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Bring to volume with lab water. Store frozen at -20
°
C for up to 11 months.
2.
Glucoheptose Internal Standard –
Weigh 0.1 ± 0.005g of glucoheptose and transfer to a 100 mL volumetric. Bring to volume with
lab water.
It is recommended to portion out aliquots of this solution to increase stability.
Store frozen at -20
°
C for up to 6
months.
3.
Working standards –
Store frozen at -20
°
C for up to 6 months.
Table 2-1
Working Standard Preparation
Fructose (µg/mL)
WS4 = 50 mL stock + 5 mL IS→ 100 mL
50
WS3 = 25 mL stock +5 mL IS→ 100 mL
25
WS2 = 5 mL stock +5 mL IS→ 100 mL
5
WS1 = 5 mL Stock + 10 mL IS → 200 mL
2.5
To prepare WS:
1. Add fructose stock solution to each flask according to the procedure above
2. Add IS solution to each flask
3. Dilute to volume with lab water
4.
Fructan Spikes
Table 2-2
Fructan Spike Overview
Spike Level
0.03%
0.3%
0.3%
0.3%
3%
3%
3%
Matrix
Lab water and
9% sucrose*
Product #4
Product #4
Product #4
Product #4
Product #4
Product #4
Fructan Type
scFOS,
Ingredion
scFOS,
Ingredion
Oligofructose,
Beneo Orafti
P95
Oligofructose
and Inulin,
Beneo Orafti
Synergy 1
scFOS,
Ingredion
Oligofructose,
Beneo Orafti
P95
Oligofructose
and Inulin,
Beneo Orafti
Synergy 1
*No additional matrix used in this spike to very precisely control the amount of sucrose in the samples.
Sample Preparation
1.
If running in parallel with Part 1 above an aliquot from the diluted solution can be used in this method. If so skip to step 4.
below. Otherwise proceed to step 2.
2.
Powder sample reconstitution (if needed) –
Accurately weight 5.0 g ± 10% into a 100 mL beaker (record
powder weight)
. Tare
and deliver 40 g of lab water to beaker (record
water weight
). Allow to stir for 30 minutes, or until dissolved.
3.
Sample dilution –
As with the qualitative analyses, samples require different dilutions according to their individual fructan
content, again following the guidance in Table 1-1 (in Part 1). Record all weights to four decimal places. This aliquot can be
shared with the qualitative ID methodology in Appendix 1.
4.
Removal of inherent glucose, fructose, and sucrose -
Transfer 0.2 g ± 10% of the diluted sample from step 1 or 3 above, as
appropriate (as per sample table guidelines) to a glass screw cap scintillation vial (record weight to 4 decimal places, SW
2
). Add
200 µL of sucrase solution (reagent 16) to vial. Cap, swirl gently, and incubate at 40
°
C for 2 hours (do not use foil lined caps).
After the sucrase incubation, add 700 µL lab water to scintillation vial. Then add 200 µL of the sodium borohydride solution
(reagent 14). Cap, swirl, and incubate at 40
°
C for 1 hour. After the borohydride reduction is complete, neutralize the excess
reagent with 500 µL of 0.2 M acetic acid (reagent 12). Swirl gently (leave uncapped to allow gas generated to vent safely) and
allow samples to sit at room temperature for 15 minutes.
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 is recommended.
5.
Fructan hydrolysis –
Add 100 µL of glucoheptose internal standard solution (Standards 2) and 100 µL of fructanase solution
(reagent 18). Cap, swirl, and incubate at 40
°
C for 30 minutes. 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)
.
Instrument Conditions
1.
Gradient –
Fructose and glucoheptose are eluted isocratically using 50 mM NaOH at 1.0 mL/min for 20 minutes. The column is
then washed for 10 minutes with 400 mM NaOH and 60 mM sodium acetate. Following the wash step, the column is re-
FOS-04
FOR ERP USE ONLY
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