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LC Technical Note

9
GL Sciences Inc.

Analysis of Vitamin C in Food by HPLC


Chemical Structure
CH2OH H C OH O O H HO OH
Reduction
H CH2OH C OH O O H O O

This is an application data of analyzing L-ascorbic acid and Dehydroascorbic acid, which are known to have a Vitamin C activity and Isoascorbic acid by HPLC using PDA. Dehydroascorbic acid is a Vitamin C compound like Ascorbic acid. Dehydroascorbic acid (DHAsA) is an oxidized form of Ascorbic acid (AsA). AsA can be detected by an UV Detector, but DHAsA can not. Therefore, it is necessary to convert the structure of the compound to make it detected by an UV Detector analyzing the total amount of Vitamin C. Also, there is an isomer of AsA known as Isoascorbic acid (ErA), which is a food additive. This application was conducted based on the Japanese Food Sanitation Inspection Guideline.

CH2OH HO C H O O H HO OH

Oxidation

L-ascorbic acid Reduced form existent UV absorption

Dehydro ascorbic acid Oxdized form non-existent UV absorption

Isoascorbic acid (D-ascorbic acid) existent UV absorption

Vitamin C

D-enantiomer

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 can be measured by a Homocysteine NO 2 reudction method. COOH
COOH C C C C OH H
CH2OH H C OH O O H

O O

C C

R = NO 2

RHNN

Hydrolysis
H C C OH H HO

Derivatization

RHNN H HO

Analysis

HPLC
DNPH Derivatization Method

at Oxid

ion

Dehydroascorbic acid

CH3OH

CH3OH

Sample
CH2OH

2,3-diketo-L-gulonic acid

Osazones

Redu ct

ion

OH O O

H HO OH

Analysis

HPLC
Homocysteine Reduction Method

Ascorbic acid Structures are created using Chemistry 4-D Draw which is provided by ChemInnovayion Software, Inc.

Analysis of Standard Solution


DNPH Derivatization Method
1.L-Ascorbic acid 1mg/L
4

Homocysteine Reduction Method


1. Isoascorbic acid 5mg/L
0.8

2. L-Ascorbic acid 5mg/L 1 2

mAU 2

-0.2 0

0.0

0.2

mAU 0.4

0.6

Time (min)

10

8 Time (min)

10

12

14

Analytical Conditions
: Inertsil SIL-100A (5m, 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 Injection Volume : 20 L Column

Analytical Conditions
: Inertsil NH2 (5m, 250 x 4.6 mm I.D.) Mobile Phase : A) CH3CN B CH3OH C) 0.01M phosphoric Buffer D) 0.03% homocystein solution A/B/C/D = 600/30/100/30, v/v/v/v Flow Rate : 1.0 mL/min Column Temp. : 40 Detection : PDA 270 nm Injection Volume : 5 L Column

GL SciencesLCTechnicalNote

DNPH Derivatization Method


Pretreatment Conditions
Sample
5g 5%Metaphosphoric acid 30mL grinding extraction Dilute to 50mL with 5%Metaphosphoric acid
4500000 4000000
DNPH-AsA
R 2 = 1.000

3500000 3000000

Area

2500000 2000000 1500000 1000000 500000 0 0 10 20 30 40 50

Filtration
Centrifugation 3000rpm, 10min 0.45m Filter

Fractionation
2mL Fraction

Derivatization
5%Metaphosphoric acid 1mL 2,6-dichloroindophenol 3 drop 2%thioureaMetaphosphoric acid solution 2mL 2% 2,4-DNPH4.5M Sulfuric acid 0.5mL Heating (50, 90min) Water cooling

Conc.mg/L

Calibration Curve1
1 The calibration sample was prepared by diluting L-Ascorbic acid in steps and pretreating it. The concentration described above is the concentration after diluting the sample.

liquid-liquid extraction
Ethyl acetate 2mL Shake 1hr

Supernatant liquid Lower layer 0.5mL Fraction Dilute to 1mL with Hexane Waste

Measurement sample

Analysis of food (Analytical Conditions )

Tea leaf
mAU 80 100 120 140

Sausage
mAU 80 100 120 140

1. L-Ascorbic acid

60

40

20

20

40

60

Time (min)

10

Time (min)

10

Baby formula
mAU 80 100 120 140

Spinach
mAU 80 100 120 140

1
mAU 0 2 4

60

40

40

60

20

20

0 2 4 6 8 10

Time (min)

10

Time (min)

GL SciencesLCTechnicalNote

Homocysteine Reduction Method


Pretreatment Conditions
Liquid Sample
Solid Sample
900000 800000 700000 600000
ErA AsA

R2 = 0.9974 R2 = 0.998

Area

10g 10g 4% Metaphosphoric acid 10mL 4% Metaphosphoric acid 10mL 2% Metaphosphoric acid 30mL Dilute to 50mL with 2% Metaphosphoric acid ultrasonic extraction 10min Dilute to 50mL with 2%Metaphosphoric acid

500000 400000 300000 200000 100000 0 0 10 20 30 40 50

Filtration
Centrifugation 3000rpm 10min 0.45m Filter

2mL Fraction 2mL Fraction

Reduction
0.1% Homocystein 1mL 10% Disodium Hydrogen Phosphate 1mL Heating ( 40, 20min )

Conc. mg/L

Calibration Curve2
2 On this figure, standard solution is diluted by 2% Metaphosphoric acid.

Measurement Sample

Measurement Sample
8

Effect of Reduction Method

AsA

L-ascorbic acid + Iso ascorbic acid

Total Ascorbic acid + Total Iso ascorbic acid

mAU

Reduction

6 8 10 Time (min)

12

14

mAU

6 8 10 Time (min)

12

14

Before

After

Analysis of food (Analytical Conditions ) Tea leaf

Sausage
40

1. Iso ascorbic acid 2. L-Ascorbic acid

40

mAU

20

20

mAU

8 Time (min)

10

12

14

8 Time (min)

10

12

14

Beer
2
40
0.8 0.6

Fish sausage
40

mAU 0.4

mAU

0.2

1
mAU 20

20

0.0

0.2

0
0

8 Time (min)

10

12

14

8 Time (min)

10

12

14

GL SciencesLCTechnicalNote

Modified analytical conditions by Homocysteine Reduction Method


1. Iso ascorbic acid 5mg/L 2. L-Ascorbic acid 5mg/L
4

Analytical Conditions
Column : Inertsil NH2 (5m, 250 x 4.6 mm I.D.) Mobile Phase : A) CH3CN B) H2O C) CH3COOH A/B/C = 87/11/2 , v/v/v Flow Rate : 2.0 mL/min Column Temp. : 40 Detection : PDA 243 nm Injection Volume : 20 L

1 2
mAU 2 0 0

4 Time (min)

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