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Analytica Chimica Acta 646 (2009) 128–140

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Analytica Chimica Acta

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eadspace solid-phase microextraction–gas chromatography–masspectrometry determination of the characteristic flavourings menthone,somenthone, neomenthol and menthol in serum samples with and withoutnzymatic cleavage to validate post-offence alcohol drinking claims

atja Schulz a,∗, Martin Bertau b, Katja Schlenz c, Steffen Malt c, Jan Dreßler a, Dirk W. Lachenmeier d

Institut für Rechtsmedizin, Technische Universität Dresden, Fetscherstraße 74, D-01307 Dresden, GermanyTechnische Universität Bergakademie Freiberg, Fakultät Chemie und Physik, Institut für Technische Chemie, Leipziger Str. 29, D-09596 Freiberg, GermanyHochschule Zittau/Görlitz (FH), Fakultät für Mathematik and Naturwissenschaften, Fachbereich Chemie, Theodor-Körner-Allee 16, D-02763 Zittau, GermanyChemisches und Veterinäruntersuchungsamt (CVUA) Karlsruhe, Weißenburger Straße 3, D-76187 Karlsruhe, Germany

r t i c l e i n f o

rticle history:eceived 11 February 2009eceived in revised form 4 May 2009ccepted 9 May 2009vailable online 18 May 2009

eywords:ongener analysiseverage-characteristic aroma compoundsenthoneenthol

eppermint liqueureadspace solid-phaseicroextraction–gas

hromatography–mass spectrometry

a b s t r a c t

A rapid HS-SPME–GC–MS (headspace solid-phase microextraction–gas chromatography–mass spectrom-etry) method has been developed for determination of menthone, isomenthone, neomenthol and mentholin serum samples with and without enzymatic cleavage. These flavour compounds are characteristicmarkers for consumption of peppermint liqueurs as well as certain digestif bitters, herbal and bitterliqueurs.

This method enabled the detection of the four compounds with a limit of detection (LOD) of 2.1 ng mL−1

(menthone and isomenthone), 2.8 ng mL−1 (neomenthol) and 4.6 ng mL−1 (menthol), and a limit of quan-tification (LOQ) of 3.1 ng mL−1 (menthone and isomenthone), 4.2 ng mL−1 (neomenthol) and 6.8 ng mL−1

(menthol) in serum samples. The method shows good precision intraday (3.2–3.8%) and interday(5.8–6.9%) and a calibration curve determination coefficient (R2) of 0.990–0.996.

Experiments were conducted with a volunteer, who consumed peppermint liqueur on three dif-ferent days under controlled conditions. At defined intervals, blood samples were taken, and theconcentration–time profiles for serum menthone, isomenthone, neomenthol and menthol, as free sub-stances as well as glucuronides, were determined. Both menthol and neomenthol underwent a rapidphase II metabolism, but minor amounts of free substances were also detected. Menthone and isomen-thone were rapidly metabolised and were found in lower concentrations and over a shorter time spanthan the other analytes.

In blood samples taken from 100 drivers who claimed to have consumed peppermint liqueur priorto the blood sampling, menthone, isomenthone, neomenthol and menthol were detected in the serumas free substances in concentrations between 3.1 and 7.0 ng mL−1 in eight cases (menthone), 3.1 and

−1 −1
11.3 ng mL in eight cases (isomenthone), 5.3 and 57.8 ng mL in nine cases (neomenthol) and 8.0 and92.1 ng mL−1 in nine cases (menthol). The sum values of free and conjugated substances ranged between4.2 and 127.8 ng mL−1 in 35 cases for neomenthol and 11.0 and 638.2 ng mL−1 in 59 cases for menthol.Menthone and isomenthone were not conjugated.
These test results confirmed that the analysis of characteristic beverage aroma compounds, such asmenthone, isomenthone, neomenthol and menthol, can be used for specific verification of post-offence

ms.
alcohol consumption clai
. Introduction

The determination of congener substances of alcoholic bever-ges in serum samples, the so-called ‘congener analysis’, is used

∗ Corresponding author. Tel.: +49 351 4584940; fax: +49 351 4584397.E-mail address: [email protected] (K. Schulz).

003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2009.05.010

© 2009 Elsevier B.V. All rights reserved.

in forensic toxicology for the verification of post-offence drinkingclaims, especially in cases of road accidents. In these situations, thedefendant claims to have drunk the alcohol after the offence and
was sober at the time of accident. With information given by thedefendant regarding to type, quantity and time of consumption, theplausibility of this claim can sometimes be verified by analysis ofcongener substances in serum samples and comparison to theo-retically expected congener concentrations. If these values do not
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himica Acta 646 (2009) 128–140 129

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orrespond, the post-offence drinking claim is considered to be dis-roved. Congener analysis was introduced by Machata and Prokop1] and extended by Bonte and Busse [2]. In the past, its appli-ation was limited to the determination of volatile alcohols andetones (methanol, 1-propanol, 2-butanone, 2-butanol, isobutanol,-butanol, 2-methyl-1-butanol and 3-methyl-1-butanol). Theseubstances are contained in nearly all alcoholic beverages and differnly in their concentration pattern between each type of beverage,ut they are not characteristic of any specific alcoholic beverage.

Recently, we reported the determination of anethole [3] andugenol [4] in serum samples as beverage-characteristic congenerubstances. Anethole is a characteristic marker of aniseed spiritsnd was detectable in serum samples after consumption of theseeverages. Eugenol is contained in bitters, and in herbal and spice

iqueurs, and was present in serum of consumers of these bever-ges after enzymatic cleavage. The determination of these typesf beverage-specific aroma compounds in serum samples clearlyllows conclusions about the type of consumed beverage.

In this study, the suitability of menthone, isomenthone, neo-enthol and menthol, as characteristic markers for consumption

f peppermint liqueurs as well as digestif bitters, herbal and bitteriqueurs [5], is evaluated for the first time.

The peppermint herb (Mentha × piperita L.) is a hybridetween the green spearmint (Mentha spicata L.) and water mintMentha aquatica L.) [6]. Menthol is contained in the essentialils of the peppermint plant at a concentration level of 30–55%,eomenthol at 2–6%, menthone at 14–32%, and isomenthone at.5–10% [7,8]. Furthermore, different methyl esters, terpenes (e.g.ulegone), and polyphenols are contained in peppermint oil [9,10].atural extracts of this plant are used as aromatic substances in theforementioned liqueurs and spirits [5]. Menthol levels have beenetermined in biological material, for example in plasma and urinef rats [11,12] and humans [11,13–19]. The measurement methods

ncluded liquid/liquid-extraction (LLE) with n-hexane and GC-FID-etection [13], LLE with tetrachloromethane and GC–MS [14] andS-SPME–GC–MS [11]. Unchanged (free) menthol in human sub-

ects was only reported by Valdez et al. [13] following applicationf dermal patches. In all other studies, menthol was determined

n biological material as the sum of menthol and its glucuronideonjugate, following enzymatic hydrolysis with �-glucuronidase.nvestigations of menthone-metabolism by yeast and yeast-likeungi [20] have indicated that (−)-menthone is reduced to (+)-eomenthol. The metabolism of (−)-menthone by human livericrosomes was investigated by Miyazawa and Nakanishi [21], who

ound that (−)-menthone was metabolised to (+)-neomenthol and-hydroxymenthone. Reports of the metabolism of (−)-menthone

n mammals and humans are not available, to our knowledge.Because of its three asymmetric C-atoms (23 = 8), menthol

ccurs in eight optically active stereoisomers (four diastereomernantiomeric pairs).

These four diastereomeric pairs are (−)- and (+)-menthol,−)- and (+)-neomenthol, (−)- and (+)-isomenthol and (−)-nd (+)-neoisomenthol. (−)-Menthol, (1R,2S,5R)-(−)-2-isopropyl--methylcyclohexanol; C10H20O; CAS 2216-51-5, Fig. 2a, is the mainomponent of peppermint oil. The biosynthesis of (−)-mentholn peppermint has been described in detail by Croteau et al.6] and Davis et al. [22]. All steps of its biosynthesis are stere-selective. The last steps of biosynthesis are the transformationrom (+)-pulegone by (+)-pulegone-reductase to (−)-menthonend (+)-isomenthone. Subsequently (−)-menthone is reduced to−)-menthol and (+)-neomenthol and (+)-isomenthone into (+)-
somenthol and (+)-neoisomenthol by stereoselective menthoneeductases.
Menthone has two asymmetric C-atoms and therefore occursn four optic active stereoisomers (two diastereometric enan-iomeric pairs). These are (−)- and (+)-menthone and (−)- and

Fig. 1. Chemical structures of (a) (−)-menthone [14073-97-3] and (b) (+)-isomenthone [001196-31-2], C10H18O both.

(+)-isomenthone. The chemical structures of (−)-menthone((2S,5R)-2-isopropyl-5-methylcyclohexanone; C10H18O; CAS14073-97-3) and (+)-isomenthone ((2R,5R)-2-isopropyl-5-methylcyclohexanone; C10H18O; CAS 001196-31-2) are shownin Fig. 1.

The chemical structures of (−)-menthol, (1R,2S,5R)-(−)-2-isopropyl-5-methylcyclo-hexanol, (+)-neomenthol, (1S,2S,5R)-(−)-2-isopropyl-5-methylcyclo-hexanol, (+)-isomenthol, (1S,2R,5R)-(−)-2-isopropyl-5-methylcyclo-hexanol, and (+)-neoisomenthol,(1R,2R,5R)-(−)-2-isopropyl-5-methylcyclo-hexanol, are shown inFig. 2.

Menthol is commonly used in toothpaste, mouthwash, oralpharmaceutical preparations, chewing gum and shaving cream[23]. Menthol can also be detected in a number of other mate-rials, for example in tobacco products [24,25], in pharmaceuticalproducts [26,13], in Chinese medicinal herbs [27] and in honey [28].

2. Materials and methods

2.1. Reagents and standards

(−)-Menthol, (+)-neomenthol and (−)-menthone were pur-chased from Fluka (Taufkichen, Germany) and dicyclohexyl-methanol as internal standard was obtained from Sigma–Aldrich(Steinheim, Germany). (±)-Isomenthone and (+)-isomenthol werepurchased from Roth (Karlsruhe, Germany). (+)-Isomenthol wasused for comparing of retention times. Neoisomenthol was notcommercially available. �-Glucuronidase for enzymatic cleavagewas purchased from Roche Diagnostic, Boehringer Mannheim(Mannheim, Germany). Na2SO4 and ethanol were obtained fromMerck (Darmstadt, Germany). All chemicals were of analyticalgrade. Water was deionised. Negative control serum samples forspiking with menthone, isomenthone, neomenthol and mentholwere taken from the authors.

2.2. Preparation of standard solutions

A standard solution of 200 and 2000 ng mL−1 of menthone,isomenthone, neomenthol and menthol was made in water withethanol as solubilizer. The concentration of ethanol was approx-imately 0.2 % vol. The aqueous standard solution must be freshlyprepared, because it is instable. The serum stock solution ofmenthone, isomenthone, neomenthol and menthol was made byaddition of 1 mL menthone, menthol and neomenthol solution(aqueous standard 200, 2000 and 20,000 ng mL−1, respectively) to9 mL of negative control serum sample. Subsequent solutions forcalibration curves and validation parameters were accomplished
by adding of standard stock solution to negative control serum inresulting concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, 50,100, 200, 1000, 2000 ng mL−1 menthone, isomenthone, mentholand neomenthol each. The serum stock solutions were also freshlyprepared. For accuracy and precision tests interday, a serum stock

130 K. Schulz et al. / Analytica Chimica Acta 646 (2009) 128–140

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ig. 2. Chemical structures of (a) (−)-menthol [2216-51-5] (b) (+)-neomenthol [2216ll.

olution with 20 ng mL−1 menthone, isomenthone, menthol andeomenthol was frozen at −18 ◦C. During the routine use of thessay, this 20 ng mL−1 sample was also applied as quality controlample.

.3. Headspace-SPME procedure

SPME experiments were performed using a manual fibre holderupplied from Supelco (Taufkirchen, Germany). Four commerciallyvailable fibres, carbowax/divinylbenzene (CW/DVB, 65 �m),table-Flex carboxen/polydimethylsiloxane (CAR/PDMS, 85 �m),table-Flex polydimethylsiloxane/divinylbenzene (PDMS/DVB,5 �m) and Stable-Flex divinylbenzene/Carboxen/polydimethyl-iloxane (DVB/CAR/PDMS, 50/30 �m) were purchased fromupelco. Before use, each fibre was conditioned in the GC injectionort under helium flow in accordance with the temperature andime recommended by the manufacturer. Fibre blanks were runeriodically to ensure the absence of contaminants or carryover.

The SPME-procedure without enzymatic cleavage was asollows: 200 �L serum and 200 �L internal standard solution100 ng mL−1 dicyclohexylmethanol) were placed in a 22 mLeadspace-vial containing a 8 mm × 3 mm PTFE-coated stir bar and.1 g Na2SO4. The samples were immediately sealed with silicone-TFE septa. Before HS-SPME analysis, the sample vial was stirredor 1 min and conditioned for 1 min in a thermostatic water batht a temperature of 50 ◦C. Then the sample was extracted usingDMS/DVB (60 �m) fibre for 30 min at 50 ◦C using a magnetic agi-ation rate of 700 rpm. The thermal desorption of the analytes wasarried out by exposing the fibre in the GC injection port at 250 ◦Cor 3 min. To prevent a memory effect, the fibre was kept in the injec-ion port for an additional time of 7 min in the split mode (purgen).

For sample preparation with enzymatic cleavage, 20 �L internaltandard solution (1000 ng mL−1 dicyclohexylmethanol), 175 �Luffer (phosphate buffer, 0.1 M, pH 6.0) and 5 �L �-glucuronidaseere used instead of 200 �L internal standard solution. The enzymeas added immediately before the SPME-enrichment. The incuba-

ion time corresponds to the extraction time of 30 min.

.4. Beverage analysis

The testing of spirits for menthone, neomenthol and men-hol content was conducted using the headspace-trap procedure.he headspace analysis was performed with the PerkinElmer Tur-oMatrix HS 110-trap automatic headspace sampler with trapnrichment and flame ionization detector (PerkinElmer, Shelton,T, USA). A capillary column Rtx 1701 (60 m × 0.530 mm I.D.;
.5 �m film thickness) with phenylcyanopropyl phase from Restekas used. Data acquisition and integration were carried out with
otalChrom (Version 6.2.1) software. The enrichment conditionsnd chromatographic conditions were previously described inetail [29].

], (c) (+)-isomenthol [23283-97-8] and (d) (+)-neoisomenthol [20752-34-5] C10H20O

2.5. GC–MS conditions

The GC–MS system used for analysis was a Hewlett PackardGC 5890 series II with a 5971 mass selective detector (Wald-bronn, Germany). Data acquisition and analysis were performedusing standard software supplied by the manufacturer. Sub-stances were separated on a fused silica capillary column HP-5MS(30 m × 0.25 mm I.D.; 0.25 �m film thickness) supplied by Agilent(Waldbronn, Germany). Temperature programme: 40 ◦C hold for5 min, 5 ◦C min−1 up to 160 ◦C, 20 ◦C min−1 up to 220 ◦C, hold for3 min.

The temperatures for the injection port and detector were setat 250 and 280 ◦C, respectively. Splitless injection mode (splitlesstime 3 min) and helium with a flow rate of 1.15 mL min−1 as carriergas were used. A narrow bore inlet liner (0.75 mm I.D.) for SPMEapplications was used.

To determine the retention times and characteristic mass frag-ments, electron impact (EI) mass spectra of the analytes wererecorded by total ion monitoring. All investigations (optimisation,statistical parameters and original serum samples) were monitoredin full scan mode with a scan range of 33–250 m/z and a scan rateof 3.1 scans/s. For evaluation, diagnostic mass fragments of men-thol and neomenthol (m/z = 71, 81, 95, 123 and 138) from full scanmode with target ion m/z = 95 (menthol) and m/z = 138 (neomen-thol) were selected. In the case of menthone and isomenthone thediagnostic mass fragments (m/z = 69, 112, 139 and 154) from fullscan mode with target ion m/z = 112 were selected.

The retention times were as follows: menthone = 17.71 min,isomenthone = 18.02 min, neomenthol = 18.04 min and men-thol = 18.29 min. Because of the same retention times ofisomenthone and neomenthol, the more characteristic targetion m/z = 138 must be used for the quantification of neomenthol.Isomenthol – for comparison – has a retention time of 18.76 min. Ithas nearly the same mass fragments as menthol and neomenthol.The four diastereomeric menthol-derivates only differ in theirretention times, but only marginally in their mass spectra. Becauseof using an achiral column, the enantiomeric pairs were notseparated.

2.6. Experimental design

The conduct of the drinking experiments was approved by theethics committee of Medical Faculty Carl Gustav Carus, Dresden,Germany (registration number: EK 16012007).

The drinking experiments were carried out by a volunteersubject using the beverage “Bramsch Pfefferminzlikör” (“Bramschpeppermint liqueur”) at three different dosages: 4 × 40 mL (corre-
sponding to the consumption of 5.6 mg of menthone abs., 4.5 mgof isomenthone abs., 1.1 mg of neomenthol abs. and 12.8 mg ofmenthol abs.; test 1); 8 × 40 mL (corresponding to the consump-tion of 11.2 mg of menthone abs., 9.0 mg of isomenthone abs.,2.2 mg of neomenthol abs. and 25.6 mg of menthol abs.; test 2)

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On this basis, the statistical data of menthone, isomenthoneneomenthol and menthol were determined and are summarisedin Table 1.

No interfering peaks were found with the same retention timeas menthone, isomenthone neomenthol, menthol and the internal

Table 1Results of method validation with HS-SPME–GC–MS of menthone, isomenthone,neomenthol and menthol in serum samples.

Statistical parameter Menthone Isomenthone Neomenthol Menthol

Retention time (min) 17.71 18.02 18.04 18.29LODa (ng mL−1) 2.1 2.1 2.8 4.6LOQa (ng mL−1) 3.1 3.1 4.2 6.8R2 0.996 0.996 0.994 0.990Precision intradayb (%) 3.2 3.2 3.7 3.8Precision interdayb (%) 6.1 6.1 5.8 6.9Linear range (ng mL−1) 2–2000 2–2000 2–2000 2–2000

K. Schulz et al. / Analytica C

nd 14 × 40 mL (corresponding to the consumption of 19.6 mg ofenthone abs., 15.7 mg of isomenthone abs., 3.9 mg of neomen-

hol abs. and 44.8 mg of menthol abs.; test 3). The interval betweenhe individual tests was seven days in each case. The consumptionime was 1 h. The following personal data relating to the subject30–32] were established: male, 29 years of age, 92 kg, 1.90 m, leanuild, rindiv [Ulrich/Cramer/Zink] = 0.75, (The factor rindiv [Ulrich/Cramer/Zink]escribes the individual distribution factor. It considers the indi-idual body weight and height for the ethanol calculation. Thealculation of individual factors is more precise than using averageistribution factors, which are 0.7 for men and 0.6 for women.).

The blood samples were taken at defined times, see Table 3.The blood samples were centrifuged, the serum separated and

he blood–alcohol level established according to forensic guide-ines. The serum samples were then frozen and stored at −18 ◦Cntil HS-SPME–GC–MS analysis for the detection of menthone, iso-enthone neomenthol and menthol.

.7. Method validation

All statistical data (limit of detection, LOD; limit of quan-ification, LOQ; determination coefficient, R2; relative standardeviations intraday and interday, RSD; and linear range) were eval-ated using SQS2.0 (PerkinElmer, Shelton, CT, USA) by analysingenthone, isomenthone, neomenthol and menthol standard solu-

ion using the method described above.The applicability of this method was also tested by means of

outine analysis of samples from drivers found to be under thenfluence of alcohol who claimed to having consumed peppermintiqueur.

. Results and discussion

.1. HS-SPME optimisation

The optimisation of the HS-SPME parameters was conductedsing a similar protocol to that used for anethole and eugenol inur previous studies [3,4]. An optimised method was devised thatllowed the simultaneous determination of all substances of inter-st: anethole, eugenol, menthone, isomenthone, neomenthol andenthol.

At first step, four fibres (65 �m CW/DVB, 85 �m CAR/PDMS,5 �m PDMS/DVB and 50/30 �m DVB/CAR/PDMS) were evaluated

n order to obtain the best sensitivity and selectivity for men-hone, isomenthone, neomenthol and menthol determination. Forhis experiment, the extraction time in the GC injector port waset at 10 min and the extraction temperature was fixed to 50 ◦C.he desorption temperatures were the recommended condition-

ng temperatures for each fibre (220 ◦C for CW/DVB, 300 ◦C forAR/PDMS, 250 ◦C for PDMS/DVB and 270 ◦C for DVB/CAR/PDMS),hich ensured complete desorption of menthone, isomenthone

eomenthol and menthol from the fibres. No carryover on sec-nd desorption was found for any of the fibres, indicating completeemoval of analytes at these temperatures. The highest extractionield was detected with the 65 �m PDMS/DVB fibre, and it washerefore used for all subsequent investigations.

Next, the effect of sampling temperature from 25 to 80 ◦C onhe menthone, isomenthone neomenthol and menthol extractionields was investigated using PDMS/DVB fibre. Slightly differentffects could be observed for each substance: the maxima for men-
hone and isomenthone are at 25 ◦C and the yield continuouslyecreases to 80 ◦C. For neomenthol, the maximum extraction yield
s at a temperature of 40 ◦C. For menthol, the maximum extractionield is at temperatures between 40 and 50 ◦C. At temperaturesigher than 50 ◦C, a decreasing yield results for all compounds,

Acta 646 (2009) 128–140 131

because the equilibrium between the fibre and the headspaceis shifted increasingly towards the headspace. For the enrich-ment of anethole, the best results were achieved at a samplingtemperature of 50 ◦C; for eugenol of 60 ◦C [3,4]. Therefore, as a com-promise, a sampling temperature of 50 ◦C was chosen for all sixsubstances.

The extraction time of menthone, isomenthone, neomentholand menthol was investigated between 2 and 60 min using theoptimised conditions previously established. The highest extrac-tion yield was at 30 min for menthone and isomenthone and at60 min for neomenthol and menthol, but these amounts were onlymarginally higher than for a 30 min extraction time and so, oncemore, a compromise sampling time of 30 min – analogous to thatused for extraction of anethole and eugenol – was used.

The manufacturer’s recommended hydrolysis time for the enzy-matic cleavage of the majority of glucuronide conjugates was75 min at 37 ◦C or 45 min at 46 ◦C. The incubation time of �-glucuronidase was therefore tested in the range of 30–180 minat the sampling temperature of 50 ◦C. Similar to the increase ofSPME extraction yield at longer extraction times, increases in enzy-matic cleavage were observed above 30 min. From our experience,blood samples may require longer hydrolysis times than usuallyrecommended, and for future studies (e.g. more detailed elimi-nation kinetics) deuterated internal standards of the glucuronideswould be preferable to control for the completeness of hydroly-sis, however, such standards are not commercially available to ourknowledge. Nevertheless, as in our previous study [4], we decidedto use 30 min for combined cleavage and SPME extraction, espe-cially because of time efficiency reasons for manual SPME operation(we aimed to conduct the next SPME extraction in the time of theGC-run to measure as many samples as possible during the work-ing day). The validation data shown below prove that the assayprovides reproducible data even if we slightly work outside theoptima for both SPME and enzymatic cleavage. Both extraction yieldand amount of enzymatic cleavage are sufficient for our purpose tovalidate alcohol drinking claims.

