1、BRITISH STANDARD BS EN ISO 14565:2001 BS 5766-26:2001 Animal feeding stuffs Determination of vitamin A content Method using high- performance liquid chromatography The European Standard EN ISO 14565:2000 has the status of a British Standard ICS 65.120 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERM
2、ITTED BY COPYRIGHT LAWBS EN ISO 14565:2001 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 March 2001 BSI 03-2001 ISBN 0 580 36934 X Nation
3、al foreword This British Standard is the official English language version of EN ISO 14565:2000. It is identical with ISO 14565:2000. The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs, which has the responsibility to: A list of organizations re
4、presented on this committee can be obtained on request to its secretary. Cross-references Attention is drawn to the fact that CEN and CENELEC Standards normally include an annex which lists normative references to international publications with their corresponding European publications. The British
5、 Standards which implement these international or European publications may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purpor
6、t to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible internatio
7、nal/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN ISO ti
8、tle page, the EN ISO foreword page, the ISO title page, pages ii and iii, a blank page, pages 1 to 11, the annex ZA page, an inside back cover and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No.
9、 Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM ENISO14565 December2000 ICS06.012 Englishversion AnimalfeedingstuffsDeterminationofvitaminAcontent Methodusinghighperformanceliquidchromatography(ISO 14565:2000) AlimentsdesanimauxDterminationdelateneuren vitamineAMthodeparchromatographiel
10、iquidehaute performance(ISO14565:2000) FuttermittelBestimmungdesGehaltsanVitaminA VerfahrenmitHochleistungsFlssigchromatographie(ISO 14565:2000) ThisEuropeanStandardwasapprovedbyCENon1December2000. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthi
11、sEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherla
12、nguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Iceland,Ireland,Italy,Luxembourg,Netherl
13、ands,Norway,Portugal,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2000CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.
14、No.ENISO14565:2000EForeword ThetextoftheInternationalStandardISO14565:2000hasbeenpreparedbyTechnicalCommittee ISO/TC34“Agriculturalfoodproducts“incollaborationwithTechnicalCommitteeCEN/TC327 “AnimalfeedingstuffsMethodsofsamplingandanalysis“,thesecretariatofwhichisheldbyNEN . ThisEuropeanStandardshal
15、lbegiventhestatusofanationalstandard,eitherbypublicationofan identicaltextorbyendorsement,atthelatestbyJune2001,andconflictingnationalstandardsshallbe withdrawnatthelatestbyJune2001 . AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizationsofthe followingcountriesareboundtoimpl
16、ementthisEuropeanStandard:Austria,Belgium,Czech Republic,Denmark,Finland,France,Germany,Greece,Iceland,Ireland,Italy,Luxembourg, Netherlands,Norway,Portugal,Spain,Sweden,SwitzerlandandtheUnitedKingdom. Endorsementnotice ThetextoftheInternationalStandardISO14565:2000wasapprovedbyCENasaEuropean Standa
17、rdwithoutanymodification. NOTE:NormativereferencestoInternationalStandardsarelistedinannexZA(normative). ENISO14565:2000 Reference number ISO 14565:2000(E) INTERNATIONAL STANDARD ISO 14565 First edition 2000-12-01 Animal feeding stuffs Determination of vitamin A content Method using high- performanc
18、e liquid chromatography Aliments des animaux Dtermination de la teneur en vitamine A Mthode par chromatographie liquide haute performance ENISO14565:2000 ii ENISO14565:2000 ISO 56541:(0002)Eiii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national st
19、andards 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 established has the right to be represented on that committee. International o
20、rganizations, 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 standardization. International Standards are drafted in accordance with the rules giv
21、en 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 Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibili
22、ty 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. International Standard ISO 14565 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal
23、feeding stuffs. Annex A of this International Standard is for information only. ENISO14565:2000 ENISO14565:2000 INTENRATIONAL TSANDADR ISO 56541:(0002)E1 Animal feeding stuffs Determination of vitamin A content Method using high-performance liquid chromatography 1 Scope This International Standard s
24、pecifies a method for the determination of the total vitamin A (retinol) content of animal feeding stuffs and pet foods using high-performance liquid chromatography. The vitamin A content is the content of all-trans-retinyl alcohol and cis-isomers determined by the method described in this Internati
25、onal Standard, and is expressed in International Units per kilogram (IU/kg). 2 Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or rev
26、isions of, any of these publications do not apply. However, parties to agreements based on this International 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 norm
27、ative documents referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 3696:1987, Water for analytical laboratory use Specification and test methods. ISO 6498, Animal feeding stuffs Preparation of test samples. 3 Term and definition For the pur
