AOAC SPIFAN Review Team (August 2016)

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Maltodextrin Hydrolysis
To each tube add 1 mL of ammonium acetate buffer and mix well. Transfer 500 µL of this solution
to a 2-mL microtube and add 200 µL of AMG solution (60 U/mL). Mix well and incubate in a water
bath at 50°C for 30 min. After incubation, cool sample to room temperature and prepare sample
for chromatography.
Preparation for Chromatography
Add 700 µL of acetonitrile to the sample or standard and mix well. Centrifuge this solution at
10000 × g for 5 min, then transfer 1 mL of the supernatant to a vial suitable for the LC
autosampler.
5. PREPARATION OF STANDARD CURVE
Prepare the solutions for the 6-level standard curve by diluting the maltotriose stock solution (4
µmol/mL) as described in the following table:
Table 2: Dilution Scheme for the Preparation of the Standard Curve
Calibration Curve Dilutions in PHOSPHATE BUFFER (0.2 mol/L, pH 6.0)
Carbohydrate # Std
Maltotriose
Stock volume
Final Volume
Standard
Concentration
[µL] [mL] [µmol/mL]
Maltotriose
Level #1 50 20 0.0100
Level #2 250 20 0.0500
Level #3 750 20 0.1500
Level #4 1120 10 0.4480
Level #5 1750 10 0.7000
Level #6 2250 10 0.9000
Preparation Procedure for Standards:
For each standard, transfer 1000 µL of solution to a 2-mL microtube, add 400 µL of laminaritriose
solution (0.3 µmol/mL) then take a 500 µL aliquot and follow the non-treated procedure but stop
before the maltodextrin hydrolysis. Follow the maltodextrin hydrolysis procedure but add 200 µL
of ammonium acetate buffer (0.1 mol/L, pH 5.5) instead of AMG solution (i.e. no hydrolysis should
occur). Then continue with the preparation for chromatography.
6. CHROMATOGRAPHIC CONDITIONS
Analyse the prepared samples and standards on a UHPLC system equipped with a FLD under the
following conditions:
FLD: Excitation λ: 330 nm Emission λ: 420 nm
Trap Column: Acquity BEH Amide (2.1 mm x 5 mm; 1.7 µm) or equivalent (see for
plumbing)
Column: Acquity UPLC BEH Glycan (2.1 mm x 150 mm; 1.7 µm) or equivalent
Column temperature: 25°C ± 1 °C
Injection volume: 3 µL
Eluent A: Acetonitrile
Eluent B: Ammonium formate 0.1 mol/L, pH 4.4
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE