Analysis of Resveratrol in Wineby HPLC

• Introduction

• Resveratrol

o Discovery

o Biosynthesis

• HPLC separation

• Results

• Conclusion

Outline

• Composition of flavoring, coloring and

other characteristic ingredients in

wine is largely dependent on make

and storage

• White and red wine differ not only in

color of the grapes, but also in the

way they are produced

Introduction

• For white wine, grapes are pressed and the resulting must is

fermented into an alcoholic beverage “Mostgärung”

• To produce red wine the grapes are squashed to produce a slurry

of grape juice, pulp and skin. During fermentation of this mash, the

colorants are extracted into solution. Pressing takes place after

fermentation “Maischegärung”.

• Only fermentation from mash can result in extraction of

compounds from grape skin and seeds

• Therefore red wine contains a number of compounds

that are not found in white wine.

• Also after fermentation, the

process of “aging” during the

period of storage in a wooden

cask results in distinct changes

and a unique profile of over 1000

different compounds in each lot.

Introduction

5

Introduction

Apart from water and ethanol, red wine contains in average:

Component Amount Charecteristic

Glycerol 1 % Oily, sweetness

Organic acids 0.4 % Acidity

Anthocyanins

0.1 %

Red colour

CatechinsBitterness (> 20 mg/L), antioxidants

Tannins Astringency, bitterness

Flavonols antioxidants

Other compounds 0.5 %

• Another molecule in red wine, however, has been the main

focus of health benefits in recent years: Resveratrol

• It has been shown in studies that resveratrol, as well as

having antioxidant properties, can help prevent high blood

pressure (hypertension) in mice and also has anti-

inflammatory effects. It’s also suspected to have anticancer,

and chemopreventive abilities.

Resveratrol

• They collected veratrum grandiflorum,

a stout, exotic and flowering plant

from the Hokkaido Island.

A phenolic compound was obtained by crystallization and

the molecular formula was identified.

• The new compound was named RES (resorcinol family),

VERATR (from veratrum grandiflorum) OL (used to

indicate hydroxyl groups)

Resveratrol - Discovery

• Reseveratrol was first discovered byJapanese

researcher Michio Takoaka and his group in

1939.

Resveratrol - Biosynthesis

• Resveratrol is found in the skin of red

grapes, peanuts, in many berries and a

few other plants… but how does it get

there?

• Resveratrol is derived from p-coumaric

acid which is an intermediate in lignin

production. The two key enzymes are

Coenzyme A (CoA) Ligase (4CL) and

Stilbene Synthase (STS)

• Resveratrol is produced in plants when

they are exposed to stress such as

UV-light, disease and pests.

• Reversed phase HPLC is most appropriate to separate

the cis- and trans isomers from each other.

• Fluorescent detection is used for high sensitivity

(LOQ ≤ 0.015 mg/L1 vs. 0.3 mg/L2 using DAD)

• Wine sample complexity requires

gradient optimization to resolve the

trans-resveratrol peak from the rest of

fluorescent stilbenes and polyphenols.

• Fast analysis using Nexera X2 UHPLC system

equipped with PDA and RF-20 AXS Fluorescent detector.

HPLC separation

1 J. Sep. Sci. 2007, 30, 669 – 6722 J. Agric. Food Chem., Vol. 51, No. 18, 200

Method Development

Columnn: GIST C18, 100 x 2.1 mm, 2 µm

Mobile Phase:

