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Weigh 10 g (m) of the reconstituted sample in to a 50-mL (V) volumetric flask and adjust to the
volume with phosphate buffer (0.2 mol/L, pH 6.0). Depending on GOS concentration and
oligosaccharide profile the weight (m) can be adjusted between 0.5 – 15 g to optimise the
chromatography signals.
For analysis of ready-to-feed products
Into a 50-mL (V) volumetric flask, weigh 10 g (m) of liquid sample and add a magnetic stirring bar
and 20 mL of phosphate buffer (0.2 mol/L, pH 6.0.). Place the sample in a water bath at 70 °C for
20-25 min with constant stirring. Cool the solution to the room temperature, remove the magnetic
stirring bar and adjust to the final volume with phosphate buffer (0.2 mol/L, pH 6.0). Depending on
GOS concentration and oligosaccharide profile the weight (m) can be adjusted between 0.5 – 15 g
to optimise the chromatography signals.
For analysis of powder products without prior reconstitution:
Weigh 1.5 g (m) of powder in to a 50 mL (V) volumetric flask. Add a magnetic stirring bar and 35
mL of phosphate buffer (0.2 mol/L, pH 6.0.). Place the sample in a water bath at 70 °C for 20-25
min with constant stirring. Cool the solution to the room temperature, remove the magnetic
stirring bar, and adjust to the final volume with phosphate buffer (0.2 mol/L, pH 6.0). Depending
on GOS concentration and oligosaccharide profile the weight (m) can be adjusted between 0.2 – 3
g to optimise the chromatography signals.
Addition of Internal Standard
Transfer 1000 µL of diluted sample into a 2-mL microtube. Add 400 µL of laminaritriose (0.3
µmol/mL) and mix well. Take an aliquot of 500 µL and transfer to a microtube for the “non-treated
procedure”. Take a 2
nd
aliquot of 500 µL and transfer to a microtube for the “enzyme-treated
procedure”.
Non-treated Procedure
To the microtube containing 500 µL of sample solution add 25 µL of phosphate buffer (0.2 mol/L,
pH 6.0). Mix well and place in a water bath at 60°C for 60 min. Cool sample to room temperature
and continue with 2AB Labelling.
Enzyme-treated Procedure
To the microtube containing 500 µL of sample solution add 25 µL of β-galactosidase solution. Mix
well and place in a water bath at 60°C for 60 min. Cool sample to room temperature and continue
with 2AB Labelling.
2AB Labelling
Transfer 20 µL of sample to a 2-mL microtube. Add 200 µL of 2AB labelling reagent and mix well.
Place the sample in a water bath at 65 °C for 2 hours. After incubation, mix well then cool sample
at 4°C for 10 min.
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE