Journal of the American Society of Brewing Chemists

The Science of Beer

ISSN: 0361-0470 (Print) 1943-7854 (Online) Journal homepage: https://www.tandfonline.com/loi/ujbc20

Determination of 3-Methyl-2-Butene-1-Thiol in
Beer

Henry Goldstein, Sydney Rader & Aki A. Murakami

To cite this article: Henry Goldstein, Sydney Rader & Aki A. Murakami (1993) Determination of
3-Methyl-2-Butene-1-Thiol in Beer, Journal of the American Society of Brewing Chemists, 51:2,
70-74, DOI: 10.1094/ASBCJ-51-0070

To link to this article: https://doi.org/10.1094/ASBCJ-51-0070

Published online: 06 Feb 2018.

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Determination of 3-Methyl-2-Butene-l-Thiol in Beer
Henry Goldstein, Sydney Rader, and Aki A. Murakami, Miller Brewing Company, Research Department, Milwaukee,
WI53208
ABSTRACT The beer was extracted with a flow of high-purity helium for
90 min at a temperature of 40° C and a flow rate of 600 ml/
Three-methyl-2-butene-l-thiol, the compound responsible for the light- min. The gas and non-thiol organics passed through the thiol
struck character in beer, was determined at levels below 1 ng/ L in beer. trap while thiols were trapped in the Hg (II) cyanide-containing
The sensory threshold in American lagers is 1-5 ng/L. Beer was first glass wool (Fig. 2).
gas extracted, thiols were trapped on an Hg (II) cyanide trap, and thiols
were freed from the trap with acid and extracted into dichloromethane. The glass wool containing the complexed thiols was carefully
An aliquot of the dichloromethane thiol solution then was injected (on- transferred to a 10-ml vial that contained 5 mg of dithiothreitol
column injection) into a gas chromatograph. Detection was by flame (1) (to prevent oxidation of the thiols), 5 ml of hydrochloric acid
ionization, flame photometry, mass spectrometry, or sulfur chemilumines- solution, and 200 n\ of dichloromethane. It is very important
cence detectors. to use 10-ml "serum bottles" (No. 037-754, Curtin Matheson
Keywords: Light-struck, 3-Methyl-2-butene-l-thiol, Mercaptan, Thiol Scientific, Houston, TX) because their geometry is suited to the
extraction, centrifugation, and phase separation described below.
Through the investigations of Kuroiwa and Hashimoto (5), The contents of the vial were sealed with two Teflon-lined septa
3-methyl-2-butene-l-thiol (3M2B1T) was determined to be the (Curtin Matheson No. 386-227) and mixed on a vortex mixer
primary cause of the light-struck character in beer. A simple to extract the thiols into the dichloromethane phase. The phases
mechanism for the formation of this compound was proposed then were separated by centrifuging the vial in an inverted position
(1,2,6,7,9) and has been generally accepted. According to this to bring the organic layer into the neck of the vial (Fig. 3). The
view, the carbon skeleton of 3M2B1T is derived from the iso- organic layer was removed with a 25-jul syringe, while care was
hexenoyl side chain of the iso-a-acids.
Despite the practical and theoretical importance of 3M2B1T
$ 29/42 inner joint
in beer, analytical observations of the compound have been few
(1,2,8,12) and, for the most part, qualitative and at levels far
above the sensory threshold (13). It was to remedy the lack of
a specific quantitative analytical method for 3M2B1T that the A -r
present work was undertaken. To be useful, such a quantitative
method would have to be accurate and precise both at and below
the sensory threshold in beer. At the time that this work was $ 29/42
outer joint
initiated, the sensory threshold of 3M2B1T (in beer) was reported
as 50-3,200 ng/L (10,11,14), but our preliminary research indi-
cated the threshold for 3M2B1T in beer was much less than 50
ng/L, although at the time we did not know how much less.
For this reason, we set out to develop an analytical procedure
for 3M2B1T in beer at levels between 1 and 10 ng/L.
The analytical approach that was eventually chosen was modeled
after Knarr and Rappaport's (4) determination of methanethiol 7.6 cm i.d. 128 cm
in air. Some of the key elements of the working method are 8.3 cm o.d 123 cm
large sample size, isolation of thiols from most other organics,
concentration of the thiols, gas chromatographic analysis, sensi-
tive and selective detection, and use of an internal standard thiol.

EXPERIMENTAL

Summary of Experimental Method

A gas washing bottle (Fig. 1) was filled with 4.26 L of beer,
three to four drops of tributyl phosphate (to prevent overfoaming),
the internal standard working solution, and/ or 3M2B1T standard.

