β-thujone and the bitter components in Artemisia absinthium

Food Chemistry xxx (2016) xxx–xxx

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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 Schönenberger a, Stephane Burgos a, Julien Ducruet a, Armelle Vallat b a b

CHANGINS, Viticulture and Oenology, University of Applied Sciences and Arts Western Switzerland, Route de Duillier 50, 1260 Nyon, Switzerland University of Neuchâtel, NPAC, avenue de Bellevaux 51, 2000 Neuchâtel, Switzerland

a r t i c l e

i n f o

Article history: Received 15 December 2015 Received in revised form 5 June 2016 Accepted 15 June 2016 Available online xxxx Keywords: Absinthe Artemisia absinthium Thujone Absinthin Artemisetin Dihydro-epi-deoxyarteannuin B SPME-GC–MS UHPLC-HR-MS

a b s t r a c t The concentrations of a/b-thujone and the bitter components of Artemisia absinthium were quantified from alcoholic wormwood extracts during four phenological stages of their harvest period. A solidphase micro-extraction method coupled to gas chromatography–mass spectrometry was used to determine the concentration of the two isomeric forms of thujone. In parallel, the combination of ultra-high pressure liquid chromatography and high resolution mass spectrometry allowed to quantify the compounds absinthin, artemisetin and dihydro-epi-deoxyarteannuin B. This present study aimed at helping absinthe producers to determine the best harvesting period. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Absinthe is an alcoholic (45–75%) beverage containing mostly herb extracts. It is an anise-flavoured spirit derived from botanicals, 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 maximum limit of 35 mg/kg (sum of a/ b isomers) was introduced for thujone in the European Union (Lachenmeier, Nathan-Maister, Breaux, Luauté, & Emmert, 2010). Therefore, it is required to precisely 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 extraction or solid phase extraction, but these techniques are time-

⇑ Corresponding author. E-mail address: [email protected] (B. Bach).

consuming and require large amounts of samples and solvents. In 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 liquid chromatography coupled to high resolution mass spectrometry was developed and validated for the quantification of three compounds, i.e. absinthin, artemisetin (or artemetin) and dihydroepi-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/Ó 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

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B. Bach et al. / Food Chemistry xxx (2016) xxx–xxx

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 inflorescence 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, 60–61 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 protocol 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 different 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 GC–MS 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 spectrometry. 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 PTFEsilicon septum and heated at 45 °C for 5 min on a heating platform with agitation at 500 rpm. The SPME 50/30 lm fiber, preconditioned according to the manufacturer’s 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.

2.3.3. GG-MS instrumentation and conditions The GC–MS 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, Switzerland) 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 oven’s 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 maximum ionization time 15,000 ls. For analysis, the mass spectrometer 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, precision, limit of detection (LOD) and quantification (LOQ), and accuracy. Using Selected Ion Monitoring (SIM), the specificity was confirmed based on the presence of the ions (quantifier and qualifier, 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 construct 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 GC–MS response versus analyte concentration were found to have good linearity in the tested concentration range, with R2 values >0.990. Limits of detection and quantification were estimated following the IUPAC approach which consisted of analysing the blank sample to establish 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 displayed in Table 1 were obtained by analysing 3 different spiked concentrations in wormwood macerates during 6 different days using SPME-GC–MS 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 simplified 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 overestimations are indicated. Precision and trueness contributions are combined together as follows to obtain the overall uncertainty.

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

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B. Bach et al. / Food Chemistry xxx (2016) xxx–xxx 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 Level 1 Level 2 Level 3

0.27 1.60 31.90

mg/L mg/L mg/L

<10% 8.1 3.5 3.8

<15% 12.1 3.1 5.5

80–120% 119.5 94.0 98.6

<20% 16.6 4.2 8.8

The results in Table 1 show good uncertainty, with results <20% for each spike level. All statistical tests were performed using the XLSTAT software, version 2014 (Addinsoft – France).

Table 2 Method validation of artemisetin, absinthin and dihydro-epi-deoxyarteannuin B, quantification curves, coefficient of determination R2, limit of quantification (LOQ), precision (intra- and interday) and accuracy. Method validation

2.4. Experimental part for UHPLC-HRMS analysis 2.4.1. Chemicals and reagents Formic acid, ULC/MS grade acetonitrile and water were purchased from Biosolve BV (Valkenswaard, the Netherlands). Artemisetin (CAS 479-90-3, 98% HPLC purity) was purchased from Stanford chemicals (Irvine CA, USA). Absinthin and dihydro-epi-deoxyarteannuin B were purified in our laboratory by SPE (DiscoveryÒ DSC-18, 5 g, Supelco, Bellefonte, PA, USA) followed by semi-preparative liquid chromatography (LC). The SPE was performed as follows: The cartridge was conditioned and equilibrated with 20 mL methanol and 20 mL: MilliQ water:methanol (95:5, v/v) respectively, then 2.4 g of dried plant material extract was deposed on cartridge, the cartridge was washed with 40 mL MilliQ water:methanol (95:5, v/v) and eluted by 40 mL methanol:MilliQ water (85:15, v/v). The eluted fractions were evaporated under vacuum and further fractionated by semipreparative LC using a 1525 EF binary HPLC pump coupled to a 2487 UV detector (Waters, Milford, MA, USA). The chemical structures were confirmed by 1H NMR spectrum (Avance 400 MHz, Bruker) and the purity assessed by UHPLC-HRMS and NMR analyses, 96% for absinthin and 95% for dihydro-epi-deoxyarteannuin B. The characteristic resonances observed in 1H NMR spectra were compared with the previous papers (Sy & Brown, 2001; Dudley 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 compounds 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; 5–70% B in 7 min; 70–100% 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 temperature 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 temperatures 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 solutions 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

Chemical formula Quantification ion (m/z ± 0.010 Da) Curvea

R2 LOQ (lg/mL) Intraday Precision (%) 0.08 lg/mL Intraday Precision (%) 2 lg/mL Interday Precision (%) 0.08 lg/mL Interday Precision (%) 2 lg/mL Accuracy (%) 6.25 lg/mL Accuracy (%) 4 lg/ mL Accuracy (%) 0.05 lg/mL a b c

Analytes Artemisetin

Absinthin

Dihydro-epideoxyarteannuin B

C20H20O8 389.124

C30H40O6 497.290

C15H22O2 235.170

Y = 15.61 X2 + 33296.50 X  3.30 0.999 0.02 6.26

b

Y = 16.96 X2 + 327.36 X  1.30 0.999 0.05 6.31

b

1.97

1.96

1.31

7.73

10.54

13.80

4.32

7.89

3.10

92.3 ± 10.9c

103.7 ± 11.2

99.0 ± 11.8

103.1 ± 6.4

110.7 ± 7.8

107.6 ± 8.8

106.2 ± 0.4

97.0 ± 0.3

95.4 ± 0.3

Y = 7276.3 X + 66.6

0.998 0.02 5.47

Concentration range 0.08–10 lg/mL. A weighting factor of 1/X was applied. Mean ± SD (n = 4).

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 GC–MS analyses (see above) for each analyte. Precision (intra- and interday) was measured by the relative standard 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 samples (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 climate 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 environment. 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

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B. Bach et al. / Food Chemistry xxx (2016) xxx–xxx

amount of thujone. The objective of this study was to propose analytical methods to the absinthe producers that thy better determine harvest dates. In this context, samples were collected at different phenological stages on a parcel following a defined protocol. After drying, the samples were macerated in ethanol according to a protocol similar to that used for absinthe production (cf. Material and methods). Fig. 1 summarizes the quantitative results of a- and b-thujone detected in the ethanolic extracts at each phenological stage. bthujone was more abundant than a–thujone 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, certainly 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 concentrations 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 characteristics 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, hexane, was not documented although flavonoids are notoriously better 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

