Food Anal.

Methods
DOI 10.1007/s12161-015-0224-5

Application of Polyamide Nanofibers, SPME/GC-MS,

and Chemometrics for Comprehensive Analysis of Volatiles
in Thymus vulgaris L. and Thymus serpyllum L
M. Asadollahi-Baboli 1 & A. Aghakhani 2 & V. Bikdeloo 1

Received: 8 February 2015 / Accepted: 8 June 2015

# Springer Science+Business Media New York 2015

Abstract Garden thyme (Thymus vulgaris L.) and wild

thyme (Thymus serpyllum L.) volatile components have been
extracted using solid-phase microextraction (SPME) with
polyamide nanofibers, prepared by electrospun technique.
The polyamide nanofibers were used for SPME because of
its high porosity and high surface to volume ratio. The homogeneity and porosity of the prepared polyamide nanofibers
was investigated by scanning electron microscopy having diameters of 50100 nm. Gas chromatography-mass spectrometry (GC-MS) together with chemometrics was implemented
to resolve the co-eluted components and improve analytical
performance. After applying multivariate curve resolutionalternating least squares (MCR-ALS) to resolve the overlapped and embedded peak clusters, 32 and 43 components have
been identified in garden and wild thyme, respectively.
Furthermore, principal component analysis (PCA) was implemented to characterize hidden structure and to identify volatiles differentiating between garden and wild thyme. The proposed strategy of SPME/GC-MS together with chemometrics
may be useful for comprehensive and rapid analysis of complex natural extracts such as garden and wild thyme.
Keywords Chemometrics . Solid-phase microextraction .
Multivariate curve resolution-alternating least squares .
Polyamide nanofibers . Thyme

* M. Asadollahi-Baboli
asadollahi@nit.ac.ir
1

Department of Science, Babol University of Technology,

Babol 47148-71167, Mazandaran, Iran

Department of Semiconductors, Materials and Energy Research

Center, Karaj 31787-316, Iran

Introduction
Thyme is an evergreen aromatic and important plant in North
America, Europe, Middle East, and Asia with many industrial
and medical applications (Stahl-Biskup and Saez 2003). The
extracts and oil of thyme were reported to have antibacterial,
antifungal, carminative, and expectorant activities (Daniel
2006; Jouki et al. 2014; Sellamuthu et al. 2013). Garden
thyme (Thymus vulgaris L.) and wild thyme (Thymus
serpyllum L.) are well-known spices as flavoring agent and
herbal medicine. The commercial value of these species depends on their chemical composition of volatiles fraction.
Garden and wild thyme are used in many fields such as aromatherapy, spices, nutrition, perfumes, and cosmetics (StahlBiskup and Saez 2003). The volatile compositions of thyme
extracts from different origins have been reported in literature.
Thymol and carvacrol have been reported as the major components in both garden and wild thyme (Giordiani et al. 2008;
Golmakani and Rezaei 2008; Nikolic et al. 2014; Rota et al.
2008). The fragrance volatiles of garden thyme also have been
reported in literature using head space solid-phase
microextraction (SPME) sample preparation with different
sorbents (Bicchi et al. 2006; Staszek et al. 2014).
Nevertheless, the minor components are extremely important
factors determining the spicy aroma and sensory quality of
thyme. Moreover, these minor components may have important therapeutic value (Giordiani et al. 2008). In all previous
studies, the minor and major components in complex gas
chromatography-mass spectrometry (GC-MS) data have been
determined using direct similarity searches. Due to presence
of baseline drift, noise, and co-eluted components (embedded/
overlapping peaks) in real complex samples such as thyme,
similarity matches in the MS library can mislead to wrong
identification and quantification of volatiles (AsadollahiBaboli and Mani-Varnosfaderani 2014). To solve these

Food Anal. Methods

problems, chemometric resolution techniques were utilized to

enhance the analysis of thyme. To the best of authors knowledge, the volatile components of thyme species have not been
analyzed with chemometric resolution techniques so far.
SPME has many benefits such as simple sample preparation, simple to use, low solvent consumption, low temperature
extraction and cost-effectiveness (Jiang and Pawliszyn 2012).
In this work, polyamide was electrospun into the fibrous sheet
with nanoscale dimensions to improve the performance of
extraction due to possessing high porosity and high surface
to volume ratio (Jiang and Pawliszyn 2012). Then, the prepared polyamide nanofibers were used for the SPME analysis
of garden and wild thyme. Therefore, a new strategy based on
SPME, GC-MS, and chemometrics was applied for the comprehensive analysis of garden and wild thyme. In the present
contribution, volatiles of garden and wild thyme were extracted by the help of ultrasonic and SPME. At the same time, the
analytical performance of polyamide fibers was also investigated. Moreover, a strategy based on chemometric resolution
techniques such as multivariate curve resolution-alternating
least square (MCR-ALS) (Jaumot et al. 2015) was proposed
to improve the resolution, qualification, and quantification of
co-eluted compounds and to generate the informative secondorder data. Finally, principal component analysis (PCA)
(Xanthopoulos et al. 2013) was used to identify the changes
in chemical compositions of both garden and wild thyme.

Experimental
Electrospinning of Polyamide Nanofibers
In order to obtain a homogenous polymer solution, 0.2 g of
nylon 6 was dissolved in 1 mL of formic acid. Then, 0.5 mL of
this solution was withdrawn into a 2.5-mL syringe which was
eventually located in a syringe pump. The nanofiber sheet was
fabricated by electrospinning of polyamide on a piece of aluminum foil (55 cm). Under this condition, the aluminum foil
and the polymer containing syringe needle were connected to
the high-voltage power supply terminals (15 kV). The distance between the needle and the collector was set at 10 cm,
while a flow rate of 1.5 L min1 was set for the syringe pump
to deliver the polyamide polymer solution. All the
electrospinning processes were performed under the ventilation for 8 h. After the electrospinning experiment, a sheet with
a typical dimension of 11 cm was cut from the central part of
the foil employed for SPME. A Brandenburg (West Midlands,
England)-regulated power supply and a KDS100 syringe
pump (KdScientific Co., Holliston, MA, USA) were used
for electrospinning and the polymer solution delivery in the
electrospinning process, respectively. The image of the surface of polyamide nanofibers was obtained by scanning

electron microscopy (SEM) using a FE-SEM S-4800II instrument (Hitachi, USA).

SPME and GC-MS Analysis
The garden and wild thyme leaves were collected from
Mazandaran Province located in the north of Iran (south of
Caspian Sea). The air-dried plant material was grinded to obtain a homogenous fine-grade powder and was kept at 5 C in
the absence of light. Extraction process using SPME method
was carried out as following: 2.0 g of thyme leaves and 10 mL
of hexane were placed in a 20-mL glass vial. Afterward, the
glass vial was placed in the sonication bath with polyamide
nanofiber sheet inside it to collect volatiles for 30 min at room
temperature (25 C). After the extraction, the polyamide nanofiber sheet was folded and inserted inside a 5-mL glass vial for
solvent desorption using 1 ml of acetone for 10 min. The
obtained solution was concentrated by a gentle flow of nitrogen up to 200 L. An aliquot of the 1 L for each organic
extract from garden and wild thyme was injected into an
Agilent HP-6890 gas chromatograph coupled with Agilent
HP5973 mass selective detector equipped with a 30 m
0.25 mm HP-5MS fused silica column (0.25 m film thickness). The MS was operated in the EI mode (70 eV). Helium
(99.999 %) was employed as a carrier gas with flow rate of
1 mL min1. The GC column temperature was programmed
from 50 to 240 C at 3 C/min and held for 20 min. Mass
range scanned from 40m/z to 400m/z. The injector temperature was set at 230 C in the split ratio of 1:5. Above process
were also carried out using typical polydimethylsiloxane
(PDMS) for the sake of comparison.

