1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58Method using high-performance liquid chromatography ICS 67.100.10Heat-treated milk Determination of
2、 lactulose content BRITISH STANDARDBS ISO 11868:2007BS ISO 11868:2007This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 May 2007 BSI 2007ISBN 978 0 580 50740 3Amendments issued since publicationAmd. No. Date Commentscontract. Users are respon
3、sible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.National forewordThis British Standard was published by BSI. It is the UK implementation of ISO 11868:2007. It supersedes BS 1741-13:1998 which is withdrawn.The UK participation in its
4、preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products.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 Reference numbersISO 118
5、68:2007(E)IDF 147:2007(E)INTERNATIONAL STANDARD ISO11868IDF147Second edition2007-03-15Heat-treated milk Determination of lactulose content Method using high-performance liquid chromatography Lait trait thermiquement Dtermination de la teneur en lactulose Mthode par chromatographie liquide haute perf
6、ormance BS ISO 11868:2007ii iiiContents Page Foreword iv 1 Scope . 1 2 Terms and definitions. 1 3 Principle. 1 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
7、 Recovery 6 9 Calculation and expression of results 6 9.1 Calibration . 6 9.2 Calculation of lactulose content . 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) Resu
8、lts of interlaboratory test 9 Bibliography . 10 BS ISO 11868:2007iv 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 technica
9、l 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 organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely wit
10、h the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. 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 Inter
11、national 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 possibility that some of the elements of this document may
12、 be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent 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 publi
13、shed jointly by ISO and IDF. This edition of ISO 11868IDF 147 cancels and replaces ISO 11868:1997, of which it constitutes a minor revision. BS ISO 11868:2007vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member cou
14、ntry. Every National Committee has the right to be represented on the IDF Standing Committees 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 Tea
15、ms and Standing Committees are circulated to the National Committees for voting. Publication 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 subj
16、ect of patent rights. IDF shall not be held responsible for identifying any or all such 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
17、by IDF and ISO. All work was carried out by a former Joint ISO/IDF/AOAC Group of Experts 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 147
18、A:1994, of which it constitutes a minor revision. BS ISO 11868:2007blank1Heat-treated milk Determination 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, skim
19、med, partially skimmed or whole milk, by high-performance liquid chromatography, in order to distinguish 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-tre
20、ated milks. The method described in this International Standard is to be used in cases of dispute. 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
21、the procedure specified in this International Standard NOTE The lactulose content is expressed as 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 chro
22、matography (HPLC). The result obtained for the sample is evaluated by reference to standard samples 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
23、water of equivalent purity. 4.1 Lactose monohydrate. 4.2 Lactulose, at least 99 % pure. 4.3 Sample 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
24、000 ml volumetric flask and dilute to the mark with water. BS ISO 11868:20072 4.4 Eluent. 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. he
25、lium sparging) may be used instead of boiling water. NOTE These alternatives are usually 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
26、with water. 4.5.2 Pasteurized skimmed milk, lactulose free, as determined using the 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, re
27、spectively, of the lactulose standard solution (as described in 8.2) to the pasteurized 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
28、.3.1 Filter papers, medium grade, of diameter about 12,5 cm. 5.3.2 Cellulose acetate 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.
