DETERMINATION OF PHYTOESTROGENS IN BEER BY HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY WITH DIODE ARRAY DETECTION

J. Bernal1. C. Martínez García-Mauriño1, F. R. Marin2, G. Reglero2, A. Cifuentes1, E. Ibañez1

1Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain2Sección Departamental Ciencias de la Alimentación, Universidad Autónoma de Madrid,

Campus de Cantoblanco, 29049 Madrid, Spain

Introduction

Hop (Humulus lupulus L)Hop (Humulus lupulus L)

Sleeping and nervous disorders

Mild sedative

Bitter stomachic

MEDICAL PURPOSES

Estrogenic activity

Cancer related bioactivity

PHARMACOLOGICAL PROPERTIES

PRENYLFLAVONOIDSPRENYLFLAVONOIDS

Introduction

PRENYL SIDE CHAINPRENYL SIDE CHAIN

Increases the lipophilicityIncreases the lipophilicity

Strong affinity

to biological membranes

Strong affinity

to biological membranes

Interesting biological

activities

Interesting biological

activities

Introduction

> 15> 15HOP´S PRENYLFLAVONOIDSHOP´S PRENYLFLAVONOIDS

XanthohumolXanthohumol 8-prenylnaringenin8-prenylnaringenin

• Atherosclerosis

• Inhibit HIV-1 and the replication of Plasmodium falciparum

• Anticancer activity

• Atherosclerosis

• Inhibit HIV-1 and the replication of Plasmodium falciparum

• Anticancer activity

• The most potent phytoestrogen• The most potent phytoestrogenTHERAPEUTICAL PROPERTIESTHERAPEUTICAL PROPERTIES

Herbal supplements

Herbal supplements

FUNCTIONAL FOODSFUNCTIONAL FOODS Fortified food

(enriched beer)

Fortified food

(enriched beer)

Introduction

Brewing IndustryBrewing Industry

HopHop

BitternessBitterness

AromaAroma

Functional

properties

Functional

properties

BeerBeer

Prenylflavonoid content

Prenylflavonoid content

• Wort boiling (converts Xanthohumol into inactive isoxanthohumol)

• The form in which hop bitterness and aroma is added

• Wort boiling (converts Xanthohumol into inactive isoxanthohumol)

• The form in which hop bitterness and aroma is added

SHOULD BE DETERMINEDSHOULD BE DETERMINED

Objectives

BIBLIOGRAPHIC REVISION

BIBLIOGRAPHIC REVISION

HPLC-MS

METHODS

HPLC-MS

METHODS

Extremely sensitive

Robust and reliable

Not affordable for all

laboratories

Not so fast

Extremely sensitive

Robust and reliable

Not affordable for all

laboratories

Not so fast

TO DEVELOP AND VALIDATE A FAST, PRECISE AND SIMPLE HPLC-DAD

METHOD TO ANALYZE XANTHOHUMOL (XN), ISOXANTHOHUMOL (IXN)

AND 8-PRENYNARINGENIN (8PN) IN BEER SAMPLES

TO DEVELOP AND VALIDATE A FAST, PRECISE AND SIMPLE HPLC-DAD

METHOD TO ANALYZE XANTHOHUMOL (XN), ISOXANTHOHUMOL (IXN)

AND 8-PRENYNARINGENIN (8PN) IN BEER SAMPLES

Scheme

Chromatographic conditions

Detection conditions

Column

Mobile phase composition

Temperature

Injection volume

Sample treatment Solid phase extraction (SPE)

Validation of the method

Selectivity

Linearity

Accuracy

Detection limit (LOD)

Quantitation limit (LOQ)Application to samples

Chromatographic conditions

DAD-SPECTRA

Chromatographic conditions

• Chromatographic Column: ODS Spherisorb C18 5μ 80Å (250 x 4.0 mm i.d)

• Mobile phase : (A) 1% acetic acid in acetonitrile , (B) 1% acetic acid in water and (C) 1% acetic acid in methanol in a gradient elution analysis:

• Mobile phase flow-rate: 1 mL /min

• Separation temperature: 25ºC

• Injection volume: 20 μL

Sample treatment

SPE PROCEDURE

DegassedDegassed

FilteredFiltered

HPLC-DAD Chromatograms

HPLC-DAD chromatograms at 280 nm of a non spiked and spiked with IXN and 8PN at 10 mg/L (a, d) alcohol free, (b, e) dark and (c, f) pale (golden) beer samples

HPLC-DAD Chromatograms

HPLC-DAD chromatograms at 370 nm of a non spiked and spiked with XN at 2 mg/L (a, d) alcohol free, (b, e) dark and (c, f) pale (golden) beer samples

