1、BRITISH STANDARD BS5766-17: 1999 ISO14797: 1999 Methods for analysis of animal feeding stuffs Part 17: Determination of furazolidone content Method using high-performance liquid chromatography ICS 65.120BS5766-17:1999 This BritishStandard, having been prepared under the directionof the Consumer Prod
2、ucts and Services Sector Committee, was published underthe authority of the Standards Committee and comesinto effect on 15June1999 BSI 03-2000 ISBN 0 580 32339 0 National foreword This BritishStandard reproduces verbatim ISO14797:1999 and implements it as the UK national standard. The UK participati
3、on in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs, 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 UK in
4、terests 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 BritishStandards which implement international or European publications ref
5、erred 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 of a
6、 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, pagesii
7、 toiv, pages1 to8, an inside back cover and abackcover. 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 CommentsBS5766-17:1999 BSI
8、03-2000 i Contents Page National foreword Inside front cover Foreword iv Text of ISO14797 1ii blankBS5766-17:1999 ii BSI 03-2000 Contents Page Foreword iv 1 Scope 1 2 Normative reference 1 3 Principle 1 4 Reagents 1 5 Apparatus 2 6 Sampling 2 7 Preparation of test sample 2 8 Procedure 2 8.1 General
9、2 8.2 Preparation of a spiked sample 2 8.3 Extraction 2 8.3.1 Feeding stuffs with a furazolidone content of25mg/kg to2500mg/kg 2 8.3.2 Feeding stuffs with a furazolidone content of2500mg/kg to5000mg/kg 3 8.3.3 Premixtures with a mass fraction of furazolidone of0,5% to7%0,5%(m/m) to7%(m/m) 3 8.3.4 Pr
10、emixtures with a mass fraction of furazolidone of7% to10%7%(m/m) to10% (m/m) 3 8.3.5 Premixtures with a mass fraction of furazolidone of10% to20%10%(m/m) to20%(m/m) 3 8.4 Column chromatography 3 8.5 HPLC analysis 3 8.5.1 HPLC conditions 3 8.5.2 Procedure 4 9 Confirmation 4 9.1 General 4 9.2 Co-chrom
11、atography 4 9.3 Diode array detector 4 9.3.1 Conditions 4 9.3.2 Procedure 4 9.3.3 Evaluation 5 9.3.4 Confirmation criteria 5 10 Calculation of results 5 10.1 General 5 10.2 Feeding stuffs with a furazolidone content of25mg/kg to5000mg/kg 5 10.3 Premixtures with a mass fraction of furazolidone of up
12、to20% 20% (m/m) 5 11 Precision 5 11.1 Interlaboratory test 5 11.2 Repeatability 6 11.3 Reproducibility 6 12 Test report 6 Annex A (informative) Example of a chromatogram 7 Annex B (informative) Results of an interlaboratory trial 7 Annex C (informative) Bibliography Inside back coverBS5766-17:1999 B
13、SI 03-2000 iii Page Figure A.1 7 Table B.1 Statistical results of the interlaboratory test on feeding stuffs 8 Table B.2 Statistical results of the interlaboratory test with premixtures 8 Descriptors: Agricultural products, food, animal feeding products, chemical analysis, determination of content,
14、furazolidonum, high performance liquid chromatography.BS5766-17:1999 iv BSI 03-2000 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 thro
15、ugh 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 organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collabor
16、ates closely with the 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
17、 least75% of the member bodies casting a vote. International Standard ISO14797 was prepared by Technical Committee ISO/TC34, Agricultural food products, Subcommittee SC10, Animal feeding stuffs. Annex A to Annex C of this International Standard are for information only.BS5766-17:1999 BSI 03-2000 1 1
18、 Scope This International Standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of the furazolidone content of premixtures and animal feeding stuffs. The method is applicable to animal feeding stuffs with a furazolidone content of25mg/kg to5000mg/kg and to
19、premixtures with a mass fraction of furazolidone of up to20% formerly written as20% (m/m). NOTE 1For animal feeding stuffs, the furazolidone content is expressed in milligrams per kilogram; for premixtures, as a mass fraction in percent%(m/m). NOTE 2Furazolidone is a chemotherapeuticum belonging to
20、the group of nitrofuranes. Nitrofuranes are bacteriostatic or bactericidal against Gram-positive and Gram-negative microorganisms and against some moulds and protozoa. 2 Normative reference The following standard contains provisions which, through reference in this text, constitute provisions of thi
21、s International Standard. At the time of publication, the edition indicated 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 bel
22、ow. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 6498:1998, Animal feeding stuffs Preparation of test sample. 3 Principle Furazolidone is extracted from the sample with a mixture of acetonitrile and methanol. Animal feeds are pre-wetted with water. The ex
23、tract of animal feeds is purified through a short aluminum oxide column and subsequently diluted with water. Theextract of premixtures is directly diluted with a mixture of water, acetonitrile and methanol. The final extract is analysed by reverse-phase HPLC with UV detection at a wavelength of365nm
24、 (seereferences1 to3). 4 Reagents Use only reagents of recognized analytical grade. 4.1 Water, demineralized or deionized, with resistivity of at least10M7cm, or water of at least equivalent purity. 4.2 Extraction solvent: mixture of acetonitrile and methanol(1:1 by volume). Combine equal volumes of
25、 acetonitrile and methanol. Mix well and allow to adjust to room temperature before use. 4.3 Dilution solvent: mixture of extraction solvent(4.2) and water(4.1)(35:65 by volume). Mix350ml of extraction solvent(4.2) with650ml of water(4.1). 4.4 Acetic acid (CH 3 CO 2 H), volume fraction of10%10% (V/V
26、). Dilute10ml of glacial acetic acid to100ml with water(4.1). 4.5 Sodium acetate buffer solution, c(CH 3 CO 2 Na)=0,01mol/l, pH=6,0. Weigh0,82g of sodium acetate into a1000ml one-mark volumetric flask. Dissolve in700ml of water. Adjust the pH to6,0 with acetic acid(4.4). Dilute to the mark with wate
27、r and mix. 4.6 Mobile phase for HPLC Combine800ml of sodium acetate buffer solution(4.5) and200ml of acetonitrile and mix. Filter the eluent through a0,224m filter using a solvent filtration system(5.2), and degas for10min in an ultrasonic bath(5.3) before use. 4.7 Furazolidone standard material: N-
28、(5-nitro-2- furfurylidene)-3-amino-2-oxazolidone; CAS number67-45-8 according to Chemical Abstracts Registry. WARNING Because of the sensitivity of furazolidone to light, conduct all operations in the absence of daylight or artificial white light. Avoid inhalation of and exposure to the toxic furazo
29、lidone standard material and solutions thereof. Work in a fumehood when handling the solvents and solutions. Wear safety glasses and protective clothing. 4.8 Furazolidone stock solution (approximately2504g/ml). Weigh25mg1mg of furazolidone(4.7) to the nearest0,1mg into a100ml one-mark volumetric fla
30、sk. Dissolve in extraction solvent(4.2), dilute to the mark and mix. Calculate the concentration taking into account the purity of the standard material. Prepare fresh every month. Store in the dark at0 C to8 C. 4.9 Furazolidone working solutions (approximately54g/ml and12,54g/ml). Pipette2,0ml and5
31、,0ml respectively of the furazolidone stock solution(4.8) into separate100ml one-mark volumetric flasks. Add65ml of water, dilute to the mark with extraction solvent(4.2) and mix. Prepare fresh for each series of samples. 4.10 Neutral aluminum oxide, activity1. NOTE0% to1% of water is necessary for
32、total de-activation.BS5766-17:1999 2 BSI 03-2000 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 pH-meter 5.2 Solvent filtration system, all-glass apparatus suitable for0,224m filters. 5.3 Ultrasonic bath 5.4 Rotary shaker, horizontal rotation, rotation frequency250min
33、1to300min 1 . 5.5 Glass microfibre filter, of diameter15cm. 5.6 Glass wool 5.7 Glass column for chromatography, of length30cm, internal diameter10mm, restricted at the end and fitted with a wad of glass wool(5.6). 5.8 Filtration system, equipped with poly(vinylidene difluoride) (PVDF) filters or pol
34、ytetrafluorethylene (PTFE) filters of pore size0,454m. 5.9 HPLC system, comprising the following. 5.9.1 Pump, pulse free, capable of maintaining a volume flow rate of0,1ml/min to2,0ml/min. 5.9.2 Injection system with loop suitable for204l to504l injections. 5.9.3 UV detector, suitable for measuremen
35、ts at a wavelength of365nm. If available, a diode array detector may be used for confirmation purposes. 5.9.4 Recorder 5.9.5 Guard column: silica bonded C 18packing with particle size374m to1004m, of length20mm, internal diameter3,9mm, or a guard column of equivalent quality. 5.9.6 Analytical column
36、: silica bonded C 18packing with particle size54m, of length200mm, internal diameter3,0mm, or an analytical column of equivalent quality. For furazolidone a capacity factor (K) of at least1,0 shall be obtained. NOTEThe capacity factor is defined as: 5.10 Disposable syringe, of capacity5ml. 6 Samplin
37、g Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO64974. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. 7 Preparation of test
38、 sample Prepare the test sample in accordance with ISO6498. Grind the laboratory sample (usually500g) so that it passes completely through a sieve with1mm apertures. Mix thoroughly. 8 Procedure 8.1 General In conjunction with the analysis of the test sample (or a series of test samples), analyse a b
39、lank sample, a spiked blank sample and, if available, a reference sample. NOTEBlank samples are homogenates of comparable feeds with a furazolidone content of less than10mg/kg. Spiked blank samples are blank feed samples to which furazolidone is added. Blank samples and reference samples can be kept
40、 for a year, ifstored at a temperature of0 C to8 C. The analysis should be repeated when the recovery is lower than94% or higher than106%. 8.2 Preparation of a spiked sample The furazolidone content of the spiked sample should be approximately equal to the expected furazolidone content of the sample
41、. For a spiked sample with a furazolidone content of250mg/kg, use the following procedure. Pipette5,0ml of the furazolidone stock solution(4.8) into a250ml conical flask. Under a flow of nitrogen, evaporate to a volume of approximately0,5ml and add5g of blank feed. Mix thoroughly and allow to stand
42、for at least10min before proceeding with the extraction(8.3). 8.3 Extraction 8.3.1 Feeding stuffs with a furazolidone content of25mg/kg to2500mg/kg Weigh5,0g of the prepared test sample to the nearest0,05g in a250ml conical flask. Add15,0ml of water, mix and allow to stand for5min. Add35,0ml of extr
43、action solvent(4.2), stopper and shake vigorously for30min on the rotary shaker(5.4). Filter the solution through a glass microfibre filter(5.5) and use the filtrate for column chromatography according to8.4. where K is the capacity factor; t R is the retention time, in minutes, of furazolidone; t 0
44、 is the retention time, in minutes, of the unretained furazolidone peak. K t R t 0 -1 =BS5766-17:1999 BSI 03-2000 3 8.3.2 Feeding stuffs with a furazolidone content of2500mg/kg to5000mg/kg Weigh5,0g of the prepared test sample to the nearest0,1g in a250ml conical flask. Add30,0ml of water, mix and a
45、llow to stand for5min. Add70,0ml of extraction solvent(4.2), stopper and shake vigorously for30min on the rotary shaker(5.4). Filter the solution through a glass microfibre filter(5.5) and use the filtrate for column chromatography according to8.4. 8.3.3 Premixtures with a mass fraction of furazolid
46、one of0,5% to7%0,5%(m/m) to7%(m/m) Weigh1,0g of the prepared test sample to the nearest0,01g in a250ml conical flask. Add100,0ml of extraction solvent(4.2), stopper and shake vigorously for30min on the rotary shaker(5.4). Filter the solution through a glass microfibre filter(5.5). Dilute the fitrate
47、 with dilution solvent(4.3) to obtain a final solution with a furazolidone content between54g/ml and104g/ml. The dilution factor isf. Mix well and filter the solution using the filtration system(5.8). Use the filtrate for HPLC analysis according to8.5. NOTEThe required dilution factor (f) can be est
48、imated by using the equation: 8.3.4 Premixtures with a mass fraction of furazolidone of7% to10% 7%(m/m) to10% (m/m) Weigh1,0g of the prepared test sample to the nearest0,01g in a500ml conical flask. Add200,0ml of extraction solvent(4.2), stopper and shake vigorously for30min on the rotary shaker(5.4
49、). Filter the solution through a glass microfibre filter(5.5). Dilute the fitrate with dilution solvent(4.3) to obtain a final solution with a furazolidone content between54g/ml and104g/ml. The dilution factor isf. Mix well and filter the solution using the filtration system(5.8). Use the filtrate for HPLC analysis according to8.5. NOTESee the note in8.3.3 for the calculation of the dilution factor. 8.3.5 Premixtures with a mass fraction of furazolidone of10% to20%10%(m/m) to20%(m/m) Weigh0,5g of the prepared test sample
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