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DETERMINATION OF PHYTOESTROGENS IN BEER BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE ARRAY DETECTION J. Bernal 1. C. Martínez García-Mauriño 1,

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Presentation on theme: "DETERMINATION OF PHYTOESTROGENS IN BEER BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE ARRAY DETECTION J. Bernal 1. C. Martínez García-Mauriño 1,"— Presentation transcript:

1 DETERMINATION OF PHYTOESTROGENS IN BEER BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE ARRAY DETECTION J. Bernal 1. C. Martínez García-Mauriño 1, F. R. Marin 2, G. Reglero 2, A. Cifuentes 1, E. Ibañez 1 1 Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, Madrid, Spain 2 Sección Departamental Ciencias de la Alimentación, Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain

2 Introduction Hop (Humulus lupulus L) Sleeping and nervous disorders Mild sedative Bitter stomachic MEDICAL PURPOSES Estrogenic activity Cancer related bioactivity PHARMACOLOGICAL PROPERTIES PRENYLFLAVONOIDS

3 Introduction PRENYL SIDE CHAIN Increases the lipophilicity Strong affinity to biological membranes Strong affinity to biological membranes Interesting biological activities Interesting biological activities

4 Introduction > 15 HOP´S PRENYLFLAVONOIDS Xanthohumol 8-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 THERAPEUTICAL PROPERTIES Herbal supplements FUNCTIONAL FOODS Fortified food (enriched beer) Fortified food (enriched beer)

5 Introduction Brewing Industry Hop Bitterness Aroma Functional properties Functional properties Beer 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 DETERMINED

6 Objectives 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

7 Scheme Chromatographic conditions Detection conditions Column Mobile phase composition Temperature Injection volume Sample treatmentSolid phase extraction (SPE) Validation of the method Selectivity Linearity Accuracy Detection limit (LOD) Quantitation limit (LOQ) Application to samples

8 Chromatographic conditions DAD-SPECTRA

9 Chromatographic conditions Chromatographic Column: ODS Spherisorb C 18 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

10 Sample treatment SPE PROCEDURE Degassed Filtered

11 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

12 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

13 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

14 Validation of the method LINEARITY MATRIX-MATCHED CALIBRATION STANDARD CURVES (6 beers, 1 standard) IXN and 8PN STANDARD ADDITION METHOD R 2 > 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

15 Validation of the method PRECISION AND RECOVERY STUDIES COMPOUND SPIKED LEVEL (mg/L) PRECISION STUDIES (n=5) RECOVERY STUDIES (n=5 ) RepeatabilityIntermediate precision Measured concentration (mg/L) RSD (%) Recovery (%) Measured concentration (mg/L) RSD (%) Measured concentration (mg/L) RSD (%) XN IXN PN %RSD< 5% Recoveries (75-98%) %RSD< 5%

16 Application of the method Beer 1-2 (dark); Beer 3-5 (golden/pale); Beer 6 non alcohol SAMPLEXNIXN8PN Beer 1< LOD Beer 2< LOD5.3< LOD Beer 3< LOD Beer 4< LOD3.7< LOQ Beer 5

17 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.

18 ACKNOWLEDGMENTS This work has been funded by a CICYT project (AGL /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 /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

19 DETERMINATION OF PHYTOESTROGENS IN BEER BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE ARRAY DETECTION J. Bernal 1. C. Martínez García-Mauriño 1, F. R. Marin 2, G. Reglero 2, A. Cifuentes 1, E. Ibañez 1 1 Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, Madrid, Spain 2 Sección Departamental Ciencias de la Alimentación, Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain


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