INTERNATIONAL STANDARD ISO 14502-1:2005(E) Determination of substances characteristic of green and black tea— Part Content of total polyphenols in tea — Colorimetric method using Fol calteu reagent 1 Scope This part of ISO 14502 specifies a method for the determination of the total polyphenol content of leaf tea and instant tea by a colorimetric assay using Folin-Ciocalteu phenol reagent"). It is applicable to both green and black 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. ISO 1872, Tea — Preparation of ground sample of known dry matter content 1S0 3696:1987, Water for analytical laboratory use — Specification and test methods 1S0 7513, Instant tea in solid form — Determination of moisture content (loss in mass at 103 °C) 3. Principle Polyphenols are extracted with 70 % methanol from a test portion of finely ground leaf tea at 70°C. Instant teas are dissolved in hot water with 10% acetonitrile (volume fraction) added to stabilize the extract. The Polyphenols in the extract are determined colorimetrically using Folin-Ciocalteu phenol reagent. The reagent ‘contains phospho-tungstic acids as oxidants, which on reduction by readily oxidized phenolic hydroxy groups yield a blue colour with a broad maximum absorption at 765 nm. This is due to the formation of so-called tungsten and molybdenum blues. The Folin-Ciocaiteu phenol reagent reacts with a wide range of polyphenol ‘compounds and, although the response can vary with the individual components, selection of gallic acid as a calibration standard enables useful total polyphenol data to be obtained, 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 Acetonitril 4.3 Methanol.44 Methanollwater extraction ture, 70 % methanol (volume fraction). [Add 700 ml of the methanol (4.3) toa 1 litre one-mark volumetric flask. Dilute to the mark with water (4.1) and mix, 45. Folin-Ciocalteu phenol reagent, commercially available ready prepared, Itis advisable to check the calibration linearity with respect to gallic acid in order to ascertain the suitability of the supplied reagent. 4.6 Dilute Folin-Ciocalteu phenol reagent, 10 % (volume fraction) Using a pipette, transfer 20 ml of Foi Dilute to the mark with water and mix CCiocalteu phenol reagent (4.5) to a 200 ml one-mark volumetric flask. Prepare fresh reagent solution dally. To avoid contamination of the concentrated Folin-Ciocalteu reagent, discard any unused dispensed reagent. 4.7 Sodium carbonate solution, 7,5 % (mass concentration. \Weigh (37,50 + 0,01) g of anhydrous sodium carbonate (NazCOs) into a 600 ml one-mark volumetric flask, Add sufficient warm water to half-fil the flask. Swirl to dissolve the sodium carbonate, cool to room temperature, dilute to the mark with water ang mx NOTE This eoluton will remain stabe at room temperature for up to t month. 48 Gallic acid stock standard solution, corresponding to approximately 1 000 yg/ml of anhydrous gallic acid, Weigh (0,110 Dissolve in water, 001) g of gallic acld monohydrate (1Y'= 188,14) into a 100 ml one-mark volumetric flask, lute to the mark and mix. Prepare a fresh standard solution daly NOTE Gallic acid monohydrate is preferred over anhydrous, due to its greater solubility. reduced hygroscopic Properties and availabilty of certfied reagent grades, e.g. ACLS. which is used to denole chemicals that meet Specifications autined by the American Chemical Society. f nat knovn, the moisture content oes In mass at 103 °C) on a portion of the standard material should be determines. The concentration of the stock standard solution on a gale acid ‘annyérous basie can ten be calculated 4.9 Gallic acid standard solutions A to E Using pipettes, transfer the volumes of gallc acid stock standard solution (4.8) given in Table 1 to 100-ml one- mark volumetric flasks. Dilute to the mark with water and mix. These dilute standard solutions should be prepared on the day of us Table 1 — Gallic acid dilute standard solutions. solution | Volume of gallic acid stock solution ml5 Apparatus Usual laboratory apparatus and, in particular, the follwing 5.1 Analytical balance, capable of weighing to an accuracy of + 0,001 g. 5.2 Water bath, capable of being maintained at 70 °C 1°. 5.3 Dispenser, for methanol/water extraction mixture (44), and set at 5,0 ml 54 Centrifuge, capable of 2000 Relative Centrifugal Force (R.C.F) (typically 3 509 r/min). 5.5 Spectrophotometer, set at 785 nm and able to accommodate 10 mm path length cells, preferably in an ‘automatic flow cell assembly, 5.6 Pipettes, glass or automatic, o cover the volume range for standard and sample extract dilutions 5.7 Onesmark volumetric flasks, of capacities 100 mil, 200 ml, 500 ml, and 1 ire 5.8 Vortex mixer, for efficient mixing during extraction, 5.9 Extraction tubes, glass, of 10 ml capacity, stoppered and able to withstand being centrifuged. 5.10 Graduated tubes, glass, of 10 ml capacity with 0.1 ml graduations, As the assay is sensitive to traces of organic impurities, extraction tubes (5.9) and graduated tubes (5.10) should all be taken through an additional cleaning procedure of washing in approx. 15% (volume fraction) nitric acid, followed by rinsing thoroughly in water and drying in an air oven at 100 °C. The use of disposable plastic tubes as an altemative to the graduated tubes in the final colorimetie assay is recommended, as ‘adcitional cleaning has not been found necessary 6 Sampling A representative sample should have 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 — 180 1839 for leaf tea, and — 180 7516 for instant tea. 7 Preparation of test samples To ensure homogeneity, grind the sample of leaf tea in accordance with ISO 1572 and store samples in well- ‘sealed containers protected from light. Grinding of instant tea is only required on samples of a coarse granular structure.8 Procedure 8.1 General If itis required to check whether the repeatabilty limit (10.2) is met, carry out two single determinations in ‘accordance with 8.2 to 8.6 under repeatability conditions. 8.2 Determination of dry matter content Caleulate the dry matter content from the moisture content (loss in mass at 103 °C) determined on a portion of, the test sample (Clause 7) in accordance with — 180 1572 for leat tea, or — 1807813 for instant tea 8.3. Test portion 8.3.1 Instant tea Weigh (0,500 + 0,001) g of the test sample (Clause 7) into @ 50 ml one-mark volumetric flask 832 Leaftea Weigh (0,200 + 0,001) g of the test sample (Clause 7) into an extraction tube (6.8) 8.4 Extraction of polyphenols 8.4.1 Instant tea 8.4.1.4 Add to the instant tea in the flask (8.3.1) approximately 25 ml of hot water (maximum temperature 60 °C). Mix to dissolve the sample and allow to coo! to room temperature. 844.2 Add 5.0 ml of acetonitrile (4.2). Dilute to the mark with water and mix. 842 Leaftea 84.2.4 Place the methanol/water extraction mixture (4.4) contained in the dispenser (5.3) in the water bath (6.2) set at 70°C, and allow at least 30 min for the extraction mixture to equlibrate. 84.2.2 Place the extraction tube containing the sample (8.3.2) n the water bath set at 70°C. Dispense 5,0 mi of hot methanol/water extraction mixture from 8.4.2.1 into the extraction tube, stopper and mix on the vortex mixer (5.8). 84.2.3 Continue heating the extraction tube in the water bath for 10 min, mixing on the vortex mixer after 5 min and 10 min, tis Important to mix the samples thoroughly to ensure complete extraction. 84.2.4 Remove the extraction tube from the water bath and allow it to cool to room temperature. Remove the stopper and place the tube in the centrifuge (5.4) at 3 500 rimin for 10 min 84.2.5 Carefully decant the supernatant into a graduated tube (5.10). 84.2.6 Repeat extraction steps 8.4.2.2 to 8.4.2.5. Combine the extracts, make up to 10 ml with cold ‘methanol! water extraction mixture (4.4) and mox the contents.84.2.7 The extract from 8.4.2.6 is stable for at least 24 h if stored at 4 °C. Allow the extract to attain room temperature before proceeding with the assay. Resuspension of the small amount of particulate material that may settle during storage is not necessary. 8.5 Dilution Using a pipette, transfer 1,0 ml of the sample extract (instant tea extract from 84.1.2 or leaf tea extract from 8.4.26) into a one-mark 100 mi volumetric flask. Dilute to the mark with water and mix. 8.6 Determination 8.6.1 Using a pipette, transfer 1,0 ml of the gal into separate plastic or graduated tubes (5.10). acid standard solutions A, B, ©, D and E (4.9), in duplicate, NOTE Thos correspond to approximately 10g, 20 ug. 30 ug. 40 bg and 50 ug of anhydrous gallic acid 8.6.2 Using a pipette, transfer 1,0 ml of water, in duplicate, into separate tubes (5.10). These are reagent blanks. 8.6.3 Using a pipette, transfer 1,0 ml of diluted sample extract (8.5), in duplicate, into separate tubes. 