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BS EN ISO 13904:2016 pdf free

BS EN ISO 13904:2016 pdf free.Animal feeding stuffs – Determination of tryptophan content
For the determination of the total tryptophan, the sample is hydrolysed under alkaline conditions with saturated barium hydroxide solution and heated to 110℃ for 20 h. After hydrolysis, an internal standard is added.
For the determination of free try ptophan, the sample is extracted under mild acidic conditions in the presence of an internal standard. For commercial pure substances and premixtures containing more than 2 % of tryptophan, it is possible to add the internal standard after the extraction.
The tryptophan and the internal standard in the hydrolysate or in the extract are determined by reversed phase C18 HPLC with fluorescence detection.
For verification of the calibration standard solution, it is possible to use a control solution of tryptophan.
This control solution is prepared and analyzed as it is described for the calibration standard solution, but shall come from another manufacturer than the standard substance (3.2). The recovery of tryptophan in the control solution sample shall be between 99 % and 101 %.
Dissolve 3,00 g of acetic acid in 900 ml of water (3.1) and add 50,0 ml of 1,1,1-trichloro-2-methyl-2 -propanol solution (3.19). Adjust the pH to 5,00 using ethanolamine (3.18). Make up to 1 000 ml with water (3.11).
Shelf life of the mobile phase (especially stability of the mixture of acetic acid and ethanolamine) has to be checked by the retention times.
See also Annex B for an alternative mobile phase: The mixture of phosphate buffer and methanol is cheaper and harmless; pH adjustment is not necessary. The mixture is very stable.
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 (4.7).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)].BS EN ISO 13904 pdf free.

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