ANNEX IIIU.K.METHODS OF ANALYSIS TO CONTROL THE COMPOSITION OF FEED MATERIALS AND COMPOUND FEED
F.DETERMINATION OF AMINO ACIDS (EXCEPT TRYPTOPHANE)U.K.
3.ReagentsU.K.
Double distilled water or water of equivalent quality must be used (conductivity < 10 μS).
3.1.Hydrogen peroxide, w (w/w) = 30 %.U.K.
3.2.Formic acid, w (w/w) = 98 %-100 %.U.K.
3.3.Phenol.U.K.
3.4.Sodium disulphite.U.K.
3.5.Sodium hydroxide.U.K.
3.6.5-Sulfosalicylic acid dihydrate.U.K.
3.7.Hydrochloric acid, density approximately 1,18 g/ml.U.K.
3.8.tri-Sodium citrate dihydrate.U.K.
3.9.2,2'-Thiodiethanol (thiodiglycol).U.K.
3.10.Sodium chloride.U.K.
3.11.Ninhydrin.U.K.
3.12.Light petroleum, boiling range 40-60 oC.U.K.
3.13.Norleucine, or other compound suitable for use as internal standard.U.K.
3.14.Nitrogen gas (< 10 ppm oxygen).U.K.
3.15.1-Octanol.U.K.
3.16.Amino acids.U.K.
3.16.1.Standard substances listed under paragraph 1. Pure compounds containing no water of crystallisation. Dry under vacuum over P2O5 or H2SO4 for 1 week prior to use.U.K.
3.16.2.Cysteic acid.U.K.
3.16.3.Methionine sulphone.U.K.
3.17.Sodium hydroxide solution, c = 7,5 mol/l:U.K.
Dissolve 300 g NaOH (3.5) in water and make up to 1 litre.
3.18.Sodium hydroxide solution, c = 1 mol/l:U.K.
Dissolve 40 g NaOH (3.5) in water and make up to 1 litre.
3.19.Formic acid — phenol solution:U.K.
Mix 889 g formic acid (3.2) with 111 g water and add 4,73 g phenol (3.3).
3.20.Hydrolysis mixture, c = 6 mol HCl/l containing 1 g phenol/l:U.K.
Add 1 g phenol (3.3) to 492 ml HCl (3.7) and make up to 1 litre with water.
3.21.Extraction mixture, c = 0,1 mol HCl/l containing 2 % thiodiglycol: Take 8,2 ml HCl (3.7), dilute with approximately 900 ml water, add 20 ml thiodiglycol (3.9) and make up to 1 litre with water, (do not mix 3.7 and 3.9 directly).U.K.
3.22.5-Sulfosalicylic acid, ß = 6 %:U.K.
Dissolve 60 g 5-sulfosalicylic acid (3.6) in water and make up to 1 l with water.
3.23.Oxidation mixture (Performic acid — phenol):U.K.
Mix 0,5 ml hydrogen peroxide (3.1) with 4,5 ml formic acid-phenol solution (3.19) in a small beaker. Incubate at 20-30 oC for 1 hour in order to form performic acid, then cool on an ice-water bath (15 min.) before adding to the sample.
Caution: Avoid contact with skin and wear protective clothing.
3.24.Citrate buffer, c = 0,2 mol Na+/l, pH 2,2:U.K.
Dissolve 19,61 g sodium citrate (3.8), 5 ml thiodiglycol (3.9), 1 g phenol (3.3) and 16,5 ml HCl (3.7) in approximately 800 ml water. Adjust pH to 2,2. Make up to 1 litre with water.
3.25.Elution buffers, prepared according to conditions for the analyser used (4.9).U.K.
3.26.Ninhydrin reagent, prepared according to conditions for the analyser used (4.9).U.K.
3.27.Standard solutions of amino acids. These solutions shall be stored below 5 oC.U.K.
3.27.1.Stock standard solution of amino acids (3.16.1).U.K.
c = 2,5 μmol/ml of each in hydrochloric acid.
May be obtained commercially.
3.27.2.Stock standard solution of cysteic acid and methionine sulphone, c = 1,25 μmol/ml.U.K.
Dissolve 0,2115 g cysteic acid (3.16.2) and 0,2265 g methionine sulphone (3.16.3) in citrate buffer (3.24) in a 1 litre graduated flask and make up to mark with citrate buffer. Store below 5 oC for not more than 12 months. This solution is not used if the stock standard solution (3.27.1) contains cysteic acid and methionine sulphone.
3.27.3.Stock standard solution of the internal standard e.g. norleucine, c = 20 μmol/ml.U.K.
Dissolve 0,656 g norleucine (3.13) in citrate buffer (3.24) in a graduated flask and make up to 250 ml with citrate buffer. Store below 5 oC for no more than 6 months.
3.27.4.Calibration solution of standard amino acids for use with hydrolysates, c = 5 nmol/50 μl of cysteic acid and methionine sulphone and c = 10 nmol/50 μl of the other amino acids. Dissolve 2,2 g sodium chloride (3.10) in 100 ml beaker with 30 ml citrate buffer (3.24). Add 4,0 ml stock standard solution of amino acids (3.27.1), 4,0 ml stock standard solution of cysteic acid and methionine sulphone (3.27.2) and 0,5 ml stock standard solution of internal standard (3.27.3) if used. Adjust pH to 2,2 with sodium hydroxide (3.18).U.K.
Transfer quantitatively to a 50 ml graduated flask and make up to the mark with citrate buffer (3.24) and mix.
Store below 5 oC for not more than 3 months.
See also observation 9.1.
3.27.5.Calibration solution of standard amino acids for use with hydrolysates prepared according to paragraph 5.3.3.1 and for use with extracts (5.2). The calibration solution is prepared according to 3.27.4 but omitting sodium chloride.U.K.
Store below 5 oC for not more than 3 months.