Introduction. Absinthe is a highly alcoholic (45 to 75%) drink containing mostly herb extracts. It is an anise-flavoured spirit derived from botanicals, including ...
Characterization of wormwood extract by HS-SPMEGC-MS and UHPLC-HR-MS Marilyn CLEROUX 1, Benoit BACH 1, Mayra SAILLEN 1, Stephane BURGOS 1, Julien DUCRUET 1 and Armelle VALLAT 2 1 University of Applied Sciences and Arts Western Switzerland, CHANGINS, route de Duillier 50, 1260 Nyon, Switzerland 2 University of Neuchâtel, NPAC, avenue de Bellevaux 51, 2000 Neuchâtel, Switzerland
Introduction Absinthe is a highly alcoholic (45 to 75%) drink 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. Thujone is a natural essence typically associated with common wormwood. This compound (β-thujone in particular) is an epileptiform convulsant and is widely regarded as the “active” ingredient in absinthe and since 1990, within the whole European union, a thujone maximum limit of 35 mg/kg (sum of α/ β thujone) was introduced. In consequence, a precise determination of the concentration of α- and β-thujone in this beverage is presently needed, according to international food safety regulations. A lot of conventional techniques were already developed to analyse thujone in absinthe or in wormwood extract, such as liquid-liquid extraction or solid phase extraction, but these techniques are time consuming and require large amounts of samples and solvents. Such, headspace analysis is a very attractive methodology for analysing volatiles, because it requires minimal sample preparation and can be automated. Of the headspace methods, solid-phase micro-extraction (SPME) is now probably one of the most widely used method in food analysis, offering many benefits over other headspace techniques. Although SPME is very well established for the analysis of flavours, but this technique is not commonly used for the analysis of thujone in absinthe or in wormwood extract. In the present study, a SPME method coupled GC-MS technique is proposed for determining the concentration of thujone in wormwood extract. In parallel sesquiterpene lactones and flavonoids, which account for the characteristic bitterness of wormwood were profiled by ultrahigh pressure liquid chromatography- high resolution mass spectrometry (UHPLC-HR-MS). Two identified compounds for which reference standards were available, absinthin and artemisetin, were quantified by external calibration.
Figure 1: botanical drawing of wormwood (Artemisia absinthium)
GC Method Validation
Figure 3 : Structure of α- and β-thujone Figure 2 : SPME extraction for heated and agitated samples. Standard 50/30µm DVB/CAR/PDMS SPME fibers were used. Molecule
RT (min)
MW
Quantifier ion (m/z)
Qualifier ion (m/z)
α-thujone
23.517
152.23
95
109
β-thujone
24.279
152.23
95
109
1,6-heptadien-4-ol (ISTD)
19.967
112.17
71
121
Validation was carried out in terms of specificity, linearity, precision, limit of detection (LOD) and quantification (LOQ) and accuracy. Specificity. Using Selected Reaction Monitoring, the specificity is confirmed based on the presence of the ions (quantifier and qualifier) at the correct retention times corresponding to thujone standards. The measured peak area ratios of qualifier/quantifier ion have to be in close accordance with the ion ratios of the standards as indicated in Table 1. Linearity and Calibration Curve. Linearity of the method was evaluated using calibration curves with 8 calibration levels during 6 different days. The linearity of calibration curves is assessed over the range from 0.1–100 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 linear least-squares 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. LOD and LOQ. 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 respectively 0.01 mg/L and 0.05 mg/L. Precision. Precision results displayed in table 3 were obtained by analysing 3 different spike concentrations in wormwood macerates during 6 different days using SPME-GC-MS on same and different SPME DVB/CAR/PDMS fibre. Accuracy. Under the conditions described above, accuracy was evaluated by comparing found values with spikes by standard addition in wormwood macerates. The results in Table 3 show good accuracy, with recoveries between 80% and 120% for each spike level. Measurement Uncertainty. Measurement uncertainty is estimated using the simplified approach based on existing validation data proposed by Bartwick & Ellison, 2000. Precision and trueness contributions are combined together as followed to obtain the overall uncertainty. The results in Table 3 show good uncertainty, with results below < 20% for each spike level.
Table 1: GC-MS parameters of thujones analysis
Profiling by UHPLC-HR-MS Alcoholic solution of absinthe was injected directly in Acquity BEH C18 column (1.7 μm, 2.1 x 50mm). The compounds were separated by a water/acetonitrile+0.05%FA gradient. The calculated LOD and LOQ for Artemisetin were found to be respectively 3 ng/mL and 10 ng/mL Other compounds present • Artemisinic acid, C15H22O2, RT 3.99 min. • Flavonoid Casticin, C19H18O8, RT 4.71 min. • Unknown, C30H36O5, RT 6.13 min.
Conclusion HS-SPME-GC–MS methodology was found to be fully suitable for the analysis of thujones in wormwood macerate due to its selectivity and sensitivity. The headspace method, in conjugation with GC–MS analysis in SIM mode, provides a high recovery, good linearity over a wide range of concentration and high sensitivity. This method offers a number of practical advantages: small sample volume (only 10 µL), simplicity of extraction and low cost when compared to other methods currently in use. UHPLC-HR-MS permits by one injection to obtain the quantification of major bitterness compounds. The method is robust and any particular preparation of samples is necessary.
