DeterminationofGalactooligosaccharides in Infant Formulaand
AdultNutritionals
High-Performance LiquidChromatographyafter 2-Aminobenzamide
Derivatization
SeanAustinandThierryBénet
NestléResearchCentre, Vers-Chez-Les-Blanc, POBox44, CH-1000 Lausanne26, Switzerland
Note:
1.
Themethoddescribedanddatapresentedarealreadypublished in Austin, S., et.al (2014)
Int. J.Anal. Chem.
2014
: Article ID768406. (dx.doi.org/ 10.1155/2014/768406)
A.
ApplicabilityStatement
Thismethod is suitable for thedeterminationof galactooligosaccharidesonly in theabsenceof other
reducingoligosaccharides. Exceptions aremade formaltooligosaccharidesormaltodextrinswhich
canbe removedenzymatically, and for reducingoligosaccharideswithaketoseat the reducingend
(e.g. partiallyhydrolysed inulin)whichwill not bederivatisedunder theexperimental conditions. It
hasbeenapplied to infant formula, but hasnot yet been validated for adult nutritional products.
B.
Principle
Oligosaccharides (OS) present in samples are extracted in water at 70 °C. An internal standard
(laminaritriose) is added to the extracted OS and they are fluorescently labeled with 2-
aminobenzamide (2AB, also known as 2-anthranilic acid amide ) via formation of a Schiff base. The
double bondof the Schiff base is reducedusing sodium cyanoborohydride to give a stable derivative
(Figure1).
Figure 1: Reaction scheme for 2AB labeling of oligosaccharides
If products containmaltooligosaccharides, these are removed by hydrolysis with amyloglucosidase
before proceeding to the chromatographic step. Labeled extracts are dilutedwith acetonitrile prior
to injection on an HPLC system equippedwith HILIC trapping and analytical columns. The samples
O
H
H H
H
OH
OH
H
OH
OH
OH
OH
H
H H
H
OH
OH
H
OH
O
OH
OH
H
H H
H
OH
OH
H
OH
N
OH
O NH
2
O
NH
2
NH
2
OH
H
H H
H
OH
OH
H OH
OH
NH
O NH
2
H
NaBH
3
CN
Schiff base
GOS-01
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
