A P P L I C AT I O N N O T E

Liquid Chromatography

Authors:
Andrew P. Zdzieblo
Wilhad M. Reuter
PerkinElmer, Inc.
Shelton, CT

The Qualitative and

Quantitative Analysis of Introduction

α-Acids in Hops and Beers by Alpha acids (α-acids) are a class of

chemical compounds of primary

UHPLC with UV Detection importance in the production of beer.

They are found in the resin glands of
the flowers of the hop plant (Humulus
lupulus) and are normally added to the
boil after mashing the grains, providing beers with their aroma and bitter taste.1
The α-acids found in hop resins are isomerized to form the iso-α-acids during prolonged boiling
in the wort. The degree of isomerization and the amount of bitter taste produced by the
addition of hops is highly dependent on the type of hop and the length of time the hops are
boiled. Longer boil times will result in isomerization of more of the available α-acids, making
the beer more bitter.2 The α-acid percentages vary within specific varieties of hops, depending
on the growing conditions, drying methods, age of hops, climate and other factors. Figure 1
shows the common α-acids and iso-α-acids involved in the beer brewing process.
 Since the quality and quantity of α-acids is so important in Solvents, Standards and Samples
consistently providing individual beers with their recognizable All solvents, reagents, and diluents used were HPLC-grade or ACS
taste, it is essential to monitor their amount in hops and beers grade and filtered via 0.45-µm filters. For all dilutions, 8:2
and to monitor the formation of the iso-α-acids during the beer methanol/0.1% trifluoroacetic acid (TFA) was used.
brewing process. The focus of this application note is to provide
The α-acids (ICE-3, Hops Extract Standard) and iso-α-acids
an easy, straightforward, and robust analytical method for
(DCHA-Iso, ICS-I3 Standard) were obtained from the American
establishing the type and amount of α-acids in hops pellets, as
Society of Brewing Chemists (ASBC), St. Paul, MN.3 ICE-3
well as determining the amount of α-acids and iso-α-acids in was reported to contain 13.88% cohumulone and 30.76%
various beers. humulone/adhumulone (N+adhumulone). ICS-I3 was reported
Method conditions and performance data, including linearity and to contain 62.3% iso-α-acids, including trans-isocohumulone,
repeatability, are presented. trans-isohumulone and trans-isoadhumulone, all in
dicyclohexylamine salt form.
The samples included three different hop pellets (English East,
Kent Goldings, American Simcoe and New Zealand Galaxy) and
four different beers from a colleague’s private stock (English Bitter,
India Pale Ale (Double IPA), Extra Special Bitter and Flanders Red.
Experimental
Standard Preparation
Two stock solutions, 7.3 mg/mL of ICE-3 standard (α-acids) and
α-acids cis/trans-iso-α-acids 1.4 mg/mL of ICS-I3 standard (iso-α-acids), were prepared as stock
cohumulone; R = CH(CH3)2 iso-cohumulone
humulone; R = CH2CH(CH3)2 iso-humulone solutions by transferring the appropriate weight of each standard
adhumulone; R = CH2(CH3)C2H5 iso-adhumulone into an individual 10-mL volumetric flask. Note: each initial
Figure 1. Isomerization path of hop α-acids to iso-α-acids during brewing, showing the standard contained different amounts of the individual
representative α-acids. analytes, reflected in the concentrations shown in Table 3.
6 mL of methanol was then added to each flask, which was
Experimental sonicated for 10 minutes and filled to volume with methanol.
Hardware/Software The ICE-3 stock solution was filtered through a 0.45 µm nylon
For the chromatographic separations, a PerkinElmer Altus™ filter; however, the ICS-I3 stock solution was not filtered due to
UPLC® System was used, including the Altus A-30 Solvent/ potential retention of iso-α-acids on the filter.3 Both stock
Sample Module, integrated vacuum degasser, A-30 column solutions were then transferred into clear glass containers and
heater, and Altus A-30 UV detector. For spectral confirmation, stored under refrigeration until further use. For the working
Altus-30 SQ and PDA (photodiode array) detectors were standard, 2 mL of the α-acids stock solution and 6 mL of the
used. All instrument control, analysis and data processing was iso-α-acids stock solution were transferred into a 25-mL volumetric
performed using the Waters® Empower® 3 Chromatography flask, which was then filled to volume with diluent. Six calibration
Data Software (CDS) platform. levels were prepared via serial dilution of the working standard
Method Parameters solution. Each standard level was injected in triplicate.
The LC method parameters are shown in Table 1. Hops Preparation
Each hop sample, coming in either pellets or coarse powder,
Table 1. LC Method Parameters.
was weighed (weight ranged from 0.2 g to 0.5 g) in duplicate
HPLC Conditions and crushed into a fine powder using a mortar and pestle.
PerkinElmer Brownlee™ SPP C18 2.7 µm, 3.0 x 100-mm
Column: The powdered samples were transferred into individual 50-mL
(Part# N9308410)
Mobile Phase A (MPA): 0.1% Phosphoric Acid (H3PO4) centrifuge tubes. Following, 6 mLs of diluent was added to each
plus 0.2 mM Disodium EDTA tube, which was then stored at room temperature to soak for four
Mobile Phase:
Mobile Phase B (MPB): Acetonitrile (ACN) hours. Every hour, each tube was vortex-mixed for 60 seconds.
Solvent mix: 35 % MPA/ 65% MPB (isocratic) Afterwards, the tubes were sonicated for 15 minutes in water and
Analysis Time: 6 min.
then filled to the 10-mL mark using diluent. All tubes were then
Flow Rate: 1.0 mL/min.
hand-shaken and centrifuged at 7000 rpm for 10 minutes. Each
Pressure: 4200 psi/280 bar (maximum)
Oven Temp.: 40 ºC supernatant was transferred into an individual 25-mL volumetric
Detection: 270 nm flask. 10 mL of diluent was then added into each centrifuge tube
Injection Volume: 4 µL containing the remaining precipitate, followed by vortex-mixing
Sampling (Data) Rate: 5 pts./sec for two minutes and centrifuging for 10 minutes at 7000 rpm.
The supernatant from each tube was then added to the
corresponding 25-mL flask. Finally, the flasks were filled to volume
with diluent, transferred into HPLC vials and injected in triplicate.

2
Beer Preparation
of nitrogen. The residue was re-dissolved in 2.0 mL of diluent,
The four beer samples were prepared by liquid-liquid extraction
transferred into HPLC vials and injected in triplicate.
(LLE). Duplicate 10-mL aliquots of each beer were transferred into
individual centrifuge tubes followed by the addition of 0.5 mL
Results and Discussion
of phosphoric acid and 10 mL of trimethylpentane. A blank
Using the optimized chromatographic conditions described above,
sample was prepared by substituting 10 mL of water for the
Figure 2 shows the HPLC separation of the level-4 working standard
10 mL of beer. The tubes were vortexed for one minute. To
containing three α-acids along with their three isomers, all well
diminish the layer of foam that developed between the water
resolved in less than four minutes.
and trimethylpentane layers, 1-2 mL of methanol was added to
the centrifuge tube. Subsequently, each tube was sonicated in Figure 3 shows the overlay of 12 replicate injections of the level-4
water for 15 minutes, followed by centrifuging for five minutes working standard containing α-acids and iso-α-acids, demonstrating
at 2000 rpm. 5 mLs of clear supernatant was then collected in high reproducibility. The retention time precision for all compounds
an evaporating dish and evaporated to dryness under a stream was < 0.18% RSD.

isohumulone
isocohumulone

0.30

0.25

humulone
0.20
cohumulone
AU

0.15
isoadhumulone

0.10
adhumulone

0.05

0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Figure 2. Chromatogram of the level-4 working standard, containing α-acids and iso-α-acids; by UV at 270 nm.

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Figure 3. Overlay of 12 replicates of level-4 working standard, by UV at 270 nm. 3

 100000

-100000
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00

A summary of the method performance, including %RSD, USP Figure 4 shows the calibration results for the six analytes (α-acids
220000.0
900000
200000.0

800000

tailing, USP plate count and USP resolution is presented in Table 2. and iso-α-acids). All six analytes had an exceptional linear fit, with
180000.0

700000 160000.0

R2 values > 0.9999 (n = 3 at each level).

600000 140000.0

The calibration set was based on a series of working standard

600000
120000.0

A r ea
500000
500000 900000
100000.0

dilutions. The individual analyte concentrations are shown for each

400000
Table 3. Concentrations of α-acids and iso-α-acids in working standard at each
80000.0
A r ea

800000
600000
400000
60000.0

level in Table 3.
300000
concentration level.
700000
40000.0
500000
300000
200000 600000
20000.0

Level 1 Level 2 Level 3 Level 4 Level 5 Level 6

Area

Compound
100000 0.0
500000
400000

(µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL)

200000
Table 2. Method performance.
0
-20000.0
400000

A r ea
-40000.0
300000

isocohumulone 3.64 7.28 14.6 21.8 36.4 72.8

100000
-100000 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

RT Mean USP Mean USP USP

300000

Area
Peak Name 0.00 10.00 20.00 %RSD
30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00

(min) Tailing Plate Count Resolution

200000
200000
0
isohumulone
100000
5.35 10.7 21.4 32.1 53.5 107
isocohumulone
-100000
0.00
1.582
10.00
0.150
20.00 30.00
1.04 40.00 50.00
15645 60.00
-----
70.00
isoadhumulone
100000
0 1.40 2.79 5.59 8.38 14.0 27.9
isohumulone 2.031 0.165 1.06 17172 7.98 cohumulone
-1000000
4.39 8.77 17.5 26.3 43.9 87.7
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00

isoadhumulone 2.203 0.164 0.98 15551 2.59 humulone

-100000

500000 0.00 10.00

7.48 20.00
14.930.00
29.9
40.00 50.00
44.9 60.00
74.8 70.00
149
cohumulone 220000.0
2.662 0.176 1.00 17986 6.13 adhumulone 2.24 4.49 8.98 13.5 22.4 44.9
400000
humulone 200000.0
3.472 0.176 1.02 18893 8.97 Total iso-α-acids 10.4 20.8 41.6 62.3 103 207
180000.0

adhumulone 160000.0 3.725 0.177 0.96 18431 2.40 Total α-acids

300000
14.1 28.2 56.4 84.6 141 282
140000.0

A r ea
900000

120000.0 200000
800000 220000.0
A r ea

100000.0
200000.0
700000
80000.0
100000
180000.0
60000.0 900000
600000
600000
160000.0
40000.0
500000 isocohumulone 800000
140000.0
0 isohumulone
20000.0
500000
120000.0
700000
400000
A r ea

0.0

A r ea
100000.0
-20000.0 600000
400000
300000 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00
80000.0
-40000.0 500000
200000 60000.0
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
300000
40000.0
400000

A r ea
100000
Area

20000.0
2000000 300000
0.0
200000
-100000 -20000.0
100000
-40000.0
100000
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

0 0

R = 0.99997
2
-100000
800000
R = 0.99998
2
-100000
500000
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00
700000

400000 Concentration (µg/mL) Concentration (µg/mL)

600000

500000
300000

220000.0
isoadhumulone 500000
400000
cohumulone
A r ea

A r ea

200000.0
200000
300000
180000.0
400000
160000.0 200000
100000 220000.0
140000.0
100000
300000
200000.0
900000
120000.0
0 180000.0
A r ea

A r ea

100000.0 0
800000
160000.0
200000
80000.0
-100000
700000 140000.0
60000.0
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00
600000 120000.0 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00
40000.0 100000
A r ea

100000.0
20000.0
500000
80000.0
0.0 0
400000
A r ea

R2 = 0.99997 R2 = 0.99999
60000.0
-20000.0

300000 40000.0
-40000.0
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 20000.0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00
200000

100000
Concentration (µg/mL) 0.0
Concentration (µg/mL)
-20000.0

0 -40000.0
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
800000
-100000 240000.0
humulone 220000.0
adhumulone
700000 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00
200000.0

600000 180000.0

500000 160000.0
500000
140000.0
800000
400000 120000.0
A r ea
A r ea

400000
100000.0
700000
300000
500000
80000.0
300000 600000
200000 60000.0

40000.0
500000
A r ea

220000.0 400000
100000
200000 20000.0
200000.0 400000
0 0.0
A r ea

180000.0
R2 = 0.99999 R2 = 0.99998
300000
-20000.0
300000
100000
-100000
160000.0
-40000.0
A r ea

140000.0 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 200000 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
200000
120000.0
0
Concentration (µg/mL) 100000 Concentration (µg/mL)
A r ea

100000.0
100000
80000.0 0

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00

Figure 4. Results of 6-level calibration set for α-acids and iso-α-acids.

60000.0

40000.0
-100000
0

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00

20000.0

0.0
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00
-20000.0

-40000.0
240000.0 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 1.6x106

220000.0
1.4x106
200000.0

180000.0
1.2x106
800000
160000.0
1.0x106
140000.0
700000
240000.0
120000.0
8.0x105
A r ea

600000 220000.0
A r ea

100000.0
800000 5
200000.0
80000.0 6.0x10
500000
500000 180000.0
60000.0 700000 5
4.0x10
400000 160000.0
40000.0
A r ea

140000.0
600000 5
400000
20000.0 2.0x10
300000
120000.0
0.0
A r ea

500000
0.0
100000.0
200000
-20000.0
300000
80000.0
400000
-40000.0 -2.0x105
A r ea

100000
60000.0
A r ea

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
300000 0.00 50.00 100.00 150.00 200.00 250.00
200000 40000.0
0
20000.0
200000

4 -100000
100000
0.0
100000
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 -20000.0

-40000.0
0
0 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
Table 4 presents the estimated limits of quantitation and detection Table 4. Calculated estimated LOQ and estimated LOD for α-acids and iso-α-acids.
(LOQ, LOD) for all tested α-acids and iso-α-acids. These limits were LOD (µg/mL) LOQ (µg/mL)
Compound
derived using the signal-to-noise (s/n) results obtained during (s/n ≥ 3/1) (s/n ≥ 10/1)
calibration, using an average of three replicates per level. cohumulone 0.09 0.30
humulone 0.13 0.42
As can be seen, the LODs and LOQs for the three iso-α-acids are
quite similar. Also, they are over twice as low as those of the three adhumulone 0.19 0.46

α-acids. isocohumulone 0.04 0.14

isohumulone 0.05 0.17
By applying our chromatographic conditions, we were able to
isoadhumulone 0.06 0.20
separate all six α-acids/iso-α-acids that were analyzed.
347.29
Figure 5 shows the UV spectrum and corresponding mass
0.22
197.7
mass of humulone and adhumulone (negative ion mode). Based
1.0x10 7 347.29
197.7 1.0x10 7
0.20
spectrum for the peaks eluting at 3.47 and 3.73 minutes. The on the above, as well as the elution order of isohumulone and
0.22

0.18 8.0x10 6
0.20
8.0x10 6
UV spectra of both peaks are very similar and, as the peak at
0.16
0.18

236.6
isoadhumulone, it is likely that the peak eluting at 3.47 minutes is
6.0x10 6

Intensity
0.14
0.16

3.47 minutes is known to contain humulone, this suggests that humulone and the peak eluting at 3.73 minutes is adhumulone.
6.0x10 6

Intensity
0.12
0.14 236.6
AU

0.10
0.12 284.1 4.0x10 6
AU

322.3
the peak at 3.73 minutes is also related to humulones. The mass
0.08
0.10 284.1
322.3
Having access to pure individual standards of humulone and
4.0x10 6

0.06
0.08 2.0x10 6

spectra of both peaks are identical and match the expected M-H
0.04
0.06 adhumulone would help to confirm the identity of these peaks.
2.0x10 6

0.02
0.04 0.0
0.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00
0.02 0.0 m/z
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00
0.00 nm m/z
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm

Apex UV spectrum of peak at 3.47 min. Mass spectrum of peak at 3.47 min.
0.30
197.7 1.2x107 361.34
0.30
197.7 1.2x107 361.34
0.25
1.0x107
0.25
1.0x107
0.20
8.0x106
236.6
0.20 8.0x106
Intensity

236.6
AU AU

0.15
6.0x106
Intensity

0.15 285.3 323.5 6.0x106

0.10 4.0x106
285.3 323.5
0.10 4.0x106

0.05 2.0x106

0.05 2.0x106

0.00 0.0
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00
0.00 nm 0.0 m/z
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
m/z
nm

197.7 Apex UV spectrum of peak at 3.73 min. 4.5x106 Mass spectrum of peak at 3.73 min.
361.35
0.070 197.7 4.5x106 361.35
4.0x106
0.070
0.060 4.0x106
3.5x106

0.060 3.5x106
0.050 3.0x106
236.6
0.050 3.0x106
2.5x106
Intensity
AU AU

0.040
236.6
2.5x106
Intensity

0.040 285.3 2.0x106

0.030 322.3
285.3 2.0x106
322.3 1.5x106
0.030
0.020
1.5x106
1.0x106
0.020
0.010 1.0x106
5.0x105
0.010
0.000 5.0x105
0.0
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00
0.000 nm 0.0 m/z
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00
nm m/z

Figure 5. The UV spectra and corresponding mass spectra (neg. ion mode) of the peaks eluting at 3.47 min. and 3.73 min.

5
 Figure 6 shows the chromatograms for each of the three different hops samples. Comparing the chromatograms, there are clear differences
between each hops sample. Also, of note is that they all show very low iso-α-acids content, compared to the α-acids, as expected.

0.50
0.50
0.50
GALAXY

cohumulone
0.45
0.45

humulone
0.45
0.40
0.40
0.40
0.35
0.35
0.35
0.30
0.30
0.30
AUAUAU

0.25
0.25
0.25

iso-adhumulone

adhumulone
0.20

iso-humulone
0.20
0.20
0.15
0.15
0.15
0.10
0.10
0.10
0.05
0.05
0.05
0.00
0.00
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Minutes 3.50 4.00 4.50 5.00 5.50 6.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Minutes 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

1.10
1.10
1.10
SIMCOE

humulone
1.00
1.00
1.00
0.90
0.90
0.90
0.80
0.80
0.80
0.70
0.70
0.70
cohumulone

0.60
0.60
0.60
AUAUAU

0.50
0.50
0.50
isoadhumulone

adhumulone
0.40
iso-humulone

0.40
0.40
0.30
0.30
0.30
0.20
0.20
0.20
0.10
0.10
0.10
0.00
0.00
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Minutes 3.50 4.00 4.50 5.00 5.50 6.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Minutes 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

EAST KENT
humulone

0.50
0.50
0.50

GOLDINGS
cohumulone

0.45
0.45
0.45
0.40
0.40
0.40
0.35
0.35
0.35
0.30
0.30
0.30
AUAUAU

0.25
adhumulone
iso-cohumulone

0.25
0.25
iso-humulone
iso-adhumulone

0.20
0.20
0.20
0.15
0.15
0.15
0.10
0.10
0.10
0.05
0.05
0.05
0.00
0.00
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Minutes 3.50 4.00 4.50 5.00 5.50 6.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Minutes 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Figure 6. Chromatograms of the three analyzed hop species.

The quantitative results of the three analyzed hops samples are Table 5. Quantitative results for α-acids and iso-α-acids in the four different hops samples.
presented in Table 5, reflecting an average of two sample Concentration (mg/g) in 3 species of Hops
Compound
preparations and three injections per sample. As expected, the Galaxy Simcoe East Kent Goldings
concentrations of α-acids vary from hop to hop species. cohumulone 15.9 11.1 9.12
Although the proportions of α-acids in both Simcoe and East humulone 19.7 45.4 21.5
Goldings hops are similar, the Simcoe hop contains about twice adhumulone 4.67 6.93 4.62
the overall amount of α-acids. In addition, the concentration of isocohumulone ND ND NQ
iso-α-acids in each of the hops is very low or not detected, which isohumulone 0.18 NQ 0.17
is to be expected as the iso-α-acids only arise as part of the isoadhumulone 0.28 0.25 0.46
brewing process. Total α-acids 40.3 63.5 35.2
Total iso-α-acids 0.46 0.25 0.63
ND = not detected; NQ = detected, but not quantitatable
6
Figure 7 shows the chromatograms of the four different beer samples, with all six analytes well resolved. The amount of α-acids
in Double IPA beer is similar to the iso- α-acids, while in Extra Special Bitter, the α-acid amounts are much lower in comparison.
Of particular note is the apparent absence of α-acids in English Bitter and in Flanders Red beers.
0.080

0.080
0.070
0.080
0.070
0.080
0.060
0.070
DOUBLE IPA

isoadhumulone
0.060
0.070
0.050

isocohumulone
0.060
0.050
0.060
0.040
AU AU AU AU

cohumulone
isohumulone
0.050
0.040
0.050
0.030
0.040

humulone
0.030
0.040
0.020
0.030
0.020
0.030
0.010

adhumulone
0.020
0.010
0.020
0.000
0.010
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
0.000
0.010 Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
0.000 Minutes
0.0000.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

0.030

0.030

0.025
0.030 ENGLISH BITTER
isoadhumulone

0.025
0.030

0.020
0.025
isocohumulone

0.020
0.025
AU AU AU AU

0.015
0.020

0.015
0.020
isohumulone

0.010
0.015
cohumulone

0.010
0.015

0.005
0.010

0.005
0.010

0.000
0.005

0.0000.00
0.005
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
0.000
Minutes
0.0000.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.028

0.026
0.028
isoadhumulone

0.024
0.026
0.028 EXTRA SPECIAL
0.022
0.024
0.028
0.026
0.020
BITTER
0.022
0.026
0.024
0.018
isohumulone

0.020
0.024
0.022
isocohumulone

0.016
0.018
0.022
0.020
0.014
0.016
AU AU AU AU

0.020
0.018
0.012
0.014
0.018
0.016
0.010
0.012
cohumulone

0.016
0.014
0.008
0.010
0.014
0.012
0.006
humulone

0.008
0.012
0.010
0.004
0.006
0.010
0.008
0.002
0.004
0.008
0.006
0.000
0.002
0.006
0.004
0.0000.00
0.004
0.002
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.0020.00
0.000 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.0000.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.0055

0.0055
0.0050 FLANDERS RED
0.0055
0.0050
0.0045
isoadhumulone

0.0055
0.0050
0.0045
0.0040

0.0050
0.0045
0.0040
0.0035

0.0045
0.0040
0.0035
0.0030

0.0040
AU AU AU AU

0.0035
0.0030
0.0025

0.0035
0.0030
0.0025
0.0020

0.0030
0.0025
0.0020
0.0015

0.0025
0.0020
0.0015
0.0010

0.0020
0.0015
0.0010
0.0005

0.0015
0.0010
0.0005
0.0000

0.0010
0.00050.00
0.0000 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.0005
0.00000.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
0.0000
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

Figure 7. Chromatograms
0.00 of the 0.50
four analyzed1.00
beers. 1.50 2.00 2.50
Minutes
3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
7
Table 6. Quantitative results for α-acids and iso-α-acids in four different beers. Conclusion
Extra Special Flanders Red
English Bitter Double IPA
Compound Bitter 2014 The results obtained confirm the applicability of this method for the
µg/mL µg/mL µg/mL µg/mL efficient, routine, and robust chromatographic analysis of the six
cohumulone NQ 5.51 0.66 ND investigated α-acids including their isomers. All compounds are
humulone ND 5.90 0.50 ND completely separated in under six minutes by UHPLC® using UV
adhumulone ND 1.02 ND ND detection. The results showed excellent retention time repeatability
isocohumulone 0.90 2.94 0.74 ND as well as exceptional linearity over the tested concentration ranges.
isohumulone 0.69 3.00 1.13 ND Thereupon and considering the very lower LOQ levels for these
isoadhumulone 2.03 6.94 2.57 0.23 α-acids and their corresponding isomers, this application can be
Total α-acids ND 12.4 1.16 ND
considered very effective for the monitoring of these acids during
Total iso-α-acids 3.62 12.9 4.44 0.23
beer production.
NQ = detected, but not quantitatable; ND = not detected
References
The results for the analysis of the four tested beers are presented
1. [Online]. Available: https://en.wikipedia.org/wiki/Alpha_acid.
in Table 6 and reflect an average of two sample preparations and
[Accessed 12 06 2015].
three injections per sample. The concentration and proportion
of α-acids and iso-α-acids differ markedly from beer to beer. 2. International Calibration Standards, American Society of Brewing
Compared to the concentrations found in hops, the concentrations Chemist, 2009.
of iso-α-acids are much higher. This was to be expected, as these 3. ASBC, Method of Analysis of the American Society of Brewing
acids increase markedly during the brewing process.5 Chemists, St. Paul: American Society of Brewing Chemists, 1996.
4. The Technical Committee and the Editorial Committee of the
ASBC, "Iso-a-, a-, and b-acids in Hop extracts and isomerized
Hop extracts by HPLC," in Methods of Analysis of the American
Society of Brewing Chemists, St. Paul, American Society of
Brewing Chemists, 2009, pp. 1-4, chapter Hops-16.
5. S. Hieronymus, For the Love of Hops. The practical Guide to
Aroma, Bitterness and the Culture of Hops, Boulder: Brewers
Publications, 2012

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