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This standard is developed in accordance with the rules given in GB/T 1.1-2009.
This standard replaces GB/T 8313-2008 Determination of total polyphenols and catechins content in tea. The following main technical changes have been made with respect to GB/T 8313-2008:
——Chemical reagents (see 3.3.6 of this standard; 5.5 of Edition 2008);
——The abbreviations of catechin standards are modified (see 3.3.10.3 of this standard; 5.9.3 of Edition 2008);
——Liquid chromatogram of catechin and caffeine standards is added (see Figure A.1).
This standard is redrafted by reference to but is not equivalent to ISO 14502-1:2005 Determination of substances characteristic of green and black tea - Part 1: Content of total polyphenols in tea - Colorimetric method using Folin-Ciocalteu reagent and ISO 14502-2:2005 Determination of substances characteristic of green and black tea - Part 2: Content of catechins in green tea - Method using high-performance liquid chromatography
This standard was proposed by the All China Federation of Supply and Marketing Cooperatives.
This standard is under the jurisdiction of the Technical Committee 339 on Tea of Standardization Committee of China (SAC/TC 339).
The previous editions of this standard are as follows:
——GB/T 8313-1987, GB/T 8313-2002, and GB/T 8313-2008.
Determination of total polyphenols and catechins content in tea
1 Scope
This standard specifies the methods of determining catechins in tea by high-performance liquid chromatography (HPLC) and polyphenols in tea by spectrophotometry.
This standard is applicable to the determination of catechins and polyphenols in tea and tea products.
2 Normative 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.
GB/T 6682 Water for analytical laboratory use - Specification and test methods
GB/T 8302 Tea - Sampling (GB/T 8302-2013, ISO 1839:1980, NEQ)
GB/T 8303 Tea - Preparation of ground sample and determination of dry matter content (GB/T 8303-2013, ISO 1572:1980, MOD)
3 Determination of catechins in tea - HPLC method
3.1 Principle
Extract the catechins in ground tea specimens with 70% methanol aqueous solution in a water bath at 70℃. Determine the catechins by C18 column with detection wavelength of 278 nm, gradient elution and HPLC analysis, and directly quantify it by external standard method of catechin standards. The relative response factor (RRFStd) of catechins and caffeine from ISO international ring test results may also be used for quantification.
3.2 Apparatus
3.2.1 Analytical balance: with an accuracy of 0.0001 g.
3.2.2 Water bath: 70 ℃±1 ℃.
3.2.3 Centrifuge: with a rotating speed of 3,500 r/min.
3.2.4 High-performance liquid chromatograph (HPLC): with gradient elution function and equipped with ultraviolet detector and chromatographic workstation.
3.3 Reagents
3.3.1 Unless otherwise specified, analytically-pure reagents and Grade III water defined in GB/T 6682 are adopted for the analysis.
3.3.2 Acetonitrile: chromatographically pure.
3.3.3 Methanol.
3.3.4 Acetic acid: chromatographically pure.
3.3.5 70% methanol aqueous solution.
3.3.6 Ethylenediamine tetraacetic acid disodium (EDTA-2Na) solution: 10 mg/mL (freshly prepared before use).
3.3.7 Ascorbic acid solution: 10 mg/mL (freshly prepared before use).
3.3.8 Stable solution: Add 25 mL of EDTA-2Na solution (3.3.6), 25 mL of ascorbic acid solution (3.3.7) and 50 mL of acetonitrile (3.3.2) into a 500 mL volumetric flask, dilute to the scale with water, and shake well.
3.3.9 Liquid chromatographic mobile phase
3.3.9.1 Mobile phase A: Add 90 mL of acetonitrile (3.3.2), 20 mL of acetic acid (3.3.4) and 2 mL of EDTA-2Na solution (3.3.6) into a 1,000 mL volumetric flask, dilute to the scale with water, and shake well. The solution shall pass through a 0.45 μm filter membrane (3.3.12).
3.3.9.2 Mobile phase B: Add 800 mL of acetonitrile (3.3.2), 20 mL of acetic acid (3.3.4) and 2 mL of EDTA-2Na solution (3.3.6) into a 1,000 mL volumetric flask, dilute to the scale with water, and shake well. The solution shall pass through a 0.45 μm filter membrane (3.3.12).
3.3.10 Stock standard solution
3.3.10.1 Caffeine stock solution: 2.00 mg/mL.
3.3.10.2 Gallic acid (GA) stock solution: 0.100 mg/mL.
3.3.10.3 Catechins stock solution: catechin (+C) 1.00 mg/mL, epicatechin (EC) 1.00 mg/mL, epigallocatechin (EGC) 2.00 mg/mL, epigallocatechin gallate (EGCG) 2.00 mg/mL, epicatechin gallate (ECG) 2.00 mg/mL.
3.3.11 Standard working solution: Prepared with stabilizing solution (3.3.8).
The concentration of standard working solution: Gallic acid 5 μg/mL ~ 25 μg/mL, caffeine 50 μg/mL ~ 150 μg/mL, +C 50 μg/mL ~ 150 μg/mL, EC 50 μg/mL ~ 150 μg/mL, EGC 100 μg/mL ~ 300 μg/mL, EGCG 100 μg/mL ~ 400 μg/mL, ECG 50 μg/mL ~ 200 μg/mL.
3.3.12 0.45 μm organic phase filter membrane.
3.4 Operation method
3.4.1 Sampling
The sampling shall be carried out according to the requirements of GB/T 8302.
3.4.2 Preparation of specimen
The specimen shall be prepared according to the requirements of GB/T 8303.
3.4.3 Determination procedures
3.4.3.1 Determination of dry matter content
It shall be determined according to the requirements GB/T 8303.
3.4.3.2 Preparation of test solution
3.4.3.2.1 Spent liquor: weigh 0.2 g (accurate to 0.0001 g) of evenly ground specimen (3.4.2) into a 10 mL centrifuge tube, add 5 mL of 70% methanol aqueous solution (3.3.5) preheated at 70 ℃, mix well with a glass rod to moisten, and immediately move it to the water bath (3.2.2) at 70 ℃, and extract for 10 min (stir it once every 5 min). After the extraction, cool it to room temperature, transfer it to a centrifuge and centrifuge for 10 min at a speed of 3,500 r/min, and transfer the supernatant to a 10 mL volumetric flask. Extract the residue once with 5 mL of 70% methanol aqueous solution, and repeat the above operation. Combine the extracting solution and dilute to 10 mL, shake well, and filter them with the 0.45 μm filter membrane (3.3.12) for later use (the extracting solution may be stored for up to 24 h at 4 ℃).
3.4.3.2.2 Test solution: Pipette 2 mL of the spent liquor (3.4.3.2.1) into a 10 mL volumetric flask, dilute to the scale with the stable solution (3.3.8), shake well, and filter it with the 0.45 μm filter membrane (3.3.12) for test.
Foreword i
1 Scope
2 Normative references
3 Determination of catechins in tea - HPLC method
4 Determination of polyphenols in tea
Annex A (Informative) Liquid chromatogram of catechin and caffeine standard samples
