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Review of Methods of
Analysis for
Biologically Active
Principles (A01041)
Prepared for the Food Standards Agency
by
Dr Jayne S. Turner and Professor David E. G. Shuker
Department of Chemistry
The Open University
Walton Hall
Milton Keynes
MK6 7AA.
Contents
Introduction........................................................................................................... - 7 -
Search Strategy ..................................................................................................... - 7 -
Scope of Summary................................................................................................ - 7 -
Abbreviations........................................................................................................ - 8 -
β-Asarone.................................................................................................................. - 9 -
1. Chemical and physical data ............................................................................. - 9 -
1.1 Synonyms and trade names........................................................................ - 9 -
1.2 Numbers..................................................................................................... - 9 -
1.3 Structural and molecular formulae............................................................. - 9 -
1.4 Chemical and physical properties of the pure substance ........................... - 9 -
2. Source, Background, Usage and Limits......................................................... - 10 -
2.1 Source ...................................................................................................... - 10 -
2.2 Background .............................................................................................. - 10 -
2.3 Usage........................................................................................................ - 11 -
2.4 Limits ....................................................................................................... - 11 -
3. Biochemical Aspects...................................................................................... - 11 -
3.1 Metabolism .............................................................................................. - 11 -
4. Analysis.......................................................................................................... - 11 -
4.1 Extraction Methods.................................................................................. - 12 -
4.2 Quantitation.............................................................................................. - 13 -
5. Summary ........................................................................................................ - 14 -
6. References...................................................................................................... - 14 -
Coumarin................................................................................................................. - 16 -
1. Chemical and physical data ........................................................................... - 16 -
1.1 Synonyms and trade names...................................................................... - 16 -
1.2 Numbers................................................................................................... - 16 -
1.3 Structural and molecular formulae........................................................... - 16 -
1.4 Chemical and physical properties of the pure substance ......................... - 16 -
2.1 Source ...................................................................................................... - 17 -
2.2 Background .............................................................................................. - 17 -
2.3 Usage........................................................................................................ - 17 -
2.4 Limits ....................................................................................................... - 18 -
3. Biochemical Aspects....................................................................................... - 18 -
3.1 Metabolism .............................................................................................. - 18 -
4. Analysis.......................................................................................................... - 19 -
4.2 Quantitation.............................................................................................. - 20 -
5. Summary ........................................................................................................ - 21 -
6. References...................................................................................................... - 22 -
Estragole ................................................................................................................. - 25 -
1.2 Numbers................................................................................................... - 25 -
2. Background, Usage and Limits...................................................................... - 26 -
2.1 Source ...................................................................................................... - 26 -
2.2 Background .............................................................................................. - 26 -
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2.3 Usage........................................................................................................ - 27 -
2.4 Limits ....................................................................................................... - 27 -
3.1 Metabolism .............................................................................................. - 27 -
4. Analysis.......................................................................................................... - 27 -
4.1 Extraction methods .................................................................................. - 27 -
4.2 Quantitation.............................................................................................. - 29 -
5. Summary ........................................................................................................ - 29 -
6. References...................................................................................................... - 30 -
Hydrocyanic Acid ................................................................................................... - 32 -
1.2 Numbers................................................................................................... - 32 -
2.1 Source ...................................................................................................... - 33 -
2.2 Background .............................................................................................. - 33 -
2.3 Usage........................................................................................................ - 33 -
2.4 Limits ....................................................................................................... - 33 -
3.1 Metabolism .............................................................................................. - 33 -
4. Analysis.......................................................................................................... - 34 -
5. Summary ........................................................................................................ - 36 -
6. References...................................................................................................... - 36 -
Isosafrole................................................................................................................. - 38 -
1.2 Numbers................................................................................................... - 38 -
2.1 Source ...................................................................................................... - 39 -
2.2 Background .............................................................................................. - 39 -
2.3 Usage........................................................................................................ - 39 -
2.4 Limits ....................................................................................................... - 40 -
3. Biochemical Aspects....................................................................................... - 40 -
3.1 Metabolism .............................................................................................. - 40 -
4. Analysis.......................................................................................................... - 40 -
4.1 Extraction................................................................................................. - 41 -
4.2 Quantitation.............................................................................................. - 41 -
5. Summary ........................................................................................................ - 42 -
6. References...................................................................................................... - 42 -
Menthofuran............................................................................................................ - 44 -
1.2 Numbers................................................................................................... - 44 -
2.1 Source ...................................................................................................... - 45 -
2.2 Background .............................................................................................. - 45 -
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2.3 Usage........................................................................................................ - 45 -
2.4 Limits ....................................................................................................... - 45 -
3.1 Metabolism .............................................................................................. - 46 -
4. Analysis.......................................................................................................... - 46 -
4.2 Quantitation.............................................................................................. - 47 -
5. Summary ........................................................................................................ - 47 -
6. References...................................................................................................... - 47 -
Methyleugenol ........................................................................................................ - 49 -
1.2 Numbers................................................................................................... - 49 -
2.1 Source ...................................................................................................... - 50 -
2.2 Background .............................................................................................. - 50 -
2.3 Usage,....................................................................................................... - 50 -
2.4 Limits ....................................................................................................... - 51 -
3.1 Metabolism .............................................................................................. - 51 -
4. Analysis.......................................................................................................... - 51 -
4.2 Quantitation.............................................................................................. - 53 -
5. Summary ........................................................................................................ - 54 -
6. References...................................................................................................... - 55 -
Pulegone.................................................................................................................. - 57 -
1. Chemical and physical data ............................................................................ - 57 -
1.2 Numbers................................................................................................... - 57 -
2.1 Source ...................................................................................................... - 58 -
2.2 Background .............................................................................................. - 58 -
2.3 Usage........................................................................................................ - 58 -
2.4 Limits ....................................................................................................... - 58 -
3.1 Metabolism .............................................................................................. - 59 -
4. Analysis.......................................................................................................... - 59 -
4.2 Quantitation.............................................................................................. - 61 -
5. Summary ........................................................................................................ - 62 -
6. References...................................................................................................... - 62 -
Quassine.................................................................................................................. - 65 -
1.2 Numbers................................................................................................... - 65 -
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2.1 Source ...................................................................................................... - 66 -
2.2 Background .............................................................................................. - 66 -
2.3 Usage........................................................................................................ - 66 -
2.4 Limits ....................................................................................................... - 66 -
4. Analysis.......................................................................................................... - 66 -
4.2 Quantitation.............................................................................................. - 67 -
5. Summary ........................................................................................................ - 67 -
6. References...................................................................................................... - 67 -
Safrole ..................................................................................................................... - 68 -
1.2 Numbers................................................................................................... - 68 -
2.1 Source ...................................................................................................... - 69 -
2.2 Background .............................................................................................. - 69 -
2.3 Usage........................................................................................................ - 69 -
2.4 Limits ....................................................................................................... - 69 -
3.1 Metabolism .............................................................................................. - 70 -
4. Analysis.......................................................................................................... - 71 -
4.1 Extraction................................................................................................. - 71 -
4.2 Quantitation.............................................................................................. - 72 -
5. Summary ........................................................................................................ - 74 -
6. References...................................................................................................... - 75 -
Teucrin A ................................................................................................................ - 78 -
1.2 Numbers................................................................................................... - 78 -
2.1 Source ...................................................................................................... - 79 -
2.2 Background .............................................................................................. - 79 -
2.3 Usage........................................................................................................ - 79 -
2.4 Limits ....................................................................................................... - 79 -
3.1 Metabolism .............................................................................................. - 79 -
4. Analysis.......................................................................................................... - 79 -
4.2 Quantitation.............................................................................................. - 80 -
5. Summary ........................................................................................................ - 80 -
6. References...................................................................................................... - 80 -
Thujone ................................................................................................................... - 81 -
1.2 Numbers................................................................................................... - 81 -
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2.1 Source ...................................................................................................... - 83 -
2.2 Background .............................................................................................. - 83 -
2.3 Usage........................................................................................................ - 83 -
2.4 Limits ....................................................................................................... - 83 -
3.1 Metabolism .............................................................................................. - 83 -
4. Analysis.......................................................................................................... - 84 -
4.2 Quantitation.............................................................................................. - 86 -
5. Summary ........................................................................................................ - 87 -
6. References...................................................................................................... - 88 -
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Introduction
The purpose of this project was to carry out a comprehensive literature search and to
give a full and critical evaluation of previously reported analytical methods for twelve
biologically active principles (BAPs) as required by the Food Standards Agency. The
active principles in question are β-asarone, coumarin, pulegone, safrole, isosafrole,
thujone, menthofuran, estragole, hydrocyanic acid, methyleugenol, Teucrin A and
quassine. Particular attention is given to the extraction and quantification of these
compounds in food products.
In the UK, the use of flavourings is currently controlled by the Flavourings in Food
Regulations (SI 1992 No. 1971) as amended. These regulations are based on the
European Council and Commission Directives on flavourings for use in foodstuffs
(88/388/EEC amended by 91/71/EEC). These Directives regulate the use of
flavourings in food across the European Union (EU) and lay down limits for certain
BAPs which may be present in flavourings. A new regulation to replace Directive
88/388/EEC (as amended) is currently being discussed and is expected to be
published in summer 2006.
Biologically active principles (BAPs) are chemical substances that occur naturally in a
number of natural flavouring source materials. They cannot be added as such into
foodstuffs or flavourings due to concerns over their toxicity. They are listed in Annex
II of the Framework Directive 88/388/EEC.
Search Strategy
A search for each compound was first made via the on-line publications of World
Health Organisation (WHO) and of relevant EC documents. Chemical and physical
data, or references to such data, was obtained from the Beilstein CrossFire
Information System and from the Merck Index. Further information was obtained
from the INCHEM and IARC databases. The following databases were searched, ISI
Web of Science (1945-2005), Science Citation Index (SCI) (1945-2005), Science
Direct, SwetsWise and PubMed, using >compound name< as search string, with the
exception of the SCI search for coumarin when >coumarin analys*< was used. The
online search facilities of American Chemical Society (ACS) Publications and the
Royal Society of Chemistry (RSC) journals archive were searched as above. All the
relevant refereed articles were retrieved; key papers/authors were entered into the SCI
and cross-referenced.
Scope of Summary
The purpose of the summary in each monograph is to give a critical evaluation of the
existing analytical methods for each of the named compounds. This includes
extraction and quantification techniques that have been used previously for the
determination of each compound from varying sources. Where applicable, there is an
indication for further work that may be required for the enforcement of the proposed
EC regulations on flavouring.
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Abbreviations
AOAC Association of Official Analytical IOFI International Organization of the

Chemists Flavour Industry
AP active principle IUPAC International Union of Pure and
Applied Chemistry
bw body weight JECFA Joint Expert Committee on Food
Additives
CAS Chemical Abstract Service KBr potassium bromide
CDCl3 chloroform-d LD50 dose at which 50% lethality occurs
DAD diode array detector m/z mass to charge ratio

DNSH dansylhydrazine MS mass spectrometry
EINECS European Inventory of Existing µg microgram

Commercial Chemical Substances
ELISA enzyme linked immunosorbent mg milligram
assay
FDA Food and Drug Administration nm nanometre
FEMA Flavor and Extract Manufacturers NMR nuclear magnetic resonance

Association
FID flame ionization detector NOEL no effect level
GC gas chromatography SDE simultaneous distillation extraction
GC/MS Gas chromatography/ mass SPME solid phase micro

spectrometry extraction
GRAS generally regarded as safe SD standard deviation
HCN hydrocyanic acid TLC thin layer chromatography
HPLC high performance liquid TDI tolerated daily intake

chromatography
Hz hertz UV ultra violet
id internal diameter v/v volume/volume
IR infra red λem wavelength of emission
Petr Petroleum
-8-
β-Asarone
1. Chemical and physical data
1.1 Synonyms and trade names

Cis-asarone
Cis-2,4,5-trimethoxy-1-propenyl benzene
2,4,5-trimethoxypropen-1-ylbenzene
1,2,4-trimethoxy-5-prop-1-enyl-benzene (IUPAC name)
Z-asarone
Cis-isoasarone
Asarin
Asarin camphor
Asarabacca camphor
1.2 Numbers
CAS 5273-86-9, 494-40-6, 2883-98-9
Beilstein Registry Number 1910605
Merck Monograph Number 0000831
1.3 Structural and molecular formulae
6 H
H3CO 5
C 2'
1
1'
CH
2 3'
CH3
4
H3CO OCH3
3
Table 1. Structural and molecular information
Molecular Formula C12H16O3

Molecular Weight 208.26
Elemental Composition C 69.21%, H 7.74%, O 23.05%
Class Isocyclic
1.4 Chemical and physical properties of the pure substance

A summary of chemical and physical properties of pure β-asarone is given in Table 2.
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Table 2. Chemical and physical properties
Physical appearance Yellow liquid

Density 1.0933
Fluorescence excitation 310 nm emission 355 nm 1
UV absorbance 254 nm2
IR (KBr) vmax 1608, 1580, 1508 (aromatic) and 857
(cis-double bond)3
Known to be soluble in alcohol, ether, glacial acetic acid, carbon
tetrachloride, chloroform and petr. ether4.
Insoluble water
Boiling point 281-283°
(CDCl3) δ 1.84 (3H, dd, J = 7.3 Hz and J
1
H NMR
= 1.8 Hz, -CH=CH-CH3), 3.81, 3.83 and
3.89 (each 3H, s, three OCH3), 5.76 (1H,
dq, J = 12Hz and 6.8 Hz, H-2’), 6.49 (1H,
dq, J = 11.4 Hz and 1.83 Hz, H-1’), 6.53
(1H, s, H-3) and 6.84 (1H, s, H-6)5
(CDCl3) δ 151.5 s (C-4), 148.5 s (C-2),
13
C NMR
125.8 d (C-1’), 124.7 d (C-2’), 118.0 s
(C-5), 114.1 d (C-6), 56.6 q, 56.4 q, 56.0
q (OCH3), 14.6 q (C-3’)5
β-asarone is a stable compound and in mass spectra a strong response for the parent
ion (m/z 208 M+) is observed, with consistent fragments, 193, 165, 140 and 692,3,5,6. In
the absence of light, β-asarone is stable, at 30°C, for at least 40 days7. β-asarone is a
ninhydrin positive substance.8
2. Source, Background, Usage and Limits
2.1 Source
Acorus calamus L-calamus – sweet flag
Asarum europaeum L.- hazelwort
Oil from the tetraploid (48 chromosomes) ‘Indian’ form of Calamus contains up to
90% β-asarone, the ‘Kashmir’ form is hexaploid (72 chromosomes) and oils from this
source contains approximately 5% β-asarone. The ‘European’ form is triploid (36
chromosomes) and contains less than 10% β-asarone. A diploid (24 chromosomes)
form exists, with virtually no β-asarone and although this has been used to flavour
alcoholic beverages9, it is not commercially available as a flavouring.
2.2 Background
The rhizomes of A. calamus have been used by traditional herbalists in the treatment
of intestinal disorders and asthma, used as a stimulant and it has been shown to have
antibacterial properties. Extracts of the tetraploid form of A. calmus should be
considered unfit for human consumption in any amounts. In the US calamus oil and
its extracts are prohibited from all use in food. β-asarone may also be a potent
antifungal agent against some plant pathogens6.
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2.3 Usage
There is no data for the use of hazelwort oil in foodstuff but calamus oil is used
mainly for flavouring alcoholic beverages including bitters, liqueurs and vermouths10.
2.4 Limits
Annex II of Directive 88/388/EEC limits the amount of β-asarone from flavourings
and other food ingredients to:
food 0.1 mg/kg,

beverages 0.1 mg/kg11
The exceptions are alcoholic beverages and seasonings used in snack food where a
level of 1 mg/kg has been set10. It is proposed in the current version of the EC
flavouring regulation (WGF/002/02 rev 3) that β-asarone should be limited to 0.5
mg/kg in alcohol beverages12. In addition the tetraploid form of calamus should not be
used for the production of flavourngs.
A value of 2 µg/kg bw/day has been estimated for human intake of β-asarone in the
UK. This value is derived from limited data on levels of β-asarone present in certain
foods and the subsequent consumption of these foods and alcoholic beverages11
3. Biochemical Aspects
3.1 Metabolism
The metabolism of beta-asarone has yet to be established, but it is a 1-
propenylbenzene compound and as such its metabolism is thought to be different from
those of the allylbenzenes, whose metabolic pathways are better known10. Scheme 1
shows the products of oxidation following the conversion of β-asarone into the trans
isomer13. The aldehyde is a main metabolite13. Following epoxidation of the double
bond an alcohol functionality is added to either C-1’ or to C-2’ depending on the ring
opening of the epoxide. These compounds are present in their more stable keto form9.
4. Analysis
The AOAC official method of analysing β-asarone is given in method 977.09, which
refers to wine. The apparatus used is a gas chromatograph (GC) fitted with a flame
ionization detector with a glass or stainless steel column (1.8 m x 2 mm id), this is
packed with 10 % SP-1000 on Chromosorb W HP 80/100. Ethyl palmitate, in a 1
mg/mL solution in hexane, is used as an internal standard and the β-asarone standard
is 1 mg/mL in ethanol. A standard curve, using concentrations over 1 – 5 mg/L, is
prepared. The sample (100 mL of sample mixed with 50 mL of water) is distilled to
obtain 100 mL of distillate. This is extracted with 10 mL of hexane and a 5 µL sample
is injected into the GC14.
In 1983 LGC15 investigated the then current International Organization of the Flavour
Industry (IOFI) method of analysing β-asarone, in which a 100 g sample of alcoholic
beverage is diluted to 250 mL with water, it is then distilled and 200 mL of the
distillate collected. The distillate is saturated with sodium chloride and then extracted
three times with n-pentane (3 x 50 mL). The extract is dried with anhydrous sodium
sulphate and evaporated to a smaller volume, this is then analysed by GC (Carbowax
20 M column). Tricosane (1.032 g/L n-pentane) was used as an internal standard. It
- 11 -
was concluded that the method worked satisfactorily and gave good sensitivity, with a
limit of detection of 0.001 mg/mL, when the internal standard was added directly to
the distillate15.
IOFI recommends the use of GC in β-asarone determination, the recommended

method (21 (1987)) suggests using a glass or fused silica column packed with
Carbowax 20 M or similar and the use of a flame ionization detector16.
H H
H3CO C H3CO C CH3
CH C
H
CH3
H3CO OCH3 H3CO OCH3
β-asarone α-asarone
O
O
CH3
H3CO CH
H3CO CH C
H
H3CO OCH3
H3CO OCH3
2,4,5-trimethylbenzaldehyde
H2
H3CO C CH3 H
H3CO C CH3
C
C
O
OH
H3CO OCH3
H3CO OCH3
2,4,5-trimethoxyphenylacetone
+
O OH
H3CO C CH3 H3CO C CH3

C
H
H3CO OCH3 H3CO OCH3
2,4,5-trimethoxypropiophenone
Scheme 1. The proposed metabolic pathways for β-asarone9,13.
4.1 Extraction Methods

β-asarone has been extracted from wine samples following distillation and hexane
extraction. One extraction with hexane was sufficient to extract β-asarone from the
initial distillate when an equal volume of saturated sodium chloride solution was
added2. In spirits β-asarone was extracted by solid-phase extraction on phenyl sorbent
using elution with pentane-diethyl ether (7 + 3 by volume)17.
- 12 -
Five compounds, including β-asarone, were extracted from 10 mL samples of
alcoholic beverages. The samples were pipetted into test tubes containing 1.1 g
sodium sulphate. After neutralizing with sodium bicarbonate, known amounts of the
five compounds (0, 0.2 0.5, 1.0 and 2.0 mg/kg dissolved in ethanol) and 0.5 mg/kg
tetralin (as an internal standard) were added to the sample tubes. These samples were
extracted three times with diethyl ether and concentrated to 3 mL. The resulting
samples were then analysed by selected ion monitoring mass spectrometry (MS)18.
4.2 Quantitation
The oils of Daucus carota spp. maximus have been characterised by retention indices
and mass spectral information19. The most reliable technique to date for the
quantitation of asarone isomers from essential oils is GC/MS20. Thin layer
chromatography (TLC) could not separate the two asarone isomers but it could be
used for rapid screening of the total asarone content of ‘vegetable extracts, foodstuffs,
drinks and drugs’20.
Levels of β-asarone were determined in wine samples by GC, following steam

distillation and hexane extraction. Standards of β-asarone were extracted from
ethanol-water (1:4. v/v) and wine samples. Recoveries of added β-asarone (1-5 mg/L)
were 100.3% (SD 2.94%, n=10). No β-asarone was detected in unspiked wine
samples with a detection limit of 0.5 mg/L2.
The same GC method was evaluated in a collaborative study between nine

laboratories. Ten samples of wine, with added known amounts of β-asarone, and
standard of β-asarone (4 mg/L) in ethanol-water (1:4, v/v) were sent to each
laboratory along with GC column packing material, samples of β-asarone and ethyl
palmitate (as internal standard). The results from this study are summarised in Table 1
and show that the GC method was reliable enough to be proposed as an "official first
action" for β-asarone21. [Comment: The authors did not indicate whether the identity
of the samples was concealed from the collaborating laboratories prior to the
statistical analyses].
Table 3. Summary of statistical analysis of results from a collaborative study of β-

asarone in wine21.
Value All samples

Amount β-asarone added (mg/L) 2-5
Mean recovery (%) 89.27
Reproducibility 16.014
(CV, %) 17.9
Repeatability 7.377
(CV, %) 8.3
Steam distillation of alcoholic beverages (adjusted with water to an ethanol

concentration of 20%) afforded a fraction which could be analysed directly on
reversed-phase (C-18) high performance liquid chromatography (HPLC) with
fluorescence detection (λex = 310nm, λem = 355 nm) for the presence of β-asarone.
The detection limit was 0.01 mg/L with recoveries of 93-105%. β-asarone was not
detected in 16 commercial samples of alcoholic beverages1.
- 13 -
β-asarone was also quantitated in spirits using reversed-phase (C-18) HPLC with
fluorescence detection (λex = 310nm, λem = 355 nm) following solid phase extraction
on phenyl absorbent and elution with pentane-diethyl ether (7:3. v/v). 3,3'-
dimethoxybiphenyl was used as an internal standard. The detection limit for β-asarone
was 0.08 mg/L and recoveries were 106.4% (SD, 11.3%). Twelve samples of spirits
were analysed and levels of β-asarone ranged from not-detectable to 3.63 mg/L17.
Reversed-phase HPLC was also used to measure β-asarone in alcoholic beverages

using a stainless steel column (25 cm x 2.6 mm id) packed with octadecylsilane, with
a fluorimeteric detector (λex 310 nm, λem 355 nm)22. This enhanced sensitivity and
lowered interference, and so detection limits down to 0.001 mg/L were achieved, with
recovery rates ranging from 100% to 108%22.
The two commonly occurring α- and β- isomers of asarone can be separated by GC

