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Single Lab Validation of a Method for the Determination of β‐
Galactooligosaccharides in Infant Formula & Adult Nutritionals / Sean Austin (NRC,
Lausanne)
25 Jul 2016
CONFIDENTIAL
∙ This document may not be reproduced or disclosed to third parties without prior authorization
Page 7 / 35
NOTE: This method has been validated with β‐galactosidase from Megazyme (# E‐BGLAN). Other sources of β‐
galactosidase must be evaluated to ensure that there are no side activities on other oligosaccharides which may be
present in the sample (including the internal standard) and that under the conditions described complete hydrolysis of
the GOS is achieved (if a significant lactose peak is still apparent in the chromatogram this indicates that there may have
been insufficient β‐galactosidase activity).
Amyloglucosidase solution (60 U/mL in 0.1 mol/L ammonium acetate pH 5.5):
Weigh the amount of
amyloglucosidase corresponding to 180 Units ± 20 U and dissolve with 3.0 mL of ammonium acetate
buffer (0.1 mol/L pH 5.5).
NOTE: This method has been validated with amyloglucosidase from Roche (#11202367001). Other sources of
amyloglucosidase must be evaluated in terms of baseline interference and maltodextrin hydrolysis efficiency. The
evaluation is performed by running a blank with amyloglucosidase only, and a test with a pure maltodextrin in place of
the sample
.
LC Eluent B: Ammonium formate (0.1 mol/L, pH 4.4):
Add 4.6 g
0.1 g (3.78 mL) of formic acid
(100 %) in a beaker containing 800 mL of water. Adjust the pH to 4.40
0.05 with ammonium
hydroxide (25 %). Transfer quantitatively to a 1000‐mL volumetric flask, and dilute to the volume
with water.
2AB labelling reagent: 2AB (0.35 mol/L) ‐ NaBH
3
CN (1.0 mol/L) in DMSO ‐ acetic acid (30 %) solution.
Depending on the number of samples to be labelled, pipette the appropriate volume of DMSO and
acetic acid in to a 10‐mL glass tube (see Table 2 below for quantities). Mix the solution using a
vortex mixer. In a second 10‐mL glass tube weigh the amount of anthranilamide (2AB) and sodium
cyanoborohydride (NaBH
3
CN) indicated in Table 2 below, then add the corresponding volume of
30 % acetic acid in DMSO. Mix using a vortex mixer, and if necessary use an ultrasonic bath to
achieve complete dissolution of the solids (about 10 min).
Table 2: Examples of quantities for 2AB reagent preparation.
Max. number
of tests
30 % Acetic acid in DMSO
2AB (0.35 mol/L), NaBH
3
CN(1 mol/L)
in 30 % Acetic acid/DMSO
DMSO
(mL)
Acetic acid
100 % (mL)
30 % Acetic Acid
in DMSO (mL)
2AB
(mg)
NaBH
3
CN
(mg)
11
2.10
0.90
2.50
118
5
157
5
22
4.20
1.80
5.00
236
10
314
10
35
6.30
2.70
7.50
354
10
471
10
47
7.70
3.30
10.00
708
10
942
10
VALIDATION REPORT
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