® 1993 American Society of Brewing Chemists, Inc. Fig. 1. Gas extraction apparatus.

taken not to remove any of the aqueous layer. A 10-jul syringe,
fitted with a fused silica needle (to prevent chemical reactions
with metal), was filled to the 1- to 2-/xl mark from the back
end of the 25-jul syringe. On-column injection into the gas
chromatograph immediately followed.
Reagents
Dichloromethane was high-performance liquid chromatog-
raphy (HPLC) glass-distilled grade supplied by EM Science
(Gibbstown, NJ). Methanol was HPLC reagent grade supplied
by Baker (Philipsburgh, NJ). Hg (II) cyanide was 99% pure
supplied by Aldrich (Milwaukee, WI) and dissolved in methanol
(1 g/100 ml). Hydrochloric acid was reagent grade diluted to
25% by weight and saturated with sodium sulfate (99% reagent
grade) and washed several times with dichloromethane to remove
any organics that might be present.
Dithiothreitol was 99% pure and supplied by Aldrich (Mil-
waukee, WI). 3M2B1T (neat) was supplied by Parish (Orem, UT),
and 3M2B1T stock solution (2.13 X 10' ng/L) was prepared by
adding 0.24 ml of neat 3M2B1T to a 100-ml volumetric flask
containing about 50 ml of dichloromethane and diluting to the
mark with dichloromethane. 3M2B1T intermediate solution (2.13
X 107 ng/L) was prepared by adding 1.00 ml of the stock solution
to a 100-ml volumetric flask containing about 50 ml of methanol
and diluting to the mark with methanol. 3M2B1T working
solution (2.13 X 105 ng/L) was prepared by adding 1.00 ml of
the intermediate solution to a 100-ml volumetric flask containing
about 50 ml of methanol and diluting to the mark with methanol.
1-Hexanethiol (neat) was supplied by Fluka (Ronkonkoma, NY).
1-Hexanethiol stock solution (2.12 X 10' ng/L) was prepared
by adding 0.26 ml of neat 1-hexanethiol to a 100-ml volumetric
flask containing about 50 ml of dichloromethane and diluting
to the mark with'dichloromethane. 1-Hexanethiol intermediate
solution (2.12 X 107 ng/L) was prepared by adding 1.00 ml of
the stock solution to a 100-ml volumetric flask containing about
50 ml methanol and diluting to the mark with methanol. 1-
Hexanethiol working solution (2.12 X 10s ng/L) was prepared
by adding 1.00 ml of the intermediate solution to a 100-ml
volumetric flask containing about 50 ml of methanol and diluting
to the mark with methanol. Tributyl phosphate was obtained
from Aldrich (Milwaukee, WI). Silane-treated glass wool was
obtained from Supelco (Bellefonte, PA).
Finally, a word of caution concerning the handling of 3M2B1T.
The threshold in air for 3M2B1T is extremely low. Although
it is easy to synthesize this compound, we have never attempted
it. If an outside source for this material is available (see Experi-
mental for one source), purchase it from this source. Neat and
especially concentrated solutions (stock solution) should be
handled with extreme care.
Pyrex Tube
8mm O.D.
6mm i.d. Tygon Sleeve
II >
Thiol Trap Silanized Glass Wool
10 cm long 35-40mg
He
29/42 Outer Joint
Fig. 2. Gas extraction apparatus head, including Hg (II) cyanide trap.
ASBC Journal 71
Apparatus
The extraction of the beer with gas was carried out in a 5-
L gas washing bottle (Fig. 1). The body of the washing bottle
was 123 cm tall, 7.6 cm i.d., 8.3 cm o.d., and contained an inner
$29/42 joint (Pope Scientific, Menomonee Falls, WI). The gas
extraction bottle was fitted with a gas dispersion tube (6 mm
i.d., 8 mm o.d.) with a Pyrex frit (ASTM 40-60). A $29/42 ground
glass outer joint was fitted appropriately at the top of the gas
dispersion tube. The length of the gas dispersion tube, from the
top of the joint to the bottom of the frit, was 128 cm. The thiol
trap consisted of a piece of 10-cm-thick borosilicate glass tube
(8 mm o.d., 6 mm i.d.). Through one end of the tube, 35 mg
of silanized glass wool wetted with 100 ;ul of Hg (II) cyanide
solution was inserted. The other end of the tube was attached
by a Tygon sleeve or a standard taper joint to the outlet portion
of the gas dispersion tube.
Chromatography and Detection
For gas chromatography/flame ionization detector (GC/FID)
analysis, a Hewlett Packard HP 5890A GC equipped with an
FID, cool on-column injector, and an HP 3396A integrator were
used. The column was a DBS fused silica capillary column (J&W
Scientific, Folsom, CA) 30 m in length, 0.324 mm i.d., and 0.25
/im thick. The helium carrier gas was set at a head pressure of
7.5 psi and a vent flow of 60 ml/min. This gave a carrier flow
velocity of 29 cm/sec. The oven was set at 28° C for 3 min and
then temperature programmed to 60° C at a rate of 4°C/min
and continued to a final temperature of 175°C at a rate of 10°C/
min. The final oven time was 16 min. The FID temperature was
