1、Reference number ISO 6867:2000(E) ISO 2000 INTERNATIONAL STANDARD ISO 6867 First edition 2000-12-01 Animal feeding stuffs Determination of vitamin E content Method using high- performance liquid chromatography Aliments des animaux Dtermination de la teneur en vitamine E Mthode par chromatographie li
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7、 iii Foreword ISO (the International Organization 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 whic
8、h a technical committee has been established 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 ma
9、tters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Stand
10、ard 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 International Standard may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Internation
11、al Standard ISO 6867 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs. Annex A of this International Standard is for information only.INTERNATIONAL STANDARD ISO 6867:2000(E) ISO 2000 All rights reserved 1 Animal feeding stuffs Determination of v
12、itamin E content Method using high-performance liquid chromatography 1 Scope This International Standard specifies a method for the determination of the vitamin E (DL- -tocopherol) content of animal feeding stuffs and pet foods using high performance liquid chromatography. 2 Normative references The
13、 following normative documents contain provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this Interna
14、tional Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid Intern
15、ational Standards. ISO 3696:1987, Water for analytical laboratory use Specifications and test methods. ISO 6498, Animal feeding stuffs Preparation of test samples. 3P r i n c i p l e A test portion of the sample is saponified with ethanolic potassium hydroxide solution and the vitamin E is extracted
16、 into light petroleum. The light petroleum is removed by evaporation and the residue is dissolved in hexane. The vitamin E concentration in the hexane extract is determined by normal-phase liquid chromatography using conditions that separate DL- -tocopherol from other tocopherols. 4 Reagents Use onl
17、y reagents of recognized analytical grade, unless otherwise stated. 4.1 Water, complying with at least grade 3 in accordance with ISO 3696. 4.2 Potassium hydroxide solution. Dissolve 500 g of potassium hydroxide in water (4.1) and dilute to 1 litre. 4.3 Ethanol, w(C 2 H 5 OH) = 95 % (by volume), or
18、equivalent industrial methylated spirit. 4.4 Hexane, HPLC grade. 4.5 Light petroleum, boiling range 40 Ct o6 0C; the residue on evaporation shall be less than 20 mg/l. 4.6 Vitamin E standard substance: DL- -tocopherol, minimum purity not less than 96,0 %. The purity of the standard substance should
19、be checked spectrophotometrically (see 8.5.2).ISO 6867:2000(E) 2 ISO 2000 All rights reserved 4.7 1,4-Dioxan, HPLC grade. 4.8 Sodium sulfate (Na 2 SO 4 ), anhydrous. 4.9 Sodium ascorbate solution, =1 00g /l . 4.10 Inert gas,e .g .ni tro ge n . 4.11 Mobile phase for liquid chromatography. Mix 30 ml 1
20、,4-dioxan (4.7) with 970 ml hexane (4.4). Filter through a membrane filter (5.5) before use. 4.12 Ethanol, w(C 2 H 5 OH) = 96 % (by volume). 4.13 Methanol (CH 3 OH), HPLC grade. 5 Apparatus Using laboratory apparatus and, in particular, the following. 5.1 High-performance liquid chromatograph, consi
21、sting of the following. 5.1.1 Pump, set to deliver a constant eluent volume flow rate of 1,5 ml/min. 5.1.2 HPLC injection device. 5.1.3 Column, length 250 mm, internal diameter 4,6 mm, packed with a stationary phase consisting of silica. A column with at least 5 000 theoretical plates and a k value
22、of 0,8 m, both with respect to DL- -tocopherol, has been found to be satisfactory. The particle size should not be smaller than 5 m and not greater than 10 m. Other systems may be used provided that a satisfactory separation of vitamin E from other co-extractives is achieved. 5.1.4 Detector, allowin
