SPIFAN Nutrients ERP Book_9-29-15

FOS-04 FOR ERP USE ONLY DO NOT DISTRIBUTE

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

Fructan Spikes

4.

Table 2-2 Fructan Spike Overview

Spike Level

0.03%

0.3%

0.3%

0.3%

3%

3%

3%

Lab water and 9% sucrose*

Product #4

Product #4

Product #4

Product #4

Product #4

Product #4

Matrix

Oligofructose and Inulin, Beneo Orafti Synergy 1

Oligofructose and Inulin, Beneo Orafti Synergy 1

Oligofructose, Beneo Orafti P95

Oligofructose, Beneo Orafti P95

scFOS, Ingredion

scFOS, Ingredion

scFOS, Ingredion

Fructan Type

*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-

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