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Dual Lab Validation of a Method for the Determination of Fructans in Infant
Formula & Adult Nutritionals /
25 Jul 2016
48
A‐4
Chemicals
Water, Millipore Q quality
Acetonitril, HPLC‐grade
Maleic acid, purity ≥ 99%
Acetic acid glacial, 100% anhydrous
Trifluoroacetic acid
Hydrochloric acid (36.5 – 38%)
Sodium hydroxide 33 % m/m
Sodium hydroxide 50 % m/m
Potassium cyanohexaferrate(II)trihydrate
Zinc acetate
Sodium chloride
Sodium azide
Mix of pure enzymes sucrase + β‐amylase + pullulanase + maltase from Megazyme fructan assay kit (K‐
FRUC)
D‐(+)‐glucose, purity ≥ 99%
D‐(+)‐ fructose purity ≥ 99%
Chitobiose
A‐5
Reagents
Sodium azide 0.5%,
dissolve 1 g sodium azide in 200 ml demineralized water
Liquid chromatographic eluent A
,
1M sodium acetate solution
, dissolve in the eluent bottle 136 ± 1 g
sodium acetate trihydrate in 1000 ml carbonate free Millipore‐Q water and purge directly with helium.
Keep the eluent continuously under a blanket of helium
Liquid chromatographic eluent B, 0.2 M Sodium hydroxide solution
, weight 3846 ± 5 g carbonate free
Millipore‐Q water in the eluent bottle and purge with helium for at least 15 min. Pipet carefully 40 ml
50% sodium hydroxide in the water in the flask and purge with helium for another 15 min. Keep the
eluent continuously under a blanket of helium.
Liquid chromatographic eluent C
,
Millipore Q water with sodium azide
, fill a 4 l eluent bottle with 3900
ml carbonate free Millipore‐Q water. Add with a graduated glass 100 ml 0.5% sodium azide solution
and purge with helium for 15 min. Keep the eluent continuously under a blanket of helium.
Post column eluent
,
0.3M sodium hydroxide solution
, liquid chromatographic eluent, weight 3762 ± 5 g
carbonate free Millipore‐Q water in the eluent bottle and purge with helium for at least 15 min. Pipet
carefully 60 ml 50% sodium hydroxide in the water in the flask and purge with helium for another 15
min. Keep the eluent continuously under a blanket of helium.
Sodium maleate buffer, 0.1M pH = 6.5
: dissolve In a 1000 ml volumetric flask 11.6 maleic acid in 900
ml water. Adjust to pH = 6.5 ± 0.05 with 33 % sodium hydroxide solution. Make up with demineralized
water to the mark and homogenize.
1M sodium hydroxide:
dissolve 40 ± 1 g in 500 ml water in a 1000 ml volumetric flask. After cooling
down to room temperature, make up with demineralized water to the mark and homogenize.
Sodium acetate buffer, 0.1M pH = 4.5,
dissolve in a1000 ml volumetric flask 5,8 ml glacial acetic acid in
900 ml water. Adjust to pH = 4.5 ± 0.05 with 1M sodium hydroxide solution. Make up with
demineralized water to the mark and homogenize.
VALIDATION REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE