Food Chemistry xxx (2016) xxxxxx

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

A new chemical tool for absinthe producers, quantification

of a/b-thujone and the bitter components in Artemisia absinthium
Benoit Bach a,, Marilyn Cleroux a, Mayra Saillen a, Patrik Schnenberger a, Stephane Burgos a,
Julien Ducruet a, Armelle Vallat b
a
CHANGINS, Viticulture and Oenology, University of Applied Sciences and Arts Western Switzerland, Route de Duillier 50, 1260 Nyon, Switzerland
b
University of Neuchtel, NPAC, avenue de Bellevaux 51, 2000 Neuchtel, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: The concentrations of a/b-thujone and the bitter components of Artemisia absinthium were quantified
Received 15 December 2015 from alcoholic wormwood extracts during four phenological stages of their harvest period. A solid-
Received in revised form 5 June 2016 phase micro-extraction method coupled to gas chromatographymass spectrometry was used to deter-
Accepted 15 June 2016
mine the concentration of the two isomeric forms of thujone. In parallel, the combination of ultra-high
Available online xxxx
pressure liquid chromatography and high resolution mass spectrometry allowed to quantify the com-
pounds absinthin, artemisetin and dihydro-epi-deoxyarteannuin B. This present study aimed at helping
Keywords:
absinthe producers to determine the best harvesting period.
Absinthe
Artemisia absinthium
2016 Elsevier Ltd. All rights reserved.
Thujone
Absinthin
Artemisetin
Dihydro-epi-deoxyarteannuin B
SPME-GCMS
UHPLC-HR-MS

1. Introduction consuming and require large amounts of samples and solvents. In

Absinthe is an alcoholic (4575%) beverage containing mostly
herb extracts. It is an anise-flavoured spirit derived from botani-
cals, including wormwood (Artemisia absinthium), star anise
(Illicium verum), fennel seed (Foeniculum vulgare), angelica
(Angelica archangelica), peppermint (Mentha piperita) and other
medicinal and culinary herbs (Goud, Dwarakanath, & Chikka
Swamy, 2015). Thujone is a natural essence typically associated
with common wormwood (Lachenmeier et al., 2008). In particular,
b-thujone is known to be an epileptiform convulsant and is
regarded as the active ingredient in absinthe. Since 1990, a max-
imum limit of 35 mg/kg (sum of a/ b isomers) was introduced for
thujone in the European Union (Lachenmeier, Nathan-Maister,
Breaux, Luaut, & Emmert, 2010). Therefore, it is required to pre-
cisely determine the concentrations of a- and b-thujone in
absinthe, in accordance to international food safety regulations.
Many conventional techniques have been used to isolate thujone
from absinthe or wormwood extracts, such as liquid-liquid extrac-
tion or solid phase extraction, but these techniques are time-
Corresponding author.
E-mail address: benoit.bach@changins.ch (B. Bach).
contrast, headspace analysis requires minimal sample preparation
and can be automated, and thus is a very attractive methodology
for analysing volatile compounds. Among headspace methods,
solid-phase micro-extraction (SPME) is currently one of the most
widely used method in food analysis, offering many benefits over
other headspace techniques. Although SPME is well established
for the analysis of flavours, this technique is not commonly used
for the analysis of thujone in absinthe or in wormwood extracts.
In the present study, a SPME method coupled to a gas
chromatography-mass spectrometry technique was developed for
determining the concentration of thujone.
Apart from thujone, wormwood contains also high amounts of
bitter components. The main bitter molecules in wormwood are
sesquiterpene lactones including absinthin and its isomeric form
anabsinthin, anabsin, and artabsin (Goud et al., 2015; Turak, Shi,
Jiang, & Tu, 2014). In this study, the bitter components from an
ethanolic extract were investigated at defined phenological stages
of Artemisia absinthium. A method based on ultra-high pressure liq-
uid chromatography coupled to high resolution mass spectrometry
was developed and validated for the quantification of three com-
pounds, i.e. absinthin, artemisetin (or artemetin) and dihydro-
epi-deoxyarteannuin B. Dihydro-epi-deoxyarteannuin B was found
previously in Artemisia annua (Brown, 1992; Sy & Brown, 2001) but

http://dx.doi.org/10.1016/j.foodchem.2016.06.045
0308-8146/ 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Bach, B., et al. A new chemical tool for absinthe producers, quantification of a/b-thujone and the bitter components in
Artemisia absinthium. Food Chemistry (2016), http://dx.doi.org/10.1016/j.foodchem.2016.06.045
2 B. Bach et al. / Food Chemistry xxx (2016) xxxxxx
not in A. absinthium. During in vivo transformations, this compound
was an important metabolite in the biosynthesis of artemisinin
(Brown & Sy, 2007), which is one of the most efficient drugs against
Plasmodium species involved in malaria (Potawale et al., 2008).
2. Material and methods
2.1. Plants and phenological stages
Artemisia absinthium L. seeds originating from Fenaco/UFA
(Berne, Switzerland) were planted in cultivated soil with geotextile
at the Val-de-Travers (Switzerland, X:543/177 Y:201/603, altitude
1060 m), in 2013. The planting distance was 40
50 cm, which
represented 4 plants per m2. The harvest period (2014) were
defined by four phenological stages: S1, first flower bud or inflores-
cence visible; S2, first flower petals visible and the flower still
closed; S3, early flowering with 10% of flowers open; S4, full bloom
with 50% of flowers open and the first dried petals corresponding
to the BBCH scale, stages 51, 59, 6061 and 65, respectively.
2.2. Sample preparation
Three replicates of 10 wormwood plants of each phenological
stage were dried at 30 C during 36 h in a dryer, following the pro-
tocol used by absinthe producers. The dried aerial parts, petals and
inflorescences, which were separated manually from the stalk,
were then crushed and mixed. Preliminary results obtained by dif-
ferent plant quantities, different solvents with different ethanol
concentrations for maceration, and different extraction times were
used to determine the optimum extraction yield. The highest
extraction of thujone and bitter compounds was obtained from
the maceration of 2 g of dried plants in 100 mL of ethanol (96%,
v/v) for 24 h. Each sample was treated in triplicates.
2.3. Experimental GCMS analysis
2.3.1. Chemicals and reagents
All chemicals were of analytical quality a-thujone standard:
a- and b-thujone standard, 1,6-heptadien-4-ol, phosphate buffered
saline tablet, sodium chloride (NaCl, 99.5%), and sodium hydrogen
phosphate (Na2HPO4, 99.0%) were purchased from Sigma (Buchs,
Switzerland). Methanol (99.9%) and ethanol (99.9%) were obtained
from Merck (Darmstadt, Germany). The individual stock solutions
were prepared in methanol at concentration of 1000 mg/L and
stored at 4 C. Ultra-pure water from Milli-Q system (Millipore,
Bedford, USA) with a conductivity of 0.055 lS/cm was used
throughout. The 20 mL glass vials came from BGB Analytik
(Geneva, Switzerland). SPME fiber (Divinylbenzene/Carboxen/Poly
dimethylsiloxane, DVB/CAR/PDMS) was purchased from Supelco
(Bellefonte, PA, USA).
2.3.2. Sample preparation and SPME conditions
Thujone from wormwood macerate samples were extracted by
HS-SPME and analysed using gas chromatography/mass spectrom-
etry. 10 lL of standard or sample material and 1 mL of PBS buffer
0.2 M pH7.5 were pipetted into a 20 mL SPME vial and 100 lL of
an ethanolic solution of 1,6-heptadien-4-ol at 10 mg/L were added
as an internal standard. The vial was tightly capped with a PTFE-
silicon septum and heated at 45 C for 5 min on a heating platform
with agitation at 500 rpm. The SPME 50/30 lm fiber, precondi-
tioned according to the manufacturers instructions, was then
inserted into the headspace, where the extraction did last40 min
at 45 C and agitation at 250 rpm. The fiber was then desorbed in
the GC injector for 5 min at 250 C.
Please cite this article in press as: Bach, B., et al. A new chemical tool for absinthe producers, quantification of a/b-thujone and the bitter components in
Artemisia absinthium. Food Chemistry (2016), http://dx.doi.org/10.1016/j.foodchem.2016.06.045
2.3.3. GG-MS instrumentation and conditions
The GCMS system used was a Varian CP3800 equipped with a
Saturn 2000 ion trap mass spectrometer and STAR version 5.52
chromatography software (Varian, Palo Alto, CA). The GC was fitted
with a Combi-Pal Autosampler (CTC Analytics, Zwingen, Switzer-
land) used in SPME mode throughout validation. The column was
a 30 m
0.25 mm DB-WAX capillary with 0.25 lm film thickness
(J & W Scientific, Folsom, CA, USA). The carrier gas was helium at
a 1 mL/min flow rate. Samples were injected by placing the SPME
fiber at the GC inlet for 5 min in the splitless mode. The ovens
starting temperature was 35 C, which was kept for 2 min, then
raised to 210 C at a rate of 3 C /min and then 20 C/min until
250 C and kept at 250 C for 2 min for a total runtime of
64.33 min. The injector was kept at 250 C. Trap temperatures
were the following: manifold 50 C, transfer line 250 C, and trap
200 C. To determine the characteristics of desired compounds,
the mass spectrometer was set in electron ionization mode using
a scan time of 0.37 s/scan and covering a mass-to-charge (m/z)
range from 35 to 200. The emission current was 10 lA; the maxi-
mum ionization time 15,000 ls. For analysis, the mass spectrome-
ter was operated in the selective ion mode (SIM). Analyses were
carried out in duplicate.
2.3.4. Method validation
Validation was carried out in terms of specificity, linearity, pre-
cision, limit of detection (LOD) and quantification (LOQ), and accu-
racy. Using Selected Ion Monitoring (SIM), the specificity was
confirmed based on the presence of the ions (quantifier and qual-
ifier, respectively 95 and 109 m/z) at the correct retention times
corresponding to thujone standards (23.517 for a-thujone and
24.279 for b-thujone). b-thujone, where no high purity standard
is available, was calculated later with the response factor of
a-thujone. The measured peak area ratios of qualifier/quantifier
ions were about 0.2, and had to be in close accordance with the
ion ratios of the standards. Linearity of the method was evaluated
using calibration curves with 8 calibration levels during 6 different
days. The linearity of the calibration curves was assessed over the
range from 0.1 to 100 mg/L. The mean values were used to con-
struct the calibration graphs by plotting the peak area ratio against
the standard concentration. Regression, slope and origin intercept
were calculated by a linear least-square regression. The resulting
calibration curves obtained by plotting the GCMS response versus
analyte concentration were found to have good linearity in the
tested concentration range, with R2 values >0.990. Limits of detec-
tion and quantification were estimated following the IUPAC
approach which consisted of analysing the blank sample to estab-
lish noise levels and then estimating LOD and LOQ for signal/noise,
3 and 10 respectively. The calculated LOD and LOQ were found to
be 0.01 mg/L and 0.05 mg/L, respectively. Precision results dis-
played in Table 1 were obtained by analysing 3 different spiked
concentrations in wormwood macerates during 6 different days
using SPME-GCMS on the same and different SPME (DVB/CAR/
PDMS) fibers. Despite a significant variability in the response signal
between the fibers, satisfactory results were obtained for precision,
in terms of reproducibility intra-analysis with the same SPME fiber
and inter-analysis between different SPME fibers, thanks to the
internal standard. Under the conditions described above, accuracy
was evaluated by comparing found values with spikes by standard
addition in wormwood macerates. The results in Table 1 show
good accuracy, with recoveries between 80% and 120% for each
spiked level. Measurement uncertainty is estimated using the sim-
plified approach based on existing validation data proposed by
Barwick & Ellison, 2000. The results in Table 1 show good accuracy,
except in the case of very low concentrations for which overesti-
mations are indicated. Precision and trueness contributions are
combined together as follows to obtain the overall uncertainty.
 B. Bach et al. / Food Chemistry xxx (2016) xxxxxx 3

