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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/Paediatric Nutritional Formula
Following Enzymatic Hydrolysis
Author:
Abbott Nutrition
Summary of Method:
Internal standard, 5-methyl-DL-tryptophan, and protease enzyme solution, (containing at
least 10 proteolytic enzymes), are added to weighed samples diluted in trisma buffer. Duplicate blanks are
included with each analysis set. The samples set is incubated overnight at 50
O
C, cooled and made up to 50 mLs
with methanol/water. Tryptophan is quantitated on a C-8 RP column on an isocratic HPLC system using a 4 point
ISTD calibration with FLD detection, (Excitation 295nm, Emission 345nm).
Method Scope/Applicability:
This method is applicable to the determination of total tryptophan in infant
formula and adult/pediatric nutritional formulas.
The LOQ of a typical ready to feed formula is 0.18 mg/100g and the analytical range of the method is 0.18 – 300
mg/100g.
General comments about the method:
Method is clearly written but needs more detail; part numbers or at minimum, CAS numbers, safety section and
more detail in the sample preparation section re sample weight
Method Clarity:
A 25 g in 200 g water should have been performed as per SLV protocol to ensure homogeneity. Possible two
weighing options, one for homogeneous and one for non-homogenous products.
The following calculation could be used so that analyst is clear on the sample weight to use:
w = 1000 * 200
P m
W
= amount of sample to be weighed
1000 = factor to convert from g to mg
P
= amount of protein in the sample (g/100g)
M
= Sample wt (~ 25g)
200
= Wt of dilution water
Method is for total tryptophan but free tryptophan is mentioned in the method also!
Recommend to add chromatograms to method – can use ones from SLV report.
Method Safety Concerns: