Quantitative analysis of α-pinene and β-myrcene in mastic gum oil using FT-Raman spectroscopy

Abstract

α-Pinene and β-myrcene are compounds that are contained in mastic gum in high concentrations. The β-myrcene percentage determines the marketability of mastic gums. The chemical composition of mastic gum oil of a representative resin quality was evaluated by gas chromatography–mass spectrometry (GC–MS) technique. FT-Raman spectroscopy, based on band intensity measurements, was used for the determination of α-pinene and β-myrcene content in mastic gum. Bands at 1658 and 1633 cm−1 were used for the calibration of α-pinene and β-myrcene, respectively. Calibration curves were linear (correlation coefficient for α-pinene was 0.992 and 0.997 for β-myrcene) in the range 30–80 and 3–45%, respectively. Normalization of calibration curves, against the 802 cm−1 cyclohexane band, minimized the effect of laser beam power fluctuations. The proposed method is rapid and simple. Accordingly, mastic gum oils from Chios island (Greece) contained 38.1–69.5% α-pinene and 4.5–57.9% β-myrcene.

Full text

Analytical, Nutritional and Clinical Methods Section
Quantitative analysis of a-pinene and b-myrcene in mastic gum oil
using FT-Raman spectroscopy
D. Daferera, C. Pappas, P.A. Tarantilis, M. Polissiou*
Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 75 Iera Odos, 118 55 Athens, Greece
Received 7 August 2001; received in revised form 22 October 2001; accepted 22 October 2001
Abstract
a-Pinene and b-myrcene are compounds that are contained in mastic gum in high concentrations. The b-myrcene percentage
determines the marketability of mastic gums. The chemical composition of mastic gum oil of a representative resin quality was
evaluated by gas chromatography–mass spectrometry (GC–MS) technique. FT-Raman spectroscopy, based on band intensity
measurements, was used for the determination of a-pinene and b-myrcene content in mastic gum. Bands at 1658 and 1633 cm
1
were used for the calibration of a-pinene and b-myrcene, respectively. Calibration curves were linear (correlation coefficient for a-
pinene was 0.992 and 0.997 for b-myrcene) in the range 30–80 and 3–45%, respectively. Normalization of calibration curves,
against the 802 cm
1
cyclohexane band, minimized the effect of laser beam power fluctuations. The proposed method is rapid and
simple. Accordingly, mastic gum oils from Chios island (Greece) contained 38.1–69.5% a-pinene and 4.5–57.9% b-myrcene. #2002
Elsevier Science Ltd. All rights reserved.
Keywords: a-Pinene, b-Myrcene, Determination, FT-Raman
1. Introduction
Pistacia lentiscus var. Chia, is a tree belonging to the
Anacardiaceae family, which is traditionally cultivated
in south Chios, a Greek island of the east Aegean sea.
Every year, from July to October, on the trunk of the
tree, cuttings are made and a resinous liquid substance
is exuded. This material, remaining under the tree for
many days, is coagulated by the local environmental
conditions. The coagulated product is collected and is
called gum mastic or ‘‘masticha’’ (Perikos, 1993). Mas-
ticha has numerous usages itself, but is also used in
producing gum oil by the steam-distillation method.
Gum mastic oil is used in cosmetics and perfumery, as a
flavouring in food technology and for its antimicrobial
activity, and especially against Helicobacter pylori
(Huwez, Thirwell, Cockayne, & Ala’Alden, 1998;
Magiatis, Melliou, Skaltsounis, Chinou, & Mitaku,
1999; Perikos, 1993).
The gum essential oil chemical composition varies and
depends on the gum quality. The gum quality is influ-
enced by its purity, the collection time and the duration
between exudation from the trunk and the collection.
