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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 0.125 ± 0.010 grams to 25 mL with Trizma buffer.
Scale volume as needed with proportional weight. Expiration: 6 hours.
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.
h)
Working Standard Solutions:
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)
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 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.
AMINO-02 (FEBRUARY 2017) - METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE
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