1、BRITISH STANDARD BS4401-17: 1998 ISO 13493: 1998 Methods of test for meat and meat products Part 17: Determination of chloramphenicol content (liquid chromatographic method) ICS67.120.10BS4401-17:1998 This British Standard, having been prepared under the direction of the Consumer Products and Servic
2、es Sector Board, was published under theauthority of the Standards Board and comes into effect on 15 November1998 BSI05-1999 ISBN 0 580 30422 1 National foreword This British Standard reproduces verbatim ISO13493:1998 and implements it as the UK national standard. The UK participation in its prepara
3、tion was entrusted to Technical Committee AW/6, Chemical analysis of meat and meat products, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the U
4、K interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publication
5、s referred to in this document 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 purport to include all the necessary provisions
6、 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. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, theISO title page, pa
7、ge ii, pages1 to5 and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No. Date CommentsBS4401-17:1998 BSI 05-1999 i Contents P
8、age National foreword Inside front cover Foreword ii 1 Scope 1 2 Normative reference 1 3 Definition 1 4 Principle 1 5 Reagents 1 6 Apparatus 1 7 Sampling 2 8 Preparation of test sample 2 9 Procedure 2 10 Calculation 3 11 Precision 3 12 Test report 4 Annex A (informative) Bibliography 5ii blankBS4401
9、-17:1998 ii BSI05-1999 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
10、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 the International Electrotechnical Commissi
11、on (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 least75% of the member bodies casting a vote. International
12、Standard ISO13493 was prepared by Technical Committee ISO/TC34, Agricultural food products, Subcommittee SC6, Meat and meat products. Annex A of this International Standard is for information only. Descriptors: Agricultural products, food products, animal products, meat, meat products, chemical anal
13、ysis, determination of content, food additives, chromatographic analysis.BS4401-17:1998 BSI 05-1999 1 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 suit
14、able for the determination of chloramphenicol contents greater than6,54g/kg. Test samples which have deteriorated cannot be analysed with this method. NOTEThis International Standard may be applicable for the determination of the chloramphenicol content of all kinds of meat and meat products. Howeve
15、r, materials other than muscle tissue were not included in 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 in
16、dicated was valid. All standards are subject to revision, 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 Int
17、ernational Standards. ISO3696:1987, Water for analytical 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 deter
18、mined according to the procedure specified in this International Standard NOTEThe chloramphenicol content is expressed in micrograms per kilogram. 4 Principle A test portion is extracted with water. Filtration and solid-phase extraction are used to isolate the lipophilic components from the aqueous
19、solution. The chloramphenicol is eluted from the cartridge with dichloromethane. 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 reage
20、nts of recognized analytical grade, unless otherwise specified. 5.1 Water, complying with at least grade3 in accordance with ISO3696. 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(CH 3 CO 2 Na) =
21、 0,01 mol/l, pH = 4,3. Dissolve0,82g of anhydrous sodium acetate in about970ml of water. Adjust the pH to4,3 with50% (m/m) dilute acetic acid (CH 3 CO 2 H) using the pH-meter(6.1). Transfer the solution to a1000ml one-mark volumetric flask. Dilute to the mark with water and mix. 5.6 Acetonitrile, su
22、itable for UV spectroscopy. 5.7 Mobile phase. Add750ml of acetate buffer(5.5) to250ml of acetonitrile(5.6) and mix thoroughly. Before use, filter the eluent through a0,224m filter(6.2) and degas. 5.8 Chloramphenicol stock solution, 1004g/ml. Weigh, to the nearest0,1mg,10mg of chloramphenicol and tra
23、nsfer it to a100ml one-mark volumetric flask. Dilute to the mark with methanol and mix. This stock solution is stable for1month when stored in the dark. 5.9 Chloramphenicol standard solutions. Pipette5,0ml of the stock solution(5.8) into a100ml one-mark volumetric flask. Dilute to the mark with wate
24、r and mix. Prepare four standard solutions by diluting1,0ml,2,0ml,5,0ml and15,0ml of this solution to100ml with water to obtain solutions with a chloramphenicol content of0,054g/ml, 0,104g/ml,0,254g/ml and0,754g/ml respectively. These standard solutions are stable for1week when stored in the dark. 6
25、 Apparatus Usual laboratory apparatus and, in particular, the following. 6.1 pH-meter. 6.2 Membrane filter, of low dead volume and pore size0,224m. 6.3 Mechanical or electrical equipment capable of homogenizing the laboratory sample.BS4401-17:1998 2 BSI 05-1999 This includes a high-speed rotational
26、cutter, or a mincer fitted with a plate with apertures not exceeding4,0mm in diameter. 6.4 Laboratory blender (e.g.Stomacher blender 1)or vortex type). 6.5 Filter paper, quantitative, fast filtration rate, of diameter about15cm. NOTEFor example, Whatman41 proved to be suitable 1) . 6.6 Extraction ca
27、rtridges, of capacity20ml, containing diatomaceous earth that extracts lipophilic 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 f
28、or drying with nitrogen(5.2); or rotary vacuum evaporator. 6.8 Centrifuge tubes, of capacity25ml. 6.9 Vortex mixer, operating at a rotation frequency of about700min 1 . 6.10 Centrifuge, operating at a radial acceleration of about1000g. 6.11 Micropipettes, of capacity3004l. 6.12 Liquid chromatograph,
29、 equipped with: a constant-flow pump; an injector; a reverse-phase C 8or C 18column with an internal diameter of3mm, length of20cm, and particle size of54m, or a column of equivalent quality; a UV/VIS detector suitable for measurements at a wavelength of285nm; if available, a diode array detector (f
30、or confirmation purposes); a recorder with variable measuring 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 ISO3100-1 1. It is important that the laboratory receive a sample which is truly rep
31、resentative and has not been damaged or changed during transport or storage. Proceed from a representative sample of at least200g. Store the sample in such a way that deterioration and change in composition are prevented. 8 Preparation of test sample Allow the sample to reach room temperature. Remov
32、e excess of fat and inedible parts. Homogenize the laboratory sample with the appropriate equipment(6.3). Take care that the temperature of the sample material does not rise above25C. If a mincer is used, pass the sample at least twice through the equipment. Fill a suitable airtight container with t
33、he prepared sample. Close the container and store in such a way that deterioration and change in composition are prevented. Store the sample, if necessary, at a temperature below 18C. Analyse the sample as soon as practicable, but always within24h after homogenization. 9 Procedure NOTEIf it is requi
34、red to check whether the repeatability limit (see11.2) is met, carry out two single determinations in accordance with9.1 to9.6. 9.1 General In conjunction with the analysis of the test solution (or a series of test solutions), analyse a spiked blank sample with a chloramphenicol content of104g/kg an
35、d a blank sample. 9.2 Test portion Weigh10g (m) of the prepared test sample (seeclause8) to the nearest0,1g in a100ml conical flask. 9.3 Preparation of extract Add40,0ml of water and mix vigorously for3min with the laboratory blender(6.4). The volume (V 1 ) of the water phase obtained is40,0ml plus
36、the volume of water in the test portion (normally about7,5ml water in10g ofsample). Filter the sample through a filter paper(6.5). 9.4 Solid-phase extraction Transfer20,0ml (V 2 ) of the filtrate to an extraction cartridge(6.6). After(15 0,2)min, elute the chloramphenicol with70ml of dichloromethane
37、(5.3). Evaporate the organic phase to a volume of about1ml under a gentle stream of nitrogen(5.2) in the water bath(6.7) or using the rotary vacuum evaporator(6.7). Transfer the residue to a centrifuge tube(6.8) with about10ml of dichloromethane(5.3). 1) These are examples of suitable products avail
38、able commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of these products.BS4401-17:1998 BSI 05-1999 3 Evaporate carefully to absolute dryness. 9.5 Liquid-liquid extraction Add4004l (V 3 ) of water and2,0m
39、l of toluene(5.4) to the residue and mix gently for1min at a rotation frequency of about700min 1on the vortex mixer(6.9). Centrifuge for5min at a radial acceleration of1000g in the centrifuge(6.10). Remove as much as possible of the organic phase with a pipette and discard it. Add1,5ml of toluene an
40、d mix gently for1min at a rotation frequency of about700min 1on the vortex mixer(6.9). Centrifuge for5min at a radial acceleration of1000g in the centrifuge(6.10). Remove as much as possible of the organic phase with a pipette and discard it. Transfer3004l of the aqueous phase to a suitable containe
41、r using a micropipette(6.11). 9.6 Chromatographic analysis 9.6.1 Chromatographic conditions NOTEThe injection volume and the volume flow 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
42、spiked blank sample, the four chloramphenicol standard solutions(5.9), the test solution obtained in9.5, and again the chloramphenicol standard solutions(5.9). Check for chloramphenicol signals in the sample chromatograms at the retention time of chloramphenicol. 9.6.3 Measurement Measure the chlora
43、mphenicol peak heights or peak areas of the test solution and the chloramphenicol standard solutions. The responses obtained for the chloramphenicol standard solutions shall be linearly related to the chloramphenicol contents of these solutions. NOTEConfirmation can be carried out with a diode array
44、 detector for chloramphenicol contents exceeding104g/kg. 10 Calculation Calculate the chloramphenicol content of the test sample using the equation: where Report the result rounded to the nearest0,14g/kg. The result shall not be corrected for recovery. The recovery shall be specified in the test rep
45、ort (clause12). 11 Precision 11.1 Interlaboratory tests The precision of the method was established by an interlaboratory test carried out in accordance with ISO5725 2 2) . The results of this interlaboratory test have been published (see reference 5). The values derived from this test may not be ap
46、plicable to concentration ranges and matrices other than those given. Parameter Setting Wavelength 285nm Detector range absorbance of0,005 to0,01 at full scale of recorder Recorder range 10mV Paper speed 1,0cm/min Mobile phase(5.7) volume flow rate 0,6ml/min Injection volume 1004l w is the chloramph
47、enicol content, in micrograms per kilogram, of the test sample; h is the peak height or peak area, in length or area units, found for the test solution; h s is the peak height or peak area, in length or area units, found for one of the standard solutions(5.9); is the chloramphenicol content, in micr
48、ograms per millilitre, of the standard solution; m is the mass, in grams, of the test portion(9.2); V 1 is the volume, in millilitres, of the water phase obtained after mixing in9.3 (V 1 =40ml +the volume of water in the test portion); V 2 is the volume, in millilitres, of filtrate transferred in9.4
49、 to the extraction cartridge (V 2= 20ml); V 3 is the volume, in microlitres, of water added in9.5 to the residue (V 3= 4004l). 2) ISO5725:1986 (now withdrawn) was used to obtain the precision results. w h V 1 V 3 h s m V 2 - =BS4401-17:1998 4 BSI 05-1999 The results of another interlaboratory test, carried out in accordance with ISO5725, show that recovery for meat, meat products and poultry is reproducible and approximately55%. 11.2 Repeatability The absolute difference between two independent
