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Quantification of Eugenol in Hydro-Distilled Clove Oil (Eugenia

caryophyllus) and Its Marketed Products by Validated GC-MS
Method

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DOI: 10.1080/10496475.2013.807902

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Quantification of Eugenol in Hydro-

Distilled Clove Oil (Eugenia caryophyllus)
and Its Marketed Products by Validated
GC-MS Method
a a a
MD Tanwir Athar , Ennus Tajuddin Tamboli , S. H. Ansari &
a
Sayeed Ahmad
a
Bioactive Natural Product Laboratory, Department of
Pharmacognosy and Phytochemistry, Faculty of Pharmacy , Hamdard
University , Hamdard Nagar , New Delhi , India

To cite this article: MD Tanwir Athar , Ennus Tajuddin Tamboli , S. H. Ansari & Sayeed Ahmad (2013)
Quantification of Eugenol in Hydro-Distilled Clove Oil (Eugenia caryophyllus) and Its Marketed
Products by Validated GC-MS Method, Journal of Herbs, Spices & Medicinal Plants, 19:4, 365-376, DOI:
10.1080/10496475.2013.807902

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 Journal of Herbs, Spices & Medicinal Plants, 19:365–376, 2013
Copyright © Taylor & Francis Group, LLC
ISSN: 1049-6475 print/1540-3580 online
DOI: 10.1080/10496475.2013.807902

Quantification of Eugenol in Hydro-Distilled

Clove Oil (Eugenia caryophyllus) and Its
Marketed Products by Validated GC-MS Method

MD TANWIR ATHAR, ENNUS TAJUDDIN TAMBOLI,

S. H. ANSARI, and SAYEED AHMAD
Bioactive Natural Product Laboratory, Department of Pharmacognosy and Phytochemistry,
Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi, India
Downloaded by [89.237.153.223] at 22:53 29 September 2013

A gas chromatography/mass spectrometric (GC-MS) method was

developed and validated for the analysis of eugenol in clove
( Eugenia caryophyllus) in hydro-distilled oils and marketed for-
mulations. Separation was done on HP5 MS column (5% phenyl
polymethyl siloxane; 30 m × 0.25 mm i.d. and 0.25-µm film thick-
ness), using the EI/CI MSD as the detector and helium at a flow rate
of 1 mL.min−1 as the carrier gas. The method was validated as per
the ICH guidelines for linearity, precision, accuracy, robustness,
LOD, and LOQ. The eugenol in marketed formulations of clove oil
analyzed and validated by this method ranged from 3% to 48%.

KEYWORDS quantitative analysis, volatile oil, steam distillation

INTRODUCTION

Clove oil is distilled from the flowers, stems, and leaves of the clove tree
(Eugenia aromatica or E. caryophyllus, Myrtaceae) (3,13). It is colorless or
pale yellow liquid when freshly prepared, becoming thicker and darker after
exposure to air, containing not less than 85% and not more than 95% w/w of
phenolic substance (10). The chief constituent is eugenol [(2-methoxy-4-(2-
propenyl) phenol], which has annual worldwide production of ∼22,000 kg
(5). It is the main active substance of the clove oil (6), categorized as a
safe food additive by the FDA (3). Eugenol is slightly water-soluble and is

Received October 24, 2012.

Address correspondence to Sayeed Ahmad, Bioactive Natural Product Laboratory,
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Hamdard
University, Hamdard Nagar, New Delhi, India-110062. E-mail: sahmad_jh@yahoo.co.in

365
 366 M. T. Athar et al.

miscible with alcohol. It is as potent and antiseptic as phenol, with fewer

irritant properties. It also acts as an antibacterial agent. E. jambolan is a
constituent of antidiabetic capsule patented in the U.S. patent office (U.S.
Patent no 20020025349) (11). Eugenol has been used in periodontal packs
in applied to denuded areas of tissue after gingivectomy (15).
Methods reported for the estimation of eugenol include those based
on non-aqueous titration (7), high-performance liquid chromatography (8),
HPTLC (14), and GC (12,16). The present method was an attempt to develop
a fully validated gas chromatography mass spectroscopy (GC-MS) method
for quantification of eugenol in herbal formulations. GC-MS is a useful tool
for identification and quantification of volatile constituents because of its
simplicity, non-requirement of liquid mobile phase, cost–effectiveness, and
direct matching and identification of each constituent from the library.
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MATERIAL AND METHODS

Chemicals and Reagents
Eugenol standard 99.8% (Sigma), methanol (MS grade Fluka), and inert
helium gas (Sigma Gases and Services Ltd, New Delhi) were used for the
analysis. The dried flower buds of E. caryophyllus were from the Khari baoli,
New Delhi.

Instrumentation
GSMS analysis of the standard eugenol and formulations were carried out
on a GC-MS system (Agilent 7890A series, Germany) equipped with split-
splitless injector and CTC-PAL auto-sampler attached to an apolar HP-5MS
(5% phenyl polymethyl siloxane) capillary column (30 m × 0.25 mm i.d. and
0.25-µm film thickness) and fitted to a mass detector. Carrier gas flow rate
(He) was 1 mL.min−1 , split ratio 1:100, injector temperature at 260◦ C, detector
temperature at 300◦ C, while column temperature was kept at 70◦ C for 2 min
followed by linear programming from 70◦ to 230◦ C (at 5◦ C.min−1 ), and kept
isothermal for 2 min. A transfer line was heated at 280◦ C. Mass spectra were
acquired in EI mode (70 eV); in range 30 to 600 m/z, and 2 µL of sample
in methanol was injected. The components of the oil and standard were
identified by comparison of mass spectra to those from NIST/NBS libraries,
using search engines.

Extraction of Volatile Oil by Steam Distillation

A Clavenger apparatus was used to extract volatiles; 120 g of coarsely ground
dried flower buds were taken in a 250-mL round-bottom flask, and water
was added to approximately three-fourths full. The flask was heated using
 Eugenol in Hydro-Distilled Clove Oil 367

a heating mantle in order to boil the water. The volatile oil along with the
water vapor condensed in the condenser and accumulated in a graduated
side arm of the Clavenger apparatus. Distillation was continued until there
was no difference in successive readings of the oil volume. The oil was
drained, dried over anhydrous sodium sulfate, filtered through 0.22-µM filter
paper, and kept at 4◦ C in sealed vials in dark.

Formulation Sample Preparation

Twenty-five µL of clove oil was diluted up to 5 mL using MS-grade methanol
followed by vortex shaking for 1 min. Samples were transferred to GC-MS
vials and placed on tray 1 of CTC PAL for injection.
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Preparation of Standard Drugs Solution

Concentrations of standard eugenol (99.8%) at 1,000, 800, 400, 200, 100, 80,
40, 20, and 10 µL.L−1 were used to develop a standard plot taking total ion
chromatogram (TIC) of each spectrum.

RESULTS
Optimization of GC-MS Method
GC method was optimized by varying the column type and oven tempera-
ture. The HP-5MS column produced good resolution of eugenol in a short
time using gradient oven temperature. The fragmented ions were separated
by the analyzer, according to their mass-to-charge ratio.

Interpretation of Electron Ionization Mass Spectra

A reliable interpretation of EI mass spectra was made for standard eugenol
with a retention time of 4.93 min. The mass spectrum of the eugenol
(Figure 1) showed a molecular ion at m/z 164. This spectrum was compared
with NIST Library (Figure 2). The base peak was generated by the loss of
electron from eugenol. Subsequent elimination of methyl group (–15 u) led
to a fragment ion at m/z. 149 The release of a methyl group and water group
(–33 u) from the molecular ion produced the fragment ion at m/z 131. The
possible formation of tropylium ion yielded the fragment ion at m/z 91 that
signified the presence of a benzyl group in the molecule (Figure 3). The
peak at m/z 65 was due to loss of an acetylene group from the tropylium
ion. On the basis of the mass-spectrometric data, three diagnostic ions of
eugenol (m/z 164, 149, and 131) were chosen and acquired for quantitative
analyses by using chemstation software.
 368 M. T. Athar et al.

FIGURE 1 Mass spectra of standard eugenol.

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FIGURE 2 Head-to-tail comparison of mass spectra of standard eugenol with library spectra.

Validation of the Method

Validation of the method was performed as per the requirement of ICH
guidelines (12) for linearity, precision, accuracy, specificity, robustness, LOD,
and LOQ similar to methods previously reported by laboratory (1,2,4).
 Eugenol in Hydro-Distilled Clove Oil 369
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FIGURE 3 Possible mass fragmentation pattern of eugenol.

Linearity
Range of concentration studied was planned according to approximate
response factors obtained from preliminary experiments with standard solu-
tions. The calibration plots showed good linearity for 10 to 1,000 µL.L−1 .
The slope, intercept, and correlation coefficient values were derived from
liner least-square regression treatment (y = 13578.2x –4E+06). The correla-
tion coefficient values of Eugenol (0.9962) indicated the best linearity of the
method (Table 1).

