24 / 258
H
aselberger
&
J
acobs
:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
. 6, 2016
1579
C. Reagents
(a)
Laboratory water.—
ASTM Type 1.
(b)
NaOH solution
.—50%, w/w
(Fisher Part No. SS254-500
or equivalent). If an alternate vendor is selected, it is imperative
that the carbonate level be equivalent to or less than Fisher
(≤0.10%).
(c)
Sodium acetate trihydrate.—
Reagent grade (Sigma Part
No. 71188 or equivalent).
(d)
Helium gas.—
Ultra-high purity (UHP).
(e)
Fructan commodities.—
For use as retention time (RT)
standards
.
(1) Short-chain Fructooligosaccharides (scFOS,
derived from sucrose).
—Used to identify GF
3
and GF
4
(which will be the second and third/last major peaks present).
This commodity is a representative of a category 1 fructan.
Alternately one may use discrete GF
3
and GF
4
reagents (e.g.,
Wako Part No. 295-73401).
(2) Beneo Orafti HP Inulin.—
Used to identify category 3
type fructans.
D. Preparation of Standards and Solutions
(a)
Fructan RT marker standards.—
Weigh 0.4 ± 0.004 g
of each fructan reference material, scFOS (or ~0.050 g each
of individual GF
3
and GF
4
standards) and Beneo Orafti HP.
Transfer to a 1000 mL volumetric flask. Bring to volume with
laboratory water and mix to ensure complete dissolution.
Transfer ~1 mL of each to autosampler vials. Store at –20°C for
up to 6 months.
(b)
Mobile phase A.—
Deliver laboratory water to an
acceptable container and sparge with UHP helium for 10 min.
Store under ~3–5 psi blanket of helium on the instrument.
Expiration is 30 days at room temperature.
(c)
Mobile phase B.—
Deliver 1000 mL laboratory water to
an acceptable container and sparge with UHP helium for 10 min.
Add 40 ± 0.1 g of 50% (w/w) NaOH and continue to sparge for
2 additional min. Store under ~3–5 psi blanket of helium on the
instrument. Expiration is 30 days at room temperature.
(d)
Mobile phase C.—
Deliver 1000 mL laboratory water
to an acceptable container and sparge with UHP helium for
10 min. Add 40.8 ± 0.1 g sodium acetate trihydrate and continue
to sparge for 5 additional min (or until dissolved). Then filter
the solution through the membrane filter. Store under ~3–5 psi
blanket of helium on the instrument. Expiration is 14 days at
room temperature.
E. Sample Preparation
(a)
Powder sample
.—Reconstitution (if needed). Accurately
weigh 5.0 ± 0.025 g into a 100 mL plastic beaker [record powder
weight (PW)]. Tare and deliver 40 ± 0.2 g laboratory water to
beaker [record water weight (WW)]. Allow to stir for 30 min,
or until dissolved.
(b)
Sample
dilution.—
Samples
require
differing
dilutions according to their individual fructan content as per
Table
2016.06D
. For example, a liquid ready-to-drink (RTD)
sample containing 0.5% fructan would be diluted at a rate of 7 g
to 200 mL. Record all weights to 4 decimal places. This solution
can be used also with the quantitative fructan methodology in
Part II. This is SW
1
.
(c)
Filtration.—
After bringing the samples to volume, filter
through a 0.45 µm nylon syringe filter into an autosampler vial
(prepared samples can be stored in vials at 2–10°C for 5 days).
F. Instrumental Analysis
(a)
Gradient.—
Fructans are eluted using a gradient of NaOH
and sodium acetate at 1.0 mL/min as per Table
2016.06E
.
Column and detector compartment are maintained at 20°C.
Note:
A CarboPac PA1 guard is used for this procedure rather
than the borate trap used in Part II for quantitative fructan
determination. Therefore, if same day analysis is desired, two
separate HPAEC-PAD systems are needed.
The gradient program recommended in Table
2016.06E
has
Table 2016.06D. Sample size guidelines
Sample type
Fructan level
(as-is), % SW
1
, g
Volumetric flask
size, mL
a
Powder product
0.27–1.0
10 g ± 10%
50
1.1–10
7 g ± 10%
200
11–45
4 g ± 10%
500
RTF product
b
0.03–0.20 10 g ± 10%
50
0.21–1.0
7 g ± 10%
200
1.1–5.0
4 g ± 10%
500
Commodity
45–100
0.4 g ± 10%
1000
a
For the sake of procedural simplification, the density of this solution
is treated as 1 g/mL, so that the weight of the diluted solution in
grams can be assumed equal to the flask volume (in mL). This
incurs a <0.3% error, which is inconsequential for a result reported to
3 significant digits.
b
For concentrated liquid (CL) products dilute 10 mL of product with
10 mL of laboratory water and then treat the diluted material per RTF
guidelines.
Table 2016.06E. Qualitative ID (Part I) gradient
Time, min
Flow, mL/min
A (laboratory water), % B (500 mM NaOH), %
C (300 mM NaOAc), % Curve
0
1.0
67.0
8.0
25.0
NA
0
1.0
67.0
8.0
25.0
5
40.0
1.0
0.0
8.0
92.0
5
45.0
1.0
0.0
8.0
92.0
5
45.1
1.0
67.0
8.0
25.0
5
55.0
1.0
67.0
8.0
25.0
5
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