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Determination of anethole in serum samples by headspace solid-phase microextraction-gas chromatography–mass spectrometry for congener analysis
Journal of Chromatography A, 1200 (2008) 235–241
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Determination of anethole in serum samples by headspace solid-phase microextraction-gas chromatography–mass spectrometry for congener analysis Katja Schulz a,∗ , Katja Schlenz c , Robert Metasch d , Steffen Malt c , b ¨ Wolfgang Romhild , Jan Dreßler a a
Institut f¨ ur Rechtsmedizin, Technische Universit¨ at Dresden, Fetscherstr. 74, D-01307 Dresden, Germany Institut f¨ ur Rechtsmedizin, Otto-von-Guericke-Universit¨ at Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany c Hochschule Zittau/G¨ orlitz (FH), Fakult¨ at f¨ ur Mathematik and Naturwissenschaften, Fachbereich Chemie, Theodor-K¨ orner-Allee 16, D-02763 Zittau, Germany d Hochschule f¨ ur Technik und Wirtschaft (FH), Fakult¨ at Maschinenbau/Verfahrenstechnik, Fachbereich Chemieingenieurwesen, Friedrich-List-Platz 1, D-01169 Dresden, Germany b
a r t i c l e
i n f o
Article history: Received 6 February 2008 Received in revised form 25 April 2008 Accepted 20 May 2008 Available online 28 May 2008 Keywords: Congener analysis Beverage-characteristic aroma compounds Anethole Ouzo HS-SPME-GC–MS
a b s t r a c t A rapid headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPMEGC–MS) method has been developed for the determination of anethole in serum samples. Anethole is a characteristic marker for the consumption of aniseed spirits. This method enabled the detection of anethole with a limit of detection (LoD) of 3.6 ng/ml and a limit of quantification (LoQ) of 5.3 ng/ml in serum samples with a good degree of precision intraday (2.8%) and interday (4.5%). Experiments were conducted with one volunteer, in which the subject consumed the alcoholic drink ouzo on 3 different days under controlled conditions. At defined intervals, blood samples were taken from the subject. Using these blood samples, the concentration–time profiles for anethole were determined. In blood samples taken from 50 drivers who claimed to have consumed drinks containing anethole (ouzo, raki and the German aniseed ¨ liqueur “Kustennebel”) before the taking of the blood sample, anethole was detected in the serum in concentrations of between 5.4 and 17.6 ng/ml in 10 cases. This is the first report describing the qualitative and quantitative determination of a beverage-characteristic aroma compound – in this case anethole – in serum samples after consumption of alcoholic beverages. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The congener analysis is used to check the plausibility of postoffence drinking claims in forensic toxicology. There the defendant claims to have drunk the alcohol only after the offence and has been sober at the time of the accident. In congener analysis, the information given by the defendant regarding the type, quantity and time of consumption of the respective beverage is used to calculate the theoretically expected congener concentration in the blood and this is compared with the analytically determined concentrations in the blood sample. If these values do not correspond, the post-offence drinking claim is deemed to be disproved.
∗ Corresponding author. Tel.: +49 351 4584940; fax: +49 351 4584397. E-mail address: [email protected] (K. Schulz). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.05.066
The analysis of congeners in alcoholic beverages was introduced by Machata and Prokop [1]. On this basis, Bonte developed the current standard of analysis: static headspace sampling in combination with cryofocussing gas chromatography and flame ionisation detection [2]. Before headspace analysis, the alcoholglucuronides in blood samples were hydrolysed, followed by ultracentrifugation [3–5]. Congener analysis is currently limited to the congener substances methanol, 1-propanol, 2-butanon, 2-butanol, isobutanol, 1-butanol and 2-methyl-1-butanol and 3-methyl-1-butanol, which can be detected qualitatively and quantitatively in blood and/or serum samples [6]. None of these congeners is characteristic of a particular alcoholic beverage. The aim of the present paper was to detect congener substances that are characteristic of certain alcoholic beverages in serum samples, in order to be able to draw conclusions as to the type of beverage consumed. One of these congener substances is anethole, which is contained in relatively high concentrations in ouzo and
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validation parameters were made by adding standard stock solution to negative control serum in resulting concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 50, 100 and 200 ng/ml (ppb) anethole. 2.3. Headspace-SPME procedure Fig. 1. Chemical structure of trans-anethole [4180-23-8] C10 H12 O.
raki as well as pastis, sambuca and mistra [7]. Whereas an official reference method exists for detecting anethole in spirits [8] and its detection in spirits by means of HS-SPME [9] and by means of LC was recently described [10], anethole was not yet determined in serum samples. The SPME procedure was developed by Pawliszyn and coworkers [11–16]. Headspace-sampling techniques (e.g. headspaceSPME) offer many advantages for sample preparation in the GC analysis of volatile and semi-volatile organic compounds. Volatile compounds are separated from the sample matrix (i.e. serum) prior to their introduction into the GC without solvent extraction. Only minimal sampling handling is required. The HS-SPME method also makes it possible to extract and concentrate volatile analytes from small to large volumes of sample whilst achieving a high degree of sensitivity. This technique is used for many applications; e.g. for beverage [17] and food samples [18], for bioanalytical applications [19] and environmental analysis [20]. (E)-1-Methoxy-4-(1-propenyl)benzene (trans-anethole; C10 H12 O; CAS 4180-23-8), Fig. 1, is a volatile compound that is found naturally in many herbs. Anethole is contained in the ethereal oils of the plants aniseed (Pimpinella anisum) at a concentration level of 80–90%, star anise (Illicium verum) at a level of >90% and fennel (Foeniculum vulgare) at a level of 50–80% [7]. Natural extracts of these plants are used as aromatic substances in the aforementioned spirits. The detection of anethole is relatively specific for alcoholic beverages containing this substance. The investigations of the authors came to the result, that anethole could not be detected in serum samples after the consumption of anise and fennel tea in high amounts. Furthermore, anethole is used in medicine as expectorant, antitussive and antispasmodic drug for gastrointestinal tract and therefore contained in few pharmaceuticals. The incorporated amounts for therapeutic dosages of anethole are much lower than in the case of moderate ouzo consumption. For the present study, more than 500 serum samples from drivers who claimed to have consumed other alcoholic beverages than ouzo and raki were analysed for anethole. In no case anethole could be detected.
