1、January 2011 Translation by DIN-Sprachendienst.English price group 9No part of this translation 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
2、 67.140.20!$lm“1739174www.din.deDDIN ISO 20481Coffee and coffee products Determination of the caffeine content using high performance liquidchromatography (HPLC) Reference method (ISO 20481:2008)English translation of DIN ISO 20481:2011-01Kaffee und Kaffee-Erzeugnisse Bestimmung des Coffeingehaltes
3、mit Hochleistungs-Flssigchromatographie (HPLC) Referenzverfahren (ISO 20481:2008)Englische bersetzung von DIN ISO 20481:2011-01Caf et drivs du caf Dtermination de la teneur en cafine par chromatographie liquide haute performance(CLHP) Mthode de rfrence (ISO 20481:2008)Traduction anglaise de DIN ISO
4、20481:2011-01SupersedesDIN 10777-2:2000-06www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.140 .11 2DIN ISO 20481:2011-01 A comma is used as the decimal marker. Contents Page National foreword .3 National Annex NA (informative) Bibl
5、iography.4 1 Scope 5 2 Normative references 5 3 Principle5 4 Reagents.5 5 Apparatus .6 6 Sampling.7 7 Preparation of test samples7 7.1 Green coffee .7 7.2 Roasted coffee .7 7.3 Soluble coffee 8 7.4 Mixed beverages8 8 Procedure .8 8.1 Optional determination of coffee dry matter (see 9.2)8 8.2 Test po
6、rtion 8 8.3 Extraction of caffeine 8 8.4 Determination.9 9 Calculation9 9.1 Caffeine content in the sample.9 9.2 Optional determination of caffeine content in the sample on the dry basis (see 8.1) 10 10 Precision.10 10.1 Interlaboratory test 10 10.2 Repeatability.11 10.3 Reproducibility.11 11 Test r
7、eport 11 Annex A (informative) Precision data of validation study on caffeine determination in coffee and coffee products.12 Bibliography 14 2 DIN ISO 20481:2011-01 National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Subcom
8、mittee SC 15 “Coffee” (Secretariat: ABNT, Brazil), under German leadership. Germany also led the relevant interlaboratory test, the results of which led to the publication of International Standard ISO 20481 with an expanded scope, which has been adopted into the German body of standards as DIN ISO
9、20481 as a replacement for DIN 10777-2:2000-06. The responsible German body which was involved in the preparation of this standard was the Normen-ausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-05-09 AA Kaffee. Th
10、e text of International Standard ISO 20481:2008 has been adopted in this standard without any modification. In the text of ISO 20481:2008 a reference to item 10 listed in the Bibliography has apparently been inadvertently left out. The reference that was omitted in 10.1 of the ISO Standard has been
11、added to DIN ISO 20481. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 565 DIN ISO 565 ISO 648 DIN EN ISO 648 ISO 1042 DIN EN ISO 1042 ISO 3696 DIN ISO 3696 ISO 3726 DIN 10764-2 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 ISO 5
12、725-6 DIN ISO 5725-6 ISO 6673 DIN ISO 6673 ISO 11817 DIN 10772-1 Amendments This standard differs from DIN 10777-2:2000-06 as follows: a) the scope has been extended to include green coffee and soluble coffee; b) a new international interlaboratory test has been carried out and new/additional precis
13、ion data for green coffee and soluble coffee have been obtained; c) Clause 7 “Preparation of test samples” has been extended to include green coffee; d) the standard has been brought up-to-date with the current rules of presentation. Previous editions DIN ISO 10777-2: 1994-03, 2000-06 3 DIN ISO 2048
14、1:2011-01 National Annex NA (informative) Bibliography DIN 10764-2, Analysis of coffee and coffee products Determination of loss in mass of soluble coffee Part 2: Method using vacuum oven (routine method) DIN 10772-1, Analysis of coffee and coffee products Karl Fischer method for the determination o
15、f water content Part 1: Reference method for roasted coffee DIN ISO 565, Test sieves Metal wire cloth, perforated metal plate and electroformed sheet Nominal sizes of openings DIN EN ISO 648, Laboratory glassware Single-volume pipettes DIN EN ISO 1042, Laboratory glassware One-mark volumetric flasks
16、 DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part
17、2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN ISO 5725-6, Accuracy (trueness and precision) of measurement methods and results Part 6: Use in practice of accuracy values DIN ISO 6673, Green coffee Determination of loss in mass at 105
18、C 4 Coffee and coffee products Determination of the caffeine content using high performance liquid chromatography (HPLC) Reference method 1 Scope This International Standard specifies a high performance liquid chromatography (HPLC) method for the determination of the caffeine content of: green coffe
19、e; roasted coffee; soluble coffee, regular and decaffeinated; and mixed instant coffee products (e. g. coffee/chicory mix or cappuccino-type coffee drink). 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the
20、 edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696, Water for analytical laboratory use Specification and test methods ISO 3726, Instant coffee Determination of loss in mass at 70 C under reduced pressure ISO 667
21、3, Green coffee Determination of loss in mass at 105 C ISO 11817, Roasted ground coffee Determination of moisture content Karl Fischer method (Reference method) 3 Principle Caffeine is extracted from samples with water at 90 C in the presence of magnesium oxide. After filtration, the caffeine conten
22、t of the extract is determined by HPLC on a RP-18 column using isocratic elution with UV detection at approximately 272 nm. Wherever appropriate, the caffeine content may be given on dry basis which requires a moisture determination by a suitable standard method. 4 Reagents Unless otherwise specifie
23、d, use only reagents of recognized analytical grade, and only water conforming to the requirements of ISO 3696, grade 1. 4.1 Methanol, HPLC grade. 4.2 Magnesium oxide (MgO), heavy, high grade1). 1) Merck 105867 is an example of a suitable, commercially available product. This information is given fo
24、r the convenience of users of this International Standard and does not constitute an endorsement of this product by ISO. 5 DIN ISO 20481:2011-01 NOTE The use of MgO is important for the lifetime of the analytical column, especially for green coffee. The amount of MgO used depends on the equipment an
25、d on the type of coffee product. The precision data in Annex A were acquired using the procedure described in this International Standard. The appearance of interfering peaks in the chromatogram may be due to improper adsorption. In these cases, subject the MgO used to examination. 4.3 Caffeine (1,3
26、,7-trimethylxanthine; 1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione; methyltheobromine; C8H10N4O2), pure anhydrous. 4.4 Mobile phase, 24 % volume fraction methanol in water. SAFETY PRECAUTIONS Wear gloves, eye protection and dispense reagents in a fume cupboard (fume hood). Transfer 240 ml methanol (4.
27、1) to a 1 l one-mark volumetric flask (5.11). Make up to the mark with water, mix and filter through a 0,45 m filter (5.2). NOTE By changing the ratio of methanol to water, the retention time of caffeine can be adjusted to optimize the HPLC separation on the column used. 4.5 Caffeine standard soluti
28、ons. 4.5.1 Stock solution, corresponding to 200 mg/l. Weigh (0,200 0,001) g anhydrous caffeine (4.3) into a 1 l one-mark volumetric flask (5.11). Add sufficient warm water to half fill the flask. Swirl to dissolve the caffeine, cool to room temperature, make up to the mark with water and mix. The so
29、lution is stable at +4 C for one month. Store in a refrigerator. 4.5.2 Dilute standard solution for regular coffee, corresponding to approximately 40 mg/l. Pipette (5.12) 50 ml of the caffeine standard stock solution (4.5.1) to a 250 ml one-mark volumetric flask (5.11). Make up to the mark with wate
30、r and mix. Prepare fresh dilute standard solution daily. 4.5.3 Dilute standard solution for decaffeinated coffee, corresponding to approximately 4 mg/l. Pipette (5.12) 5 ml of the caffeine standard stock solution (4.5.1) to a 250 ml one-mark volumetric flask (5.11). Make up to the mark with water an
31、d mix. Prepare fresh dilute standard solution daily. 4.5.4 Calibration plot. The use of a three to five point calibration plot is optional. Recommended concentration range is 5 mg/l to 25 mg/l for regular coffee and 0,5 mg/l to 2,5 mg/l for decaffeinated samples. 5 Apparatus Usual laboratory apparat
32、us and, in particular, the following. 5.1 Analytical balance, capable of weighing to an accuracy of 0,1 mg. 5.2 Membrane filter units of pore size 0,45 m, for filtration of mobile phases and diluted sample extracts. 5.3 High performance liquid chromatograph, equipped to perform an isocratic elution,
33、 with a UV detector set at 272 nm (270 nm to 280 nm) or a filter detector (254 nm), and with a data collection/integration system. The use of a degasser is optional. 5.4 Chromatographic column for HPLC, of minimum length 125 mm, packed with C18 material, preferentially 5 m spherical particles, with
34、an efficiency of separation of at least 5 000 theoretical plates. The 6 DIN ISO 20481:2011-01 theoretical number of plates, Nth, of the column may be calculated by determining peak shape after injection of the caffeine standard solution by Equation (1): 2rth5,54tNb=(1) where tris the retention time,