8.6.4 Using a pipette, add 5,0 ml of dilute Folin-Ciocalteu phenol reagent (4.6) into each tube and mix, 8.6.5 Within 3 min to 8 min after the addition of the dilute Folin-Ciocalteu phenol reagent, pipette 4,0 mi of ‘sodium carbonate solution (4.7) into each tube. Stopper and mix. 8.6.6 Allow to stand at room temperature for 60 min, and then measure the optical densities in 10-mm path length cells against water on the spectrophotometer (6.5) set at 765 nm. 8.6.7 The reagent blank (8.6.2) should have an optical density of <0,010. Higher values indicate ‘contamination problems from poor quality water, reagents or glassware. It is also important that the sample optical density be within the calibration range. Repeat the colorimetric assay with an increased dilution (8.5) if the sample optical density is higher than the 50 ig gallic acid standard E. 9 Calculation 9.1 Calculate, to the nearest 0,1 yg, the mass of anhydrous gallic acid, m, in the 1,0 ml aliquots of the standard solutions A, B, C, D and E (4.8) taken in 8.6.1, using the formula: mx wpm, ta 10 000 “400.100 where mg is the mass of gallic acid monohydrate, in grams, used to prepare the stock standard solution (48); is the volume of gallic acid stock standard solution, in militres, used to prepare the standard solutions A, B, C, D and E (4.9 ); ‘wont sts is the dry matter content, expressed as amass fraction, in percent, ofthe gallic acid 9.2 Construct a best-fit inear calibration graph from the mass of anhydrous gallic acid in standards A, B, C, D and E (49 ), as calculated in 9.1, against the gallic acid standard optical densities after subtracting the reagent blank optical density,Obtain the calibration line slope and intercept value. A typical galic acid calibration graph is given in Annex A. ‘A linear calibration should be obtained. Calculate the calibration line slope value to the nearest 0,000 1 for use in subsequent calculations. The linear callbration should also have an intercept close to the origin. Intercept values on the optical density y-axis > 0,04 should be investigated. For example, check the gallic acid moisture content, standard preparation or pipette calibration. The total polyphenol content, 1-7, expressed as a percentage by mass on a sample dry matter basis, is given by the formula: (Psample ~ Pintercapt )* sample * d * 100 Seid * ample “10 000% Wo, sample where Dangle isthe optical density obtained for the sample test solution; Dywescegt 18 the optical density at the point the best-ft near calibration line intercepts the y-axis; Sea isthe slope obtained from the best-fit near calibration ‘msanple isthe mass, in grams, of the sample test portion: oorpie (8 the sample extraction volume, in mililtres (50 ml for instant tea and 10 ml for eat tea): a is the dition factor used prior to the colorimetic determination (typcally 1,0 mi to 100 mi, thus a dilution factor of 100); ‘pm, sample 8 the dry matter content, expressed as a mass fraction in percent, of the test sample, determined in accordance with 8.2. 10 Precision 10.1 Interlaboratory test Details of the interlaboratory test to determine the precision of the method are summarized in Annex B. The ‘values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those aiven, 10.2 Repeatability The absolute difference between two independent single test results, obtained using the same method on Identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases be greater than the repeatabiity limit values, 7, given in Table B.1 10.3 Reproducibility ‘The absolute difference between two single test results, obtained using the same method on identical test material in different laboratories with different operators using different equipment, vill In not more than 5 % of ‘cases be greater than the reprodueibilty limit values, 2, given in Table B.1.Annex A (informative) Gallic acid calibration graph 10 20 30 40 50 X._ Content of anhydrous galle acid, ugrml Y Optical density, 785 nm = 0,132 =0,0113 R= 0.9985 Calibration ine slope = 0132 Intercept value = 0,011 3 Figure A.1 — Best-fit linear calibration graph for gallic acidAnnex B (informative) Results of interlaboratory test {An interlaboratory test, carried out in 2001 under the auspices of the International Organization for Standardization, gave the statistical results (evaluated in accordance with ISO 5725-2) shown in Table B.1. Table 8.1 — Precision data samme uentneaton Greonleaties | Blackeaftea | Black wat A 3 c aero patcipatng aborts 2 ze Ay numer of accepted est resus 20 7 Fy mas nator" ono iy mater bai 2435 El ‘398 Repestabity sindard devon ome oate eat Rapes costco vr, % 126 148 159 Rapestoity mt r(#28 2) oss ast aso Reproaucmy standard deviation, 18 1188 1028 Reproaucay coemlent of vari, 5 ae sat 738 Reprod It. R= 28.9) ars 222 2088