SPIFAN Nutrients ERP Book_9-29-15

Amino-02 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

c) Protease Solution - Weigh 0.200 ± 0.010 grams to 50 mL with Trizma buffer. Expiration: 6 hours.

d) 5-Methyl-DL-tryptophan, Internal Standard Solution - 0.0400 ± 0.0020 grams to 50 mL with water.

e) Tryptophan Standard Solution - (0.0100 ± 0.0010 grams per 100 mL) Store frozen in vials. Expiration: 6 months.

h) Working Standard Solutions:

Combine the appropriate aliquot of tryptophan and internal standard stock solutions in separate 50 mL volumetric flasks for three working levels.

Standard

TRP Stock Sol.

Int. Std. Stock

LOW MID HIGH

1.0 mL 5.0 mL 10.0 mL

0.5 mL 0.5 mL 0.5 mL

Add 12.5 mL of methanol to each of the three 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: (20:80, Methanol/0.05 M Phosphate Buffer)

h) Column Rinse Solution: (25% methanol in water)

F. 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 directly into tarred centrifuge tubes using a disposable transfer pipette. For powder samples weigh into a tarred tube with a spatula. Record weights.

d) Add 0.5 mL (500  L) of protease enzyme solution to all samples.

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