1、December 2007DEUTSCHE NORM English price group 14No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.140.10!$Jja“139716
2、2www.din.deDDIN ISO 14502-2Determination of substances characteristic of green and black tea Part 2: Content of catechins in green tea Method using high-performance liquid chromatography(ISO 14502-2:2005 + Corrigendum 1:2006)English version of DIN ISO 14502-2:2007-12Bestimmung von charakteristischen
3、 Substanzen von grnem und schwarzem Tee Teil 2: Gehalt an Catechinen in grnem Tee Corrigendum 1:2006)Englische Fassung DIN ISO 14502-2:2007-12www.beuth.deDocument comprises 26 pagesVerfahren mit Hochleistungs-Flssigchromatographie (ISO 14502-2:2005 +DIN ISO 14502-2:2007-12 2 Contents Page National f
4、oreword 3 National Annex NA (informative) Bibliography . 3 1 Scope 4 2 Normative references . 4 3 Principle . 4 4 Reagents 4 5 Apparatus . 8 6 Sampling 8 7 Preparation of test samples 9 8 Procedure . 9 8.1 General 9 8.2 Determination of dry matter content . 9 8.3 Test portion . 9 8.4 Extraction of p
5、olyphenols. 9 8.5 Dilution. 10 8.6 Determination . 10 9 Calculation. 11 9.1 General. 11 9.2 Quantitation using catechin standards . 11 9.3 Quantitation using a caffeine standard and catechin Relative Response Factors (RRFs) . 12 10 Precision . 13 10.1 Interlaboratory test 13 10.2 Repeatability 13 10
6、.3 Reproducibility . 13 11 Test report . 13 Annex A (informative) Results of interlaboratory tests . 14 Annex B (informative) Assessment of purity of standards . 13 Annex D (informative) The effect of ferric ions on catechin RRFs . 21 Annex E (informative) Quantitative comparison Use of catechin sta
7、ndards or a Bibliography 26 caffeine standard in conjunction with catechin RRFs. 24 Annex C (informative) Typical HPLC chromatograms .14 .DIN ISO 14502-2:2007-12 3 National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products”, Subcommittee SC 8 “Tea” (Secretariat
8、: BSI, United Kingdom). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und land-wirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee Tee. Technical Corrigendum 1:2006 to ISO 14502-2:2005 has been taken into a
9、ccount in this standard. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. DIN shall not be held responsible for identifying any or all such patent rights. The DIN Standard corresponding to the International Standard referred to in
10、this document is as follows: ISO 3696 DIN ISO 3696 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods 1 Scope This part of ISO 14502 specifies a high-performance liquid chromatographic (HPLC) method for the determination of t
11、he total catechin content of tea from the summation 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 Normat
12、ive references The following referenced documents 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 groun
13、d sample of known dry matter content ISO 3696:1987, 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
14、 extracted with 70 % methanol at 70 C. Instant teas 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. Externa
15、l standards are used for quantitation. External 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 interlaborato
16、ry test. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. 4.1 Water, conforming to grade 1 of ISO 3696:1987. 4.2 Acetonitrile, HPLC grade. 4 Determination of substances characteristic of green and black tea Part 2: Content of catechins in green tea Method usin
17、g high-performance liquid chromatography DIN ISO 14502-2:2007-12 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 (4.3) to a 1 litre one-mark volumetric flask. Dilute to the mark with water (4.1) and
18、 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. Swirl to dissolve the EDTA, cool to room temperature, dilute to the mark with water and
19、 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 fresh solution daily. 4.8 Stabilizing solution, 10 % (volume fraction) acetonitrile wit
20、h 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 mark with water and mix. Prepare a fresh solution daily. 4.9 HPLC mobile phases. SAFET
21、Y 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 acetonitrile (4.2) and 40 ml of acetic acid (4.4) to a 2 litre one-mark volumetric fla
22、sk. 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 fraction) acetonitrile, 2 % (volume fraction) acetic acid with 20 g/ml EDTA. Transfer 8
23、00 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 through a filter of 0,45 m pore size (5.10). 4.10 Stock standard solutions. 4.10.1 Gene
24、ral. 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 levels of water of crystallization will not be accounted for in the HPLC assessment. T
25、he 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 as marker compounds to aid identification. In these circumstances, quantitation may be
