178 / 220
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 (se
e Table 1below 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 i
n Table 1below, 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 1: 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
4.
SAMPLE PREPARATION
For analysis of products on a ready to feed basis:
Reconstitute powder or liquid concentrates according to instructions (for SPIFAN samples powder
samples (25 g) were weighed in to a bottle and water (200 g) was added. For SRM 1849a, the
powder (10 g) was weighed in to a bottle and water (100 g) was added). The mixture was placed in
a water bath at 70°C with constant agitation for 20-25 min then cooled to room temperature.
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE