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Table 2-3
SPIFAN Fructan Spike Overview
Spike Level
50% 150% 50% 150%
Matrix
410457651Z 410457651Z 00859RF00 00859RF00
Fructan Type
SPIFAN HP SPIFAN HP Synergy 1
Synergy 1
Table 2-4
SPIFAN Fructan Overspike Overview
Matrix
Fructan Used to Spike
Child Formula Powder
SPIFAN scFOS
Toddler formula powder milk based
SPIFAN P95
Infant formula powder, milk based
SPIFAN P95
Child formula powder
SPIFAN scFOS
Infant formula powder FOS/GOS based
SPIFAN P95
Adult nutritional RTF high fat
SPIFAN scFOS
(50% and 100% levels made for each product)
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 sample is RTF, skip this step)
–
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-equilibrated with 50 mM NaOH for 15 minutes (see Figure 2-1 and Table 2-5 below). Column and
detector compartment are maintained at 20 °C.
It is recommended to clean the column and trap approximately every
five quantitative analytical sequences (method part 2) per the conditions outlined in Appendix C (5 sequences would
roughly equate to ~130 sample and/or control injections. Failure to regularly clean the column/trap set may result in
Fos-04 (February 2016)
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