TABLE 1 Calibration Data for Eugenol (n = 3)

Concentration
(µL.L−1 ) Response ± SD %RSD

10 819,340 ± 8733.3 1.07

20 1,676,182 ± 30,583.9 1.82
40 3,346,992 ± 51,248.8 1.53
80 6,549,392 ± 41,427.6 0.63
100 8,290,965 ± 32,394.8 0.39
200 18,181,327 ± 149,756.3 0.82
400 47,640,065 ± 592,924.0 1.24
800 108,868,615 ± 3,485,085.1 3.20
1,000 134,160,174.7 ± 3,574,785.0 2.66
 370 M. T. Athar et al.

TABLE 2 Repeatability of the Method (n = 3) as Determined by GC-MS

TIC1 RT2

Concentration
(µL.L−1 ) Mean TIC ± SD % RSD3 Mean RT ± SD % RSD

80 6,542,596 ± 39478 0.60 4.939 ± 0.039 0.79

100 8,291,847 ± 25428 0.31 4.868 ± 0.093 1.92
200 18,625,924 ± 281708 1.51 4.894 ± 0.061 1.24
1 Total ion chromatogram.
2 Retention time.
3 Relative standard deviation.

Precision
The precision of the method was evaluated in terms of repeatability and
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intermediate precision. The repeatability was determined by injecting freshly

prepared 80, 100, and 120 µL.L−1 solutions of eugenol and the %RSD values
of TIC and RT calculated. The repeatability of experiment was satisfactory,
and the %RSD values varied from 0.61 to 1.51 and 0.79 to 1.24 for TIC and
retention time, respectively (Table 2).
For intermediate precision, three QC samples (80, 100, and 200 µL.L−1 )
of eugenol were injected in triplicate on 3 days (inter-day precision) and
three times on the same day (intraday precision). The inter- and intraday
precision were satisfactory at the three concentrations, and the %RSD values
were within accepted variable limit of ±2%. The %RSD of TIC ranged from
0.61 to 1.52 and 0.44 to 2.08 for inter- and intraday, respectively. The %RSD
of RT ranged from 0.36 to 1.69 and 0.20 to 1.61 for inter-day and intraday,
respectively (Table 3). The low %RSD values via TIC and RT confirmed the
good precision of the developed method.

Accuracy
The accuracy of the method was determined by spiking eugenol three times
at three concentrations (50%, 100%, and 150%) to sample containing 200
µL.L−1 of eugenol. The average recovery of low, medium, and high were
104.8, 97.6, and 100.7, respectively (Table 4).

Robustness
Robustness of the method was carried out by introducing very small but
deliberate changes in the analytical methodology. For the newly proposed
method, robustness studies were carried out by varying gas chromatographic
conditions—deviation of ±10% on the carrier gas flow, ±2◦ C on the initial
oven temperature, and ±1◦ C. Min−1 on the ramp rate. The retention times
 Downloaded by [89.237.153.223] at 22:53 29 September 2013

TABLE 3 Intermediate Precision of the Method (n = 3) as Determined by GC-MS

Inter-day precision Intra-day precision

1 2
TIC RT (Min) TIC RT (Min)

Conc
(µL.L−1 ) Mean area ± SD %RSD3 Mean RT ± SD %RSD Mean area ± SD %RSD Mean RT ± SD %RSD

371
80 6,546,917 ± 40,134 0.61 4.924 ± 0.018 0.36 6,596,917 ± 96,344 1.46 4.962 ± 0.010 0.20
100 8,317,704 ± 58,254 0.70 4.910 ± 0.022 0.44 8,274,371 ± 36,431 0.44 4.930 ± 0.056 1.13
200 18,538,146 ± 282,701 1.52 4.897 ± 0.083 1.69 18,308,146 ± 381,696 2.08 4.889 ± 0.079 1.61
1 Total ion chromatogram.
2 Retention time.
3 Relative standard deviation.
 372 M. T. Athar et al.

TABLE 4 Accuracy of Method as Determined by GC-MS, (n = 3)

% of standard Theoretical Amount of drug recovered % of drug %

spiked content (µL.L−1 ) (µL.L−1 ± SD) recovered RSD∗

0 200 2,021 ± 40.1 101.1 1.98

50 300 3, 143.7 ± 128.4 104.8 4.1
100 400 3, 905.7 ± 71.3 97.6 1.8
150 500 5, 036.3 ± 54.6 100.7 1.1
∗ Relative standard deviation.

and TIC of method were almost the same, indicating the robustness of the
method (Table 5).
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LOD and LOQ

The present method was validated as per ICH Q2 guidelines (9). Eugenol
solution was injected individually, and the limit of detection (LOD) and
the limit of quantification (LOQ) for values were determined at the lowest
concentrations at which signal to noise ratio was 3 and 10, respectively.

TABLE 5 Robustness of the Method (n = 3) as Determined by GC-MS

Carrier gas flow

(mL.Min−1 )

Actual Used Mean area ± SD Mean RT1 ± SD % RSD2 of area % RSD of RT

0.9 8,267,404 ± 56,862 4.947 ± 0.052 0.69 1.06

1.0 1.0 8,290,738 ± 96,090 4.902 ± 0.116 1.16 2.37
1.1 8,301,075 ± 74,545 4.877 ± 0.081 0.90 1.65

Oven
temperature (◦ C)

Actual Used Mean area ± SD Mean RT ± SD % RSD of area % RSD of RT

70 68 8,292,071 ± 15,875 4.937 ± 0.038 0.19 0.77

70 830,4271 ± 102,598 4.928 ± 0.079 1.24 1.61
72 8,331,075 ± 92,346 4.887 ± 0.095 1.11 1.94

Ramp rate
(◦ C.Min−1 )

Actual Used Mean area ± SD Mean RT ± SD % RSD of area % RSD of RT

5.0 4.0 8308771 ± 90367 4.907 ± 0.004 1.09 0.09

5.0 8287604 ± 59848 4.872 ± 0.065 0.72 1.32
6.0 8297742 ± 89668 4.913 ± 0.030 1.08 0.60
1 Retention time.
2 Relative standard deviation.
 Eugenol in Hydro-Distilled Clove Oil 373

A LOD and LOQ value for the compound was 3 and 10 µL.L−1 , respectively.

Identification and Characterization of Reference Standard

Relative percentage amounts of eugenol constituents were calculated from
TIC response by Agilent Chemstation Software. Identification of eugenol
in the reference standard was based on comparison of their mass spectra
with those of the matching NIST library spectra and by comparison of the
fragmentation pattern of the mass spectral data with those reported in the
literature.

Identification and Quantification of Eugenol in Samples

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Eugenol was analyzed in hydro-distilled oil and marketed formulations in

the same way as the reference standard. The eugenol peaks in the samples
were identified by comparing the RT of reference standard and matching
with mass fragmentation pattern as in the NIST library (Figure 4). There
was no interference of impurities at the eugenol’s retention time in samples
(Table 6).

FIGURE 4 Total ion chromatogram (TIC) of clove oil showing eugenol as major peak (RT
4.95).
 Downloaded by [89.237.153.223] at 22:53 29 September 2013

TABLE 6 Eugenol Concentrations in Hydrodistilled Clove Oil and Marketed Products (n = 3)

Concentration
obtained Assay by
S.NO Company Address Batch No Appearance (µL.L−1 ) GC-MS

1. Fluka Sigma-Aldrich Corporation CAS NO 97-53-0 Transparent dark yellow 5,000.00 99.99
Bangalore, India
2. Rex pharma Kasna, GB nagar UP 288/007 Clear Transparent 1,600.40 32.01
3. Dabur pharma Sahibabad, UP SB 187 Clear Transparent 1,916.73 38.33
4. Agarwal pharmaceuticals Narela, Delhi RP 1369 Tranparent Light Yellow 2,045.4 40.91

374
5. Arora pharmaceuticals Lawrence road, New Delhi R 144 Clear Transparent Dark 2,425.32 48.51
Yelllow
6. SD Fine chmicals pvt. Ltd. 315/317, Industrial state, TV — Clear Transparent 1,601.13 32.02
tower 248 Worli, Mumbai Brownish color
7. Divine laboratories P.box 9015, Delhi 0914 Transparent Dark Yellow 906.31 18.13
8. Parul chemicals Shashtri Nagar, Delhi 016 Clear Transparent 169.85 3.40
9. Sisla laboratories Narela, Delhi 11 Transparent, Dark Yellow 818.35 16.37
10. UK pharma Bawana, Delhi 006 Transparent Yellow 1,686.72 33.73
11. Hydro-distilled clove oil BNPL, Jamia Hamdard 001 Transparent Dark Yellow 1,891.24 37.82
 Eugenol in Hydro-Distilled Clove Oil 375

DISCUSSION

GC-MS data showed eugenol as the major constituent of clove oil having
molecular ion peak at m/z 164, which corresponded to the relative molecu-
lar mass of eugenol. Similarly, the fragment at m/z 149 corresponded to loss
of the methyl group from the side chain and peak at m/z 91 was typical ion
of a tropylium signifying the presence of a benzyl group in the molecule.
The peaks signified stepwise loss of hydrocarbons from the parent molecule.
This method was superior in linearity, recovery, and sensitivity compared to
a validated GC-FID method reported earlier (16) for quantitative estimation
of eugenol along with other components. This method can be used for the
routine analysis of the eugenol in formulations as well as crude drugs con-
taining eugenol, either as a principal bioactive component or as a minor
component.
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