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Research Article
DEVELOPMENT AND VALIDATION OF GC METHOD FOR THE ESTIMATION OF EUGENOL IN
CLOVE EXTRACT
Clove oil (Syzygium aromaticum) is widely used as a perfume and The GC-FID parameters used in the method development were
food flavoring as a medicine for the treatment of asthma and various based on the boiling point of the drug. Eugenol has a boiling point of
allergic disorders in Korea and as a general antiseptic in medical about 254˚C. The injection port and detector temperature were set
dental practices. The essential ingredient responsible for its to 225˚C and 270˚C, respectively.
antifungal activity is eugenol from the clove. Clove oil, applied to a Manual split less injection of approximately 1 μL sample was
cavity in a decayed tooth, also relieves toothache [2]. performed at an inlet temperature of 225˚C. The detector
Eugenol, the major constituent of clove oil, has been widely used for temperature was set to 270˚C. After injection, the oven temperature
its anesthetic and analgesic action in dentistry. Eugenol exhibits was increased quickly from 110˚C and then programmed within 8
pharmacological effects on almost all systems. It possesses min to 220˚C at a rate of 15˚C per min for 2 min. Nitrogen at a flow
significant antioxidant, anti-inflammatory and cardiovascular rate of 1.18 mL/min was used as a carrier gas. Synthetic air (flow
properties, in addition to analgesic and local anesthetic activity [3,4]. rate of 100 mL/min), hydrogen (25 mL/min) were fed to the FID. All
Eugenols treat cold, dental abscesses, gum disease, ear ache and the gases used in these studies were of pharmacopoeial purity.
arthritis pain. It also act as anti-fungal, anticonvulsant and Procurement of plant materials
anticarcinogenic and antimutagenic activities [5].From the literature
review it suggests that there have been some methods developed for The clove buds were collected from local market of Mysore.
clove by gas chromatography [6-9], HPLC [10]. This work describes
Preparation of extract
a simple, sensitive, and precise gas chromatographic method for the
analysis of eugenol from clove extract. The method validated Collected clove buds were washed with water and dried in shade,
according standard ICH guidelines [11] and used for the then coarsely powdered in a blender. From the coarse powder 50gm
determination of eugenol in alcoholic and aqueous extract. was subjected to reflux for 2 hours at temperature 80°C to 90°C. The
solvents was decanted and filtered with filter paper and recovered
by distillation. The extracts were dried under water bath at 60° C to
70° C respectively.
Preparation of standard Sample
A standard stock solution of eugenol (20mg/mL) was prepared by
dissolving 9.3 mL of accurately weighed eugenol in 99.9% ethanol
Fig. 1: Chemical structure of eugenol and volume was made up to the 10 mL. From the standard stock
Gurupadayya et al.
Int J Pharm Pharm Sci, Vol 6, Issue 2, 473-476
solution, working standard (200-1000 ng/mL) was prepared for the System suitability
gas chromatography method.
After the method conditions were established as described above,
Preparation of Extract Sample method was validated for precision, accuracy, and linearity.
Precision was measured as the repeatability of a series of results
10 mg of ethanolic and aqueous extracts of clove was weighed (n=6) and was also checked inter-day. Accuracy was determined as
accurately in 10 mL volumetric flasks and it was dissolved in percentage recovery (n=3) at three concentrations (80,100 and
with 99.9% of methanol followed by sonication for 5 to 10 120% of the amount expected) achieved by spiking placebo with
minutes and volume was made up using the same solvent. reference standard. Linearity was established by chromatography of
Solution was filtered using 2 µm syringe filter. From the stock a series of solutions (n=5) of decreasing concentrations. The limit of
solution 1 μl was injected on the head of the injector. This detection (LOD) and quantification (LOQ) were determined. These
concentration was used for the estimation of eugenol from the values are summarized in Table 1
extract of clove.
Quantitative analysis of the drugs was performed under the
RESULTS AND DISCUSSION conditions established. The selectivity of the method was evaluated
by comparing retention time values in chromatograms obtained
The proposed method was validated according to the International from the analyzed product with those in the chromatograms
Conference on Harmonization (ICH) guidelines. obtained from reference standard.
200 16545.2
300 53020.2
400 87515.4
500 127767.6
600 168977
700 209869.4
800 284019.2
900 320433.7
1000 365856.2
474
Gurupadayya et al.
Int J Pharm Pharm Sci, Vol 6, Issue 2, 473-476
Accuracy 3.0
5.832/225107
amount of standard drugs to pre analyzed commercial 1.5
9.865/3043
times. These values are listed in table 5. 0.5
0.0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
(ng/mL)
1.763/123500028
2.25
7.160/41910
1.25
5.814/38663
0.50
Limit of detection and limit of quantitation
6.719/1137
0.25
standard deviation (SD) of the response and slope was use for the -0.25
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
3.0
σ = Standard deviation of response
2.5
S = Slope of calibration curve
2.0
The LOD & LOQ was found to be 64.310 ng and 194.88 ng for
Eugenol. 1.5
6.232/10986
6.321/16199
6.158/2614
0.5
sample solution 1 μL were injected on the head of the injector. Fig. 5: Chromatogram showing aqueous extract of clove
475
Gurupadayya et al.
Int J Pharm Pharm Sci, Vol 6, Issue 2, 473-476
uV(x10,000)
3. Ayoola GA, Lawore FM, Adelowotan T, Aibinu IE, Adenipekun E,
3.251/520490
5.836/213281
9.0 Coker HAB, Odugbemi TO, Chemical analysis and antimicrobial
8.0 activity of the essential oil of Syzigium aromaticum (clove).
7.0 African J Microbiology Research 2008; 2: 162-166.
6.0 4. Jing SX, Yao L, Yun C, The effect of Eugenol on the Cariogenic
5.0
properties of Streptococcus Mutans and Dental Caries
development in rats. Experimental and Therapeutic Medicine
6.469/42180
4.0
2013; 5: 1667 -16670.
3.0
5. Bishnu J, Govind PS, Buddha BB, Megh RB, Dinita S,
Phytochemical extraction and antimicrobial properties of
6.758/4613
2.0
476