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1、 Reference numbers ISO 11868:2007(E) IDF 147:2007(E) ISO and IDF 2007 INTERNATIONAL STANDARD ISO 11868 IDF 147 Second edition 2007-03-15 Heat-treated milk Determination of lactulose content Method using high- performance liquid chromatography Lait trait thermiquement Dtermination de la teneur en lac
2、tulose Mthode par chromatographie liquide haute performance Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 05/08/2007 08:12:03 MDTNo reproduction or networking permitted without lice
3、nse from IHS -,-,- ISO 11868:2007(E) IDF 147:2007(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the comput
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6、blem relating to it is found, please inform the ISO Central Secretariat at the address given below. ISO and IDF 2007 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopyin
7、g and microfilm, without permission in writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax +
8、 41 22 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2007 All rights reserved Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=IHS Emplo
9、yees/1111111001, User=Wing, Bernie Not for Resale, 05/08/2007 08:12:03 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 11868:2007(E) IDF 147:2007(E) ISO and IDF 2007 All rights reserved iii Contents Page Foreword iv 1 Scope . 1 2 Terms and definitions. 1 3 Principle. 1
10、4 Reagents 1 5 Apparatus 2 6 Sampling 3 7 Preparation of test sample. 4 8 Procedure 4 8.1 Test portion. 4 8.2 Preparation of calibration samples. 4 8.3 Chromatographic determination. 5 8.4 Recovery 6 9 Calculation and expression of results 6 9.1 Calibration . 6 9.2 Calculation of lactulose content .
11、 6 10 Precision 7 10.1 Interlaboratory test. 7 10.2 Repeatability 7 10.3 Reproducibility 7 11 Test report . 7 Annex A (informative) Example of a chromatogram. 8 Annex B (informative) Results of interlaboratory test 9 Bibliography. 10 Copyright International Organization for Standardization Provided
12、by IHS under license with ISO Licensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 05/08/2007 08:12:03 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 11868:2007(E) IDF 147:2007(E) iv ISO and IDF 2007 All rights reserved Foreword ISO (the International O
13、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
14、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
15、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
16、. 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
17、atent rights. ISO 11868IDF 147 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. This edition of ISO 11868IDF 147 cancels and replaces ISO 11868:1997, of
18、 which it constitutes a minor revision. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 05/08/2007 08:12:03 MDTNo reproduction or networking permitted without license from IHS -,-,- I
19、SO 11868:2007(E) IDF 147:2007(E) ISO and IDF 2007 All rights reserved v Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Comm
20、ittees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Public
21、ation as an International Standard requires approval by at least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such
22、 patent rights. ISO 11868IDF 147 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by a former Joint ISO/IDF/AOAC Group of Expert
23、s on Characterization of milk and milk products according to heat treatment (E704), under the aegis of its project leader, Mr M.A.J.S. van Boekel (NL). This edition of ISO 11868IDF 147 cancels and replaces IDF 147A:1994, of which it constitutes a minor revision. Copyright International Organization
24、for Standardization Provided by IHS under license with ISO Licensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 05/08/2007 08:12:03 MDTNo reproduction or networking permitted without license from IHS -,-,- Copyright International Organization for Standardization Provided by IHS under
25、 license with ISO Licensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 05/08/2007 08:12:03 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 11868:2007(E) IDF 147:2007(E) ISO and IDF 2007 All rights reserved 1 Heat-treated milk Deter
26、mination of lactulose content Method using high-performance liquid chromatography 1 Scope This International Standard specifies a method for the determination of the lactulose content of heated milk, skimmed, partially skimmed or whole milk, by high-performance liquid chromatography, in order to dis
27、tinguish milk sterilized by ultra-heat treatment (UHT) from in-bottle sterilized milk. The method has been tested over a lactulose content range of 200 mg/l to 1 500 mg/l and is applicable to all heat-treated milks. The method described in this International Standard is to be used in cases of disput
28、e. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 lactulose content of skimmed, partially skimmed or whole milk mass of substances determined by the procedure specified in this International Standard NOTE The lactulose content is expressed a
29、s milligrams per litre of sample. 3 Principle Fat and protein are removed from a sample of milk, which is then filtered. The lactulose content of the filtrate is determined by high-performance liquid chromatography (HPLC). The result obtained for the sample is evaluated by reference to standard samp
