45.4.06A
AOACOfficial Method 997.08
Fructans inFoodProducts
IonExchangeChromatographicMethod
First Action 1997
Final Action1999
(Applicable to the determination of added fructans in processed
foods.)
See
Table
997.08A
for the results of the interlaboratory study
supporting acceptance of themethod.
A. Principle
Fructans are extracted from the product with boiling water.
Aliquot of extract is hydrolyzed using lyophilized
amyloglucosidase to remove starch. Part of that hydrosylate is
treatedwith inulinasefollowedbydeterminationofreleasedsugars.
Theinitialtestportionandfirstandsecondhydrosylatesareanalyzed
usinghighperformance anion exchange chromatographywithpulsed
amperometric detection (HPAEC–PAD). In sugar analysis 1, free
fructoseand sucrosearedetermined in initial extract. Insugaranalysis
2, sumof free glucose andglucose frommaltodextrins and starch are
determined in the firsthydrosylate. Insugaranalysis3, total amountof
glucoseand totalamountof fructose from thehydrosylateplusglucose
and fructose from the secondhydrosylate aredetermined.
Fructansarecalculatedfromconcentrationsofglucoseandfructose.
B. Apparatus
(
a
)
Anion exchange chromatograph (HPAEC)
.—Liquid
chromatograph gradient pump with eluent degas module,
micro-injection valve, pulsed electrochemical detector working in
pulsed amperometric detection mode (PAD), or equivalent, and
automated sampler.
HPAEC conditions: column temperature, 40
°±
0.5°C; flow rate,
1.0mL/min; injection volume, 50
m
L; detector sensitivity, analog
range 1–3
m
C.
See
Table
997.08B
for eluent gradient and
Table
997.08C
fordetector timeprogram. (
Note
:Varyparameters to
optimize chromatography.)
(
b
)
HPAEC column
.—250
´
4 mm id with pellicular
anion-exchange resinwith similar guard column (50
´
4mm id).
(
c
)
Data integrator
.—Carbopac PA1.
(
d
)
Analytical balance.
—Accuratelyweighing to0.1mg.
(
e
)
Membrane filters
.—0.2
m
mporosity.
(
f
)
Column oven
.—Maintaining 40
° ±
0.5°C.
(
g
)
Vacuum oven
.—Maintaining 55
° ±
3°C.
(
h
)
Reaction tubes
.—15mL, equippedwith screw caps.
(
i
)
pH meter
.—Temperature compensated, standardized with
pH 4.0, 7.0, and 9.0 buffer solutions.
(
j
)
Glassmicrofiber filters
.—Retaining0.7
m
mparticle size.
(
k
)
Filterholder
.—25mmdiameter, for lowpressure syringe, or
equivalent.
(
l
)
Syringes
.—Luer lock, 10mL, low pressure.
(
m
)
Desiccator
.—With SiO
2
, or equivalent desiccant. Every
2weeks dry desiccant overnight at 130°C.
(
n
)
Glass bottles
.—100 or 150mLwith screw caps.
(
o
)
Magnetic stirrers and stir bars.
(
p
)
Water baths
.—With shaker, maintaining 85
° ±
2°C and
60
° ±
2°C.
(
q
)
Volumetric flasks
.—100mL, and 1 and 2L.
(
r
)
Mortarwith pestle
.
(
s
)
Blender
.
(
t
)
Knife and scissors
.
(
u
)
Mixing rods
.
(
v
)
Vortexmixer.
(
w
)
Pasteur pipets
.—3 mL plastic pipets with large tip, or
equivalent.
C. Reagents
Forextraction,andformobilephaseandreagentspreparation,use
H
2
O,ASTMqualityType1,orACS(reagentgrade),orequivalent.
(
a
)
Acetate buffer
.—pH 4.5. Pipet 28.0 mL 0.2M acetic acid
(11.46mL/L) and 22.0mL0.2MNa acetate (28.23 g trihydrate/L)
into 100mLvolumetric flask. Dilute to volumewithH
2
O.
(
b
)
Hydrochloric acid solution
.—0.05M. Dilute stock solution
of known titer, e.g., 50mL1MHCl (86mL/L) to 1LwithH
2
O.
(
c
)
Potassium hydroxide solution
.—0.05M. Dilute stock
solution of known titer, e.g., 50mL1MKOH (56 g/L) to 1Lwith
H
2
O.
(
d
)
Lyophilized amyloglucosidase
.—Containing <0.02%
glucose, fructose, andsucrose.Storeat4
°
Cwhennot inuse. (Sigma
A7420, or equivalent.)
(
e
)
Inulinasesolution
.—Containing<0.005%glucose, fructose,
and sucrose (available as Fructanase-powder ref. nr. E-FRMXPD
and as Fructanase-liquid ref. nr. E-FRMXLQ from Megazyme
International Ireland Ltd., Bray Business Park, Bray, County
Wicklow, Ireland, Tel: +35312861220, Fax: +35312861264,
. Store at 4°C.
ã
2013AOAC INTERNATIONAL
Table 997.08A. Interlaboratorystudy results for determinationof fructans in foodand foodproductsby ionexchange
chromatography
Sample
a
Mean, g/100g
initial product
s
r
s
R
RSD
r
,% RSD
R
,% r
b
R
c
HorRat
Low-fat spread
8.1
0.23 0.39
2.9
4.8
0.65
1.10
1.65
Cheese spread
4.6
0.16 0.51
3.4
11.1
0.44
1.4
3.50
Chocolate
9.4
0.54 0.86
5.8
9.2
1.5
2.4
3.23
Wine gum
41.6
1.7
2.8
4.0
6.7
4.6
7.8
2.94
Powder drinkmix
15.5
0.68 0.72
4.4
4.7
1.9
2.0
1.78
Biscuits
12.2
0.55 0.94
4.5
7.7
1.5
2.6
2.81
a
Blind duplicates.
b
r = 2.8
´
s
r
.
c
R= 2.8
´
s
R
.
FOS-01
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