SPME experiments were performed using a manual fibre holder supplied by Supelco (Taufkirchen, Germany). Four commercially available fibres, Carbowax/divinylbenzene (CW/DVB, 65 m), Stable-Flex Carboxen/polydimethylsiloxane (CAR/PDMS, 85 m), Stable-Flex polydimethylsiloxane/divinylbenzene (PDMS/DVB, 65 m) and Stable-Flex divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 m) were purchased from Supelco. Before use, each fibre was conditioned in the GC injection port under helium flow in accordance with the temperature and time recommended by the manufacturer. Fibre blanks were run periodically to ensure the absence of contaminants or carryover. The SPME procedure was as follows: 200 l serum and 200 l internal standard solution (100 ng/ml dicyclohexylmethanol) were placed in a 22 ml headspace-vial containing an 8 mm × 3 mm PTFEcoated stir bar and 0.1 g Na2 SO4 . The samples were immediately sealed with silicone-PTFE septa. Before HS-SPME analysis, the sample vial was stirred for 1 min and conditioned for 1 min in a thermostatic water bath at a temperature of 50 ◦ C. Then the sample was extracted using PDMS/DVB (65 m) fibre for 30 min at 50 ◦ C and a magnetic agitation rate of 700 rpm. The thermal desorption of the analyte was carried out by exposing the fibre in the GC injection port at 250 ◦ C for 3 min. To prevent a memory effect, the fibre was kept in the injection port for an additional time of 7 min in the split mode (purge on). 2.4. Beverage analysis The testing of spirits for anethole content was conducted using the headspace-trap procedure. The headspace analysis was performed with the PerkinElmer TurboMatrix HS 110-trap automatic headspace sampler with trap enrichment and flame ionisation detector (PerkinElmer, Shelton, USA). A capillary column Rtx 1701 (60 m × 0.530 mm i.d.; 1.5 m film thickness) with phenylcyanopropyl phase from Restek was used. Data acquisition and integration were carried out with TotalChrom (Version 6.2.1) software. The enrichment conditions and chromatographic conditions were described in our recently published article [21]. 2.5. GC–MS conditions
2. Experimental 2.1. Reagents and standards Anethole was purchased from Fluka (Taufkichen, Germany) and dicyclohexylmethanol as an internal standard was obtained from Sigma–Aldrich (Steinheim, Germany). Na2 SO4 and ethanol were obtained from Merck (Darmstadt, Germany). All chemicals were of analytical grade. Water was deionised. Negative control serum samples for spiking with anethole were obtained from the authors. 2.2. Sample preparation A standard solution of 200 and 2000 ng/ml of anethole was made in water with ethanol as solubilizer. The serum stock solution of anethole was made by addition of 1 ml anethole solution (aqueous standard 200 and 2000 ng/ml, respectively) to 9 ml of negative control serum sample. Subsequent solutions for calibration curves and
The GC–MS system used for analysis was a Hewlett–Packard GC 5890 series II with a 5971 mass selective detector (Waldbronn, Germany). Data acquisition and analysis were performed using standard software supplied by the manufacturer. Substances were separated on a fused silica capillary column HP-5MS (30 m × 0.25 mm i.d.; 0.25 m film thickness) supplied by J&W Scientific (California, USA). Temperature program: 40 ◦ C hold for 5 min, 5 ◦ C/min up to 160 ◦ C, 20 ◦ C/min up to 220 ◦ C, hold for 3 min. The temperatures for the injection port and detector were set at 250 and 280 ◦ C, respectively. Splitless injection mode (splitless time 3 min) and helium with a flow rate of 1.15 ml/min as carrier gas were used. To determine the retention times and characteristic mass fragments, electron impact (EI) mass spectra of the analytes were recorded by total ion monitoring. All investigations (optimisation, statistical parameters and original serum samples) were monitored in full scan mode with a scan range of 33–250 m/z. In order to search for possibly existing metabolites in the mass spectra at later time,
K. Schulz et al. / J. Chromatogr. A 1200 (2008) 235–241
Fig. 2. Peak areas of spiked serum samples (anethole 20 ppb each; M = 148) by HSSPME extraction with four different fibres; extraction temperature = 50 ◦ C, sampling time = 30 min, desorption time = 10 min, n = 3.
the full scan mode was selected. For evaluation, diagnostic mass fragments of anethole (m/z = 77, 117, 147 and 148) from full scan mode with target ion m/z = 148 were selected. 2.6. Structure of the experiment The conduct of the drinking experiments was approved by the ethics committee. The drinking experiments were carried out on a volunteer subject using the beverage “Helenas Ouzo” at three different dosages: 3 × 40 ml (corresponding to the consumption of 56.0 mg of anethole abs.; test 1); 5 × 40 ml (corresponding to the consumption of 93.4 mg of anethole abs.; test 2) and 9 × 40 ml (corresponding to the consumption of 168.1 mg of anethole abs.; test 3). The interval between the individual tests was 7 days in each case. The consumption time was 1 h. The following personal data relating to the subject were established: male, 22 years of age, 66 kg, 1.78 m, lean build. The blood samples were taken at defined times, see Table 3. The blood samples were centrifuged, the serum separated and the blood alcohol level measured according to forensic guidelines. The serum samples were then frozen and stored at −18 ◦ C until HS-SPME-GC–MS analysis for the detection of anethole. 2.7. Method validation All statistical data (limit of detection, LoD; limit of quantification, LoQ; correlation coefficient, R2 ; relative standard deviations intraday and interday, RSD; linear range) were evaluated according to a German standard procedure (DIN 32645) by analysing anethole standard solution using the method described above. The method was also tested by means of routine analysis of samples from drivers found to be under the influence of alcohol who claimed to have consumed beverages containing anethole.
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desorption was found for any of the fibres, indicating complete removal of the analyte at these temperatures. The highest extraction yield was found with the 65 m PDMS/DVB fibre. The fibre provided an extraction yield higher than the other fibres, from 2-fold for CW/DVB to 3-fold for DVB/CAR/PDMS and CAR/PDMS. Remarkably, both Carboxen fibres showed a lower capacity to retain anethole. Carboxen fibres seem to be ideal for the SPME analysis of very small molecules (C2 –C6 ). The micropore diameter of Carboxen ˚ [18] for the enrichment of anetlayer appears too small (10–17 A) hole (C10 ). Therefore, the 65 m PDMS/DVB fibre was used for all subsequent investigations. The effect of sampling temperature on the anethole extraction yield was investigated from 25 to 80 ◦ C using PDMS/DVB fibre. A maximum at 50 ◦ C could be observed. At temperatures from 25 to 50 ◦ C an increasing concentration of anethole in the headspace was observable. At temperatures higher than 50 ◦ C, the equilibrium between fibre and headspace is more and more shifted towards the headspace. An extraction temperature of 50 ◦ C was chosen since at this temperature the best anethole response was obtained. The extraction time of anethole was investigated between 2 and 60 min using the optimal conditions previously established. The time required to reach equilibrium was 30 min, and so this time was considered the optimal value. To summarise, the HS-SPME optimal conditions for the analysis of anethole were: 65 m PDMS/DVB fibre, extraction temperature 50 ◦ C, sampling time 30 min. In addition, a desorption temperature of 250 ◦ C and a desorption time of 10 min (3 min splitless time and an additional time at 7 min in the split mode), a stirring speed of 700 rpm and 0.1 g Na2 SO4 were chosen. Calibration of the method was performed using the standard solution in the concentration range of 2–200 ng/ml serum. Statistical data for the calibration functions, calculated according to a German standard procedure (DIN 32645), are summarised in Table 1. No interfering peaks were found in serum samples with the same retention time as anethole and the internal standard. The relative standard deviations (RSD), both intra- and interday, of 2.8% and 4.5% indicate a very good reproducibility of the method. The correlation coefficient (R2 ) of 0.990 of the calibration graphs emphasise good linearity in the concentration range investigated. The LoD of 3.6 ng/ml and LoQ of 5.3 ng/ml show the excellent sensitivity of the method (Table 1). 3.2. Beverage analysis The anethole concentration in five German products (four ouzo ¨ and one aniseed liqueur “Kustennebel”) and three original Greek ouzos was determined using the headspace-trap technique [21]. The results are shown in Table 2. The anethole concentrations detected in the German products lay in the region of 400–800 mg/l. The anethole concentration in
3. Results and discussion 3.1. HS-SPME optimisation First, the selection of the appropriate fibre was carried out. Four fibres (65 m CW/DVB, 85 m CAR/PDMS, 65 m PDMS/DVB and 50/30 m DVB/CAR/PDMS) were evaluated in order to obtain the best sensitivity and selectivity for anethole determination (Fig. 2). In addition, extraction time in the GC injector port was 10 min and the extraction temperature was fixed at 50 ◦ C. The desorption temperatures were the recommended conditioning temperatures for each fibre: 220 ◦ C for CW/DVB, 300 ◦ C for CAR/PDMS, 250 ◦ C for PDMS/DVB, and 270 ◦ C for DVB/CAR/PDMS to ensure complete desorption of anethole from the fibres. No carryover on second
Table 1 Results of method validation according to a German standard procedure (DIN 32645) Headspace SPME-GC–MS of anethole in serum samples 3.6 LoDa (ng/ml) LoQa (ng/ml) 5.3 0.990 R2 2.8 Precision intradayb (%) 4.5 Precision interdayb (%) Linear range (ng/ml) 2–200 All measurements were made in triplicate. a Limit of detection and quantitation were determined by establishing a specific calibration curve from samples containing the analyte in the range of LoQ. The limits were calculated from the residual standard deviation of the regression line. b Precisions are expressed as RSD (%), intraday (n = 7), interday (n = 5).
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Table 2 Results of investigation of different spirits containing anethole Spirit
Origin
Ethanol (vol.%)
canethole (g/ml)
Ouzo Helenas Ouzo Zeus Ouzo Mirios Ouzo 12 ¨ ¨ Kustennebel “Anis&Kom” Ouzo Etairetikis Poiotitos Ouzo Giokarinis Ouzo Knossos
Germany, Saxonia Germany, Saxonia Germany, Saxonia Germany, Saxonia Germany, Saxonia Greece, Santorin Greece, Samos Greece, Crete
37.5 37.5 37.5 38.0 21.8 38.5 42.0 40.0
467 641 794 549 497 1062 1238 390
the selected Greek products varies from 300 to 1300 mg/l. In beverages containing large numbers of congener substances such as whisky, brandy and certain fruit spirits, similar concentrations of 1-propanol, isobutanol and 2/3-methylbutanol are found, which are detectable in serum samples for differing lengths of time, even after consumption of moderate amounts. The anethole concentration in the selected alcoholic beverages needed to be high enough to be detectable in serum samples after consumption. A further important factor in enabling detection is the elimination kinetic of the congener substance concerned. Among the congener substances that are detectable at present, the rate of elimination rises with increasing carbon chain length, i.e. with decreasing polarity of the alcohols.
Fig. 3. Concentration–time profiles of anethole after drinking tests with 120 ml (intended maximum BAC = 0.5‰), 200 ml (intended maximum BAC = 1.0‰) and 360 ml (intended maximum BAC = 1.5‰) Helenas Ouzo.
It is not possible to make detailed statements regarding elimination kinetics as the number of measuring points was too small. Concentration–time profiles with rapid resorption- and elimination phases – as in this case – are ideal for the verification of post-offence drinking claims. In congener analysis, the detection time for the target analyte should extend over a few hours (approx. 0.5–4 h), since post-offence drinking claims are usually made for this period of time. A detection time for the analytes in the serum of more than 12 h would not be suitable for the verification of post-offence drinking claims, since the intention is not to record drinking that took place at an earlier time.
3.3. Drinking tests with volunteer In order to be able to make statements regarding the concentration–time profile of anethole, the drinking experiments were conducted with differing amounts of the beverage. The male subject (22 years, lean build) consumed the German product “Helenas Ouzo” in three separate tests, drinking 120, 200, and 360 ml respectively. During each test, eight blood samples were taken. The blood-alcohol concentrations and the corresponding anethole concentrations are listed in Table 3. The corresponding concentration–time profile of the anethole is depicted in Fig. 3. The anethole concentrations detected show rapid resorption of anethole and also rapid elimination. (In each case, the maximum anethole concentration corresponds to the maximum blood alcohol concentration.) Even immediately after cessation of drinking, anethole concentrations can clearly be measured. Anethole at concentrations above the detection level of 3.6 ng/ml serum can be detected in the selected volunteer for 3 h after ceasing consumption of 120 ml of Helenas Ouzo and 200 ml ouzo, and for 7 h after ceasing consumption of 360 ml ouzo.
3.4. Serum samples from drivers Serum samples taken in 2005 and 2006 from drivers who were found to be under the influence of alcohol and who claimed to have consumed beverages containing anethole were tested for anethole. These cases were not post-offence drinking claims but rather cases of drivers who had consumed beverages containing anethole and who could usually give information about the drinking time and the amount consumed. The results are listed in Table 4. In 10 out of 50 serum samples anethole was detected at concentration levels of between 5.4 and 17.6 ng/ml serum. Of these, eight were cases of ouzo consumption, one of raki consumption ¨ and one of German aniseed liqueur “Kustennebel” consumption.
Table 3 Test conditions, blood-alcohol concentrations and anethole concentrations in the serum samples from the drinking experiment Duration of drinking 1 h
Blank sample before testing 0 h after CDb , 1 h after BDc 0.5 h after CD, 1.5 h after BD 1 h after CD, 2 h after BD 2 h after CD, 3 h after BD 3 h after CD, 4 h after BD 7 h after CD, 8 h after BD 23 h after CD, 24 h after BD a b c
Drinking test 1 (120 ml ouzo and 56.0 mg anethole)
Drinking test 2 (200 ml ouzo and 93.4 mg anethole)
Drinking test 3 (360 ml ouzo and 168.1 mg anethole)
BACa (‰)
canethole (ng/ml)
BACa (‰)
canethole (ng/ml)
BACa (‰)
canethole (ng/ml)
0.00 0.49 0.48 0.41 0.23 0.11 0.00 0.00
0 17.5 8.36 7.57 7.14 4.55 <3.6 <3.6
0.00 0.59 0.74 0.72 0.60 0.41 0.00 0.00
0 15.9 25.7 17.5 12.0 4.49 <3.6 <3.6
0.00 0.78 1.47 1.75 1.62 1.43 0.69 0.00
0 15.1 24.9 73.0 34.4 15.5 6.10 <3.6
BAC, blood alcohol concentration. CD, cessation of drinking. BD, begin of drinking.
K. Schulz et al. / J. Chromatogr. A 1200 (2008) 235–241
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Table 4 Anethole concentrations in serum samples from drivers No.
Data
Alleged consumption
Time between CDa and TBSb
BACc (‰)
canethole (ng/ml)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
38 years, male, 90 kg, 164 cm 44 years, female, 71 kg, 171 cm 36 years, female, 65 kg, 158 cm 42 years, male, 82 kg, 182 cm 37 years, female, 62 kg, 163 cm 49 years, male, 72 kg, 175 cm 17 years, male, 61 kg, 179 cm 37 years, male, 104 kg, 187 cm 41 years, male, 62 kg, 182 cm 19 years, male, 65 kg, 186 cm 40 years, female, 75 kg, 165 cm 22 years, male, 70 kg, 176 cm 25 years, female, 59 kg, 156 cm 45 years, male, 80 kg, 174 cm 45 years, male, 72 kg, 175 cm 31 years, male, 90 kg, 175 cm 34 years, male, 80 kg, 176 cm 17 years, male, 68 kg, 178 cm 44 years, male, 62 kg, 176 cm 45 years, male, 80 kg, 175 cm 18 years, male, 75 kg, 184 cm 47 years, male, 72 kg, 174 cm 26 years, male, 95 kg, 186 cm 54 years, male, 92 kg, 170 cm 16 years, male, 72 kg, 177 cm 45 years, female, 78 kg, 170 cm 50 years, male, 110 kg, 178 cm 48 years, male, 80 kg, 193 cm 43 years, male, 90 kg, 180 cm 20 years, male, 80 kg, 180 cm 33 years, male, 78 kg, 168 cm 23 years, female, 50 kg, 165 cm 19 years, male, 75 kg, 175 cm 50 years, female, 59 kg, 166 cm 19 years, male, 75 kg, 175 cm 30 years, female, 48 kg, 160 cm 29 years, male, 80 kg, 175 cm 46 years, male, 69 kg, 175 cm 32 years, female, 65 kg, 158 cm 44 years, male, 82 kg, 171 cm 34 years, male, 65 kg, 173 cm 63 years, male, 75 kg, 170 cm 38 years, male, 69 kg, 170 cm 24 years, male, 85 kg, 163 cm 23 years, male, 79 kg, 186 cm 20 years, male, 106 kg, 193 cm 24 years, male, 75 kg, 173 cm 27 years, male, 85 kg, 187 cm 43 years, male, 85 kg, 185 cm 57 years, male, unknown
60 ml ouzo/0.5 l beer 40 ml ouzo/cola-whisky 40 ml ouzo/250 ml red wine 40 ml ouzo/1.5 l beer 40 ml ouzo/200 ml wine 40 ml ouzo/1.0 l beer 40 ml ouzo/0.5 l beer/0.25 l martini Ouzo#/1.0 l beer 40 ml ouzo/0.5 l beer Ouzo#/vodka-cola 80 ml ouzo/1.5 l beer 0.3 l ouzo Raki# ¨ Kustennebel# 40 ml ouzo 20 ml ouzo/4.0 l beer 40 ml ouzo/1.5 l beer Ouzo#/2.5 l beer 60 ml ouzo/3.5 l beer 80 ml ouzo/2.0 l beer 0.5 l ouzo/beer 80 ml ouzo/2.0 l beer 60 ml ouzo/4.0 l beer 40 ml ouzo/1.5 l beer Ouzo#/beer 80 ml ouzo/1.0 l beer 120 ml raki/1.0 l beer 120 ml ouzo/1.0 l beer/0.75 l sparkling wine Ouzo#/beer 200 ml raki/1.0 l beer/cocktail 20 ml ouzo/40 ml herb liqueur/1.0 l beer 120 ml ouzo 120 ml ouzo 120 ml ouzo 200 ml ouzo Ouzo#/white wine 120 ml ouzo/3.0 l beer/0.2 l herb liqueur 40 ml raki/1.0 l beer/0.7 l red wine Ouzo#/0.2 l red wine/0.1 l sparkling wine 40 ml ouzo/1.75 l beer 40 ml ouzo 40 ml ouzo/0.5 l beer/400 ml wine 40 ml ouzo 750 ml ouzo 100 ml ouzo/3.0 l beer/300 ml apple grain 40 ml ouzo/2.0 l beer/60 ml tequila/20 ml herb liqueur Ouzo# 80 ml raki/600 ml beer/800 ml cuba-libre 120 ml ouzo ¨ Kustennebel#/2.5 l beer/peppermint-liqueur
3 h 30 min 50 min 2h 1 h 35 min 1 h 10 min 1 h 25 min 2 h 45 min 1 h 5 min 7 h 15 min 2 h 05 min 1 h 10 min 5 h 45 min 2h 40 min 1 h 25 min 35 min 1 h 10 min 2 h 55 min 1 h 55 min 5 h 25 min 6 h 5 min 1 h 20 min 50 min 45 min 1 h 45 min 3 h 45 min 1 h 55 min 50 min 1 h 25 min 6h 2h 60 min 60 min 60 min 60 min 7 h 10 min 4 h 25 min 2 h 40 min 55 min 1 h 20 min 1 h 40 min 4h 2 h 45 min 2 h 25 min 2h 1 h 35 min >2 h 1 h 15 min 55 min 4 h 10 min
2.11 2.69 0.49 1.11 2.82 1.60 1.45 2.21 1.06 1.52 1.60 1.02 1.49 2.23 1.88 2.72 1.45 1.46 2.37 2.17 1.11 1.34 1.70 2.08 2.18 2.13 2.02 1.73 2.51 0.57 2.08 0.52 0.20 0.25 0.46 1.63 2.16 1.06 1.44 2.41 1.97 1.77 0.36 1.67 1.84 1.70 1.77 1.41 0.18 2.45
n.d. n.d. n.d. n.d. n.d. n.d. n.d. 10.8 n.d. n.d. 11.0 n.d. 13.6 12.6 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 14.6 n.d. n.d. n.d. 17.6 n.d. 16.4 5.4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 12.9 n.d. n.d. n.d. n.d. 7.35 n.d.
n.d., not determined; (#) amount of consumed beverage not known. Only the positive cases of anethole are bold-lines. a CD, cessation of drinking. b TBS, taking of blood samples. c BAC, blood alcohol concentration.
In the positive cases, the time difference between the cessation of drinking and the taking of blood samples was 40 min (12.6 ng/ml anethole; case 14) at the minimum and 2 h 25 min (12.9 ng/ml anethole; case 44) at the maximum. The corresponding blood alcohol concentrations lay between 0.18‰ (7.35 ng/ml anethole; case 49) and 2.23‰ (12.6 ng/ml anethole; case 14). Where given, the information about the time of drinking and the amount consumed accounts for the anethole concentrations detected in the positive cases. Even where the blood alcohol concentration was low (cases 32, 34, 35, and 49 with 0.52, 0.25, 0.46, and 0.18‰, respectively) and only spirits containing anethole had been consumed, anethole concentrations above the limit of quantification could be detected (Fig. 4). It is evident from the results of the investigations conducted in the drinking experiments and from the samples taken from drivers
who were under the influence of alcohol that anethole is very sensitive and can be reliably detected even in authentic samples after consumption of spirits containing anethole, such as ouzo, raki and ¨ the German aniseed liqueur “Kustennebel”. In no case was a positive result for anethole found where 40 ml or less of spirits containing anethole had been consumed or where the time difference between the cessation of drinking and the taking of the blood sample was greater than 4 h (except in the experiment involving a very large amount of ouzo—360 ml). It is intended to develop a method of differentiating positive from negative samples through the time difference between cessation of drinking and the taking of a blood sample, in order to be able to distinguish between pre-offence and post-offence drinking claims and thus to be able to reliably disprove or confirm post-offence drinking claims which are of significance in cases of traffic offences.
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Fig. 4. (a) GC–MS chromatogram (scan mode) of case 32 (21.50 min = anethole; 29.26 min = dicyclohexylmethanol, internal standard, BAC = 0.52‰) and (b) corresponding mass spectrum of peak at 21.50 min (anethole).
The blood alcohol level detected is not, however, decisive for a positive anethole finding. Information about the amount consumed and the time of drinking the beverage containing anethole are absolutely essential for verifying the plausibility of claims.
is characteristic of certain alcoholic beverages Hitherto, it was only possible to detect this substance in the beverages themselves and not in blood samples. Hence, improved verification of post-offence drinking claims is now possible. Acknowledgments
4. Conclusions The developed HS-SPME-GC–MS method has shown itself to be ideal for the detection of anethole in serum samples taken from drivers under the influence of alcohol owing to the simplicity of the preparation procedures required for the samples, the excellent degree of sensitivity and its other very good statistical parameters. The selection of fibre coating allows for optimal adjustment to specific substances or substance classes. Therefore, the PDMS/DVB fibre was used for subsequent investigations. Based on the drinking experiments, concentration–time profiles were drawn up which enable statements to be made about changes in detection levels over time and the duration of the detectability of anethole in serum. It was possible to conclude that this method of verifying post-offence drinking claims relating to spirits containing anethole is very suitable within an approximate time-frame of 30 min to 4 h and in cases of consumption of relatively small amounts of spirits containing anethole (upwards of 40 ml ouzo). Thus, it was possible to extend the range of analytes detectable through congener analysis by that of the substance anethole, which
The authors would like to thank Dr. Uwe Schmidt and Uwe Hanisch for the taking of blood samples. References [1] G. Machata, O. Prokop, Blutalkohol 8 (1971) 349. [2] W. Bonte, J. Busse, Blutalkohol 17 (1980) 49. ¨ [3] W. Bonte, R. Sprung, H. Jurgens, J. Manigold, P. Olbrich, W. Pahl, Zbl. Rechtsmed. 25 (1983) 68. ¨ ¨ [4] W. Bonte, G. Stoppelmann, E. Rudell, R. Sprung, Blutalkohol 18 (1981) 303. ¨ [5] B. Kuhnholz, W. Bonte, Blutalkohol 20 (1983) 399. ¨ ¨ ¨ [6] W. Bonte, Begleitstoffe alkoholischer Getranke. Max Schmidt-Romhild, Lubeck, 1987. [7] E. Kolb, Spirituosentechnologie, B. Behr’s Verlag, Hamburg, 2004. [8] Verordnung (EG) Nr. 2091/2002 der Kommission vom 26. November 2002 zur ¨ Anderung der Verordnung (EG) Nr. 2870/2000 mit gemeinschaftlichen Referen¨ Spirituosen, ABI. EU L322, 2002, pp. 11. zanalysenmethoden fur [9] K. Lachenmeier, F. Mußhoff, B. Madea, E.M. Sohnius, W. Frank, D.W. Lachenmeier, Deut. Lebensm. -Rundsch. 101 (2005) 187. [10] J.M. Jurado, A. Alcazar, F. Pablos, M.J. Martin, Chromatographia 64 (2006) 223. [11] C.L. Arthur, J. Pawliszyn, Anal. Chem. 62 (1990) 2145. [12] C.L. Arthur, D.W. Potter, K.D. Buchholz, S. Motlagh, J. Pawliszyn, LC–GC 10 (1992) 656.
