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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 5 / 35
2.2. Method
2.2.1
Principle
Samples are reconstituted in water and further diluted until the concentration of GOS in solution is
appropriate such that the chromatographic signals will be within the range covered by the standard
curve. An aliquot of the sample solution is taken and an internal standard is added, this is then split
into two aliquots. One aliquot is treated with a β‐galactosidase the other is not. Then both aliquots
are labelled with a fluorescent tag, 2‐aminobenzamide (2AB). After labelling both aliquots are
treated with amyloglucosidase to remove maltodextrins, then they are analysed by hydrophilic
interaction liquid chromatography (HILIC) with fluorescence detection (FLD). Since it is the 2AB
label that is detected, each oligosaccharide has an equivalent molar response in the detector. It is
thus necessary to know the molecular weight of each signal in the chromatogram to convert the
molar quantities analysed to mass quantities. This can be done by coupling a mass spectrometer
(but once a GOS ingredient profile has been characterized by HILIC‐FLD‐MS, future samples can be
analysed without the MS).
2.2.2
Equipment
‐ 2‐mL microtubes safe lock or screw cap
‐ 5‐mL plastic tubes with caps
‐ Floating rack for microtubes
‐ Centrifuge for 2‐mL microtubes able to operate at 10000
g
‐ Water bath(s) for 2‐mL tubes able to operate at 65 °C and 50 °C ± 1.0 °C
‐ Vortex mixer
‐ Micropipettes with tips (0.1 to 1) mL (e.g. Eppendorf Multipipette plus)
‐ Analytical balance reading down to 0.1 mg (e.g. Mettler AT200)
‐ Ultrasonic bath
‐ UHPLC column: BEH‐Glycan (2.1 × 150 mm, 1.7 µm) or equivalent.
‐ UHPLC guard column: BEH‐Amide (2.1 × 5 mm, 1.7 µm) or equivalent
‐ Ultra high performance liquid chromatograph (UHPLC) equipped with the following modules:‐
‐ Gradient pump
‐ On‐line degasser
‐ Autosampler with a cooled sample compartment (8 ‐ 10°C)
‐ Temperature controlled column compartment
‐ Fluorescence detector
‐ Ultra High Pressure switching valve (2‐way 6‐port, 2‐way 10‐port, or column selection valve)
2.2.3
Chemicals & Reagents
‐ Dimethylsulfoxide, puriss p.a.
‐ Anthranilic acid amide, purum (2‐aminobenzamide, 2AB)
‐ Ammonium acetate, p.a.
‐ Sodium cyanoborohydride
‐ Formic acid, GR for analysis
‐ Acetic Acid anhydrous, GR for analysis
VALIDATION REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE