To determine the amount of vitamin C that is present in certain commercial food products by the titration method.
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LC Technical Note
9 GL Sciences Inc.
This is an application data of analyzing L-ascorbic acid and Dehydroascorbic
Chemical Structure
acid, which are known to have have a Vitamin C activity and Isoascorbic acid by HPLC
CH2OH
using PDA.
H
C
Dehydroasc Dehydroascorbic orbic acid is a Vitamin Vitamin C compound compound like Ascorbi Ascorbicc acid.
OH O
H
C
Oxidation
O
Dehydroascorbic acid (DHAsA) is an oxidized form of Ascorbic Ascorbic acid (AsA). AsA
Reduction
OH
L-ascorbic
acid
H O O
O
H HO
O
OH
Dehydro ascorbic ascorbic acid
Isoascorbic Isoascorbic acid
（Reduced form）
（Oxdized form）
( D-ascorbic acid)
existent UV absorption
non-existent UV absorption
existent UV absorption
analyzing the total amount of Vitamin C. C. Also, there is an isomer of AsA known as Isoascorbic acid (ErA), which is a food additive.
C
H
HO
to convert the structure of the compound to make it detected by an UV Detector
HO
OH O O
H
can be detected detected by an UV Detector, but DHAsA can not. Therefore, it is necessary necessary
CH2OH
CH2OH
This application was conducted based on the Japanese Food Sanitation
D-enantiomer
Vitamin C
Inspection Guideline.
Outline The total amount of Ascorbic acid can be measured by a DNPH Derivatization method. Simultaneous analysis of Isoascorbic acid and Reduced L-ascorbic acid acid can be measured by a Homocysteine NO2 reudction reudction method. COOH COOH
CH2OH H
C
OH O O
O
O
C
O
C
H
C
OH
HO
C
H
Hydrolysis
H
a t i o n O x i d
R = NO 2
O
Derivatization
RHNN
C
RHNN
C
H
C
OH
HO
C
H
CH3OH
Dehydroascorb Dehydroascorbic ic acid
Sample
HPLC
Analysis
（DNPH Derivatization Method ）
CH3OH
Osazones
2,3-diketo-L-gulonic acid CH2OH
R e d du c u ct t i i o o n n
H
C
OH O O
HO
HPLC
Analysis
H
（Homocysteine Reduction Method ）
OH
Ascorbic acid Structures are created using Chemistry 4-D Draw which is provided by ChemInnovayion Software, Inc.
Analysis of Standard Solution Homocystei Homocysteine ne Reduction Reduction Method
DNPH Derivatiz Derivatization ation Method
1. Isoascorbic Isoascorbic acid 5mg/L
1. L-Ascorbic acid 1mg/L
2. L-Ascorbic acid 5mg/L
8 . 0
4
6 . 0
U A m 2
1 2
U 4 A . m 0
1
2 . 0 0 . 0 0
2 . 0 -
0
2
4
6
8
10
0
2
4
Time (min)
6
8 Time (min)
10
Analytical Conditions
Analytical Conditions
: Inertsil SIL-100A (5μm, 250 x 4.6 mm I.D.) Mobile Phase : A) CH3COOC2H5 B) n-Hexane C) CH3COOH A/B/C = 50/40/10, v/v/v Flow Rate : 1.5 mL/min Column Temp. : 40 ℃ Detection : PDA 495 nm Inject Injection ion Volum Volumee : 20 μL
: Inertsil NH2 (5μm, 250 x 4.6 mm I.D.) Mobile Phase : A) CH3CN B） CH3OH C) 0.01M phosphoric Buffer D) 0.03% homocystein homocystein solution A/B/C/D = 600/30/100/30, v/v/v/v Flow Rate : 1.0 mL/min Column Te Temp. : 40 ℃ Detection : PDA 270 nm Inject Injection ion Volum Volumee : 5 μL
