AOAC SPIFAN Nutrients ERP-Final Review (Jan. 2019)

2017.03 (JAN 2019) Amino-02-(ERP Review for Pubs) Tryptophan by enz hyd_Abbott Labs_Method_10172018

FOR ERP USE ONLY DO NOT DISTRIBUTE

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Jonathan Draher and Norman White Abbott Nutrition, Division of Abbott Laboratories

a) Methanol, HPLC grade b) Sodium Phosphate Monobasic, HPLC grade. c) Phosphoric Acid, (85%) d) Trizma Base e) Hydrochloric Acid, 6N.

f) Laboratory Water – minimum of 18 mega ohm conductivity g) 5-Methyl-DL-tryptophan Sigma Cat # M-0534, or equivalent. h) Protease from Streptomyces griseus : Type XIV (enzyme): Sigma Cat # P- 5147, or equivalent. 3. Solutions a) Hydrochloric Acid, 1.0N Solution. Aliquot 167 mL of 6N HCL (standardized) to a 1000 mL volumetric flask containing approximately 700 mL of laboratory water. Bring to volume with Laboratory Water. Storage: Room temperature. Expiration date: 2 years. b) Trizma Buffer, 0.1M, pH 8.5. Weigh 6.055 grams Trizma base, and transfer to a 400 mL beaker. Bring volume to 300 mL with Laboratory Water. While stirring, add 1.0N HCL drop wise until the pH of the solution reaches 8.5. Quantitatively transfer solution from beaker to a 500 mL volumetric flask and bring to volume with water. Storage: Room temperature. Expiration date: 6 months. such that 10 Sigma Units is delivered in 500 uL of solution. Transfer the appropriate weight of Protease into a volumetric flask of chosen volume according to amount needed for the assay (see below). Rinsing the weighing paper and the sides of the flask, add about 3/4 of volume with 0.1M Trizma buffer, pH 8.5. Swirl the solution briefly (avoid foaming), and allow it to stand for about 5 minutes until all the powder dissolves. Bring to volume mark with 0.1M Trizma buffer, pH 8.5. Make solution homogeneous by GENTLY inverting flask several times. DO NOT SHAKE, as this will cause foaming to occur. Alternate preparations: Prepare to 50, 25, or 10 mL volume as is adequate for each run at 0.5 mL per sample preparation. The Protease solution is stable for 6 c) Protease Solution. Weigh an amount of Protease enzyme material to be diluted to the desired volume

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