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

Put the required test portion weight, accurately weighted at 0.1 mg, in an erlemeyer flask and disperse

in 40 ml demineralized water. In case of lumps, use an ultraturrax to disperse to sample.

Check the pH and, if necessary, adjust the pH within the range 5.0 – 9.0 with respectively 1M

hydrochloric acid or 1M sodium hydroxide solutiom.

Heat the sample solutions during 20 min. in a waterbath adjusted to 80 ± 2 °C

Cool down to room temperature in a cold water or icebath.

Transfer the sample quantitatively in a 100 ml volumetric flask. Make up with demineralized water to

the mark and homogenize.

Filter the sample solution over black band filter paper.

Diluted the filtered sample solution with demineralized water according to the dilution scheme in

table 2

Table 2

Dilution scheme

Expected fructan

content (%)

Dilution factor

ml prepared sample

solution

ml demineralized

water

0 ‐ 5

5 ‐ 10

1.00

10 – 20

1.00

4.00

20 ‐ 100

0.25

4.75

Pipet 0.2 ml of the according table 2 diluted sample solution in a 2 ml micro centrifuge tube

Add 0.10 ml chitobiose internal standard solution

Add 0.20 ml enzyme mix A and homogenize

Heat the obtained solution in the micro centrifuge tubes during 90 min. in a waterbath adjusted to 40

± 2 °C

Add 10 μl Carrez I solution and homogenize

Add 10 μl Carrez II solution and homogenize

Centrifuge the prepared samples in the micro centrifuge tubes at 14000 rpm during 10 min..

Optional determination of the free sugars

Mix 400 μl of the centrfugated sample with 400 μl 0.1M sodium acetate buffer pH = 4.5.

Filter the mixture with a 0.2 μm syringe filter in a chromatographic vial and close the vial with a screw

cap.

The sample is now ready for the chromatographic analysis of the free sugars.

A‐7 GC‐SPE removal of monosaccharides

A‐7‐a Actrivation of the GC‐SPE columns

Mount the required number of GC‐SPE column on the vacuum manifold.

Flush the GC‐SPE 4 times with 400 μl 80% acetonitrile + 0.1% TFA followed by three times flushing with

400 μl demineralized water

NOTE Perform all flushing steps at gravity conditions (without applying vacuum or positive pressure)

and take care that at each flushing step the entire flushing solution passes the GS‐SPE

VALIDATION REPORT

FOR ERP USE ONLY

DO NOT DISTRIBUTE