ISO 14718 pdf download.Animal feeding stuffs — Determination of aflatoxin B 1 content of mixed feeding stuffs — Method using high-performance liquid chromatography
1 Scope
This International Standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of aflatoxin B 1 content of animal feeding stuffs including those containing citrus pulp. The lower limit of determination is 1 mg/kg. NOTE 1 This International Standard may be applicable for the determination of the aflatoxin B 1 content of a number of raw materials and straight feeding stuffs such as corn gluten, groundnut, palm kernel, copra, citrus pulp, tapioca, soya bean, rice bran, pollard, rape seed, niger seed and cotton seed (see references [1 ] and [2]). These materials were, however, not included in the collaborative testing of the method. NOTE 2 This International Standard may also be applicable for the determination of the content of the sum of the aflatoxins B 1 , B 2 , G 1 and G 2 . However, the method has not been validated for this parameter by collaborative testing.
2 Normative reference
The following normative document contains provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subseqent amendments to, or revisions of, this publication do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the normative document indicated below. For undated references, the latest edition of the normative document referred to applies. Members of IEC and ISO editions maintain registers of currently valid International Standards. ISO 6498:1 998, Animal feeding stuffs — Preparation of test sample .
3 Principle
The sample is extracted with chloroform. The extract is filtered and an aliquot portion is purified on a Florisil® 1 ) cartridge and a C 1 8 cartridge. The final separation and determination is achieved by high-performance liquid chromatography (HPLC) using a reverse-phase C 1 8 column, followed by post-column derivatization with iodine or bromine, and fluorescence detection.
4 Reagents and materials
Use only reagents of recognized analytical grade.4.4 Control sample. Prepare a control sample of about 2 kg of compound feed with an aflatoxin B 1 content of about 5 mg/kg by combining samples of previous determinations with an aflatoxin B 1 content of about 5 mg/kg. Mix thoroughly. The aflatoxin B 1 content of the control sample should be determined five times by two analysts following the procedure described in clause 8. From the results the mean aflatoxin B 1 content, the standard deviation and the coefficient of variation should be calculated.4.11 Chloroform, stabilized with ethanol (mass fraction 0,5 % to 1 ,0 %). WARNING: Chloroform is a toxic substance. Avoid inhalation of and exposure to chloroform. Work in a fumehood when handling the solvent and solutions thereof. The adsorption characteristics of the Florisil® cartridge (4.6) may change if stabilizers other than ethanol are used. When chloroform as described is not available, the adsorption characteristics should be verified in accordance with clause 8.4.18.1 Mobile phase for HPLC with iodine derivatization. Combine 1 20 ml of acetonitrile (4.1 0), 21 0 ml of methanol (4.9) and 390 ml of water (4.1 ) and mix. Filter the eluent through a 0,45 mm PTFE membrane filter using the solvent filtration system (5.1 ) and degas for 1 0 min in the ultrasonic bath (5.2) before use. NOTE The composition of the mobile phase solvent may need adjustment depending on the characteristics of the HPLC column used. 4.18.2 Mobile phase for HPLC with bromine derivatization. Combine 400 ml of acetonitrile (4.1 0), 700 ml of methanol (4.9) and 1 300 ml of water (4.1 ) and mix. Add to the mixture 286 mg of potassium bromide (4.1 7) and 1 52 ml of concentrated nitric acid (4.1 6). Mix well and degas with a stream of inert gas for 1 5 min. 4.19 Saturated iodine solution for HPLC with iodine derivatization. Add 2 g of iodine to 400 ml of water. Mix for at least 90 min and filter through a 0,45 mm PTFE membrane filter (see 5.1 ). Prepare the solution fresh on the day of use. Protect the saturated solution from light to prevent photodegradation. 4.20 Sodium hypochlorite solution (household quality), r(active chlorine) = 1 00 g/l. 4.21 Sodium hypochlorite solution, volume fraction 1 %. Dilute 1 0 ml of sodium hypochlorite solution (4.20) with 990 ml of water-acetone mixture (4.1 4).