1、BRITISH STANDARDBS ISO 14502-2:2005Incorporating Corrigendum No. 1 Determination of substances characteristic of green and black tea Part 2: Content of catechins in green tea Method using high-performance liquid chromatographyICS 67.140.10g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g
2、44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS ISO 14502-2:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 7 April 2005 BSI 2006ISBN 0 580 4577
3、7 XNational forewordThis British Standard reproduces verbatim ISO 14502-2:2005, including Technical Corrigendum April 2006 and implements it as the UK national standard.The UK participation in its preparation was entrusted to Technical Committee AW/8, Tea, which has the responsibility to: aid enquir
4、ers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK.A list of organizations represen
5、ted on this committee can be obtained on request to its secretary.Cross-referencesThe British Standards which 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
6、 “Search” facility of the BSI Electronic Catalogue or of British Standards Online.This publication does not purport 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 leg
7、al obligations.Summary of pagesThis document comprises a front cover, an inside front cover, the ISO title page, pages ii to iv, pages 1 to 23 and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No.
8、Date Comments16366Corrigendum No. 131 July 2006 Changes to Clauses 4.10.1, 9.1, 9.2.3 and Annex E, Table E.2Reference numberISO 14502-2:2005(E)INTERNATIONAL STANDARD ISO14502-2First edition2005-03-01Determination of substances characteristic of green and black tea Part 2: Content of catechins in gre
9、en tea Method using high-performance liquid chromatography Dtermination des substances caractristiques du th vert et du th noir Partie 2: Dosage des catechins dans le th vert Mthode par chromatographie en phase liquide haute performance BS ISO 145022:2005ii BS ISO 145022:2005iiiContents Page Forewor
10、d iv 1 Scope 1 2 Normative references . 1 3 Principle . 1 4 Reagents 1 5 Apparatus. 5 6 Sampling 5 7 Preparation of test samples. 6 8 Procedure. 6 8.1 General. 6 8.2 Determination of dry matter content. 6 8.3 Test portion . 6 8.4 Extraction of polyphenols 6 8.5 Dilution. 7 8.6 Determination 7 9 Calc
11、ulation. 8 9.1 General. 8 9.2 Quantitation using catechin standards 8 9.3 Quantitation using a caffeine standard and catechin Relative Response Factors (RRFs) . 9 10 Precision 10 10.1 Interlaboratory test . 10 10.2 Repeatability 10 10.3 Reproducibility 10 11 Test report 10 Annex A (informative) Resu
12、lts of interlaboratory tests 11 Annex B (informative) Assessment of purity of standards 13 Annex C (informative) Typical HPLC chromatograms 14 Annex D (informative) The effect of ferric ions on catechin RRFs 18 Annex E (informative) Quantitative comparison Use of catechin standards or a caffeine sta
13、ndard in conjunction with catechin RRFs. 21 Bibliography . 23 BS ISO 145022:2005iv 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 throu
14、gh 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 collabora
15、tes 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 2. The main task of technical committees is to prepare International Standar
16、ds. 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 th
17、is document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 14502-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 8, Tea. ISO 14502 consists of the following parts, under the general title Deter
18、mination of substances characteristic of green and black tea: Part 1: Content of total polyphenols in tea Colorimetric method using Folin-Ciocalteu reagent Part 2: Content of catechins in green tea Method using high-performance liquid chromatography BS ISO 145022:20051Determination of substances cha
19、racteristic of green and black tea Part 2: Content of catechins in green tea Method using high-performance liquid chromatography 1 Scope This part of ISO 14502 specifies a high-performance liquid chromatographic (HPLC) method for the determination of the total catechin content of tea from the summat
20、ion of the individual catechins. It is applicable to both leaf and instant green tea, and with precision limitations to black tea (see Annex A). Gallic acid and caffeine can also be determined by this method, as can theogallin and theaflavins. 2 Normative references The following referenced document
21、s are indispensable for the application 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 3696:19
22、87, 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 Principle The total catechin content from a test portion of finely ground leaf tea is extracted with 70 % methanol at 70 C. Instant t
23、eas are dissolved in hot water with 10 % (volume fraction) acetonitrile added to stabilize the extract. The individual catechins in the extract are determined by HPLC on a phenyl-bonded column using gradient elution with UV detection at 278 nm. External standards are used for quantitation. External
24、catechin standards of defined purity and moisture content may be used directly. Alternatively, caffeine may be used as a standard in conjunction with individual catechin Relative Response Factors (RRFs) established by an ISO international interlaboratory test. 4 Reagents Use only reagents of recogni
25、zed analytical grade, unless otherwise specified. 4.1 Water, conforming to grade 1 of ISO 3696:1987. 4.2 Acetonitrile, HPLC grade. BS ISO 145022:20052 4.3 Methanol. 4.4 Acetic acid, glacial HPLC grade. 4.5 Methanol/water extraction mixture, 70 % methanol (volume fraction). Add 700 ml of the methanol
26、 (4.3) to a 1 litre one-mark volumetric flask. Dilute to the mark with water (4.1) and mix. 4.6 EDTA solution, 10 mg/ml. Weigh (1,00 0,01) g of EDTA (ethylenediaminetetraacetic acid disodium salt, dihydrate) into a 100 ml one-mark volumetric flask. Add sufficient warm water to half-fill the flask. S
27、wirl to dissolve the EDTA, cool to room temperature, dilute to the mark with water and mix. Prepare a fresh solution daily. 4.7 Ascorbic acid solution, 10 mg/ml. Weigh (1,00 0,01) g of L-ascorbic acid into a 100 ml one-mark volumetric flask. Dissolve in water, dilute to the mark and mix. Prepare a f
28、resh solution daily. 4.8 Stabilizing solution, 10 % (volume fraction) acetonitrile with 500 g/ml of EDTA and ascorbic acid. Using a pipette, transfer 25 ml of EDTA solution (4.6), 25 ml ascorbic acid solution (4.7) and 50 ml of acetonitrile (4.2) to a 500 ml one-mark volumetric flask. Dilute to the
29、mark with water and mix. Prepare a fresh solution daily. 4.9 HPLC mobile phases. SAFETY PRECAUTIONS Wear gloves, eye protection and dispense reagents in a fume cupboard. 4.9.1 Mobile phase A, 9 % (volume fraction) acetonitrile, 2 % (volume fraction) acetic acid with 20 g/ml EDTA. Transfer 180 ml of
30、acetonitrile (4.2) and 40 ml of acetic acid (4.4) to a 2 litre one-mark volumetric flask. Add sufficient water to half-fill the flask and add 4,0 ml of EDTA solution (4.6). Dilute to the mark with water, mix and filter through a filter of 0,45 m pore size (5.10). 4.9.2 Mobile phase B, 80 % (volume f
31、raction) acetonitrile, 2 % (volume fraction) acetic acid with 20 g/ml EDTA. Transfer 800 ml of acetonitrile (4.2) and 20 ml of acetic acid (4.4) into a 1 litre one-mark volumetric flask. Add approximately 100 ml water and 2,0 ml of EDTA solution (4.6). Dilute to the mark with water, mix and filter t
32、hrough a filter of 0,45 m pore size (5.10). 4.10 Stock standard solutions. 4.10.1 General. If catechins of known purity are available, they may be used directly as external standards. In addition to the normally quoted HPLC purity, it is important that their moisture contents be also known, as high
33、levels of water of crystallization will not be accounted for in the HPLC assessment. The purity and moisture content data on standards used in the interlaboratory test are given in Annex B. If comprehensive purity data are unavailable or cannot be determined, catechin materials should only be used a
34、s marker compounds to aid identification. In these circumstances, quantitation may be achieved using a caffeine external standard in conjunction with BS ISO 145022:20053consensus individual catechin RRF values (with respect to caffeine) obtained from interlaboratory tests (see 4.10.2 Caffeine stock
35、standard solution, corresponding to 2,00 mg/ml. Weigh (0,200 0,001) g of anhydrous caffeine into a 100 ml one-mark volumetric flask. Add sufficient warm water to half-fill the flask. Swirl to dissolve the caffeine then cool to room temperature. Dilute to the mark with water and mix. 4.10.3 Gallic ac
36、id stock standard solution, corresponding to approximately 1,00 mg/ml of anhydrous gallic acid. Weigh (0,110 0,001) g of gallic acid monohydrate (M.W. 188,14) into a 100 ml one-mark volumetric flask. Dissolve in water, dilute to the mark and mix. Prepare fresh standard solution daily. Gallic acid mo
37、nohydrate is preferred over anhydrous, due to its greater solubility, reduced hygroscopic properties and availability of certified reagent grades, e.g. A.C.S., which is used to denote chemicals that meet specifications set by the American Chemical Society. If not known, the moisture content (loss in
38、 mass at 103 C) should be determined on a portion of the standard material. 4.10.4 Preparation of individual catechin stock standard solutions Accurately weigh the amounts of standards given in Table 1 into separate one-mark volumetric flasks. Dissolve in stabilizing solution (4.8), gently warming i
39、f necessary (max. 40 C). Cool to room temperature, dilute to the mark with stabilizing solution and mix. Table 1 Catechin stock standard solutions Mass of standard Volume of stabilizing solution Nominal concentration of stock standard Standard component g ml mg/ml (+)-Catechin 0,020 0,001 20 1,0 ()-
40、Epicatechin 0,020 0,001 20 1,0 ()-Epigallocatechin 0,040 0,001 20 2,0 ()-Epigallocatechin gallate 0,040 0,001 20 2,0 ()-Epicatechin gallate 0,040 0,001 20 2,0 Where sufficient quantities (i.e. 20 mg) are available, an analytical balance capable of weighing to an accuracy of at least 0,1 mg is requir
41、ed for the preparation of the individual stock standard solutions. For limited quantities (i.e. 20 mg), an analytical balance capable of weighing to 0,01 mg is required. 4.11 Dilute standard solutions. 4.11.1 Dilute gallic acid standard solution, corresponding to approximately 200 g/ml. Using a pipe
42、tte, transfer 20 ml of the gallic acid stock standard solution (4.10.3) to a 100 ml one-mark volumetric flask. Dilute to the mark with stabilizing solution (4.8) and mix. BS ISO 145022:20059.3 and Reference 3). 4 4.11.2 Mixed standard solutions. Prepare the three mixed standard solutions A, B and C
43、containing caffeine, gallic acid and the catechins being used for external standardization or as marker compounds. Carefully pipette the volumes given in Table 2 of caffeine stock standard solution (4.10.2), dilute gallic acid standard solution (4.11.1) and any available individual catechin stock st
44、andard solutions (4.10.4) into three separate 20 ml one-mark volumetric flasks. Dilute to the mark with stabilizing solution (4.8) and mix. Pipette 1,0 ml aliquots of each mixed standard solution into labelled small amber glass vials. Gently flush with nitrogen prior to sealing and store frozen at 2
45、0 C. The nominal concentrations of components of standard solutions A, B and C are given in Table 3. With catechins of unknown purity it is essential that an individual HPLC assessment be first carried out to check for other potentially interfering components. NOTE The nominal concentrations of the
46、mixed standard solutions A, B and C are given in Table 2 and have been selected to cover the range typically found in tea. Calculate the actual anhydrous concentrations from the masses used for preparation of the stock standard solutions along with the standard moisture contents. The mixed working s
47、tandard solutions A, B and C will remain stable for at least 2 months when stored frozen at 20 C. Only thaw sufficient mixed working standard solution vials for each batch of analysis. Discard any remaining solution, and do not refreeze. Table 2 Preparation of mixed standard solutions A, B and C Ali
48、quots required for the preparation of 20 ml of mixed standard solution ml Component Solution A B C Caffeine 2,00 mg/ml caffeine stock standard solution (4.10.2) 0,5 1,0 1,5 Gallic acid 200 g/ml dilute gallic acid standard solution (4.11.1) 0,5 1,0 2,5 +C 1,00 mg/ml +C stock standard solution (4.10.4
49、) 1,0 2,0 3,0 EC 1,00 mg/ml EC stock standard solution (4.10.4) 1,0 2,0 3,0 EGC 2,00 mg/ml EGC stock standard solution (4.10.4) 1,0 2,0 3,0 EGCG 2,00 mg/ml EGCG stock standard solution (4.10.4) 1,0 2,0 4,0 ECG 2,00 mg/ml ECG stock standard solution (4.10.4) 0,5 1,0 2,0 Table 3 Nominal concentrations of mixed standard solutions A, B and C Nominal concentration g/ml Component A B C Gallic acid 5 10 25 Caffeine 50 100 150 +C 50 100 150 EC 100 150 EGC 100 200 300 EGCG 100 200 400 ECG 50 100 200 BS IS
