Method Submissions-5th Set of Nutrients_6-30-14 - page 62

(dd) SPE elute solution - Into a 100mL flask, add 25mL of acetonitrile and 50 µL of
TFA.Makeup to themarkwithwater.
(ee)Fructose stock solution (1.0 mg/mL) –Weigh 100 mg of fructose(s)into a 100mL
volumetric flaskweigh, dissolveandmadeup to themarkwithwater.
(ff) Fructose standard solution (7.0µg /mL) – Pipette 700µL of the fructose stock
solution intoa100mL standard flaskandmadeup to themarkwithwater.
E. SamplePreparation
FOSExtraction –Accuratelyweighabout 1.0gof infant fromula intoa100mL standard flask
andadd70mLof hot (80°C)water. Heat at 80°C for 20minwith constant stirring. Allow
sample to cool to room temperatureandmakeup to themarkwithwater, this solution is
termed the “sample solution”. Takeanaliquot of the sample solutionand filter througha
folded filter paper. This is termed the “sample filtrate”.
Removal of interfering carbohydrates –Pipette200μLof sample filtrate intoaglass test tube.
Add0.2mLof theSucrase /α-amylase) / pullulanase /maltase solution (aa),vortex and
incubateat 40°C for 60min. Add0.2mLof the sodiumborohydride solution (x),vortex and
incubateat 40°C for 30min. After coolingexcessborohydride isdestroyedbyadding0.5mL
of 0.2Maceticacid(y).The resulting solution is termed “solutionA”
SPE cleanup- Thegraphitised carbonSPE column ispreparedbypassing through3 x400μL
ofwash solution (cc)followedby3 x400μLofwater. A400μLaliquot of solutionA is then
applied to theSPE columnandwashedbypassing through6 x600μLofwater. Fructans are
eluted in3 x400μLof elute solution (dd),collected ina2mLmicrocentrifuge tube, anddried
ina vacuum centrifugeat 40°C.
Fructanhydrolysis –Thedried fructans are resuspended in0.2mLof sodiumacetatebuffer
(v)and0.1mLof the fructansepreparatio n (bb)isadded. Themixture is incubatedat 40°C
for 20min. After cooling the sample is filtered thougha0.22μmmembrane filter andplaced
ina100μLglass insert fitting intoa suitableautosampler vial.
F. Determination
Mobilephase– fructose is eluted isocraticallyusing5.1mM sodiumhydroxide flowingat 1.0
mL/min for 45min, the column is thenwashed for10minwith300mM sodiumhydroxide
FOS-03
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE