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1、A Reference number ISO 14797:1999(E) INTERNATIONAL STANDARD ISO 14797 First edition 1999-03-15 Animal feeding stuffs Determination of furazolidone content Method using high-performance liquid chromatography Aliments des animaux Dosage de la furazolidone Mthode par chromatographie liquide haute perfo
2、rmance Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 07:04:27 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14797:1999(E) ISO 1999 All rights re
3、served. Unless 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
4、CH-1211 Genve 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 tec
5、hnical 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 closel
6、y 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 least 75 %
7、 of the member bodies casting a vote. International Standard ISO 14797 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 10, Animal feeding stuffs. Annexes A to C of this International Standard are for information only. Copyright International Organization fo
8、r Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 07:04:27 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISOISO 14797:1999(E) 1 Animal feeding stuffs Determination of f
9、urazolidone content Method using high-performance liquid chromatography 1 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
10、 feeding stuffs with a furazolidone content of 25 mg/kg to 5 000 mg/kg and to premixtures with a mass fraction of furazolidone of up to 20 % formerly written as 20 % (m/m). NOTE 1For animal feeding stuffs, the furazolidone content is expressed in milligrams per kilogram; for premixtures, as a mass f
11、raction in percent % (m/m). NOTE 2Furazolidone is a chemotherapeuticum belonging to 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 con
12、tains 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, and parties to agreements based on this International Standard are encouraged to investiga
13、te 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 6498:1998, Animal feeding stuffs Preparation of test sample. 3 Principle Furazolidone is extracted from the sample with a
14、mixture of acetonitrile and methanol. Animal feeds are pre-wetted with water. The extract of animal feeds is purified through a short aluminum oxide column and subsequently diluted with water. The extract of premixtures is directly diluted with a mixture of water, acetonitrile and methanol. The fina
15、l extract is analysed by reverse-phase HPLC with UV detection at a wavelength of 365 nm (see references 1 to 3). 4 Reagents Use only reagents of recognized analytical grade. 4.1 Water, demineralized or deionized, with resistivity of at least 10 Mcm, or water of at least equivalent purity. 4.2 Extrac
16、tion solvent: mixture of acetonitrile and methanol (1:1 by volume). Combine equal volumes of 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). Mix 350 ml of extraction s
17、olvent (4.2) with 650 ml of water (4.1). Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 07:04:27 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 14
18、797:1999(E) ISO 2 4.4 Acetic acid (CH3CO2H), volume fraction of 10 % 10 % (V/V). Dilute 10 ml of glacial acetic acid to 100 ml with water (4.1). 4.5 Sodium acetate buffer solution, c(CH3CO2Na) = 0,01 mol/l, pH = 6,0. Weigh 0,82 g of sodium acetate into a 1 000 ml one-mark volumetric flask. Dissolve
19、in 700 ml of water. Adjust the pH to 6,0 with acetic acid (4.4). Dilute to the mark with water and mix. 4.6 Mobile phase for HPLC. Combine 800 ml of sodium acetate buffer solution (4.5) and 200 ml of acetonitrile and mix. Filter the eluent through a 0,22 m filter using a solvent filtration system (5
20、.2), and degas for 10 min in an ultrasonic bath (5.3) before use. 4.7 Furazolidone standard material: N-(5-nitro-2-furfurylidene)-3-amino-2-oxazolidone; CAS number 67-45-8 according to Chemical Abstracts Registry. WARNING Because of the sensitivity of furazolidone to light, conduct all operations in
21、 the absence of daylight or artificial white light. Avoid inhalation of and exposure to the toxic furazolidone 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 (approxima
22、tely 250 g/ml). Weigh 25 mg 1 mg of furazolidone (4.7) to the nearest 0,1 mg into a 100 ml one-mark volumetric flask. 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. Stor
23、e in the dark at 0 C to 8 C. 4.9 Furazolidone working solutions (approximately 5 g/ml and 12,5 g/ml). Pipette 2,0 ml and 5,0 ml respectively of the furazolidone stock solution (4.8) into separate 100 ml one-mark volumetric flasks. Add 65 ml of water, dilute to the mark with extraction solvent (4.2)
24、and mix. Prepare fresh for each series of samples. 4.10 Neutral aluminum oxide, activity 1. NOTE 0 % to 1 % of water is necessary for total de-activation. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 pH-meter. 5.2 Solvent filtration system, all-glass apparatus suitab
25、le for 0,22 m filters. 5.3 Ultrasonic bath. 5.4 Rotary shaker, horizontal rotation, rotation frequency 250 min1 to 300 min1. 5.5 Glass microfibre filter, of diameter 15 cm. 5.6 Glass wool. 5.7 Glass column for chromatography, of length 30 cm, internal diameter 10 mm, restricted at the end and fitted
26、 with a wad of glass wool (5.6). 5.8 Filtration system, equipped with poly(vinylidene difluoride) (PVDF) filters or polytetrafluorethylene (PTFE) filters of pore size 0,45 m. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Stand
27、ards 1/9972545001 Not for Resale, 04/20/2007 07:04:27 MDTNo reproduction or networking permitted without license from IHS -,-,- ISOISO 14797:1999(E) 3 5.9 HPLC system, comprising the following. 5.9.1 Pump, pulse free, capable of maintaining a volume flow rate of 0,1 ml/min to 2,0 ml/min. 5.9.2 Injec
28、tion system with loop suitable for 20 l to 50 l injections. 5.9.3 UV detector, suitable for measurements at a wavelength of 365 nm. If available, a diode array detector may be used for confirmation purposes. 5.9.4 Recorder. 5.9.5 Guard column: silica bonded C18 packing with particle size 37 m to 100
29、 m, of length 20 mm, internal diameter 3,9 mm, or a guard column of equivalent quality. 5.9.6 Analytical column: silica bonded C18 packing with particle size 5 m, of length 200 mm, internal diameter 3,0 mm, or an analytical column of equivalent quality. For furazolidone a capacity factor (K) of at l
30、east 1,0 shall be obtained. NOTE The capacity factor is defined as: =K t t R 0 1 where Kis the capacity factor; tRis the retention time, in minutes, of furazolidone; t0is the retention time, in minutes, of the unretained furazolidone peak. 5.10 Disposable syringe, of capacity 5 ml. 6 Sampling Sampli
31、ng is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 6497 4. 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 sampl
32、e Prepare the test sample in accordance with ISO 6498. Grind the laboratory sample (usually 500 g) so that it passes completely through a sieve with 1 mm 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 bl
33、ank sample, a spiked blank sample and, if available, a reference sample. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 07:04:27 MDTNo reproduction or networking permitted witho
34、ut license from IHS -,-,- ISO 14797:1999(E) ISO 4 NOTE Blank samples are homogenates of comparable feeds with a furazolidone content of less than 10 mg/kg. Spiked blank samples are blank feed samples to which furazolidone is added. Blank samples and reference samples can be kept for a year, if store
35、d at a temperature of 0 C to 8 C. The analysis should be repeated when the recovery is lower than 94 % or higher than 106 %. 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. For a spiked
36、 sample with a furazolidone content of 250 mg/kg, use the following procedure. Pipette 5,0 ml of the furazolidone stock solution (4.8) into a 250 ml conical flask. Under a flow of nitrogen, evaporate to a volume of approximately 0,5 ml and add 5 g of blank feed. Mix thoroughly and allow to stand for
37、 at least 10 min before proceeding with the extraction (8.3). 8.3 Extraction 8.3.1 Feeding stuffs with a furazolidone content of 25 mg/kg to 2 500 mg/kg Weigh 5,0 g of the prepared test sample to the nearest 0,05 g in a 250 ml conical flask. Add 15,0 ml of water, mix and allow to stand for 5 min. Ad
38、d 35,0 ml of extraction solvent (4.2), stopper and shake vigorously for 30 min on the rotary shaker (5.4). Filter the solution through a glass microfibre filter (5.5) and use the filtrate for column chromatography according to 8.4. 8.3.2 Feeding stuffs with a furazolidone content of 2 500 mg/kg to 5
39、 000 mg/kg Weigh 5,0 g of the prepared test sample to the nearest 0,1 g in a 250 ml conical flask. Add 30,0 ml of water, mix and allow to stand for 5 min. Add 70,0 ml of extraction solvent (4.2), stopper and shake vigorously for 30 min on the rotary shaker (5.4). Filter the solution through a glass
40、microfibre filter (5.5) and use the filtrate for column chromatography according to 8.4. 8.3.3 Premixtures with a mass fraction of furazolidone of 0,5 % to 7 % 0,5 % (m/m) to 7 % (m/m) Weigh 1,0 g of the prepared test sample to the nearest 0,01 g in a 250 ml conical flask. Add 100,0 ml of extraction
41、 solvent (4.2), stopper and shake vigorously for 30 min on the rotary shaker (5.4). 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 between 5 g/ml and 10 g/ml. The dilution factor is f.
42、 Mix well and filter the solution using the filtration system (5.8). Use the filtrate for HPLC analysis according to 8.5. NOTE The required dilution factor (f) can be estimated by using the equation: f m w V e exp r = r where feis the estimated required dilution factor of the sample extract; mis the
43、 mass, in grams, of the test portion; wexpis the expected furazolidone content, in milligrams per kilogram, of the sample; rris the required furazolidone content, in micrograms per millilitre, of the final solution; Vis the total volume, in millilitres, of extraction solvent added to the test portio
44、n (see also note in 8.5.2). 8.3.4 Premixtures with a mass fraction of furazolidone of 7 % to 10 % 7 % (m/m) to 10 % (m/m) Weigh 1,0 g of the prepared test sample to the nearest 0,01 g in a 500 ml conical flask. Add 200,0 ml of extraction solvent (4.2), stopper and shake vigorously for 30 min on the
45、rotary shaker (5.4). Filter the solution through a glass microfibre filter (5.5). Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 07:04:27 MDTNo reproduction or networking permit
46、ted without license from IHS -,-,- ISOISO 14797:1999(E) 5 Dilute the fitrate with dilution solvent (4.3) to obtain a final solution with a furazolidone content between 5 g/ml and 10 g/ml. The dilution factor is f. Mix well and filter the solution using the filtration system (5.8). Use the filtrate f
47、or HPLC analysis according to 8.5. NOTE See the note in 8.3.3 for the calculation of the dilution factor. 8.3.5 Premixtures with a mass fraction of furazolidone of 10 % to 20 % 10 % (m/m) to 20 % (m/m) Weigh 0,5 g of the prepared test sample to the nearest 5 mg in a 500 ml conical flask. Add 200,0 m
48、l of extraction solvent (4.2), stopper and shake vigorously for 30 min on the rotary shaker (5.4). 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 between 5 g/ml and 10 g/ml. The dilution factor is f. Mix well and filter the solution using the filtration system (5.8). Use the filtrate for HPLC analysis according to 8.5. NOTE See the note in 8.3.3 for the calculation of the dilution factor. 8.4 Column