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B. Bach et al. / Food Chemistry xxx (2016) xxx–xxx OH CH3 H3 C

CH3 O

H3 C

H O

O H3 C

H3 C

CH3 alpha-thujone

CH3 beta-thujone

H

H

H

OH

H

H

H

O

H

O absinthin O

OCH3 H H3CO

O OCH3

H3CO

OCH3 OH

O

artemisetin

O 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 temperatures 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 demonstrated 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 information 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. References Aberham, A., Cicek, S. S., Schneider, P., & Stuppner, H. (2010). Analysis of sesquiterpene lactones, lignans, and flavonoids in wormwood (Artemisia absinthium L.) using high-performance liquid chromatography (HPLC)-mass

spectrometry, reversed phase HPLC, and HPLC-solid phase extraction-nuclear magnetic resonance. Journal of Agricultural and Food Chemistry, 58, 10817–10823. Baraldi, R., Isacchi, B., Predieri, S., Marconi, G., Vincieri, F. F., & Bilia, A. R. (2008). Distribution of artemisinin and bioactive flavonoids from Artemisia annua L. during plant growth. Biochemical Systematics and Ecology, 36, 340–348. Barwick, V. J., & Ellison, S. L. R. (2000). The evaluation of measurement uncertainty from method validation studies – Part 1. Description of a laboratory protocol ACCRED Q A, 5(2), 47–53. Brown, G. D. (1992). Two new compounds from Artemisia annua. Journal of Natural Products, 55, 1756–1760. Brown, G. D., & Sy, L.-K. (2007). In vivo transformations of artemisinic acid in Artemisia annua plants. Tetrahedron, 63, 9548–9566. Dudley, G. B., Engel, D. A., Ghiviriga, I., Lam, H., Poon, K. W., & Singletary, J. A. (2007). Synthesis of +-dihydro-epi-deoxyarteannuin B. Organic Letters, 15, 2839–2842. Goud, B. J., Dwarakanath, V., & Chikka Swamy, B. K. (2015). A review on history, controversy, traditional use, ethnobotany, phytochemistry and pharmacology of Artemisia absinthium Linn. International Journal of Advanced Research in Engineering and Applied Sciences, 4, 77–107. Lachenmeier, D. W. (2007). Assessing the authenticity of absinthe using sensory evaluation and HPTLC analysis of the bitter principle absinthin. Food Research International, 40, 167–175. Lachenmeier, D. W., Nathan-Maister, D., Breaux, T. A., Sohnius, E.-M., Schoeberl, K., & Kuballa, T. (2008). Chemical composition of vintage pre-ban absinthe with special reference to thujone, fenchone, pinocamphone, methanol, copper, and antimony concentrations. Journal of Agricultural and Food Chemistry, 56, 3073–3081. Lachenmeier, D. W., Nathan-Maister, D., Breaux, T. A., Luauté, J., & Emmert, J. (2010). Absinthe, Absinthism and Thujone – New Insight into the Spirit’s Impact on Public Health. The Open Addiction Journal, 3, 32–38. Potawale, S. E., Waseem, Md., Md, S., Mehta, U. K., Dhalawat, H. J., Luniya, K. P., ... Mantri, R. A. (2008). Research and medicinal potential of Artemisia annua: A review. Pharmacologyonline, 2, 220–235. Sy, L.-K., & Brown, G. D. (2001). Deoxyarteannuin B, dihydro-deoxyarteannuin B and trans-5-hydroxy-2-isopropenyl-5-methylhex-3-en-1-ol from Artemisia annua. Phytochemistry, 58, 1159–1166. Turak, A., Shi, S.-P., Jiang, Y., & Tu, P.-F. (2014). Dimeric guaianolides from Artemisia absinthium. Phytochemistry, 105, 109–114. Zhang, W., Luo, S., Fang, F., Chen, Q., Hu, H., Jia, X., & Zhai, H. (2005). Total synthesis of absinthin. Journal of the American Chemical Society, 127, 18–19.

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