Identification of Hop-Derived Bitter Compounds in Beer

Identification of Hop-Derived Bitter
Compounds in Beer Using LC-HR-MS/MS
André Schreiber
SCIEX, Concord, Ontario (Canada)
RUO-MKT-11-4016-A For Research Use Only. Not for use in diagnostic procedures.
Overview – 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
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Hop-Derived Bitter Compounds*
Xanthohumol
H3C
Isoxanthohumol
CH3
H3C
OH
HO
OH
wort-boiling
OH
O
HO
O
CH3
O
OH
O
trans- and cis-iso-α-acids
(such as isocohumulone and isohumulone)
CH3
CH3
CH3
CH3
H3C
H3C
CH3
O
OH
O
OH
HO
CH3
O
H3C
CH3
H
HO
O
O
H3C
CH3
O
O
H3C
α-acids (such as cohumulone and humulone)
OH
O
O
H3C
CH3
CH3
CH3
H
O
O
HO
CH3
OH
H3C
HO
OH
H3C
H3C
H3C
H3C
β-acids (such as colupulone)
H3C
CH3
CH3
CH3
CH3
O
O
H3C
O
CH3
H
CH3
OH
O
HO
H3C
CH3
O
OH
HO
OH
CH3
HO
O
H3C
H3C
H3C
CH3
CH3
CH3
3
CH3
H3C
* D. Intelmann, G. Haseleu, and Th. Hofmann: J. Agric. Food Chem. 57 (2009) 1172-1182
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CH3
H
CH3
O
CH3
O
H3C
Experimental
• Store-bought beer samples
‒ Degassed and diluted 2x with water
• ExionLC™ AD system
‒
‒
‒
‒
Phenomenex Luna Omega 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 QTOF 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
‒ TOF-MS 100-1000 Da (200 msec) DP = 80 V
‒ 10x MS/MS 50-1000 Da (50 msec) CE = 35 V and CES = 15 V
‒ Dynamic background subtraction on with intensity threshold of 10 cps
• SCIEX OS Software for data acquisition and processing
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SCIEX X500R QTOF System powered by SCIEX OS Software
SCIEX X500R QTOF system
SCIEX ExionLC™ AD system
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SCIEX OS software
Profiling of 26 Hop-Derived Bitter Compounds using HR-MS
Retention times were
established by evaluating
data of ~40 beer samples.
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Identification based on mass error, isotope ratio and RT matching.
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Xanthohumol (X) and Isoxanthohumol (IX) and X/IX Ratio
IX
IX
IX
X
X/IX = 0.021
X
0.169
X
0.247
German pilsner
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German Weissbier
Dry-hoped IPA
Xanthohumol and Isoxanthohumol in Different Beers
X/IX Ratio Depends on Boiling Conditions and Time of Hop Addition
India Pale Ale and Pale Ale
4.00E+06
Stout
3.50E+06
3.00E+06
2.50E+06
2.00E+06
German and Czech Pilsner
American
Lager
Isoxanthohumol
Xanthohumol
1.50E+06
X/IX ratio
1.00E+06
Weissbier
5.00E+05
Sgu
WHP
SCo
Pwe
Pwa
PUQ
Pra
PSt
PKr
Pje
Pko
PHP
Pbi
PCz
Pbe
LPA
IRU
LAS
Ioc
IMT
Iim
IMD
IHV
IHR
IHSt
IGI
IHB
IDE
Ibo
ID9
IbL
I10
Bta
Bur
Aco
BBm
Abu
ACL
Ablu
ABL
0.00E+00
1.00E+00
9.00E-01
8.00E-01
7.00E-01
6.00E-01
Isoxanthohumol
5.00E-01
Xanthohumol
4.00E-01
X/IX ratio
3.00E-01
2.00E-01
1.00E-01
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WHP
Sgu
SCo
Pwe
Pwa
PUQ
PSt
Pra
PKr
Pje
Pko
PHP
PCz
Pbi
Pbe
LPA
IRU
LAS
Ioc
IMT
IMD
Iim
IHV
IHR
IHSt
IHB
IGI
IDE
ID9
Ibo
IbL
I10
Bta
Bur
Aco
© 2016 AB Sciex
BBm
ACL
Abu
Ablu
ABL
0.00E+00
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
humulone
cis-iso
cohumulone
trans-iso
cohumulone
trans-iso
humulone
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cohumulone
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
humulone
Structural Evaluation using HR-MS/MS in SCIEX OS
TOF-MS/MS
ChemSpider search and in-silico fragmentation of Isoxanthohumol
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Add HR-MS/MS Spectrum to Library in SCIEX OS
TOF-MS/MS
HR-MS/MS spectra can be added to mass spectral libraries in SCIEX OS software.
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Increasing Confidence in Identification using HR-MS/MS
XIC
TOF-MS
MS/MS
Spectra acquired in TOF-MS-IDA-MS/MS mode are automatically searched against
the MS/MS library and flagged by evaluating the library FIT.
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Summary
• Profiling of hop-derived bitter compounds in beer using LC-HRMS/MS
• Use SCIEX X500R QTOF system with SCIEX OS software
‒ High resolution TOF-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
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For Research Use Only. Not for use in diagnostic procedures.
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Thank you for your attention!
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