To summarise, the HS-SPME optimal conditions for theanalysis of menthone, isomenthone, neomenthol and menthol werethe same as those used previously for the analysis of anethole andeugenol: a 65 �m PDMS/DVB fibre, extraction temperature 50 ◦C,sampling and incubation time 30 min. In addition, a desorptiontemperature of 250 ◦C and a desorption time of 10 min (3 min split-less time and an additional time at 7 min in the split mode), astirring speed of 700 rpm and 0.1 g Na2SO4 to facilitate a salting-out-effect were also applied.

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 limitswere calculated from the residual standard deviation of the regression line.

b Precision are expressed as RSD (%), intraday (n = 7) and interday (n = 5).

1 himica Acta 646 (2009) 128–140

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Fig. 3. Concentration–time profiles of menthone using drinking tests with differentamounts of “Bramsch Pfefferminzlikör”. The results with and without enzymaticcleavage were exactly identical.

Fig. 4. Concentration–time profiles of isomenthone using drinking tests with differ-ent amounts of “Bramsch Pfefferminzlikör”. The results with and without enzymaticcleavage were exactly identical.

32 K. Schulz et al. / Analytica C

tandard in serum samples. The RSD’s, both intra- and interday,f 3.2% and 6.1% for menthone and isomenthone, 3.7% and 5.8%or neomenthol and 3.8% and 6.9% for menthol indicate a veryood reproducibility of the method. The determination coefficientsR2) of 0.990 and better of the calibration graphs emphasise goodinearities in the concentration range investigated. The LOD with.1 ng mL−1 (menthone and isomenthone) to 4.6 ng mL−1 (men-hol) and LOQ with 3.1 ng mL−1 (menthone and isomenthone) to.8 ng mL−1 (menthol) show the excellent sensitivity of the methodTable 1).

Finally, during the course of our investigation, we analysed auality control sample (spiked serum sample) at least once weeklynd documented the results in a quality control chart. The resultshowed that the interday accuracy of the assay is sufficient, as theeviations between the quality control samples were below 10% invery case.

.2. Beverage analysis

The menthone, isomenthone, neomenthol and menthol concen-ration in three German and five Italian and one Irish products wereetermined using the headspace-trap technique [29]. The resultsre shown in Table 2.

The menthone, isomenthone, neomenthol and menthol con-entrations detected in the German and Italian products lay inhe region of 20–80 mg L−1 for menthone, 15–70 mg L−1 for iso-

enthone, 5–20 mg L−1 for neomenthol and 30–150 mg L−1 forenthol. The peppermint liqueurs “Eis Mint”, “Goldene Aue” and

Ramazzotti Menta” contain the highest concentrations of theseubstances and “Fernet branca” and “Baileys Mint” the lowest con-entrations. The “Ramazzotti” brand liqueurs contain very differentmounts of peppermint with the four analytes only detectable inMenta” but not in “Amaro” types. We decided to use “Bramsch Pfef-erminzlikör” for our drinking experiment, because this beverages preferred in our region (Saxony). From our experience with con-ener analysis, the concentrations in at least some of the beveragesnalysed are high enough to facilitate detection in blood sera fol-owing consumption. For example, anethole in an aniseed beverage500 mg L−1) and also eugenol in the aromatic bitter “Underberg”350 mg L−1) could be clearly detected in serum samples taken dur-ng drinking experiments [3,4].

In serum samples of drivers who had consumed several aniseedpirits and aromatic bitters, anethole and eugenol could be reliablyetected.

In the previously investigated beverages containing anetholend eugenol, the flavouring compounds were found in approx-mately the same order of magnitude in comparison to theeppermint liqueurs analyzed in this study. These substances wereetectable in serum samples taken after different time spans, evenfter only moderate consumption. So we were confident to detecthe aroma compounds menthone, isomenthone, neomenthol and

enthol in serum samples.

.3. Drinking tests

In order to establish the concentration–time profiles ofenthone, isomenthone, neomenthol and menthol, drinking

xperiments were conducted with different amounts of a pep-ermint liqueur. A male subject (29 years, rindiv = 0.75) consumedhe German product “Bramsch Pfefferminzlikör” in three separateests, drinking 160, 320 or 560 mL. During each test, eight blood
amples were taken. The blood–alcohol concentrations and theorresponding menthone, isomenthone, neomenthol and mentholoncentrations, with and without enzymatic cleavage, are listed inables 3–5. The corresponding concentration–time profiles of theseavour compounds are depicted in Figs. 3–6.
Fig. 5. Concentration–time profiles of neomenthol with and without enzymaticcleavage using drinking tests with different amounts of “Bramsch Pfefferminzlikör”.

K. Schulz et al. / Analytica Chimica Acta 646 (2009) 128–140 133

Table 2Results of investigation of different peppermint and herbal liqueurs.

Spirit Origin Alcoholicstrength (% vol)

Menthone(mg L−1)

Isomenthone(mg L−1)

Neomenthol(mg L−1)

Menthol(mg L−1)

Bramsch Pfefferminzlikör (peppermint liqueur) Germany, Saxony 20.0 35 28 7 80Goldene Aue Pfefferminzlikör (peppermint liqueur) Germany, Thuringia 20.0 64 39 12 143Eis-Mint Pfefferminzlikör (peppermint liqueur) Germany, Saxony-Anhalt 20.0 68 68 18 150Ramazzotti Menta (mint liqueur) Italy 32.0 62 41 13 128Ramazzotti Amaro (herb liqueur) Italy 30.0 <1 <1 <1 1Fernet Branca (digestif bitter) Italy 40.0 19 17 4 35Branca Menta (digestif bitter) Italy 35.0 73 29 10 107Volare (peppermint liqueur) Italy 22.0 58 20 5 106Baileys Mint (cream liqueur) Ireland 17.0 6 3 <1 12

Table 3Test conditions, blood–alcohol concentrations and menthone, isomenthone, neomenthol and menthol concentrations without and with enzymatic cleavage in the serumsamples from the drinking experiment with 160 mL peppermint liqueur.

Duration of drinking 1 h BACa (‰) Drinking test 1 (160 mL peppermint liqueur)

5.6 mg menthone 4.5 mg isomenthone 1.1 mg neomenthol 12.8 mg menthol

cmenthone (ng mL−1) without/with enzymatic cleavage

cisomenthone (ng mL−1) without/with enzymatic cleavage

cneomenthol (ng mL−1) without/with enzymatic cleavage

cmenthol (ng mL−1) without/with enzymatic cleavage

Blank sample before testing 0.00 0/0 0/0 0/0 0/00 h after CDb 1 h after BDc 0.18 <2.1/<2.1 <2.1/<2.1 <4.2/34.1 5.1/324.20.5 h after CD 1.5 h after BD 0.13 0/0 <2.1/<2.1 <4.2/28.4 <4.6/251.11 h after CD 2 h after BD 0.10 0/0 0/0 <4.2/20.4 <4.6/209.22 h after CD 3 h after BD 0.04 0/0 0/0 0/13.9 <4.6/136.73 h after CD 4 h after BD 0.00 0/0 0/0 0/9.3 0/94.37 h after CD 8 h after BD 0.00 0/0 0/0 0/<4.2 0/14.123 h after CD 24 h after BD 0.00 0/0 0/0 0/0 0/0

a Blood–alcohol concentration.b Cessation of drinking.c Begin of drinking.




