Commission Regulation (EC) No 152/2009Show full title

3. Reagents

3.1. Methanol.

3.2. Acidified methanol.

Transfer 4,0 ml hydrochloric acid (ρ20 = 1,18 g/ml) into a 500 ml graduated flask, make up to the mark with methanol (3.1) and mix. This solution shall be freshly prepared before use.

3.3. Acetonitrile, equivalent to HPLC grade.

3.4. Molecular sieve.

Type 3A, 8 to 12 mesh beads (1,6-2,5 mm beads, crystalline alumino-silicate, diameter of pores 0,3 mm).

3.5. Aluminium oxide acidic activity grade I for column chromatography.

Transfer 100 g aluminium oxide into a suitable container and add 2,0 ml of water. Stopper and shake for approximately 20 minutes. Store in a well stoppered container.

3.6. Potassium dihydrogen phosphate solution, c = 0,025 mol/l.

Dissolve 3,4 g of potassium dihydrogen phosphate in water (HPLC grade) in a 1 000 ml graduated flask, make up to the mark and mix.

3.7. Di-sodium hydrogen phosphate solution, c = 0,025 mol/l.

Dissolve 3,55 g of anhydrous (or 4,45 g of dihydrate or 8,95 g of dodecahydrate) di-sodium hydrogen phosphate in water (equivalent to HPLC grade) in a 1 litre graduated flask, make up to the mark and mix.

3.8. HPLC mobile phase.

Mix together the following reagents:

• 650 ml acetonitrile (3.3),

• 250 ml water (equivalent to HPLC-grade),

• 50 ml potassium di-hydrogen phosphate solution (3.6),

• 50 ml di-sodium hydrogen phosphate solution (3.7).

Filter through a 0,22 μm filter (4.6) and degas the solution, (e.g. by ultrasonification for 10 minutes).

3.9. Standard substance.

Pure robenidine: 1,3-bis [(4-chlorobenzylidene)amino]guanidine — hydrochloride.

3.9.1. Robenidine stock standard solution: 300 μg/ml

Weigh to the nearest 0,1 mg, 30 mg of robenidine standard substance (3.9). Dissolve in acidified methanol (3.2) in a 100 ml graduated flask, make up to the mark with the same solvent and mix. Wrap the flask with aluminium foil and store in a dark place.

3.9.2. Robenidine intermediate standard solution: 12 μg/ml

Transfer 10,0 ml of the stock standard solution (3.9.1) into a 250 ml graduated flask, make up to the mark with the mobile phase (3.8) and mix. Wrap the flask with aluminium foil and store in a dark place.

3.9.3. Calibration solutions

Into a series of 50 ml calibrated flasks, transfer 5,0, 10,0, 15,0, 20,0 and 25,0 ml of the intermediate standard solution (3.9.2). Make up to the mark with mobile phase (3.8) and mix. These solutions correspond to 1,2, 2,4, 3,6, 4,8 and 6,0 μg/ml of robenidine respectively. These solutions must be freshly prepared before use.

3.10. Water equivalent to HPLC grade.