A: Water incl. 0.2 % Formic Acid

B: Acetonitrile

GE Program

0 min BCONC 20%

4 min BCONC 50%

4.01 min BCONC 20%

Flow rate: 0.6 ml/min

Temperature: 50 °C

PDA detection:190 – 360 nm

Detection: RF-20AXS – Excitation λ = 300 nm Emission λ = 386 nm SENS = Med

Cell Temperature: 20 °C

Inj. Vol.: 2 µl

Results

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 min

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90 trans-Resveratrol/0.889

cis-Resveratrol/1.241

• 17.5 mg /L trans-resveratrol standard – PDA detection 291 nm

Results

• 9 mg /L trans-resveratrol standard – RF - Ex:300nm Em:386nm

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 min

0

10

30

50

70

90

110

130

150

170

190

210

mV

trans-Resveratrol

cis-Resveratrol

13

1.60 1.65 1.70 1.75 min

0

25000

50000

75000

100000

125000

150000

175000

200000

225000

250000

275000

300000

325000

350000

uV

Data3:Trans_Res_20ppm_MeOH-Water_20deg_001.lcd Detector A:Ex:300nm,Em:386nmData2:Trans_Res_20ppm_MeOH-Water_30deg_001.lcd Detector A:Ex:300nm,Em:386nmData1:Trans_Res_20ppm_MeOH-Water_40deg_001.lcd Detector A:Ex:300nm,Em:386nm

trans-Resveratrol cis-Resveratrol

2.20 2.25 2.30 2.35 2.40 min

0

25000

50000

75000

100000

125000

150000

175000

200000

225000

250000

uV

Data3:Cis_Res_20ppm_MeOH-Water_20deg_001.lcd Detector A:Ex:300nm,Em:386nmData2:Cis_Res_20ppm_MeOH-Water_30deg_001.lcd Detector A:Ex:300nm,Em:386nmData1:Cis_Res_20ppm_MeOH-Water_40deg_001.lcd Detector A:Ex:300nm,Em:386nm

Method Development

• Cell temperature affects sensitivity

• improve baseline stability and eliminate the effect of

temperature

40 ºC

30 ºC

20 ºC

40 ºC

30 ºC

20 ºC

Sample preparation

Sample preparation

• Wine sample, LL extracted – PDA detection 280 nm

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 min

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

mAU

trans-Resveratrol = 7.7 mg/L

cis-Resveratrol = 1.6 mg/L

Results

Example: Filtered red wineDatafile Name:Shim_Spätburgunder_002.lcd

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

0

25

50

75

100

125

150

175

200

225

mAU

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

barB.Conc

Pump A Degassing Unit Pressure(x -1000)Pump A Pressure

291nm,4nm

tra

ns-

Re

sve

ratr

ol

cis-

Re

sve

ratr

ol

17

Datafile Name:Shim_Spätburgunder_002.lcd

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

0

50

100

150

200

250

300

350mV

0.0

25.0

50.0

75.0

barPump A PressureDetector A Ex:300nm,Em:386nm

tra

ns-R

esve

ratr

ol

cis

-Re

sve

ratr

ol

LL extraction vs filtered (0,2 µm)

sample RF Signal

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

0

250000

500000

750000

1000000

1250000

1500000

1750000

2000000

2250000

2500000

2750000

uV

Data2:Shim-Wine-LL-extract_2ul_IV_GE_002.lcd Detector A:Ex:300nm,Em:386nmData1:Shim-Wine-filtered_GE_.lcd Detector A:Ex:300nm,Em:386nm

18

LL extraction vs filtered (0,2 µm)

sample PDA signal

19

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

0

25000

50000

75000

100000

125000

150000

175000

200000

225000

uV

Data2:Shim-Wine-LL-extract_2ul_IV_GE_002.lcd PDA Ch1 280nm,4nm Data1:Shim-Wine-filtered_GE_.lcd PDA Ch1 280nm,4nm

Conclusion

• The proposed method allows the determination of trans-

resveratrol in wine in a one step procedure

• Fluorescence detection is highly sensitive

and allows for the detection of resveratrol in

concentrations present in red wine

• Animal studies suggest as much as 500 mg daily may be

necessary to provide any health benefits, even if a 40 mg

daily dose is sufficient, as suggested elsewhere, red wine

contains at most 12 mg resveratrol per liter, you'd need to

drink a little over 3 liters of wine daily to get that much

resveratrol.

Cheers !