LC Technical Note

Simultaneous Analysis of 12 Soybean Isoflavones

LT166

GL Sciences Inc.

Isoflavones are contained in several products made of soybeans. They are known to be functional compoundswith antioxidant properties and for their metabolites for having estrogenic properties. Soybean isoflavones
contain 15 types of isoflavones: 3 types of isoflavones (daidzein, glycitein and genistein) called Aglycone (nonglycoside),3 types of glycosides (daidzin, glycitin, genistin), 3 types of acetylated glycosides, 3 types of malonylated
glycosides and 3 types of succinylated isoflavone glycosides.
In this note InertSustainSwift C18 column was used to analyze 12 types of soybean isoflavones (the 3 types ofsuccinylated isoflavone glycosides are not been considered in this analysis).
(R. Hirano)
R5
OH
R1O

OHO

R2R3

HO

R4O

HOHOO

OH

Isoflavone

HPLC Conditions
ColumnFlow rate
Column temp.
Detection
Injection Vol.

OO

O

Soybean Isoflavone Glycosides: InertSustainSwift C18(5 μm, 250 x 4.6 mm I.D).
: 1.0 mL/min: 35 ℃: PDA 254 nm (PD7752 PDA
Detector): 10 μL: A) CH3CN/H2O/CH3COOH = 150/850/1, v/v/v
B) CH3CN/H2O/CH3COOH = 350/650/1, v/v/v

EluentTime
時 間 (min.)（分 ）010606180

溶Eluent
離 液 ＡA（(%)％ ）1001000100100

溶Eluent
離 液 ＢB（(%)％ ）0010000

Standard Solution
PDA

12409mAU
20

3145

2

1011876

1. Daidzin (D)2. Glycitin (Gl)
3. Genistin (G)4. 6’’-O-Malonyldaidzin (MD)
5. 6’’-O- Malonylglycitin (MGl)
6. 6’’-O- Acetyldaidzin (AD)
7. 6’’-O- Malonylgenistin (MG)
8. 6’’-O- Acetylglycitin (AGl)
9. Daizein (De)
10. Glycitein (Gle)11. 6’’-O- Acetylgenistin (AG)
12. Genistein (Ge)
10 mg/L each

0Sample preparation method:
Dissolve each isoflavone withdimethylsulfoxide (DMSO) to 1 mg/mL.
Mix the 12 isoflavones together anddilute with 70 % aqueous ethanol
solution to 50 mg/L. Dilute with more
70% aqueous ethanol solution ifnecessary.

02040
Time [min]6080

GL Sciences LC Technical NoteCalibration Curve (0.5 - 50 mg/L)

2500000

2000000

D1500000

AreaGlGMD1000000

MGIAD
50000000102030
Concentration [mg/L]

4050604000000
3500000
3000000
MG

Area

2500000

AGI
2000000

D e
Gle

1500000

AG1000000

Ge

50000000102030
Concentration [mg/L]

405060

Correlation Coefficient (R2)R2

D
0.9996Gl
0.9998G0.9996MD
0.9995MGI
0.9995AD
0.9997

MG0.9996

AGI
0.9996De
0.9996Gle
0.9996AG
0.9996

Ge0.9996

GL Sciences LC Technical Note
Example of Pretreatment Method

Liquid Sample

Solid Sample
— 1g

— 1g

Extraction

Extraction— dilute with 25mL of 70 % aqueous ethanol solution— shake for 30 min— centrifuge at 10,000 rpm for 10min— collect the supernatant— dilute with 70% of aqueous ethanol solution
to 100 mL— centrifuge at 10,000 rpm for 10 min
and collect the supernatant

Residue—dilute with 25mL of 70%
aqueous ethanol solution— filtrate the supernatant by GL Chromato Disk
25P 0.45 μm—shake for 30 min—centrifuge at 10000 rpm for10 min

HPLCRepeat
thisprocedure twice—collect the supernatant

Dilute to Volume— dilute with aqueous ethanol solution to 100 mL— filtrate by GL Chromato Disk 25P 0.45μm

HPLC

Examples of Sample Analysis37110116mAU
42T i m e

310mAU
429T i m e4 0( m i n )2 02

Enlarged view
拡大7520630
T i m e (min)126 08 0

9 1140125060

Enlarged view

6 034

1mAU
-0.4 -0.20.00.20.40.60.8

2105060

8 0

7Tofu

4250

73
1

12Unflavored Soy Milk

11640

1

10511630
T i m e (min)34

42

Flavored Soy Milk

124 0( m i n )70520mAU-0.4 -0.20.00.20.40.60.8

052 002420734mAU-0.4 -0.20.00.20.40.60.8

mAU
4211169T i m e4 0( m i n )2 052012630
T i m e (min)

