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SUPPLEMENTARY MATERIAL
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Rapid analysis of Achillea tenuifolia Lam essential oils by polythiophene/hexagonally
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ordered silica nanocomposite coating as a solid-phase microextraction fiber
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Marzieh piryaeia,b, Mir Mahdi Abolghasemic , Hossein Nazemiyeha,b*
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a
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Sciences, Tabriz, Iran
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b
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c
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
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical
Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Chemistry, Faculty of Science, University of Maragheh, Maragheh, Iran
Corresponding author:
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Hossein Nazemiyeh
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Tel: +98 411 336 7014; Fax: +98 411 336 7929
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E-mail address: hosseinNazemiyeh@yahoo.com
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Abstract
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In this work, a highly porous fiber coated with polythiophene/hexagonally ordered silica
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nanocomposite (PT/SBA-15) was prepared and used for extraction of essential oils with
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microwave assisted distillation headspace solid phase microextraction (MA-HS-SPME)
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method. The prepared nanomaterials were immobilized on a stainless steel wire for
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fabrication of the SPME fiber. Using microwave assisted distillation headspace solid
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phase microextraction followed by GC-MS, 24 compounds were separated and identified
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in Achillea tenuifolia, which mainly included limonene (28.6%), α-cadinol (12.7%),
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borneol(6.7%), caryophyllene oxide(3.2%), bornyl acetate(4.3%), camphene(3.2%) and
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para-cymene(2.3%). The experimental results showed that the polythiophene/hexagonally
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ordered silica nanocomposite fibres were suitable for the semi-quantitative study of the
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composition of essential oils in plantmaterials andmonitoring the variations in the volatile
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components of the plants.
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Keywords: Polythiophene, Hexagonally ordered silica nanocomposite, Solid-phase
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microextraction (SPME), Essential oil, Achillea tenuifolia, Microwave-assisted extraction
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Experimental
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Plant material
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The aerial parts of Achillea tenuifolia were harvested during the flowering season from
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the Tabriz- Ahar Road (Northwest of Iran; July 2013) and a voucher specimen (Tbz- PhF
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752) was deposited in the Herbarium of the Faculty of Pharmacy, Tabriz, Iran. The plant
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materials were dried in air and stored in sealed bags in a cool place.
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Chemicals and Reagents
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Poly (ethylene glycol)-block-poly (propylene glycol)-block-poly(ethylene glycol)
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(EO20– PO70–EO20 or Pluronic P123) as surfactant and tetraethoxyorthosilicate (TEOS)
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were purchased from Aldrich. Thiophene, FeCl3, hydrogen peroxide and all chemical
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solvents were obtained from the Fluka or Merck companies. All solvents used in this
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study were of analytical reagent grade. All reagents used in this study were of analytical
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grade: n-alkanes (C6–C24) and sodium hydroxide which were purchased from Merck
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(Darmstadt, Germany) and used as received.
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Apparatus
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A Hewlett-Packard Agilent 7890A series GC equipped with a split/splitless injector and
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an Agilent 5975C mass-selective detector system were used for determination. The MS
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was operated in the EI mode (70 eV). Helium (99.999%) was employed as a carrier gas,
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and its flow rate was adjusted to 1.1 mL min-1. The separation of PAHs was performed on
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a 30 m×0.25 mm HP-5 MS column with 0.25 μm film thickness. The column was held at
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50°C and increased to 180°C at a rate of 15°C min-1 and then raised to 260°C at 20°C
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min-1 and kept at this temperature for 5 min. The injector temperature was set at 260°C,
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and all injections were carried out on the splitless mode for 2 min. The GC–MS interface,
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ion source and quadrupole temperatures were set at 280, 230 and 150°C, respectively.
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Compounds were identified using the Wiley 7N (Wiley, New York, NY, USA) Mass
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Spectral Library. The mass spectra of target compounds were acquired and quantified in
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the selected-ion monitoring (SIM) mode. Ions were selected after injection of
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concentrated solution of compounds and recording the total ion chromatogram. The
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highest abundance ion was selected as the quantitative ion; two other ions were used for
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confirmation of individual analytes (Table 1). GC–MS was tuned before each analysis
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with HP Chem-Station Standard Spectra Auto tune routine with perfluorotributylamine
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(PFTBA). A homemade SPME device was used for holding and the injection of the
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proposed fiber into the GC–MS injection port. Before its use for SPME experiment, the
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fiber was heated at 100°C for 20 min in oven, and finally conditioned at 260°C in a GC
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injection port under helium gas for 2 h until a clean blank was obtained. Moreover,
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blanks were run periodically during the analysis to confirm the absence of contaminants.
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Synthesis of PT/SBA-15 nanocomposites
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Highly ordered mesoporous SBA-15 was synthesized using a procedure reported by
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Gholivand and co-workers (Gholivand, Piryaei, & Abolghasemi, 2013). In a typical
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synthesis, 3.2 g of triblock EO20–PO70–EO20 (Pluronic P123, mw 5800) as a template
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was dissolved through stirring in 125 mL of 2 M HCl, at ambient temperature (25–30°C)
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for 1 h. Finally, 6.8 g of silica source (TEOS) was added to the homogeneous solution
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under stirring to form a gel at 25°C for 24 h, and this was allowed to stand for
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crystallization under static hydrothermal conditions at 100°C for 48 h in a Teflon reactor.
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The crystallized product was filtered off, washed with warm deionized water, dried at
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80°C, and finally calcined at 600°C in the air for 6 h to remove the template. The
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polythiophene/hexagonally ordered silica, defined as PT/SBA-15, was synthesized
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following the procedures described elsewhere (Abolghasemi, &Yousefi, 2013). The
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synthesis of PT/SBA-15 composites was carried out in a 250 mL three-necked round-
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bottomed flask equipped with a magnetic stir bar, by the oxidation of thiophene monomer
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with ferric chloride (FeCl3) used as oxidant. In a typical synthesis, SBA-15 was thermally
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treated at 120°C in a vacuum oven to remove the physically adsorbed water. Then, 0.50 g
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of SBA-15 was dispersed in 10 mL of CHCl3, and then thiophene (5 mL) was added. The
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mixture was sonicated at ambient temperature for 1 h. When the methylene chloride and
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unadsorbed thiophene had been slowly evaporated at 30°C for 24 h under a vacuum oven,
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the mixture was added to a solution of H2O/ethanol (volume ratio: 5:1), which contained
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5 ml 30% hydrogen peroxide aqueous solution and 4 mg FeCl3. The polymerization
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proceeded under nitrogen atmosphere at pH 2 for over 12 h at 50°C. The product was
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directly precipitated into vigorously stirred methanol (six volumes), then filtered off and
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washed with methanol several times, and eventually dried under vacuum at 50°C for 12 h
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to remove the physically adsorbed water molecules.
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Preparation of the SPME fiber
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A piece of stainless-steel wire with a 200 μm diameter was twice cleaned with methanol
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in an ultrasonic bath for 20 min and dried at 70°C. One centimeter of the wire was limed
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with epoxy glue and the PT/SBA-15 nanocomposite was immobilized onto the wire. The
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coated wire was heated to 50°C for 48 h in an oven, gently scrubbed to remove
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nonbonded particles and assembled in the SPME holder device. Figure S3 shows the
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SEM image of a fabricated SPME fiber. Finally, the prepared SPME fiber was inserted
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into the GC injection port to be cleaned and conditioned at 260°C for 2 h in a helium
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environment (Abolghasemi, &Yousefi, 2013).
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MA–HS-SPME of essential oil
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The microwave oven with a maximum delivered power of 900W (model of
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GE614ST/GE614W, Samsung Company, Korea) was used as a heating device. In order
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to prevent from microwave leaking, aluminum foil was tacked onto the inner wall and the
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outer wall of the microwave in the interface part. MA-HS-SPME extraction was
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performed according to the following procedure. Weighed amount (2 g) of powdered
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herb was transferred into a 25 ml round bottom flask. After assembling a condenser, the
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SPME fiber was exposed to the sample headspace, the herb was heated by microwave
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power (450 W). After the extraction time (4min), the fiber was withdrawn from the bottle
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and inserted into the GC-MS injection port for analysis.
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Essential oil hydrodistillation (HD)
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Air-dried aerial parts of Achillea tenuifolia (50 g) were ground and subjected to
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hydrodistillation for 2 h, using a Clevenger-type apparatus as recommended. Briefly, the
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plant was immersed in water and heated to boiling, after which the essential oil was
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evaporated together with water vapor and finally collected in a condenser. The distillate
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was isolated and dried over anhydrous sodium sulfate. The oil was stored at 4 °C until
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analysis by GC–MS. The yield of the yellowish oil from the aerial parts of Achillea
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tenuifolia was 0.32 % (w/w), based on dry weight of the sample.
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References
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Abolghasemi, M. M., &Yousefi, V. (2013). Polythiophene/hexagonally ordered silica
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nanocomposite coating as a solid-phase microextraction fiber for the determination of
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polycyclic aromatic hydrocarbons in water. Journal of Separation Science, 1, 1–7.
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Gholivand, M.B., Piryaei, M., & Abolghasemi, M. M.)2013). Analysis of volatile oil
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composition of Citrus aurantium L. by microwave-assisted extraction coupled to
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headspace solid-phase microextraction with nanoporous based fibers. Journal of
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Separation Science, 36, 872–877.