Studies on the chemical composition of the gum oil
showed the predominant presence of monoterpenes,
a-pinene and b-myrcene, which constitute the majority
of the oil. The a-pinene and b-myrcene contents in
mastic gum oil are typically measured by gas chroma-
tography (GC) (Papageorgiou, Mellidis, & Argyriadou,
1991). Time-consumption and the prior sample hand-
ling are the basic disadvantages of the method. There is
a proportion between the concentrations of a-pinene
and b-myrcene, which characterizes the authenticity of
the gum essential oil. Percentages of about 60–80% for
a-pinene and 7–20% for b-myrcene represent an accep-
table oil quality. Increases of b-myrcene in the oil mix-
ture devalue its quality.
FT-Raman spectroscopy is an analytical technique
based on the interaction of an incident monochromatic
radiation with vibrational energy levels of molecules. It
is widely used for qualitative comparisons between
samples. The concentration of a compound in a mixture
is relatable to the Raman intensities if an appropriate
0308-8146/02/$ - see front matter #2002 Elsevier Science Ltd. All rights reserved.
PII: S0308-8146(01)00382-X
Food Chemistry 77 (2002) 511–515
www.elsevier.com/locate/foodchem
* Corresponding author: Tel.: +30-152-942-41; fax: +30-152-942-65.
E-mail address: mopol@aua.gr (M. Polissiou).

reference material is used to determine its value (Free-
man & Mayo, 1969; Hancewicz & Petty, 1995; Skoulika,
Georgiou, & Polissiou, 2000; Sun, Ibrahim, Oldham,
Schultz, & Conners, 1997). FT-Raman spectra of a-pinene
have been recorded in order to determined the quanti-
tative relationship between vibrational circular dichro-
ism (VCD) and Raman optical activity (ROA) spectra
(Qu, Lee, Yu, Freedman, & Nafie, 1996).
This paper describes the development of a method for
quantitative analysis of a-pinene and b-myrcene in
mastic gum oil by FT-Raman spectroscopy. The recor-
ded FT-Raman spectra of the gum mastic oils allow us
to correlate their chemical compositions with the per-
centages of a-pinene and b-myrcene. The proposed
method is simple, rapid and nondestructive for the
sample and all samples are measured as neat liquids
without further treatment.
2. Materials and methods
2.1. Materials
A sample of first quality mastic gum and 10 mastic
gum oils from resins collected over different durations
were kindly provided by the Chios gum mastic growers’
association. The 10 samples were randomly character-
ized as ‘‘oil 1’’, ‘‘oil 2’’, ‘‘oil 3’’, ‘‘oil 4’’, ‘‘oil 5’’, ‘‘oil 6’’,
‘‘oil 7’’, ‘‘oil 8’’, ‘‘oil 9’’ and oil 10’’. Standards of
a-pinene and b-myrcene were purchased from the
Sigma-Aldrich Co.
2.2. Isolation of essential oil from the mastic gum
A typical essential oil was isolated according to the
Likens–Nickerson’s method, using a micro steam dis-
tillation extraction apparatus for organic solvents
lighter than water (Daferera, Ziogas, & Polissiou, 2000).
In that way, all the aroma constituents were con-
centrated in the extracting solvent. The procedure was
protected by placing inert gas (N
2
) in the main body of
the apparatus in order to avoid creating oxidized by-
products. The diethylether extract was stored at 4 C
until its analysis by gas chromatography–mass spectro-
metry (GC–MS).
2.3. GC–MS analysis conditions
The analysis of the typical gum essential oil was per-
formed using a Hewlett Packard 5890 II GC, equipped
with a HP-5 capillary column (30 m, 0.25 mm id, 0.25
mm film thickness) and a mass spectrometer 5971 A as
detector. The carrier gas, helium, was stable at 2.5 psi.
Column temperature was initially kept for 3 min at
40 C, then gradually increased to 180 Cat3C/min
and finally increased to 270 Cat30C/min. Injector
and detector (MS transfer line) temperatures were set at
220 and 290 C, respectively. For GC–MS detection, an
electron ionization system was used with ionization
energy of 70 eV. The extract diluted 1:5 (v/v) with di-
ethylether and 1.0 ml of the diluted sample was injected
automatically and split-less.
The samples from the Chios gum mastic growers’
association were also analyzed by the above GC–MS
method in order to determine the a-pinene and b-myr-
cene. The samples diluted with dichloromethane (1:100,
v/v) and 1 ml of the diluted sample was injected auto-
matically and split less.
2.4. FT-Raman spectroscopy
Standard solutions were prepared using the stan-
dards, a-pinene and b-myrcene, in dichloromethane.
FT-Raman spectra of cyclohexane, standard solu-
tions, and ten mastic gum oils from the Chios gum
mastic growers’ association were recorded with a Nico-
let 750 FT-Raman spectrometer, equipped with a
Nd:YAG laser source that emits at 1064 nm. A calcium
fluorine (CaF
2
) beam splitter, an indium-gallium
arsenide (InGaAs) detector and 180backscattering
geometry are used in the spectrometer. An optical bench
alignment was performed before each batch of mea-
surements to ensure that the spectrometer was fine-
tuned and the detector signal maximized. Sample cells
used were cut to 6 cm from Wimad WG-5M NMR
tubes of 4.97 mm outer diameter and 0.38 mm wall
thickness. A motorized positioner focuses the laser
beam to the sample and a manual side-to-side adjuster
allows sample adjustment for maximum optical effi-
ciency. Spectra were accumulated from 100 scans col-
lected during 3 min at a resolution of 4 cm
1
.
The FT-Raman spectra were smoothed and their
baselines were corrected using the ‘‘automatic smooth’’
and the ‘‘automatic baseline correct’’ functions of the
built-in software of the spectrophotometer (OMNIC
3.1). Then the intensities of the 1658 and 1633 cm
1
peaks were measured. The intensity of the 802 cm
1
cyclohexane peak was measured as well.
3. Results and discussion
3.1. GC–MS results
A typical total ion chromatogram (TIC) of the mastic
gum oil, isolated by the Likens-Nickerson method, is
presented in Fig. 1. This mastic gum oil is characterized
by the presence of a-pinene (72.1%), b-pinene (2.9%),
b-myrcene (16.5%), limonene (1.0%), linalool (1.0%)
and caryophyllene (1.1%). All other compounds were
present at relative concentrations less than 1.0% in the
mixture (Table 1).
512 D. Daferera et al. / Food Chemistry 77 (2002) 511–515

Table 2 shows the percentages of a-pinene and
b-myrcene in the samples from the Chios gum mastic
growers’ association, as determined by the GC–MS
analysis. The contents (%) of a-pinene and b-myrcene
in mastic gum oils were 33.7–72.8 and 3.8–63.5,
respectively.
The chemical composition of the oils was determined
by comparing the mass spectra of oil components with
those of mass spectra from the NBS75K data library.
The main components of the oil (a-pinene and b-myr-
cene) were also determined by comparison of their rela-
tive retention times with those of standards.
3.2. FT-Raman results
The FT-Raman spectrum (Fig. 2) of a-pinene showed
characteristic peaks at 1658 cm
1
(C¼C of cyclohex-
ene), 1436 cm
1
(C–H) and 667 cm
1
(C–C ring
breathing), b-myrcene at 1633 cm
1
(R
1
R
2
C¼CH
2
and
R
1
R
2
C¼CHR
3
) (Bour, 1998; Freeman & Mayo, 1969)
and of cyclohexane at 802 cm
1
(C–C, cyclohexane
chair form). The FT-Raman spectrum of mastic gum oil
showed characteristic peaks at 1658 cm
1
, assigned
mainly to a-pinene and at 1633 cm
1
, assigned to b-
myrcene. It was observed that the Raman intensities of
Fig. 1. A representative TIC of gum oil isolated with diethylether by the Likens–Nickerson method.
Table 1
Chemical composition of the essential oil from gum of Pistacia lentiscus var. chia isolated by the Likens–Nickerson method
A/A Retention time (min) Component Composition (%)
1 11.70 4-methylene-1-(1-methylethyl)-bicyclo[3.1.0]hexane(sabinene or 4(10) thujene) 0.6
2 12.82 2,6,6-trimethyl-bicyclo[3.1.1]hept-2-ene(a-pinene) 72.1
3 13.22 2,2-dimethyl-3-methylene-bicyclo[2.2.1]heptane(camphene) 0.7
5 14.62 6,6-dimethyl-2-methylene-bicyclo[3.1.1]heptane(b-pinene) 2.9
6 15.67 7-methyl-3-methylene-1,6-octadiene (b-myrcene) 16.5
7 16.44 1-methoxy-2-methyl-benzene (o-cresol- methyl-ether) 0.7
8 17.16 1-methyl-4-(1-methylethyl)-benzene(p-cymene) 0.2
9 17.36 1-methyl-4-(1-methylethenyl)cyclohexene (limonene) 1.0
10 21.21 3,7-dimethyl-1,6-octadien-3-ol (linalool) 1.0
11 22.48 2,2,3-trimethyl-3-cyclopentene-1-acetaldehyde(a-campholene aldehyde) 0.3
12 23.10 6,6-dimethyl-2-methylene-bicyclo[3.1.1]heptan-3-ol 0.3
13 23.27 4,6,6-trimethyl-bicyclo[3.1.1]hept-3-en-2-ol <0.1
17 25.79 a,a,4-trimethyl-3-cyclohexene-1-methanol (a-terpineol) 0.1
18 26.07 6,6-dimethyl-bicyclo[3.1.1]hept-2-ene-2-methanol 0.2
19 26.74 4,6,6-trimethyl-bicyclo[3.1.1]hept-3-en-2-one(d-verbenone) 0.2
20 30.41 1-methoxy-4-(1-propenyl)-benzene (anethole) 0.1
21 36.63 4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene(caryophyllene) 1.1
22 38.11 2,6,6,9-tetramethyl-1,4,8-cycloundecatriene(a-caryophyllene or a-humulene) 0.1
23 39.96 1,2-dimethoxy-4-(1-propenyl)-benzene(methyl-isoeugenol) <0.1
Others not identified 1.7
Total 100
D. Daferera et al. / Food Chemistry 77 (2002) 511–515 513

the same sample fluctuated after closing–opening of the
Raman source. Normalization minimizes the effect of
laser power fluctuations (Skoulika et al., 2000). So the
percent relative intensities of 1658 and 1633 cm
1
to the
802 cm
1
cyclohexane were measured for every stan-
dard solution and oil sample. Percent normalized inten-
sities of 1658 cm
1
(I
1
) and 1633 cm
1
(I
2
) were
correlated with a-pinene and b-myrcene contents,
respectively. There were two linear relationships: one
between I
1
and a-pinene content, and a second between
I
2
and b-myrcene (Tables 3 and 4; Figs. 3 and 4).
Empirical equations of calibration curves are,
Table 2
The percentages of a-pinene and b-myrcene in samples from the Chios
gum mastic growers’ association, as determined by GC–MS analysis
Mastic gum oil a-pinene (%) b-myrcene (%)
Oil 1 70.4 22.9
Oil 2 72.8 6.0
Oil 3 65.6 26.9
Oil 4 59.1 33.5
Oil 5 63.2 28.6
Oil 6 54.0 3.8
Oil 7 68.4 25.4
Oil 8 64.9 28.3
Oil 9 71.5 19.4
Oil 10 33.7 63.5
Fig. 2. FT-Raman spectra (1800–600 cm
1
) of mastic gum oil, a-pinene, b-myrcene and cyclohexane.
Fig. 4. The calibration curve of b-myrcene.Fig. 3. The calibration curve of a-pinene.
Table 3
Contents (%) of a-pinene standards correlated with normalized inten-
sity at 1658 cm
1
Content (%)
of a-pinene
(%) Normalized intensity
at 1658 cm
1
S.D. (n=3)
30.0 10.90.4
40.0 13.00.8
50.0 14.50.2
60.0 16.70.6
70.0 19.10.4
80.0 21.80.9
514 D. Daferera et al. / Food Chemistry 77 (2002) 511–515

for a-pinene:
I1¼ð4:20:6Þþð0:21 0:01Þa-pinene ð%Þ
ðr¼0:882;n¼3Þ
for b-myrcene:
I2¼ð3:30:8Þþð1:11 0:03Þb-myrcene ð%Þ
ðr¼0:997;n¼3Þ
The % RSD fluctuated from 1.4 to 6.2% for the cali-
bration curve of a-pinene and 1.3 to 2.9% for the cali-
bration curve of b-myrcene.
Contents (%) of a-pinene and b-myrcene of mastic
gum oils of Chios were measured by using the earlier
empirical equations (Table 5). According to the pro-
posed method, the mastic gum oils contained 38.1–
69.5% a-pinene and 4.5–57.9% b-myrcene. The % RSD
fluctuated from 1.5 to 5.3% for a-pinene and 1.2 to
3.1% for b-myrcene.
The large range of a-pinene and b-myrcene percen-
tages was due to the collection time of resin and the
duration between its exudation from the trunk and the
collection. The concentration of b-myrcene was
increased and exceeded a-pinene in resins collected
immediately (oil 10), decreased to less than 20%, in
resins left to mature physiologically over a maximum
time of 2 months (‘‘oil 1’’, ‘‘oil 2’’, ‘‘oil 6’’, ‘‘oil 9’’) and
was intermediate in other cases: ‘‘oil 3’’, ‘‘oil 4’’, ‘‘oil 5’’,
‘‘oil 7’’, ‘‘oil 8’’.
In conclusion, comparison of the GC–MS and FT-
Raman methods shows that the results are similar. The
quantitative analysis of a-pinene and b-myrcene in gum
oil can be determined by FT-Raman spectroscopy. The
main advantage of this method over the existing GC–
MS method is its simplicity, immediacy, speed and
being non-destructive to the sample.
Acknowledgements
We thank ‘‘The Chios Gum Mastic Growers Asso-
ciation’’ for providing of samples and the Hellenic
Ministry of Development, General Secretarial of
Research and Technology for financial support.
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Table 4
Contents (%) of b-myrcene standards correlated with normalized
intensity at 1633 cm
1
Content (%) of
b-myrcene
(%) Normalized intensity
at 1633 cm
1
S.D. (n=3)
3.0 6.20.1
10.0 14.10.4
18.0 23.80.7
25.0 32.60.5
35.0 41.00.7
45.0 53.30.7
65.0 81.20.3
Table 5
Contents (%) of a-pinene and b-myrcene of mastic gum oils measured
by the FT-Raman spectroscopic method
Mastic
gum oil
Normalized
intensity
at 1658 cm
1
Normalized
intensity
at 1633 cm
1
a-Pinene
content (%)
b-Myrcene
content (%)
Oil 1 17.5 23.8 63.31.4 18.5 0.2
Oil 2 18.8 11.3 69.51.9 7.2 0.8
Oil 3 17.8 34.3 64.81.1 27.9 0.7
Oil 4 17.7 39.0 64.33.4 32.2 1.0
Oil 5 16.4 32.5 58.11.2 26.3 0.5
Oil 6 14.5 8.3 49.0 0.8 4.5 0.1
Oil 7 18.1 31.7 66.21.0 25.6 0.6
Oil 8 17.8 33.5 64.81.6 27.2 0.5
Oil 9 16.4 22.4 58.11.7 17.2 0.2
Oil 10 12.2 67.6 38.11.2 57.9 1.7
D. Daferera et al. / Food Chemistry 77 (2002) 511–515 515