ISO 14939 pdf download.Animal feeding stuffs — Determination of carbadox 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 carbadox content in premixtures and animal feeding stuffs. The method is applicable to animal feeding stuffs with a mass fraction of carbadox of 0,5 mg/kg (limit of quantification) to 1 00 mg/kg, and to premixtures with a mass fraction of carbadox up to 1 0 %. The lower limit of detection is 0,1 mg/kg. NOTE 1 For animal feeding stuffs the mass fraction of carbadox is expressed in milligrams per kilogram, and for premixtures as a percentage by mass. NOTE 2 Carbadox is a chemotherapeuticum belonging to the quinoxaline group. Carbadox is used as a growth-promoting feed additive for piglets.
2 Normative reference
The following normative document contains provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 6498:1 998, Animalfeeding stuffs — Preparation oftestsamples
3 Principle
Carbadox is extracted from the sample with a mixture of acetonitrile and methanol. Animal feeds are prewetted with water. The extract of animal feeds is purified through a short aluminium oxide column. The extract 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 of 365 nm (see references [1 ] to [3]). The presence of dimetridazole, nitrofurazone or sulfadimidine sodium can interfere with the determination of carbadox. Alternatively, carbadox may be determined after post-column derivatization with sodium hydroxide with detection at a wavelength of 420 nm.
4 Reagents
Use only reagents of recognized analytical grade. 4.1 Water, demineralized or deionized, with resistivity of at least 1 0 M? ? cm, or water of at least equivalent purity. 4.2 Extraction 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 ) (70:30 by volume). Mix 70 ml of extraction solvent (4.2) with 30 ml of water (4.1 ). 4.4 Acetic acid, volume fraction, w(CH 3 CO 2 H) = 1 0 %. Dilute 1 0 ml of glacial acetic acid to 1 00 ml with water. 4.5 Sodium acetate solution, c(C 2 H 3 NaO 2 ) = 0,01 mol/l, pH = 6,0. Weigh 0,82 g of water-free sodium acetate into a 1 000 ml one-mark volumetric flask. Dissolve in 700 ml of water. Adjust the pH to pH = 6,0 with acetic acid (4.4). Dilute to the mark with water and mix. 4.6 Mobile phase for HPLC. Combine 825 ml of sodium acetate solution (4.5) and 1 75 ml of acetonitrile and mix. Filter the eluent through a 0,22 µm filter using a solvent filtration system (5.2), and degas for 1 0 min in an ultrasonic bath (5.3) before use. 4.7 Carbadox standard material, 3-(2-quinoxalinyl methylene) carbazic acid methy ester N, ? N -dioxide (CAS number 6804-07-5). WARNING — Because of the sensitivity of carbadox to light, conduct all operations in the absence of daylight or artificial white light. Avoid inhalation of and exposure to the toxic carbadox standard material and solutions thereof. Work in a fume cupboard when handling the solvents and solutions. Wear safety glasses and protective clothing.4.8 Carbadox stock solution (approximately 1 00 µg/ml). Weigh 1 0 mg ? 1 mg of carbadox (4.7), to the nearest 0,1 mg, into a 1 00 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. Store in the dark at 0 °C to 8 °C.