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5
PROCEDURE
5.1
Sample preparation for hydrolysis
SRM 1849a (NIST), reference infant formula 04-1426 (Bipea)
Weigh out an amount of sample corresponding to about 5-6 mg protein into a 10 mL glass tube with
screw cap. Complete to 100 mg with water.
Infant and adult/pediatric nutritional formula powder
Dilute 25 g of powder formula in 200 g water (225 g final). Transfer an amount of the diluted sample
corresponding to 5-6 mg of protein into a glass tube with screw cap (e.g. for products containing 15
% of protein, this corresponds to 350 to 400 µL (A in Table 7).
Preparation of sample hydrolysis solution
Add 3,3’-dithio-dipropionic acid (DDP), phenol and HCl to the tubes as described in Table 7.
10 pmol/µL of Nva (final concentration after derivatization) are added to the sample mixture.
Table 7: Preparation of sample hydrolysis solution
Solution
SRM 1846a &
Ref. IF. 04-1426
Infant/nutritional
formulas
Sample solution
100 mg
A µL
1 % DDP in 0.2 M NaOH
600 µL
600 µL
0.1
% phenol in 12 M HCl
2500 µL
2500 µL
10 mM Nva in 0.1 M HCl
200 µL
200 µL
0.2 M HCl
600 µL
600 µL
H2O
1000 µL
1100 – A* µL
*A: Volume of sample dissolved in water corresponding to 5 -6 mg protein.
Close the tubes with a screw cap and place them in an oven at 110°C for 24h.
5.2
Preparation of cystine calibration standards
Table 8 provides an example of how to prepare calibration standards for cystine containing 0 to 10
pmol/µL and 10 pmol/µL of Nva (final concentration after derivatization). Other concentrations can
be used; however a minimum of three different concentrations is required.
AMINO-01
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE