground glass neck. 5.6 Anti-bumping granules. 5.7 Condenser, water-cooled with ground glass joint to fit a 1 litre round-bottom flask. 5.8 Analytical balance, capable of weighing to an accuracy of . 6 Sampling It is important that the laboratory
39、 receive a sample that is truly representative and has not been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. Recommended sampling methods are given in ISO 1839 for regular green tea and black tea; ISO 7516 for instant te
40、a. 7 Preparation of test sample Grind the sample of green tea or black tea in accordance with ISO 1572. Grinding is not required for instant tea samples. 8 Procedure 8.1 General If it is required to check whether the repeatability requirement is met, carry out two single determinations in accordance
41、 with 8.2 to 8.6 under repeatability conditions. 8.2 Determination of dry matter content Calculate the dry matter content from the moisture content (loss in mass at ) determined on a portion of the test sample in accordance with: ISO 1572 for green tea or black tea; ISO 7513 for instant tea. t W 0,5
42、 110 mm 4,6 mm 0,45 m 0,001 g 103 C BSISO10727:2002ISO :72701(2002)E 4 ISO 2002 Ar llithgr seresvde 8.3 Test portion 8.3.1 Green tea and black tea Weigh, to the nearest , of the test sample (clause 7) into a round-bottom flask (5.5). 8.3.2 Instant tea Weigh, to the nearest , of the test sample (clau
43、se 7) into a round-bottom flask (5.5). 8.4 Extraction of caffeine 8.4.1 Add, to the tea in the flask, of magnesium oxide (4.5), a few anti-bumping granules (5.6) andof water. Weigh, to the nearest , the flask and contents. 8.4.2 Mix the contents. Attach the condenser (5.7) and place the flask in the
44、 electric heating mantle (5.4) and rapidly bring to boiling. Reduce the heat and gently simmer for with occasional swirling. 8.4.3 Remove the flask and contents from the heating mantle and cool to room temperature. Dry the outside of the flask and weigh to the nearest . The mass of the cooled flask
45、and contents shall be equal to the mass determined in 8.4.1. If the masses differ, add sufficient water to increase the mass to that determined in 8.4.1 and then mix contents. 8.4.4 Leave the flask to stand to allow the contents to settle, then remove approximately of the supernatant solution and pa
46、ss it through the filter (5.3). 8.5 Dilution (for regular green tea and black tea and for regular instant tea only) Using a pipette, transfer of the filtrate obtained in 8.4.4 to a one-mark volumetric flask. Dilute to the mark with water and mix. For decaffeinated products, no dilution is necessary.
47、 8.6 Determination 8.6.1 Adjustment of the apparatus Set up the chromatograph (5.1) in accordance with the manufacturers instructions and adjust it as follows: flow rate of the mobile phase (4.3): to depending upon the column used (see 5.2); temperature of the column (5.2): optimally (but see 4.3).
48、8.6.2 HPLC analysis Once the flow rate of the mobile phase (4.3) and the temperature are stable, inject onto the column of each of the caffeine standard solutions A, B, C and D (4.7.1 to 4.7.4) followed by an equal volume of the test solution (obtained using the procedure in 8.4.4 for decaffeinated
49、tea and the procedure in 8.5 for regular leaf tea and regular instant tea). Repeat injection of standard solutions at regular intervals (typically after six test solutions). Collect and record the data for the peaks of all standards and test samples. 0,001 g 1,0 g 0,001 g 0,5 g 4,5 g 0,5 g 300 ml 0,1 g 20 min 0,1 g 10 ml 1,0 ml 10 ml 0,5 ml/min 1,5 ml/min 40 C 20 l BSISO10727:2002ISO 27012002:7)E( IS