set at 250° C. Two microliters of the extract was injected. Under
these conditions, the 3M2B1T was expected to elute at 9.2 min
and the 1-hexanethiol at 13.0 min. Peak integration parameters
were optimized before the expected elution time of the 3M2B1T
peak to help facilitate accurate integration of this peak.
For GC/flame photometric detector (FPD) analysis, the same
conditions used for the GC/FID above were used except that
the FPD temperature was 180°C.
For the GC/sulfur chemiluminescence analysis, a Hewlett
Packard HP 5890 Series II GC equipped with an FID, a cool
on-column injector, an electronic flow controller, and a Sievers
350B sulfur chemiluminescence detector (SCD) were used. A DB1
fused silica capillary column (J&W Scientific, 30 m, 0.32 mm
i.d., 0.25 nm film) was used. The helium carrier was set at the
head pressure of 5.7 psi, which gave flow velocity of 26 cm/
sec. The detector used 460 ml of air and 220 ml of hydrogen
Borosilicate glass vial
Hydrochloric acid layer
Glass wool from trap
Dichloromethane extract layer
Septum seal, teflon lined
Fig. 3. Liberation and dichloromethane extraction of thiols.
 72 Vol. 51, No. 2
per minute. The oven was set at 40° C for 4 min and then tempera-
ture programmed to 120°C at a rate of 10° C/min and continued
to a final temperature of 175° C at a rate of 25° C/min. The final
oven time was 20 min. The injection volume was 2 pi.
For GC/mass spectrometry (MS) analysis, a Varian 3400 GC
and Finnigan MAT (Cincinnati, OH) TSQ 700 mass spectrometer
were used. The GC was equipped with a J&W on-column injector.
The mass spectrometer was operated in electron impact (El) mode
scanning only two ions, m/z 102 and 118, for 0.5 sec each for
maximum sensitivity and selectivity. The m/z 102 and 118 ions
were characteristic of 3M2B1T and 1-hexanethiol, respectively.
Other mass spectrometer parameters included: electron energy,
70 eV; electron multiplier, 1,500 V; electron current, 200 pA.;
and ion source temperature, 150°C. The same DB1 column used
for the chemiluminescence analysis was used with the helium
carrier set at 1 psi head pressure. The oven was set at 40° C for
3 min and then temperature programmed to 175°C at a rate
of 20° C/min. The final oven time was 20 min. The injection
volume was 2 pi.
Standard Addition Calibration
The standard addition and calibration were performed using
the previously mentioned GC-MS conditions and equipment. An
unexposed beer, known to contain only trace amounts of 3M2B1T,
was spiked with the 3M2B1T working solution (see above) to
obtain 4.54, 2.27, 1.14, 0.57, 0.23, and 0.0 ng/L 3M2B1T in the
beer sample. The 1-hexanethiol working solution was added to
each of the above beers to obtain a concentration of 10 ng/L
(internal standard). The standard curve was produced by plotting
peak area ratios of 3M2B1T and 1-hexanethiol against added
3M2B1T.
B
Detector
Response
uJ
o 8 12
Minutes
Fig. 4. Flame ionization detector gas chromatogram of a light-exposed
beer. A = 3-methyl-2-butene-l-thiol, B = 1-hexanethiol, C =
dichloromethane (solvent).
RESULTS AND DISCUSSION
The development of an analytical procedure for 3M2B1T was
from the outset plagued by our inaccurate understanding of the
sensory threshold for this compound in the beers of interest. Any
analytical method for a beer flavor constituent must be sensitive
for concentrations below the sensory flavor threshold of that
constituent. This is especially important for 3M2B1T, because
sensory panelists have difficulty in quantitatively distinguishing
among high concentrations and tend to level them. The panelists
undergo "burn-out" even when the levels are extremely low. The
sensiory threshold for 3M2B1T in water is 0.2-0.3 ng/L (3), and
we have determined (unpublished) that in an American lager beer,
the threshold of added 3M2BlTis 1.25-2.50 ng/L. For this reason,
an analytical method that cannot detect 3M2B1T lower than
around 5 ng/L in beer is useless. Ideally, the method should
be accurate and precise at 0.5 ng/L. To achieve this using the
method under discussion, several steps in the procedure require
special care.
Gas Extraction
The gas extraction flask should be conditioned before use
because there are "active sites" in the flask that cause low recoveries
of 3M2B1T by sorption or chemical reaction. The conditioning
step involves the addition of 1 ml of a 2.0 X 107 ng/ L solution
of isoamylthiol to a beer followed by gas extraction as described
in the Experimental section. This should be done on a daily basis
before any sample or standard is analyzed.
Detector
Response
\
0 10
Minutes
Fig. 5. Flame photometric detector gas chromatogram of a light-exposed
beer. A = 3-methyl-2-butene-l-thiol, B — 1-hexanethiol, C = 1-
butanethiol.

The time required for gas extraction (90 min) is important.
The majority of thiols are extracted after 60 min. Extraction times
longer than 90 min are not needed and are detrimental because
of the instability of the thiols, especially 3M2B1T, on the thiol
trap.
GC Injection
Cool, on-column injection is very important. This is due to
the extreme reactivity of 3M2B1T on hot metallic and glass
surfaces. The use of conventional flash vaporization injectors will
decrease sensitivity by about one order of magnitude.
During the course of this work, several different types of
detectors were used. These included FID, PD, an SCD, and MS
detectors. Acceptable results were achieved with all of these, but
some advantages and disadvantages will be cited for each.
Flame lonization Detection
Figure 4 is a reproduction of an FID chromatogram of a light-
exposed (three weeks, 25 ft-c) beer employing the method that
has been described. In addition to the peak for 3M2B1T and
for 1-hexanethiol (added internal standard), several other peaks
are visible, the largest for dichloromethane (solvent). Although
quantitation of 3M2B1T is possible, chromatographic conditions
must be adjusted carefully so that coelution of important peaks
with non-thiol compounds is avoided. The advantage of the FID
is that it probably is the most common gas chromatographic
detector, and most instruments incorporate this type of detector.
A linear standard addition calibration is achieved. The detection
limit for FID is about 2 ng/L.
41.1
L/ 6 9.2
J r\ /—SH~I+
\=y
68.: 10 2.1
39.1 53.1 67.1
45.0
I 47.1 59.1 71.1 87.1 99.1
»-l, I Ihl II i.
40 60 80 100
m/z
Fig. 6. Electron impact mass spectrum of 3-methyl-2-butene-l-thiol.
\3M2B1T
102 m/z
118 m/z
, I
Fig. 7. Gas chromatography/mass spectrometry chromatographic traces
of beer spiked with 3-methyl-2-butene-l-thiol (3M2B1T) at 3 ng/L and
1-hexane thiol at 10 ng/L. Top: Selected ion monitoring at m/z 102
(molecular ion of 3M2B1T); Middle: selected ion monitoring at m/z 118
(molecular ion of 1-hexanethiol); Bottom: reconstructed ion chro-
matogram of 3M2B1T.
ASBC Journal 73
Flame Photometric Detection
Figure 5 is a chromatogram of a similar light-exposed beer
employing FPD. It should be noted that in contrast to Figure
4, there are few potentially interfering peaks. In this experiment,
both 1-butanethiol and 1-hexanethiol were employed as internal
standards, but this practice was discontinued after it was estab-
lished that only one internal standard was necessary. The standard
addition calibration for 3M2B1T is achieved when the square
root of the thiol peak area ratios are plotted to overcome the
nonlinearity of FPD. A disadvantage of FPD for 3M2B1T is
its lower sensitivity compared with that of the other detectors
evaluated. This is true with compounds that have high carbon-
to-sulfur ratios. For example, this detector is more sensitive for
methanethiol than for 3M2B1T. Whereas the major advantage
of FPD is selectivity, the detection limit for FPD is about the
same as that for FID (i.e., 2 ng/L).
Mass Spectrometric Detection
Figure 6 is an El mass spectrum of 3M2B1T. Assignments
for the various major ions are given. The molecular ion for
3M2B1T is at m/z 102. Ions of m/z 69 and 41 are of slightly
higher abundances than m/z 102, but because of their occurrence
for many other common compounds, m/z 102 was chosen for
selective ion monitoring.
To illustrate this technique, an unhopped beer was spiked with
3M2B1T (3 ng/L) and 1-hexanethiol (10 ng/L). Figure 7 shows
chromatograms at m/z 102 for 3M2B1T (selected ion monitoring),
Detector
Response
103.2
ll , I
120
0 10
Minutes
Fig. 8. Sulfur chemiluminescence detector gas chromatogram of a light-
exposed beer. A = 3-methyl-2-butene-l-thiol, B = 1-hexanethiol.
Hexanethiol
(3M2B1Tp.h.) 20.
Added to GC column
Added to beer
Nanograms of 3M2B1T Added
Fig. 9. Recovery of 3-methyl-2-butene-l-thiol (3M2B1T) in beer. Peak
Height (p.h.) vs. 3M2B1T added. A = 3M2B1T injected on to column.
B = 3M2B1T added to the beer and isolated by the standard gas extraction
procedure.
74 Vol. 51, No. 2
m/z 118 for 1-hexanethiol (selected ion monitoring), and the
reconstructed ion chromatogram, which sums both ions. The
3M2B1T peak in the first trace is dominant, as is the 1-hexanethiol
peak in the second trace. The very small peak for 3M2B1T in
the third scan is small because the GC-MS computer adjusts the
scale to the largest peak. Figure 7 illustrates the advantage of
GC-MS (selected ion monitoring) for the method under discus-
sion. GC-MS is the most sensitive and by far the most selective.
Its major disadvantage is its very high cost when compared with
every other detector that was evaluated. A linear standard addition
calibration employing GC-MS was produced. It should be noted
that with GC-MS, one can easily qiiantitate 3M2B1T at levels
at or lower than 1 ng/L in beer.
Chemiluminescence Detection
We evaluated the Sievers SCD. Figure 8 is a typical gas chro-
matogram of a light-exposed beer (similar conditions as pre-
viously) using this detector. The level of 3 M2B1T in this particular
beer sample was 7 ng/L. Although the sensitivity and selectivity
of this detector (detection limit of 1 ng/L) is not as good as
for GC-MS, its cost is very low compared with GC-MS. A linear
standard addition calibration using this detector was produced.
Indigenous 3M2B1T and Preparation of Standards
When making up standard solutions, it is important to be aware
that most beers containing iso-a-acids contain a small quantity
of what we call "indigenous" 3M2B1T. We have discovered that
this indigenous 3M2B1T originates from a thermal reaction that
occurs after kettle, hopping. Indigenous levels vary from 1 to 5
ng/L depending on the beer..For this reason, it is advantageous
to prepare standards employing unhopped beer, if possible.
It was essential to determine the efficiency of the isolation/
concentration procedure. To accomplish this, known amounts
of 3M2B1T were directly injected into the GC and compared
to the detector (FID) responses when similar quantities of
3M2B1T were added to beer and analyzed in the usual manner.
Figure 9 illustrates the results of this experiment. Recoveries of
3M2B1T were quite acceptable.
CONCLUSIONS
3M2B1T was detected and quantitated in beer at levels slightly
below and above 1 ng/L depending on the detector. Because
the sensory threshold of American lagers is 1.25-2.50 ng/L, it
is important to be able to detect and quantify at or below 1
ng/L. At these levels or slightly below, 3M2B1T may play a
positive role in beer flavor as it possibly does in other beverages
(3) because most, if not all, conventionally hopped beers contain
indigenous 3M2B1T.
The technique involved gas extraction of sample beers, trapping
of the thiols (3M2B1T and 1-hexanethiol) on glass wool containing
Hg (II) cyanide, freeing the thiols from the mercury complex
with acid, extracting into dichloromethane, and analyzing by gas
chromatography. FID, FPD, SCD, and selective ion monitored
MS have been used as GC detectors. Because of the low levels
of detection, the analyst must be experienced and competent.
Care must be exercised when handling even dilute solutions of
3M2B1T because of its extremely low sensory level.
ACKNOWLEDGMENTS
We thank Etzer Chicoye and Vince Bavisotto for their support and
encouragement during the developmental stages of this research. We are
also grateful to Art Rehberger for permission to both present and publish
our work. We are also grateful to Velta Young for her excellent GC-
MS and SCD chromatography. Finally, we would like to acknowledge
Joe Hoff for his creativity and his leadership of two of us (S.R.R and
A.A.M.).
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[Received October 15, 1992. Accepted January 25, 1993.]