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Part 2
Quantitative Fructan Determination
Materials
Fructans:
1.
scFOS: Ingredion Incorporated Nutraflora (lots 7642339101 and SPIFAN 2 kit lots used)
2.
oligofructose: Beneo Orafti P95(lots PCRMA0AMA0 and SPIFAN 2 kit lots used)
3.
oligofructose + inulin: Beneo Orafti Synergy 1 (YAGKL7HAF8 lot used)
4.
Enriched inulin: Beneo Orafti HP (SPIFAN 2 kit lot used)
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
a)
Placebo Child Formula Powder (00847RF00)
b)
Toddler Formula Powder Milk-Based (4052755861)
c)
Infant Formula Powder Milk Based (4044755861)
d)
Child Formula Powder (00866RF00)
e)
Infant Formula Powder FOS/GOS Based (50350017W1)
f)
Adult Nutritional RTF High Fat (00729RF00)
g)
Infant formula powder partially hydrolyzed soy based (41457651Z), spiked with Beneo HP
h)
Adult Nutritional powder low fat (00859RF00), Spiked with Beneo Synergy 1
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 (p/n 079850) and combination pH Ag/AgCl reference electrode (p/n 061879). 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
1.
Lab water
- ASTM Type 1 water.
2.
50% w/w NaOH
(Fisher p/n SS254-500).
3.
Sodium Acetate trihydrate
(Sigma p/n 71188).
4.
Glucoheptose
(Sigma p/n G4375).
This reagent has been discontinued by Sigma. Potential alternate suppliers
include: 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)
Æ
NO SUBSTITUTIONS!
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
Fos-04 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE