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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 8 / 35
2.2.4
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.
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.
VALIDATION REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE