EUROPEAN PHARMACOPOEIA 5.

0 Eugenol
helium for chromatography R as the carrier gas at a flow
rate of 1.5 ml/min,
a flame-ionisation detector,
a split ratio of 1:100,
maintaining the temperature of the column at 60 C for
5 min, then raising the temperature at a rate of 5 C/min to
200 C and maintaining at 200 C for 5 min; maintaining
the temperature of the injection port and that of the detector
at 220 C.
Inject about 0.5 l of the reference solution. When the
chromatogram is recorded in the prescribed conditions the
components elute in the order indicated in the composition
of the reference solution. Record the retention times of
these substances.
The assay is not valid unless: the number of theoretical
plates calculated for the peak due to limonene at 110 C
is at least 30 000; the resolution between the peaks
corresponding to limonene and cineole is at least 1.5.
Inject about 0.5 l of the test solution. Using the
retention times determined from the chromatogram obtained
with the reference solution, locate the components of the
reference solution on the chromatogram obtained with the
test solution.
Determine the percentage content of these components by
the normalisation procedure.
The percentages are within the following ranges:
-pinene: traces to 9.0 per cent,
-pinene: less than 1.5 per cent,
-phellandrene: less than 1.5 per cent,
limonene: traces to 12.0 per cent,
1,8-cineole: minimum 70.0 per cent,
camphor: less than 0.1 per cent.
STORAGE
In a well-filled, airtight container, protected from light and
at a temperature not exceeding 25 C.
01/2005:1100
EUGENOL
Eugenolum
C
10
H
12
O
2
M
r
164.2
DEFINITION
Eugenol is 2-methoxy-4-(prop-2-enyl)phenol.
CHARACTERS
A colourless or pale yellow, clear liquid, darkening on
exposure to air, with a strong odour of clove, practically
insoluble in water, freely soluble in alcohol (70 per cent V/V),
practically insoluble in glycerol, miscible with glacial acetic
acid, with alcohol, with fatty oils and with methylene
chloride.
IDENTIFICATION
First identification: B.
Second identification: A, C, D.
A. It complies with the test for refractive index (see Tests).
B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
eugenol CRS.
C. Examine by thin-layer chromatography (2.2.27), using a
TLC silica gel F
254
plate R.
Test solution. Dissolve 50 l of the substance to be
examined in alcohol R and dilute to 25 ml with the same
solvent.
Reference solution. Dissolve 50 l of eugenol CRS in
alcohol R and dilute to 25 ml with the same solvent.
Apply to the plate 5 l of each solution. Develop over a
path of 15 cm using a mixture of 10 volumes of ethyl
acetate R and 90 volumes of toluene R. Dry the plate in
a current of cold air and examine in ultraviolet light at
254 nm. The principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution. Spray with anisaldehyde solution R.
Heat at 100-105 C for 10 min. The principal spot in the
chromatogram obtained with the test solution is similar
in position, colour and size to the principal spot in the
chromatogram obtained with the reference solution.
D. Dissolve 0.05 ml in 2 ml of alcohol R and add 0.1 ml
of ferric chloride solution R1. A dark green colour is
produced which changes to yellowish-green within 10 min.
TESTS
Relative density (2.2.5) : 1.066 to 1.070.
Refractive index (2.2.6) : 1.540 to 1.542.
Dimeric and oligomeric compounds. Dissolve 0.150 g of
the substance to be examined in ethanol R and dilute to
100.0 ml with the same solvent. The absorbance of the
solution (2.2.25) at 330 nm is not greater than 0.25.
Related substances. Examine by gas chromatography
(2.2.28).
Test solution. Dissolve 1.00 g of the substance to be
examined in ethanol R and dilute to 5.0 ml with the same
solvent.
Reference solution (a). Dilute 1.0 ml of the test solution to
100.0 ml with ethanol R.
Reference solution (b). Dissolve 50 mg of vanillin R in 1 ml
of the test solution and dilute to 5 ml with ethanol R.
The chromatographic procedure may be carried out using:
a fused-silica capillary column 30 m long and
0.25 mm in internal diameter coated with a film of
polymethylphenylsiloxane R (film thickness 0.25 m),
helium for chromatography R as the carrier gas at a flow
rate of 1 ml/min,
a flame-ionisation detector,
a split ratio of 1:40,
Time
(min)
Temperature
(C)
Rate
(C/min)
Comment
Column 0 - 2 80 isothermal
2 - 27 80 280 8 linear gradient
27 - 47 280 isothermal
Injection port 250
Detector 280
Inject 1 l of the test solution and 1 l each of reference
solutions (a) and (b). The test is not valid unless in the
chromatogram obtained with reference solution (b), the
relative retention time of the peak due to vanillin is at least
1.1 with respect to the peak due to eugenol. Calculate the
percentage content of related substances from the areas
General Notices (1) apply to all monographs and other texts 1571
Eugenol EUROPEAN PHARMACOPOEIA 5.0
of the peaks in the chromatogram obtained with the test
solution by the normalisation procedure; disregard the
solvent peak and any peak with an area less than 0.05 times
that of the principal peak in the chromatogram obtained
with reference solution (a). The content of related substances
with a relative retention time greater than 2.0 in respect to
the main peak is not greater than 1.0 per cent ; the content
of any related substances is not greater than 0.5 per cent ;
the total content of related substances is not greater than
3.0 per cent.
Hydrocarbons. Dissolve 1 ml in 5 ml of dilute sodium
hydroxide solution R and add 30 ml of water R in a
stoppered test-tube. Examined immediately, the solution is
yellow and clear (2.2.1).
Sulphated ash (2.4.14). Not more than 0.1 per cent,
determined on 1.0 g.
STORAGE
Store in a well-filled container, protected from light.
IMPURITIES
A. (1R,4E,9S)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]un-
dec-4-ene (-caryophyllene),
B. (1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene
(-humulene, -caryophyllene),
C. (1R,4R,6R,10S)-4,12,12-trimethyl-9-methylene-5-
oxatricyclo[8.2.0.0
4,6
]dodecane (-caryophyllene oxide),
D. R1 = H, R2 = H, R3 = CH
2
-CH=CH
2
: 4-(prop-2-enyl)phenol,
E. R1 = CH
3
, R2 = OCH
3
, R3 = CH
2
-CH=CH
2
:
1,2-dimethoxy-4-(prop-2-enyl)benzene (eugenol methyl
ether),
F. R1 = H, R2 = OCH
3
, R3 = CH=CH-CH
3
(cis) :
2-methoxy-4-[(Z)-prop-1-enyl]phenol (cis-isoeugenol),
G. R1 = H, R2 = OCH
3
, R3 = CH=CH-CH
3
(trans) :
2-methoxy-4-[(E)-prop-1-enyl]phenol (trans-isoeugenol),
H. R1 = H, R2 = OCH
3
, R3 = CHO: 4-hydroxy-3-
methoxybenzaldehyde (vanillin),
I. R1 = CO-CH
3
, R2 = OCH
3
, R3 = CH
2
-CH=CH
2
:
2-methoxy-4-(prop-2-enyl)phenyl acetate (acetyleugenol),
J. R1 = H, R2 = OCH
3
, R3 = CO-CH=CH
2
:
1-(4-hydroxy-3-methoxyphenyl)prop-2-enone,
K. R1 = H, R2 = OCH
3
, R3 = CH=CH-CHO:
(E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enal
(trans-coniferyl aldehyde),
L. 2-methoxy-4-[3-methyl-5-(prop-2-enyl)-2,3-
dihydrobenzofuran-2-yl]phenol (dehydrodi-isoeugenol),
M. 3,3-dimethoxy-5,5-bis(prop-2-enyl)biphenyl-2,2-diol
(dehydrodieugenol),
N. O. two further unknown dimeric compounds,
P. toluene.
1572 See the information section on general monographs (cover pages)