SPIFAN Nutrients ERP Book_9-29-15

FOS-04 FOR ERP USE ONLY DO NOT DISTRIBUTE

Part 2 Quantitative Fructan Determination

Materials

Fructans:

scFOS  Ingredion Incorporated (Nutraflora) oligofructose  Beneo Orafti P95 and L95 oligofructose + inulin  Beneo Orafti Synergy 1

1. 2. 3.

Product Matrices

1. Powdered pediatric formula (milk based with oligofructose) 2. Powdered infant formula (soy based with scFOS) 3. Powdered adult product (milk based with Synergy 1) 4. Powdered infant formula (low lactose, milk based with GOS, used for spiking experiments) 5. Abbott internal custom control 2010_R (milk based with scFOS). 6. SPIFAN material  Placebo Child Formula Powder (00847RF00) 7. SPIFAN material  Toddler Formula Powder Milk-Based (4052755861) 8. SPIFAN material  Infant Formula Powder Milk Based (4044755861) 9. SPIFAN material  Child Formula Powder (00866RF00) 10. SPIFAN material  Infant Formula Powder FOS/GOS Based (50350017W1) 11. SPIFAN material  Adult Nutritional RTF High Fat (00729RF00)

Apparatus

1. LC system - Dionex ICS-3000 or 5000 consisting of a SP, DP, WPS-3000 (TB, P or S), and an electrochemical cell with a gold working electrode. Utilizing Chromeleon 6.80 or equivalent. 2. Analytical Column – Analytical column, Thermo CarboPac PA1, 250x4mm (p/n 035391); borate trap (placed instead of a guard column), Thermo p/n 047078. 3. Analytical Balance – readability to 0.01 mg recommended. 4. pH meter – readability to ±0.01. 5. Water bath, 40 ° C – Maintained at 40 ± 2 ° C. 6. Syringe filter – 0.45 µm nylon (Whatman p/n 6752-2504 or equivalent).

Reagents

Lab water - ASTM Type 1 water.

1. 2. 3.

50% w/w NaOH (Fisher p/n SS254-500). Sodium Acetate trihydrate (Sigma p/n 71188).

4. Glucoheptose (Sigma p/n G4375).  This reagent has been discontinued by Sigma. Potential alternate suppliers include: Spectrum Chemical, MPI, Krackeler, and Cedarlane Labs. 5. Fructose (Sigma p/n PHR 1002 or USP 1286504). 6. Sodium borohydride (Sigma p/n 452882 or 452173). 7. Maleic Acid (Sigma p/n M0375). 8. NaOH 1N (JT Baker p/n 5635-02). 9. Acetic Acid (Sigma p/n A6283). 10. Fructanase (Megazyme p/n E-FRMXPD). 11. Sucrase (Megazyme p/n E-SUCR). 12. 0.2 M Acetic acid solution – Transfer 2.9 mL acetic acid to a 250 mL flask. Dilute to volume with lab water. 13. 50 mM NaOH solution – Transfer 5 mL of 1N NaOH into a 100 mL flask. Bring to volume with lab water. 14. Alkaline Borohydride solution ~10 mg/mL – Immediately before use weigh 100 mg of sodium borohydride into a 15 mL tube. Dissolve in 5 mL of 50 mM NaOH. 15. 0.1M, pH 6.5, Sodium Maleate buffer – Weigh 2.9 ±1% of maleic acid in a 250 mL beaker. Dissolve in ~ 150 mL lab water. Adjust pH to 6.5 with 1N NaOH. Transfer to 250 mL flask; bring to volume with lab water. 16. Sucrase Solution – Measure 10 mL of maleate buffer in a graduated cylinder. Deliver to sucrase vial. Cap and swirl gently to dissolve. Divide into ~450 µL aliquots and store frozen at -20 ° C (each tube is enough for 2 samples) for up to a year. 17. Acetate Buffer – Combine 2.9 mL of glacial acetic acid with 450 mL of lab water. Adjust pH to 4.5 with 1N NaOH. Bring total volume to 500 mL with lab water. 18. Fructanase solution – Dissolve contents of fructanase vial in 22 mL of acetate buffer. Swirl gently to dissolve. Divide into ~1 mL aliquots and store frozen at -20 ° C for up to a year (each tube should contain enough for 9 samples).

Preparation of Standards and Spikes

1. Fructose stock solution 100 µg/mL – Weigh 0.05 ± 0.0025 g of fructose reference material and transfer to a 500 mL volumetric.

Page 6 of 14