Validation of the method

Injected extracts of non spiked beer samples

SELECTIVITY

All samples with IXN and 8PN

No real blank of beer Check purity

Compare DAD spectra

LOD&LOQ

Injected extracts of non spiked beer samples (n=20)

LOD 3xS/N

LOQ 10xS/N

Validation of the methodLINEARITY

MATRIX-MATCHED CALIBRATION STANDARD CURVES (6 beers, 1 standard)

IXN and 8PNSTANDARD ADDITION METHOD

R2 > 0.99

Quantitation of XN with a standard calibration curve

Slope and intercept values within the same range for standards and beer samples

Quantitation of IXN and 8PN with and specific calibration curve

Slope within the same range for standards and beer samples but different intercept value for each beer sample and standards

Validation of the methodPRECISION AND RECOVERY STUDIES

COMPOUNDSPIKED LEVEL(mg/L)

PRECISION STUDIES (n=5)RECOVERY STUDIES (n=5)

Repeatability Intermediate precision

Measured concentratio

n (mg/L)

RSD(%)

Recovery(%)

Measured concentration

(mg/L)

RSD(%)

Measured concentration

(mg/L)

RSD(%)

XN 0.5 0.5 2.7 0.5 2.8 0.5 2.3 98.09.9 9.4 2.4 9.3 2.5 9.3 2.1 94.5

45.8 44.3 2.3 44.2 2.4 44.3 1.9 96.8

IXN1.1 0.9 2.9 0.9 3.3 0.9 3.1 86.7

10.8 10.0 4.1 9.9 2.9 10.0 3.4 92.445.2 40.3 3.7 40.4 3.2 40.4 2.9 89.4

8PN10.1 7.6 3.1 7.5 2.7 7.6 2.8 75.220.5 16.5 2.5 16.4 2.3 16.6 2.5 80.944.8 36.8 2.9 36.5 3.1 36.8 3.2 82.1

%RSD< 5%Recoveries (75-98%)

%RSD< 5%

Application of the method

Beer 1-2 (dark); Beer 3-5 (golden/pale); Beer 6 non alcohol

SAMPLE XN IXN 8PN

Beer 1 < LOD 3.2 8.0

Beer 2 < LOD 5.3 < LOD

Beer 3 < LOD 6.2 5.5

Beer 4 < LOD 3.7 < LOQ

Beer 5 <LOD 7.1 11.8

Beer 6 <LOD 0.8 9.4

Absence of XN

Transformation

into IXN

Loss of the

medical properties

IXN presence

No influence of type and origin of the beer except for

the alcoholic content

8PN presence

No influence of type and origin of the beer except for

the alcoholic content

Free alcohol beer could be a good nutritional source of 8PN

CONCLUDING REMARKS

• A fast, simple and precise HPLC-DAD method for the determination of XN, IXN and 8PN has been developed

• The use of Sep-Pak cartridges avoids interferences from matrix and provides good recoveries in a short period of time

• The quantitation of XN could be done with a standard curve, meanwhile for IXN and 8PN it must be done with their own matrix matched calibration curve.

• No XN was detected in the analyzed beers and the content of IXN and 8PN only showed differences for the non-alcoholic beers.

• A fast, simple and precise HPLC-DAD method for the determination of XN, IXN and 8PN has been developed

• The use of Sep-Pak cartridges avoids interferences from matrix and provides good recoveries in a short period of time

• The quantitation of XN could be done with a standard curve, meanwhile for IXN and 8PN it must be done with their own matrix matched calibration curve.

• No XN was detected in the analyzed beers and the content of IXN and 8PN only showed differences for the non-alcoholic beers.

ACKNOWLEDGMENTS

• This work has been funded by a CICYT project (AGL 2007-64198/ALI).

• J.Bernal would like to thank the Spanish Ministry for a contract “Juan de la Cierva”.

• Special thanks to the members of the IFI and UAM research groups

• This work has been funded by a CICYT project (AGL 2007-64198/ALI).

• J.Bernal would like to thank the Spanish Ministry for a contract “Juan de la Cierva”.

• Special thanks to the members of the IFI and UAM research groups

DETERMINATION OF PHYTOESTROGENS IN BEER BY HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY WITH DIODE ARRAY DETECTION

J. Bernal1. C. Martínez García-Mauriño1, F. R. Marin2, G. Reglero2, A. Cifuentes1, E. Ibañez1

1Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain2Sección Departamental Ciencias de la Alimentación, Universidad Autónoma de Madrid,

Campus de Cantoblanco, 29049 Madrid, Spain