SPIFAN Nutrients ERP Book_9-29-15

FOS-04 FOR ERP USE ONLY DO NOT DISTRIBUTE

FOS-04 Determination of Fructans in Infant and Adult/Pediatric Nutritional Formula as well as ingredient commodities High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection

Principle

The basic principle of this method is as follows. A two part analysis is performed. Part 1 is the qualitative determination of the type of fructan present in the sample, if not already known. Part 2 is the quantitative determination of fructan in the sample. The sample to be analyzed is weighed and diluted with lab water, as appropriate. Part 1 of the analysis is then performed. Part 2 can be run in parallel, whereby an aliquot of the diluted product is treated with sucrase to hydrolyze any sucrose present. Glucose and fructose released by the enzymatic hydrolysis, as well as inherent glucose and fructose, are then reduced to sugar alcohols by the addition of sodium borohydride. Excess borohydride is neutralized by the addition of acetic acid. Lastly an aliquot of internal standard (glucoheptose) and an aliquot of fructanase are added to the sample solution. After the fructanase hydrolysis is completed the samples are analyzed for fructose on HPAEC-PAD instrumentation. The total fructan content is calculated from the peak areas, adjusted by a commodity factor ( CF, determined by part 1 )..

Part 1 Qualitative Fructan ID

Materials

Fructans:

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

1. 2. 3.

enriched inulin  Beneo Orafti HP

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)  Spiked with inulin (HP) 5. SPIFAN material  Placebo Child Formula Powder (00847RF00) 6. SPIFAN material  Toddler Formula Powder Milk-Based (4052755861) 7. SPIFAN material  Infant Formula Powder Milk Based (4044755861) 8. SPIFAN material  Child Formula Powder (00866RF00) 9. SPIFAN material  Infant Formula Powder FOS/GOS Based (50350017W1)

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 Set – Analytical column, Thermo CarboPac PA1, 250x4mm (p/n 035391); PA1 guard column (Thermo p/n 043096). 3. Analytical Balance – readability to 0.01 mg recommended. 4. 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).

Preparation of Standards and Spikes

1. Fructan RT marker standards – Weigh 0.4 ± 0.004 g of each fructan reference material (one each of scFOS, Synergy 1, and HP) and transfer to a 1000 mL volumetric. Bring to volume with lab water. Transfer to autosampler vials. Store at -20 ° C for up to 6 months.