Identification of Hop-Derived Bitter Compounds in Beer Using LC-HR-MS/MS

Transcription

1 Identification of Hop-Derived Bitter Compounds in Beer Using LC-HR-MS/MS André Schreiber SCIEX, Concord, ntario (Canada) RU-MKT A For Research Use nly. Not for use in diagnostic procedures.

2 verview Hop-Derived Bitter Compounds in Beer Beer has been attracting consumers over centuries due to its Refreshing character Attractive aroma Typical bitter taste. Typical beer bitterness is caused by adding cones, pellets, or extracts of hops during wort boiling. During the wort-boiling process, a number of isomerization processes have been identified to be of major importance for bitter taste development in the final beer. Xanthohumol Isoxanthohumol α-acids trans- and cis-iso-α-acids β-acids AB Sciex

3 Hop-Derived Bitter Compounds* Xanthohumol Isoxanthohumol H H H wort-boiling H H C H 3 C H 3 α-acids (such as cohumulone and humulone) H H trans- and cis-iso-α-acids (such as isocohumulone and isohumulone) H H H H H H H H H H C H 3 CH3 C H 3 CH3 β-acids (such as colupulone) H H H H H H H H C H 3 CH3 C H 3 CH AB Sciex * D. Intelmann, G. Haseleu, and Th. Hofmann: J. Agric. Food Chem. 57 (2009)

4 Experimental Store-bought beer samples Degassed and diluted 2x with water ExionLC AD system Phenomenex Luna mega 1.6 µm Polar C18 (50 x 2.1 mm) Gradient of water/methanol + 5 mm ammonium formate Flow 0.5 ml/min Injection of 5 µl SCIEX X500R QTF system Turbo V source with ESI probe, TEM = 450ºC Mass calibration using the integrated calibrant delivery system (CDS) with the TwinSprayer probe (dual ESI needle). Information dependent acquisition (IDA) in negative polarity TF-MS Da (200 msec) DP = 80 V 10x MS/MS Da (50 msec) CE = 35 V and CES = 15 V Dynamic background subtraction on with intensity threshold of 10 cps SCIEX S Software for data acquisition and processing AB Sciex

5 SCIEX X500R QTF System powered by SCIEX S Software SCIEX X500R QTF system SCIEX ExionLC AD system SCIEX S software AB Sciex

6 Profiling of 26 Hop-Derived Bitter Compounds using HR-MS Retention times were established by evaluating data of ~40 beer samples AB Sciex Identification based on mass error, isotope ratio and RT matching.

7 Xanthohumol (X) and Isoxanthohumol (IX) and X/IX Ratio IX IX IX X X/IX = X X German pilsner German Weissbier Dry-hoped IPA AB Sciex

8 Xanthohumol and Isoxanthohumol in Different Beers X/IX Ratio Depends on Boiling Conditions and Time of Hop Addition 4.00E E+06 India Pale Ale and Pale Ale Stout 3.00E E E E E E+05 American Lager German and Czech Pilsner Weissbier Isoxanthohumol Xanthohumol X/IX ratio 0.00E+00 ABL Ablu Abu ACL Aco BBm Bta Bur I10 IbL Ibo ID9 IDE IGI IHB IHR IHSt IHV Iim IMD IMT Ioc IRU LAS LPA Pbe Pbi PCz PHP Pje Pko PKr Pra PSt PUQ Pwa Pwe SCo Sgu WHP 1.00E E E E E E E E-01 Isoxanthohumol Xanthohumol X/IX ratio 2.00E E E+00 ABL Ablu Abu ACL Aco BBm Bta Bur I10 IbL Ibo ID9 IDE IGI IHB IHR IHSt IHV Iim IMD IMT Ioc IRU LAS LPA Pbe Pbi PCz PHP Pje Pko PKr Pra PSt PUQ Pwa Pwe SCo Sgu WHP AB Sciex

9 Peak Areas of trans- and cis-iso-α-acids and α-acids Czech pilsner (trans/cis = 0.06 and iso-α/α = 121) cis-iso cohumulone cis-iso humulone trans-iso cohumulone trans-iso humulone cohumulone humulone AB Sciex trans/cis = 0.02 to 0.15 (American Lager < Pilsner ~ IPA) iso-α/α = 20 to >200 (IPA < American Lager ~ Pilsner) Highest iso-α/α in Irish stout

10 Structural Evaluation using HR-MS/MS in SCIEX S TF-MS/MS ChemSpider search and in-silico fragmentation of Isoxanthohumol AB Sciex

11 Add HR-MS/MS Spectrum to Library in SCIEX S TF-MS/MS HR-MS/MS spectra can be added to mass spectral libraries in SCIEX S software AB Sciex

12 Increasing Confidence in Identification using HR-MS/MS XIC TF-MS MS/MS Spectra acquired in TF-MS-IDA-MS/MS mode are automatically searched against the MS/MS library and flagged by evaluating the library FIT AB Sciex

13 Summary Profiling of hop-derived bitter compounds in beer using LC-HR- MS/MS Use SCIEX X500R QTF system with SCIEX S software High resolution TF-MS and MS/MS data acquired in IDA mode Target peak finding based on molecular formula (suspect screening) Automatic identification using mass accuracy and isotope pattern, retention times of isomers were assign by comparing results of beer samples with literature value After structural elucidation HR-MS/MS spectra were added to a spectral library for future work 40 beer samples were analyzed Xanthohumol / Isoxanthohumol ratio was compared to conditions of beer making for different styles of beer (i.e. pilsner vs. IPA) Trans / cis ratio and iso-α / α ratio was evaluated for different beers AB Sciex

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15 AB Sciex Thank you for your attention!