30、les consisting of lactulose-free skimmed milk with known amounts of added lactulose. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and double-distilled water or water of equivalent purity. 4.1 Lactose monohydrate. 4.2 Lactulose, at least 99 % pure. 4.3 Samp
31、le pretreatment solution. Dissolve 91,0 g of zinc acetate dihydrate, Zn(CH3COO)22H2O, 54,6 g of phosphotungstic acid tetracosahydrate, H3P(W3O10)424H2O, and 58,1 ml of glacial acetic acid in water in a 1 000 ml volumetric flask and dilute to the mark with water. Copyright International Organization
32、for Standardization Provided by IHS under license with ISO Licensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 05/08/2007 08:12:03 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 11868:2007(E) IDF 147:2007(E) 2 ISO and IDF 2007 All rights reserved 4.4 E
33、luent. Filter the water, HPLC grade, through a membrane filter with a 0,45 m pore diameter (5.8) and, prior to use, boil to remove dissolved air. To remove dissolved air, other methods giving the same results (e.g. helium sparging) may be used instead of boiling water. NOTE These alternatives are us
34、ually more expensive. 4.5 Standard samples. 4.5.1 Lactulose standard solution. Weigh, to the nearest 0,1 mg, about 75 mg of lactulose (4.2) in a 100 ml volumetric flask (5.6). Dissolve in water and dilute to the mark with water. 4.5.2 Pasteurized skimmed milk, lactulose free, as determined using the
35、 method specified below. Use identical pasteurized skimmed milk samples containing approximately 250 mg, 500 mg, 750 mg and 1 000 mg of lactulose per litre, obtained by the addition of 5 ml, 10 ml, 15 ml and 20 ml, respectively, of the lactulose standard solution (as described in 8.2) to the pasteur
36、ized skimmed milk. 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Analytical balance, capable of weighing to the nearest 0,1 mg. 5.2 Glass funnels, of diameter about 7 cm. 5.3 Filters. 5.3.1 Filter papers, medium grade, of diameter about 12,5 cm. 5.3.2 Cellulose acetat
37、e membranes, with 0,45 m pore diameter. 5.4 Measuring cylinder, of capacity 25 ml. 5.5 Graduated pipette, of capacity 10 ml, graduated in 0,1 ml. 5.6 One-mark volumetric flasks, of capacity 50 ml, 100 ml and 1 000 ml. 5.7 One-mark pipettes, capable of delivering 5 ml, 10 ml, 15 ml and 20 ml. 5.8 Gla
38、ss filtration equipment, with 0,45 m pore diameter filter. 5.9 Glass flasks, of capacity 20 ml, with stopcock. 5.10 Ultrasonic water bath. 5.11 Water vacuum pump. 5.12 HPLC equipment, as follows. 5.12.1 Magnetic stirrer and heater, for keeping the eluent at a temperature of 90 C 2 C before it is tra
39、nsported to the precolumn for analysis. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 05/08/2007 08:12:03 MDTNo reproduction or networking permitted without license from IHS -,-,- I
40、SO 11868:2007(E) IDF 147:2007(E) ISO and IDF 2007 All rights reserved 3 5.12.2 Pump, capable of delivering a volume flow rate of between 0,3 ml/min and 0,6 ml/min, with a pulsation of less than 1 % of the pressure drop over the column (1,5 MPa to 4 MPa). 5.12.3 HPX-87 P column (Bio-Rad, 30 cm 0,78 c
41、m)1), or an equivalent column packed with sulfonic ion exchanger in the lead form, based on a polystyrene divinylbenzene 8 % crosslinked polymer. The pre-column consists of the Bio-Rad de-ashing system1) (a cartridge, 3 cm 0,46 cm, packed with a cation-exchange resin in the hydrogen form and a cartr
42、idge, 3 cm 0,46 cm, packed with an anion-exchange resin in the carbonate form) or a system of equivalent effectiveness. The pre-columns extend both the life and the length of the analytical column, minimizing separation problems and substantially reducing quantization errors. When the HPLC system be
43、gins to lose resolution, replace the spent pre-column before contamination extends to the main column. 5.12.4 Thermostatic column oven, capable of being maintained at a temperature of 75 C 1 C. The pre-columns should be placed outside the oven. The inlet tubing to the main column should have a lengt
44、h of 10 cm to 15 cm in the oven to equilibrate the eluent temperature to 75 C, otherwise peak distortion may occur. 5.12.5 Refractive index detector, highly sensitive, with a noise level of less than 5 109 refractive index units (RIU), measured in water. The internal thermostat should be set at a te
45、mperature above room temperature, sufficient to obtain a stable baseline. A temperature of 35 C to 40 C is advisable in most cases. NOTE Highly sensitive monitoring of the refractive index is hampered by baseline drift due to thermal changes. To minimize the baseline drift, it is advisable to locate
46、 the HPLC equipment in a conditioned room to avoid temperature changes. 5.12.6 Integrator, capable of peak height measurements. The integration control parameters should be carefully chosen (e.g. peak width, slope drift, peak threshold). The integrator should be forced to drop a perpendicular betwee
47、n the lactose and the lactulose peaks. (Skimming leads to inaccuracy due to the presence of varying amounts of glucose in the milk.) The integrator shall be inhibited against finding the baseline between the lactose and the lactulose peaks, unless the valley reaches the baseline at all lactulose con
48、centrations. Many integrators automatically vary peak integration parameters during the run. If possible, this feature should be disconnected in order to obtain more repeatable results. 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 International Standard. A recommended sampling method is given in ISO 707IDF 50. Stor