9 1140125060

Enlarged view

6 08 0GL Sciences LC Technical Note
Examples of Sample Analysis
1mAU
10

734

Soybeans

7mAU-0.4 -0.20.00.20.40.60.8

54103150

204 0( m i n )3740220

9T i m e

4 0( m i n )2 074630T i m e12mAU2040

1210802 0T i m e

1360

8 0

6 8 9 11RoastedSoybean
Flour12129 10405060

拡大Enlarged view
6 04 0( m i n )8 0

Supplement containing soybean isoflavones
36 910812mAU-0.4 -0.20.00.20.40.60.8

mAU
100 200

50

拡大Enlarged view30
T i m e (min)

20

122

100

3040

mAU
-0.4 -0.20.00.20.40.60.8

60

11

68 0

Natto
12 *(Fermentedsoybeans)
*

10

3260

6 04 0( m i n )

11150

拡大
Enlarged view30
T i m e (min)20

92 0409mAU-0.4 -0.20.00.20.40.60.8

mAU
100

96 012116121210

28 0

Boiled Soybeans1130
T i m e (min)3160

Enlarged view

320

11650

6 0

1

101

124030T i m e (min)12mAU-0.4 -0.20.00.20.40.60.8

T i m e920

92 0mAU
102

6 8

9 10

T i m e4 0( m i n )

2 020

1230
T i m e (min)405060拡大
Enlarged view
6 08 0GL Sciences LC Technical NoteO

Chemical Structure and UV Spectrum
1. Daidzin

2. Glycitin

OOHOHOOOHHO

3. Genistin

OH

4. Malonyldaidzin
OHOHO

R2 ：O

HOOH

O

OHOOO
H 3C
C 6H11O 5

C 6H11O5250300

OO

250300mAU

200350O

O250300

R1mAU

O

mAUO

400 200

350O

400 200350

400 200

250300350

Wave length (nm)Wave length (nm)Wave length (nm)Wave length (nm)5. Malonylglycitin

6. Acetyldaidzin

7. Malonylgenistin

8. Acetylglycitin

OHOH

O

OH

O

O
O

H 3C

H 3C

250300OO

350250300OO

250300OmAU

400 200

Wave length (nm)9. Daidzein

350OmAUO

R2R1mAU

O

R2mAU

R1400

OH

OHO

O

200

O

mAU

C6H11O5400 200

350

400 200

250300350

Wave length (nm)Wave length (nm)Wave length (nm)10. Glycitein

11. Acetylgenistin

12. Genistein

OH400

OH

O

OH

OH
CH3O

OH

O

OH

O

O
O

O

250300350

Wave length (nm)

400 200

250300350

Wave length (nm)※

400 200

OO

mAU

O

mAU

mAU
200

HO

R2HOmAU

HO

250

300

350

Wave length (nm)

400 200

250300350

Wave length (nm)The absorbance scale (Y axis) is the same in every UV spectrum.

400

GL Sciences LC Technical NoteCoffee BreakHPLC Column
InertSustainSwift C18 5 μm, 250 x 4.6 mm I.D.
Cat.No. 5020-88027

The soybean isoflavones are one type of Polyphenols. Theyare also known as estrogen-like since the chemical structure of
the isoflavones metabolites is similar to female hormones one(estrogens: e.g. estradiol) and some researches has also
reported that their behavior is similar to that of female
hormones. For this reason, among their applications they canalso be used in the human body as substitutes of female
hormones in case of deficiency.
CH3Daidzein
Genistein
Glycitein

GL Chromato DiskFor both hydrophilic andhydrophobic samples 25P
0.45 μm
Cat.No. 5040-28542

R1： H： OH： H

Daidzin
Genistin
Glycitin6’’-O-Acetyldaidzin
6’’-O-Acetylgenistin
6’’-O-Acetylglycitin
6’’-O-Malonyldaidzin
6’’-O-Malonylgenistin
6’’-O-Malonylglycitin
6’’-O-Succinyldaidzin
6’’-O-Succinylgenistin
6’’-O-Succinylglycitin

R2： H
： H
： OCH3

R3：H： OH：H：H： OH：H：H： OH：H：H： OH：HOH

HOEstradiol
R4：H：H： OCH3：H：H： OCH3：H：H： OCH3：H：H： OCH3R5：H：H：H： COCH3： COCH3： COCH3： COCH2COOH： COCH2COOH： COCH2COOH： COC2H4COOH： COC2H4COOH： COC2H4COOH

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly. We reserve theright to amend this information or data at any time and without any prior announcement.

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