cmenthone (ng mL−1) without/with enzymatic cleavage

cisomenthone (ng mL−1) without/with enzymatic cleavage

cneomenthol (ng mL−1) without/with enzymatic cleavage

cmenthol (ng mL−1) without/with enzymatic cleavage

Blank sample before testing 0.00 0/0 0/0 0/0 0/00 h after CDb 1 h after BDc 0.49 3.3/3.3 3.1/3.1 4.5/85.2 7.1/738.10.5 h after CD 1.5 h after BD 0.52 3.2/3.2 <2.1/<2.1 4.2/92.1 <4.6/740.41 h after CD 2 h after BD 0.45 <2.1/<2.1 <2.1/<2.1 < 4.2/63.2 <4.6/473.62 h after CD 3 h after BD 0.33 0/0 0/0 0/28.7 <4.6/216.43 h after CD 4 h after BD 0.18 0/0 0/0 0/10.8 <4.6/88.67 h after CD 8 h after BD 0.00 0/0 0/0 0/<4.2 0/29.923 h after CD 24 h after BD 0.00 0/0 0/0 0/0 0/0





cmenthone (ng mL−1)without/with enzymaticcleavage

cisomenthone (ng mL−1)without/with enzymaticcleavage

cneomenthol (ng mL−1)without/with enzymaticcleavage

cmenthol (ng mL−1)without/with enzymaticcleavage

Blank sample before testing 0.00 0/0 0/0 0/0 0/00 h after CDb 1 h after BDc 0.76 5.9/5.9 3.9/3.9 7.4/126.2 19.7/931.00.5 h after CD 1.5 h after BD 1.04 3.7/3.7 3.3/3.3 8.1/118.1 10.9/859.51 h after CD 2 h after BD 1.05 2.9/2.9 <2.1/<2.1 6.7/104.3 6.3/759.02 h after CD 3 h after BD 0.90 2.2/2.2 <2.1/<2.1 4.3/83.0 <4.6/575.43 h after CD 4 h after BD 0.78 <2.1/<2.1 0/0 <4.3/35.5 <4.6/243.97 h after CD 8 h after BD 0.28 0/0 0/0 0/7.2 0/51.223 h after CD 24 h after BD 0.00 0/0 0/0 0/0 0/0


134 K. Schulz et al. / Analytica Chimica

Fa

ricaiMlvlplwtctHtoci

aWvdiaa3tcirs

aaeht

gts

triad in �-glucuronidase active sites is partially disrupted for

ig. 6. Concentration–time profiles of menthol with and without enzymatic cleav-ge using drinking tests with different amounts of “Bramsch Pfefferminzlikör”.

The concentration changes in all four substances indicated showapid resorption of these aroma compounds as well as rapid elim-nation. In the majority of cases, the maximum concentrationsorresponded to the maximum blood–alcohol concentration. Event the first measuring point, immediately after cessation of drink-ng, neomenthol and menthol concentrations were detectable.

enthone and isomenthone, at concentrations above the detectionevel of 2.1 ng mL−1 serum, could not be detected in the selectedolunteer after cessation of consumption of 160 mL of peppermintiqueur. When the volume consumed was increased to 320 mL ofeppermint liqueur, menthone was detected above the detection

imit at the cessation of drinking as well as 0.5 h later. Isomenthoneas only detected at the cessation of drinking. After the consump-

ion of 560 mL peppermint liqueur, menthone was detected fromessation of drinking to 2 h after cessation of drinking and isomen-hone at cessation of drinking to 0.5 h after cessation of drinking.owever, the highest concentrations of menthone and isomen-

hone were shown at the first measuring point of the test. Becausef their chemical structures, menthone and isomenthone cannot beonjugated directly. Therefore, they only occur as free substancesn the same concentrations and require no enzymatic cleavage.

Neomenthol and menthol were detectable in serum samplesfter drinking tests in very low concentrations as free substances.

hen 560 mL peppermint liqueur was consumed, the maximumalue for neomenthol was 8.1 ng mL−1 (0.5 h after cessation ofrinking) and for menthol was 19.7 ng mL−1 (at cessation of drink-

ng). Both substances were rapidly conjugated with glucuroniccid. Conjugates occurred in serum samples at concentrations ofbout 30 ng mL−1 (drinking test 1) to 130 ng mL−1 (drinking test) as neomenthol glucuronide and 320 ng mL−1 (drinking test 1)o 930 ng mL−1 (drinking test 3) as menthol glucuronide. The con-entration of menthol in the beverage “Bramsch Pfefferminzlikör”s approximately 10-fold higher than that of neomenthol, and thisatio was also observable for the conjugated substances in serumamples after consumption of this beverage.

We also were able to detect the mass fragments m/z = 95, 123nd 138 in traces at the retention time of isomenthol (18.76 min)nd a retention time of 18.55 min (probably neoisomenthol). How-ver, the concentrations were too low for quantification and wead no standard substance of neoisomenthol available to confirmhe identity of the peak.

Furthermore, the ratio of conjugated neomenthol to unconju-ated neomenthol was smaller than the ratio of conjugated mentholo unconjugated menthol. It can be assumed, therefore, that theteric obstruction for neomenthol is greater than that for menthol.

Acta 646 (2009) 128–140

Concentration–time profiles with rapid resorption and elimina-tion phases, as seen in this case, are ideal for the verification ofpost-offence drinking claims. In congener analysis, the detectiontime for the target analyte should extend over a few hours (approx.0.5–4 h), since post-offence drinking claims are usually made forthis period of time.

A detection time of more than 12 h for analytes in the serumwould not be suitable for the verification of post-offence drinkingclaims, since the intention is not to record drinking that took placeat an earlier time, prior to the offence.

3.4. Insights into human metabolism of menthol and relatedcompounds after oral ingestion

In the literature [13], the concentration of unchanged menthol4 h after application of dermal patches containing 37.44 mg men-thol was 32 ng mL−1 in plasma. We detected concentrations of thesame magnitude, however by oral application.

Other articles [11,14] have described higher concentrations asthe determination was the sum value of menthol and menthol glu-curonide after enzymatic hydrolysis with �-glucuronidase. Afteringestion of 100 mg menthol, a plasma level of 1200 ng mL−1

menthol glucuronide could be measured [14], and 1492 ng mL−1

could be measured in plasma after ingestion of 180 mL pepper-mint oil [11] in humans. Our observations on menthol are inagreement with these data. However, we were unable to detect7-hydroxymenthone, one of the proposed metabolites of (−)-menthone detected in human liver microsomes [21]. We were alsonot able to find any metabolites of isomenthone (neoisomenthol orisomenthol) above the detection limit.

Obviously menthone and isomenthone are reduced rapidlyto give menthol and neomenthol as well as isomenthol andneoisomenthol, respectively. Both compounds were found to bemetabolised completely. The carbinolic phase I-metabolites areinstantly glucuronidated by UDP-glucuronyl transferases (UGT),and they subsequently enter the gut via biliary excretion. Inthe intestine however, the glucuronidated species are subjectto bacterial �-glucuronidase activity which releases the phaseI-metabolites from their glucuronic acid esters. Yet, there is adiscrimination between menthol stereoisomers, namely between(2S)- and (2R)-configured species. While (2S)-configured mentholand neomenthol are readily liberated from their conjugates, thisreaction is highly unfavourable for (2R)-configured isomentholand neoisomenthol glucuronides. Consequently isomenthol andneoisomenthol were detected only in trace amounts in plasma sam-ples, and for the same reason there was an almost unaltered ratioof the phase I-metabolites.

The cause is enterohepatic cycling which involves thesequence events hepatic glucuronidation, biliary excretionand intestinal hydrolysis of the glucuronide (glucuronidation–deglucuronidation-cycle) and reabsorption. It is effective only forthe (2S)-carbinols menthol and neomenthol which effect can beunderstood in terms of substrate stereogeometry. From what isknown from lipase-catalysed hydrolyses of methyl esters and fromX-ray studies of the respective transition state analogues, it is thestereogeometry at C-2 which determines whether hydrolysis mayoccur [33]. In isomenthol and neoisomenthol the isopropyl sub-stituent is (2R)-configured, what means that it is directed towardshistidine and pushes against the imidazole ring which rotates inresponse to steric interaction. For menthyl ester hydrolyses torsionangles up to 60◦ are reported [33]. As a consequence the catalytic

which reason the ability of histidine to form a bifurcated hydrogenbond with both serine-O and (neo)isomenthyl-O gets lost. In facthydrogen bonding takes place only between histidine and serine-O,while imidazole torsion in response to steric hindrance impedes


F ptiong

ifhenreds

netafms

3

fctdd(ti

c

ig. 7. Enterohepatic cycling of menthol and neomenthol leads to steady reabsorlucuronides via the faeces.

nteraction with the substrate. Since the latter effect is detrimentalor the leaving group properties of the glucuronides, enzymaticydrolysis is not favoured and (neo)isomenthyl glucuronidexcretion is the preferred process. On the contrary, menthol andeomenthol are readily restored from their glucuronides ande-uptake from the gut is predominant over competing faecalxcretion. In addition, since UGT and �-glucuronidases work withifferent enzymatic mechanisms, the UGT catalysed reaction istereogeometry invariant while the latter is not (Fig. 7).

With this in mind it is easily conceivable why isomenthol andeoisomenthol were not found but near the detection limit. And thisxplanation holds true as well for the �-glucuronidase treatment ofhe samples, where for the same reasons potentially present tracemounts of (neo)isomenthyl glucuronides were not cleaved. It isor the same reasons that menthol-�-d-glucuronide instead of free

enthol is administered in the treatment of the irritable bowelyndrome [34].

.5. Serum samples from drivers

Serum samples taken in 2005 and 2006 from drivers who wereound to be under the influence of alcohol, and who claimed to haveonsumed peppermint liqueur, were tested for menthone, isomen-hone, neomenthol and menthol. These cases were not post-offencerinking claims but were simply standard traffic control cases ofrivers who had consumed beverages containing these substances
peppermint liqueur) and who could usually give information abouthe drinking time and the amount consumed. The results are listedn Table 6.
In 8 out of 100 serum samples, menthone was detected atoncentration levels between 3.1 and 7.0 ng mL−1 serum and iso-

of the free carbinols while isomenthol and neoisomenthol are excreted as their

menthone was also detected in eight cases at concentration levelsbetween 3.1 and 11.3 ng mL−1 serum. These positive menthone andisomenthone findings were mostly the same cases. Menthone andisomenthone were only detected in cases that also gave positiveneomenthol and menthol results.

Out of 100 serum samples, neomenthol, as a sum value of freeand conjugated forms, was detected in 35 cases and free neomen-thol in nine cases. The concentrations in sera ranged from 5.3 to57.8 ng mL−1 as the free substance and from 4.2 to 127.8 ng mL−1 asthe sum values of free and conjugated neomenthol.

Out of 100 serum samples, menthol, as a sum value of free andconjugated substance, was detected in 59 cases and free mentholalso in nine cases. The concentrations in sera ranged from 8.0 to92.1 ng mL−1 as the free substance and from 11.0 to 638.2 ng mL−1

as the sum values of free and conjugated neomenthol. In all prob-ability, the high concentrations of unconjugated neomenthol andmenthol resulted from a partial cleavage of the glucuronides in theblood sample itself prior to analysis.

In all positive cases of neomenthol, menthol was also positive.In some cases, menthol glucuronide was the only detectable sub-stance (n = 21). In all of these cases, the concentration of mentholglucuronide was below 50 ng mL−1 serum and free menthol wasnot detectable. It can then be assumed that the beverage consump-tion had been more than 5 h previous to the blood sampling and/orthe amount of consumed beverage was very small (80 mL or less).Nearly all other positive cases of menthol glucuronide were com-
bined with positive cases of neomenthol glucuronide.
For all cases with positively detected substances, the time dif-ference between the cessation of drinking and the taking of bloodsamples was 30 min at the minimum (case 14) and 3 h 55 min(case 30) at the maximum. This indicates a recent consumption


Table 6Menthone, isomenthone, neomenthol and menthol, and concentrations in serum samples from drivers.

No. Data Alleged consumption Time betweenCDa and TBSb

BACc (‰) Cmenthone

(ng mL−1)dCisomenthone

(ng mL−1)eCneomenthol

(ng mL−1)fCmenthol

(ng mL−1)g

1 24 y, m, 86 kg, 172 cm 0.3 L peppermint liqueur/2.0 L beer 1 h 50 min 2.40 n.d. n.d. n.d., 13.3 n.d., 21.72 25 y, m, 75 kg, 170 cm Peppermint liqueur*/wine*/4.0 L

beer50 min 2.33 n.d. n.d. n.d., n.d. n.d., n.d.

3 25 y, m, 75 kg, 170 cm Peppermint liqueur/wine/4.0 Lbeer

1 h 30 min 2.22 n.d. n.d. n.d., n.d. n.d., n.d.

4 40 y, m, 95 kg, 186 cm 60 mL peppermint liqueur/7.5 Lbeer



1 h 10 min 1.96 n.d. n.d. n.d., 16.6 n.d.,61.3

6 45 y, m, 56 kg, 164 cm Peppermint liqueur*/1.5 L beer 1 h 30 min 1.12 5.7 5.0 28.2, 80.6 13.7, 203.67 23 y, f, 72 kg, 168 cm Peppermint liqueur*/herb

liqueur*/1.5 L beer2 h 50 min 1.25 n.d. n.d. n.d., 9.0 n.d., 80.8



9 35 y, m, 109 kg, 187 cm 2.8 L peppermint liqueur/5.0 L beer 1 h 40 min 2.48 n.d. n.d. n.d., n.d. n.d., n.d.10 26 y, m, 63 kg, 171 cm 0.7 L peppermint liqueur/0.5 L beer 1 h 45 min 2.24 n.d. n.d. n.d., n.d. n.d., 21.611 25 y, m, 85 kg, 163 cm 80 mL peppermint liqueur/1.0 L

beer60 min 1.09 n.d. n.d. n.d., n.d. n.d., 28.6

12 28 y, m, 85 kg, 180 cm 0.35 L peppermint liqueur/4.0 Lbeer

30 min 3.02 n.d. n.d. n.d., n.d. n.d., n.d.

13 19 y, m, 85 kg, 173 cm 0.35 L peppermint liqueur/0.5 Lbeer/80 mL herb liqueur

1 h 50 min 1.23 n.d. n.d. n.d., 16.8 n.d., 49.6


30 min 2.52 6.5 3.8 6.0, 20.2 9.3, 265.4

15 18 y, m, 75 kg, 181 cm Peppermint liqueur*/beer* 2 h 15 min 1.28 n.d. n.d. n.d., n.d. n.d., 42.216 23 y, m, 75 kg, 182 cm 40 mL peppermint liqueur/1.0 L


17 44 y, m, 80 kg, 176 cm 0.7 L peppermint liqueur/6.0 L beer 5 h 15 min 1.92 n.d. n.d. n.d., n.d. n.d., n.d.18 34 y, m, 80 kg, 180 cm Peppermint liqueur*/2.5 L beer 1 h 10 min 2.27 n.d. n.d. n.d., n.d. n.d., n.d.19 21 y, m, 71 kg, 178 cm 0.35 L peppermint liqueur 1 h 20 min 1.68 n.d. n.d. n.d., 9.7 n.d., 66.120 46 y, m, 75 kg, 177 cm 1.4 L peppermint liqueur/1.0 L

sangria2 h 40 min 3.11 n.d. n.d. n.d., 15.1 n.d., 47.5


1 h 20 min 1.64 n.d. n.d. n.d. < 4.2 n.d., 35.3


1 h 40 min 1.54 n.d. n.d. n.d. < 4.2 n.d., 29.8

23 43 y, m, 77 kg, 172 cm 0.7 L peppermint liqueur/5.0 L beer 2 h 35 min 2.26 n.d. n.d. n.d., n.d. n.d., n.d.24 26 y, m, un-known, 174 cm 120 mL peppermint liqueur/2.0 L

beer/40 mL herb liqueur2 h 15 min 1.27 n.d. n.d. n.d., 11.2 n.d., 71.2


1 h 20 min 2.28 n.d. n.d. n.d., 5.0 n.d., 40.8

26 19 y, m, 80 kg, 177 cm 0.5 L peppermint liqueur/0.1 L beer 1 h 35 min 1.29 n.d. n.d. n.d., 11.1 n.d., 61.827 27 y, m, 80 kg, 175 cm Peppermint liqueur*/1.5 L beer 1 h 45 min 1.31 n.d. n.d. n.d., n.d. n.d., n.d.28 22 y, m, 95 kg, 190 cm 0.3 L peppermint liqueur/4.5 L beer 60 min 2.18 n.d. n.d. n.d., n.d. n.d., n.d.29 23 y, m, 66 kg, 166 cm 0.2 L peppermint liqueur/2.0 L beer 2 h 30 min 0.89 n.d. n.d. n.d., 7.4 n.d., 40.330 22 y, m, 66 kg, 175 cm 0.5 L Peppermint liqueur/3.0 L beer 3 h 55 min 1.38 < 3.1 3.3 13.1, 28.3 <6.8, 134.631 29 y, m, 76 kg, 174 cm Peppermint liqueur*/beer* 1 h 40 min 2.09 n.d. n.d. n.d., n.d. n.d., n.d.32 20 y, m, 80 kg, 178 cm 350 mL peppermint liqueur/4.0 L

beer13 h 55 min 2.37 n.d. n.d. n.d., n.d. n.d., 17.7



34 22 y, m, 80 kg, 181 cm Peppermint liqueur*/5.0 L beer 1 h 45 min 1.92 n.d. n.d. n.d., n.d. n.d., n.d.35 40 y, m, 59 kg, 172 cm 40 mL peppermint liqueur/3.0 L

beer1 h 10 min 2.95 n.d. n.d. n.d., n.d. n.d., n.d.

