AOAC SPIFAN Review Team (August 2016)

Dual Lab Validation of a Method for the Determination of Fructans in Infant

Formula & Adult Nutritionals /

25 Jul 2016

Introduction

Presently 2 different official AOAC methods are available for the determination of the total fructan

content in food products, being AOAC 997.08 [1] and AOAC 999.03 [2]. Both analytical methods for

inulin/FOS have typical different fields of application in the analysis of (food) samples. The different

underlying principles of both analytical methods are the reason that there is not one ‘golden standard’

analytical method for the fructan determination. It is necessary to choose the best method for

different matrices.

The principles and the pro’s and contra’s of both fructan analysis methods are described below.

In the

AOAC 997.03 method

[1] first the free fructose and free sucrose content are quantified. In the

next step after enzymatic conversion of starch and maltodextrins, the total glucose is measured. The

last step includes total conversion of inulin/FOS and sucrose into glucose and fructose and the

quantitative analysis of the glucose and fructose after these enzymatic conversions. After three high

performance anion exchange chromatographic (HPAEC‐PAD) measurements the fructan content is

calculated by subtracting the glucose, sucrose and fructose contents as measured in step 1 and 2 from

the total fructose and glucose content as measured in step 3.This implies that large corrections have

to be made for samples containing large quantities of fructose, glucose, sucrose, maltodextrin and/or

starch. Subtraction of two large values in order to calculate much lower inulin/FOS values generally

results in less precise data with a large standard deviation.

The principle of the

AOAC 999.03 method

[2] differs from AOAC 997.08 in that here all

monosaccharides present after the combined amylases and sucrase treatment are removed by

converting them into alditols (borohydride reduction). So, also samples with high contents of

monosaccharides, sucrose, maltodextrin and/or starch can be accurately measured in this procedure

because subtractions are not needed. However, there is a major drawback in this method since also

the reducing end groups of the oligo‐fructose molecules F

are reduced into alditol end groups and so

these end groups escape the analysis. This implies that recovery of difructose (F

) is only 50%, for F

%, for F

75% etcetera. GF

molecules do not have a reducing end group, so these are recovered

completely. FOS material prepared by depolymerization of inulin contains in general high amounts of

and F

, so total recovery will be about 80% or even lower for these preparations.

In the new developed method a solution has been found for both the drawback of the AOAC 997.08

method (determination by difference) and the AOAC 999.03 method (reduction of end‐standing

fructose unit in fructo‐oligosaccharides). Following the idea of Cuany etal [3] a graphitized carbon solid

phase extraction (GC‐SPE) column is used to eliminate the interfering monosaccharides glucose and

fructose before the enzymatic hydrolysis of the fructans.

Principle analytical method

The investigated fructan analysis protocol is based on an aqueous extraction of the fructan

constituents, followed by enzymatic hydrolysis of possibly present maltodextrins/malto‐

oligosaccharides and free sucrose. Graphitized Carbon Solid Phase Extraction (GC‐SPE) columns are

applied to concentrate the fructan constituents and to remove the free monosaccharides. The free

monosaccharides are removed from the GC‐SPE column by washing with aqueous sodium chloride

solution. The concentrated fructan constituents are eluted from the GC‐SPE with

VALIDATION REPORT

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