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Determination of β-Galactooligosaccharides in Infant
Formula & Adult Nutritionals
Sean Austin, Denis Cuany, Catherine Murset-Mounoud
Nestlé Research Centre, Vers-Chez-Les-Blanc, 1000 Lausanne, Switzerland
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. 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)
3. CHEMICALS & REAGENTS
- Dimethylsulfoxide, puriss p.a.
- Anthranilic acid amide, purum (2-aminobenzamide, 2AB)
- Ammonium acetate, p.a.
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE