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J. Bernal 1 . C. Martínez García-Mauriño 1 , F. R. Marin 2 ,

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

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J. Bernal 1 . C. Martínez García-Mauriño 1 , F. R. Marin 2 ,

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  1. 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, Spain 2Sección Departamental Ciencias de la Alimentación, Universidad Autónoma de Madrid, Campus de Cantoblanco, 29049 Madrid, Spain

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

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

  4. Introduction HOP´S PRENYLFLAVONOIDS > 15 THERAPEUTICAL PROPERTIES Xanthohumol 8-prenylnaringenin Herbal supplements • The most potent phytoestrogen • Atherosclerosis • Inhibit HIV-1 and the replication of Plasmodium falciparum • Anticancer activity FUNCTIONAL FOODS Fortified food (enriched beer)

  5. Introduction Bitterness Functional properties Aroma Brewing Industry Beer Hop • Wort boiling (converts Xanthohumol into inactive isoxanthohumol) • The form in which hop bitterness and aroma is added Prenylflavonoid content SHOULD BE DETERMINED

  6. Objectives Extremely sensitive Robust and reliable Not affordable for all laboratories Not so fast HPLC-MS METHODS BIBLIOGRAPHIC REVISION 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. Detection conditions Column Mobile phase composition Temperature Injection volume Chromatographic conditions Sample treatment Solid phase extraction (SPE) Selectivity Linearity Accuracy Detection limit (LOD) Quantitation limit (LOQ) Validation of the method Scheme Application to samples

  8. Chromatographic conditions DAD-SPECTRA

  9. Chromatographic conditions • Chromatographic Column: ODS Spherisorb C185μ 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 SELECTIVITY No real blank of beer Check purity Compare DAD spectra Injected extracts of non spiked beer samples All samples with IXN and 8PN LOD&LOQ LOD 3xS/N LOQ 10xS/N Injected extracts of non spiked beer samples (n=20)

  14. Validation of the method LINEARITY R2 > 0.99 MATRIX-MATCHED CALIBRATION STANDARD CURVES (6 beers, 1 standard) IXN and 8PN STANDARD ADDITION METHOD Slope and intercept values within the same range for standards and beer samples Slope within the same range for standards and beer samples but different intercept value for each beer sample and standards Quantitation of IXN and 8PN with and specific calibration curve Quantitation of XN with a standard calibration curve

  15. Validation of the method %RSD< 5% PRECISION AND RECOVERY STUDIES Recoveries (75-98%) %RSD< 5%

  16. Application of the method Absence of XN Transformation into IXN Loss of the medical properties 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 IXN presence No influence of type and origin of the beer except for the alcoholic content Beer 1-2 (dark); Beer 3-5 (golden/pale); Beer 6 non alcohol

  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.

  18. 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

  19. 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, Spain 2Sección Departamental Ciencias de la Alimentación, Universidad Autónoma de Madrid, Campus de Cantoblanco, 29049 Madrid, Spain

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