Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance) (c. 152)

ANNEX IV U.K. METHODS OF ANALYSIS TO CONTROL THE LEVEL OF AUTHORISED ADDITIVES IN FEED

G. DETERMINATION OF LASALOCID SODIUM U.K.

3. Reagents U.K.

3.1. Potassium dihydrogen phosphate (KH₂PO₄). U.K.

3.2. Orthophosphoric acid, w (w/w) = 85 %. U.K.

3.3. Orthophosphoric acid solution, c = 20 %. U.K.

Dilute 23,5 ml of orthophosphoric acid (3.2) to 100 ml with water.

3.4. 6-Methyl-2-heptylamine (1,5-dimethylhexylamine), w (w/w) = 99 %. U.K.

3.5. Methanol, equivalent to HPLC grade. U.K.

3.6. Hydrochloric acid, density = 1,19 g/ml. U.K.

3.7. Phosphate buffer solution, c = 0,01 mol/l. U.K.

Dissolve 1,36 g of KH₂PO₄ (3.1) in 500 ml of water (3.11), add 3,5 ml of orthophosphoric acid (3.2) and 10,0 ml of 6-methyl-2-heptylamine (3.4). Adjust the pH to 4,0 with orthophosphoric acid solution (3.3) and dilute to 1 000 ml with water (3.11).

3.8. Acidified methanol. U.K.

Transfer 5,0 ml of hydrochloric acid (3.6) into a 1 000 ml graduated flask, make up to the mark with methanol (3.5) and mix. This solution must be prepared freshly before use.

3.9. HPLC mobile phase, phosphate buffer-methanol solution 5 + 95 (V + V). U.K.

Mix 5 ml of phosphate buffer solution (3.7) with 95 ml of methanol (3.5).

3.10. Lasalocid sodium standard substance with guaranteed purity, C₃₄H₅₃O₈Na (sodium salt of a polyether monocarboxylic acid produced by Streptomyces lasaliensis), E763. U.K.

3.10.1. Lasalocid sodium stock standard solution, 500 μg/ml U.K.

Weigh to the nearest 0,1 mg, 50 mg of lasalocid sodium (3.10) into a 100 ml graduated flask, dissolve in acidified methanol (3.8), make up to the mark with the same solvent and mix. This solution must be freshly prepared before use.

3.10.2. Lasalocid sodium intermediate standard solution, 50 μg/ml U.K.

Pipette 10,0 ml of stock standard solution (3.10.1) into a 100 ml graduated flask, make up to the mark with acidified methanol (3.8) and mix. This solution must be prepared freshly before use.

3.10.3. Calibration solutions U.K.

Into a series of 50 ml graduated flasks transfer 1,0, 2,0, 4,0, 5,0 and 10,0 ml of the intermediate standard solution (3.10.2). Make up to the mark with acidified methanol (3.8) and mix. These solutions correspond to 1,0, 2,0, 4,0, 5,0 and 10,0 μg of lasalocid sodium per ml respectively. These solutions must be prepared freshly before use.

3.11. Water, equivalent to HPLC grade. U.K.