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FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
1
Expert Review Panel for Infant Formula and Adult Nutrition
Evaluation of Method AMINO-02
Title:
HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional
Formula Following Enzymatic Hydrolysis
Author:
Abbott Nutrition, Division of Abbott Laboratories
Summary of Method:
Sample preparation consists of adding a weighed sample, the enzyme solution, internal standard (5-
methyl-DL-tryptophan) and Trizma buffer into a tube. The preparation is mixed and incubated at 50°C
for sixteen hours (overnight), to assure complete hydrolysis of all sample types. After hydrolysis, the
sample-enzyme mixture is diluted to 50mL with methanol/water and filtered.
The sample is injected onto a C-8 column with reference standards and enzyme blank preparations
and the Tryptophan and 5-Methyl-DL-tryptophan are detected by native fluorescence at an
excitation
wavelength of 295 nm and emission wavelength of 345 nm. Tryptophan is quantified by internal standard
calibration mode using reference standard to internal standard amount and response ratios.
Method Scope/Applicability:
This HPLC method allows for the determination of total tryptophan in infant, pediatric, and adult nutritional
products.
General comments about the method:
The method is clear, simple and easy to follow.
Method Safety Concerns:
No specific concerns on safety.
Pros/Strengths
Method ruggedness were established run the samples on two HPLC systems, use of two different
stationary phase batches, precisions runs prepared by two analysts. The results obtained show the
ruggedness of the method.
Supporting Data