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

ANNEX A Detailed method description

A‐1 Scope of the method

Determination of the fructan content in all forms of infant, adult and/or pediatric formula (powder,

ready‐to‐feed liquids, and liquid concentrates.

A‐2

Principle of the method

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

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 water/acetonitrile/TFA. Thereafter the eluted fructans

are hydrolyzed with fructanase into their monosaccharides glucose and fructose. The glucose and

fructose contents are determined with gradient elution High Performance Anion Exchange

Chromatography with Pulsed Amperometric Detection (HPAEC‐PAD).

A‐3

Apparatus

Metal free alkaline resistant anion exchange chromatograph with post column derivatization unit,

consisting of

‐ gradient pump

‐ autosampler with 20 μl injector

‐ column oven adjusted at 20°C

‐ CarboPak PA‐1 guard column 2 x 50 mm

‐ CarboPak PA‐1 analytical column 2 x 250 mm

‐ post column pump with pulse damping restriction

‐ mixing coil

‐ pulsed amperometric detector

‐ integration software

Analytical balance, accurately weighting to 0.1 mg

pH‐meter

Micro centrifuge tubes 2 ml

Centrifuge for 2 ml micro tubes

Waterbath at 80 ± 2°C

Waterbath at 40 ± 2°C

Ultraturrax

black band filter paper

Multipipet

Graphitized carbon solid phase extraction columns (GC‐SPE)

Vacuum manifold applicable for the GC‐SPE

Single –use syringes

Syringe filters 0.2 μl

Screw cap closed 15 ml polypropylene tubes

VALIDATION REPORT

FOR ERP USE ONLY

DO NOT DISTRIBUTE