29、 5.7 One-mark pipettes, capable of delivering 5 ml, 10 ml, 15 ml and 20 ml. 5.8 Glass 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 stirr
30、er and heater, for keeping the eluent at a temperature of 90 C 2 C before it is transported to the precolumn for analysis. BS ISO 11868:200735.12.2 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 col
31、umn (1,5 MPa to 4 MPa). 5.12.3 HPX-87 P column (Bio-Rad, 30 cm 0,78 cm)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,
32、packed with a cation-exchange resin in the hydrogen form and a cartridge, 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
33、 substantially reducing quantization errors. When the HPLC system begins 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 outsi
34、de the oven. The inlet tubing to the main column should have a length 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 109refractive index units (RIU
35、), measured in water. The internal thermostat should be set at a temperature 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
36、 changes. To minimize the baseline drift, it is advisable to locate 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 thresho
37、ld). The integrator should be forced to drop a perpendicular between 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
38、 peaks, unless the valley reaches the baseline at all lactulose concentrations. 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
39、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. Store the sample in such a way that deterioration and change in c
40、omposition are prevented. 1) The HPX-87 P column (Bio-Rad, 30 cm 0,78 cm) and Bio-Rad de-ashing system are examples of suitable products available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO or IDF
41、of these products. BS ISO 11868:20074 7 Preparation of test sample Bring the sample to 25 C 5 C and mix carefully. If the fat is not evenly dispersed, heat the sample slowly to 40 C, mix gently by inversion only and cool quickly to 20 C 2 C. 8 Procedure 8.1 Test portion 8.1.1 Pipette 15 ml of the te
42、st sample (Clause 7) into a 50 ml volumetric flask (5.6). Add 20 ml of water using a graduated cylinder (5.4) and swirl. Add 5,5 ml of the sample pretreatment solution (4.3) with a graduated pipette (5.5) and swirl. Dilute to the mark with water and mix. 8.1.2 After leaving to stand for 1 h at 25 C
43、5 C, filter using a glass funnel (5.2) through a filter (5.3.1 or 5.3.2). Discard the first 5 ml of filtrate. Collect the rest of the filtrate in a clean glass vessel. 8.2 Preparation of calibration samples 8.2.1 General Pipette 15 ml of lactulose-free skimmed milk (4.5.2) into each of four 50 ml vo
44、lumetric flasks. 8.2.2 Standard A 8.2.2.1 Pipette 5 ml of the standard lactulose solution (4.5.1) into the first 50 ml volumetric flask containing the lactulose-free skimmed milk (8.2.1) and swirl. 8.2.2.2 Add 15 ml of water using a measuring cylinder (5.4) and swirl. 8.2.2.3 Add 5,5 ml of the sampl
45、e pretreatment solution (4.3) with a graduated pipette (5.5) and swirl. Dilute to the mark with water and mix. After allowing the solution to stand for 1 h at 25 C 5 C, filter using a glass funnel (5.2) through a filter (5.3.1 or 5.3.2). Discard the first 5 ml of filtrate. Collect the rest of the fi
46、ltrate in a clean glass vessel. 8.2.3 Standard B Pipette 10 ml of the standard lactulose solution (4.5.1) into the second 50 ml volumetric flask containing the lactulose-free skimmed milk (8.2.1) and swirl. Add 10 ml of water using a measuring cylinder (5.4) and swirl. Proceed as in 8.2.2.3. 8.2.4 S
47、tandard C Pipette 15 ml of the standard lactulose solution (4.5.1) into the third 50 ml volumetric flask containing the lactulose-free skimmed milk (8.2.1) and swirl. Add 5 ml of water using a measuring cylinder (5.4) and swirl. Proceed as in 8.2.2.3. BS ISO 11868:200758.2.5 Standard D Pipette 20 ml
48、 of the standard lactulose solution (4.5.1) into the fourth 50 ml volumetric flask containing the lactulose-free skimmed milk (8.2.1). Proceed as in 8.2.2.3. 8.3 Chromatographic determination 8.3.1 Pipette about 3 ml of filtrate from the test portion (8.1.2) and from the calibration samples (8.2) in
49、to separate glass flasks (5.9). Remove dissolved air from the filtrate by attaching the flask stopcock to the water vacuum pump (5.11) and carrying out an ultrasonic bath treatment (5.10) for about 30 s at room temperature. If possible, avoid foaming. NOTE Inclusion of air in the sample may cause the appearance of a negative peak after the lactulose retention time. 8.3.2 Inject 10 l to 30 l (accurately measured) of filtrate into the HPLC apparatus (5.12) operating at a volume flow
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