36 19 y, m, 65 kg, 175 cm Peppermint liqueur*/3.0 L beer 55 min 1.62 7.0 8.2 43.1, 108.8 11.0, 638.237 30 y, m, 68 kg, 176 cm 80 mL peppermint liqueur/2.5 L

beer30 min 2.18 n.d. n.d. n.d., 7.6 n.d., 35.8


1 h 10 min 2.04 n.d. n.d. n.d., n.d. n.d., 11.0

39 18 y, m, 85 kg, 189 cm Peppermint liqueur*/herbliqueur*/grain*/beer*


40 47 y, m, 100 kg, 185 cm Peppermint liqueur*/3.0 L beer 2 h 30 min 1.99 n.d. n.d. n.d., 9.1 n.d., 60.941 26 y, m, 67 kg, unknown Peppermint liqueur*/beer* 45 min 2.32 < 3.1 4.0 18.1, 33.1 9.8, 96.842 35 y, m, 63 kg, 164 cm 100 mL peppermint liqueur/2.5 L


43 49 y, m, 68 kg, 170 cm 0.7 L peppermint liqueur/0.35 Lbrandy, 5.0 L beer

16 h 1.83 n.d. n.d. n.d., 5.3 n.d., 38.8




3 h 1.34 n.d. n.d. n.d., n.d. n.d., 37.3

46 19 y, m, 80 kg, 184 cm Peppermint liqueur*/5.0 L beer 1 h 15 min 1.38 n.d. n.d. n.d., n.d. n.d., 33.347 27 y, m, 81 kg, 189 cm 0.4 L peppermint liqueur/2.0 L beer 1 h 10 min 2.30 n.d. n.d. n.d., n.d. n.d., 18.648 40 y, m, 83 kg, unknown 700 mL peppermint liqueur/1.5 L



Table 6 (Continued )






(ng mL−1)g

49 66 y, m, 84 kg, 172 cm 700 mL peppermint liqueur Unknown; >1 h30 min

0.77 n.d. n.d. n.d., n.d. n.d., 19.4

50 66 y, m, 84 kg, 172 cm 700 mL peppermint liqueur Unknown; >2 h 0.73 n.d. n.d. n.d., n.d. n.d., 22.051 30 y, m, 85 kg, 195 cm 350 mL peppermint liqueur/4.5 L

beer/0.5 L apple liqueur1 h 30 min 2.44 n.d. n.d. n.d., n.d. n.d., n.d.

52 30 y, m, 73 kg, 175 cm 60 mL peppermint liqueur/60 mLgrain/0.75 L beer

Unknown; >1 h 1.59 n.d. n.d. n.d., n.d. n.d., n.d.



54 18 y, m, 85 kg, 182 cm Peppermint liqueur*/1.25 Lbeer/0.6 L sangria

50 min 1.74 n.d. n.d. n.d., n.d. n.d., 33.3






2 h 1.10 n.d. n.d. n.d., n.d. n.d., 14.6

58 26 y, m, 65 kg, 180 cm Peppermint liqueur */0.5 L beer 1 h 30 min 1.79 n.d. n.d. n.d., n.d. n.d., n.d.59 26 y, m, 65 kg, 180 cm Peppermint liqueur */0.5 L beer 1 h 45 min 1.68 n.d. n.d. n.d., n.d. n.d., n.d.60 23 y, m, 78 kg, 189 cm Peppermint liqueur */2.5 L beer 1 h 30 min 1.76 n.d. n.d. n.d., n.d. n.d., n.d.61 52 y, m, 90 kg, 170 cm 0.7 L peppermint liqueur/1.0 L beer 19 h 1.75 n.d. n.d. n.d., 4.2 n.d., 15.062 47 y, f, 59 kg, 170 cm 0.4 L peppermint liqueur/0.5 L

white wine/0.5 L sangria1 h 50 min 2.64 3.7 n.d. n.d., 16.8 92.1, 459.1

63 61 y, m, 95 kg, 160 cm 2.1 L peppermint liqueur/4.0 L beer 60 min 2.95 4.3 6.8 46.7, 127.8 22.1, 422.064 31 y, m, 75 kg, 175 cm 100 mL peppermint liqueur/1.6 L


65 24 y, m, 70 kg, 170 cm Peppermint liqueur*/10.0 L beer 45 min 1.96 n.d. n.d. n.d., n.d. n.d., n.d.66 37 y, m, 80 kg, 180 cm 350 mL peppermint liqueur/3.5 L

beerUnknown 1.70 n.d. n.d. n.d., 4.6 n.d., 51.9


2 h 10 min 2.10 n.d. n.d. n.d. n.d. n.d. 18.8

68 21 y, m, 64 kg, 171 cm Peppermint liqueur*/1.0 L beer 45 min 1.87 n.d. n.d. n.d., n.d. n.d., n.d.69 36 y, m, 63 kg, 170 cm 0.175 L peppermint liqueur/3.5 L

beer5 h 0.97 n.d. n.d. n.d., n.d. n.d., n.d.

70 27 y, m, 70 kg, 172 cm 0.1 L peppermint liqueur/4.0 L beer 2 h 5 min 2.16 n.d. n.d. n.d., n.d. n.d., n.d.71 27 y, m, 70 kg, 172 cm 0.1 L peppermint liqueur/4.0 L beer 2 h 25 min 2.11 n.d. n.d. n.d., n.d. n.d., n.d.72 38 y, m, 75 kg, 185 cm 40 mL peppermint liqueur/4.0 L

beer50 min 2.16 n.d. n.d. n.d. n.d. n.d. 20.6

73 30 y, m, 76 kg, 178 cm Peppermint liqueur*/5.0 L beer 1 h 10 min 3.67 n.d. n.d. n.d., n.d. n.d., n.d.74 50 y, m, 100 kg, 172 cm 0. 3 L peppermint liqueur 5 h 35 min 2.39 n.d. n.d. n.d., 9.3 n.d,. 26.675 44 y, m, 60 kg, 165 cm 1.4 L peppermint liqueur 1 h 5 min 2.24 3.1 < 3.1 9.7, 16.8 <6.8, 122.176 18 y, m, 56 kg, 163 cm Peppermint liqueur*/3.5 L beer 1 h 35 min 1.04 n.d. n.d. n.d., n.d. n.d., 19.577 35 y, m, 102 kg, 183 cm Peppermint

liqueur*/vodka*/liqueur*45 min 2.18 n.d. n.d. n.d. < 4.2 n.d., 53.2

78 42 y, m, 71 kg, 170 cm Peppermint liqueur*/2.5 L beer 1 h 30 min 1.82 n.d. n.d. n.d., n.d. n.d., n.d.79 37 y, m, 78 kg, 180 cm 80 mL peppermint liqueur/1.0 L

white wine/3.0 L beerUnknown > 1 h 3.78 n.d. n.d. n.d., n.d. n.d., n.d.

80 53 y, m, 87 kg, 190 cm 0.5 L peppermint liqueur/5.0 L beer Unknown 1.90 4.0 3.1 <4.2, 18.3 24.9, 124.881 42 y, m, unknown, unknown 0.33 L peppermint liqueur/2.5 L


82 25 y, m, 84 kg, 186 cm 0.2 L peppermint liqueur/5.0 L beer 1 h 15 min 2.98 3.2 11.3 57.8, 67.8 27.4, 149.183 19 y, m, 65 kg, 160 cm 0.175 L peppermint liqueur/1.2 L


84 49 y, m, 70 kg, 163 cm Peppermint liqueur*/2.0 L beer 5 h 25 min 2.61 n.d. n.d. n.d. n.d. n.d. 12.685 27 y, m, 100 kg, 180 cm 0.25 L peppermint liqueur/5.0 L

beer/0.15 L vodka25 min 1.61 n.d. n.d. n.d., n.d. n.d., n.d.


2 h 55 min 0.65 n.d. n.d. n.d. n.d., n.d.


2 h 50 min 1.16 n.d. n.d. n.d. n.d. n.d. n.d.


Uunknown 2.45 n.d. n.d. n.d., n.d. n.d., n.d.

89 52 y, m, 95 kg, 183 cm 0.5 L peppermint liqueur/2.5 L beer 1 h 30 min 3.14 n.d. n.d. n.d., n.d. n.d., 38.890 35 y, m, 81 kg, 178 cm 0.7 L peppermint liqueur 10 min 2.49 n.d. n.d. n.d., 6.6 n.d., 28.491 25 y, m, 68 kg, 175 cm Peppermint liqueur*/4.0 L

beer/herb liqueur*1 h 50 min 2.02 n.d. n.d. n.d., 13.0 n.d., 78.6

92 19 y, m, 103 kg, 185 cm Peppermint liqueur*/beer*/whisky* 2 h 15 min 1.40 n.d. n.d. n.d., 12.6 n.d., 38.593 50 y, m, 82 kg, 173 cm 0.7 L peppermint liqueur 5 h 30 min 3.22 n.d. n.d. n.d., 4.7 n.d., 21.594 28 y, m, 99 kg, 180 cm 1.0 L peppermint liqueur/5.5 L beer 55 min 2.17 n.d. n.d. n.d., 9.0 n.d., 40.295 57 y, m, unknown Peppermint liqueur*/2.5 L

beer/ouzo*4 h 10 min 2.45 n.d. n.d. n.d., n.d. n.d., n.d.

96 48 y, f, 70 kg, 174 cm 0.2 L peppermint liqueur/1.0 L beer 55 min 1.67 n.d. n.d. 5.3, 13.0 8.0, 240.897 39 y, m, 107 kg, 185 cm 0.12 L peppermint liqueur/5.0 L



Table 6 (Continued )






(ng mL−1)g


1 h 05 min 2.80 n.d. n.d. n.d., 9.1 n.d., 40.9

99 22 y, f, 54 kg, 163 cm 0.1 L peppermint liqueur/1.0 L beer 2 h 20 min 1.05 n.d. n.d. n.d., n.d. n.d., 27.2100 21 y, f, 52 kg, 160 cm 0.12 L peppermint liqueur/2.0 L

beer/0.06 L fig liqueur2 h 15 min 1.19 n.d. n.d. n.d., n.d. n.d., 24.1

a CD cessation of drinking.b TBS taking of blood samples.c BAC blood–alcohol concentration.

d,e

otc8

sii1

t

F(m

Without/with enzymatic cleavage (same results).f Results without/with enzymatic cleavage.g Results without/with enzymatic cleavage.* Amount of consumed beverage not known.

f peppermint liqueur, in accordance with our findings gathered byhe drinking tests. In these cases, the corresponding blood–alcoholoncentrations were between 1.12‰ (case 6) and 2.98‰ (case2).

The gas chromatogram and mass spectra of an authentic bloodample (case 63) after peppermint liqueur consumption are shownn Fig. 8. Fig. 8b shows the mass spectra of the co-eluting substances,
somenthone (m/z = 112, 139, 154) and neomenthol (m/z = 95, 123,38). Therefore, these target ions have been used.
It is evident from the results of the investigations conducted inhe drinking experiments, and from the samples taken from drivers

ig. 8. (a) GC–MS chromatogram (scan mode) of an authentic blood sample a17.71 min = menthone, 18.05 min = isomenthone and neomenthol, 18.29 min = menthol, 29

ass spectra of peaks at (b) 17.71 min (menthone), (c) 18.05 min (isomenthone and neom

who were under the influence of alcohol, that menthone, isomen-thone, neomenthol and menthol are very sensitive and specificmarkers that can be reliably detected even in authentic samples,after consumption of peppermint liqueur.

Although the free substances neomenthol and menthol aredetectable in greater amounts in cases with current consumption ofpeppermint liqueur, an enzymatic hydrolysis is necessary for analy-
sis. After the consumption of volumes of about 200 mL peppermintliqueur or less, the free substances, neomenthol and menthol, arenot detectable in serum samples and the menthone and isomen-thone concentrations are too low to detect reliably.
fterpeppermint liqueur consumption (case 63) without enzymatic cleavage.29 min = dicyclohexylmethanol, internalstandard, BAC = 2.95‰) and correspondingenthol) and (d) 18.29 min (menthol).


(Conti

csc

fsacoo

iao

fomusoeiaus

Fig. 8.

However, after enzymatic cleavage, it is possible to clearly detectonsumed amounts of as low as 20 mL peppermint liqueur in timepans of about 3 h after cessation of drinking due to the elevatedoncentrations of menthol in the serum samples.

Our intention is to develop a method for differentiating positiverom negative samples through the time difference between ces-ation of drinking and the taking of a blood sample, in order to beble to distinguish between pre-offence and post-offence drinkinglaims. Thus, we will be able to reliably disprove or confirm post-ffence drinking claims that are of significance in cases of trafficffences.

Information about the amount consumed and the time of drink-ng the beverage containing menthone, isomenthone, neomentholnd menthol are absolutely essential for verifying the plausibilityf claims.

A limitation of the suitability of neomenthol and mentholor validating alcohol drinking claims could be their commonccurrence in other sources of human exposure (e.g. toothpaste,outhwash, chewing gum). Furthermore, menthol is also often

sed in pharmaceutical products. Therefore, the possibility thaterum menthol levels could be altered through consumptionr use of these other products in relevant amounts cannot be
xcluded. This would also have to be considered in expert opin-ons and demands further research. Consequently, we considereds positive only those cases with menthol concentrations (sum val-es of free and conjugated substance) of more than 10 ng mL−1
erum.

nued ).

4. Conclusions

The HS-SPME–GC–MS method presented here was ideal for thedetermination of menthone, isomenthone, neomenthol and men-thol in serum samples taken from drivers under the influence ofalcohol.

The simplicity of the preparation procedures required for thesamples, the excellent degree of sensitivity and its other verygood validation results are the main advantages of this tech-nique. The PDMS/DVB fibre, found to be the best suited fibre forthe enrichment of anethole and eugenol in earlier investigations,was also very well suited for the substances investigated in thisstudy; namely, menthone, isomenthone, neomenthol and men-thol.

Based on the results of the drinking experiments,concentration–time profiles were constructed to enable state-ments to be made about changes in detection levels over time,as well as the duration of the detection in serum. This method ofverifying post-offence drinking claims relating to spirits contain-ing these flavour materials appeared to be very suitable withinan approximate time-frame of 30 min to 4 h, even in cases ofconsumption of relatively small amounts of spirits.

Thus, it was possible to extend the range of analytesdetectable through congener analysis to now include the sub-stances menthone, isomenthone, neomenthol and menthol, whichare characteristic of peppermint liqueur and certain other alcoholicbeverages (mint liqueurs, digestif bitters).

1 himica

vv

A

H

R

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40 K. Schulz et al. / Analytica C

These characteristic beverage flavourings had not been pre-iously determined for congener analysis. Hence, improvederification of post-offence drinking claims is now possible.

cknowledgments

The authors would like to thank Dr. Uwe Schmidt and Uweanisch for the taking of blood samples.

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headspace solid-phase microextraction–gas chromatography–mass spectrometry determination of the...

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