23、g the measurement of fluorescence emitted at a wavelength of 326 nm when the column eluent is irradiated with ultraviolet light at a wavelength of 293 nm, with integrator/recorder. 5.2 Boiling water bath. 5.3 Rotary vacuum evaporator, with water bath at 40 C. 5.4 Extraction apparatus (see Figure 1)
24、consisting of the following: a cylinder of 1 litre capacity fitted with a ground glass neck and stopper; a ground glass joint, fitting the cylinder and equipped with an adjustable tube passing through the centre; and a side-arm. The adjustable tube should have a U-shaped lower end and a jet at the o
25、pposite end so that the upper liquid layer in the cylinder may be transferred to a separating funnel of 1 litre capacity. Other extraction equipment such as conical flasks and separating funnels may be used in place of the apparatus shown in Figure 1, provided that satisfactory recoveries of vitamin
26、 E are achieved. 5.5 Membrane filter, 0,45 m pore size, for filtration of mobile phase (4.11) and sample test solutions.ISO 6867:2000(E) ISO 2000 All rights reserved 3 5.6 Grinding apparatus, capable of grinding the sample so that it passes through a sieve with 1 mm apertures. 5.7 UV (or UV/Visible)
27、 spectrometer, capable of measuring absorbance at the wavelengths defined in 8.5.2, equipped with quartz cells of 10 mm path length. Key 1 Cylinder, of capacity 1 litre, with ground-glass neck 5 Bottle, of capacity 1 litre, with ground-glass joint 2 Light petroleum layer 6 Side-arm 3 Aqueous layer +
28、 saponified feed 7 Adjustable tube 4J e t Figure 1 Example of extraction apparatusISO 6867:2000(E) 4 ISO 2000 All rights reserved 6 Sampling It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. Sampling is
29、 not part of the method specified in this International Standard. A recommended sampling method is g iv e ni nI S O64 97 1 . Store the sample in such a way that deterioration and change in its composition are prevented. 7 Preparation of test samples Prepare the test sample in accordance with ISO 649
30、8. Just prior to starting the analysis, grind a portion of the well-mixed laboratory sample so that it passes through a sieve with 1 mm apertures. Mix thoroughly. Homogenize canned pet foods. Pass semi-moist pet foods through a mincer with 4-mm apertures. 8 Procedure 8.1 General Because of the sensi
31、tivity of vitamin E to UV radiation and air, perform all operations away from natural and strong fluorescent light and as rapidly as is consistent with accurate working. Use amber glassware where possible. Complete each assay within one working day. 8.2 Saponification Weigh, to the nearest 0,1 g, ap
32、proximately 50 g of the prepared sample (see clause 7) into a 1 litre conical flask. Add to the test portion 200 ml of ethanol (4.3) whilst swirling the flask to disperse the sample. Add 2 ml of sodium ascorbate solution (4.9), mix by swirling and then add 50 ml of potassium hydroxide solution (4.2)
33、 and swirl again. Fit a reflux condenser to the flask and immerse the flask in the boiling water bath (5.2). Allow the contents of the flask to reflux for 30 min, swirling occasionally. NOTE In exceptional cases some products may require a longer saponification time. Cool the flask to room temperatu
34、re under a stream of cold water. Transfer the contents of the flask into the extraction cylinder (see 5.4). 8.3 Extraction of vitamin E Rinse the saponification flask with two 25 ml portions of ethanol (4.3) and transfer the rinsings to the cylinder. Repeat the rinsing of the flask with two 125 ml p
35、ortions of light petroleum (4.5) and one 250 ml portion of water (4.1), each time transferring the rinsings to the cylinder. Stopper the cylinder and shake well for 1 min, releasing the pressure from time to time. Cool the cylinder under a stream of cold water while waiting for the two liquid phases
36、 to separate, before removing the stopper.ISO 6867:2000(E) ISO 2000 All rights reserved 5 When the layers have separated, remove the stopper, wash the sides of the stopper with a few millilitres of light petroleum (4.5) and insert the adjustable tube (see 5.4), positioning the lower open end so that
