HPLC ANALYSIS OF PHENOLIC HPLC ANALYSIS OF PHENOLIC COMPOUNDS IN NORWAY SPRUCE COMPOUNDS IN NORWAY SPRUCE

WITH DIODE ARRAY AND MASS WITH DIODE ARRAY AND MASS SPECTROMETRIC DETECTIONSPECTROMETRIC DETECTION

Gloria Cosco Salguero, Jan Fähnrich

Institute of Chemical Technology, Dept. of Analytical Institute of Chemical Technology, Dept. of Analytical Chemistry,Chemistry,

Technická 5, 166 28 Praha 6Technická 5, 166 28 Praha 6

Subdivision of Ultraviolet RadiationSubdivision of Ultraviolet Radiation

1. UV-A 320 - 400 nmPenetrates atmosphere at nearly full intensity

Probably only weakly harmful

2. UV-B 280 - 315 nmOnly partially absorbed in stratosphere

Very harmful - probably forest decline disease

3. UV-C 200 - 280 nmCompletely absorbed by ozone layer and oxygen (O2)

Hazardous

According to International Commission on Illumination

OZONE (OOZONE (O33) ABSORPTION ) ABSORPTION

SPECTRA SPECTRA

(a)(a) Spectrum from 200 to 300 nm Spectrum from 200 to 300 nm(b)(b) Spectrum from 295 to 325 nm Spectrum from 295 to 325 nm

OzoneOzone concentration reduction concentration reduction allows more UV light to penetrate allows more UV light to penetrate

to earth’s surface.to earth’s surface.

Norway spruce needles

UV-B Radiation Effects on PlantsUV-B Radiation Effects on Plants

- Decrease in photosynthetic activity.

- Susceptibility to disease.

- Changes in plant structure and

pigmentation.

- Growth retardation.

Aim of study:

Identify and quantify phenolic compounds in Norway spruce needles that absorb UV radiation and may contribute to UV light screening.

HPLC ANALYSES SYSTEMSHPLC ANALYSES SYSTEMS

IDENTIFICATION

A) DAD - LC system Waters

B) DAD - Spectra Monitor 500 (Watrex). Software

SM5000.

C) LC system HP 1100 Series - MS ion trap detector

with electrospray ionization.

QUANTIFICATION

D) UV Detection - LC system Dionex. Software

Chromeleon.

SAMPLE PREPARATIONSAMPLE PREPARATION

1. Hot water extraction:

50 g sample was mixed with 400 ml of water boiled in a round bottom flask under reflux for 1 hour. Samples were cooled to room temperature; aqueous layer was separated, centrifuged, filtered and analysed by HPLC.

2. Methanolic extraction:

Needles about 2.7 g were cut with a pair of scissors, then extracted three times in ultrasonic bath for 45 min with 8 ml of 80% methanol in water. Combined extracts were collected and the volume was adjusted to 25 ml. The supernatant was centrifuged, filtered and analysed by HPLC.

ANALYSED COMPOUNDS :ANALYSED COMPOUNDS :

FERULIC ACID FERULIC ACID pp-COUMARIC ACID CATECHIN -COUMARIC ACID CATECHIN pp-HYDROXY- -HYDROXY- ACETOPHENONE ACETOPHENONE

BENZOIC ACID BENZOIC ACID pp-HYDROXY- 3,4-DIHYDROXY- VANILLIC ACID-HYDROXY- 3,4-DIHYDROXY- VANILLIC ACID BENZOIC ACID BENZOIC ACID BENZOIC ACID BENZOIC ACID

OH

COOH

OMe

OH

COOH

COOH

OH

OMe

O

OH

OH

OH

OHH

OHH

OH

COOH

OMe

OH

COOH

OH

COOH

OH

d

Phenolic compound identification (A,B) (A)(A)Solvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 minSolvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 min

