# 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), Division 5.3: Determination of total tryptophan (hydrolysate)

Extent: E+W+S+N.I.

Weigh to the nearest 0,2 mg from 0,1 to 1 g of the prepared sample (5.1) into the polypropylene flask (4.4). The weighed sample portion shall have a nitrogen content of about 10 mg. Add 8,4 g barium hydroxide octa-hydrate (3.4) and 10 ml water. 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 water. Put on the screw cap and close the flask loosely. Transfer to an autoclave (4.6) with boiling water and steam for 30-60 minutes. Close the autoclave and autoclave at 110 (± 2) oC for 20 hours.
Before opening the autoclave reduce the temperature to just under 100 oC. In order to avoid crystallisation of Ba(OH)2 · 8 H2O, add to the warm mixture 30 ml water which is at room temperature. Shake or stir gently. Add 2,0 ml concentrated internal standard (α-methyl-tryptophan) solution (3.16). Cool the vessels on water/ice bath for 15 minutes.
Then, add 5 ml ortho-phosphoric acid (3.14). Keep the vessel in the cooling bath and neutralise with HCl (3.11) whilst stirring and adjust the pH to 3,0 using HCl (3.12). Add sufficient methanol 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 to the defined volume necessary for the chromatography (for example 100 ml). The addition of methanol shall not cause precipitation.
Filter a few ml of the solution through a 0,45 μm membrane filter (4.5) before injection on the HPLC column. Proceed to the chromatography step according to paragraph 5.4.
Protect standard solution and hydrolysates against direct sunlight. If it is not possible to analyse the hydrolysates the same day, they may be stored at 5 oC for a maximum of 3 days.

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Source: https://www.legislation.gov.uk/eur/2009/152/annex/III/division/G/division/5/division/5.3 (the official version)
Page: https://openlegislation.uk/eu-regulation-2009-152/annex-III/division-G/division-5/division-5.3
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.