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

2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method

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

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. c) Protease Solution - Weigh . c) Weigh an amount of Protease enzyme material to be diluted to the desired volume 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. 125 ± 0.010 grams to 25 mL with 1M Trizma buffer . Scale volume , 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 needed with proportional weight. Expiration: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 hours . at room temperature, but blanks and samples must be prepared with the same enzyme solution at the same time. (Note: One Sigma Unit is defined to hydrolyze casein to produce 1.0 micromole (181 µ g) of Tyrosine per minute at pH 7.5 and 37 deg. Sigma P5147 is approximately 4 Sigma units per mg solid material. To add Ten Units per sample preparation: 10/4 U/mg or 2.5 mg enzyme needed per 0.5mL addition to samples, or 50 mg/10 mL volume, 125 mg/25mL, and 250 mg/50mL. d) 5-Methyl-DL-tryptophan, Internal Standard Solution -.

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