28、poses of this International Standard, the following term and definition apply. 3.1 vitamin A content content of all-trans-retinyl alcohol and cis-isomers determined in accordance with this International Standard NOTE The vitamin A content is expressed in International Units per kilogram (IU/kg); 1 I
29、U of vitamin A is equal to 0,300 g of all-trans-retinol. 4P r i n c i p l e The sample is saponified with ethanolic potassium hydroxide solution and the vitamin A is extracted into light petroleum. The light petroleum is removed by evaporation and the residue is dissolved in 2-propanol. The vitamin
30、A concentration in the 2-propanol extract is determined by reverse-phase liquid chromatography using conditions that give a single peak for all retinol isomers. ENISO14565:2000 ISO 56541:(0002)E 2 5 Reagents Use only reagents of recognized analytical grade. 5.1 Water, complying with at least grade 3
31、 in accordance with ISO 3696. 5.2 Potassium hydroxide solution (KOH). Dissolve 500 g of potassium hydroxide in water (4.1) and dilute to 1 litre. 5.3 Ethanol, w(C 2 H 5 OH) = 95 % (by volume), or equivalent industrial methylated spirit. 5.4 2-Propanol (C 3 H 7 OH). 5.5 Light petroleum, boiling range
32、 40 Cto60 C. The residue on evaporation shall be less than 20 mg/l. 5.6 Vitamin A standard substances 5.6.1 All-trans-retinyl acetate, vitamin A acetate (C 22 H 32 O 2 ), 328.5 g/mol, with a purity of at least 90 %. 5.6.2 All-trans-retinol, vitamin A alcohol (C 20 H 30 O), 286,5 g/mol, with a purity
33、 of at least 90 % 5.7 Methanol,HP L Cg ra de . 5.8 Mobile phase for liquid chromatography. Mix together methanol (5.7) and water (4.1) in the proportions 770 + 30 (by volume). If necessary, filter through a membrane filter (6.6). 5.9 Sodium sulfate (Na 2 SO 4 ), anhydrous. 5.10 Sodium ascorbate solu
34、tion, = 100 g/l. 5.11 Inert gas,e .g .ni tro ge n . 6 Apparatus Usual laboratory apparatus and, in particular, the following. 6.1 High-performance liquid chromatograph, consisting of the following. 6.1.1 Pump, set to deliver a constant eluent volume flow rate of 1 ml/min. 6.1.2 HPLC injection device
35、. 6.1.3 Column, length 250 mm, internal diameter 4,6 mm, packed with a stationary phase consisting of octadecyl (C 18 ) groups bonded to silica. A column with at least 4 000 theoretical plates and a k value of 0,6, both with respect to all-trans-retinol, has been found to be satisfactory. The partic
36、le size shall be not smaller than 5 m and not greater than 10 m. Other systems may be used provided that a satisfactory separation of vitamin A from other co-extractives is achieved. ENISO14565:2000 ISO 56541:(0002)E3 6.1.4 Detector, allowing the measurement of ultraviolet radiation at 325 nm, and e
37、quipped with integrator/data- handling system. 6.2 UV (or UV-Visible) spectrometer, capable of measuring absorbance at the wavelengths defined in 9.6, equipped with quartz cells of 10 mm path length. 6.3 Boiling water bath. 6.4 Rotary vacuum evaporator, with water bath at 40 C. 6.5 Extraction appara
38、tus (see Figure 1) 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
39、 and a jet at the opposite 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 re
40、coveries of vitamin A are achieved. 6.6 Membrane filter, 0,45 m pore size, for filtration of mobile phase (5.8) and sample test solutions. 7 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. Sa
41、mpling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 6497 4. Store the sample in such a way that deterioration and change in its composition are prevented. 8 Preparation of test sample Prepare the test sample in accordance with ISO
42、6498. 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. Mince semi-moist pet foods to pass through a plate with 4 mm apertures. ENISO14565:2000 ISO 56541:(0002)E
43、 4 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 + saponified feed 7 Adjustable tub 4J e t Figure 1 Example of extraction apparatus ENISO14565:2000 ISO 56541:(0002)E5 9 Procedure 9
44、.1 General Because of the sensitivity of vitamin A 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. Carry out the sapon
45、ification and extraction of the all-trans-retinyl acetate standard and the feeding stuff samples at the same time. 9.2 Saponification Weigh, to the nearest 0,1 g, approximately 50 g of the prepared test sample (see clause 8) into a 1 litre conical flask. Add 200 ml of ethanol (5.3). Swirl the flask
46、contents to disperse the sample. Add 2 ml of sodium ascorbate (5.10) and 50 ml of potassium hydroxide solution (5.2) and mix by swirling. Fit a reflux condenser to the flask and immerse the flask in the boiling water bath (6.3). Allow the contents of the flask to reflux for 60 min, swirling occasion
47、ally. Remove and cool the flask to room temperature as rapidly as possible under a stream of cold water. 9.3 Extraction of vitamin A (retinol) Transfer the contents of the flask to the extraction cylinder (see 6.5). Rinse the flask with two 25 ml portions of ethanol or industrial methylated spirit (
48、5.3) and transfer the rinsings to the cylinder. Repeat the rinsing of the flask with two 125 ml portions of light petroleum (5.5) 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, taking care to release any pressure from time to time. Cool the cylinder under a stream of cold water while waiting for the two liquid phases to separate, before removin