Column Separon SGX C-18, 7µm, 3 x 150 mmColumn Separon SGX C-18, 7µm, 3 x 150 mm(B)(B) Phosphoric acid 10mM/Methanol (70:30) Isocratic elution Phosphoric acid 10mM/Methanol (70:30) Isocratic elution

Column Separon SGX C-18, 7µm, 3 x 150 mm Column Separon SGX C-18, 7µm, 3 x 150 mm

( ( + )+ ) IDENTIFIED COMPOUNDS IDENTIFIED COMPOUNDS

p -Hydroxyacetophenone + +Catechin + +p -Hydroxybenzoic acid - +Vanillic acid - -3,4-Dihydroxybenzoic acid - -Benzoic acid - -Ferulic acid - -p -Coumaric acid + +

PHENOLIC COMPOUNDSAQUEOUS EXTRACT

(A,B)

METHANOLIC EXTRACT

(A,B)

Phenolic compound identification (A,B,C) (A)(A)Solvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 minSolvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 min

Column Separon SGX C-18, 7µm, 3 x 150 mmColumn Separon SGX C-18, 7µm, 3 x 150 mm(B)(B) Phosphoric acid 10mM/Methanol (70:30) Isocratic elution Phosphoric acid 10mM/Methanol (70:30) Isocratic elution

Column Separon SGX C-18, 7µm, 3 x 150 mm Column Separon SGX C-18, 7µm, 3 x 150 mm(C)(C) Solvent A (Water) / Solvent B (Acetonitrile)Solvent A (Water) / Solvent B (Acetonitrile)

Linear Gradient from 5 to 100%B in 40 minLinear Gradient from 5 to 100%B in 40 min

AQUEOUS EXTRACT

METHANOLIC EXTRACT

p -Hydroxyacetophenone + + + +Catechin + + + +p -Hydroxybenzoic acid - + + +Vanillic acid - - + +3,4-Dihydroxybenzoic acid - - + +Benzoic acid - - + +Ferulic acid - - + +p -Coumaric acid + + + -

PHENOLIC COMPOUNDSAQUEOUS EXTRACT

(A,B)

METHANOLIC EXTRACT

(A,B)

ANALYSIS BY SPECTROMETRIC MASS (C)

ChromatogramChromatogram of methanolic of methanolic

extractextract

SAMPLE PREPARATIONSAMPLE PREPARATION

3. Acid hydrolysis:

Aliquots of water and methanolic extraction were filtered, then mixed with HCl 6N. Final solution had concentration of 1.7N. Solution was heated at 70oC at temperature bath for 2 hours. Then solutions were cooled, centrifuged, filtered and analysed by HPLC.

Phenolic compound identification (A,B) (A)(A)Solvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 minSolvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 min

Column Separon SGX C-18, 7µm, 3 x 150 mmColumn Separon SGX C-18, 7µm, 3 x 150 mm(B)(B) Phosphoric acid 10mM/Methanol (70:30) Isocratic elution Phosphoric acid 10mM/Methanol (70:30) Isocratic elution

Column Separon SGX C-18, 7µm, 3 x 150 mm Column Separon SGX C-18, 7µm, 3 x 150 mm

p -Hydroxyacetophenone + +Catechin - -p -Hydroxybenzoic acid - +Vanillic acid - -3,4-Dihydroxybenzoic acid - -Benzoic acid - -Ferulic acid - +p -Coumaric acid - +

PHENOLIC COMPOUNDSAQUEOUS EXTRACT

(A,B)

METHANOLIC EXTRACT

(A,B)

Phenolic compound identification (A,B,C) (A)(A)Solvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 minSolvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 min

Column Separon SGX C-18, 7µm, 3 x 150 mmColumn Separon SGX C-18, 7µm, 3 x 150 mm(B)(B) Phosphoric acid 10mM/Methanol (70:30) Isocratic elution Phosphoric acid 10mM/Methanol (70:30) Isocratic elution

Column Separon SGX C-18, 7µm, 3 x 150 mm Column Separon SGX C-18, 7µm, 3 x 150 mm(C)(C) Solvent A (Water) / Solvent B (Acetonitrile)Solvent A (Water) / Solvent B (Acetonitrile)

Linear Gradient from 5 to 100%B in 40 minLinear Gradient from 5 to 100%B in 40 min

AQUEOUS EXTRACT

METHANOLIC EXTRACT

p -Hydroxyacetophenone + + + +Catechin - - + +p -Hydroxybenzoic acid - + + +Vanillic acid - - + +3,4-Dihydroxybenzoic acid - - + +Benzoic acid - - + +Ferulic acid - + + +p -Coumaric acid - + + -

PHENOLIC COMPOUNDSAQUEOUS EXTRACT

(A,B)

METHANOLIC EXTRACT

(A,B)

ANALYSIS BY SPECTROMETRIC MASS (C)

Chromatogram Chromatogram of methanolic of methanolic extract after extract after hydrolysishydrolysis

Phenolic compound quantification(D)Solvent A (Phosphoric acid 0.01mol/l) Solvent B (Methanol)

Linear Isocratic (70:30)

Phenolic compounds in different extract (μg/g)

AQUEOUS EXTRACT

METHANOLIC EXTRACT

p -Hydroxyacetophenone 44.4 164.5 564.5 1497.6Catechin - - - -p -Hydroxybenzoic acid - - 45 56Vanillic acid - - - -3,4-Dihydroxybenzoic acid - - - -Benzoic acid 5.3 - - -Ferulic acid - 63 12.2 -p -Coumaric acid 12.4 67.3 36.4 102.3

PHENOLIC COMPOUNDSAQUEOUS EXTRACT

METHANOLIC EXTRACT

ANALYSIS AFTER HYDROLYSIS

SAMPLE FRACTIONATION (1)

-1. Separation using principle of ionic exclusion

-- Compounds studied : Phenolic acids (e.g. hydroxybenzoic, - p- coumaric acids) and neutral compounds (catechin,- p-hydroxyacetophenone).-- HPLC system consisted of an isocratic pump LCP 4000. Diode- array detector Spectro Monitor 500. Software SM5000.-- Column Separon SGX C18 7µm, 3 x 150 mm.-- Mobile phase, methanol: water 80:20 (v/v). Aqueous phase was- proved with various concentrations of sodium dodecylsulfate- from 0.01 mM to 30 mM.

1.5

2.0

2.5

3.0

3.5

0 1 2 3 4 5 6

p-Hydroxyacetophenone

Phloroglucinol

Catechin

p-Cinnamic acid

Ferulic acid

p-Hydroxybenzoic acid

Protocatechuic acid

Benzoic acid

p-Coumaric acid

Vainillic acid

m-Coumaric acid

min,Rt

2

1

2

1

Lmmol,)SDS(

c

Phenolic compound retention behaviour under ion-exclusion conditionsColumn 150 x 3.2 mm, Separon SGX C18 7µm, mobile phase methanol-aqueous sodium dodecylsulfate (SDS) 0.1 to 30 mmol l-1, 0.4 ml min-1

SAMPLE FRACTIONATION (2)

2. Separation using principle of hydrophilic interaction

- Compounds studied: Phenolic acids (e.g. hydroxybenzoic, - p-coumaric acids) and neutral compounds (catechin,- p-hydroxyacetophenone).-- HPLC system consisted of an isocratic pump LCP 4000. Diode- array detector Spectro Monitor 500. Software SM5000.

- Column Lichrosorb Si-60, 7µm, 3 x 150 mm.

- Mobile phase, acetonitrile:water (v/v). Organic phase in

percentage from 50 to 100%.

Phenolic compound retention behaviour under hydrophilic interaction conditionsColumn Lichrosorb Si-60, 7µm, 3 x 150 mm, mobile phase acetonotrile-aqueous, 0.4 ml min-1

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

50 60 70 80 90 100 110

% acetonitrile

t R, min

p-Hydroxybenzoic acid

p-Coumaric acid

Benzoic acid

Catechin

p-Hydroxyacetophenone

Fenol

Floroglucin

Pyrocatechin

Resorcin

Hydrochinon

Ethylenglycol

Benzyl alcohol

CONCLUSIONS

1. Phenolic compounds occur in Norway spruce needles in the free form and as conjugates, probably glycosides or esters.

2. In extracts p-hydroxyacetophenone, vanillic, 3,4-dihydroxybenzoic, ferulic and p-coumaric acids were identified as UV-B radiation absorbing substances.

3. Sample requires clean up and fractionation in order to simplify chromatograms and eliminate interfering components.

CONCLUSIONS

5. Systems operating on the principle of ionic exclusion can separate the phenolic acids and neutral compounds like catechin and p-hydroxyacetophenone. They are not directly applicable to samples containing a large amount of acids or salts like hydrolyzed samples.

6. Hydrophilic chromatography can separate phenolic acids from compounds without carboxyl groups. Sample must be extracted with acetonitrile (or solvent with similar properties) or transferred to this solvent.

ACKNOWLEDGEMENTS

Financial support of the Ministry of Environment of the Czech Republic (grant VaV340/1/01)

and of the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 223400008)

is gratefully acknowledged.

Chromatographic conditions for Chromatographic conditions for LC system HP 1100 SeriesLC system HP 1100 Series

- Column Luna LC-18, 3μm, 150 x 2.0 mm I.D. from Phenomenex (USA), and a security guard C-18 (2.0 x 4 mm I.D.). - Flow = 0,2 ml/min.

- Lineal gradient from 5:95 to 100:0 (v/v) water:acetonitrile in 40 min. - Injection volume 3 µl. - Temperature 20 °C - MS ion trap detector with electrospray ionization (Finnigan,Deca). - Capillary temperature 350 °C. - MS operated in negative-ion. [M-H]-

Chromatograms of phenolic acidsChromatograms of phenolic acids

0

50

100

0

50

100

Rel

ativ

e A

bund

ance

15,19 min

137,46

15,84 min

167,39

NL: 3,81E5

m/z= 136,9-137,9

F: - c ESI Full ms

[ 60,00-1000,00]

NL: 9,24E5

m/z= 166,9-167,9

F: - c ESI Full ms

[ 60,00-1000,00]

p-hydroxybenzoic acid

vanillic acid

Time (min)

0

50

100

193,37

m/z= 192,9-193,9 F: - c ESI Full ms [ 60,00-1000,00]

ferulová

18,96 min

0 5 10 15 20 25 30 35 40

Behaviour of several phenolic acids and neutral compounds

ACN : 50%H2O : 50%

ACN : 80%H2O : 20%

ACN : 95%H2O : 5%

ACN : 98%H2O : 2%

ACN : 100%

1.27 1.58 2.81 8.59 a

1.22 1.53 3.01 9.57 a

- - - 10.88 -

- - - 12.52 -

- - 8.4 9.94 a

a - a - a

a - a - a

1.82 1.92 2.09 - a

1.83 1.89 2.04 2.10 2.30

1.88 - 1.98 - 2.05

1.86 - 2.05 - 2.23

2.03 - 2.17 - 2.35

1.86 - 1.99 - 2.11

1.96 - 2.02 - 2.17

1.98 - - - -

1.94 - 3.66 - 7.58

1.93 - 2.00 - 2.33

Pyrocatechin

Resorcin

Hydrochinon

Time retention(min)

p -Hydroxybenzoic acid

p -Coumaric acid

p -ferulic acid

Fenol

Glycerol

Ethylenglycol

Benzyl alcohol

Vainillic acid

Benzoic acid

Catechin

Quevercetin

Rutin

p -Hydroxyacetophenone

Floroglucin

a = Compound eluted in greater times of 15 minutes