and essential oils from different plants show dramatically different ratios of isomers20.
This method does not appear to have been used to detect the two isomers in
foodstuffs.
5. Summary
Distillation, with or without coupled solvent extraction, was the basis of the most
common methods of extracting β-asarone from plants and from beverages. No
reference to the extraction of this compound from a food matrix could be found. As
limits are to be applied to alcoholic beverages only, steam distillation followed by
hexane extraction would be suitable.
The methods of quantification using GC and GC/MS are acceptable for levels of β-
asarone at 1 mg/kg, however if the limit is reduced to 0.5 mg/kg, these methods
approach their limit of detection. A more sensitive approach is the use of HPLC with
fluorescence detection which has a limit of detection as low as 0.01 mg/kg, which
would be sensitive enough to reliably monitor a limit of 0.5 mg/kg that is proposedfor
alcoholic beverages sold in the EU.
6. References
(1) Curro, P.; Micali, G.; Lanuzza, F. Determination of Beta-Asarone, Safrole,
Isosafrole and Anethole in Alcoholic Drinks by High-Performance Liquid-
Chromatography. Journal of Chromatography 1987, 404, 273-278.
(2) Dyer, R. H.; Martin, G. E.; Buscemi, P. C. Gas-Liquid-Chromatographic
Determination of Beta-Asarone in Wines and Flavors. Journal of the
Association of Official Analytical Chemists 1976, 59, 675-677.
(3) Babu, K. S.; Srinivas, P. V.; Praveen, B.; Kishore, K. H.; Murty, U. S. et al.
Antimicrobial constituents from the rhizomes of Rheum emodi.
Phytochemistry 2003, 62, 203-207.
(4) The Merck Index - Monograph Number 000831. Asarones. 2004.
(5) McGaw, L. J.; Jager, A. K.; van Staden, J. Isolation of beta-asarone, an
antibacterial and anthelmintic compound, from Acorus calamus in South
Africa. South African Journal of Botany 2002, 68, 31-35.
(6) Lee, J. Y.; Lee, J. Y.; Yun, B.-S.; Hwang, B. K. Antifungal Activity of beta-
asarone from Rhizomes of Acorus gramineus. Journal of Agricultural and
Food Chemistry 2004, 52, 776-780.
(7) Streloke, M.; Ascher, K. R. S.; Schmidt, G. H.; Neumann, W. P. Vapor-
Pressure and Volatility of Beta-Asarone, the Main Ingredient of an Indigenous
- 14 -
Stored-Product Insecticide, Acorus- Calamus Oil. Phytoparasitica 1989, 17,
299-313.
(8) Oswald, E. o.; Fishbein, L.; Corbett, B. J. Metabolism of naturally occurring
propenylbenzene derivatives : I. Chromatographic separation of ninhydrin-
positive materials of rat urine. Journal of Chromatography A 1969, 45, 437-
445.
(9) Mazza, G. Gas chromatographic and mass spectrometric studies of the
constituents of the rhizome of calamus : I. The volatile constituents of the
essential oil. Journal of Chromatography A 1985, 328, 179-194.
(10) Opinion of the Scientific Committee on Food on the Presence of Beta-asarone
in flavourings and other food ingredients with flavouring properties.
SCF/CS/FLAVOUR/9 ADD1 Final January 2002. 2002.
(11) EEC 1988, Council Directive 88/388/EEC of 21 June 1988 on the
approximation of the laws of the Member States relating to flavourings for use
in foodstuffs and to source materials for their production. Official Journal of
the European Communities, 15.7.1988.
(12) Proposal of the European Parliament and of the Council on Flavourings and
certain food ingredients with flavouring properties for use in and on foods.
2004, WGF/002/02-rev 2.
(13) Mazza, G. Identification of Oxidation-Products of Beta-Asarone in Acorus-
Calamus L by Gas-Chromatography and Mass-Spectrometry. Sciences Des
Aliments 1984, 4, 473-481.
(14) AOAC Official Methods of Analysis of AOAC International. 17th edition,
2000.
(15) The Investigation of Methods for the Determination of Active Flavouring
Principles in Food and Drink. Report prepared at the Laboratory of the
Government Chemist for the Food Science Division of the Ministry of
Argiculture Fisheries and Food. 1983.
(16) International Organization of the Flavor Industry (IOFI) Recommended
Methods. Method 21. Beta-asarone: determination by capillary gas
chromatography. 1987.
(17) Lander, V.; Worner, M.; Kirchenmayer, C.; Wintoch, H.; Schreier, P. Use of
Solid-Phase Extraction for Rapid Sample Preparation in the Determination of
Food Constituents .2. Asarone, Quinine, Coumarin, and Quassine in Spirits.
Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung 1990, 190, 410-
413 (German).
(18) Galli, C. L.; Galli, G.; Tragni, E.; Caruso, D.; Fiecchi, A. Quantitative-
Analysis of Alpha,Beta-Thujone, Pulegone, Safrole, Coumarin and Beta-
Asarone in Alcoholic Beverages by Selected- Ion Monitoring. Journal of
Applied Toxicology 1984, 4, 273-276.
(19) Saad, H.-E. A.; El-Sharkawy, S. H.; Halim, A. F. Essential oils of Daucus
carota ssp. maximus. Pharmaceutica Acta Helvetiae 1995, 70, 79-84.
(20) Oprean, R.; Tamas, M.; Roman, L. Comparison of GC-MS and TLC
techniques for asarone isomers determination. Journal of Pharmaceutical and
Biomedical Analysis 1998, 18, 227-234.
(21) Dyer, R. H. Gas-Liquid-Chromatographic Determination of Beta-Asarone in
Wine - Collaborative Study. Journal of the Association of Official Analytical
Chemists 1977, 60, 1041-1043.
(22) Micali, G.; Curro, P.; Calabro, G. Reversed-phase high-performance liquid
chromatography for the determination of [beta]-asarone. Journal of
Chromatography A 1980, 194, 245-250.
- 15 -
Coumarin

2H-1-benzopyran-2-one
1,2-benzopyrone
5,6-benzo-2-pyrone
Benzo-α-pyrone
Cis-ortho-coumarinic acid lactone
Coumarinic anhydride
ortho-Hydroxycinnamic acid lactone
chromen-2-one (IUPAC name)
hydrocoumarin
1.2 Numbers
CAS 91-64-5
O O
Molecular formula C9H6O2

Molecular weight 146.15
Elemental composition C 73.97%, H 4.14%, O 21.90%
Class Heterocyclic, naturally occurring
benzopyrone1

A summary of chemical and physical properties of pure coumarin is given in Table 2.
- 16 -
Table 2. Chemical and physical properties.
Physical appearance Colourless flakes with a vanilla-like

odour2
Volatility Intermediate3
Melting point 69-73°
Boiling point 298°
UV absorbance λmax (nm) in
CH3OH 273, 311
KOH 274-5, 311
AlCl3 273-4, 3114
Vapour pressure, kPa at 106°C 0.135
Octanol/water partition coefficient as log 1.395
Po/w
Freely soluble Alcohol, chloroform, ether
1g dissolves in 400 mL cold water
50 mL boiling water
(CDCl3) δ6.44 (d, J = 9.5 Hz, 1H, vinyl),
1
H NMR
7.32 (m, 2H, aryl), 7.50 (m, 2H aryl),
7.73 (d, J = 9.5 Hz, 1H vinyl)6
(CDCl3) δ 116.7, 116.9, 118.8, 124.5,
13
C NMR
127.9, 134.8, 143.5, 154.0, 160.86
M/Z 1466,7
2.1 Source
Coumarin has been extracted from fruits, roots, bark, stalks and branches of a wide
variety of plants including tonka bean, cassia, lavender, lovage, yellow sweet clover,
deer tongue, woodruff, cabbage, radish and spinach.
2.2 Background
Coumarin has a vanilla-like taste, smell and appearance8. The amount of coumarin in
a plant increases significantly when the plant becomes the object of external
aggression9 and different coumarin derivatives have been used to develop
classification patterns10. Coumarin is not included in the list of active principles in the
proposed EC regulation on flavourings following EFSA’s opinion that a TDI can be
set for this substance. Accordingly coumarin has been added to the register for
flavouring substances.
2.3 Usage
Cassia extract is often used in the place of true vanilla but true vanilla does not
contain coumarin. The presence or absence of coumarin is used to distinguish between
the two3.
- 17 -
2.4 Limits
Annex II of Directive 88/388/EEC11 limits the amount of coumarin from flavourings
food 2 mg/kg,
beverages 2 mg/kg
Exceptions include alcoholic beverages and certain types of caramel confectionery
where a limit of 10 mg/kg is set and chewing gum with a limit of 50 mg/kg.
Coumarin does not appear in the proposed EC flavouring regulation as a listed active
principle as mentioned previously12.
The European Committee of Experts have stated that as there is no quantitative

information on the levels of coumarin that occur in both foodstuffs and in natural
flavourings no rough estimates on the daily human intake can be established1.
However daily intakes in a German study have been estimated to be 0.02 mg/kg bw
from food13.
3.1 Metabolism
Coumarin is rapidly absorbed by the body following administration, either orally or
topically. It tends to be metabolised via one of two pathways, which can be
distinguished by the presence of particular metabolites in urine14 and appears to be
species specific14. In humans and baboons coumarin is catalysed predominately by
cytochrome P450 isoenzyme CYP2A6 to form 7-hydroxycoumarin (umbelliferone)
(see Figure 1), which is metabolised further to form glucuronide or sulphate
conjugates13. The results of in vitro studies on human live microsomes suggest that at
high coumarin concentrations the 7-hydroxycoumarin pathway becomes saturated and
then the 2-hydroxyphenylacetaldehyde pathway may be favoured15,16.
Figure 1. Coumarin and associated metabolites.
O O
O O OH
Coumarin 7-hydroxycoumarin
(umbelliferone)
O O
OH
O
HO
O
3,4 coumarin epoxide 2-hydroxyphenylacetic acid
- 18 -
4. Analysis
The most up to date method of analysing coumarin, according to the AOAC17,
involves the determination of coumarin in vanilla extract (method number 955.31).
Spectrophotometrically, the absorbance is recorded at 270 nm and 325 nm for samples
that are less than 0.4 g/100 mL, samples greater than this concentration are diluted
accordingly. This follows careful separation of the sample from the vanilla extract by
chromatographic column. Method 976.12, gives a gas chromatographic (GC) method
of analysing coumarin in wines in which a flame ionization detector and a 1.8 m x 2
mm id glass column is used. The column is packed with 10% SP-1000 Carbowax 20
M-TPA on 100-200 mesh Chromosorb W AW17.
In 1983 LGC21 found the gas chromatographic methods being used by the
International Organization of the Flavour Industry (IOFI) for the determination of
coumarin were satisfactory, with a limit of detection of 0.2 mg/kg. The column used
was 2 m x 4 mm id glass column packed with 105 Carbowax on Chromsorb W, 60/8
mesh and dibenzyl ether was used as an internal standard. It was also concluded that
the various extraction methods used were poor at extracting coumarin and the steam
distillation extraction process also gave poor results, particularly for wine samples18.

Extracting coumarin from powdered cassia using a miniaturized steam distillation
process and coupled with solvent extraction proved to be inefficient3, even using a
range of solvents including pentane, dichloromethane, chloroform, ethyl acetate and
methyl tert-butyl ether. However a liquid-liquid extraction of the first aqueous
solution from the steam distillation using pentane provided samples pure enough for
analysis3.
Methanol extraction of plant material prepared for micro high performance liquid
chromatography (HPLC)/ mass spectrometry (MS) was achieved by manual shaking
of the plant (0.15g) with 10 mL of methanol. The crude extract was concentrated (0.5
mL), then separated by thin layer chromatography (TLC) (silica gel 60; eluent
toluene-diethyl ether-acetic acid 50:45:5) and the spot with Rf = 0.6 was recovered
and again extracted with methanol. This underwent a second TLC clean-up (silica gel
60; eluent chloroform-methanol 85:15) and the spot with a Rf = 0.2 was recovered and
again extracted with methanol9. Methanol extraction of a dried Chilean herb that had
been immersed in dichloromethane also clearly showed coumarins following TLC
separation (silica gel 60; eluent dichloromethane-methanol 98:2) under UV light at
365nm19. [Comment. The authors make no reference to which coumarin derivatives
were detected.] Coumarin has also been extracted from the leaves of grape vines by
extraction with ethanol20. A single-step liquid extraction of 1 mL of a blood plasma
sample (treated with 200 µL of Tris buffer pH8) was achieved with hexane (6 mL).
Coumarins have also been extracted from sagebrush by soaking 0.5 g of the dried
plant in 5 mL of ethanol-water (70-30 v/v) for 24 hours, this was then extracted three
times with 5 mL ethanol-water (70-30 v/v) and finally filtered22.
Five compounds, including coumarin, were extracted from 10 mL samples of

alcoholic beverages. The samples were pipetted into test tubes containing 1.1 g of
Na2SO4. After neutralizing with sodium bicarbonate known amounts of the five
compounds (0, 0.2 0.5, 1.0 and 2.0 mg/kg dissolved in ethanol) and 0.5 mg/kg tetralin
(as an internal standard) were added to the samples. These samples were extracted
- 19 -
three times with diethyl ether and concentrated to 3 mL. The resulting samples were
then analysed by selected ion monitoring MS7.
Simultaneous distillation-extraction (SDE) processes make use of the equipment

designed by Likens and Nickerson in 1964, which was designed to isolate and
concentrate volatiles in one step23. It was shown that adding salt to the aqueous phase
increased the recovery of some compounds, including coumarin23.
Solid phase micro extraction (SPME) is a solvent free method of extraction in which
volatile analytes in the headspace are absorbed onto fibres (65 µm Carbowax
divinylbenzene) and are recovered by thermal desorption. This method has been used
to isolate coumarin found in tobacco products24,25 and coumarin derivatives in human
plasma, urine and breast milk26. In spirits coumarin was extracted by solid-phase
extraction on phenyl sorbent using elution with pentane-diethyl ether (7 + 3 by
volume)27.
Various methods of extraction of a medicinal plant, ‘guaco’, were compared with

regards to the coumarin containing hydroalcoholic extracts. Maceration with ethanol,
maceration under sonication with ethanol, infusion and a range of supercritical fluid
extractions (CO2:EtOH (95:5), (90:10), (85:15), and CO2:EtOH:H2O (95:2.5:2.5))
were all tested followed by HPLC-UV. It was concluded that maceration under
sonication was the most convenient method of extraction28. Extracts of the ‘guaco’
plant were obtained by both maceration/hexane extraction and exhaustive extraction;
there was no significant difference in the results obtained with the two extraction
methods29.
4.2 Quantitation
Gas-chromatography (GC) and GC/mass spectrometry (MS) are the most common
methods of analysing samples that contain coumarin, usually following SPME. In one
method, the GC was fitted with 30-m DB-5MS column and compound identification
was confirmed by retention time and coumarin had a retention time of 12.56 minutes
under the conditions used. Standards in the range of 0.0091-1.7 µg/g were analysed
and a linear reponse was obtained. The mean recovery at higher concentrations (0.22
and 1.7 µg) was 100% and 106% respectively but at the lower concentration of 0.018
the mean recovery was 159% and the authors set the limit of detection at 0.013 µg/g24.
The same method was used four years later to study the coumarin content of Indian
bidi cigarette tobacco. Small volumes of extracts (0.4 µL) of the ‘guaco’ plant were
analysed by GC coupled with a flame ionisation detector (FID). Analyses gave
recoveries of 96.5 (+/- 0.5%), a linear calibration curve (regression equation
r=0.9965) and good repeatability (average standard error following multiple injections
< 5%). The detection limit was found to be 96 ng (0.4 µL of a 0.240 mg/mL
solution)29.
Reverse-phase high performance liquid chromatography (HPLC) was used in the

early 1980’s to analysis the phenolic compounds in the leaves of grape vines. Samples
were extracted by ethanol and the stationary phase was LiChrosorb RP-18 using a
mobile phase of methanol-acetic acid (1%)-water with a methanol gradient of 5 to
80% over 40 minutes. Detection was carried out at 275 nm and the limit of detection
was 32 µg/g of coumarin 30. A similar HPLC set-up was used with varying eluents to
separate phenolics and coumarins in complex mixtures. The UV detector was set at
280 nm and coumarin was cleanly separated with a retention time of 15.88 min30.
- 20 -
An isocratic HPLC method was used to monitor the levels of coumarin in vanilla-
flavouring products. Recovery rates ranged between 99.4-101.3% over 0.500 and 1.50
mg/mL from vanilla extracts and vanilla flavourings. The minimum detectable
quantity was estimated to be 0.6 ng31.
Levels of coumarin in blood plasma were determined by measuring UV absorbance at

275 nm. This method used acetonitrile-0.01 M phosphate buffer (pH 3) (60:40) with
0.02 M tetramethylammonium chloride, the internal standard was flunitrazepam. A
consistent linear detection response was found over the range 0-10 µg/L with a
detection limit of 0.3 µg/L21. HPLC- ultraviolet diode array (UV/DAD) was used to
analyse the hydroalcoholic extracts of plants with a sensitivity of 5 µg/L. The analysis
can be completed in 6 min and good repeatability was found as the standard error of
replicate injections was less than 5%29. HPLC-UV/DAD has also been used to detect
coumarin and derivatives, such as warfarin, in biological samples, using a methanol
and acetic acid mixture (8:2) as eluent. The photodiode array detector was set at 280
nm, the correlation coefficient was r= 0.989 and the limit of detection was 0.02
µg/mL35.
Concerns that analytical methods ‘based on chromatographic techniques and

conventional detectors are inadequate to accurately analyse coumarins in complex
matrices’ were raised in 19959. To address these inaccuracies a method of analysis
was determined using a micro HPLC/MS approach with a particle beam interface for
a range of coumarin derivatives. An additional clean up step needed to be introduced
to be able to detect some of the compounds that were being analysed and some
compounds fell below the limits of detection9.
A comparison of HPLC and spectrofluorimetry was carried out in distilled beverages.

However, three coumarin derivatives analysed are all more fluorescent than coumarin
itself. The study showed that both methods were suitable for the recovery of these
compounds from alcoholic spirits, the HPLC method proved to be accurate based on
the recovery rate but less precise than spectrofluorimetry. This method proved to be
more accurate than HPLC under the chromatographic conditions used. It was thought
that improving the conditions would improve the precision of the HPLC technique,
which was easier and faster to use33.
Coumarin is a poorly fluorescent molecule that is known to convert to the highly

fluorescent 7-hydroxycoumarin in the presence of aqueous OH radicals34. Polyclonal
antibodies for 7-hydroxycoumarin have been used to measure coumarin levels
indirectly38. However as other products are formed, (3OH-, 4OH-, 5OH-, 6OH-, and
8OH-coumarin35), this characteristic probably could not be exploited with regards to
quantative detection.
5. Summary
Coumarin is not included in as an active principle in the proposed flavouring
regulation following EFSA’s opinion that a TDI can be set for this substance. The
current limit of 2 mg/kg is set for both food and beverages with two exceptions, where
the limit is higher at 10 mg/kg. Although there are a number of methods for detecting
coumarin, including GC, GC/MS and HPLC, the most sensitive method of detection
appeared to be HPLC-UV/DAD. This method had a limit of detection of 0.02 µg/mL,
with a correlation coefficient of 0.989.
- 21 -
Investigations have been carried out to try to improve the detection methods and these
have included micro HPLC/MS and spectrofluorimetry, particularly with regards to
detecting coumarin that has been extracted from complex matrices. Extraction
methods from plants are carried out by steam distillation and/or liquid extraction and
SPME has been used to extract coumarin from plasma and breast milk. However, no
specific reference to the extraction of coumarin from food matrices could be found.
6. References
(1) Opinion on Coumarin. Reports of the Scientific Committee for Food (Thirty-
sixth series). December 1994.
(2) The Merck Index - Monograph Number 0002588. Coumarin. 2004.
(3) Jayatilaka, A.; Poole, S. K.; Poole, C. F.; Chichila, T. M. P. Simultaneous
micro steam distillation/solvent extraction for the isolation of semivolatile
flavor compounds from cinnamon and their separation by series coupled-
column gas chromatography. Analytica Chimica Acta 1995, 302, 147-162.
(4) Mendez, J.; Lojo, M. I. Spectral analysis of coumarins. Microchemical
Journal 1968, 13, 506-512.
(5) Coumarin. ICSC 1105. International Programme on Chemical Safety and the
Commission of the European Communities. 1999.
(6) Shi, Z.; He, C. Efficient Functionalization of Aromatic C-H Bonds Catalyzed
by Gold (III) under Mild and Solvent-Free Conditions. Journal of Organic
Chemistry 2004, 69, 3669-3671.
(8) Sparks, L.; Bleasdell, B. D. A Comparison of Separation Techniques: Analysis
of Vanilla for Coumarin Contamination. Journal of Chemical Education 1986,
63, 638-639.
(9) Cappiello, A.; Famiglini, G.; Mangani, F.; Tirillini, B. Analysis of Coumarins
by Micro High-Performance Liquid Chromatography-Mass Spectrometry with
a Particle Beam Interface. Journal of the American Society for Mass
Spectrometry 1995, 6, 132-139.
(10) Yan, F.; Liang, Z.; Jianna, C.; Zhengtao, W.; Losahan, X. et al. Analysis of
Cnidium monnieri fruits in different regions of China. Talanta 2001, 53, 1155-
1162.
2004, WGF/002/02-rev 2.
(13) Eisenbrand, G.; Otteneder, M.; Tang, W. Synthesis of N-acetyl-S-(3-
coumarinyl)-cysteine methyl ester and HPLC analysis of urinary coumarin
metabolites. Toxicology 2003, 190, 249-258.
(14) IARC Summary and Evaluation (Coumarin). 2000, 77.
(15) Moran, E.; O'Kennedy, R.; Thornes, R. D. Analysis of coumarin and its
urinary metabolites by high-performance liquid chromatography. Journal of
Chromatography: Biomedical Applications 1987, 416, 165-169.
- 22 -
(16) Ward, E. M.; Smyth, M. R.; O'Kennedy, R.; Lunte, C. E. Application of
capillary electrophoresis with pH-mediated sample stacking to analysis of
coumarin metabolites in microsomal incubations. Journal of Pharmaceutical
and Biomedical Analysis 2003, 32, 813-822.
2000.
(19) Vogel, H.; Gonzalez, M.; Faini, F.; Razmilic, I.; Rodriguez, J. et al.
Antioxidant properties and TLC characterization of four Chilean
Haplopappus-species known as bailahuen. Journal of Ethnopharmacology
2005, 97, 97-100.
(20) Villeneuve, F.; Abravanel, G.; Moutounet, M.; Alibert, G. General scheme of
analysis of phenolic compound in plant extracts by reversed-phase high-
performance liquid chromatography. Journal of Chromatography A 1982, 234,
131-140.
(21) Lamiable, D.; Vistelle, R.; Trenque, T.; Fay, R.; Millart, H. et al. Sensitive
high-performance liquid chromatographic method for the determination of
coumarin in plasma. Journal of Chromatography: Biomedical Applications
1993, 620, 273-277.
(22) Tamma, R. V.; Miller, G. C.; Everett, R. High-performance liquid
chromatographic analysis of coumarins and flavonoids from section
tridentatae of Artemisia. Journal of Chromatography A 1985, 322, 236-239.
(23) Chaintreau, A. Simultaneous distillation-extraction: from birth to maturity-
review. Flavour and Fragrance Journal 2001, 16, 136-148.
(24) Stanfill, S. B.; Ashley, D. L. Solid phase microextraction of alkenylbenzenes
and other flavor-related compounds from tobacco for analysis by selected ion
monitoring gas chromatography-mass spectrometry. Journal of
(25) Stanfill, S. B.; Calafat, A. M.; Brown, C. R.; Polzin, G. M.; Chiang, J. M. et al.
Concentrations of nine alkenylbenzenes, coumarin, piperonal and pulegone in
Indian bidi cigarette tobacco. Food and Chemical Toxicology 2003, 41, 303-
317.
(26) de Vries, J. X.; Schmitz-Kummer, E. Determination of the coumarin
anticoagulant phenprocoumon and metabolites in human plasma, urine and
breast milk by high-performance liquid chromatography after solid-phase
extraction. Journal of Chromatography B: Biomedical Sciences and
Applications 1994, 655, 63-71.
Food Constituents .2. Asarone, Quinine, Coumarin, and Quassine in Spirits.
413 (German).
(28) Celeghini, R. M. S.; Vilegas, J. H. Y.; Lancas, F. M. Extraction and
Quantitative HPLC Analysis of Coumarin in Hydroalcoholic Extracts of
Mikania glomerata Spreng. ('guaco') Leaves. Journal of the Brazilian
Chemical Society 2001, 12.
(29) Vilegas, J. H. Y.; Marchi, E. d.; Lancas, F. M. Determination of Coumarin and
Kaurenoic Acid in Mikania glomerata ('Guaco') Leaves by Capillary Gas
Chromatography. Phytochemistry Analysis 1997, 8, 74-77.
- 23 -
(30) Casteele, K. V.; Geiger, H.; Sumere, C. F. V. Separation of phenolics (benzoic
acids, cinnamic acids, phenylacetic acids, quinic acid esters, benzaldehydes
and acteophenones, miscellaneous phenolics) and coumarins by reversed-
phase high performance liquid chromatography. Journal of Chromatography
1983, 258, 111-124.
(31) Thompson, R. D.; Hoffmann, T. J. Determination of coumarin as an adulterant
in vanilla flavoring products by high-performance liquid chromatography.
Journal of Chromatography A 1988, 438, 369-382.
(32) Park, S. W.; Seo, B.; Kim, E.; Kim, D.; Paeng, M. S. et al. Purification and
Determination Procedure of Coumarin Derivatives. Journal of Forensic
Science 1996, 41, 685-688.
(33) Fernandez Izquierdo, M. E.; Quesada Granados, J.; Villalon Mir, M.; Lopez
Martinez, M. C. Comparison of methods for determining coumarins in
distilled beverages. Food Chemistry 2000, 70, 251-258.
(34) Louit, G.; Foley, S.; Cabillic, J.; Coffigny, H.; Taran, F. et al. The reaction of
coumarin with the OH radical revisited: hydroxylation product analysis
determined by fluorescence and chromatography. Radiation Physics and
Chemistry 2005, 72, 119-124.
(35) Egan, D. A.; Deasy, B.; Dempsey, E.; Smyth, M. R.; Okennedy, R. Antibody-
Based Approaches to Coumarin Analysis. Journal of Cancer Research and
Clinical Oncology 1994, 120, S28-S29.
- 24 -
Estragole

