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1578
H
aselberger
&
J
acobs
:
J
ournal of
AOAC I
nternational
V
ol
. 99, N
o
. 6, 2016
AOAC Official Method 2016.06
Fructans in Infant, Pediatric,
and Adult Nutritional Formulas
HPAEC/PAD
First Action 2016
[Applicable to the determination of the total fructan content
in infant, pediatric, and adult nutritional products (as well as
commodities) as defined in SMPR 2014.02 (1)].
See
Tables
2016.06A–C
for matrixes for which SLV data has
been generated, supporting acceptance of the method.
A. Principle
A two-part analysis is performed. Part I is a qualitative
classification of the fructan present in the sample. This
classification is based on rules related to presence/absence of
GF
3
(nystose) and/or GF
4
(fructofuranosylnystose), specifically,
and higher oligomers, generally. It groups the fructans into one
of three DP ranges and allows assignment of an appropriate
commodity factor for calculating the total fructan concentration
based on the amount of fructose released by enzymatic
hydrolysis of the fructans.
Part II is the quantitative determination of the fructan-derived
fructose, from which the total fructan in calculated. The sample
to be analyzed is weighed and diluted with laboratory water, as
appropriate. Part I of the analysis is then performed. Part II can
be run in parallel, whereby an aliquot of the diluted sample is
treated with sucrase to hydrolyze any sucrose present. Glucose
and fructose released by the sucrase treatment, as well as
inherent glucose and fructose, are then reduced to sugar alcohols
by the addition of sodium borohydride. Excess borohydride is
neutralized by the addition of acetic acid. Lastly, an aliquot of
internal standard (IS; glucoheptose) and an aliquot of fructanase
are added to the sample solution. After the fructanase hydrolysis
is completed, the samples are analyzed for fructose on HPAEC-
PAD instrumentation. The total fructan content is calculated
from the fructose, adjusted by the commodity factor,
CF,
determined by Part I (if not known).
Part I - Fructan Classification for Determination of
Commodity Factor
B. Apparatus and Materials
(a)
LC system
.—One biocompatible gradient pump;
biocompatible refrigerated autosampler capable of injecting
4 μL; electrochemical detector with gold electrode
capable of pulsed amperometric detection. For example,
Dionex ICS-3000 or 5000 (Dionex Corp., Sunnyvale,
CA) consisting of an SP, DP, WPS-3000 (TB; P or S type),
and an electrochemical cell with a conventional gold working
electrode and combination pH Ag/AgCl reference electrode or
equivalent.
(b)
Analytical column set.—
Analytical column, Thermo
CarboPac PA1, 250 × 4 mm PA1 guard column.
(c)
Analytical balance.—
Readable to 0.01 mg.
(d)
Syringe filter.—
0.45 µm nylon.
(e)
Volumetric flasks
.—
Glass, Class A, assorted sizes.
(f)
Eluent filtration apparatus with disposable membrane
filter
.—
0.2 μm polyethersulfone (PES).
(g)
Helium sparge
.—
Tubing and frit assembly.
Table 2016.06B. Total fructan single-laboratory validation data: Recovery from overspiked SPIFAN samples
50% Overspike
100% Overspike
Sample type
No. of
replicates
a
Spiking
material
b
Native fructan
level, g/100 g
c
Avg. % rec.
RSD, % Avg. % rec.
RSD, %
Child formula powder
6
scFOS
0.277
97.2
6.28
100
3.74
Toddler formula powder, milk based
6
Oligofructose
0.233
108
4.38
105
15.0
Infant formula powder, milk based
6
Oligofructose
0.283
101
8.53
98.7
4.82
Child formula powder
6
scFOS
0.277
102
4.78
96.0
5.75
Infant formula powder FOS and GOS
6
Oligofructose
0.036
97.4
11.1
97.6
7.91
Adult nutritional RTF high fat
6
scFOS
0.500
92.9
4.91
94.2
12.6
a
Duplicates on each of 3 days.
b
From SPIFAN kits. scFOS = Ingredion Nutraflora FOS powder. Oligofructose = Beneo Orafti P95 powder.
c
Mean from previous precision determination, duplicates on each of 6 days.
Table 2016.06C. Total fructan single-laboratory validation data: Recovery from spiked SPIFAN placebo samples
Sample type
No. of replicates
a
Spiking material
b
Spike level, g/100 g RTF Avg. % rec.
RSD, %
Infant formula powder partially hydrolyzed soy based
6
Inulin
0.120
97.4
4.25
Infant formula powder partially hydrolyzed soy based
6
Inulin
0.353
102
4.25
Adult nutritional powder low fat
6
Oligofructose/inulin
0.133
98.1
4.65
Adult nutritional powder low fat
6
Oligofructose/inulin
0.378
95.9
3.82
a
Duplicates on each of 3 days.
b
Inulin = Beneo Orafti HP powder (SPIFAN kit). Oligofructose/inulin = Beneo Orafti Synergy 1.
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