1、BRITISH STANDARDBS ISO 20481:2008Coffee and coffee products Determination of the caffeine content using high performance liquid chromatography (HPLC) Reference methodICS 67.140.20g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g
2、54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS ISO 20481:2008This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2008 BSI 2008ISBN 978 0 580 57553 2National forewordThis British Standard is the UK implem
3、entation of ISO 20481:2008. The UK participation in its preparation was entrusted to Technical Committee AW/15, Coffee.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a cont
4、ract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.Amendments/corrigenda issued since publicationDate CommentsReference numberISO 20481:2008(E)INTERNATIONAL STANDARD ISO20481First edition2008-05-01Coffee and coffee
5、 products Determination of the caffeine content using high performance liquid chromatography (HPLC) Reference method Caf et drivs du caf Dtermination de la teneur en cafine par chromatographie liquide haute performance (CLHP) Mthode de rfrence BS ISO 20481:2008ii iiiForeword ISO (the International O
6、rganization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been estab
7、lished 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 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardizat
8、ion. 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 Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting
9、. 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 this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such p
10、atent rights. ISO 20481 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 15, Coffee. BS ISO 20481:2008blank1Coffee and coffee products Determination of the caffeine content using high performance liquid chromatography (HPLC) Reference method 1 Scope This International St
11、andard specifies a high performance liquid chromatography (HPLC) method for the determination of the caffeine content of: green coffee; roasted coffee; soluble coffee, regular and decaffeinated; and mixed instant coffee products (e. g. coffee/chicory mix or cappuccino-type coffee drink). 2 Normative
12、 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 565, Test sieves Metal wire cloth
13、, perforated metal plate and electroformed sheet Nominal sizes of openings ISO 648, Laboratory glassware Single volume pipettes ISO 1042, Laboratory glassware One-mark volumetric flasks ISO 3696, Water for analytical laboratory use Specification and test methods ISO 3726, Instant coffee Determinatio
14、n of loss in mass at 70 C under reduced pressure ISO 6673, 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 o
15、f magnesium oxide. After filtration, the caffeine content 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 suitab
16、le standard method. 4 Reagents Unless otherwise specified, use only reagents of recognized analytical grade, and only water conforming to the requirements of ISO 3696, grade 1. 4.1 Methanol, HPLC grade. BS ISO 20481:20082 4.2 Magnesium oxide (MgO), heavy, high grade1). NOTE The use of MgO is importa
17、nt for the lifetime of the analytical column, especially for green coffee. The amount of MgO used depends on the equipment and 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
18、in the chromatogram may be due to improper adsorption. In these cases, subject the MgO used to examination. 4.3 Caffeine (1,3,7-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. SAFE
19、TY PRECAUTIONS Wear gloves, eye protection and dispense reagents in a fume cupboard (fume hood). Transfer 240 ml methanol (4.1) 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, th
20、e retention time of caffeine can be adjusted to optimize the HPLC separation on the column used. 4.5 Caffeine standard solutions. 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
21、half fill the flask. Swirl to dissolve the caffeine, cool to room temperature, make up to the mark with water and mix. The solution 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 m
22、l 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 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
23、 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 and 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
24、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 apparatus 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 mo
25、bile phases and diluted sample extracts. 1) Merck 105867 is an example of a suitable, commercially available product. This information is given for the convenience of users of this International Standard and does not constitute an endorsement of this product by ISO. BS ISO 20481:200835.3 High perfor
26、mance liquid chromatograph, equipped to perform an isocratic elution, 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, pa
27、cked with C18 material, preferentially 5 m spherical particles, with an efficiency of separation of at least 5 000 theoretical plates. The 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): 2
28、rth5,54tNb=(1) where tris the retention time, 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
29、 with cogged wheel, with cooling jacket, or analytic 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 565, R20 series. 5.11 One-mark volumetric flasks, of capac
30、ities 1 l and 250 ml, complying with the requirements of ISO 1042, class A. 5.12 Pipettes, of capacities 50 ml and 5 ml, complying with the requirements of ISO 648, class A. 6 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during
31、transport or storage. Sampling is not part 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 beve
32、rage powders, a minimum of 50 g of the product 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 f
33、rom the well-mixed total ground coffee. BS ISO 20481:20084 7.2 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. 7.3 Soluble coffee Gri
34、nding of soluble coffee is not required. 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 b
35、e used in 9.2 from the moisture content 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
36、subject to agreement between the interested 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)
37、 to the nearest milligram, and transfer 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, 8.2.1 or 8.2.3)
38、 in the flask, 5 g magnesium oxide (4.2) 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
39、flask from the water bath, cool to room 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 HP
40、LC separation. BS ISO 20481:200858.4 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
41、.3) in accordance with the manufacturers 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
42、more than one standard to the peak 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 (
43、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 analysis, thoroughly flush the chromatographic system and column with 50 % volume fraction
44、 methanol (4.1) and water, and replace column sealing plugs if disconnected for storage. 9 Calculation 9.1 Caffeine content in the sample Calculate the caffeine content, wx, expressed as a percentage by mass, numerically equivalent to grams per 100 g coffee as is, according to Equation (2): cccs cs1
45、00 25xxxAV AwAm Am= (2) where Acis the area, in arbitrary area units, of the HPLC caffeine peak of the caffeine standard solution; Axis the area, in arbitrary area units, of the HPLC caffeine peak of the sample solution; msis the mass, in grams, of the test portion; V is the volume, in litres, of th
46、e extracted sample solution, as defined in 8.2 (V = 0,25 l); cis the mass concentration, in grams per litre, of the caffeine standard solution. NOTE Peak height can be used as an alternative to peak area. If more than one calibration standard is used, construct a linear calibration graph for the caf
47、feine standards, to obtain the slope and intercept value. Use this calibration plot to calculate the concentration of caffeine in the sample. Take as the result the arithmetic mean of two determinations carried out under repeatability conditions (10.2). BS ISO 20481:20086 9.2 Optional determination
48、of caffeine content in the sample on the dry basis (see 8.1) Calculate the caffeine content, xw expressed as a percentage by mass, numerically equivalent to grams per 100 g on the dry basis, by Equation (3): cccsd csd100 2 500100xxxAV AwAmw Amw= = (3) where wdis the dry matter content of the sample
49、(grams per 100 g), as determined in accordance with 8.1. Take as the result the arithmetic mean of two determinations carried out under repeatability conditions (10.2). 10 Precision 10.1 Interlaboratory test Details of the interlaboratory test to determine the precision of the method are summarized in Annex A. The caffeine results were taken based on coffee as is. The repeatability and reproducibility limits, as defined in ISO 5725-1:19942, 3.16 and 3.20, respectively, are calculated according to IS
