Page 13 - Application Notebook - Solution for Food Development

Application No.L507
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Q HPLC Analysis of Low Molecular Weight Soluble Table 1 HPLC Analytical Conditions
Dietary Fiber System : Prominence-i, RID-20A
The two beverages were prepared according to the Column : Shim-pack SPR-Na (250 mm L. × 7.8 mm I.D., 8 μm) × 2
Guard Column : Shim-pack SPR-Na (50 mm L. × 7.8 mm I.D., 8 μm)
procedure shown in Fig. 1, and analyzed by HPLC. The Mobile Phase : Water
results of HPLC analysis are shown in Fig. 2 and 3 Flowrate : 0.50 mL/min
alongside the results of HPLC analysis of maltotriose. Column Temp. : 80 ˚C
Injection Volume : 20 μL
Analytical conditions are shown in Table 1. The
Detection : RID-20A
saccharides detected were divided into monosaccharides,
disaccharides, and trisaccharides or larger on the
chromatograms, with trisaccharides or larger being μRIU
regarded as the dietary fiber fraction. The published Dietary fiber
1)
analytical method states to analyze the trisaccharide
maltotriose under the same conditions as the sample, 75
then to include all peaks that elute equal to or earlier
than maltotriose in the dietary fiber fraction. The
1)
published analytical method states to use either a ligand 50 Glucose
exchange column or gel filtration column for HPLC.
Analysis was performed using a Shim-pack SPR-Na ligand 25
exchange column. Ligand exchange columns are mainly
Drink A
used in carbohydrate analysis, and are excellent at Maltotriose
separating carbohydrates. Ligand exchange columns can 0
easily separate glucose from other carbohydrates, and 0 5 10 15 20 25 30 35 min
are therefore suited to analyses using glucose as an
internal reference standard as shown below. Fig. 2 Analysis of Low Molecular Weight Soluble
Dietary Fiber in Beverage A
The actual quantification analysis was performed using
glucose produced by hydrolysis of starch as an internal μRIU
reference standard. The amount of glucose in each 200 Dietary fiber
sample was calculated by a separate pyranose oxidase Glucose
method, and the amount of low molecular weight 150
soluble dietary fiber was obtained by multiplying the
peak area ratio of the dietary fiber fraction to the
glucose peak by the amount of glucose. Depending on 100
the sample and HPLC column type, glucose may not
separate from accompanying components. If glucose 50
does not separate from other components, Drink B
quantification can be performed by adding a known Maltotriose
concentration of glycerin or another internal reference 0
standard after the enzyme pretreatment steps, then 0 5 10 15 20 25 30 35 min
calculating amounts based on the peak area ratio of the
dietary fiber fraction to the internal reference standard. Fig. 3 Analysis of Low Molecular Weight Soluble
Analytical sensitivity will differ when a compound other Dietary Fiber in Beverage B
than glucose is used as the internal reference standard,
and results will need to be corrected accordingly by
calculating a coefficient relative to analytical sensitivity
for glucose.
The amount of dietary fiber is shown by adding the
amount of low molecular weight soluble dietary fiber
calculated by HPLC analysis to the result obtained by a
separate prosky method. This method of calculation
includes oligosaccharides in the dietary fiber fraction, so
to show the amount of dietary fiber in the sample minus 1) Food Labeling Standards
indigestible oligosaccharides, the amount of indigestible — Appendix: Methods of analysis of nutritional composition
oligosaccharides must be deleted from the results. Please (Notification No. 139 from the Deputy Secretary
General of the Consumer Affairs Agency, dated
1)
refer to the analytical method for further information. March 30, 2015) [In Japanese]

First Edition: Oct. 2016

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