AOAC OMB Final Action Recommendation (December 2019)-2016.14

FOS-03 FOR WORKING GROUP/ERP USE ONLY DO NOT DISTRIBUTE

(dd)

SPE elute solution - Into a 100 mL flask, add 25 mL of acetonitrile and 50 µL of

TFA. Make up to the mark with water.

(ee)Fructose stock solution (1.0 mg/mL) – Weigh 100 mg of fructose (s) into a 100 mL

volumetric flask weigh, dissolve and made up to the mark with water.

(ff) Fructose standard solution (7.0 µg /mL) – Pipette 700 µL of the fructose stock

solution into a 100mL standard flask and made up to the mark with water.

E. Sample Preparation

FOS Extraction – Accurately weigh about 1.0 g of infant fromula into a 100 mL standard flask

and add 70 mL of hot (80°C) water. Heat at 80°C for 20 min with constant stirring. Allow

sample to cool to room temperature and make up to the mark with water, this solution is

termed the “sample solution”. Take an aliquot of the sample solution and filter through a

folded filter paper. This is termed the “sample filtrate”.

Removal of interfering carbohydrates – Pipette 200 μL of sample filtrate into a glass test tube.

Add 0.2 mL of the Sucrase / α-amylase) / pullulanase / maltase solution (aa), vortex and

incubate at 40°C for 60 min. Add 0.2 mL of the sodium borohydride solution (x), vortex and

incubate at 40°C for 30min. After cooling excess borohydride is destroyed by adding 0.5 mL

of 0.2 M acetic acid (y). The resulting solution is termed “solution A”

SPE cleanup - The graphitised carbon SPE column is prepared by passing through 3 x 400 μL

of wash solution (cc) followed by 3 x 400 μL of water. A 400 μL aliquot of solution A is then

applied to the SPE column and washed by passing through 6 x 600 μL of water. Fructans are

eluted in 3 x 400μL of elute solution (dd) ,collected in a 2 mL microcentrifuge tube, and dried

in a vacuum centrifuge at 40°C.

Fructan hydrolysis – The dried fructans are resuspended in 0.2 mL of sodium acetate buffer

(v) and 0.1 mL of the fructanse preparation (bb) is added. The mixture is incubated at 40°C

for 20min. After cooling the sample is filtered though a 0.22 μm membrane filter and placed

in a 100 μL glass insert fitting into a suitable autosampler vial.

F. Determination

Mobile phase– fructose is eluted isocratically using 5.1 mM sodium hydroxide flowing at 1.0

mL/min for 45 min, the column is then washed for 10 min with 300 mM sodium hydroxide