1-allyl-4-methoxy-benzene (IUPAC name)
1-methoxy-4-(2-propenyl) benzene
4-allyl-anisole
4-methoxy-1-(2-propenyl) benzene
Chavicyl methyl ether
Esdragol
Esdragole
Esdragon
Estragol
Estragon
Isoanethole
Methyl chavicol
p-allylanisole
p-methoxy-allylbezene
Tarragon
1.2 Numbers
CAS Number 140-67-0
FEMA Number 2411
C of E 184
EINECS 205-427-8
Molecular Formula C10H12O

Elemental Composition C 81.04 %, H 8.16 %, O 10.80 %
Class Isocyclic, substituted allylbenzene

A summary of chemical and physical properties of pure estragole is given in table 2.
- 25 -
Physical appearance Colourless liquid at room temperature

with a distinct anise odour
Boiling point 215-216°
Density 0.965 g/L
Soluble in Alcohol and chloroform1
In water Forms azeotropic mixtures1
Insoluble in Water
IR vmax 3030, 1620, 15002
m/z 149 [M+1]+, 123 [M + 1 – 28]+, 205 [M+
57]+3
δ ppm: 7.06 (2H, d, aromatic, J = 8.6 Hz),
1
H NMR
6.79 (2H, d, aromatic, J = 8.6 Hz), 6.01 –
5.81 (1H, CH2CH=CH2, m), 5.06 – 4.97
(2H, m, CH2CH=CH2), 3.70 (3H, s,
OCH3), 3.28 (2H, d, CH2CH=CH2, J =
6.6Hz )2
δ ppm:157.9 (C1), 137.8 (C2’), 131.9
13
C NMR
(C4), 129.3(C3/5), 115.2 (C3’), 113.7
(C2/6), 55.0 (C methyl), 39.2 (C1’)2.
In biosynthesized estragole, the carbon atoms adjacent to the methoxy group have a
relative high abundance of deuterium atoms, which is caused by a natural isotope
hydrogen shift that occurs during the hydroxylation of the benzene ring4.
2. Background, Usage and Limits
2.1 Source
Estragole is a constituent of the essential oil found in many herbs that are regularly
used in food products, including those shown in Table 3.
Table 3. A list of commonly used herbs and the percentage of estragole found in their
essential oils.
Plant oil % of estragole found in essential oil

Anis vert 15
Bay leaves 326
Star anise 5-65
Sweet basil 20-435
Sweet fennel 5-205
Tarragon 60-755
2.2 Background
In the US, estragole was given Generally Regarded As Safe (GRAS) status in 1965,
this was changed in 1999 when estragole was added to the list of chemicals known to
cause cancer6. A study that investigated the stability of estragole, in aqueous
solutions, found that it remained stable for more than twenty days by measuring the
UV absorbance at 279 nm7.
- 26 -
2.3 Usage
The essential oils of many plants containing estragole are used in the flavouring of
food products, perfumes, soaps and detergents6.
2.4 Limits
Estragole was not listed in Annex II of the Council Directive 88/388/EEC8 as a
compound for concern, despite some proposals for limits in estragole content in foods.
In the proposed EC flavouring regulation estragole will be limited in cheese and fish
and fish products to 50 mg/kg. This limit will also be applied to processed fruits,
vegetables (including mushrooms, fungi, roots, tubers, pulses and legumes), nuts and
seeds and alcoholic beverages. A 10 mg/kg limit is to be set for non-alcoholic
beverages.
The evaluation of estragole intake levels is generally poor due to the lack of data on
the concentrations found in foodstuffs. That said, the intake average has been
estimated to be 4.3 mg/day (European data), with an upper percentile (97.5%) of 8.7
mg/day. However, it is not possible to estimate the relative contributions of total
exposure from food accurately9.
3.1 Metabolism
Estragole belongs to the class of compounds called alk-2-enylbenzenes, which also
includes safrole, isosafrole and methyleugenol, and all these compounds are
metabolised in a similar manner. There are three known metabolic pathways (shown
in Scheme 1), O-demethylation, 1’-hydroxylation and epoxidation; epoxidation is
considered to be the least significant of the three10. The extent that the other pathways
predominate appears to be dose dependant. At lower doses O-demethylation is
predominant but as the dose increases the extent of O-demethylation decreases and 1’-
hydroxylation inceases10. The metabolites formed are electrophiles that have the
capability of reacting with DNA and other cellular macromolecules11.
4. Analysis
4.1 Extraction methods

The most common method of extraction of essential oils from plants containing
estragole is by steam distillation. By coupling this with liquid-liquid continuous
extraction (using dichloromethane) the levels of estragole in basil and thyme were
found to be 2.03 mg/g and 0.011 mg/g respectively12.
Simultaneous distillation-extraction with a Likens-Nickerson apparatus and

dichloromethane was used for the extraction of food products including bologna,
Vienna sausage and cola tasting drinks. The solid samples (150-500 g) were finely
ground then homogenised and liquid samples (between 100-200 mL), were placed in
the Likens-Nikerson apparatus for 1.5 hours with 50 mL dichloromethane at 50°.
- 27 -
OMe OMe OH
epoxidation O-demethylation
Hydroxy-
O allyl-benzene
Estragole
Estragole 2',3'-oxide
1'-hydroxylation
OMe
OMe
OMe
O3SO
1' sulphooxyestragole HO
1'hydroxyestragole-
2',3'-oxide O
HO
1',hydroxyestragole
OMe
carbonium ion
DNA adducts
Scheme 1. A summary of the metabolism of estragole6,10,11.
Samples were held at 90° under agitation and the cold finger was maintained at 5°.
The extracts were dried using anhydrous sodium sulphate and concentrated under
vacuum to 2-5 mL. These samples were analysed by gas chromatography (GC)
coupled to ion trap mass spectrometry (MS), where the limit of detection was 10
ng/mL of injected sample13.
volatile analytes in the headspace are absorbed onto fibres (65 µm Carbowax
divinylbenzene) and are recovered by thermal desorption. This method has been used
to isolate compounds, including estragole, in various tobacco products14,15. Estragole
was also found to be present in the leaves of avocado following SPME, but it was not
present in avocado puree. The extracted volatile compounds were analysed by GC/MS
for identification however these measurements were not quantative16.
- 28 -
Super critical carbon dioxide extraction of volatile compounds from the leaves of
Croton zehntneri Pax et Hoff showed that the levels of recovered estragole decreased
with increasing extraction time (0-50 min gave 2.1 % of total, whereas 260-300 min
gave 0.64 %). This was compared to steam distillation which gave a higher value of
2.59 % of the total extract being estragole17. Supercritical CO2 extraction of fennel
resulted in essential oil that had comparable composition to those extracted by steam
distillation18.
4.2 Quantitation
The SDE extracts of food products were analysed by GC with a flame ionization
detector, with electronic pressure control and split/splitless inlet system, containing 2
mm id silcosteel liner. A fused silica capillary column (30 m x 0.25 mm id with 0.25
µm film) of 5% diphenyl/95% dimethylpolysiloxane was used and 3-
methylphenylacetate was used as an internal standard to calibrate GC retention times.
Percentage recovery rates were stated as being ‘essentially 100%’ and estragole was
found (as an average) in the following food matrices:
tomato sauce 0.81 mg/kg

pesto sauce 5.26 mg/kg
fresh basil 13.17 mg/kg,
estragole was found not to be present in cola tasting soft drinks, bologna sausage and
Vienna sausage13. Following SPME, the tobacco derived samples were analysed by
GC/MS. The retention time in this given system for estragole was 6.71 min and the
mean recovery was between 100-116%. The detection limit was given as being
0.0029 µg/g14. The same method was used four years later to study the estragole
content of Indian bidi cigarette tobacco, when the limit of detection was given as
0.027 µg/g with a correlation coefficient of 0.925 over a concentration range of 0.022
– 7.6 µg15.
Chemotypes are the range of compounds that make up the characteristic flavour
and/or odour of plants. Basil chemotypes were identified by vibrational spectroscopy
methods19. Near infrared, attenuated total reflectance IR and NIR-FT Raman
spectroscopy measurements were able to determine many compounds in the essential
oil of basil, including estragole in the range of 0.55 - 98.09 g/100g19.
Stable isotope ratios have been used to authenticate the source of food ingredients20.
There are well defined patterns in the ratios of stable isotopes from a given
environment and these patterns can be used to determine between synthetic and
natural sources. On-line capillary gas chromatography isotope ratio MS was used in
the combustion and pyrolysis modes to determine δ13C values for estragole. These
actually showed that there was very little difference in the ratios of 12C and 13C in
samples from synthetic or natural sources. However there were notable differences in
the δ2H and δ18O data between the two sources of estragole21.
5. Summary
In the proposed EC flavouring regulation estragole is to be limited to a variety of
products at 50 mg/kg, and to non alcoholic beverages at 10 mg/kg.
SFE studies have shown that the extraction of estragole is not significantly improved
by this method compared to steam distillation of plant material. Although SPME has
- 29 -
been used to extract estragole from the avocado plant, the Likens-Nickerson apparatus
with dichloromethane has been reported to be have been used to extract estragole
from whole food meat based products and non-alcoholic beverages.
Quantitation has been carried out by GC, either coupled with MS for tobacco derived
samples or FID for food derived samples. No reference could be found for any other
methods of quantitation. The GC-FID method produced good recovery rates and
although no limit of detection was given, values of estragole found ranged from 0.81
mg/kg to 13.17 mg/kg, which is suitable for detecting the proposed limits.
6. References
(1) The Merck Index - Monograph Number 0003740. Estragole. 2004.
(2) Gomes, P.; Gosmini, C.; Perichon, J. New Chemical Cross-Coupling between
Aryl Halides and Allylic Acetates Using a Cobalt Catalyst. Organic Letters
2003, 5, 1043-1045.
(3) Lange, G.; Schultze, W. Some Aspects of Isobutane and Ammonia Chemical
Ionization for the Analysis of Volatile Esters and Phenylpropanoids Occurring
in Essential Oils. Organic Mass Spectrometry 1992, 27, 481-488.
(4) Manitto, P.; Monti, D.; Speranza, G. Evidence for an NIH shift as the origin of
the apparently anomalous distribution of deuterium in estragole from
Artemisia dracunculus. Journal of Natural Products 2000, 63, 713-715.
(5) Rietjens, I. M. C. M.; Martena, M. J.; Boersma, M. G.; Spiegenlenburg, W.;
Alink, G. M. Molecular Mechanism of Toxicity of Important Food-borne
Phytotoxins. Molecular Nutrition and Food Research 2005, 49, 131-158.
(6) McDonald, T. A. Evidence on the Carcinogenicity of Estragole; Reproductive
and Cancer Hazard Assessment Section Office of Environmental Health
Hazard Assessment. California Environmental Protection Agency., 1999.
(7) Samuelly, M.; Keup, W. Studies on aqueous solutions of essential oils.
Analytica Chimica Acta 1970, 51, 109-116.
the European Communities 1988, 15.7.1988.
(9) Opinion of the Scientific Committee on Food on Estragole (1-Allyl-4-
methoxybezene). SCF/CS/FLAV/FLAVOUR/6 ADD2 Final. 2001.
(10) Smith, R. L.; Adams, T. B.; Doll, J.; Freon, V. J.; Goodman, J. I. et al. Safety
assessment of allylalkoxybenzene derivatives used as flavouring substances --
methyl eugenol and estragole. Food and Chemical Toxicology 2002, 40, 851-
870.
(11) Phillips, D. H.; Miller, J. A.; Miller, E. C.; Adams, B. Structures of the DNA
Adducts Formed in Mouse Liver after Administration of the Proximate
Hepatocarcinogen 1'-Hydroyestragole. Cancer Research 1981, 41, 176-186.
(12) Lee, S.-J.; Umano, K.; Shibamoto, T.; Lee, K.-G. Identification of volatile
components in basil (Ocimum basilicum L.) and thyme leaves (Thymus
vulgaris L.) and their antioxidant properties. Food Chemistry 2005, 91, 131-
137.
(13) Siano, F.; Ghizzoni, C.; Gionfriddo, F.; Colombo, E.; Servillo, L. et al.
Determination of estragole, safrole and eugenol methyl ether in food products.
Food Chemistry 2003, 81, 469-475.
- 30 -
317.
(16) Lopez, M. G.; Guzman, G. R.; Dorantes, A. L. Solid-phase microextraction
and gas chromatography-mass spectrometry of volatile compounds from
avocado puree after microwave processing. Journal of Chromatography A
2004, 1036, 87-90.
(17) Sousa, E. M. B. D.; Martinez, J.; Chiavone-Filho, O.; Rosa, P. T. V.;
Domingos, T. et al. Extraction of volatile oil from Croton zehntneri Pax et
Hoff with pressurized CO2: solubility, composition and kinetics. Journal of
Food Engineering, In Press, Corrected Proof.
(18) Ehlers, D.; Farber, J.; Martin, A.; Quirin, K. W.; Gerard, D. Analysis of
essential fennel oils - Comparison of CO2 extracts and steam-distilled oils.
Deutsche Lebensmittel-Rundschau 2000, 96, 330-335.
(19) Schulz, H.; Schrader, B.; Quilitzsch, R.; Pfeffer, S.; Kruger, H. Rapid
classification of basil chemotypes by various vibrational spectroscopy
methods. Journal of Agricultural and Food Chemistry 2003, 51, 2475-2481.
(20) Rossmann, A. Determination of Stable Isotope Ratios in Food Analysis. Food
Reviews International 2001, 17, 347-381.
(21) Ruff, C.; Hor, K.; Weckerle, B.; Konig, T.; Schreier, P. Authenticity
assessment of estragole and methyl eugenol by on- line gas chromatography-
isotope ratio mass spectrometry. Journal of Agricultural and Food Chemistry
2002, 50, 1028-1031.
- 31 -
Hydrocyanic Acid

Cyanwasserstoff
Formonitrile
Hydrogen cyanide (IUPAC name)
Methanenitrile
Prussic acid
1.2 Numbers
CAS Number 74-90-8, 6914-07-4
Beilstein registry number 1718793
EINECS Number 200-821-6
H C N
Molecular Formula HCN

Elemental Composition C 44.44 %, H 3.73%, N 51.83 %
Class of Compound acyclic

As an aqueous solution HCN is a colourless to pale blue liquid, with a faint bitter,
almond-like odour.
A summary of chemical and physical properties of HCN is given in Table 2.
Acid strength weak (pKa 9.2)

Miscible in water, alcohol and ether
Soluble in ethanol
Boiling point 25.6°
Melting point -13.4°
Density (liquid) 0.699
Density (gas) 0.901
Log Octanol/water partition Coefficient 1.07
- 32 -
2.1 Source
Some plants synthesise cyanogenic glycosides, which decompose in the presence of
hydrolytic enzymes (β-glycosidases), to liberate free cyanide. One example of these
glycosides is amygdalin found in apricots, peaches, cherries, pears, apples and
almonds (3-5% of bitter almonds is amygdalin). The roots of the cassava, a staple in
many African diets, can contain as much as 500 mg/kg, measured as HCN, on a fresh
weight bias1.
2.2 Background
Hydrocyanic acid is hydrogen cyanide dissolved in water. Cassava contains large
amounts of the cyanogenic glycosides, linamarin and lotaustralin which are
hydrolysed by endogenous linamerase2. As long ago as 1891 there was a colorimetric
test published in The Lancet for measuring HCN in the blood, such was the concern of
cyanide poisoning; 1 mL of blood was diluted and a 1% solution of potassium
ferrocyanide was added, no colour change indicated a presence of HCN3.
The HCN contents of two cassava products, ‘gari’ and ‘lafun’, were shown to
decrease during cooking processes; soaking in water was responsible for a decrease of
up to 75%4. The HCN content of maize also decreased by 35% following roasting5
and the HCN content of mushrooms decreases during the cooking process6. It has also
been noted that in gari, the HCN content shows a positive correlation with sugar
content but a poor correlation with the starch content2.
2.3 Usage
No known food uses.
2.4 Limits
Annex II of Directive 88/388/EEC7 limits the amount of HCN from flavourings and
other food ingredients to:
food 1.0 mg/kg,
beverages 1.0 mg/kg,
the exceptions are alcoholic beverages (1 mg/% volume of alcohol), 5 mg/kg in
canned stone fruit and 50 mg/kg in nougat, marzipan or its substitutes or similar
products10.
In the proposed EC flavouring regulation the limits on nougat, marzipan and similar
products and in canned stone fruit will be retained as above and alcoholic beverages
will be restricted to 35 mg/kg8.
3.1 Metabolism
Cyanogenic glycosides can liberate HCN by the action of hydrolytic β-glycosidases.
Hydrolysis liberates the corresponding aglycone, this further breaks down non-
enzymically to give HCN and the respective carbonyls9.
- 33 -
4. Analysis
The most current methods from the AOAC10 of HCN in beans (method number
915.03) can be conducted in one of two ways; the first, acid titration method, involves
the maceration of the sample and steam distillation, collecting the distillate in 20 mL
0.002 M AgNO3 with HNO3. 150 mL of the distillate (with 0.02 M KSCN) is then
titrated with excess AgNO3 using a ferric alum indicator. 1 mL 0.02 M AgNO3 = 0.54
mg HCN. The second method involves alkaline titration, the extraction is carried out
as above except that the distillate is collected in a NaOH solution (0.5 g in 20 mL
water). 8 mL of 6 M NH4OH and 2 mL 5% KI solution is added to the sample and
titrated with 0.02 M AgNO3. 1 mL 0.02 M AgNO3 = 1.08 g HCN13. Another AOAC
method is for measuring HCN in almond extract (method number 920.144), the
qualitative test suggests adding several drops of freshly prepared 3% FeSO4⋅7H2O
solution to a sample, along with one drop 1% FeCl3⋅6H2O solution. After mixing
thoroughly 10% NaOH solution is added dropwise until no further precipitate is
formed, H2SO4 is added to dissolve the precipitate, if HCN is present a blue colour or
suspension forms. A quantitative method takes 25 mL of sample, to which 5 mL of
freshly prepared Mg(OH)2 is added, this is titrated with 0.1 M AgNO3, with K2CrO4
as an indicator, 1 mL 0.1 M AgNO3 = 0.0027 g HCN10.
In 1983 LGC11, found the then International Organization of the Flavour Industry
(IOFI) methods for the detection of HCN to be inconsistent and they recommended a
colourimetric method based on p-phenylenediamine. The method uses a homogenised
sample (which included apricot brandy, orange juice and tinned peaches), which is
mixed with 100 mL water, 100 mL pH 7 buffer and 10 mL of a suspension made from
10 sweet almonds that have been ground and suspended in 10 mL water to release the
enzymes. 50 mL of 0.5 M sodium hydroxide is added to two collecting vessels and
after 1 hour 60 mL 0.1 M orthophosphoric acid is added. The whole apparatus is left
for 18 hours under nitrogen after which the two collecting vessels are combined. 20
mL of the resulting sample is taken and 0.5 mL concentrated orthophosphoric acid is
added followed by 0.5 mL bromine water. Finally 0.5 mL arsenious acid and 20 mL
p-phenylenediamine/pyridine reagent is added and the absorbance at 515 nm is
measured against a water blank. The cyanide concentration is determined by
calculation against a calibration curve. The limit of detection is given as 0.1 mg/kg
and the recovery rate was between 96.4 and 100.8 %11.
During the 1980’s the cyanide content of plants was usually measured using alkaline
picrate based methods. Cyanide undergoes an irreversible reaction with picrate to
form a red complex that absorbs strongly at 510 nm, this complex can then undergo a
further reversible reaction that results in a complex that does not absorb at 510 nm.
The plant material underwent steam distillation or air aspiration of HCN into alkaline
picrate and the resulting colour was measured spectrophotometrically. The
disadvantages of this system include incomplete recovery of cyanide and interference
from other compounds. To reduce interference a variation was developed in which a
filter paper strip impregnated with picrate was suspended above the sample and the
appearance and intensity of the red complex gave a qualitative detection of cyanide.
In the cyanide-picrate system the results are susceptible to variation due to the pH of
the picrate and the sample, the total picrate present and the time and temperature of
the reaction. The timing of the reaction is given as significant as the irreversible
formation of the red complex is fast, but the formation of the complex that does not
absorb at 510 nm is slow. For the strip test an important consideration is the amount
of picrate on the strip and this could be problematic when comparing results between
laboratories12. This test proved useful for farmers in detecting the presence of HCN in
- 34 -
green fodder before being fed to livestock13. Improvements in computer imagery,
allowed the spectral intensity of picrate paper, in the presence of HCN, to give
standards upon which the HCN concentrations in white clover could be determined14.
A picric acid solution (5 g picric acid and 25 g sodium carbonate) was combined with
800 mL distilled water, and this was to impregnate ‘Whatman’ filter paper, which
were cut in to strips. These strips were exposed to the plant samples, which had been
lightly crushed, along with 3 drops of toluene and allowed to develop for 24 hours at
25°C. The strips were scanned and the colour spectral intensity recorded; the intensity
was compared with calibration curves that had been generated using a range of
standards (0-70 µg CN/100 µL)14.
In 1989 a study of the HCN content in gari flour (a cassava product) compared the
spectrophotometric alkaline picrate method 15 with the AOAC alkaline titration
method (1970). The results were analysed statistically (standard error, t-test and
analysis of variance) and it was seen that there was a significant difference in the
amount of measured HCN between the two methods. The alkaline titration method
gave higher HCN values than the alkaline picrate spectrophotometric method but the
level of interference from non cyanide material in the spectrophotometric method was
lower than that in the titration method. However it is deemed possible to have
incomplete take up by the alkaline picrate of the emitted HCN or to have incomplete
elution of the reaction product in the alkaline picrate method15.
The AOAC (1975) method was used to measure HCN in Nigerian vegetables; these
vegetables were dried, ground into a powder and were shown to have between 4-23
mg/100g HCN content, no recovery rates or limits of detection were given16. The
same method was used to measure HCN in three varieties of Piper species, between
80.6-85.8 mg/100 g of HCN in plant material were reported but again no information
on limits of detection were given nor any investigation on recovery rates17.
HCN has been recovered from complex food matrices18. In 1998 popular Nigerian
soup meals were made, using locally purchased ingredients and prepared as they
would be in the average kitchen. These meals were homogenized, dried and the HCN
concentration determined via the alkaline titration method of AOAC (1984). Although
concentrations were measured at between 0.17-0.42 mg/100 g dry wt, no limits of
detection nor recovery rates were given18.
HCN and cyanide react in the presence of chloramine T to form chlorocyanide, which
in turn produces a blue colour in the presence of a pyridine-pyrazolone reagent. This
can be measured spectrophotometrically at 620 nm and this method was used to
determine the HCN content of mushrooms1. The freshly collected samples were
placed in a stream of nitrogen for 4 hours with 0.1 M sodium hydroxide solution. The
samples were then adjusted to pH 6.8 with phosphate buffer and reacted with
chloramine T, to give chlorocyanide, before the addition of the pyridine-pyrazolone
reagent. The amounts of HCN found in the various mushroom species ranged from
0.5 to 268 mg/kg but no limits of detection were given nor were recovery rates
recorded1. A comprehensive evaluation of a method based on chloramine T for
detecting HCN in spirit drinks was carried out over four years and included 60
laboratories. This method involves the formation of chlorocyanide, followed by the
addition of a barbituric acid – pyridine solution which gives a red-violet colour that is
measured at 570 nm. Although no details of the study were given, as regards to
detection limits and recovery rates, the statistical analyses of the results of the main
- 35 -
trial were ‘excellent’ with ‘high precision for all matrices’. This method is being
considered for adoption into EU legislation19.
Cigarette smoke was drawn through a dilute sodium hydroxide solution, to which
picric acid was added. Following the addition of 2,6-dinitro-5-hydroxy-4-
hydroxylamino-1,3-dicyclobenzene the absorbance of the red solution was recorded at
505 nm9. An improvement of this method was published. The smokes were drawn
through dilute sodium carbonate solution with picric acid and resorcinol, and the
cyanides formed an indophenol compound that absorbed at 481 nm. The limit of
detection was given as 0.01 µg/mL CN- in the aqueous sample20.
5. Summary
In the proposed EC flavouring regulation hydrocyanic acid is to be restricted in
canned stone fruit to 5 mg/kg, in nougat, marzipan and similar products to 50 mg/kg
and in alcoholic beverages to 35 mg/kg.
For the HCN content in alcoholic drinks a comprehensive study was carried out,
which used a method based on chloramine T, and although the authors gave no details
on the limits of detection, the paper suggested that the method was suitable for the use
of detecting HCN in all spirit drinks. Although references were found that measured
HCN in cassava products and in food matrices (e.g. soups), none could be found that
related to the extraction from nougat, marzipan or similar. The only reference found
that related to canned stone fruit was the investigation carried out by LGC which used
a colourmetric method based on p-phenylenediamine. They gave the limit of detection
as 0.1 mg/kg, which is more than sensitive enough for the proposed limits.
6. References
(1) Stijve, T.; de Meijer, A. A. R. Hydrocyanic acid in mushrooms, with special
reference to wild- growing and cultivated edible species. Deutsche
Lebensmittel-Rundschau 1999, 95, 366-373.
(2) Maduagwu, E. N.; Fafunso, M. Particle size distribution of hydrocyanic acid
in gari, a cassava-based product. Toxicology Letters 1980, 7, 171-174.
(3) Anon The Detection of Hydrocyanic Acid in the Blood. The Lancet 1891, 138,
1297.
(4) Ketiku, A. O.; Akinyele, I. O.; Keshinro, O. O.; Akinnawo, O. O. Changes in
the hydrocyanic acid concentration during traditional processing of cassava
into 'gari' and 'lafun'. Food Chemistry 1978, 3, 221-228.
(5) Ayatse, J. O.; Eka, O. U.; Ifon, E. T. Chemical evaluation of the effect of
roasting on the nutritive value of maize (Zea mays, Linn.). Food Chemistry
1983, 12, 135-147.
(6) Ekanem, E. O.; Ubengama, V. S. Chemical composition, anti-nutritional
factors and shelf-life of oyster mushroom (Pleurotus ostreatus). Journal of
Food Science and Technology-Mysore 2002, 39, 635-638.
(8) Proposal for a Regulation of the European Parliament and of the Council on
flavourings and certain food ingredients with flavouring properties for use in
and on foods. 2004.
- 36 -
(9) Minutes' statement of the Scientific Committee on Food on precursors of
hydrocyanic acid and other food ingredients with flavouring properties - 137th
plenary meeting. 4th April 2003. SCF/FLAV/FLAVOUR/48 Final. 2003.
2000.
(12) Williams, H. J.; Edwards, T. G. Estimation of Cyanide with Alkaline Picrate.
Journal of Science of Food Agriculture 1980, 31, 15-22.
(13) Srinivasan, E. V. R.; Krishnamoorthy, A.; Anbumani, S. P. Detection of
Hydrocyanic Acid (HCN) in Green Fodder. Indian Journal of Animal Sciences
1994, 64, 1303-1304.
(14) Ayres, J. F.; Murison, R. D.; Turner, A. D.; Harden, S. A rapid semi-
quantitative procedure for screening hydrocyanic acid in white clover
(Trifolium repens L.). Australian Journal of Experimental Agriculture 2001,
41, 515-521.
(15) Ukhun, M. E.; Nkwocha, F. O. The hydrocyanic acid (HCN) content of garri
flour made from cassava (Manihot spp.) and the influence of length of
fermentation and location of source. Food Chemistry 1989, 33, 107-113.
(16) Udosen, E. O.; Ukpanah, U. M. The Toxicants and Phosphorus-Content of
Some Nigerian Vegetables. Plant Foods for Human Nutrition 1993, 44, 285-
289.
(17) Isong, E. U.; Essien, I. B. Nutrient and antinutrient composition of three
varieties of Piper species. Plant Foods for Human Nutrition 1996, 49, 133-
137.
(18) Akpanabiatu, M. I.; Bassey, N. B.; Udosen, E. O.; Eyong, E. U. Evaluation of
Some Minerals and Toxicants in Some Nigerian Soup Meals. Journal of Food
Composition and Analysis 1998, 11, 292-297.
(19) Brereton, P.; Hasnip, S.; Bertrand, A.; Wittkowski, R.; Guillou, C. Analytical
methods for the determination of spirit drinks. TrAC Trends in Analytical
Chemistry 2003, 22, 19-25.
(20) Drochioiu, G.; Pui, A.; Danac, R.; Basu, C.; Murariu, M. Improved
spectrophotometric assay of cyanide with picric acid. Revue Roumaine De
Chimie 2003, 48, 601-606.
- 37 -
Isosafrole

1,2-(methylenedioxy)-4-(1’-propenyl)benzene
3,4-methylenedioxy-1-propenyl benzene
3,4-(methylenedioxy)propenylbenzene
4-propenyl-1,2-methylenedioxybenzene
5(1’-propenyl)-1,3-benzodioxide
5-(1-propenyl)-1,3-benzodioxole
5-prop-1-enyl-1,3-benzodioxole
5-prop-1-enylbenzo[1,3]dioxole (IUPAC name)
isosafrol (cis and trans)
1.2 Numbers
CAS numbers 120-58-1, 4043-71-4 ((E)-isosafrole), 17627-76-8 ((Z)-isosafrole).
Beilstein Registry numbers 82640, 82642 ((E)-isosafrole), 82641 ((Z)-isosafrole).
O O
O O
(E)-isosafrole (Z)-isosafrole

Class heterocyclic, substituted allylbenzene

A summary of chemical and physical properties of pure isosafrole is given in table 2.
- 38 -
Physical appearance A liquid at room temperature with a

distinct anise odour
Melting point E: 8.2°, Z: -21.5°1
Boiling point E isomer bp760 253°, bp100 179.5°, bp20 135.6°,
bp3.4 85-86°.1
Boiling point Z isomer bp3.5 77-79°
Density 1.12
Miscible with alcohol, ether and benzene1
Soluble in 8 parts of 90% alcohol1
UV absorbance (90% alcohol) E: 305, 267, 259.5 nm, Z:
296.5, 259 nm.1
m/z 1622
IR (liquid film) Z isomer vmax 1500 (aryl-H), 1450, 1260, 1040 (C-
O), 940 and 820 cm-1 3
(CDCl3) δ 6.8 (3 H, m, aryl Hs), 6.35
1
H NMR Z isomer
(1H, dq, JHMe 2, JHH 11 Hz, CH=CHMe),
5.95 (2 H, s, OCH2), 5.7 (1 H, dq, JHMe 7,
JHH 11 Hz, CHMe), 1.9 (3 H, dd, JHMe 2, 7
Hz, Me)3.
The differences in the fragmentation patterns that occur during isobutane and
ammonia chemical ionisation have been studied. These patterns allow for the
differentiation between safrole and isosafrole that have identical molecular weight,
but not between the isomers of isosafrole. Isosafrole exhibits the protonated
molecular ion, and the adduct ion [M + 57]+. [M + 1 – 28]+ is also produced at
significantly higher abundance than for safrole (abundance 100 for isosafrole
compared to 18 for safrole) 4.
2.1 Source
The Committee of Experts on Flavouring Substances (CEFS 1999)5 stated that
isosafrole is not contained in any natural source material used by the flavouring
industry. Isosafrole was found not to be a component of sassafras teas that were
extracted by supercritical fluid (CO2)2.
Isosafrole may be present in ylang-ylang oil6, which is used as a scent and a

flavouring.
2.2 Background
In 2002 it was concluded that isosafrole does not occur in any relevant source
materials for flavourings and was present as an artefact of the extraction process7.
However, this was revised in 2003, and it was considered that isosafrole only occurs
sporadically and always with safrole8..
2.3 Usage
Isosafrole is used to enhance the perfumes in soaps, and small quantities are used in
the US, together with methyl salicylate, in root beers and sarsaparilla flavours1.
- 39 -
2.4 Limits
Annex II of Directive 88/388/EEC limits the amount of isosafrole from flavourings
food 1 mg/kg,
beverages 1 mg/kg.
The exceptions are alcoholic beverages of <25% volume 2 mg/kg, alcoholic

beverages >25% volume 5 mg/kg and 15 mg/kg in food containing mace and
nutmeg9. These limits are the same as for safrole. Isosafrole does not appear in Annex
II of the proposed EC flavouring regulation.
3.1 Metabolism
In rats 89% of the dose of isosafrole administered was excreted as a metabolite, in
urine, within 72 hours7. Isosafrole has been shown to undergo metabolism and forms
the metabolites summarised in Scheme 1.
HO
HO O o
epoxidation
HO
HO O
epoxide-diol
1,2-dihydroxy-4-(1'-propenyl)benzene isosafrole
allylic hydroxylation
at the 3' position
O
chemical
O rearrangement
OH
O
OH
O
3'-hydroxyisosafrole 1'-hydroxyisosafrole
Scheme 1. A summary of the known derivatives formed during isosafrole

metabolism7,10,11.
4. Analysis
In 1983, LGC assessed the then current International Organization of the Flavour
Industry (IOFI) methods for the analysis of isosafrole in food and drink samples12.
Liquid and solid samples were added to enough water after the addition of triethylene
glycol to facilitate stirring and steam distilled. The distillate was collected and n-
pentane added and then the sample was extracted with the same solvent. This was
dried and reduced in volume before being analysed by gas chromatography (GC).
Providing that m-tolyl acetate was added to the distillate as an internal standard, the
extraction methods were deemed to be satisfactory. The GC method, 2.5 m x 4 mm id
glass packed with 4% Carbowax 20 M on Chromsorb G, 80/100 mesh, was also
considered to be good, with a detection limit of 10 µg12.
- 40 -
The official AOAC method for determining isosafrole in non-alcoholic drinks is given
by method number 969.1313. The sample undergoes steam distillation and is detected
with a GC fitted with flame ionization detector, 1.8 m x 4 mm glass column packed
with 15% polypropylene glycol adipate on 60-80 mesh Gas-Chrom P. The working
internal standard is m-tolyl acetate13.
4.1 Extraction
In meat based products, isosafrole was extracted for 5 hours by Likens-Nikerson
extraction using hexane (30 mL) for 100g of the food matrix, then dried with sodium
sulphate and analysed by high performance liquid chromatography (HPLC). UV
maxima in water/CH3CN (55:45) was given as, E isomer 211, 262 and 303; the Z
isomer 211, 259 and 298-30014.
4.2 Quantitation
The UV absorbance at 261 nm was used in an investigation in to the stability of
isosafrole, over the course of 30 and 180 days, in aqueous solutions. No change was
observed 15.
Larry16 proposed a GC method of detection for safrole and related compounds and
although the method proved successful for methyl salicylate it was not suitable for
isosafrole as the average recovery of spiked samples proved to range from 71 – 181%
across a collaborative study involving nine different laboratories16. The method was
revised and this was subjected to another collaborative study. It had a more successful
recovery range (between 77 and 80%) and was adopted as official first action by the
AOAC as a semi-quantitative method for the determination of isosafrole17.
A method for detecting safrole and isosafrole in soft drinks was reported in 200118, in
which the GC method was suitable for the direct injection of samples of soft drinks
into the equipment. The samples were decarbonated by stirring and 1 mL of sample
had 50 µL of 0.1% (w/v, in methanol, equal to 50 µg) of an internal standard 1,4-
dihydroxybenzene added. An aliquot (0.1 µL) of this solution was directly injected
into a GC for analysis. The GC was fitted with an analytical semi-polarized column
CP SIL 8CB (30 m x 0.53 mm, 1.5 µm). Isosafrole had a retention time of 15.33 min
for the Z isomer and 17.12 for the E isomer. The percentage recovery, in the spiked
samples, was between 98-104% and the lowest quantitatively determinable
concentration was 0.25 µg/mL. Of the 25 soft drinks sampled 5 did not contain
isosafrole and in the other 20 samples only one exceeded the current Chinese
regulation (1 µg/mL). The authors concluded that the method was more accurate than
the current AOAC method of detection with regard to the coefficient of variation18.

reversed-phase (C-18) HPLC with fluorescence detection (λex = 290 nm, λem = 325
nm) for the presence of isosafrole. The detection limit was 0.01 mg/L with recoveries
of between 96-100.2%. Isosafrole was not detected in any of the commercial samples
of alcoholic beverages19. In another HPLC method20 for the determination of safrole
in sassafras derived herbal products, an analytical column (Nov-Pak C18, 300 x 3.9
mm stainless steel column) with 4 µm particle size packing material was used along
with a liquid phase that consisted of methanol – 0.025 M KH2PO4, (75 + 25) that was
adjusted to pH 3.5 with H3PO4. This system afforded retention times for isosafrole of
7.60 min (E isomer) and 8.02 min (Z isomer), and the limits of detection were given
- 41 -
as 0.0018 µg/mL and the limits of quantitation were 0.0061 µg/mL. No isosafrole was
found in any of the various samples20.
5. Summary
Isosafrole does not appear in the proposed flavouring regulation as an active principle.
However, methods for detecting safrole would also have the capacity to detect
isosafrole, a reported GC method of detection was not only able to distinguish
between safrole and isosafrole but also between the isosafrole isomers. This method
gave the lowest quantitatively determinable concentration as 0.25 µg/mL. HPLC was
also able to determine between the two isosafrole isomers and this method was more
sensitive at a limit of detection of 0.0018 µg/mL.
Extraction methods were reported for the recovery of isosafrole from both alcoholic
and non alcoholic beverages as well as meat based products. Analysis of a meat based
product utilised a Likens-Nikerson method of extraction using hexane, whereas
beverage samples were either directly injected or steam distilled.
6. References
(1) The Merck Index - Monograph Number 0005244 Isosafrole. 2004.
(2) Heikes, D. L. SFE with GC and MS Determination of Safrole and Related
Allylbenzenes in Sassafras Teas. Journal of Chromatographic Science 1994,
32, 253-258.
(3) Buss, A. D.; Warren, S. The Stereocontrolled Horner-Wittig Reaction:
Synthesis of Disubstituted Alkenes. J. Chem. Soc. Perkins Trans. 1 1985,
2307-2325.
(5) Council of Europe. Committee of Experts on Flavouring Substances. 45th
Session. Record. 1999.
(6) Mazza, G. Identification of Oxidation-Products of Beta-Asarone in Acorus-
Calamus L by Gas-Chromatography and Mass-Spectrometry. Sciences Des
Aliments 1984, 4, 473-481.
(7) Opinion of the Scientific Committee on Food on Isosafrole.
SCF/CS/FLAV/FLAVOUR/30 Final. 2003.
(8) Swist, M.; Wilamowski, J.; Parczewski, A. Determination of synthesis method
of ecstasy based on the basic impurities. Forensic Science International, In
Press, Corrected Proof.
(10) WHO Food Additives Series 16: Safrole.
2000.
- 42 -
(14) Tateo, F.; Bononi, M.; Lubian, E.; Martello, S.; Fasan, S. Determination of
safrole and isosafrole in meat based products. Industrie Alimentari 1999, 38,
941-945.
(16) Larry, D. Gas Chromatographic Determination of Methyl Salicylate, Safrole,
and Related Compounds in Non-alcoholic Beverages. Journal of the AOAC
1969, 52, 481-485.
(17) Larry, D. Gas Chromatographic Determination of Safrole and Related
Compounds in Non-alcoholic Beverages: Collaborative Study. Journal of
A.O.A.C. 1971, 54, 900-902.
(18) Choong, Y. M.; Lin, H. J. A rapid and simple gas chromatographic method for
direct determination of safrole in soft drinks. Journal of Food and Drug
Analysis 2001, 9, 27-32.
(20) Carlson, M.; Thompson, R. D. Liquid chromatographic determination of
safrole in sassafras- derived herbal products. Journal of AOAC International
1997, 80, 1023-1028.
- 43 -
Menthofuran

3,6-dimethyl-4,5,6,7-tetrahydrobenzofuran (IUPAC name)
3,6-dimethyl coumaronetetra-hydride [4,5,6,7]
4,5,6,7-tetrahydro-3,6-dimethyl benzofuran
(+)-menthofuran
(R)-3,6-dimethyl-4,5,6,7-tetrahydrobenzofuran
(R)-3,9-epoxy-p-mentha-3,8-diene
1.2 Numbers
CAS Numbers 494-90-6, 17957-94-7, 59553-66-1, 80183-38-6
Fl Number 13.035
FEMA Number 3235
C of E Number 2265
EINECS Number 207-7955
JECFA Number 758

H3C
O
CH3
Molecular formula C10H14O

Class Heterocyclic

A summary of chemical and physical properties of pure menthofuran is given in Table
2.
- 44 -
Physical appearance Colourless, clear oil.

Specific gravity 0.970-0.9759
Boiling point 196°C
Melting point -17°C
Refractive index (20°C) λ589 nm 1.48
Infrared (neat) cm-1 2940, 1650, 1560,
1460,1420,1260,1170, 11001
1
H NMR (CDCl3) 7.03 (br,s,1), 2.64 (dd, 1, J =
16.1, 5.2), 2.23-2.43 (m, 2), 2.15 (dd, 1, J
= 16.1 9.4), 1.92 (d, 3, J = 1.2), 1.76-2.00
(m, 2), 1.35 (m, 1), 1.07 (d, 3, J = 6.6)1.
13
C NMR (CDCl3) 150.6, 136.7, 119.6, 117.4, 31.4,
29.6, 21.5, 19.8, 8.11.
2.1 Source
Menthofuran is a constituent of peppermint oil, and is also found in other mint species
including spearmint and watermint2.
2.2 Background
In US premium mint oils coming from the Midwest3, the composition of peppermint
essential oil is affected by environmental conditions, particularly light. For example
exposure to blue light reduces menthol biosynthesis by 65% but leads to a 2-3 fold
increase in the menthofuran content which lowers the organoleptic and economic
value4. Menthofuran has been found to be a key compound in the regulation of the
levels in pulegone and menthol in peppermint plants5. Menthofuran levels in
peppermint oil can range between 1 and 9 %2.
2.3 Usage
Peppermint and spearmint are both used extensively in the flavouring of food and
beverages, as well as personal hygiene products such as toothpaste and soaps.
2.4 Limits
Menthofuran is not listed as an active principle in Annex II of Directive 88/388/EEC.
However it will be included in the proposed EC flavouring regulation , the limits of
menthofuran are outlined as follows:
Mint/peppermint confectionery 500 mg/kg.

Micro breath freshening confectionery 3000 mg/kg
Chewing gum 1000 mg/kg
Mint/peppermint containing alcohol beverages 200 mg/kg9.
In the US menthofuran has been given Flavor and Extracts Manufacturers Association
(FEMA) generally regarded as safe (GRAS) status and is listed as an authorised
synthetic flavouring substance7. JECFA stated that there were no safety concerns at
current levels of intake when used as a flavouring agent8.
- 45 -
The tolerated daily intake (TDI) of menthofuran is given to be 0.1 mg/kg bw, this is
based on the no effect limit (NOEL) of 20 mg/kg bw/d in a toxicity study in rats with
a safety factor of 2007.
3.1 Metabolism
While menthofuran is present in mint, it is also a metabolite of pulegone, in which
pulegone is oxidized to give menthofuran by cytochrome P4509. The metabolic
pathway of pulegone and menthofuran is complex and has been shown to include at
least 24 compounds9. Scheme 1 shows the structures of pulegone and menthofuran.
H3C H H3C H H3C H
O O O
OH
pulegone 9-hydroxypulegone
menthofuran
Scheme 1. The oxidation of pulegone into menthofuran7.
4. Analysis

Generally the peppermint plant is distilled to extract the essential oil. A variation of
the distillation process using a Likens-Nickerson apparatus was used to extract oil
components which were then analysed by gas chromatography (GC)/mass
spectrometry (MS). The extracting solvent was 5:1 diethyl ether and hexane, with the
addition of camphor as an internal standard4.
The methods of solvent extraction and solid phase microextraction (SPME) were
compared, followed again by GC/MS detection using samples from the peppermint
plant. The solvent extraction was carried out on a Dean and Stark apparatus in which
50 g of plant material was extracted in 1.5 L of water and the distillation was allowed
to continue for 2 hours. A PDMS coated fibre (100 µm) and a manual SPME holder
were used in the SPME, in which samples were taken directly from the headspace of
this apparatus. It was found that the SPME method of extraction recovered a three
fold increase in the amount of menthofuran over the solvent based extraction process.
Average amounts were determined as a percentage by peak area measurements (3.33
% from SPME compared to 1.10 % from hydrodistillation10).
Menthofuran was recorded as a percentage of the total of aroma samples that had been
extracted from peppermint leaves, peppermint tea, peppermint essential oil,
peppermint sweets and peppermint chewing gum. The samples were extracted in the
same way (simultaneous distillation and extraction (SDE)) using Marcusson’s micro-
- 46 -
apparatus with hexane (500 µL). Distillation time was two hours and no further
purification or concentration was required11.
4.2 Quantitation
Quantification of the essential oil was achieved by GC with flame ionization detection
(FID) using a 30 m fused silica column coated with Carbowax 20M. The menthofuran
content of the plants grown under varying light conditions were expressed as mg/kg
fresh weight and values ranged between 150.65 and 617.254.
It has been reported that capillary GC improved the specificity and sensitivity of the
separation and measurement of a variety of compounds in peppermint oil,
menthofuran included. A support-coated open –tubular (SCOT) glass capillary
column (43 mm x 0.5 mm internal diameter) coated with SP-1000 was utilised with a
GC with dual flame ionization detectors. The internal standard was 5% (w/v) 2-
methylnaphthalene in hexane. The recovery rates were determined by adding a known
weight of menthofuran, and were shown to be 97% (S.D. 4.0). Menthofuran was
eluted in under 30 mins and was expressed as a percentage of the total (average
10.79%) with a coefficient of variation of 3.34%12.
In a study that determined the levels of menthofuran in food samples using GC/FID,
with two fused silica capillary columns (50 mm x 0.2 mm) with bonded stationary
phases, it was considered that identification of the compounds by MS were
unnecessary. The quantitative yields of each compound was calculated using peak
area and expressed as a percentage. For menthofuran these percentages were;
peppermint leaves 3.2 %, peppermint tea 0.3 %, peppermint essential oil 4.0 %,
peppermint sweets 1.4 % and peppermint chewing gum 1.4 %11.
5. Summary
In the proposed flavouring regulation menthofuran is to be restricted in certain
confectionery products and alcoholic beverages, the lowest concentration being 200
mg/kg in alcoholic beverages and the highest in micro breath freshening
confectionery, 3000 mg/kg.
Menthofuran differs from pulegone, in that SPME proved to be three times more
efficient at extraction than solvent based extraction methods of the peppermint plant.
However, SDE proved successful at extracting menthofuran specifically from
confectionery products.
GC/FID were the only reported methods of detection found. Extraction from plants
were given values between 150.65 and 617.25 mg/kg fresh weight and other samples
were expressed as % of total. These could not be expressed as concentrations as the
injection sample size was not given.
6. References
(1) Snider, B. B.; Shi, Z. Total synthesis of (+/-)-Chondrillin, (+/-)-Plakorin, and

related peroxy ketals. Development of a general route to 3,6-dihydro-1,2-
dioxin-3-ols. Journal of the American Chemical Society 1992, 114, 1790-
1800.
- 47 -
(2) The European Agency for the Evaluation of Medicinal Products. Draft
Position Paper on the Use of Herbal Medicinal Products Containing Pulegone
and Menthofuran. 2004, EMEA/HMPWP/52/04.
(3) Economic Research Service for the Consolidated Farm Service Agency,
Office of Management. US Department of Agriculture. Peppermint and
Spearmint. 1995.
(4) Maffei, M.; Canova, D.; Bertea, C. M.; Scannerini, S. UV-A effects on
photomorphogenesis and essential-oil composition in Mentha piperita. Journal
of Photochemistry and Photobiology B: Biology 1999, 52, 105-110.
(5) Mahmoud, S. S.; Croteau, R. B. Menthofuran regulates essential oil
biosynthesis in peppermint by controlling a down stream monoterpene
reductase. Plant Biology 2003, 100, 14481-14486.
2004, WGF/002/02-rev 2.
(7) Opinion of the Scientific Committee on Food on Pulegone and Menthofuran.
2002.
(8) Summary of Evaluations Performed by the Joint FAO/WHO Expert
Committee on Food Additives. JECFA Evaluations. Menthofuran. 2001.
(9) Zhou, S.; Koh, H.-L.; Gao, Y.; Gong, Z.-y.; Lee, E. J. D. Herbal bioactivation:
The good, the bad and the ugly. Life Sciences 2004, 74, 935-968.
(10) Rolf, J. Monoterpene composition of essential oil from peppermint (Mentha x
piperita L.) with regard to leaf position using solid-phase microextraction and
gas chromatography/mass spectrometry analysis. Journal of Agricultural and
Food Chemistry 1999, 47, 3782-3786.
(11) Oral, A.; Kahn, J. Determination of Peppermint and Orange Aroma
Compounds in Foods and Beverages. Proc. Estonian Acad. Sic. Chem. 2001,
50, 217-225.
(12) Sang, J. P. Estimation of menthone, menthofuran, methyl acetate and menthol
in peppermint oil by capillary gas chromatography. Journal of
- 48 -
Methyleugenol

1-allyl-1,2-dimethoxybenzene
1,2-dimethyoxy-4-(2-propenyl) benzene
1,2-dimethoxy-4-allylbenzene
1,3-(3,4-dimethoxyphenyl)-2-propene
1,3,4-eugenol methyl ether
3,4-dimethoxybenzene
4-allyl-1,2-dimethoxybenzene (IUPAC name)
4-allyl veratrol
eugenol methyl ether
eugenyl methyl ether
veratrole methylether
1.2 Numbers
CAS number 93-15-2
Beilsten Registry Number 1910871
FEMA Number 2475
C of E Number 185

Class substituted allylbenzene
- 49 -
A summary of chemical and physical properties of pure methyleugenol is given in
table 2.
Physical appearance Colourless/pale yellow oil

Melting point -4°C
Boiling point 254-255°C
Density 1.036
(CDCl3) δ: 3.33 (2H, d, J = 6.5 Hz, H-1’),
1
H NMR
3.86 (3H, s, 3-OMe), 3.35 (3H, s, 4-
OMe), 5.08 (2H, m, H-3’), 5.95 (1H, m,
H-2’), 6.71-6.81 (3H, m, aromatic
protons)1.
(CDCl3) δ: 39.7 (C1’), 55.7 (4-OMe),
13
C NMR
55.8 (3-OMe), 111.3 (C5), 111.8 (C2),
115.4 (C3’), 120.3 (C6), 132.6 (C1),
137.6 (C2’), 147.4 (C4), 148.9 (C3)1.
m/z 178.0994 [M]+, 163.0759 [M-CH3]2
2.1 Source
Natural constituent of a number of plants including, nutmeg, pimento, lemon grass,
tarragon, basil, star anise, sweet flag, cardamom, hyssop, fennel and myrtle.3
2.2 Background
Methyleugenol is more volatile than eugenol, therefore there are significant losses of
this compound at low concentrations when hexane is used as an extraction solvent and
subsequently evaporated off4. The total concentration of methyleugenol present in a
plant is variable and dependent of factors such as age of plant at harvest, harvesting
techniques, storage conditions, processing and method of measurement5. Pesto,
prepared from fresh sweet basil, is the food product that contains the highest known
concentration of methyleugenol, with a typical serving containing 250 µg/kg5.
Methyleugenol was partially studied with regards to the stability of the compound; it
was found that when the UV was measured at 283 nm there was no change over time6.
In basil the methyleugenol content is predominant in plants up to 10 cm in height, in
plants taller than this eugenol in predominant7.
2.3 Usage
Methyleugenol is used as a flavouring agent in jellies, baked goods, non-alcoholic
beverages, chewing gum, relish and ice-creams; it is also found in several cosmetic
products3.
- 50 -
2.4 Limits
Annex II of Directive 88/388/EEC does not list methyleugenol as a prohibited
compound10. However it is intended under the proposed EC flavouring regulation to
limit methyleugenol in dairy products (20 mg/kg), meat and meat products, including
poultry and game 15 mg/kg, fish and fish products 10 mg/kg, soups and sauces 60
mg/kg, ready-to-eat savouries 20 mg/kg and non alcoholic beverages 1 mg/kg11.
At present the estimated intake of methyleugenol in the UK is reported to be 13

mg/person/day and the 97th percentile as 36 mg/person/day3. In the US the total
average daily intake is estimated to be 0.5 µg/kg bw/day and for the 90th percentile as
5.0-6.3 µg/kg bw/day5.
3.1 Metabolism
The pathways that have been identified in the metabolism of methyleugenol have
been summarised in Scheme 1. The major metabolic oxidation pathway results in the
formation of 3,4-dimethoxybenzoic acid and 3,4-dimethyoxycinnamic acid, both
being excreted as urinary lysine conjugates8, following hydroxylation at the 1’
position. 1’ hydroxymethyleugenol can undergo sulphonation that ultimately result in
the formation of the highly reactive carbonium ion that can form DNA adducts8,12.
The formation of 1’hydroxymethyleugenol appears to be the most prominent
metabolic route at high levels of methyleugenol exposure, whereas at lower does?
doses the O-demethylation route appears to be more predominant5. This results in the
formation of dihydroxyallylbenzene and/or eugenol8. The minor epoxide pathway can
result in the formation of the diol but, in the body, the epoxide is rapidly detoxified by
epoxide hydrolase and glutathione transferase5.
4. Analysis

The standard method of extraction of methyleugenol from plant material is by
hydrodistillation. This is usually followed by gas chromatography (GC)-mass
spectrometry (MS)7,13-15. Methyleugenol was found to be a compositional component
of Artemisia campestris var.glutinosa, in which 100 g of dried plant was
hydrodistilled in a Clevenger type apparatus and the resulting oil was characterised by
GC/MS. It was found that methyleugenol contributed to 4.5 and 6.6% of the total oil
composition13. A similar method of extraction was used to determine the
methyleugenol content of basil, where it was found that it contributed to 58.7% of the
total essential oil composition7.
Simultaneous distillation-extraction with a Likens-Nickerson apparatus and

dichloromethane was used for the extraction of food products including bologna and
ground and homogenised and liquid samples (100-200 mL), were placed in the
Likens-Nikerson apparatus for 1.5 hours with 50 mL dichloromethane at 50°. Samples
were held at 90° under agitation and the cold finger was maintained at 5°. The extracts
were dried in anhydrous sodium sulphate and concentrated under vacuum to 2-5 mL.
These samples were analysed by GC coupled to an ion trap MS, where the limit of
detection was 8ng/mL of injected sample16.
- 51 -
OMe OMe OH
OMe OMe OH
epoxidation O-demethylation
O
dihydroxy-
allyl-benzene
Metheugenol
2',3'-epoxidemethyleugenol
hydroxylation
OMe OMe
OMe OMe
OMe
OMe
O3SO
3,4-dimethoxy
cinnamic acid
COOH
1' sulphooxymethyleugenol HO
1',hydroxymethyleugenol
OMe
OMe
OMe
OMe
DNA adducts
COOH
3,4-dimethoxy
benzoic acid
carbonium ion
Scheme 1. A summary of the metabolic pathways that methyleugenol undergoes5,8,9,12.
analytes are absorbed on to fibres (65 µm Carbowax divinylbenzene) in the headspace
and are recovered by thermal desorption. This method has been used to isolate
methyleugenol found in tobacco products17,18. A preconditioned SPE column was
used to extract methyleugenol from human serum before highly specific analysis
using isotope dilution GC – high resolution MS2.
- 52 -
The essential oil of Laurus nobilis (bay tree) was extracted by both hydrodistillation
and supercritical carbon dioxide. With regard to the methyleugenol content, there was
no significant difference in the amounts recovered by either method.19 A further
comparison between super critical carbon dioxide fluid extraction and steam
distillation was carried out on the analysis of nutmeg and mace oils, with again little
difference between the methyleugenol content following the two different extraction
processes24. SPE followed by GC/MS analysis was used to detect methyleugenol in
unbrewed sassafras teas21. The extraction process involved using an Isco Model SFX
2-10 extractor, a 10 mL stainless steel extraction vessel and a stainless steel capillary
restrictor, which was constructed to allow a flow of 1.5 mL/min of supercritical CO2
at 345 bar at 80°C. No comparison with other extraction methods was given21.
4.2 Quantitation
GC and GC/MS are the most common methods of analysing samples that contain
methyleugenol. Following SPME the samples were analysed by GC/MS. The GC was
fitted with 30-m DB-5MS column and compound identification was confirmed by
retention time. Methyleugenol had a retention time of 11.40 minutes; over a
concentration range of 0.0022-1.5 µg a correlation coefficient of 0.972 was achieved.
The authors set the limit of detection at 0.0022 µg/g, with a recovery rate of 120%.
Methyleugenol was found in 9 of the 68 products sampled, the minimum
concentration found was 0.0032 µg/g and the maximum was 0.54 µg/g17. The same
method was used four years later to study the methyleugenol content of Indian bidi
(flavoured) cigarette tobacco. A concentration range of 0.002- 2 µg was used, which
gave a correlation coefficient of 0.967, however, the limit of detection was given as
0.04 µg/g18. [Comment: The authors stated that the minimum concentration found was
0.0032 µg/g, however they also stated that their limit of detection was 0.04µg/g,
which suggests a level of determination outside their given limits.]
µm film of 5% diphenyl/95% dimethylpolixyloxane) was used and 3-
methylphenylacetate was used as an internal standard to reference GC retention times.
Percentage recovery rates were stated as being ‘essentially 100%’ and methyleugenol
was found (as an average) in the following food matrices:
Cola tasting drink 0.04 mg/kg

Vienna sausage 0.12 mg/kg,
Tomato sauce 0.07 mg/kg
Pesto 0.12 mg/kg
Cola 0.04 mg/kg
Fresh basil 0.62 mg/kg16.
The methyleugenol content of human plasma was determined using a variation of

GC/MS in which the GC was equipped with a 30-m J& W DB-5MS [(5% phenyl)-
methyl polysiloxane 0.25 µm film thickness, 0.25 mm id] capillary column, coupled
to a high resolution MS2. Calibration curves were performed over seven
methyleugenol concentrations and the response factors were calculated by dividing
the area of the methyleugenol quantification ion (178.0994, [M]+) by the area of the
13
C3-methyleugenol ion (181.194). 13C3-methyleugenol behaves identically to
methyleugenol but has a 3 atomic-mass-unit difference which is clearly noticeable in
MS, thus the ratio between their ions can internally correct the recovery of
- 53 -
methyleugenol for each sample. The limit of detection was given as 3.1 pg/g and the
average coefficient of variation was 14% over a 200 pg/g range2. This method of
analysis was adopted by a study that measured the methyleugenol content in human
plasma following the consumption of a commercial brand of gingersnaps22.
Methyleugenol was separated from eugenol in a high performance liquid

chromatography (HPLC) system that was developed for measuring the safrole
concentrations in nutmeg, mace and nutmeg oil23. A LiChrosorb RP-8 reversed-phase
column (250 x 7 mm) was used and the mobile phase was a mixture of 500 mL
filtered de-ionised water, 175 mL acetonitrile, 125 mL methanol and 200 mL
tetrahydrofuran23. HPLC was also used for the determination of methyleugenol in
rodent plasma at concentrations that ranged from 0.050 to 10.0 µg/mL4. The mobile
phase of this system was 100% isocratic for 20 mins (water-acetonitrile (53:47 v/v)),
followed by a gradient of increasing acetonitrile4. Detection was achieved by UV at
230 nm, an internal standard, 3,4-dimethoxystyrene in acetonitrile, was used; the limit
of quantification was reported to be 0.050 µg/mL. The recovery rates were deemed to
be acceptable and the methyleugenol content was considered stable for at least 34
days following freezing and for four days at 5°C4.
Near infrared, attenuated total reflectance IR and NIR-FT Raman spectroscopy

measurements were able to determine many compounds in the essential oil of basil,
including methyleugenol in the range of 0.30-83.44 g/100g24.
Stable isotope ratios have been used to authenticate the source of food ingredients25.
There are well defined patterns in the ratios of stable isotopes from a given
environment and these patterns can be used to determine between synthetic and
natural sources. On-line capillary gas chromatography isotope ratio MS was used in
the combustion and pyrolysis modes to determine δ13C values for methyleugenol and
the values for δ2H and δ18O data, however as synthetic methyleugenol is made from
natural eugenol, the data did not allow the two sources to be differentiated30.
5. Summary
In the proposed EC flavouring regulation methyleugenol is to be limited in a range of
products, the lowest levels is 1 mg/kg in non-alcoholic beverages and the highest 60
mg/kg in soups and sauces. Other categories include fish and fish products, 10 mg/kg,
meat and meat products, 5 mg/kg and dairy products, 20 mg/kg.
No significant differences were found, with regard to the extraction of methyleugenol

from plant material, between SFE and hydrodistillation. Simultaneous distillation-
extraction using Likens-Nickerson apparatus has been reported for the extraction of
methyleugenol from meat-based products and non-alcoholic beverages.
GC-FID was used to measure the levels of methyleugenol from meat based samples
and non-alcoholic drinks. The values recorded ranged between 0.04 mg/kg and 0.62
mg/kg, these values are sensitive enough to quantify the proposed limits, although the
authors did not give a limit of detection, their percentage recovery rates were very
good.
- 54 -
6. References
(1) Meepagala, K. M.; Sturtz, G.; Wedge, D. E. Antifungal Constituents of the
Essential Oil Fraction of Artemisia dracunculus L. Var. dracunculus. Journal
of Agricultural and Food Chemistry 2002, 50, 6989-6992.
(2) Barr, D. B.; Barr, J. R.; Bailey, S. L.; Jnr, C. R. L.; Beeson, M. D. et al. Levels
of Methyleugenol in a Subset of Adults in the General U.S. Population as
Determined by High Resolution Mass Spectrometry. Environmental Health
Perspectives 2000, 108, 323-328.
(3) Opinion of the Scientific Committee on Food on Methyleugenol (4-allyl-1,2-
dimethyoxybenzene). SCF/FLAV/FLAVOUR/4 ADD1 FINAL. 2001.
(4) Graves, S. W.; Runyon, S. Determination of methyleugenol in rodent plasma
by high-performance liquid chromatography. Journal of Chromatography B:
Biomedical Sciences and Applications 1995, 663, 255-262.
(5) Smith, R. L.; Adams, T. B.; Doull, J.; Feron, V. J.; Goodman, J. I. et al. Safety
assessment of allylalkoxybenzene derivatives used as flavouring substances --
methyl eugenol and estragole. Food and Chemical Toxicology 2002, 40, 851-
870.
(7) Miele, M.; Dondero, R.; Ciarallo, G.; Mazzei, M. Methyleugenol in Ocimum
basilicum L. Cv. Genovese Gigante. Journal of Agricultural and Food
Chemistry 2001, 49, 517-521.
(8) WHO Food Additives Series 16: Eugenyl methyl ether.
2004, WGF/002/02-rev 2.
(12) De Vincenzi, M.; Silano, M.; Stacchini, P.; Scazzocchio, B. Constituents of
aromatic plants: I. Methyleugenol. Fitoterapia 2000, 71, 216-221.
(13) Juteau, F.; Masotti, V.; Bessiere, J.-M.; Viano, J. Compositional
characteristics of the essential oil of Artemisia campestris var. glutinosa.
Biochemical Systematics and Ecology 2002, 30, 1065-1070.
(14) Saad, H.-E. A.; El-Sharkawy, S. H.; Halim, A. F. Essential oils of Daucus
carota ssp. maximus. Pharmaceutica Acta Helvetiae 1995, 70, 79-84.
(15) Tam, N. T.; An, H. L.; Bighelli, A.; Muselli, A.; Casanova, J. Advances in the
chemical composition of essential oils from Illicium griffithii Hook. f. et
Thoms. from Vietnam. Journal of Essential Oil Research 2005, 17, 79-81.
Food Chemistry 2003, 81, 469-475.
- 55 -
317.
(19) Caredda, A.; Marongiu, B.; Porcedda, S.; Soro, C. Supercritical carbon
dioxide extraction and characterization of Laurus nobilis essential oil. Journal
(20) Ehlers, D.; Kirchhoff, J.; Gerard, D.; Quirin, K. W. High-performance liquid
chromatography analysis of nutmeg and mace oils produced by supercritical
CO2 extraction - comparison with steam-distilled oils - comparison of East
Indian, West Indian and Papuan oils. International Journal of Food Science
and Technology 1998, 33, 215-223.
32, 253-258.
(22) Schecter, A.; Lucier, G. W.; Cunningham, M. L.; Abdo, K. M.; Blumenthal,
G. et al. Human consumption of methyleugenol and its elimination from
serum. Environmental Health Perspectives 2004, 112, 678-680.
(23) Archer, A. W. Determination of safrole and myristicin in nutmeg and mace by
high-performance liquid chromatography. Journal of Chromatography A
1988, 438, 117-121.
(24) Schulz, H.; Schrader, B.; Quilitzsch, R.; Pfeffer, S.; Kruger, H. Rapid
classification of basil chemotypes by various vibrational spectroscopy
methods. Journal of Agricultural and Food Chemistry 2003, 51, 2475-2481.
(26) Ruff, C.; Hor, K.; Weckerle, B.; Konig, T.; Schreier, P. Authenticity
assessment of estragole and methyl eugenol by on- line gas chromatography-
isotope ratio mass spectrometry. Journal of Agricultural and Food Chemistry
2002, 50, 1028-1031.
- 56 -
Pulegone

1-isopropylidene-4-methyl-2-cyclohexanone
1-methyl-4-isopropylidene-3-cyclohexanone
2-isopropylidene-5-methyl-cyclohexan-1-one (IUPAC name)
5-methyl-2(1-methylethylidene)-cyclohexanone
(-)-pulegone
delta-4(8)-p-menthen-3-one
d-4(8)-p-methen-3-one
d-pulegone
(R) – (+) – pulegone
(R)-p-menthen-(4(8))-on-(3)
1.2 Numbers
CAS 89-82-7, 3285-04-9, 3391-90-0, 15932-80-6.
FEMA Number 2963
C of E Number 2050
JECFA Number 753
H CH3
H3C CH3
Molecular Formula C10H16O

Class isocyclic – a monoterpene ketone
- 57 -
A summary of chemical and physical properties of pure pulegone is given in Table 2.
Physical appearance An oil at room temperature with an odour

between peppermint and camphor1
Boiling point 224°
Density 0.946
UV absorbance λmax (alcohol) 253.3 nm
Miscible in: Alcohol, ether and chloroform
Practically insoluble in: water
(CDCl3) δ: 0.98 (d, J = 5 Hz, 3H, CH3),
1
H NMR
1.18-1.53 (m, 2H), 1.70–1.96 (m, 2H),
1.78 (s, 3H, =CCH3), 1.97 (s, 3H,
=CCH3), 2.06-2.96 (m, 4H)2
A photoelectron spectra of pulegone has been determined; a band a 8.8 eV

corresponds to ionization from the oxygen lone pair and exocyclic π orbital. This
assignment is supported by the reported spectra of similar terpenes3.
2.1 Source
Pulegone is a major constituent of the essential oils of peppermint and pennyroyal; it
also occurs naturally at lower levels in other foods such as oregano, beans and tea4.
2.2 Background
Pulegone is considered to be the active ingredient in pennyroyal and it is also found in
many mint species5. The amount of pulegone present in a plant can be controlled by
regulating the time of harvest6, as it is a key intermediate in the biosynthesis of the
main component of peppermint oil, menthol7 .
2.3 Usage
Pennyroyal is not used as a flavouring but peppermint is used extensively to flavour
both food and beverages.
2.4 Limits
Annex II of Directive 88/388/EEC8 limits the amount of pulegone from flavourings
food 25 mg/kg,
beverages 100 mg/kg.
The exceptions are mint flavoured beverages, 250 mg/kg, and mint confectionery 350
mg/kg.
In the proposed EC flavouring regulation the limits are:
Mint/peppermint confectionary 250 mg/kg.

Micro breath freshening confectionary 2000 mg/kg
- 58 -
Chewing gum 350 mg/kg
Mint/peppermint containing non-alcohol beverages 20 mg/kg
Mint/peppermint containing alcohol beverages 100 mg/kg9
The estimated European daily intake is 0.2 µg/kg body wt/day10.
3.1 Metabolism
The in vivo metabolism of pulegone has been extensively studied in rodents and it is
considered to undergo complicated metabolic pathways11 which contain in excess of
24 metabolites9, which has been partially summarised in Scheme 1. When it
undergoes hydroxylation at the 9- position (the predominate pathway) it is further
modified to form menthofuran, which in turn is converted in to other metabolites4. In
vitro studies have shown that pulegone converts into menthofuran via cytochrome
CYP-mediation11. Hydroxylation at the 5- position (the second major pathway) results
in the formation of piperitenone2 which undergoes many further reactions such as ring
and side chain oxidation which in turn give rise to polar urinary metabolites10. Other
fates of pulegone include epoxidation and reduction via minor pathway mechanisms4.
Conjugates of pulegone and its metabolites with glutathione and glucuronic acid have
been found in the bile of rats given pulegone4.
4. Analysis
The International Organization of the Flavour Industry (IOFI) methods of analysing
pulegone were assessed by LGC in 198512. It was found that the gas chromatographic
methods used were not acceptable and suggested improvements to the column.
However, the extraction of pulegone was considered to be satisfactory. Fat-containing
samples were steam distilled using modified Schmidt apparatus. After 150 mL
distillate had been collected, 15 mL of 15 % sodium chloride solution was added. This
was extracted three times with diethyl ether, dried over anhydrous magnesium and
reduced in volume. Other samples were diluted, or dissolved in, water, then 15 %
sodium chloride solution was added and then the sample was extracted as above12.

The most frequent method of extracting essential oils from plants is hydrodistillation
or steam distillation13. However since the development of commercial supercritical
fluid extraction (SFE) for the large-scale recovery of essential oils, the use of SFE for
analytical samples has gained importance14.
The extraction of peppermint oil by hydrodistillation and supercritical carbon dioxide

has been compared. Carbon dioxide was chosen as it the most commonly used
supercritical fluid that possesses a low critical temperature and the non-polar character
of the fluid, which are features that are required when volatile, reactive and heat-
sensitive terpenes, such as pulegone, are being isolated14. A Suprex MPS/225 system
in the SFE mode was used with varying conditions to optimise the extraction process,
with the conclusion that the best conditions for the extraction of pulegone were,
temperature = 35°C, pressure = 100 atm, dynamic time = 10 min, and modifier
volume = 0 µL. This method extracted 17.2 % more pulegone from the essential oil
than hydrodistillation20. An earlier study on the extracts of savory and peppermint also
compared hydrodistillation with supercritical fluid extraction using both carbon
dioxide and water. It was found that varying the conditions in the extraction process
- 59 -
using supercritical water would affect the concentrations of the various compounds
isolated. There was however no significant difference between the recovery values of
extracts collected by SFE, (using either carbon dioxide or water) and
hydrodistillation15.
H3C H H3C H
Glucuronic acid
conjugates
H3C H
O OH
HO
OH pulegol
2,8-dihydroxymenthone
OH
H3C OH
isopulegol epoxidation
reduction
H3C H
H3C H
hydroxylation O
hydroxylation
O 5-hydroxypulegone
O
conjugation
OH
pulegone
H3C H
9-hydroxypulegone
Glutathione
conjugates
O
H3C H
H3C H
p-mentha-1,4(8)-diene-3-one
(piperitenone)
O
O
OH
menthofuran
O H3C H
9-carboxypulegone Covalent binding to proteins
O
O
CHO
CHO
p-Cresol
OH
Scheme 1. The proposed metabolism of pulegone4,10,11.
- 60 -
One study isolated pulegone particularly from the other constituents of peppermint
essential oil; this was achieved by dissolving 1 g of peppermint oil in 15 mL menthol
and 3 g of Girard D reagent. Following two hours of reflux this was poured into 250
mL distilled water. Continuous extraction for two hours with diethyl ether precedes
extraction three times with light petroleum. The method recovered 85 +/- 2%16.
Pulegone was one of five compounds that were isolated by the liquid-liquid extraction
of 10 mL samples of alcoholic beverages. The samples were pipetted into test tubes
containing 1.1 g of Na2SO4, and after neutralizing with sodium bicarbonate, known
amounts of the five compounds (0, 0.2 0.5, 1.0 and 2.0 mg/kg) dissolved in ethanol
and 0.5 mg/kg tetralin (as an internal standard) were added to the samples. These
samples were extracted three times with diethyl ether and concentrated to 3 mL. The
resulting samples were then analysed by selected ion monitoring mass spectrometry
(MS)17.
analytes are absorbed on to fibres (65 µm Carbowax divinylbenzene) in the headspace
and are recovered by thermal desorption. This method has been used to isolate
pulegone found in various tobacco products18,19. Extracts of peppermint have been
compared following extraction by SPME with those by hydrodistillation; whilst for
most compounds the SPME sample found higher concentrations, those of pulegone
were five times higher in the sample extracted from solvent based samples20.
Pulegone was recorded as a percentage of the total of aroma samples that had been
extracted from peppermint leaves, peppermint tea, peppermint essential oil,
peppermint sweets and peppermint chewing gum. The samples were extracted in the
same way, simultaneous distillation and extraction (SDE) using micro-apparatus with
hexane (500 µL). Distillation time was two hours and no further purification or
concentration was required21.
Pulegone was one of 70 compounds extracted from honey by a method that was
developed to improved the quantification of honey flavours22. The honey sample
underwent a two step extraction process, step one removed the flavour compounds
from the sugar matrix and was achieved by using a vessel that had been purged with
high purity nitrogen. To the 100 g honey sample, 200 mL of bidistilled
dichloromethane was added and the mixture stirred for an hour under a nitrogen
stream to prevent oxidation reactions. The dichloromethane extract was concentrated
to 1 mL and this underwent step two, which involved a steam distillation-solvent
extraction process using Likens-Nickerson apparatus and the results of using
dichloromethane, pentane and acetone were compared. It was concluded that
dichloromethane was the most appropriate solvent to use22.
4.2 Quantitation
GC/MS is the most widely-used method for the analysis of essential oils and their
constituents13, 16, 21, and pulegone has been identified as a major compound found in
the oils in extracts of peppermint and pennyroyal 5,6,11,23.
The GC results following extraction via Girard D reagent were expressed as the
pulegone percentage and no actual levels of detection were recorded. Sample
percentages ranged from 0.30 % to 2.21 %, with detection being performed on a gas
chromatograph equipped with a flame-ionization detector (FID), with methyl
- 61 -
cinnamate as an internal standard16. GC-FID was the chosen method of detection in
the study that considered supercritical water extraction; the levels of pulegone were
also expressed as a percentage15.
The compounds contributing to honey flavour were quantified by GC/MS following

extraction22. The GC was fitted with a 50 m x 0.32 mm wall-coated, open tubular
(WCOT) CP-SIL5 CB capillary column and detection was obtained by FID. The GC
was directly connected to a quadrupole mass spectrometer. On this system pulegone
had a retention time of 44.5 min, a coefficient of variation of 8% and a recovery factor
of 101%22.
GC/MS was used to detect the constituents of peppermint oil following SPME, the
column used was Supelcowax (fused silica) 60 m x 0.25 mm internal diameter, with
the eluted fractions being analysed directly by MS. Average amounts of each
compound were again expressed as a percentage of the total determined by peak area
measurements plus standard deviation20. This method of detection and expression of
results was also used in the identification of the active constituents of peppermint oil,
where it was found that pulegone made up 3.2 % of the total compounds23 and
following supercritical carbon dioxide extraction (pulegone 37.8 %)14.
Following SPME the tobacco derived samples were analysed by GC/MS. The
retention time in this given system was 7.52 min and the mean recovery for pulegone
was between 97-115%. The detection limit was expressed as being 0.015 µg/g18. The
same method was used four years later to study the pulegone content of Indian bidi
cigarette tobacco, when the limit of detection was expressed as 0.19 µg/g19.
A study that considered the level of pulegone in food sample used GC/FID, with two
fused silica capillary columns (50 mm x 0.2 mm) with bonded stationary phases. It
was considered that identification of the compounds by MS were unnecessary and the
quantitative yields of each compound was calculated using peak area and expressed as
a percentage. For pulegone these percentages were; peppermint leaves 1.3 %,
peppermint tea 0.8 %, peppermint essential oil 1.6 %, peppermint sweets 0.2 % and
peppermint chewing gum 0.7 %21.
5. Summary
In the proposed EC flavouring regulation limits are set for pulegone in a variety of
confectionery products and beverages, the lowest limit is to be 20 mg/kg in non
alcoholic beverages and the highest limit is to be 2000 mg/kg for micro breath
freshening confectionery.
Pulegone has been extracted from confectionery using SDE with hexane in two hours
with no further purification and from alcoholic beverages using liquid-liquid
extraction. There appeared to be no significant advantage in using SFE.
GC/MS was the most widely used method of quantitating pulegone irrespective of the
extraction technique. GC/FID has also been used for analysis of confectionery
samples. The results were given as a percentage. As the initial sample size was not
given, the percentages could not be converted into concentrations.
6. References
(1) The Merck Index - Monograph Number 0008028 - Pulegone. 2004.

- 62 -
(2) Murahashi, Shun-Ichi; Sasao, S.; Saito, E.; Naota, T. Ruthenium-catalyzed
hydration of nitriles and transformation of [delta]-ketonitriles to ene-lactams:
total synthesis of (-)-pumiliotoxin C. Tetrahedron 1993, 49, 8805-8826.
(3) Novak, I.; Kovac, B. Photoelectron spectroscopy of natural products: terpenes.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2005,
61, 277-280.
(4) Opinion of the Scientific Committee on Food on Pulegone and Menthofuran.
2002.
(5) National Institute of Health. Medicinal Herbs: The National Toxicology
Program Extracts the Facts. Environmental Health Perspectives 1999, 107,
604-604.
(6) Final report on the safety assessment of Mentha Piperita (Peppermint) Oil,
Mentha Piperita (Peppermint) Leaf Extract, Mentha Piperita (Peppermint)
Leaf, and Mentha Piperita (Peppermint) Leaf Water. International Journal of
Toxicology 2001, 20, 61-73.
(7) Mahmoud, S. S.; Croteau, R. B. Menthofuran regulates essential oil
biosynthesis in peppermint by controlling a down stream monoterpene
reductase. Plant Biology 2003, 100, 14481-14486.
2004, WGF/002/02-rev 2.
(10) WHO Food Additives Series 46: Pulegone and related substances.
(13) Rao, B. R. R.; Bhattacharya, A. K.; Mallavarapu, G. R.; Ramesh, S. Volatile
constituents of different parts of cornmint (Mentha arvensis L.). Flavour and
Fragrance Journal 1999, 14, 262-264.
(14) Aghel, N.; Yamini, Y.; Hadjiakhoondi, A.; Pourmortazavi, S. M. Supercritical
carbon dioxide extraction of Mentha pulegium L. essential oil. Talanta 2004,
62, 407-411.
(15) Kubatova, A.; Lagadec, A. J. M.; Miller, D. J.; Hawthorne, S. B. Selective
extraction of oxygenates from savory and peppermint using subcritical water.
Flavour and Fragrance Journal 2001, 16, 64-73.
(16) Bicchi, C.; Frattini, C. Quantitative determination of minor components in
essential oils: determination of pulegone in peppermint oils. Journal of
- 63 -
317.
(20) Rohloff, J. Monoterpene composition of essential oil from peppermint
(Mentha x piperita L.) with regard to leaf position using solid-phase
microextraction and gas chromatography/mass spectrometry analysis. Journal
(21) Orav, A.; Kann, J. Determination of Peppermint and Orange Aroma
Compounds in Foods and Beverages. Proc. Estonian Acad. Sci. Chem 2001,
50, 217-225.
(22) Bouseta, A.; Collin, S. Optimized Likens-Nickerson Methodology for
Quantifying Honey Flavors. Journal of Agricultural and Food Chemistry
1995, 43, 1890-1897.
(23) Umezu, T.; Sakata, A.; Ito, H. Ambulation-promoting effect of peppermint oil
and identification of its active constituents. Pharmacology Biochemistry and
Behavior 2001, 69, 383-390.
- 64 -
Quassine

2,10 dimethyoxy-3, 8, 11a, 11c-tetramethyl 3a, 6a, 7, 7a, 8, 11a, 11b, 11c-
octahydro-4H-6-oxabenzo [de]anthracene-1, 5, 11 trione
(+)-quassine
Alloquassine
Nigakilactone D
1.2 Numbers
CAS Number 76-78-8, 96646-68-3, 21293-20-9, 135556-51-3, 75991-65-0, 139402-
22-5, 139402-23-6.
O
O
H H
O O
H
H
Table 1. Structural and molecular information.

Elemental composition C 68.03 %, H 27.0 %, O 24.71 %
Class heterocyclic, diterpene lactone

A summary of chemical and physical properties of quassine is given in Table 2.

Physical appearance Crystalline solid
Acid strength Weak acid pKa 9.61
Melting point 260-263°C
Soluble in Methanol, acetone and water1
UV absorption maxima (ethanol) 264 nm
IR (KBr) 1745 (lactone), 1702 and 1688
(ketones), 1636 (double bonds)2.
- 65 -
2.1 Source
Quassine is the dried stem wood of Quassia amara and Picrasma excelsa. Extracts
from Planch (family Simabaceae) may also be called quassine.
2.2 Background
Commercial ‘quassine’ from Q. amara is known to contain a mix of bitter principles
including quassine, whilst P. excelsa contains isoquassine but may also be referred to
as quassine.
2.3 Usage
In the proposed EC flavouring regulation flavourings and food ingredients with
flavouring properties produced from Q. amara and P. excelsa may only be used in the
production of beverages3. It is used for its bitter flavourings properties, as the extracts
are known to be 50 times more bitter than quinine.
2.4 Limits
Annex II of Directive 88/388/EEC limits the amount of quassine in flavourings and
food 5.0 mg/kg,

beverages 5.0 mg/kg,
the exceptions are alcoholic beverages (50 mg/kg) and confectionery in pastille form
(10 mg/kg)4.
In the proposed EC flavouring regulation the amount of quassine in non-alcoholic

beverages is 0.5 mg/kg3, bakery ware 1 mg/kg and alcoholic beverages 1.5 mg/kg.
No information is available.
4. Analysis
A report was prepared in 19835 that investigated the methods of detection of quassine,
with regard to the then current International Organisation of the Flavour Industry
(IOFI), and its application to food products. LGC found that the proposed IOFI gas
chromatograph (GC) method was unsuitable for the detection of quassine as it gave
inconsistent results and in a short time frame it was not possible to improve the GC
system. However they did suggest that preliminary results from a high performance
liquid chromatography (HPLC) system may prove to be more useful5.

The only reference to the extraction of quassine from any food product was from
spirits and it was extracted by solid-phase extraction on phenyl sorbent using elution
with chloroform6.
- 66 -
4.2 Quantitation
Quassine from the above extraction was quantitated using reversed-phase (C-18) high
performance liquid chromatography (HPLC) with UV detection (254 nm). The
detection limit for quassine was 0.1 mg/L and recoveries were 92.9% (SD, 1.3%).
Twelve samples of spirits were analysed, quassine was not detectable in 7 of these
samples and the other samples ranged from <0.1 mg/L to 26.1 mg/L6.
Immunoassays have been developed in which quassine can be detected using a double
antibody non competitive enzyme linked immunosorbent assay (ELISA), which had a
detection limit of 5 ng. A mixed population of antibodies was deliberately raised, as to
provide a method by which quassine and compounds that are similar in chemical
structure could be detected7.
5. Summary
The proposed limit of quassine is 0.5 mg/kg in non alcoholic beverages. The only
reference to the quantitation of quassine was in spirit beverages in which SPE was
used, followed by rp-HPLC. The detection limit was 0.1 mg/kg, which is within the
proposed limits. An immunoassay detection method has also been published for
quassine and related compounds, it may be considered that quassine is particularly
suitable for this type of detection, and the ability to raise antibodies for such a specific
structure should be relatively straightforward. A full review of the application of
immunochemical assays with regard to food analysis, published in 1992, included the
study on quassine8.
6. References
(1) Hanson, K. R.; Jaquiss, D. B.; Lamberton, J. A.; Robertson, A.; Savage, W. E.
Quassine and neoquassine. Journal of the Chemical Society 1954, 4238-4249.
(2) Valenta, Z.; Grey, A. H.; Orr, D. E.; Papadopoulos, S.; Podesva, C. The
Stereochemistry of Quassine. Tetrahedron 1962, 18, 1433-1441.
(3) Opinion of the Scientific Committee on Food on Quassine.
SCF/CS/FLAV/FLAVOUR/29 Final. 2002.
2004, WGF/002/02-rev 2.
Food Constituents .2. Asarone, Quinine, Coumarin, and Quassinee in Spirits.
413 (German).
(7) Robbins, R. J.; Morgan, M. R. A.; Rhodes, M. J. C.; Furze, J. M. An Enzyme-
Linked Immunosorbent Assay for Quassine and Closely Related Metabolites.
Analytical Biochemistry 1984, 136, 145-156.
(8) Gazzaz, S. S.; Rasco, B. A.; Dong, F. M. Application of Immunochemical
Assays to Food Analysis. Critical Reviews in Food Science and Nutrition
1992, 32, 197-229.
- 67 -
Safrole

1-allyl-3,4-methlenedioxybenzene
1,2-methylenedioxi-4-allylbenzene
1,3-benzodioxole, 5(2-propenyl)
4-allyl-1,2-methylene dioxybenzene
5-allyl-1,3-benzodioxole
5-allyl-benzo[1,3]-benzodioxole (IUPAC name)
Allyldioxybenzene methylene ether
Allylcatechin methylene ether
Allylcatechol methylene ether
Allylpyrocatechin methylene ether
m-Allylpyrocatechol, formaldehyde acetal
Allylpyrocatechol methylene ether
3,4 methylenedioxyallylbenzene
Rhyuno oil
Safrol
Shikimol
Shikimole
1.2 Numbers
CAS Number: 94-59-7
Beilstein Registry Number: 136380
Merck Monograph Number: 0008395

Class heterocyclic, substituted allylbenzene

A summary of chemical and physical properties of pure safrole is given in Table 2.
- 68 -
Physical appearance A colourless or yellow tinted liquid with

a distinct sassafras or ‘sweet-shop’ odour
Melting point 11.2°
Boiling point 232-234°1
Density 0.946
Very soluble in alcohol1
Miscible with ether and chloroform1
Insoluble in water
IR (neat) vmax 3029 (s), 1648 (w), 1592 (m) cm-1 2
(CDCl3) δ 6.75 (d, J = 7.8 Hz, 1H), 6.69
1
H NMR
(s, 1H), 6.64 (d, J = 7.8 Hz, 1H), 5.94-
5.89 (m, 3H), 5.09-5.04 (m, 2H), 3.31 (d,
J = 6.4, 2H).2
(CDCl3) δ 147.6, 145.8, 137.6, 133.8,
13
C NMR
121.3, 115.7, 109.1, 108.1, 100.8, 39.9.2
Differences in the fragmentation patterns that occur during isobutane and ammonia
chemical ionization allow for the differentiation between safrole and isosafrole that
have identical molecular weight. Safrole and isosafrole exhibit a protonated molecular
ion and an adduct ion [M + 57]+. [M + 1 – 28]+ is also produced but at a significantly
lower abundance in the case of safrole3. Mass spectrometry (MS) spectra of
commercially available safrole contains peaks that have been identified as 1-methoxy-
4-(2-propenyl)-benzene4
2.1 Source
Safrole is a natural constituent of several essential oils, in particular sassafras, in
which contributes approximately 75%1. It is also found in a number of spices such as
nutmeg, mace, cinnamon, anise, black pepper and sweet basil5.
2.2 Background
The crushed leaves of Piper auritum, the essential oil of which contains 70% safrole,
is used as a fish attractant by Panamanian fishermen6. It was found that the overall
amounts of safrole in black pepper was decreased by various cooking and/or
processing methods, these ranged from a 57% to a 99% reduction depending on the
technique used7.
2.3 Usage
Safrole occurs at varying concentrations in many spices that are routinely used to
flavour food products. Such spices are also present in numerous beverages including
cola5. It is used in the perfume industry and for denaturing fats in the manufacture of
soaps1.
2.4 Limits
Annex II of Directive 88/388/EEC limits the amount of safrole from flavourings and
- 69 -
food 1 mg/kg,
beverages 1 mg/kg.
The exceptions are alcoholic beverages of <25% volume 2 mg/kg, alcoholic

beverages >25% volume 5 mg/kg and 15 mg/kg in food containing mace and
nutmeg8,9. In the proposed EC flavouring regulation these limits are to be confined to
meat and meat products, including poultry and game 15 mg/kg, fish and fish products
15 mg/kg, soups and sauces 25 mg/kg, and non alcoholic beverages 1 mg/kg10.
Intake estimates are very poor due to the lack of data on safrole concentrations that
occur naturally in foodstuffs, although an approximate intake value in Europe is
estimated to be 2mg/person/day from various sources5.
3.1 Metabolism
Passive absorption of safrole occurs rapidly in the gastrointestinal tract and in rodents
extensive metabolism occurs5. In all species studies of the main metabolic pathways
have been identified as either, allylic hydroxylation to 1’-hydroysafrole and
isomerisation to 3’-hydroxysafrole excreted in conjugate form, or as oxidation and
cleavage of the methylene dioxy moiety, leading to the formation of 4-allyl catechol
which is in turn oxidized to 4-allyl-o-quinone5. Scheme 1 contains a summary of these
conversions.
O
O
OH
O sulphation
3'-hydroxyisosafrole O
OSO3
- OSO2
isomerisation DNA
heterolytic cleavage adducts
to carbonium ion
O O
sulphation
OH OSO3
O O
1'-hydroxysafrole
allylic
hydroxylation
O
gamma O HO
oxidation dealkylation
OH
O
O
piperonylic acid
O Safrole 4-allyl-catechol
conjugation oxidation
with epoxidation
glycine
O
O
piperonylglycine O
O
O
safrole 2',3'-oxide
4-allyl-o-quinone
Scheme 1. Proposed metabolic pathways for safrole11-13.
- 70 -
In both these pathways metabolites are formed that have the potential to react with
macromolecules, as do the products of epoxidation - however this is a minor pathway.
Another minor pathway is gamma oxidation of the allylic side chain leading to the
formation of a carboxylic acid, which is then conjugated with glycine5.
4. Analysis
In 1983, LGC assessed the then current International Organization of the Flavour
Industry (IOFI) methods for the analysis of safrole in food and drink samples14.
Liquid and solid samples were added to enough water to facilitate stirring and steam
distilled, after the addition of triethylene glycol. The distillate was collected, n-
pentane was added and extracted with the same solvent. This was dried and reduced in
volume before being analysed by gas chromatography (GC). Providing that m-tolyl
acetate was added to the distillate as an internal standard, the extraction methods were
deemed to be satisfactory. The GC method, 2.5 m x 4 mm id glass packed with 4%
Carbowax 20 M on Chromsorb G, 80/100 mesh, was also considered to be good, with
a detection limit of 10 µg14.
4.1 Extraction
Nutmeg and mace oils are produced commercially by supercritical carbon dioxide
extraction15. Supercritical fluid extraction followed by GC/MS analysis was used to
detect safrole in unbrewed sassafras teas16. The extraction process involved using an
Isco Model SFX 2-10 extractor (Lincoln NE), a 10 mL stainless steel extraction vessel
and a stainless steel capillary restrictor, which was constructed to allow a flow of 1.5
mL/min of supercritical CO2 at 345 bar at 80°C16. A comparison between super
critical fluid extraction and steam distillation was carried out on the analysis of
nutmeg and mace oils, with the following differences in yield of safrole (Table 3)15.
Table 3. A comparison of the yield of safrole (g/100g) by steam distillation and super
critical fluid extraction (CO2 at pressures 90 or 120 bar), with coefficients of variation
given in brackets15.
Sample Yield of safrole (g/100 g)

Steam distillate CO2 extract90 CO2 extract120
Nutmeg, East 3.7 (0.6) 3.0 (0.4) 2.5 (0.7)
Indies
Mace, whole blade, 3.4 (0.4) 2.8 (0.7) 2.5 (0.3)
East Indies
Mace, whole blade, 3.4 (1.3) 3.1 (0.1) 2.7 (0.1)
hand picked, East
Indies
Nutmeg, West 0.63 (0.8) 0.28 (1.7) 0.33 (3.4)
Indies
Nutmeg, 0.28 (2.0) 0.31 (1.8) 0.28 (1.2)
unassorted, West
Indies
Mace, whole 0.61 (1.1) 0.37 (0.4) 0.30 (1.7)
blades, West Indies
Mace, whole 0.47 (1.1) 0.40 (0.6) 0.39 (1.2)
blades, West Indies
Mace, whole 30.7 (0.1) 25.2 (0.1) 20.5 (0.1)
blades, Papua
- 71 -
The safrole content of Piper auritum (root beer plant, Mexican pepperleaf) was
determined as follows: the leaves were steam distilled and the distillate was extracted
with diethylether, which was dried over sodium sulphate, filtered and evaporated. The
residue was diluted with pentane and analysed by GC/MS6.
Miniaturisation of steam distillation and coupling it to a continuous liquid extraction

of the condensate has been used to extract safrole from both powered cinnamon and
cinnamon bark. A range of solvents including pentane, dichloromethane, chloroform,
ethyl acetate and methyl tert-butyl ether were used, with pentane proving to be the
best solvent for the extraction of safrole, providing samples are pure enough for
analysis 17.
Simultaneous distillation- extraction (SDE) with a Likens-Nickerson apparatus and

dichloromethane was used for the extraction of food products including bologna,
ground and homogenised and liquid samples (100-200 mL), were placed in the Likens
and Nikerson apparatus for 1.5 hours with 50 mL dichloromethane at 50°. Samples
were held at 90° under agitation and the cold finger was maintained at 5°. The extracts
were dried in anhydrous sodium sulphate and concentrated under vacuum to 2-5 mL.
These samples were analysed by GC coupled to an ion trap MS, where the limit of
detection was 5ng/mL of injected sample18.
In meat based products, safrole was extracted for 5 hours by Likens-Nikerson

extraction using hexane (30 mL) for 100g of the food matrix, dried with sodium
sulphate, followed by HPLC. UV maxima in water/acetonitrile (55:45) was given as
236 and 288 nm, with a detection limit of 0.26 mg/kg of food matrix19.
analytes are absorped on to a fibre in the headspace and are recovered by thermal
desorbtion. This method has been used to isolate the compounds found in tobacco
products, including safrole20,21.
4.2 Quantitation
Early determination of safrole made use of UV detection.22 Safrole absorbs strongly at
287 nm but weakly at 308 and it is the difference between the two that was recorded
over a range of standards (0.5 -30 mg/100 µL). The value corresponding to 1mg
safrole/100 µL from each was noted and the reciprocal of the average value
determined. The difference in absorbance between 287 and 308 (against a blank) of
each sample was then multiplied by the reciprocal, this figure was then calculated as
ppm in the original root beer samples. Safrole was found in various brands and ranged
from 8.0 – 26.7 ppm22. This method was also used in a study that investigated the
stability of safrole in aqueous solutions, in which safrole was shown to be stable for at
least 20 days23. However, concerns were raised by Genest24 that the UV absorbance
was skewed by the presence of other compounds that have a similar UV absorbance
that contribute to flavour such as eugenol and therefore any method that relies solely
on UV detection lacks specificity. Therefore a simple qualitative test was developed
that detected the presence or absence of safrole. The test was based on the Labut test
in which a green/yellow and blue ring forms at the interface between concentrated
sulphuric acid and a solution containing safrole and gallic acid. It was proposed that 5
ppm could be detected by this method and it was tested on several beverages (cola and
- 72 -
root beers), cough syrup, medicinal tonic and sassafras tea. All but one of both the
cola and root beer samples had detectable amounts of safrole but the cough syrup,
medicinal tonic and the sassafras tea all proved to be negative24. Wilson’s method was
adopted by the AOAC but it was proposed by Larry25 that a GC method of detection
could be developed. However, although the method proved successful for methyl
salicylate it was not suitable for safrole as the average recovery of spiked samples
proved to be less than 50% across a collaborative study involving nine different
laboratories25. The method was revised and this was subjected to another collaborative
study. It had a more successful recovery range (between 77 and 98%) and was
adopted as official first action by the AOAC, in which the safrole is distilled with
steam and then extracted with an organic solvent such as chloroform26. In the UK a
method was developed for the use in determining safrole in essential oil that also used
GC with a flame ionisation detector27.
The official method of analysing safrole by GC was used to determine the safrole
concentration in the essential oil of mace28. The oil was analysed by both GC and
GC/MS, peaks were identified by peak enrichment when they were co-eluted with
purchased standard compounds. Safrole was found to be present as 1.9% of the total
oil content and evidence for identification included peak enrichment, MS, NMR and
IR28. More recently (2002) GC was used to determine the safrole concentration in the
oils extracted from cinnamon leaf and nutmeg27,29. IOFI also recommends the use of
GC in safrole determination. The recommended method suggests using a glass or
fused silica column packed with Carbowax 20 M or similar and the use of a flame
ionization detector30.
Following SPME tobacco derived samples were analysed by GC/MS. The retention
time in this given system was 8.65 min and the mean recovery for safrole at spiking
concentrations 2.2 µg and 0.28 µg were 112% and 97% respectively; at low
concentrations (0.016 µg) the mean recovery was 194%, however the detection limit
was expressed as being 0.0016 µg/g30.
A method for detecting safrole in soft drinks was reported in 200131, in which the GC
method was suitable for the direct injection of samples of soft drinks into the
equipment. The samples were decarbonated by stirring and 1 mL of each sample had
50 µL of 0.1% w/v, in methanol, (equal to 50 µg) of an internal standard 1,4-
dihydroxybenzene. 0.1 µL of this solution is directly injected into a GC for analysis,
the GC was fitted with an analytical semi-polarized column CP SIL 8CB (30 m x
0.53 mm, 1.5 µ). Safrole had a retention time of 13.22 min and a recovery percentage
in spiked samples that averaged 106.5%. The lowest quantitatively determinable
concentration was 0.25 µg/mL. Of the 25 soft drinks sampled 5 did not contain safrole
and the another 20 1 µg/mL by 3-5 fold31.
µm film of 5% diphenyl/95% dimethylpolixyloxane was used and 3-
methylphenylacetate was used as an internal standard to reference GC retention times.
Percentage recovery rates were stated as being ‘essentially 100%’ and safrole was
found (as an average) in the following food matrices:
Cola tasting drink 0.02 mg/kg

Bologna sausage 2.09 mg/kg
- 73 -
Vienna sausage 0.18 mg/kg.
Safrole was found not to be present in tomato sauce and fresh basil and only one
sample of pesto sauce contained safrole (0.21 mg/kg)18.

reversed-phase (C-18) HPLC with fluorescence detection (λex = 290 nm, λem = 325
nm) for the presence of safrole. The detection limit was 0.01 mg/L with recoveries of
95.6-101.4%. Safrole was detected in 4 of the 16 commercial samples of alcoholic
beverages at levels that varied between 0.15 and 6.60 mg/L32.
Safrole concentrations in nutmeg, mace and nutmeg oil have also been determined by
high performance liquid chromatography (HPLC)33 with a variable wavelength
detector set at 282 nm (which was half way between the maxima of the two
compounds being investigated (myristin at 277 nm and safrole at 287 nm). A
LiChrosorb RP-8 reversed-phase column (250 x 7 mm) was used and the mobile
phase was a mixture of 500 mL filtered de-ionised water, 175 mL acetonitrile, 125
mL methanol and 200 mL tetrahydrofuran. Safrole standards were prepared
containing 1-6 mg safrole in 100 mL methanol with an internal standard, dibutyl
phthalate 100 mg/100 mL methanol. 10 µL of each was used to determine a standard
curve, equating to 0.1-0.6 µg of safrole. Safrole dissolved in the mobile phase of
methanol and/or acetonitrile gave UV maxima at 287 nm. The recovery rates of
safrole ranged between 94 and 97 % and safrole was found in all the samples tested at
concentrations between 0.12 and 2.16 % of the original sample33.
Another HPLC method34 was developed for the determination of safrole in sassafras-
derived herbal products and used an analytical column [(Nov-Pak C18, 300 x 3.9 mm
stainless steel column with 4 µm particle size packing material and a liquid phase that
consisted of methanol – 0.025 M KH2PO4, (75 + 25)] that was adjusted to pH 3.5 with
H3PO4. The retention time for safrole on this system was 7.10 min and the limit of
detection was given as 0.0015 µg/mL and the limit of quantitation was 0.0051 µg/mL.
Relative standard deviation for safrole (n=5) from the various samples ranged from
1.30 to 5.39% at analyte levels of 0.01-1.5%34.
A spectrophotofluorometric method was proposed for the determination of safrole in

fragrances, samples were diluted in ethanol and injected into HPLC, excitation 295
nm, emission 323 nm, the minimal detectable amount was 10 ng35.
Matrix-assisted laser desorption ionisation/time-of-flight mass spectrometry

(MALDI/TOF-MS) was thought to be a quicker and less labour intensive way of
determining safrole than GC/MS, however it failed to detect the compound and the
authors took this as an indication that safrole is not readily protonated, which is the
method of ionization in MALDI36.
5. Summary
In the proposed EC flavouring regulation safrole is to be limited in non alcoholic
beverages to 1 mg/kg, to meat, fish, poultry, game and their associated products to 15
mg/kg and to 25 mg/kg in soups and sauces respectively.
The Likens-Nickerson apparatus has been used, with either hexane or

dichloromethane, to extract safrole from food products, particularly meat based and
- 74 -
non-alcoholic beverages. No references could be found for the extraction from fish
products or soup/sauces. In a comparative study of SFE and steam distillation, there
was no significant difference in safrole extraction at various pressures in SFE and
whilst there were some differences between SFE and steam distillation, no one
method was significantly better than the other over all samples.
GC and GC/MS were the most widely used methods for quantifying safrole. Safrole
was found in quantities that ranged from 0.02 mg/kg to 2.09 mg/kg, and although no
limits of detection were given, these ranges fall within the required limits. HPLC with
fluorescence detection also gave a suitable limit of detection of 0.01 mg/kg in non
alcoholic beverages.
6. References
(1) The Merck Index - Monograph Number 0008395 Safrole. 2004.

(2) Datta, S.; Chang, C.-L.; Yeh, K.-L.; Liu, R.-S. A New Ruthenium-Catalyzed
Cleavage of a Carbon-Carbon Triple Bond: Efficient Transformation of
Ethynyl Alcohol into Alkene and Carbon Monoxide. J. Am. Chem. Soc. 2003,
125, 9294-9295.
(4) Swist, M.; Wilamowski, J.; Parczewski, A. Determination of synthesis method
of ecstasy based on the basic impurities. Forensic Science International, In
Press, Corrected Proof.
(5) Opinion of the Scientific Committee on Food on the safety of the presence of
safrole (1-allyl-3,4-methylene dioxy benzene) in flavourings and other food
ingredients with flavouring properties. SCF/CS/FLAV/FLAVOUR/6 ADD3
Final. 2002.
(6) Gupta, M. P.; Arias, T. D.; Williams, N. H.; Bos, R.; Tattje, D. H. E. Safrole,
The main component of the essential oil from piper auritum of Panama.
Journal of Natural Products 1985, 48, 330-343.
(7) Farag, S. E. A.; Abo-Zeid, M. Degradation of natural mutagenic compound
safrole in spices by cooking and irradiation. Molecular Nutrition and Food
Research 1997, 41, 359-361.
(8) General Requirements for Natural Flavourings. CAC/GL 29-1987. 1987.
2004, WGF/002/02-rev 2.
(12) Murray, M. Mechanisms of Inhibitory and Regulatory Effects of
Methylenedioxyphenyl Compounds on Cytochrome P450-Dependent Drug
Oxidation. Current Drug Metabolism 2000, 1, 67-84.
- 75 -
(15) Ehlers, D.; Kirchhoff, J.; Gerard, D.; Quirin, K. W. High-performance liquid
chromatography analysis of nutmeg and mace oils produced by supercritical
CO2 extraction - comparison with steam-distilled oils - comparison of East
Indian, West Indian and Papuan oils. International Journal of Food Science
and Technology 1998, 33, 215-223.
32, 253-258.
(17) Jayatilaka, A.; Poole, S. K.; Poole, C. F.; Chichila, T. M. P. Simultaneous
micro steam distillation/solvent extraction for the isolation of semivolatile
flavor compounds from cinnamon and their separation by series coupled-
column gas chromatography. Analytica Chimica Acta 1995, 302, 147-162.
Food Chemistry 2003, 81, 469-475.
941-945.
317.
(22) Wilson, J. B. Determination of Safrole and Methyl Salicylate in Soft Drinks.
Journal of A.O.A.C. 1959, 42, 696-698.
(24) Genest, C.; Smith, D. M.; Chapman, D. G. Detection of Safrole in Foods and
Drugs. Journal of A.O.A.C. 1961, 44, 631-632.
(25) Larry, D. Gas Chromatographic Determination of Methyl Salicylate, Safrole,
and Related Compounds in Non-alcoholic Beverages. Journal of the AOAC
1969, 52, 481-485.
(26) Larry, D. Gas Chromatographic Determination of Safrole and Related
Compounds in Non-alcoholic Beverages: Collaborative Study. Journal of
A.O.A.C. 1971, 54, 900-902.
(27) Report by the Analytical Methods Committee - Application of gas-liquid
chromatography to the analysis of essential oils. Part XVIII. Determination of
safrole in oils of cinnamon leaf, Litsea cubeba, and nutmeg. Analyst 2002,
127, 428-429.
(28) Forrest, J. E.; Heacock, R. A. Identification of the major components of
essential oil of mace. Journal of Chromatography 1972, 69, 115-121.
(29) Analytical, M. C. Application of gas-liquid chromatography to the analysis of
essential oils. Determination of safrole in oils of cinnamon leaf and nutmeg.
Analyst 2002, 127, 428-429.
- 76 -
(30) International Organization of the Flavor Industry (IOFI) Recommended
Methods. Method 22. Safrole-determination by capillary gas
chromatography. 1987.
Analysis 2001, 9, 27-32.
1988, 438, 117-121.
1997, 80, 1023-1028.
(35) Wisneski, H. H.; Yates, R. L.; Davis, H. M. High-performance liquid
chromatographic--fluorometric determination of safrole in perfume, cologne
and toilet water. Journal of Chromatography A 1983, 255, 455-461.
(36) Oltramari, A. C.; Wood, K. V.; Bonham, C.; Verpoorte, R.; Caro, M. S. B. et
al. Safrole analysis by GC-MS of prototrophic (Ocotea odorifera (Vell.)
Rohwer) cell cultures. Plant Cell Tissue and Organ Culture 2004, 78, 231-
235.
- 77 -
Teucrin A

3’,4,5,5’α,7’,8’8’α-octahydro-8’-hydroxy 7’-methylspiro(furan-3(2H))6’-
(6H)naphtha(1,8-bc)furan)-2,2’(4’H)-dione
5-(3-furanyl)-3’,4,5’,7’,8’,8’a-octahydro 8’-hydroxy-7’methyl (5aS-(5’α,6’β(R*)-
7’beta 8’,beta 8’α))(5aS-(5’α,6’β(R*)),7’β, 8’β, 8α))-5-(3- furanyl)
Spiro(furan-3(2H)6’-(6H)naphthol(1,8-bc)furan)-2,2’(4’H)dione
Teucvin
1.2 Numbers
CAS Number 12798-51-5, 88244-97-7

O
H
O
H
O
CH3
OH
H
O
O

Class heterocyclic, diterpenoid

A summary of chemical and physical properties of Teucrin A is given in Table 2.
- 78 -
(CDCl3) δ 17 (q C-17), 19.7 (t C-2), 21.6

13
C NMR
(t C-1), 24.7 (t C-3), 35.3 (t C-7), 35.7 (d
C-8), 40.6 (t C-11), 41.9 (d C-10), 53.5 (s
C-9), 71.9 (d C-12), 78.3 (d C-6),108.0 (d
C-14), 124.9 (s C-13), 126-1 (s C-
4),139.6 (d C-16), 144.2 (d C-15), 162.1
(s C-5), 173.0 (s C-18), 175.9 (s C-20)1
A proton NMR spectrum of Teucrin A shows two α protons (7.61 and 7.83 ppm) and
one β proton (6.53 ppm) from a furan nucleus. The signal from H12 (5.75 ppm, J =
8.5 Hz) show interaction with two vicinal protons on C11, which appear as a doublet at
2.6 ppm (J = 8.5 Hz) and a methyl group at the C8 position is visible at 1.2 ppm2
2.1 Source
A major component of hydroalcoholic extracts of Teucrium chamaedrys- wild
germander.
2.2 Background
Wild germander is an herbal plant that has been used for its cholerectic and antiseptic
properties3.
2.3 Usage
Extracts of wild germander are used to flavour wines, bitters and liqueurs.
2.4 Limits
Annex II of Directive 88/388/EEC does not list Teucrin A as a prohibited compound4.
However in the proposed EC flavouring regulation the amount of Teucrin A in
alcoholic beverages is restricted to 2 mg/kg5.
3.1 Metabolism
Very little is known about the metabolism of Teucrin A but bioactivation is thought to
be mediated by CYP3A46.
4. Analysis

As Teucrin A is not commercially available, standards used for the construction of
calibration curves have to be extracted from T. chamaedrys, in which the plant is
soaked for 7 days in acetone (7000 mL to 770 g of plant material). The acetone is
filtered and taken to dryness, the residue is then loaded on to a Silica gel 60 column
and eluted with ethyl acetate:petroleum ether 2:1, 4:1 and 9:1 (v/v). Teucrin A is
recovered in the 9:1 fraction and is recrystallized from acetone:ethyl ether7.
- 79 -
4.2 Quantitation
A sensitive high performance liquid chromatography (HPLC) method was given for
the analysis of Teucrin A in drinks flavoured with extracts of wild germander, which
involved the use of a reverse-phase column Adsorboshere C-18 (25 x 46 mm internal
diameter) and a UV light detector (220 nm). The mobile phase consists of a gradient
of water:acetonitrile from 99:1 to 1:99, limit of detection was given as 0.1 ppm and
the limit of quantification as 0.3 ppm7
5. Summary
In the proposed EC flavouring regulation Teucrin A is to be limited in alcoholic
beverages to the amount of 2 mg/kg, and the only reference to the quantitation of
Teucrin A was for drinks flavoured with extracts of wild germander. Direct injection
of samples on to rp-HPLC gave a limit of detection of 0.1 mg/kg, which is sensitive
enough for the proposed limits. Teucrin A may also be suitable for detection via
antibody-based methods due to the unique structure of the compound. However, there
are no repeats of such studies.
6. References
(1) Rodriguez, M. C.; Barluenga, J.; Savona, G.; Piozzi, F.; Servettaz, O. et al.
Isoteuflidin, a Neo-Clerodane Diterpenoid from Teucrium- Chamaedrys, and
Revised Structures of Teucrin-F and Teucrin-G. Phytochemistry 1984, 23,
1465-1469.
(2) Popa, D. P.; Reinbold, A. M. Absolute Teucrin-a Configuration. Khimiya
Prirodnykh Soedinenii 1974, 321-330.
(3) De Vincenzi, M.; Maialetti, F.; Silano, M. Constituents of aromatic plants:
teucrin A. Fitoterapia 2003, 74, 746-749.
2004, WGF/002/02-rev 2.
(7) Bosisio, E.; Giavarini, F.; Dell'Agli, M.; Galli, G.; Galli, C. L. Analysis by
High -performance Liquid Chromatography of Teucrin A in Beverages
Flavoured with an Extract of Teucrium chamaedrys L. Food Additives and
Contaminants 2004, 21, 407-414.
- 80 -
Thujone

1-isopropyl-4-methyl-bicyclo[3.1.0]hexan-3-one
(1S, 4R, 5R)-(-)-thujone
3-thujanone
4-methyl-1-(1-methylethyl)bicycle(3.1.0)hexan-3-one
(-)-isothujone
(-)-thujone
(+)-thujone
α-thujone
β-thujone
thujon
1.2 Numbers
CAS Numbers: (β-thujone) 471-15-8, (α-thujone) 546-80-5, 1125-12-8, 58501-78-3,
116562-20-0, 59573-80-7, 69257-13-2, 76231-76-0, 78821-49-5, 116562-18-6.
Beilstein Registry Number: 2041870, (α-thujone) 4660369, (β-thujone) 2041871.
Merck Monograph Number: 0009469
CH3 CH3
H O H O
H3C CH3 H3C CH3
(-)-α-Thujone (+)-β-Thujone
Molecular formula C10H16O

Elemental Composition C 78%, H 10.59%, O 10.51%
Class isocyclic-monoterpene ketone

A summary of chemical and physical properties of pure thujone is given in Table 2.
- 81 -
Colourless or almost colourless liquid A menthol-like odour

Soluble in ethanol, diethyl ether and chloroform
insoluble in water
UV max (isooctane) 300 nm1
Fluorescence λex 220 nm, λem 290 nm2
Boiling point α-thujone 83.8 – 84.1°
β-thujone 85.7 – 86.2°1
Density α-thujone 0.9109
β-thujone 0.91351
Index of refraction α-thujone nD15 1.4490
β-thujone 0.9135 nD25 1.45001
Optical rotation α-thujone [α]D20 -19.2°
β-thujone [α]D15 +72.5°1
m/z α-thujone 153, 135, 93
β-thujone 153, 135, 123, 933
1
H NMR α-thujone (CDCl3) δ: 0.13 (dd, H6n), 0.76 (ddd, H6x,
J6x,1 = 8.2 Hz, J6x,6n = 5.8 Hz, J6x,4x = 2.5
Hz), 0.95 (d, CH3, J = 6.7 Hz), 1.01 (d,
CH3, J =6.7 Hz), 1.09 (dd, H1, J1,6x = 8.9
Hz, J1,6n = 4.5 Hz), 1.16 (d, α-CH3, J =
7.5 Hz), 1.36 (sept., H7, J = 6.3 Hz), 2.22
(dq, H2n, J2n,CH3 = 7.5 Hz, J2n,4n = 1.0 Hz),
2.07 (dd, H4n, J4n,4x = 19.0 Hz, J4n,2n = 1.0
Hz), 2.55(ddd, H4x, J4x,4n = 19.0 Hz, J4x,6x
=2.5 Hz, J4x,1 = 1.4 Hz)4
1
H NMR β-thujone (CDCl3) δ: -0.04 (dd, H6n, J6n,6x =4.0 hz,
J6n,1= 6.0 Hz, 0.59 (bt, H6x), 0.95(d,CH3, J
= 7.0 Hz), 1.01 (d, CH3, J = 7.0 Hz), 1.04
(d, α-CH3, J = 7.0 Hz), 1.45 (ddd, H1, J1,6n
= 6.0 Hz, J1, 2x = 5.5 Hz, J1,6x = 8.0 Hz),
1.44 (sept, H7, J = 7.0 Hz), 2.13 (d, H4n, J
= 18.5 Hz), 2.55(ddd,H4x, J = 18.5 Hz,
J4x,6x = 2.0 Hz, J4x,2 J = 2.0 Hz), 2.71 (qdd,
H2x, J2x,CH3 = 7.0 Hz, J2x,1 = 5.5 Hz, J 2x,6x
= 2.0 Hz, J2x,4x = 2.0 Hz)4
1
C NMR α-thujone (CDCl3) δ: 17.99 (α-CH3), 18.58 (C6),
19.51 (CH3), 19.81 (CH3), 25.56 (C1),
29.46 (C5), 32.74 (C7), 39.46 (C4), 47.13
(C2), 220.76 (C3)4
1
C NMR β-thujone (CDCl3) δ: 12.42 (α-CH3), 14.58 (C6),
19.61 (2 x CH3), 24.53 (C1), 27.21 (C5),
32.54 (C7), 41.61 (C2), 45.30 (C4), 218.23
(C3)4
- 82 -
2.1 Source
α and β thujone occur together in the essential oils and extracts of plants such as,
Artemisia absinthuim (wormwood), Artemisia vulgarius (mugwort), Salvia officinalis
(sage), Salvia sclarea (clary), Tanacetum vuglaris (tansy), Thuja occidentalis (yellow
cedar) and species of Juniperus and Cedris. The ratio of α to β thujone isomers vary
with species.
2.2 Background
α and β thujone are diastereoisomers with synthetic α-thujone being commercially
available. The isomers are markedly different in toxicity and convulsant activity,
WHO suggested that β-thujone appeared to be significantly more toxic in animal
studies5. However most studies use materials of unspecified composition and it has
been shown that the α isomer maybe considered to be much more potent than the
β6,7.The major contributor to thujone in a diet is from sage, sage-flavoured products
and in some alcoholic drinks.
2.3 Usage
Thujone itself has been used as a solvent. However, the essential oils in which thujone
occur are used in herbal medicines, flavourings, fragrances and in rodent/mite
repellents.
2.4 Limits
Annex II of Directive 88/388/EEC limits the amount of thujone from flavourings and
food 0.5 mg/kg,

beverages 0.5 mg/kg.
The exceptions are alcoholic beverages >25% vol 10 mg/kg, alcoholic beverages
<25% vol 5 mg/kg, 35 mg/kg in bitters and 25 mg/kg in food containing sage8.
In the proposed EC flavouring regulation thujone will be limited to 10 mg/kg in

alcoholic beverages, except those produced from Artemisia species, in which case the
limit will be 35 mg/kg9.
3.1 Metabolism
The studies that investigated the metabolism of thujone diastereoisomers have
identified that, although there are differences between species, metabolism usually
involves the formation of hydroxyl and dehydro thujones6, with the 4-hydroxylation
of thujone being a product catalysed by cytochrome P450 enzymes6. A summary of
the proposed metabolites appear in Scheme 1.
- 83 -
OH
3-β−hydroxy-α-thujone
3-β−hydroxy-β-thujone
reduction
OH
O O
O
4-hydroxylation
7-hydroxylation
4−hydroxy-α-thujone thujone
4−hydroxy-β-thujone OH
7−hydroxy-α-thujone
8-hydroxylation 7−hydroxy-β-thujone
followed by
dehydration
7,8−dehydroxy-α-thujone
7,8−dehydroxy-β-thujone
Scheme 1. The proposed metabolites of α and β thujone3,6.
4. Analysis
LGC assessed the International Organization of the Flavour Industry (IOFI) method of
analysing thujone in 198510. Liquid samples were distilled and solid samples were
steam distilled into 100 mL n-pentane. The distillate was extracted three times with n-
pentane, which was then dried over anhydrous sodium sulphate and 1 mL
cyclohexane added. The solution was evaporated down and analysed by gas
chromatography (GC). These extraction processes were found to be satisfactory. The
GC method was improved using a capillary column and the detection limit was found
to be 0.2 mg/kg10.

The AOAC official method of analysis11 of thujone (method number 940.16) in
cordials and liqueurs details the extraction process which includes reflux of the
sample (500 mL) with 1 mL aniline and 1 mL of H3PO4 for 30 mins and the collection
- 84 -
of the second 100 mL distilled portion (the first is rejected). To this portion, 0.5 g
semicarbazide hydrochloride and 0.6 g anhydrous sodium acetate are added and the
mixture left overnight. The alcohol is then distilled off and essential oils removed by
steam distillation. The distillation residue is cooled and 1 mL H2SO4 (1 +1) is added.
Again this is steam distilled, collecting 20 mL, which is poured into a separator with
20 mL of ether. The mixture is shaken and separated. 10 mL of alcohol (65%) is then
added and the ether allowed to spontaneously evaporate. The odour of thujone is
apparent if more than 2 mg is present in solution11.
The most common way of extracting compounds from plant material is via
hydrodistillation, and as recently as 2004 it has been reported that this method was
used to extract thujone from Artemisia verlotiorum, in which the air dried plants were
hydrodistilled in Clevenger-like apparatus for two hours12.
Thujone was one of 70 compounds extracted from honey by a method that was
developed to improve the quantification of honey flavours13. The honey sample
underwent a two step extraction process, step one was done to remove the flavour
compounds from the sugar matrix and was achieved by using a vessel that had been
purged with high purity nitrogen. To the 100 g honey sample, 200 mL of bidistilled
dichloromethane was added and the mixture stirred for an hour under a nitrogen
stream to prevent oxidation reactions. The dichloromethane extract was concentrated
to 1 mL and this underwent step two, which involved a steam distillation-solvent
extraction process using Likens-Nickerson apparatus and the results of using
dichloromethane, pentane and acetone were compared. It was concluded that
dichloromethane was the most appropriate solvent to use13.
In the hydrodistillation of Thuja orientalis (American arborvitae), hexane was

considered to be the best apolar solvent to use as it dissolves monoterpenes but it is
volatile enough to allow for reduction of volume without appreciable losses of the
essential oil components. Thujone contributed to the total oil composition with α-
thujone at 52.28% and β-thujone at 10.30%14.
A comparison of the compounds obtained by traditional solvent and ultrasonic

assisted extraction was carried out on valerian (Valeriana officinalis L.)15. The
traditional method involved the circulation of the solvent (ethanol/water) through a
column that had been loaded with plant material. The ultrasonic assisted method was
as before but with an ultrasonic probe operating at 20 kHz, placed in the top centre of
the extractor. 25 mL samples were removed from the vessel and analysed by HPLC,
stainless steel column (250 x 4 mm internal diameter packed with Necleosil 120-5
C18) and mobile phase of acetonitrile/water (gradient of acetonitrile 20% - 85%)).
The conclusion was that there was no significant difference between the two
extraction methods. However when the method was applied to the extraction of sage
(Salvia officinalis L.), there was a difference in the amounts of α and β thujone
extracted by the ultrasonic assisted extraction. An increase of between 23.76% and
31.32% was reported depending on the exact ultrasonic method15.
The extraction of thujone from absinthe, a traditional bitter spirit that was banned in
many countries for approximately 70 years, was recently achieved by solid-phase
extraction (SPE)16. Different solid phases were evaluated including polyamide and
carbon, but the DSC-18 obtained both a good separation and a high recovery of the
analytes. The SPE tubes were first activated with 1 mL of methanol, followed by 1
mL of water, after which the sample was added. This was then washed with water (1
- 85 -
mL) and the sample eluted with 1 mL methanol which could then be analysed by GC
(as discussed below)16. SPE was also used to isolated thujone from Italian vermouth17
by 20 mL of the sample being passed through a 3 mL C18 column. The column was
washed with 0.45 mL ethanol. This was discarded, and an additional 1 mL eluted the
required compounds into a sample vial. To this solution, 25 µL of a solution of 1-
dimethylaminonaphthalene-5-sulphonylhydrazine, dansylhydrazine (DNSH) (1
mg/mL ethanol), and 25 µL ethanolic HCl (6.5 µL conc. HCl in 10 mL ethanol) was
added, allowing the formation of dansyl-α and β thujones. These could then be
separated and quantified by LC17.
Thujone was one of 251 compounds that have been detected in the air surrounding
garden waste and from kitchen waste exudates. Analysis was by GC/mass
spectrometry (MS), using dynamic and equilibrium headspace sampling.
Concentration was achieved on stainless steel tubes containing 200 g 60-80 mesh
Tenax TA followed by thermal desorption at 250°C for 20 min18.
4.2 Quantitation
Samples produced from the SPE of absinthe16 were subjected to GC determination.
The GC was fitted with a flame ionizer detector (FID) and a capillary column BGD-5,
5% diphenyl-, 95% dimethylpolysiloxane, 30 x 0.25 mm internal diameter. GC/MS
analysis was also performed on a GC fitted with the same column as above that was
directly connected to a quadrupole mass spectrometer. Identification of the
compounds was achieved by retention index and by MS data with the quantification
of α/β thujone being done by standard addition. The recovery of α-thujone depended
on the matrix of the absinthe and varied between 40-70%, thus the authors calculated
α-thujone by standard addition and as no standard of β-thujone was available, the
amount of β-thujone was calculated with the response factor of α-thujone. Using this
procedure a limit of detection was shown to be 0.1 mg/L with a precision of 5%16.
GC remains the most popular method for the analysis of thujone in essential oils19,20
and the compounds contributing to honey flavour were quantified by GC/MS
following extraction13. The GC was fitted with a 50 m x 0.32 mm wall-coated, open
tubular (WCOT) CP-SIL5 CB capillary column and detection was obtained by FID.
The GC was directly connected to a quadrupole mass spectrometer. Thujone, on this
system had a retention time of 29.8 min, a coefficient of variation of 10% and a
recovery factor of 87%13.
GC was utilized in the analysis of Thuja orientalis that had been subjected to a
Rusitec system (the simulation of rumen digestion). The GC system used a HP5 30 m
x 0.25 mm column with a flame ionisation detector and an internal standard, carvone.
This system was able to differentiate between the two isomers14.
The most recent analysis of the essential oil of Artemisia verlotiorum used GC/MS for
the identification and quantification of the component compounds, of which thujone
was one12. This system used two different columns, HP-WAX and HP-5 (30 m x 0.25
mm, 0.25 µm film thickness) with a FID dual detector. Identification was achieved by
comparison of retention times with standards and by the mean of their linear retention
indices relative to the series of n-hydrocarbons. This means of identification was also
used with regard to GC/EIMS, using a DB-5 capillary column (30 m x 0.25 mm) and
an ion-trap mass detector. All the compounds identified were confirmed by molecular
weight by GC/CIMS, using methanol as CI ionizing gas. The study was investigating
how the levels of the various compounds changed during the year in the particular
- 86 -
plant. It concluded, with regards to thujone that the levels of β-thujone were always 2-
5 times than those of α-thujone. [Comment- the systems used could clearly distinguish
between the two isomers, however the authors were investigating relative amounts of
compounds and as such their results were published as ‘ % of total’ and although
they published injection volumes (0.5 and 0.2 µL) they did not state the volume of the
distillate obtained from 200 g of dried plant. There was no mention of limit of
detection.]12
In the preparation of alcoholic beverages for thujone determination via fluorimeteric

detection2, the samples are simply diluted in methanol in 1:3 or 1:6 ratios depending
on the alcohol volume. These samples are then analysed by high performance liquid
chromatography (HPLC) but any stored sample must be in hermetically sealed bottles
as up to 75% of the sample may be lost after 25 hours and the methanol dilution must
be carried out rapidly to prevent any loss2. HPLC was used to analyse samples from
alcoholic drinks using HS-RP 18 column, the mobile phase was acetonitrile:water
(55:45) and fluorescent properties used to detect at λex 220 nm and λem 290 nm. UV
detection of thujone cannot be used owing to the low molar extinction coefficient and
although the isomers eluted as one peak this was not regarded as a problem as the
limits refer to the sum of the isomers. For the analysis coelution was used for
compound identification and a linear calibration curve was obtained by injecting
various amounts of an 8.3 x 10-3 µg/µL thujone standard. The recovery rates ranged
between 96.7 % and 106.6 % and the limit of detection was found to be 0.5 mg/kg. Of
the 22 commercially available alcoholic drinks sampled, thujone was present in 12
and two exceeded the limits as set by the EC2. At the time this was the only published
HPLC system for thujone, however it could not be repeated in another laboratory and
so an alternative method was developed17. Following derivatisation with
dansylhydrazine the thujone isomers were successfully analysed and separated on a
different HPLC system. The reversed phase LC used a Zorbax Eclipse XDB-C8 (150
x 4.6 mm internal diameter) column, with a fluorescence detector set at λex 358 nm
and λem 521 nm. The mobile phase was acetonitrile/methanol/acetic acid (60:40:0.1,
v/v/v) and 0.1% aqueous formic acid in a gradient. The recovery of α-thujone ranged
between 60-77% and the compound appeared to remain stable over a two week
period. The retention times for dansyl α-thujone and dansyl β-thujone were 18 and
19.2 min respectively17.
5. Summary
In the proposed EC flavouring regulation thujone is limited to 10 mg/kg in alcoholic
beverages, except those produced from Artemisia species, where the limit will be 35
mg/kg.
Many accounts of extracting thujone from plant material were found, most using
hydrodistillation. The most recent reported method of extracting thujone from an
alcoholic beverage was in 2004, when the extraction was achieved by SPE and this
method was successfully used to extract thujone from Italian vermouth. Although the
methods of extraction were the same, subsequent methods of detection were very
different. One used GC-FID, with a limit of detection of 0.1 mg/kg and the other used
the derivatisation of the thujone with dansylhydrazine, followed by HPLC with
fluorescence detection, however the recovery was only 60-77%. HPLC with UV
detection was reported as obtaining a limit of detection of 0.5 mg/kg, with recovery
rates of around 100%, and the samples were prepared by dilution with methanol. This
level of sensitivity is more than adequate for the proposed limits.
- 87 -
6. References
(1) The Merck Index. Monograph Number 0009469. Thujone. 2004.
(2) Micali, G.; Lanuzza, F. HPLC Determination of alpha and beta thujone,
Potentially Toxic Components of Natural Flavourings, in Alcoholic
Beverages. Flavour and Fragrance Journal 1995, 10, 329-333.
(3) Sirisoma, N. S.; Hold, K. M.; Casida, J. E. alpha- and beta-thujones (herbal
medicines and food additives): Synthesis and analysis of hydroxy and dehydro
metabolites. Journal of Agricultural and Food Chemistry 2001, 49, 1915-
1921.
(4) Lightner, D. A.; Pak, C. S.; Crist, B. V.; Rodgers, S. L.; III, J. W. G. The
Octant Rule. Conformation and Circular Dichroism of syn and anti 2-
methylbicyclo[3.1.0]hexan-3-ones, Thujone and Isothujone. Tetrahedron
1985, 41, 4321-4330.
(5) WHO Food Additives Series 16 Thujone.
(6) Opinion of the Scientific Committee on Food on Thujone.
SCF/CS/FLAV/FLAVOUR/23 ADD2 Final. 2002.
(9) Proposal for a Regulation of the European Parliment and of the Council on
flavourings and certain food ingredients with flavouring properties for use in
and on foods. 2004.
2000.
(12) Chericoni, S.; Flamini, G.; Campeol, E.; Cioni, P. L.; Morelli, I. GC-MS
analyses of the essential oil from the aerial parts of Artemisia verlotiorum:
variability during the year. Biochemical Systematics and Ecology 2004, 32,
423-429.
1995, 43, 1890-1897.
(14) Chizzola, R.; Hochsteiner, W.; Hajek, S. GC analysis of essential oils in the
rumen fluid after incubation of Thuja orientalis twigs in the Rusitec system.
Research in Veterinary Science 2004, 76, 77-82.
(15) Valachovic, P.; Pechova, A.; Mason, T. J. Towards the industrial production
of medicinal tincture by ultrasound assisted extraction. Ultrasonics
Sonochemistry 2001, 8, 111-117.
(16) Emmert, J.; Sartor, G.; Sporer, F.; Gummersbach, J. Determination of alpha-
/beta-thujone and related terpenes in absinthe using solid phase extraction and
gas chromatography. Deutsche Lebensmittel-Rundschau 2004, 100, 352-356.
(17) Scott, P. M.; Lawrence, G. A.; Lau, B. P. Y. Determination of thujone by
derivatization with dansylhydrazine and liquid chromatography. Journal of
Liquid Chromatography & Related Technologies 2004, 27, 1071-1081.
- 88 -
(18) Wilkins, C. K.; Larsen, K. Identification of Volatile (Micro)Biological
Compounds from Household Waste and Building Materials by Thermal
Desorption-Capillary Gas Chromatography-Mass Spectroscopy. Journal of
High Resolution Chromatography 1995, 18, 373-377.
(19) von Rudloff, E. Gas-Liquid Chromatography of Terpenes VI. The Volatile Oil
of Thuja Plicata Donn. Phytochemistry 1962, 1, 195-202.
(20) Vernin, G. A. GC/MS analysis of Artemisia verlotiorum Lamotte oil. Journal
of Essential Oil Research 2000, 12, 143-146.
- 89 -
Concluding Remarks
In the preparation of this series of monographs it was decided that all the synonyms
and trade names should be included with the IUPAC name being highlighted for ease
of reference. Whenever a catalogue or registry number of a compound was found this
was also included along with the catalogue name. A pictorial representation is given
in the standard ‘flying wedge notation’, along with basic molecular information, such
as formula, weight, elemental composition and class of compound. All the physical
and chemical properties that could be found were included in a table summarising
these properties. These tables are as complete as the published data will allow and
include, where possible 1H and 13C NMR data for identification purposes.
As the source of the biological active principles (BAPs) is often varied, the species of
plants that contain the highest levels of each compound has been given, other plants
that may contain levels of each BAP has also been included and the content as a
percentage of dry or wet weight of the compound given where possible. There is not
an exhaustive list of every plant that contain all the BAPs considered. The background
to each compound gives information on the biochemical properties of the compound.
The limit set on each compound is given, both as the regulations as they are now and
the proposed new regulations.
A brief summary of the metabolism of each BAP is given, and/or known metabolites;
as this may aid in the identification of any unknowns or related structures that may
occur in a food sample. The analysis section first details any current known methods
of analysis e.g. those published by the AOAC, and then goes on to discuss methods of
extraction and quantitation. The summary then briefly describes the most relevant
methods published for the quantification of BAPs in foods and beverages.
While the advancement of analytical techniques has led to the ability of the analysts
being able to quantify a wide range of compounds, it is the sample preparation that is
often problematic. The lack of publications relating directly to the extraction of
compounds of interest from foodstuffs is very apparent. This also leads to difficulties
in making comparisons between methodologies. In most cases there simply are not
any references that refer to the extraction of the BAPs from food. Exceptions include
simultaneous distillation extraction (SDE) which is the most often cited sample
preparation technique. The apparatus for the extraction was designed by Likens and
Nickerson in 1964, in which the sample is boiled and the volatile compounds are
steam-distilled and simultaneously the solvent vapours are distilled through a
connecting tube. The vapours, along with the compounds, are condensed and
collected1. Variations of this method have including microdistillation, which is more
suitable to analytical procedures. This method has been used to obtain samples
containing estragole, methyleugenol and safrole from meat based products using
dichloromethane as a solvent2. It has also been used to recover pulegone and thujone
from honey3. Hexane was used as an alternative solvent in the same apparatus, which
extracted menthofuran and pulegone from plant materials4 and safrole and isosafrole
from meat based products5.
Due to the concern of using large quantities of organic solvents in many laboratories
there is an ongoing programme of research with the aim to decrease or remove the
need for such solvents. Although there a great many references to the techniques that
are being used, again, very few directly relate to foodstuffs. One method, supercritical
fluid extraction (SFE), greatly reduces the need for solvents. However, there is usually
- 90 -
no difference in the amounts of a compound recovered from a sample using this
method than those using solvents, e.g. methyleugenol and safrole from nutmeg and
mace6. The exception being the extraction of pulegone from peppermint7, but
estragole was more efficiently recovered by steam distillation than by SFE6. Solid-
phase microextraction (SPME) has been used to extract compounds from various
sources e.g. benzene and toluene in water8, 2,4,6-trichloroanisole in wine9, aroma
compounds in Swiss cheese10 and it has been used to isolate drugs and poisons from
blood, plasma, urine, hair and breath11. With regard to the BAPs in question this
method was five times more efficient at extracting pulegone from peppermint than
solvent based extraction and three times more efficient with menthofuran12. SPME
has been used to recover safrole from tobacco products13,14, methyleugenol from
human serum15 and estragole from avocado leaves6.
Ultrasonic assisted extraction was investigated with regard to the extraction of

thujone, in valerian plants there was no significant difference between this method
and solvent extraction but in sage there was a 25% increase in the amount of thujone
recovered16.
The methods for detecting BAPs in essential oils and plant extracts are well
documented; however, the application of these methods to food samples is not always
obvious. BAPs derived from beverages are usually detected by GC, for example β-
asarone in wine17, safrole and isosafrole in soft drinks18 and GC-FID for thujone in
beverages19. GC-FID was also used to quantify the amounts of menthofuran and
pulegone in confectionery and chewing gum4 and to measure estragole, safrole and
methyleugenol in meat based products2. Tobacco products analysed by GC/MS were
able to distinguish pulegone, coumarin and safrole13,14, and this method was also able
to detect and quantify methyleugenol in blood plasma20.
HPLC has also been widely used, although no method was the same for any of the
studies. Coumarin was detected in leaves from the grape vines21, in vanilla flavouring
products22 and in blood plasma23. Methods for safrole in perfume24, in nutmeg and
mace25 and in sassafras derived products26 all used HPLC. HPLC was used to analyse
samples from beverages for β-asarone27,28, isosafrole27, teucrin A29, quassine30, and
safrole27.
Enzyme immunoassay has been used in assessing compounds in foods; the most
widely used immunological technique is the enzyme linked immunosorbent assay.
This method can be highly specific due to the antigen-antibody interaction, usually
with good sensitivity and in some cases there is minimal sample preparation31. It has
been used to determine metabolites, toxins, antibiotics, hormones and microorganisms
in many different food matrices32. However, the only reference to using this technique
towards BAPs was its use in identifying quassine from spirit beverages33.
Most of the reported methods of analysis were able to record levels of sensitivity that
were within the limits for the proposed new flavouring regulations, however it must
be stated that very few methods were subjected to interlaboratory comparisons, the
exception being the study of β-asarones in wines34.
Stable isotope analysis has been used to demonstrate the authenticity of food
ingredients, such as fruit juices, wines and honey35. This method of analysis relies on
the on-line coupling of gas chromatography with isotope ratio mass spectrometry.
Stable isotope-labelled BAPs have been used as internal standards in some cases.
- 91 -
However, none of the BAPs are commercially readily available in stable isotope
labelled forms.
In conclusion, sensitive analytical methods currently exist for all the relevant BAPs.
They are sufficient, in principle, to enable enforcement of proposed new EC
legislation. However, there is a paucity of methods available to extract many of the
BAPs from food matrices. This is likely to be the main area where future research is
required. The lack of commercially available stable isotope analogues of the BAPs
will also limit the application of potentially useful mass spectrometry-based
quantitation methods and the synthesis of such standards as part of commissioned
research might be appropriate. In this context, the source of BAPs in foodstuffs may
become an issue and one approach to address this is based on the measurement of
stable isotope ratios. Thus far, this methodology has only been applied to a small
subset of BAPs and could usefully be explored for other BAPs.
(1) Chaintreau, A. Simultaneous distillation-extraction: from birth to maturity-

review. Flavour and Fragrance Journal 2001, 16, 136-148.
Food Chemistry 2003, 81, 469-475.
1995, 43, 1890-1897.
(4) Orav, A.; Kann, J. Determination of Peppermint and Orange Aroma
Compounds in Foods and Beverages. Proc. Estonian Acad. Sci. Chem 2001,
50, 217-225.
941-945.
(6) Ehlers, D.; Farber, J.; Martin, A.; Quirin, K. W.; Gerard, D. Analysis of
essential fennel oils - Comparison of CO2 extracts and steam-distilled oils.
Deutsche Lebensmittel-Rundschau 2000, 96, 330-335.
(7) Aghel, N.; Yamini, Y.; Hadjiakhoondi, A.; Pourmortazavi, S. M. Supercritical
carbon dioxide extraction of Mentha pulegium L. essential oil. Talanta 2004,
62, 407-411.
(8) Zhang, Z.; Pawliszyn, J. Headspace Solid-Phase Microextraction. Analytical
Chemistry 1993, 65, 1843-1852.
(9) Evans, T. J.; Butzke, C. E.; Ebeler, S. E. Analysis of 2,4,6-trichloroanisole in
wines using solid-phase microextraction coupled to gas chromatography-mass
spectrometry. journal of Chromatography A 1997, 786, 293-298.
(10) Jou, K. D.; Harper, W. J. Pattern recognition of Swiss cheese aroma
compounds by SPME/GC and an electronic nose. Milchwissenschaft 1998, 53,
259-263.
(11) Junting, L.; Peng, C.; Suzuki, O. Soild-phase microextraction (SPME) of
drugs and poisons from biological samples. Forensic Science International
1998, 97, 93-100.
(12) Rohloff, J. Monoterpene composition of essential oil from peppermint
(Mentha x piperita L.) with regard to leaf position using solid-phase
microextraction and gas chromatography/mass spectrometry analysis. Journal
- 92 -
317.
(15) Barr, D. B.; Barr, J. R.; Bailey, S. L.; Jnr, C. R. L.; Beeson, M. D. et al. Levels
of Methyleugenol in a Subset of Adults in the General U.S. Population as
Determined by High Resolution Mass Spectrometry. Environmental Health
Perspectives 2000, 108, 323-328.
(16) Valachovic, P.; Pechova, A.; Mason, T. J. Towards the industrial production
of medicinal tincture by ultrasound assisted extraction. Ultrasonics
Sonochemistry 2001, 8, 111-117.
(17) Dyer, R. H.; Martin, G. E.; Buscemi, P. C. Gas-Liquid-Chromatographic
Determination of Beta-Asarone in Wines and Flavors. Journal of the
Association of Official Analytical Chemists 1976, 59, 675-677.
Analysis 2001, 9, 27-32.
(19) Emmert, J.; Sartor, G.; Sporer, F.; Gummersbach, J. Determination of alpha-
/beta-thujone and related terpenes in absinthe using solid phase extraction and
gas chromatography. Deutsche Lebensmittel-Rundschau 2004, 100, 352-356.
(20) Barr, J. R.; Barr, D. B.; Needham, L. L. Quantification of methyleugenol in
human serum. Epidemiology 1999, 10, 478O.
(21) Villeneuve, F.; Abravanel, G.; Moutounet, M.; Alibert, G. General scheme of
analysis of phenolic compound in plant extracts by reversed-phase high-
performance liquid chromatography. Journal of Chromatography A 1982, 234,
131-140.
(22) Thompson, R. D.; Hoffmann, T. J. Determination of coumarin as an adulterant
in vanilla flavoring products by high-performance liquid chromatography.
Journal of Chromatography A 1988, 438, 369-382.
(23) Lamiable, D.; Vistelle, R.; Trenque, T.; Fay, R.; Millart, H. et al. Sensitive
high-performance liquid chromatographic method for the determination of
coumarin in plasma. Journal of Chromatography: Biomedical Applications
1993, 620, 273-277.
(24) Wisneski, H. H.; Yates, R. L.; Davis, H. M. High-performance liquid
chromatographic--fluorometric determination of safrole in perfume, cologne
and toilet water. Journal of Chromatography A 1983, 255, 455-461.
1988, 438, 117-121.
1997, 80, 1023-1028.
(28) Micali, G.; Lanuzza, F. HPLC Determination of alpha and beta thujone,
Potentially Toxic Components of Natural Flavourings, in Alcoholic
Beverages. Flavour and Fragrance Journal 1995, 10, 329-333.
(29) Bosisio, E.; Giavarini, F.; Dell'Agli, M.; Galli, G.; Galli, C. L. Analysis by
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Review of Methods of

AOAC Association of Official Analytical IOFI International Organization of the

CDCl3 chloroform-d LD50 dose at which 50% lethality occurs

DAD diode array detector m/z mass to charge ratio

EINECS European Inventory of Existing µg microgram

FEMA Flavor and Extract Manufacturers NMR nuclear magnetic resonance

GC gas chromatography SDE simultaneous distillation extraction

GC/MS Gas chromatography/ mass SPME solid phase micro

HCN hydrocyanic acid TLC thin layer chromatography

HPLC high performance liquid TDI tolerated daily intake

id internal diameter v/v volume/volume

IR infra red λem wavelength of emission

1.1 Synonyms and trade names

1.3 Structural and molecular formulae

Table 1. Structural and molecular information

Molecular Formula C12H16O3

1.4 Chemical and physical properties of the pure substance

Physical appearance Yellow liquid

2. Source, Background, Usage and Limits

food 0.1 mg/kg,

IOFI recommends the use of GC in β-asarone determination, the recommended

H3CO C CH3 H3CO C CH3

H3CO OCH3 H3CO OCH3

Scheme 1. The proposed metabolic pathways for β-asarone9,13.

4.1 Extraction Methods

Levels of β-asarone were determined in wine samples by GC, following steam

The same GC method was evaluated in a collaborative study between nine

Table 3. Summary of statistical analysis of results from a collaborative study of β-

Value All samples

Steam distillation of alcoholic beverages (adjusted with water to an ethanol

Reversed-phase HPLC was also used to measure β-asarone in alcoholic beverages

The two commonly occurring α- and β- isomers of asarone can be separated by GC

1.1 Synonyms and trade names

1.3 Structural and molecular formulae

Table 1. Structural and molecular information

Molecular formula C9H6O2

1.4 Chemical and physical properties of the pure substance

Physical appearance Colourless flakes with a vanilla-like

2. Source, Background, Usage and Limits

The European Committee of Experts have stated that as there is no quantitative

Figure 1. Coumarin and associated metabolites.

4.1 Extraction Methods

Five compounds, including coumarin, were extracted from 10 mL samples of

Simultaneous distillation-extraction (SDE) processes make use of the equipment

Various methods of extraction of a medicinal plant, ‘guaco’, were compared with

Reverse-phase high performance liquid chromatography (HPLC) was used in the

Levels of coumarin in blood plasma were determined by measuring UV absorbance at

Concerns that analytical methods ‘based on chromatographic techniques and

A comparison of HPLC and spectrofluorimetry was carried out in distilled beverages.

Coumarin is a poorly fluorescent molecule that is known to convert to the highly

1.1 Synonyms and trade names

1.3 Structural and molecular formulae

Molecular Formula C10H12O

1.4 Chemical and physical properties of the pure substance

Physical appearance Colourless liquid at room temperature

2. Background, Usage and Limits

Plant oil % of estragole found in essential oil

4.1 Extraction methods

Simultaneous distillation-extraction with a Likens-Nickerson apparatus and

Scheme 1. A summary of the metabolism of estragole6,10,11.

tomato sauce 0.81 mg/kg

1.1 Synonyms and trade names

1.3 Structural and molecular formulae