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1、A Reference number ISO 13493:1998(E) INTERNATIONAL STANDARD ISO 13493 First edition 1998-09-01 Meat and meat products Determination of chloramphenicol content Method using liquid chromatography Viande et produits base de viande Dosage du chloramphnicol Mthode par chromatographie en phase liquide Cop
2、yright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/23/2007 19:11:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13493:1998(E) ISO 1998 All rights reserved. Un
3、less otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from the publisher. International Organization for Standardization Case postale 56 CH-1211 Ge
4、nve 20 Switzerland Internetisoiso.ch Printed in Switzerland ii 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 com
5、mittees. 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 with th
6、e International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. 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 m
7、ember bodies casting a vote. International Standard ISO 13493 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 6, Meat and meat products. Annex A of this International Standard is for information only. Copyright International Organization for Standardization
8、 Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/23/2007 19:11:59 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISOISO 13493:1998(E) 1 Meat and meat products Determination of chloramphenicol c
9、ontent Method using liquid chromatography 1 Scope This International Standard specifies a liquid chromatographic method for the determination of the chloramphenicol content of the muscle tissue of meat, including poultry. The method is suitable for the determination of chloramphenicol contents great
10、er than 6,5 g/kg. Test samples which have deteriorated cannot be analysed with this method. NOTE This International Standard may be applicable for the determination of the chloramphenicol content of all kinds of meat and meat products. However, materials other than muscle tissue were not included in
11、 the collaborative testing of the method. 2 Normative reference The following standard contains provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the edition indicated was valid. All standards are subject to revision,
12、and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the standard indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 3696:1987, Water for analytical
13、laboratory use Specification and test methods. 3 Definition For the purposes of this International Standard, the following definition applies. 3.1 chloramphenicol content of meat and meat products mass fraction of chloramphenicol residue determined according to the procedure specified in this Intern
14、ational Standard. NOTE The chloramphenicol content is expressed in micrograms per kilogram. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/23/2007 19:11:59 MDTNo reproduction or network
15、ing permitted without license from IHS -,-,- ISO 13493:1998(E) ISO 2 4 Principle A test portion is extracted with water. Filtration and solid-phase extraction are used to isolate the lipophilic components from the aqueous solution. The chloramphenicol is eluted from the cartridge with dichloromethan
16、e. The organic phase is evaporated and purified by liquid-liquid extraction with water and toluene. The chloramphenicol is measured with reverse-phase chromatography by ultraviolet (UV) detection. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. 5.1 Water, com
17、plying with at least grade 3 in accordance with ISO 3696. The water shall be free of organic compounds. 5.2 Nitrogen, suitable for evaporating solvents. 5.3 Dichloromethane. 5.4 Toluene. 5.5 Acetate buffer, c(CH3CO2Na) = 0,01 mol/l, pH = 4,3. Dissolve 0,82 g of anhydrous sodium acetate in about 970
18、ml of water. Adjust the pH to 4,3 with 50 % (m/m) dilute acetic acid (CH3CO2H) using the pH-meter (6.1). Transfer the solution to a 1000 ml one-mark volumetric flask. Dilute to the mark with water and mix. 5.6 Acetonitrile, suitable for UV spectroscopy. 5.7 Mobile phase. Add 750 ml of acetate buffer
19、 (5.5) to 250 ml of acetonitrile (5.6) and mix thoroughly. Before use, filter the eluent through a 0,22 mm filter (6.2) and degas. 5.8 Chloramphenicol stock solution, 100 mg/ml. Weigh, to the nearest 0,1 mg, 10 mg of chloramphenicol and transfer it to a 100 ml one-mark volumetric flask. Dilute to th
20、e mark with methanol and mix. This stock solution is stable for 1 month when stored in the dark. 5.9 Chloramphenicol standard solutions. Pipette 5,0 ml of the stock solution (5.8) into a 100 ml one-mark volumetric flask. Dilute to the mark with water and mix. Prepare four standard solutions by dilut
21、ing 1,0 ml, 2,0 ml, 5,0 ml and 15,0 ml of this solution to 100 ml with water to obtain solutions with a chloramphenicol content of 0,05 mg/ml, 0,10 mg/ml, 0,25 mg/ml and 0,75 mg/ml respectively. These standard solutions are stable for 1 week when stored in the dark. 6 Apparatus Usual laboratory appa
22、ratus and, in particular, the following. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/23/2007 19:11:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO IS
23、O 13493:1998(E) 3 6.1 pH-meter. 6.2 Membrane filter, of low dead volume and pore size 0,22 mm. 6.3 Mechanical or electrical equipment capable of homogenizing the laboratory sample. This includes a high-speed rotational cutter, or a mincer fitted with a plate with apertures not exceeding 4,0 mm in di
24、ameter. 6.4 Laboratory blender (e.g. Stomacher blender 1) or vortex type). 6.5 Filter paper, quantitative, fast filtration rate, of diameter about 15 cm. NOTEFor example, Whatman 41 proved to be suitable 1). 6.6 Extraction cartridges, of capacity 20 ml, containing diatomaceous earth that extracts li
25、pophilic components from aqueous solutions. NOTEExtrelut, manufactured by Merck, Darmstadt, Germany (No. 11737), proved to be suitable 1). 6.7 Water bath or heating block, capable of being maintained at (40 1) C, with equipment for drying with nitrogen (5.2); or rotary vacuum evaporator. 6.8 Centrif
26、uge tubes, of capacity 25 ml. 6.9 Vortex mixer, operating at a rotation frequency of about 700 min-1. 6.10 Centrifuge, operating at a radial acceleration of about 1 000 g. 6.11 Micropipettes, of capacity 300 ml. 6.12 Liquid chromatograph, equipped with: a constant-flow pump; an injector; a reverse-p
27、hase C8 or C18 column with an internal diameter of 3 mm, length of 20 cm, and particle size of 5 m, or a column of equivalent quality; a UV/VIS detector suitable for measurements at a wavelength of 285 nm; if available, a diode array detector (for confirmation purposes); a recorder with variable mea
28、suring range or an integrator. 7 Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 3100-1 1. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during
29、 transport or storage. Proceed from a representative sample of at least 200 g. Store the sample in such a way that deterioration and change in composition are prevented. 1) These are examples of suitable products available commercially. This information is given for the convenience of users of this
30、International Standard and does not constitute an endorsement by ISO of these products. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/23/2007 19:11:59 MDTNo reproduction or networking
31、permitted without license from IHS -,-,- ISO 13493:1998(E) ISO 4 8 Preparation of test sample Allow the sample to reach room temperature. Remove excess of fat and inedible parts. Homogenize the laboratory sample with the appropriate equipment (6.3). Take care that the temperature of the sample mater
32、ial does not rise above 25 C. If a mincer is used, pass the sample at least twice through the equipment. Fill a suitable airtight container with the prepared sample. Close the container and store in such a way that deterioration and change in composition are prevented. Store the sample, if necessary
33、, at a temperature below -18 C. Analyse the sample as soon as practicable, but always within 24 h after homogenization. 9 Procedure NOTE If it is required to check whether the repeatability limit (see 11.2) is met, carry out two single determinations in accordance with 9.1 to 9.6. 9.1 General In con
34、junction with the analysis of the test solution (or a series of test solutions), analyse a spiked blank sample with a chloramphenicol content of 10 mg/kg and a blank sample. 9.2 Test portion Weigh 10 g (m) of the prepared test sample (see clause 8) to the nearest 0,1 g in a 100 ml conical flask. 9.3
35、 Preparation of extract Add 40,0 ml of water and mix vigorously for 3 min with the laboratory blender (6.4). The volume (V1) of the water phase obtained is 40,0 ml plus the volume of water in the test portion (normally about 7,5 ml water in 10 g of sample). Filter the sample through a filter paper (
36、6.5). 9.4 Solid-phase extraction Transfer 20,0 ml (V2) of the filtrate to an extraction cartridge (6.6). After (15 0,2) min, elute the chloramphenicol with 70 ml of dichloromethane (5.3). Evaporate the organic phase to a volume of about 1 ml under a gentle stream of nitrogen (5.2) in the water bath
37、(6.7) or using the rotary vacuum evaporator (6.7). Transfer the residue to a centrifuge tube (6.8) with about 10 ml of dichloromethane (5.3). Evaporate carefully to absolute dryness. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technic
38、al Standards 1/9972545001 Not for Resale, 04/23/2007 19:11:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO ISO 13493:1998(E) 5 9.5 Liquid-liquid extraction Add 400 ml (V3) of water and 2,0 ml of toluene (5.4) to the residue and mix gently for 1 min at a rotation freq
39、uency of about 700 min-1 on the vortex mixer (6.9). Centrifuge for 5 min at a radial acceleration of 1 000 g in the centrifuge (6.10). Remove as much as possible of the organic phase with a pipette and discard it. Add 1,5 ml of toluene and mix gently for 1 min at a rotation frequency of about 700 mi
40、n-1 on the vortex mixer (6.9). Centrifuge for 5 min at a radial acceleration of 1 000 g in the centrifuge (6.10). Remove as much as possible of the organic phase with a pipette and discard it. Transfer 300 ml of the aqueous phase to a suitable container using a micropipette (6.11). 9.6 Chromatograph
41、ic analysis 9.6.1 Chromatographic conditions ParameterSetting Wavelength285 nm Detector rangeabsorbance of 0,005 to 0,01 at full scale of recorder Recorder range10 mV Paper speed1,0 cm/min Mobile phase (5.7) volume flow rate0,6 ml/min Injection volume 100 ml NOTEThe injection volume and the volume f
42、low rate depend on the column dimensions. 9.6.2 Chromatographic procedure Wait until the liquid chromatograph (6.12) system is stabilized. Inject the blank sample, the spiked blank sample, the four chloramphenicol standard solutions (5.9), the test solution obtained in 9.5, and again the chloramphen
43、icol standard solutions (5.9). Check for chloramphenicol signals in the sample chromatograms at the retention time of chloramphenicol. 9.6.3 Measurement Measure the chloramphenicol peak heights or peak areas of the test solution and the chloramphenicol standard solutions. The responses obtained for
44、the chloramphenicol standard solutions shall be linearly related to the chloramphenicol contents of these solutions. NOTE Confirmation can be carried out with a diode array detector for chloramphenicol contents exceeding 10 mg/kg. Copyright International Organization for Standardization Provided by
45、IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/23/2007 19:11:59 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 13493:1998(E) ISO 6 10 Calculation Calculate the chloramphenicol content of the test sample using the equation:
46、w hVV hmV = r 13 2s where wis the chloramphenicol content, in micrograms per kilogram, of the test sample; his the peak height or peak area, in length or area units, found for the test solution; hsis the peak height or peak area, in length or area units, found for one of the standard solutions (5.9)
47、; ris the chloramphenicol content, in micrograms per millilitre, of the standard solution; mis the mass, in grams, of the test portion (9.2); V1is the volume, in millilitres, of the water phase obtained after mixing in 9.3 (V1 = 40 ml + the volume of water in the test portion); V2is the volume, in m
48、illilitres, of filtrate transferred in 9.4 to the extraction cartridge (V2 = 20 ml); V3is the volume, in microlitres, of water added in 9.5 to the residue (V3 = 400 ml). Report the result rounded to the nearest 0,1 mg/kg. The result shall not be corrected for recovery. The recovery shall be specified in the test report (clause 12). 11 Precision 11.1 Interlaboratory tests The precision of the method was established by an interlaboratory test carried out in accordance with ISO 5725 22). The results of this interlaboratory tes