Enzyme activities may vary slightly from one batch to the other (Units per mg arementioned on
the label). Adapt theweight of enzyme inorder to reacha concentrationof 60U/mL
6U/mL.
Before using a new amyloglucosidase the enzyme must be evaluated in terms of baseline
interference and maltodextrin hydrolysis efficiency. The evaluation is performed by running a
blank with AMG only and a test with pure maltodextrin (AMG from Roche #11202367001 was
used for development of themethoddescribedhere).
E.
PreparationofTest Sample
Homogenize liquid laboratory samples immediatelybeforeanalysis. Powdered formulaor adult
nutritional products aregenerallyalreadyhomogenouspowders andmaybe sampleddirectly.
F.
Extraction
In a 100mL volumetric flask, weigh an amount of sample expected to contain between 100-150mg
of GOS. Add70 ± 5mLwater andplace the flask in awater bath at 70 °C ±5 °C for 20 to25minutes
under stirring. Cool the solution to room temperature before adjusting to the final volume with
water.
G.
Derivatization
Pipette 500 µL of test solution (section F) or maltotriose working standard solution (n) in a 2-mL
microtube then add to each sample and standard test, 200 µL of laminaritriose internal standard
working solution (p). Mix for a few seconds on a Vortex mixer. Transfer 20 µL of test solution
containing internal standard into 2-mL tubes (safe lock or screw cap) and add 200 µL of 2AB labeling
reagent (s) toeach tube.Mixona vortexmixer andplace the tubes inawater bath at 65 °C±1 °C for
2h±5min.Mix (Vortex) once after 20min incubation. After 2hmix the tubes (vortex) thenplace at
4 °C for at least 10min.
Witheach seriesof analyses, preparea reagent blankbyperforming the labelingprocedureon water
insteadof the sample solution.
H.
Removal ofmaltodextrin
Add 1.0mL of ammonium acetate buffer (0.1mol/L pH 5.5 (t)) to each tube after 2AB labeling and
mix (vortex). Transfer 0.5 mL of this solution to a 2-mL tube, add 200 µL of amyloglucosidase 60
U/mL (u), mix (vortex) the solution then incubate at 50 °C ± 1 °C for 30 min ±2 min. After 30 min
incubation,mix on a vortexmixer, cool to room temperature anddilutewith0.70mL of acetonitrile.
MixonaVortexmixer then centrifuge for 5minat 10000 x gbefore transferring1mL of supernatant
toan injectionvial.
IMPORTANT
: Do not treat themaltotriose standardwith the amyloglucosidase solution, addwater
insteadof amyloglucosidase solution.
GOS-01
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