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Figure S1. The response (sum area of four main peaks of Achillea tenuifolia Lam
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samples) for the designed experiments mentioned in Table 1.
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Figure S2. Effect of water addition on the extraction efficiency of the solid-phase
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microextraction method.
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Figure S3: SEM image of a fabricated SPME fiber
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Table S1. Constituents of the oil of Achillea tenuifolia Lam with the microwave assisted
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distillation headspace solid phase microextraction (MA-HS-SPME) and hydrodistillation
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methods.
Numb Compounds
RIa
er
(HD)
(MA-HS-
Repeatabilit Reproducib
PDMS
Area%b
SPME)
y R.S.D.%d
Area%e
Area%c
ility
R.S.D.%
1
α-Pinene
935
1.52
1.08
6.1
15.2
1.17
2
Camphene
954
3.73
3.21
5.4
13.7
2.72
3
Sabinene
975
2.81
1.25
7.3
14.8
2.55
4
α-Phellandrene
1004
0.85
0.67
4.1
11.2
0.51
5
α-Terpinene
1018
0.08
0.17
3.7
12.3
0.00
6
Para-cymene
1028
1.94
2.30
8.3
11.7
2.15
7
Limonene
1030
27.51
28.64
9.2
16.4
29.53
8
β-Phellandrene
1033
0.07
0.13
2.6
15.6
0.12
9
Cis-sabinene hydrate
1067
0.03
0.11
1.1
14.3
0.04
10
Meta-cresol
1075
0.07
0.05
1.0
11.5
0.03
11
Para-cymenen
1083
0.05
0.00
-
-
0.04
12
Camphor
1143
0.02
0.07
1.4
14.6
0.03
13
Isoborneol
1152
1.34
0.81
3.8
12.5
1.55
14
Trans-β-terpineol
1160
0.15
0.04
2.6
14.3
0.07
15
Borneol
1168
8.04
6.76
5.7
16.2
6.72
16
Terpin-4-ol
1177
0.04
0.05
1.7
12.7
0.00
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α-Terpineol
1187
1.27
1.83
3.6
15.6
2.17
18
2-methoxy-para-cresol
1189
0.03
0.00
-
-
0.04
19
γ-Terpineol
1198
0.18
0.14
1.4
13.4
0.24
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Trans-piperitol
1205
1.27
0.78
3.6
13.6
1.58
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Carone
1211
0.14
0.34
4.8
13.2
0.52
22
Trans-carveol
1218
0.26
0.06
6.2
14.3
0.16
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Dihydrocarveol(neo-
1226
0.02
0.00
-
-
0.00
iso)
24
Cis-carveol
1229
0.52
0.13
5.7
13.8
0.31
25
Carvone
1242
0.88
0.67
5.9
14.7
1.12
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α-Terpinen-7-al
1282
0.03
0.14
2.5
11.1
0.00
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Bornyl acetate
1285
5.22
6.31
8.6
13.5
4.52
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Trans-Linalool oxide
1288
0.17
0.04
7.1
14.1
0.00
acetate
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α-cubebene
1347
0.25
0.05
5.4
13.2
0.00
30
α-copaene
1377
0.14
0.08
4.6
16.4
0.00
31
β-caryophyllene
1417
0.41
0.22
4.3
15.7
0.56
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γ-Elemene
1438
0.34
0.08
2.7
11.3
0.74
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α-humulene
1454
4.72
3.16
9.4
12.8
5.11
34
Germacrene-D
1483
2.97
1.45
6.5
16.4
1.52
35
β-selinene
1493
0.25
0.45
4.1
14.9
0.39
36
β-guaiene
1495
0.36
0.13
3.2
12.5
0.32
37
α-selinene
1497
0.11
0.57
2.8
11.8
0.34
38
α-muurolene
1500
2.75
2.22
4.9
10.2
2.51
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Calarene
1524
0.82
1.14
3.1
11.6
0.45
40
Calacorene
1546
0.68
0.16
2.5
15.3
0.25
41
Spathulenol
1577
0.19
0.44
6.7
13.4
0.14
42
Caryophyllene alcohol
1568
0.31
0.00
-
-
0.12
43
Caryophyllene oxide
1580
2.12
3.25
2.4
14.7
2.51
44
β-copaen-4-alpha-ol
1584
0.02
0.00
-
-
0.03
45
α-cadinol
1650
16.40
12.71
8.7
17.5
11.71
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a) Retention indices using a HP-5MS column (relative retention times normalize to
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closely eluting n-alkanes).
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b) Relative area (peak area relative to total peak area) for hydrodistillation method.
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c) Relative area (peak area relative to total peak area) for MA-HS-SPME method.
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d) RSD values for MA-HS-SPME method (n = 5).
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e) Relative area (peak area relative to total peak area) for PDMS fiber.
162
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