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Single Lab Validation Report for GOS in Infant Formula and Adult Nutritionals
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Table 5.1: Preparation of sugar stock solutions
Internal standard
MW (mg/mMol) Conc (mg/1000 mL) Purity (%)
L(+)-arabinose
150.14
15
99
Sugar
MW (mg/mMol) Conc (mg/100 mL)
Purity (%)
D(+)-galactose
180.17
100
99
Lactose monohydrate 360.31
100
99
5.6.1.
Internal standard solution (15 µg/mL in 40 mM phosphate buffer pH 6.0):
Prepare the internal standard by weighing 15 ± 2 mg arabinose in a 200 mL beaker. Dissolve
the arabinose in about 100 mL Milli-Q water. Transfer analytically the arabinose stock
solution to a 1 L volumetric flask. Add 200 mL of the 0.2 M potassium phosphate buffer pH
6.0
(5.1)
and adjust to 1 L with Milli-Q water.
5.6.2.
Standard series:
Prepare the sugar stock solutions according to Table 5.1 by weighing 100 ± 5 mg lactose and
galactose to separate 100 mL volumetric flasks and adjust to 100 mL with the internal
standard solution
(5.6.1)
. The concentration of the sugar stock solution is 1000 µg/mL
(galactose or lactose) plus 15 µg/mL internal standard.
Prepare dilutions from the standard sugar stock solutions in 50 mL volumetric flasks
according to Table 5.2.
Table 5.2: Preparation of standards
Standard
Added volume stock (mL)
Concentration
galactose + lactose (µg/mL)
galactose
lactose
1
1.000
1.000
20
2
0.750
0.750
15
3
0.500
0.500
10
4
0.125
0.125
2.5
5
0.000
0.000
0.0
For each standard, add galactose and lactose stock to one volumetric flask. Adjust to 50 mL
with the diluted internal standard solution
(5.6.1)
. Due to addition of enzyme solution in the
sample (including arabinose) the standards need to be corrected by adding 25 µL buffer
solution to 400 µL standard (or by correcting for this in the calculation).
5.7.
Beta-galactosidase solution (400 U/mL):
•
Weigh 40 mg enzyme and dissolve in 1 mL with the diluted internal standard solution
(5.6.1)
.
•
Prepare fresh on daily basis.
5.8.
Carrez treated lactose standard 60 µg/mL:
Pipet 3 mL lactose stock (1 mg/mL)
(5.6.2.)
in a 50 mL volumetric flask. Add Milli-Q water
until ¾ of the flask is filled. Pipet 750 µL of Carrez 1
(5.2.)
and 750 µL of Carrez 2
(5.3.)
to the
flask and mix. Adjust the volume until 50 mL with Milli-Q water, homogenize and centrifuge
for 10 min at 13,000 rpm. Filtrate the supernatant through a 0.2 µm microfilter and transfer
to an HPLC vial. This standard is used to determine the fractionation time.
GOS-02 SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE