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HPLC Determination of Total Tryptophan in Infant Formula and
Adult/Pediatric Nutritional Formula Following Enzymatic
Hydrolysis
Abbott Nutrition, Division of Abbott Laboratories
page
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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
1.0 mL
0.5 mL
MID
5.0 mL
0.5 mL
HIGH
10.0 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.
Amino-02
FOR ERP USE ONLY
DO NOT DISTRIBUTE