26、achieved using a caffeine external standard in conjunction with 5 DIN ISO 14502-2:2007-12 consensus individual catechin RRF values (with respect to caffeine) obtained from interlaboratory tests (see 4.10.2 Caffeine stock standard solution, corresponding to 2,00 mg/ml. Weigh (0,200 0,001) g of anhydr
27、ous 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 acid stock standard solution, corresponding to approximately 1,00 mg/ml of anhydr
28、ous 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 monohydrate is preferred over anhydrous, due to its greater solubility, reduced h
29、ygroscopic 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 mass at 103 C) should be determined on a portion of the standard material. 4.1
30、0.4 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 if necessary (max. 40 C). Cool to room temperature, dilute to the mark with stab
31、ilizing 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 ()-Epicatechin 0,020 0,001 20 1,0 ()-Epigallocatechin 0,040 0,001 20 2,0 ()-Epigal
32、locatechin 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 required for the preparation of the individual stock standard solutions. For limited
33、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 pipette, transfer 20 ml of the gallic acid stock standard solution (4.10.3) to a 10
34、0 ml one-mark volumetric flask. Dilute to the mark with stabilizing solution (4.8) and mix. 6 DIN ISO 14502-2:2007-12 9.3 and Reference 3). 4.11.2 Mixed standard solutions. Prepare the three mixed standard solutions A, B and C containing caffeine, gallic acid and the catechins being used for externa
35、l 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 standard solutions (4.10.4) into three separate 20 ml one-mark volumetric f
36、lasks. 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 20 C. The nominal concentrations of components of standard solutions A, B
37、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 mixed standard solutions A, B and C are given in Table 2 and have been se
38、lected 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 standard solutions A, B and C will remain stable for at least 2 months whe
39、n 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 Aliquots required for the preparation of 20 ml of mixed standard solution ml
40、 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) 1,0 2,0 3,0 EC 1,00 mg/ml EC stock standard solution (4.10.4) 1,0 2,0 3
41、,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 Compon
42、ent 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 7 DIN ISO 14502-2:2007-12 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Analytical balances, capable of weighing to an accuracy of 0,000 1 g and
43、0,000 01 g (see 4.10.4). 5.2 Water bath, capable of being maintained at (70 1) C. 5.3 Dispenser, for methanol/water extraction mixture (4.5), and set at 5,0 ml. 5.4 Centrifuge, capable of 2 000 Relative Centrifugal Force (R.C.F.) (typically 3 500 r/min). 5.5 Vortex mixer, for efficient mixing during
44、 extraction. 5.6 Extraction tubes, glass, of 10 ml capacity, stoppered and able to withstand being centrifuged. 5.7 Graduated tubes, glass, of 10 ml capacity with 0,1 ml graduations. 5.8 One-mark volumetric flasks, of capacities 5 ml, 10 ml, 20 ml, 100 ml, 500 ml, 1 litre and 2 litres. 5.9 Pipettes,
45、 glass or automatic, to cover the volume range for standard and sample extract dilutions. 5.10 Filters, membrane filter units of 0,45 m pore size for filtration of mobile phases and diluted sample extracts. NOTE PTFE and nylon membrane filters have proven to be suitable. All membranes should be chec
46、ked to ensure that that catechin retention does not occur. 5.11 High-performance liquid chromatograph, equipped to perform binary gradient elution, with a thermostatically controlled column compartment and an ultraviolet detector set at 278 nm. 5.12 Data collection /integration system. 5.13 Chromato
47、graphic column for HPLC. NOTE Phenyl-bonded phases give additional selectivity over reversed-phase materials, and result in improved resolution of the catechins. In this part of ISO 14502 the chromatographic conditions and composition of the mobile phase specified (4.9) are suitable for a Phenomenex
48、 Luna 5 m Phenyl-Hexyl1)column of dimensions 250 mm 4,6 mm, fitted with a Phenomenex SecurityGuard2)4 mm 3,0 mm Phenyl-Hexyl cartridge. If other types of column are used, modifications to the mobile phase and chromatographic conditions may be necessary. 6 Sampling A representative sample should have
49、 been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this part of ISO 14502. A recommended sampling method is given in ISO 1839 for leaf tea, and ISO 7516 for instant tea. 1) Phenyl-Hexyl is an example of a suitable product available commercially. This information is given for the convenience of users of this part of ISO 14502 and does not constitute an endorsement by ISO of this product. 2) SecurityGuar
