2017.03 Amino Acids (Tryptophan) - Amino-02

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 Abbott Nutrition, Division of Abbott Laboratories d) 5-Methyl-DL-tryptophan, Internal Standard Solution - 0.0400 ± 0.0020 grams to 50 mL with water. Initially add ~25 mL water and stir with small stir bar. Due to slow dissolution, may stir overnight in a refrigerator. Store frozen. Expiration: 6 months e) Tryptophan Stock Standard Solution - (0.0100 ± 0.0010 grams per 100 mL) Store frozen. Expiration: 6 months. Combine the appropriate aliquot of tryptophan and internal standard stock solutions in separate 50 mL volumetric flasks for four working levels. Standard TRP Stock Sol. Int. Std. Stock VERY LOW 0.20 mL 0.5 mL LOW 1.0 mL 0.5 mL MID 5.0 mL 0.5 mL HIGH 10.0 mL 0.5 mL Add 12.0 mL of methanol to each of the four flasks containing the standard/internal standard mixtures, and bring to volume with water. Transfer diluted standards to vials for frozen storage. Expiration: 6 months. g) Mobile Phase: (18:82, Methanol/0.05 M Phosphate Buffer, pH 2.3) h) Column Rinse Solution: (25% methanol in water) PROCEDURE 1. Sample Preparation a) Set heating unit to 50°C prior to experiment. b) Using an analytical balance, tare a 50 mL centrifuge tube. c) Weigh liquid samples and reconstituted powders directly into tarred centrifuge tubes using a disposable transfer pipette. For non-reconstituted powder samples weigh into a tarred tube with a spatula. Record weights. d) Add 0.5 mL (500 µ L) of protease enzyme solution to all samples. F.E. h) Working Standard Solutions:

Page

4