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 VIII U.K. METHODS OF ANALYSIS TO CONTROL ILLEGAL PRESENCE OF NO LONGER AUTHORISED ADDITIVES IN FEED

A. DETERMINATION OF METHYL BENZOQUATE U.K.

3. Reagents U.K.

3.1. Dichloromethane U.K.

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

3.3. HPLC mobile phase U.K.

Mixture of methanol (3.2) and water (equivalent to HPLC grade) 75 + 25 (v + v).

Filter through a 0,22 μm filter (4.5) and degas the solution (e.g. by ultrasonification for 10 minutes).

3.4. Methanesulfonic acid solution, c = 2 % U.K.

Dilute 20,0 ml methanesulfonic acid to 1 000 ml with methanol (3.2).

3.5. Hydrochloric acid solution, c = 10 % U.K.

Dilute 100 ml hydrochloric acid (ρ₂₀1,18 g/ml) to 1 000 ml with water.

3.6. Cation-exchange resin Amberlite CG-120 (Na), 100 to 200 mesh U.K.

The resin is pretreated before use. Slurry 100 g resin with 500 ml hydrochloric acid solution (3.5) and heat on a hot plate to boiling, stirring continuously. Allow to cool and decant off the acid. Filter through a filter paper under vacuum. Wash the resin twice with 500 ml portions of water and then with 250 ml of methanol (3.2). Rinse the resin with a further 250 ml portion of methanol and dry by passing air through the filter cake. Store the dried resin in a stoppered bottle.

3.7. Standard substance: pure methyl benzoquate (7-benzyloxy-6-butyl-3-methoxycarbonyl-4-quinolone) U.K.

3.7.1. Methyl benzoquate stock standard solution, 500 μg/ml U.K.

Weigh to the nearest 0,1 mg, 50 mg of standard substance (3.7), dissolve in methanesulfonic acid solution (3.4) in a 100 ml graduated flask, make up to the mark and mix.

3.7.2. Methyl benzoquate intermediate standard solution, 50 μg/ml U.K.

Transfer 5,0 ml of methyl benzoquate stock standard solution (3.7.1) into a 50 ml graduated flask, make up to the mark with methanol (3.2) and mix.

3.7.3. Calibration solutions U.K.

Transfer 1,0, 2,0, 3,0, 4,0 and 5,0 ml of methyl benzoquate intermediate standard solution (3.7.2) into a series of 25 ml graduated flasks. Make up to the mark with the mobile phase (3.3) and mix. These solutions have concentrations of 2,0, 4,0, 6,0, 8,0 and 10,0 μg/ml methyl benzoquate respectively. These solutions must be freshly prepared before use.