Table 1
Summary of the validation results in wormwood macerate for thujone content.

Spiked Sample Ref. value Unit Repeatability% Intermediate reproducibility% Recovery% Standard uncertainty%
Analytical requirement <10% <15% 80120% <20%
Level 1 0.27 mg/L 8.1 12.1 119.5 16.6
Level 2 1.60 mg/L 3.5 3.1 94.0 4.2
Level 3 31.90 mg/L 3.8 5.5 98.6 8.8

The results in Table 1 show good uncertainty, with results <20% for Table 2
each spike level. All statistical tests were performed using the Method validation of artemisetin, absinthin and dihydro-epi-deoxyarteannuin B,
quantification curves, coefficient of determination R2, limit of quantification (LOQ),
XLSTAT software, version 2014 (Addinsoft France). precision (intra- and interday) and accuracy.

Method validation Analytes

2.4. Experimental part for UHPLC-HRMS analysis
Artemisetin Absinthin Dihydro-epi-
2.4.1. Chemicals and reagents deoxyarteannuin B

Formic acid, ULC/MS grade acetonitrile and water were Chemical formula C20H20O8 C30H40O6 C15H22O2
purchased from Biosolve BV (Valkenswaard, the Netherlands). Quantification ion 389.124 497.290 235.170
(m/z 0.010 Da)
Artemisetin (CAS 479-90-3, 98% HPLC purity) was purchased from Curvea Y = 15.61 b
Y = 16.96 b
Y = 7276.3 X
Stanford chemicals (Irvine CA, USA). X2 + 33296.50 X2 + 327.36 + 66.6
Absinthin and dihydro-epi-deoxyarteannuin B were purified in X  3.30 X  1.30
our laboratory by SPE (Discovery DSC-18, 5 g, Supelco, Bellefonte, R2 0.999 0.999 0.998
LOQ (lg/mL) 0.02 0.05 0.02
PA, USA) followed by semi-preparative liquid chromatography
Intraday Precision 6.26 6.31 5.47
(LC). The SPE was performed as follows: The cartridge was condi- (%) 0.08 lg/mL
tioned and equilibrated with 20 mL methanol and 20 mL: MilliQ Intraday Precision 1.97 1.96 1.31
water:methanol (95:5, v/v) respectively, then 2.4 g of dried plant (%) 2 lg/mL
material extract was deposed on cartridge, the cartridge was Interday Precision 7.73 10.54 13.80
(%) 0.08 lg/mL
washed with 40 mL MilliQ water:methanol (95:5, v/v) and eluted
Interday Precision 4.32 7.89 3.10
by 40 mL methanol:MilliQ water (85:15, v/v). The eluted fractions (%) 2 lg/mL
were evaporated under vacuum and further fractionated by semi- Accuracy (%) 92.3 10.9c 103.7 11.2 99.0 11.8
preparative LC using a 1525 EF binary HPLC pump coupled to a 6.25 lg/mL
Accuracy (%) 4 lg/ 103.1 6.4 110.7 7.8 107.6 8.8
2487 UV detector (Waters, Milford, MA, USA). The chemical struc-
mL
tures were confirmed by 1H NMR spectrum (Avance 400 MHz, Accuracy (%) 106.2 0.4 97.0 0.3 95.4 0.3
Bruker) and the purity assessed by UHPLC-HRMS and NMR analy- 0.05 lg/mL
ses, 96% for absinthin and 95% for dihydro-epi-deoxyarteannuin B. a
Concentration range 0.0810 lg/mL.
The characteristic resonances observed in 1H NMR spectra were b
A weighting factor of 1/X was applied.
compared with the previous papers (Sy & Brown, 2001; Dudley c
Mean SD (n = 4).
et al., 2007; Zhang et al., 2005).

2.4.2. UHPLC-HRMS instrumentation and conditions

The UHPLC-HRMS experiments were performed on a Synapt G2
high resolution mass spectrometer which was coupled to an
Acquity UPLCTM (Waters, Milford, MA, USA). Separation of the com-
pounds was achieved on an Acquity BEH C18 column 50
2.1 mm
i.d., 1.7 lm particle size with a guard column of identical phase
chemistry (Waters, Milford, MA, USA). The mobile phase was
0.05% formic acid in water (A)/acetonitrile (B) and the following
gradient elution program was used: 0 min 5% B; 570% B in
7 min; 70100% B in 1 min, holding at 100% during 1.9 min, and
re-equilibration at 5% B for 1.1 min. The flow rate was set to
400 lL/min, the injection volume was 2.5 lL, and the column tem-
perature was maintained at 25 C. For MS detection, ionization was
performed in positive ESI mode using a mass scan range from 50 to
600 Da. Experimental source parameters were as follows: capillary
voltage 2.8 kV, sampling cone 25 V, source and desolvation tem-
peratures 120 C and 350 C respectively, and desolvation gas flow
800 L/Hr.
2.4.3. Method validation
The method was validated for selectivity, linearity, limit of
quantification, precision and accuracy, and is shown in Table 2.
The linearity of the method was established using standard solu-
tions at concentration levels from 0.08 lg/mL to 10 lg/mL. For
dihydro-epi-deoxyarteannuin B linearity was observed over the
entire range. In contrast for artemisetin and absinthin, curves were
linear between 0.08 lg/mL and 5 lg/mL but showed a quadratic
trend above 5 lg/mL. For this reason we applied quadratic curves,
which fitted better. The limit of quantification (S/N ratio of 10) was
determined as for GCMS analyses (see above) for each analyte.
Precision (intra- and interday) was measured by the relative stan-
dard deviation (%RSD) of two different standard solutions, 0.08 and
2 lg/mL on three non-consecutive days (n = 5 for each day).
Accuracy was determined as percent of recovery by spiking sam-
ples (n = 4) with standards at three known concentrations of
0.05, 4 and 6.25 lg/mL and comparing the measured value with
the true value.
The extracts were diluted so that at final the concentrations of
the three compounds were in linearity range of calibration curves.
3. Results and discussion
Plant development and thus phenological stages show great
inter-annual variability and also large spatial differences.
Individual (genes, age) and environmental factors (weather and cli-
mate conditions in the micro and macro-scale, soil-conditions,
water supply, diseases, competition etc.) influence plants. They
can be viewed as integrative measurement devices for the environ-
ment. The seasonal cycle of plants however is influenced primarily
by temperature, photoperiod and precipitation. Therefore, the
determination of the optimum harvest date is still a challenge for
the grower, who seeks to have optimum quality with a reasonable

Please cite this article in press as: Bach, B., et al. A new chemical tool for absinthe producers, quantification of a/b-thujone and the bitter components in
Artemisia absinthium. Food Chemistry (2016), http://dx.doi.org/10.1016/j.foodchem.2016.06.045
4 B. Bach et al. / Food Chemistry xxx (2016) xxxxxx

