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H
aselberger
&
J
acobs
:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
. 6, 2016
1583
8
H
aselberger & Jacobs
: J
ournal of
AOAC I
nternati nal
Vol. 99, No. 6, 2016
is WW. Allow to stir for 30 min, or until dissolved/uniformly
suspended.
(b)
Sample dilution.—
As with the qualitative analyses,
samples require different dilutions according to their individual
fructan content (Table
2016.06D
). Record all weights to
4 decimal places. This is SW
1
.
(c)
Removal of inherent glucose, fructose, and sucrose.—
Transfer 0.2 g ± 10%of the diluted sample from step
E(b)
or
K(b)
above, as appropriate (as per guidelines in Table
2016.06D
) to a
glass screw cap scintillation vial and record weight to 4 decimal
places. This is SW
2
. Add 200 µL of sucrase solution,
J(h)
, to
vial. Cap, swirl gently, and incubate at 40°C for 2 h (do not
use foil lined caps). After the sucrase incubation, add 700 µL
laboratory water to the scintillation vial. Then add 200 µL
sodium borohydride solution,
J(f)
. Cap, swirl, and incubate
at 40°C for 1 h. After the borohydride reduction is complete,
neutralize the excess reagent with 500 µL of 0.2 M acetic acid,
J(d)
. Swirl gently (leave uncapped to allow gas generated to
vent safely) and allow samples to sit at room temperature for
15 min.
Note
: Gas bubble formation after the addition of NaBH
4
,
but prior to addition of acetic acid, may be a sign of improper
sample pH and will negatively impact final results. If this is
observed, further investigation of sucrase buffer and/or 50 mM
NaOH solution is recommended to ensure the borohydride
reagent is sufficiently basic to remain stable.
(d)
Fructan hydrolysis.—
Add 100 µL glucoheptose IS
solution,
J(b)
, and 100 µL fructanase solution,
J(j)
. Cap,
swirl, and incubate at 40°C for 30 min. After the incubation is
completed, swirl gently to ensure a homogeneous sample and
filter through a 0.45 µm nylon syringe filter into autosampler
vials (prepared samples can be stored in vials at 2–10°C for
5 days).
L. Instrumental Analysis
(a)
Gradient.—
Fructose and glucoheptose are eluted
isocratically using 50 mM NaOH at 1.0 mL/min for 20 min.
The column is then washed for 10 min with 400 mM NaOH and
60 mM sodium acetate. Following the wash step, the column is
re-equilibrated with 50 mMNaOH for 15 min (Table
2016.06G
).
Column and detector compartment are maintained at 20°C. It
is recommended to clean the column and trap approximately
every five quantitative analytical sequences, with three
complete cycles of the conditions outlined in Table
2016.06H
.
[Five sequences would roughly equate to ~130 sample and/or
control injections. Failure to regularly clean the column/trap set
may result in breakthrough of borate to the analytical column
degrading method performance (primarily observed in the peak
asymmetry of the glucoheptose).]
(b)
Injection.—
Make a single 4 µL injection of each sample
test solution. At the start of each sequence, make at least six
equilibration injections to ensure that system is stable (note that
an observed calibration error in excess of 5% on average for a
level of standard may be indicative of insufficient equilibration
time), followed by the four levels of WS. Bracket samples with
WS after every 13 injections. Maintain autosampler at 10°C.
(c)
Electrochemical detector parameters.—
This method
utilizes the carbohydrate triple waveform per Table
2016.06F
.
Note
: This waveform is not appropriate for disposable gold
electrodes. The reference electrode is set to AgCl mode.
(d)
Retention times.—
Typically fructose elutes around
10–11 min and glucoheptose around 15–18 min (
see
Figures
2016.06D
and
E
).
M. Calculations
(a)
Fructose stock standard
.—Calculate the fructose stock
concentration according to:
Fructose concn, µg/mL = fructose wt. (g) × 1000000 µg/g × purity
500 mL
where purity = %purity [from the Certificate of Analysis
(CoA)/100%].
(b)
WS
.—Calculate WS concentrations (µg/mL) as follows:
WS4 = Fructose stock × 50/100
WS3 = Fructose stock × 25/100
WS2 = Fructose stock × 5/100
WS1 = Fructose stock × 5/200
(c)
Calibration
.—Obtain the peak areas for fructose and
Figure 2016.06D Working standard (WS4) chromatogram (Part II).
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22.0
24.0
26.0
28.0
30.0
32.0
34.0
36.0
38.0
40.0
42.0
45.0
-2.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
75.0 060216-FRUCTAN #10 [modifiedbyHASELPA]
WS4
ED_1
nC
min
1 - Fructose - 9.700
2 -Glucoheptose - 15.834
28