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D.W. Potter, J. Pawliszyn, J. Chromatogr. A 625 (1992) 247. Z. Zhang, M.J. Yang, J. Pawliszyn, Anal. Chem. 66 (1994) 844. D. Louch, S. Motlagh, J. Pawliszyn, Anal. Chem. 64 (1992) 1187. Z. Zhang, J. Pawliszyn, Anal. Chem. 65 (1993) 1843. D.W. Lachenmeier, U. Nerlich, T. Kuballa, J. Chromatogr. A 1108 (2006) 116. M.S. Altaki, F.J. Santos, M.T. Galceran, J. Chromatogr. A 1146 (2007) 103.
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[19] F.M. Musteata, J. Pawliszyn, TRAC 26 (2007) 36. [20] C.L. Arthur, L.M. Killam, S. Motlagh, M. Lim, D.W. Potter, J. Pawliszyn, Environ. Sci. Technol. 26 (1992) 979. [21] K. Schulz, J. Dreßler, E.M. Sohnius, D.W. Lachenmeier, J. Chromatogr. A 1145 (2007) 204.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Determination of anethole in serum samples by headspace solid-phase microextraction-gas chromatography–mass spectrometry for congener analysis Katja Schulz a,∗ , Katja Schlenz c , Robert Metasch d , Steffen Malt c , b ¨ Wolfgang Romhild , Jan Dreßler a a
Institut f¨ ur Rechtsmedizin, Technische Universit¨ at Dresden, Fetscherstr. 74, D-01307 Dresden, Germany Institut f¨ ur Rechtsmedizin, Otto-von-Guericke-Universit¨ at Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany c Hochschule Zittau/G¨ orlitz (FH), Fakult¨ at f¨ ur Mathematik and Naturwissenschaften, Fachbereich Chemie, Theodor-K¨ orner-Allee 16, D-02763 Zittau, Germany d Hochschule f¨ ur Technik und Wirtschaft (FH), Fakult¨ at Maschinenbau/Verfahrenstechnik, Fachbereich Chemieingenieurwesen, Friedrich-List-Platz 1, D-01169 Dresden, Germany b
a r t i c l e
i n f o
Article history: Received 6 February 2008 Received in revised form 25 April 2008 Accepted 20 May 2008 Available online 28 May 2008 Keywords: Congener analysis Beverage-characteristic aroma compounds Anethole Ouzo HS-SPME-GC–MS
a b s t r a c t A rapid headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPMEGC–MS) method has been developed for the determination of anethole in serum samples. Anethole is a characteristic marker for the consumption of aniseed spirits. This method enabled the detection of anethole with a limit of detection (LoD) of 3.6 ng/ml and a limit of quantification (LoQ) of 5.3 ng/ml in serum samples with a good degree of precision intraday (2.8%) and interday (4.5%). Experiments were conducted with one volunteer, in which the subject consumed the alcoholic drink ouzo on 3 different days under controlled conditions. At defined intervals, blood samples were taken from the subject. Using these blood samples, the concentration–time profiles for anethole were determined. In blood samples taken from 50 drivers who claimed to have consumed drinks containing anethole (ouzo, raki and the German aniseed ¨ liqueur “Kustennebel”) before the taking of the blood sample, anethole was detected in the serum in concentrations of between 5.4 and 17.6 ng/ml in 10 cases. This is the first report describing the qualitative and quantitative determination of a beverage-characteristic aroma compound – in this case anethole – in serum samples after consumption of alcoholic beverages. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The congener analysis is used to check the plausibility of postoffence drinking claims in forensic toxicology. There the defendant claims to have drunk the alcohol only after the offence and has been sober at the time of the accident. In congener analysis, the information given by the defendant regarding the type, quantity and time of consumption of the respective beverage is used to calculate the theoretically expected congener concentration in the blood and this is compared with the analytically determined concentrations in the blood sample. If these values do not correspond, the post-offence drinking claim is deemed to be disproved.
∗ Corresponding author. Tel.: +49 351 4584940; fax: +49 351 4584397. E-mail address: [email protected] (K. Schulz). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.05.066
The analysis of congeners in alcoholic beverages was introduced by Machata and Prokop [1]. On this basis, Bonte developed the current standard of analysis: static headspace sampling in combination with cryofocussing gas chromatography and flame ionisation detection [2]. Before headspace analysis, the alcoholglucuronides in blood samples were hydrolysed, followed by ultracentrifugation [3–5]. Congener analysis is currently limited to the congener substances methanol, 1-propanol, 2-butanon, 2-butanol, isobutanol, 1-butanol and 2-methyl-1-butanol and 3-methyl-1-butanol, which can be detected qualitatively and quantitatively in blood and/or serum samples [6]. None of these congeners is characteristic of a particular alcoholic beverage. The aim of the present paper was to detect congener substances that are characteristic of certain alcoholic beverages in serum samples, in order to be able to draw conclusions as to the type of beverage consumed. One of these congener substances is anethole, which is contained in relatively high concentrations in ouzo and
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validation parameters were made by adding standard stock solution to negative control serum in resulting concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 50, 100 and 200 ng/ml (ppb) anethole. 2.3. Headspace-SPME procedure Fig. 1. Chemical structure of trans-anethole [4180-23-8] C10 H12 O.
raki as well as pastis, sambuca and mistra [7]. Whereas an official reference method exists for detecting anethole in spirits [8] and its detection in spirits by means of HS-SPME [9] and by means of LC was recently described [10], anethole was not yet determined in serum samples. The SPME procedure was developed by Pawliszyn and coworkers [11–16]. Headspace-sampling techniques (e.g. headspaceSPME) offer many advantages for sample preparation in the GC analysis of volatile and semi-volatile organic compounds. Volatile compounds are separated from the sample matrix (i.e. serum) prior to their introduction into the GC without solvent extraction. Only minimal sampling handling is required. The HS-SPME method also makes it possible to extract and concentrate volatile analytes from small to large volumes of sample whilst achieving a high degree of sensitivity. This technique is used for many applications; e.g. for beverage [17] and food samples [18], for bioanalytical applications [19] and environmental analysis [20]. (E)-1-Methoxy-4-(1-propenyl)benzene (trans-anethole; C10 H12 O; CAS 4180-23-8), Fig. 1, is a volatile compound that is found naturally in many herbs. Anethole is contained in the ethereal oils of the plants aniseed (Pimpinella anisum) at a concentration level of 80–90%, star anise (Illicium verum) at a level of >90% and fennel (Foeniculum vulgare) at a level of 50–80% [7]. Natural extracts of these plants are used as aromatic substances in the aforementioned spirits. The detection of anethole is relatively specific for alcoholic beverages containing this substance. The investigations of the authors came to the result, that anethole could not be detected in serum samples after the consumption of anise and fennel tea in high amounts. Furthermore, anethole is used in medicine as expectorant, antitussive and antispasmodic drug for gastrointestinal tract and therefore contained in few pharmaceuticals. The incorporated amounts for therapeutic dosages of anethole are much lower than in the case of moderate ouzo consumption. For the present study, more than 500 serum samples from drivers who claimed to have consumed other alcoholic beverages than ouzo and raki were analysed for anethole. In no case anethole could be detected.
SPME experiments were performed using a manual fibre holder supplied by Supelco (Taufkirchen, Germany). Four commercially available fibres, Carbowax/divinylbenzene (CW/DVB, 65 m), Stable-Flex Carboxen/polydimethylsiloxane (CAR/PDMS, 85 m), Stable-Flex polydimethylsiloxane/divinylbenzene (PDMS/DVB, 65 m) and Stable-Flex divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 m) were purchased from Supelco. Before use, each fibre was conditioned in the GC injection port under helium flow in accordance with the temperature and time recommended by the manufacturer. Fibre blanks were run periodically to ensure the absence of contaminants or carryover. The SPME procedure was as follows: 200 l serum and 200 l internal standard solution (100 ng/ml dicyclohexylmethanol) were placed in a 22 ml headspace-vial containing an 8 mm × 3 mm PTFEcoated stir bar and 0.1 g Na2 SO4 . The samples were immediately sealed with silicone-PTFE septa. Before HS-SPME analysis, the sample vial was stirred for 1 min and conditioned for 1 min in a thermostatic water bath at a temperature of 50 ◦ C. Then the sample was extracted using PDMS/DVB (65 m) fibre for 30 min at 50 ◦ C and a magnetic agitation rate of 700 rpm. The thermal desorption of the analyte was carried out by exposing the fibre in the GC injection port at 250 ◦ C for 3 min. To prevent a memory effect, the fibre was kept in the injection port for an additional time of 7 min in the split mode (purge on). 2.4. Beverage analysis The testing of spirits for anethole content was conducted using the headspace-trap procedure. The headspace analysis was performed with the PerkinElmer TurboMatrix HS 110-trap automatic headspace sampler with trap enrichment and flame ionisation detector (PerkinElmer, Shelton, USA). A capillary column Rtx 1701 (60 m × 0.530 mm i.d.; 1.5 m film thickness) with phenylcyanopropyl phase from Restek was used. Data acquisition and integration were carried out with TotalChrom (Version 6.2.1) software. The enrichment conditions and chromatographic conditions were described in our recently published article [21]. 2.5. GC–MS conditions
2. Experimental 2.1. Reagents and standards Anethole was purchased from Fluka (Taufkichen, Germany) and dicyclohexylmethanol as an internal standard was obtained from Sigma–Aldrich (Steinheim, Germany). Na2 SO4 and ethanol were obtained from Merck (Darmstadt, Germany). All chemicals were of analytical grade. Water was deionised. Negative control serum samples for spiking with anethole were obtained from the authors. 2.2. Sample preparation A standard solution of 200 and 2000 ng/ml of anethole was made in water with ethanol as solubilizer. The serum stock solution of anethole was made by addition of 1 ml anethole solution (aqueous standard 200 and 2000 ng/ml, respectively) to 9 ml of negative control serum sample. Subsequent solutions for calibration curves and
The GC–MS system used for analysis was a Hewlett–Packard GC 5890 series II with a 5971 mass selective detector (Waldbronn, Germany). Data acquisition and analysis were performed using standard software supplied by the manufacturer. Substances were separated on a fused silica capillary column HP-5MS (30 m × 0.25 mm i.d.; 0.25 m film thickness) supplied by J&W Scientific (California, USA). Temperature program: 40 ◦ C hold for 5 min, 5 ◦ C/min up to 160 ◦ C, 20 ◦ C/min up to 220 ◦ C, hold for 3 min. The temperatures for the injection port and detector were set at 250 and 280 ◦ C, respectively. Splitless injection mode (splitless time 3 min) and helium with a flow rate of 1.15 ml/min as carrier gas were used. To determine the retention times and characteristic mass fragments, electron impact (EI) mass spectra of the analytes were recorded by total ion monitoring. All investigations (optimisation, statistical parameters and original serum samples) were monitored in full scan mode with a scan range of 33–250 m/z. In order to search for possibly existing metabolites in the mass spectra at later time,
K. Schulz et al. / J. Chromatogr. A 1200 (2008) 235–241
Fig. 2. Peak areas of spiked serum samples (anethole 20 ppb each; M = 148) by HSSPME extraction with four different fibres; extraction temperature = 50 ◦ C, sampling time = 30 min, desorption time = 10 min, n = 3.
the full scan mode was selected. For evaluation, diagnostic mass fragments of anethole (m/z = 77, 117, 147 and 148) from full scan mode with target ion m/z = 148 were selected. 2.6. Structure of the experiment The conduct of the drinking experiments was approved by the ethics committee. The drinking experiments were carried out on a volunteer subject using the beverage “Helenas Ouzo” at three different dosages: 3 × 40 ml (corresponding to the consumption of 56.0 mg of anethole abs.; test 1); 5 × 40 ml (corresponding to the consumption of 93.4 mg of anethole abs.; test 2) and 9 × 40 ml (corresponding to the consumption of 168.1 mg of anethole abs.; test 3). The interval between the individual tests was 7 days in each case. The consumption time was 1 h. The following personal data relating to the subject were established: male, 22 years of age, 66 kg, 1.78 m, lean build. The blood samples were taken at defined times, see Table 3. The blood samples were centrifuged, the serum separated and the blood alcohol level measured according to forensic guidelines. The serum samples were then frozen and stored at −18 ◦ C until HS-SPME-GC–MS analysis for the detection of anethole. 2.7. Method validation All statistical data (limit of detection, LoD; limit of quantification, LoQ; correlation coefficient, R2 ; relative standard deviations intraday and interday, RSD; linear range) were evaluated according to a German standard procedure (DIN 32645) by analysing anethole standard solution using the method described above. The method was also tested by means of routine analysis of samples from drivers found to be under the influence of alcohol who claimed to have consumed beverages containing anethole.
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desorption was found for any of the fibres, indicating complete removal of the analyte at these temperatures. The highest extraction yield was found with the 65 m PDMS/DVB fibre. The fibre provided an extraction yield higher than the other fibres, from 2-fold for CW/DVB to 3-fold for DVB/CAR/PDMS and CAR/PDMS. Remarkably, both Carboxen fibres showed a lower capacity to retain anethole. Carboxen fibres seem to be ideal for the SPME analysis of very small molecules (C2 –C6 ). The micropore diameter of Carboxen ˚ [18] for the enrichment of anetlayer appears too small (10–17 A) hole (C10 ). Therefore, the 65 m PDMS/DVB fibre was used for all subsequent investigations. The effect of sampling temperature on the anethole extraction yield was investigated from 25 to 80 ◦ C using PDMS/DVB fibre. A maximum at 50 ◦ C could be observed. At temperatures from 25 to 50 ◦ C an increasing concentration of anethole in the headspace was observable. At temperatures higher than 50 ◦ C, the equilibrium between fibre and headspace is more and more shifted towards the headspace. An extraction temperature of 50 ◦ C was chosen since at this temperature the best anethole response was obtained. The extraction time of anethole was investigated between 2 and 60 min using the optimal conditions previously established. The time required to reach equilibrium was 30 min, and so this time was considered the optimal value. To summarise, the HS-SPME optimal conditions for the analysis of anethole were: 65 m PDMS/DVB fibre, extraction temperature 50 ◦ C, sampling time 30 min. In addition, a desorption temperature of 250 ◦ C and a desorption time of 10 min (3 min splitless time and an additional time at 7 min in the split mode), a stirring speed of 700 rpm and 0.1 g Na2 SO4 were chosen. Calibration of the method was performed using the standard solution in the concentration range of 2–200 ng/ml serum. Statistical data for the calibration functions, calculated according to a German standard procedure (DIN 32645), are summarised in Table 1. No interfering peaks were found in serum samples with the same retention time as anethole and the internal standard. The relative standard deviations (RSD), both intra- and interday, of 2.8% and 4.5% indicate a very good reproducibility of the method. The correlation coefficient (R2 ) of 0.990 of the calibration graphs emphasise good linearity in the concentration range investigated. The LoD of 3.6 ng/ml and LoQ of 5.3 ng/ml show the excellent sensitivity of the method (Table 1). 3.2. Beverage analysis The anethole concentration in five German products (four ouzo ¨ and one aniseed liqueur “Kustennebel”) and three original Greek ouzos was determined using the headspace-trap technique [21]. The results are shown in Table 2. The anethole concentrations detected in the German products lay in the region of 400–800 mg/l. The anethole concentration in
3. Results and discussion 3.1. HS-SPME optimisation First, the selection of the appropriate fibre was carried out. Four fibres (65 m CW/DVB, 85 m CAR/PDMS, 65 m PDMS/DVB and 50/30 m DVB/CAR/PDMS) were evaluated in order to obtain the best sensitivity and selectivity for anethole determination (Fig. 2). In addition, extraction time in the GC injector port was 10 min and the extraction temperature was fixed at 50 ◦ C. The desorption temperatures were the recommended conditioning temperatures for each fibre: 220 ◦ C for CW/DVB, 300 ◦ C for CAR/PDMS, 250 ◦ C for PDMS/DVB, and 270 ◦ C for DVB/CAR/PDMS to ensure complete desorption of anethole from the fibres. No carryover on second
Table 1 Results of method validation according to a German standard procedure (DIN 32645) Headspace SPME-GC–MS of anethole in serum samples 3.6 LoDa (ng/ml) LoQa (ng/ml) 5.3 0.990 R2 2.8 Precision intradayb (%) 4.5 Precision interdayb (%) Linear range (ng/ml) 2–200 All measurements were made in triplicate. a Limit of detection and quantitation were determined by establishing a specific calibration curve from samples containing the analyte in the range of LoQ. The limits were calculated from the residual standard deviation of the regression line. b Precisions are expressed as RSD (%), intraday (n = 7), interday (n = 5).
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Table 2 Results of investigation of different spirits containing anethole Spirit
Origin
Ethanol (vol.%)
canethole (g/ml)
Ouzo Helenas Ouzo Zeus Ouzo Mirios Ouzo 12 ¨ ¨ Kustennebel “Anis&Kom” Ouzo Etairetikis Poiotitos Ouzo Giokarinis Ouzo Knossos
Germany, Saxonia Germany, Saxonia Germany, Saxonia Germany, Saxonia Germany, Saxonia Greece, Santorin Greece, Samos Greece, Crete
37.5 37.5 37.5 38.0 21.8 38.5 42.0 40.0
467 641 794 549 497 1062 1238 390
the selected Greek products varies from 300 to 1300 mg/l. In beverages containing large numbers of congener substances such as whisky, brandy and certain fruit spirits, similar concentrations of 1-propanol, isobutanol and 2/3-methylbutanol are found, which are detectable in serum samples for differing lengths of time, even after consumption of moderate amounts. The anethole concentration in the selected alcoholic beverages needed to be high enough to be detectable in serum samples after consumption. A further important factor in enabling detection is the elimination kinetic of the congener substance concerned. Among the congener substances that are detectable at present, the rate of elimination rises with increasing carbon chain length, i.e. with decreasing polarity of the alcohols.
Fig. 3. Concentration–time profiles of anethole after drinking tests with 120 ml (intended maximum BAC = 0.5‰), 200 ml (intended maximum BAC = 1.0‰) and 360 ml (intended maximum BAC = 1.5‰) Helenas Ouzo.
It is not possible to make detailed statements regarding elimination kinetics as the number of measuring points was too small. Concentration–time profiles with rapid resorption- and elimination phases – as in this case – are ideal for the verification of post-offence drinking claims. In congener analysis, the detection time for the target analyte should extend over a few hours (approx. 0.5–4 h), since post-offence drinking claims are usually made for this period of time. A detection time for the analytes in the serum of more than 12 h would not be suitable for the verification of post-offence drinking claims, since the intention is not to record drinking that took place at an earlier time.
3.3. Drinking tests with volunteer In order to be able to make statements regarding the concentration–time profile of anethole, the drinking experiments were conducted with differing amounts of the beverage. The male subject (22 years, lean build) consumed the German product “Helenas Ouzo” in three separate tests, drinking 120, 200, and 360 ml respectively. During each test, eight blood samples were taken. The blood-alcohol concentrations and the corresponding anethole concentrations are listed in Table 3. The corresponding concentration–time profile of the anethole is depicted in Fig. 3. The anethole concentrations detected show rapid resorption of anethole and also rapid elimination. (In each case, the maximum anethole concentration corresponds to the maximum blood alcohol concentration.) Even immediately after cessation of drinking, anethole concentrations can clearly be measured. Anethole at concentrations above the detection level of 3.6 ng/ml serum can be detected in the selected volunteer for 3 h after ceasing consumption of 120 ml of Helenas Ouzo and 200 ml ouzo, and for 7 h after ceasing consumption of 360 ml ouzo.
3.4. Serum samples from drivers Serum samples taken in 2005 and 2006 from drivers who were found to be under the influence of alcohol and who claimed to have consumed beverages containing anethole were tested for anethole. These cases were not post-offence drinking claims but rather cases of drivers who had consumed beverages containing anethole and who could usually give information about the drinking time and the amount consumed. The results are listed in Table 4. In 10 out of 50 serum samples anethole was detected at concentration levels of between 5.4 and 17.6 ng/ml serum. Of these, eight were cases of ouzo consumption, one of raki consumption ¨ and one of German aniseed liqueur “Kustennebel” consumption.
Table 3 Test conditions, blood-alcohol concentrations and anethole concentrations in the serum samples from the drinking experiment Duration of drinking 1 h
Blank sample before testing 0 h after CDb , 1 h after BDc 0.5 h after CD, 1.5 h after BD 1 h after CD, 2 h after BD 2 h after CD, 3 h after BD 3 h after CD, 4 h after BD 7 h after CD, 8 h after BD 23 h after CD, 24 h after BD a b c
Drinking test 1 (120 ml ouzo and 56.0 mg anethole)
Drinking test 2 (200 ml ouzo and 93.4 mg anethole)
Drinking test 3 (360 ml ouzo and 168.1 mg anethole)
BACa (‰)
canethole (ng/ml)
BACa (‰)
canethole (ng/ml)
BACa (‰)
canethole (ng/ml)
0.00 0.49 0.48 0.41 0.23 0.11 0.00 0.00
0 17.5 8.36 7.57 7.14 4.55 <3.6 <3.6
0.00 0.59 0.74 0.72 0.60 0.41 0.00 0.00
0 15.9 25.7 17.5 12.0 4.49 <3.6 <3.6
0.00 0.78 1.47 1.75 1.62 1.43 0.69 0.00
0 15.1 24.9 73.0 34.4 15.5 6.10 <3.6
BAC, blood alcohol concentration. CD, cessation of drinking. BD, begin of drinking.
K. Schulz et al. / J. Chromatogr. A 1200 (2008) 235–241
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Table 4 Anethole concentrations in serum samples from drivers No.
Data
Alleged consumption
Time between CDa and TBSb
BACc (‰)
canethole (ng/ml)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
38 years, male, 90 kg, 164 cm 44 years, female, 71 kg, 171 cm 36 years, female, 65 kg, 158 cm 42 years, male, 82 kg, 182 cm 37 years, female, 62 kg, 163 cm 49 years, male, 72 kg, 175 cm 17 years, male, 61 kg, 179 cm 37 years, male, 104 kg, 187 cm 41 years, male, 62 kg, 182 cm 19 years, male, 65 kg, 186 cm 40 years, female, 75 kg, 165 cm 22 years, male, 70 kg, 176 cm 25 years, female, 59 kg, 156 cm 45 years, male, 80 kg, 174 cm 45 years, male, 72 kg, 175 cm 31 years, male, 90 kg, 175 cm 34 years, male, 80 kg, 176 cm 17 years, male, 68 kg, 178 cm 44 years, male, 62 kg, 176 cm 45 years, male, 80 kg, 175 cm 18 years, male, 75 kg, 184 cm 47 years, male, 72 kg, 174 cm 26 years, male, 95 kg, 186 cm 54 years, male, 92 kg, 170 cm 16 years, male, 72 kg, 177 cm 45 years, female, 78 kg, 170 cm 50 years, male, 110 kg, 178 cm 48 years, male, 80 kg, 193 cm 43 years, male, 90 kg, 180 cm 20 years, male, 80 kg, 180 cm 33 years, male, 78 kg, 168 cm 23 years, female, 50 kg, 165 cm 19 years, male, 75 kg, 175 cm 50 years, female, 59 kg, 166 cm 19 years, male, 75 kg, 175 cm 30 years, female, 48 kg, 160 cm 29 years, male, 80 kg, 175 cm 46 years, male, 69 kg, 175 cm 32 years, female, 65 kg, 158 cm 44 years, male, 82 kg, 171 cm 34 years, male, 65 kg, 173 cm 63 years, male, 75 kg, 170 cm 38 years, male, 69 kg, 170 cm 24 years, male, 85 kg, 163 cm 23 years, male, 79 kg, 186 cm 20 years, male, 106 kg, 193 cm 24 years, male, 75 kg, 173 cm 27 years, male, 85 kg, 187 cm 43 years, male, 85 kg, 185 cm 57 years, male, unknown
60 ml ouzo/0.5 l beer 40 ml ouzo/cola-whisky 40 ml ouzo/250 ml red wine 40 ml ouzo/1.5 l beer 40 ml ouzo/200 ml wine 40 ml ouzo/1.0 l beer 40 ml ouzo/0.5 l beer/0.25 l martini Ouzo#/1.0 l beer 40 ml ouzo/0.5 l beer Ouzo#/vodka-cola 80 ml ouzo/1.5 l beer 0.3 l ouzo Raki# ¨ Kustennebel# 40 ml ouzo 20 ml ouzo/4.0 l beer 40 ml ouzo/1.5 l beer Ouzo#/2.5 l beer 60 ml ouzo/3.5 l beer 80 ml ouzo/2.0 l beer 0.5 l ouzo/beer 80 ml ouzo/2.0 l beer 60 ml ouzo/4.0 l beer 40 ml ouzo/1.5 l beer Ouzo#/beer 80 ml ouzo/1.0 l beer 120 ml raki/1.0 l beer 120 ml ouzo/1.0 l beer/0.75 l sparkling wine Ouzo#/beer 200 ml raki/1.0 l beer/cocktail 20 ml ouzo/40 ml herb liqueur/1.0 l beer 120 ml ouzo 120 ml ouzo 120 ml ouzo 200 ml ouzo Ouzo#/white wine 120 ml ouzo/3.0 l beer/0.2 l herb liqueur 40 ml raki/1.0 l beer/0.7 l red wine Ouzo#/0.2 l red wine/0.1 l sparkling wine 40 ml ouzo/1.75 l beer 40 ml ouzo 40 ml ouzo/0.5 l beer/400 ml wine 40 ml ouzo 750 ml ouzo 100 ml ouzo/3.0 l beer/300 ml apple grain 40 ml ouzo/2.0 l beer/60 ml tequila/20 ml herb liqueur Ouzo# 80 ml raki/600 ml beer/800 ml cuba-libre 120 ml ouzo ¨ Kustennebel#/2.5 l beer/peppermint-liqueur
3 h 30 min 50 min 2h 1 h 35 min 1 h 10 min 1 h 25 min 2 h 45 min 1 h 5 min 7 h 15 min 2 h 05 min 1 h 10 min 5 h 45 min 2h 40 min 1 h 25 min 35 min 1 h 10 min 2 h 55 min 1 h 55 min 5 h 25 min 6 h 5 min 1 h 20 min 50 min 45 min 1 h 45 min 3 h 45 min 1 h 55 min 50 min 1 h 25 min 6h 2h 60 min 60 min 60 min 60 min 7 h 10 min 4 h 25 min 2 h 40 min 55 min 1 h 20 min 1 h 40 min 4h 2 h 45 min 2 h 25 min 2h 1 h 35 min >2 h 1 h 15 min 55 min 4 h 10 min
2.11 2.69 0.49 1.11 2.82 1.60 1.45 2.21 1.06 1.52 1.60 1.02 1.49 2.23 1.88 2.72 1.45 1.46 2.37 2.17 1.11 1.34 1.70 2.08 2.18 2.13 2.02 1.73 2.51 0.57 2.08 0.52 0.20 0.25 0.46 1.63 2.16 1.06 1.44 2.41 1.97 1.77 0.36 1.67 1.84 1.70 1.77 1.41 0.18 2.45
n.d. n.d. n.d. n.d. n.d. n.d. n.d. 10.8 n.d. n.d. 11.0 n.d. 13.6 12.6 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 14.6 n.d. n.d. n.d. 17.6 n.d. 16.4 5.4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 12.9 n.d. n.d. n.d. n.d. 7.35 n.d.
n.d., not determined; (#) amount of consumed beverage not known. Only the positive cases of anethole are bold-lines. a CD, cessation of drinking. b TBS, taking of blood samples. c BAC, blood alcohol concentration.
In the positive cases, the time difference between the cessation of drinking and the taking of blood samples was 40 min (12.6 ng/ml anethole; case 14) at the minimum and 2 h 25 min (12.9 ng/ml anethole; case 44) at the maximum. The corresponding blood alcohol concentrations lay between 0.18‰ (7.35 ng/ml anethole; case 49) and 2.23‰ (12.6 ng/ml anethole; case 14). Where given, the information about the time of drinking and the amount consumed accounts for the anethole concentrations detected in the positive cases. Even where the blood alcohol concentration was low (cases 32, 34, 35, and 49 with 0.52, 0.25, 0.46, and 0.18‰, respectively) and only spirits containing anethole had been consumed, anethole concentrations above the limit of quantification could be detected (Fig. 4). It is evident from the results of the investigations conducted in the drinking experiments and from the samples taken from drivers
who were under the influence of alcohol that anethole is very sensitive and can be reliably detected even in authentic samples after consumption of spirits containing anethole, such as ouzo, raki and ¨ the German aniseed liqueur “Kustennebel”. In no case was a positive result for anethole found where 40 ml or less of spirits containing anethole had been consumed or where the time difference between the cessation of drinking and the taking of the blood sample was greater than 4 h (except in the experiment involving a very large amount of ouzo—360 ml). It is intended to develop a method of differentiating positive from negative samples through the time difference between cessation of drinking and the taking of a blood sample, in order to be able to distinguish between pre-offence and post-offence drinking claims and thus to be able to reliably disprove or confirm post-offence drinking claims which are of significance in cases of traffic offences.
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Fig. 4. (a) GC–MS chromatogram (scan mode) of case 32 (21.50 min = anethole; 29.26 min = dicyclohexylmethanol, internal standard, BAC = 0.52‰) and (b) corresponding mass spectrum of peak at 21.50 min (anethole).
The blood alcohol level detected is not, however, decisive for a positive anethole finding. Information about the amount consumed and the time of drinking the beverage containing anethole are absolutely essential for verifying the plausibility of claims.
is characteristic of certain alcoholic beverages Hitherto, it was only possible to detect this substance in the beverages themselves and not in blood samples. Hence, improved verification of post-offence drinking claims is now possible. Acknowledgments
4. Conclusions The developed HS-SPME-GC–MS method has shown itself to be ideal for the detection of anethole in serum samples taken from drivers under the influence of alcohol owing to the simplicity of the preparation procedures required for the samples, the excellent degree of sensitivity and its other very good statistical parameters. The selection of fibre coating allows for optimal adjustment to specific substances or substance classes. Therefore, the PDMS/DVB fibre was used for subsequent investigations. Based on the drinking experiments, concentration–time profiles were drawn up which enable statements to be made about changes in detection levels over time and the duration of the detectability of anethole in serum. It was possible to conclude that this method of verifying post-offence drinking claims relating to spirits containing anethole is very suitable within an approximate time-frame of 30 min to 4 h and in cases of consumption of relatively small amounts of spirits containing anethole (upwards of 40 ml ouzo). Thus, it was possible to extend the range of analytes detectable through congener analysis by that of the substance anethole, which
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