37、 it is just above the level of the interface. By application of a slight pressure of inert gas (4.10) to the side arm tube, transfer the upper, light petroleum layer to a 1 litre separating funnel (see 5.4). Add 125 ml of light petroleum (4.5) to the cylinder, stopper and shake well for 1 min. Allow
38、 the layers to separate and transfer the upper layer to the separating funnel using the adjustable tube (see 5.4) as before. Again, add 125 ml of light petroleum (4.5) to the cylinder, stopper and shake well for 1 min. Again, allow the layers to separate and transfer the upper layer to the separatin
39、g funnel using the adjustable tube as before. Wash the combined light petroleum extracts with four 100 ml portions of water using at first only gentle inversion then only gentle shaking in order to keep emulsion formation to a minimum. Transfer the washed extract through a medium/fast filter paper c
40、ontaining 30 g of anhydrous sodium sulfate (4.8) into a 1 litre flask suitable for vacuum evaporation (5.3). Rinse the separating funnel with two 20 ml portions of light petroleum (4.5) and add the rinsings through the filter to the evaporation flask. Wash the filter further with two 25 ml portions
41、of light petroleum (4.5) and collect the washings in the evaporation flask. Evaporate the light petroleum extract to dryness under vacuum at a temperature not exceeding 40 C. Care should be taken to ensure that the flask is removed from the rotary evaporator immediately after reaching the point of d
42、ryness; prolonged drying may lead to loss of vitamin E from the extract residue. If the vitamin E concentration of the light petroleum extract is sufficiently high, the extract may be made up to a fixed volume with light petroleum and an aliquot part taken for the rotary evaporation stage. Restore a
43、tmospheric pressure by admitting inert gas (4.10). 8.4 Determination 8.4.1 Dissolve the residue from 8.3 in a minimum volume of hexane (4.4) and transfer quantitatively to a 25 ml volumetric flask. Rinse the evaporation flask with three small portions of hexane (4.4), transferring the rinsings to th
44、e volumetric flask. Dilute to volume with hexane and mix. If necessary, filter the sample extract through a membrane filter (5.5) or centrifuge. 8.4.2 Inject 20 l of the sample extract onto the column of the liquid chromatograph (5.1) and measure the area of the DL- -tocopherol peak. The following H
45、PLC conditions are offered for guidance; other conditions may be used provided that they give equivalent results: liquid chromatographic column (5.1.3): 250 mm 4,6 mm, silica 5 m or 10 m packing, or equivalent; mobile phase (4.11): mixture of hexane (4.4) and 1,4-dioxan (4.7), 970:30 (by volume); fl
46、ow rate: 1,5 ml/min; detector (5.1.4): fluorescence detector (excitation 295 nm, emission 330 nm).ISO 6867:2000(E) 6 ISO 2000 All rights reserved Reverse-phase chromatography may also be used provided that the efficiency of the column is sufficient to allow the separation of DL- -tocopherol from oth
47、er tocopherols and sample co-extractives. If reverse-phase chromatography is used, sample and standard solutions should be made up in an appropriate solvent, for example methanol (4.13). 8.4.3 Calculate the mean peak area from replicate injections of the sample extract and determine the DL- -tocophe
48、rol concentration in micrograms per millilitre of the extract, either a) by reference to the mean peak areas obtained from replicate injections of DL- -tocopherol standard solution of concentration within 5 % of the concentration in the sample extract, or b) by reference to a calibration curve prepa
49、red as in 8.5. 8.5 Calibration 8.5.1 Preparation of DL- -tocopherol standard solutions 8.5.1.1 DL- -tocopherol stock standard solution Dissolve approximately 100 mg, weighed to the nearest 0,1 mg, of DL- -tocopherol (4.6) in 100 ml of hexane (4.4). The stock standard solution is stable for 1 week when stored atu 4 C in an airtight amber glass flask. 8.5.1.2 DL- -tocopherol working standard solution: single-point calibration Prepare a working standard by diluting the stock standa