amount of thujone. The objective of this study was to propose ana-
lytical methods to the absinthe producers that thy better deter-
mine harvest dates. In this context, samples were collected at
different phenological stages on a parcel following a defined proto-
col. After drying, the samples were macerated in ethanol according
to a protocol similar to that used for absinthe production (cf. Mate-
rial and methods).
Fig. 1 summarizes the quantitative results of a- and b-thujone
detected in the ethanolic extracts at each phenological stage. b-
thujone was more abundant than athujone as already described
in wormwood. As results in Fig. 1B demonstrate, different extracts
contained a constant concentration of a- and b-thujone, during the
first three stages. A decrease of more than 30% was observed in the
plant collected in the fourth stage. Looking at the plant as a whole
(Fig. 1A), an increase in the amount of thujone was observed, cer-
tainly due to the growth of the plant. It should be noted that the
harvest is traditionally done by the owner at S3. According to our
results this stage corresponds to the optimum quantity of thujone.
Absinthin, artemisetin and dihydro-epi-deoxyarteannuin B
were also detected in the ethanolic extract at each phenological
stage (Fig. 2). These three components were the predominant
constituents of plant extracts prepared in distillery conditions. In
all samples absinthin was the major component, the concentra-
tions ranged from 23,450 to 31,120 mg/kg. The variations of
absinthin and dihydro-epi-deoxyarteannuin B followed similar
trends as their levels increased from S1 to S3 and then decreased
at S4. Levels were thus highest during the harvest period usually
chosen by the farmer (S3). Absinthin possesses bitter characteris-
tics and has been recently proposed as a novel criterion for the
quality of absinthe and the wormwood taste (Lachenmeier,
2007). The levels of the flavonoid artemisetin showed a different
trend with a decrease of about 25% from S1 to S4. This observation
is in contrast with Baraldi et al. (2008) who reported the highest
yield for polyphenolic compounds at the full bloom stage (S4).
No obvious reason could be found to explain this discrepancy.
However in Baraldi et al. (2008), the phenological stages were
not described with enough precision to make precise comparison
with our experiment. Also the choice of the extraction solvent, hex-
ane, was not documented although flavonoids are notoriously bet-
ter extracted in alcoholic solvents. Hexane might have extracted
only a fraction of artemisetin in the plant. Finally the production
of flavonoids by the plant depended on environmental factors such

Fig. 1. Total quantity (A) and concentration (B) (mg/kg dry weight) of thujone during the plant growth. t-Test 2 means (S1-S2, S2-S3, S3-S4), 95% confidence interval. nd = not
statistically significant difference. *0.01 < P 6 0.05, **P 6 0.01.

Fig. 2. Concentration (mg/kg dry weight) of artemisetin, absinthin and dihydro-epi-deoxyarteannuin B during the plant growth. yt-Test 2 means (S1-S2, S2-S3, S3-S4), 95%
confidence interval. nd = not statistically significant difference. *0.01 < P 6 0.05, **P 6 0.01.

Please cite this article in press as: Bach, B., et al. A new chemical tool for absinthe producers, quantification of a/b-thujone and the bitter components in
Artemisia absinthium. Food Chemistry (2016), http://dx.doi.org/10.1016/j.foodchem.2016.06.045
 B. Bach et al. / Food Chemistry xxx (2016) xxxxxx 5

OH
CH3 CH3
H H H
H3 C H3 C OH
H
O O

H H
O
H
O H
H3 C CH3 H3 C CH3 O
alpha-thujone beta-thujone absinthin

OCH3 O

H
H3CO O
OCH3

H3CO OCH3
O
OH O
artemisetin dihydro-epi-deoxyarteannuin B CH3
O

Fig. 3. Chemical structures of a/b-thujone, absinthin, artemisetin and dihydro-epi-deoxyarteannuin B.

as light, temperature, etc (Aberham, Cicek, Schneider, & Stuppner,
2010). The low levels of artemisetin which was found at the full
bloom stage might be explained by the abnormally low tempera-
tures and high rainfall during summer 2014 (Fig. 3).
4. Conclusions
The presented analytical approach of wormwood analysis for
alcoholic beverage production provides a substantial improvement
of the previously reported methods. In this study, we demon-
strated the pertinence of accurately monitoring the composition
of wormwood at different growth stages, including both thujone
and bitter compounds. Until now, absinthe producers had no infor-
mation on the composition of their material before the analysis of
the final product. In the future, we try to propose our methods to
local producers as a tool to determine the best harvest periods
more precisely. This analytical approach will also help to better
understand the impact of the environment on plants over several
years.
Acknowledgments
The authors like to thanks distillers from the Val-de-Travers
(Switzerland) for providing samples, in particular Yves Currit. They
are also grateful to the Swiss Alcohol Board for financial supporting
of this study.
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Please cite this article in press as: Bach, B., et al. A new chemical tool for absinthe producers, quantification of a/b-thujone and the bitter components in
Artemisia absinthium. Food Chemistry (2016), http://dx.doi.org/10.1016/j.foodchem.2016.06.045