35、 in seconds, of the peak; b is the peak width, in seconds, at half peak height. 5.5 Magnetic stirrer, with heater and mounted water bath. 5.6 Ultrasonic bath. 5.7 Microlitre syringe. 5.8 Coffee mill, suitable for grinding roasted coffee beans. 5.9 Grinder with cogged wheel, with cooling jacket, or a
36、nalytic grinder, with blades and cooling jacket, or any other device suitable for grinding green coffee beans. 5.10 Sieve, of nominal size of openings 630 m, complying with the requirements of ISO 5652, R20 series. 5.11 One-mark volumetric flasks, of capacities 1 l and 250 ml, complying with the req
37、uirements of ISO 10424, class A. 5.12 Pipettes, of capacities 50 ml and 5 ml, complying with the requirements of ISO 6483, class A. 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
38、 of the method specified in this International Standard. The sampling procedure shall be subject to agreement by the interested parties. Store the test sample in such a way that deterioration and change in its composition are prevented. In case of mixed beverage powders, a minimum of 50 g of the pro
39、duct is required (for portion packages: a minimum of five portions). 7 Preparation of test samples 7.1 Green coffee Grind (5.9) green coffee beans so that more than 50 % mass fraction of the sample passes through the sieve (5.10). Then take the test sample from the well-mixed total ground coffee. 7.
40、2 Roasted coffee Mill (5.8) roasted coffee beans so that more than 50 % mass fraction passes through the sieve (5.10). Use roasted ground coffee in commercial packages without further treatment except homogenization. 7 DIN ISO 20481:2011-01 7.3 Soluble coffee Grinding of soluble coffee is not requir
41、ed. 7.4 Mixed beverages Homogenize mixed coffee beverage powders using the mill (5.8) immediately before extraction in order to prevent segregation. 8 Procedure 8.1 Optional determination of coffee dry matter (see 9.2) Calculate the coffee dry matter content to be used in 9.2 from the moisture conte
42、nt determined on a portion of the test sample (Clause 7) in accordance with ISO 6673 for green coffee, ISO 11817 for roasted coffee, ISO 3726 for instant coffee. For other types of coffee and coffee-derived products, the dry matter content determination shall be subject to agreement between the inte
43、rested parties. 8.2 Test portion 8.2.1 Roasted and green coffee Weigh (5.1) about 1 g of the test sample (7.1, 7.2) to the nearest milligram, and transfer into a 250 ml volumetric flask (5.11). 8.2.2 Instant coffee Weigh (5.1) about 0,5 g of the test sample (7.3) to the nearest milligram, and transf
44、er into a 250 ml volumetric flask (5.11). 8.2.3 Mixed beverages Weigh (5.1) about 1 g of the test sample (7.4) to the nearest milligram, and transfer into a 250 ml volumetric flask (5.11). 8.3 Extraction of caffeine Add to the test portion (8.2.1, 8.2.2 or 8.2.3) in the flask, 5 g magnesium oxide (4
45、.2) and about 200 ml water. Place the flask in a water bath maintained at boiling point and wait until the solution has reached at least 90 C. Continue heating the flask in the water bath for 20 min, mix occasionally by shaking or stirring. Remove the volumetric flask from the water bath, cool to ro
46、om temperature under tap water, and make up to volume with water. Wait until the solids have settled. Take an aliquot of the supernatant solution, and filter through a 0,45 m filter (5.2), discarding the first few millilitres. The filtrate is now ready for the HPLC separation. 8 DIN ISO 20481:2011-0
47、1 8.4 Determination 8.4.1 Adjustment of the apparatus IMPORTANT The mobile phase should be degassed for 20 min in the ultrasonic bath (5.6) or purged with helium prior to use. If the HPLC equipment includes a degasser, 10 min are sufficient. Set up the chromatograph (5.3) in accordance with the manu
48、facturers instructions and adjust it as follows: a) flow rate of the mobile phase (4.4): 1,0 ml/min; b) UV detector set at 272 nm (or 254 nm in case of a filter detector). Ensure that the detector sensitivity range fits to the peak of the standard solution (in case of more than one standard to the p
49、eak of the highest one). 8.4.2 HPLC analysis Once the flow rate of the mobile phase and the corresponding pressure are stable, allow the system to equilibrate for at least 10 min. Then inject 10 l of the standard solution onto the column, using the microlitre syringe (5.7), in the next run followed by an equal volume of the sample extract (8.3). When a single standard solution is used, inject it at regular intervals (typically after six sample extracts). After each batch of analys
