BS EN ISO 13904:2016 pdf download – Animal feeding stuffs — Determination of tryptophan content
4.9 Glassware – filters.
4.10 Graduated Erlen meyers flasks: 200 ml, 250 ml, 600 ml.
4.11 Volumetric: 100 ml, 500 ml, 1 000 ml (all class A).
5 Procedure
5.1 Preparation of samples
5.1.1 Feeding stuffs
Grind the sample to pass through a 0,5 mm sieve. Samples high in moisture shall be either air-dried at a temperature not exceeding 50。C or freeze-dried prior to grinding. Samples with high fat content shall be extracted with light petroleum (3.9) prior to grinding.
5.1.2 Commercial pure substances and premixtures containing more than 2 % of tryptophan
Grind the sample to pass through to a 0,25 mm sieve and homogenize it well.
5.2 Determination of free tryptophan (extract)
5.2.1 Feeding stuffs
Weigh, to the nearest 1 mg, an appropriate amount (1 g to 5 g) of the prepared sample (5.1.1) into a conical flask. Add 100,0 ml of hydrochloric acid, (3.13) and 5,00 ml of concentrated internal standard solution (3.16)]. Shake or mix for 60 min using a mechanical shaker or a magnetic stirrer (4.Z).Allow the sediment to settle and pipette 10,0 ml of the supernatant solution into a beaker. Add 5 ml of orthophosphoric acid (3.14). Adjust the pH to 3,0 using sodium hydroxide (3.10]. Add sufficient methanol (3.8) to give a concentration of between 10 % and 30 % of methanol in the final volume.Transfer to a volumetric flask of appropriate volume and dilute with water (3.1) to a volume necessary for the chromatography [approximately, the same volume as the calibration standard solution (3.17.1]. Filter a few millilitres of the solution through a 0,45 μm or 0,22 μm membrane filter (4.5) before injection on the HPLC column. Proceed to the chromatography step according to 5.4.Protect the standard solution and extracts against direct sunlight. If it is not possible to analyze the hydrolysates the same day, they may be stored at 5。C for a maximum of three days.
5.2.2 Commercial pure substances and premixtures containing more than 2 % of tryptophan
Weigh to the nearest of 0,1 mg, 0,5 g to 5 g of well homogenized sample (5.1.2), depending on the expected concentration of tryptophan in the sample (for example, see Annex C) into a 1 000 ml volumetric flask.
Fill up to volume with 0,1 mol/l hydrochloric acid (3.13).
The mixture is stirred during 30 min on a mechanical shaker or a magnetic stirrer (4.7). Allow the particles to settle.
Transfer an aliquot of 2 ml of clear solution into a 100 ml volumetric flask. Add 2 ml of the concentrated internal standard (3.16). Fill up to the mark with 0,1 mol/l hydrochloric acid (3.13). This diluted test solution should have a tryptophan concentration as close as possible as the tryptophan concentration in the calibration standard solution (3.17.2) and the internal standard concentration shall be similar (c = 0,010 8 g/l) to the one of the calibration standard solution (c = 0,010 g/l, 3.17.2). Refer to Annex A for example of samples preparation. Filter a few millilitres of the solution through a 0,45 μm or 0,22 μm membrane filter (4.5) before injection on the HPLC column. Proceed to the chromatography step according to 5.4. Protect the standard solution and extracts against direct sunlight. If it is not possible to do the analyses the same day, then the extracts shall be stored below 5 °C for not more than three days.
5.3 Determination of total tryptophan (hydrolysates) Weigh, to the nearest 0,2 mg, from 0,1 g to 1 g of the prepared sample (5.1.1) into the polypropylene flask (4.4). The weighed test portion should have a nitrogen content of about 10 mg. Add 8,4 g of barium hydroxide octahydrate (3.4) and 10 ml of water (3.1). Mix on a vortex mixer (4.8) or magnetic stirrer (4.7). Leave the Teflon-coated magnet in the mixture. Wash down the walls of the vessel with 4 ml of water (3.1). Put on the screw cap and close the flask loosely.
Transfer to an autoclave (4.6) which contains boiling water and steam for 30 min to 60 min. Close the autoclave and autoclave at (110 ± 2) °C for 20 h. Before opening the autoclave, reduce the temperature to just under 100 °C. In order to avoid crystallization of Ba(OH) 2 8H 2 O, add to the warm mixture 30 ml of water (3.1) which is at room temperature. Shake or stir gently. Add 2,00 ml of concentrated internal standard solution (α-methyltryptophan) (3.16). Cool the vessel in a water/ice bath for 15 min.BS EN ISO 13904 pdf download.