Fig. 1 SEM image of polyamide nanofibers prepared by electrospun

technique

Food Anal. Methods

Fig. 2 Analytical performance of polyamide nanofibers and PDMS categorized into four chemical classes

Data Analysis with Chemometrics Tools

In the present contribution, the two-way data of acquired GCMS matrix was decomposed to the pure mass spectrum and
chromatographic profile of each co-eluted component using
chemometric resolution techniques. The detailed theories behind these potent techniques are described elsewhere
(Asadollahi-Baboli and Aghakhani 2014; Miao et al. 2011;
Poon and Poon 2014). However, in order to make the article
more consistent and understandable, the chemometrics procedures are described briefly in the present study. First,
denoising, background/baseline correction, and smoothing
preprocessing routines were performed for each peak cluster
to ensure that the GC-MS signal is composed of chemical

Fig. 3 The overlaid TICs of garden and wild thyme

components. Morphological score technique (Poon and

Poon 2014) was used for differentiating between the noises
and the signal channels. Least square fitting method developed by Liang and Kvalheim (Gemperline 2006) together
with congruence analysis were employed for the baseline/
background correction. The Savitsky-Golay filter
(Gemperline 2006) was also used for smoothing purpose.
Second, to determine chemical rank and local rank analysis,
morphological score and fixed-size moving-windowevolving factor analysis (FSMW-EFA) techniques (Tauler et al.
2009) were applied due to their uniformity and acceptable
performance. The morphological score is based on the fact
that the ratio of the norm of a spectrum to the norm of its first
difference is higher for a profile of a component than a profile

Food Anal. Methods

Fig. 4 The local TICs of a peak cluster A obtained from garden thyme and b peak cluster B obtained from wild thyme. c Morphological score plot and d
subspace plot of OPA-SIMPLISMA. Resolved chromatograms of e peak cluster A and f peak cluster B using MCR-ALS

generated only by noise. In FSMW-EFA technique, PCA is

implemented on fixed size windows moved row by row
downwards along the elution direction. The local rank
analysis indicates the zero components, overlap and selective regions. In the next step, MCR-ALS algorithm
was applied for resolving the co-eluted GC-MS peak
clusters into pure chromatograms and mass spectra.
This algorithm starts with initial estimates obtained by
the SIMPLISMA (Tauler et al. 2009) and applying
proper constraints until the concentration and pure

spectra optimally fit in the experimental data matrix.

Lastly, the quality and reliability of results were confirmed by comparing resolved mass spectra with those
of mass libraries. For every overlapped and embedded peak
clusters, the above procedure was performed as described.
MCRC software (Jalali-Heravi et al. 2010) was used for preprocessing, chemical rank determination, local rank analysis,
and MCR-ALS. After obtaining the MCR-ALS results for all
chromatographic segments, PCA were used to cluster data into
two clear-cut groups.

Food Anal. Methods

Results
SPME has been carried out in order to extract the volatiles of
garden and wild thyme. In this work, polyamide nanofibers
were employed for SPME because of its high surface area and
porous structure. According to the SEM image (Fig. 1), the
polyamide nanofiber sizes are in the range of 50100 nm and
possess very porous structure, which should significantly increase the surface area availability on the fiber. In addition, the
spaces among the nanofibers are in the range of 200500 nm
and this should facilitate much faster analyte diffusion, while
they are bead-free and have smooth morphology. For comparison, the effects of sorbent on the extraction efficiency, both
polyamide nanofibers and typical PDMS, were investigated to
facilitate the best performance. As shown in Fig. 2, polyamide
nanofibers show better analytical performance compared to
typical PDMS. The height of the bars in this figure represents
the relative composition of volatiles of thyme quantities in
each sorbent in terms of peak area. Volatiles were classified
into four chemical groups consisting of monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpene. Both the number of identified components and the peak area in these four chemical
groups were increased using polyamide nanofibers.
The garden and wild thyme extracts were injected in GCMS in the full scan mode. The chromatographic profiles of the

overlaid TICs of the garden and wild thyme were depicted in

Fig. 3. The real complex mixtures as shown in Fig. 3 have
several overlapped cluster peaks. Besides, there is a high
chance of finding some embedded peaks. In these cases, direct
searching in MS library without further data processing may
lead to incorrect identifications. For these chromatographic
peaks, the match indices are low using direct MS database
searching. To solve these problems and perform accurate qualitative and quantitative analyses, the overlapped/embedded
peak clusters should be resolved into pure chromatographic
profiles and mass spectra. The TICs of both garden and wild
thyme were divided into subsets using zero component regions along elution time. In this study, two peak clusters of
A (from garden thyme) and B (from wild thyme) were considered as instances to show the resolution procedure. The
local TICs of peak clusters A (highly overlapped peak cluster)
and B (embedded peak cluster) are presented in Fig. 4 panels a
and b, respectively.
Denoising, background/baseline correction, and smoothing
were initially performed for the peak clusters A and B. Then,
the chemical rank of the peak clusters A and B were determined using morphological score plot as shown in Fig. 4c. It is
clear from this plot by counting the numbers of singular vectors with the morphological score upper than that of the noise
levels that there are three and four components in peak clusters
A and B, respectively. It is often hard to arrive at safe results

Fig. 5 Resolved mass spectra (a, b) and standard mass spectra (c, d) for terpinen-4-ol and aromadendrene, respectively

Food Anal. Methods

using just one method due to the accumulation of noise in GCMS data. Consequently, subspace comparison is also utilized
for the chemical rank determination. In order to reduce the
Table 1
No.

noise effects, key spectra instead of full rank matrices are

analyzed in subspace comparison. The result of OPASIMPLISMA (Jalali-Heravi et al. 2010) is shown in Fig. 4d.

Chemical components of garden and wild thyme using proposed strategy

Chemicala

RMFb

RIc

Percentage
Garden thyme

RSDd

Wild thyme

RSD

1
2
3
4
5
6
7
8
9
10
11
12

3-Thujene
-Pinene
Camphene
-Phellandrene
-Pinene
-Myrcene
-Terpinen
p-Cymene
m-Cymene
1,8-Cineol
-Ocimene
trans-Sabinene

923
940
919
958
975
953
971
950
966
924
932
950

917
923
937
944
966
983
1008
1016
1032
1038
1041
1045

0.12
0.80
0.55
0.34
0.25
0.43
0.63
3.30
0.31

0.20

3.16
4.36
7.72
4.50
11.12
4.64
10.64
4.80
4.35

5.37

0.28
1.64
0.07
0.81
0.21
0.39
1.45

0.26
0.55
0.38

3.76
5.47
5.10
7.97
6.35
6.58
6.27

9.66
7.49
3.23

13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

-Terpinene
2-Carene
4-Thujanol
-Terpineol
Linalool
Terpinen-4-ol
cis-Chrysanthenol
Sabinol
Camphor
Citronellal
Sorborneol
p-Menth-8-en-3-ol
-Terpineol
cis-Dihydrocarvone
p-Cumic aldehyde
Thymol methyl ether
Cuminaldehyde
Anethole

971
940
926
958
948
973
954
967
910
925
969
917
957
905
915
948
920
972

1050
1062
1069
1079
1083
1089
1123
1130
1134
1156
1172
1178
1184
1190
1222
1226
1234
1245

0.29
0.13

0.34
0.48
0.64

0.21
0.28

0.19
0.37
0.28

0.18

0.39
1.09

11.30
8.53

5.90
6.38
11.04

5.45
6.41

7.31
11.37
9.90

9.41

8.67
4.00

3.95
0.11
0.08
0.12
0.83
0.23
0.34
0.43
0.04
0.65
0.28
0.59
0.89
0.17
0.39
2.28

0.34

5.11
9.04
3.84
10.52
5.91
9.50
10.34
7.34
8.86
8.52
4.27
9.62
8.79
12.80
8.91
5.60

6.00

31
32
33
34
35
36
37
38
39
40
41
42

Isobornyl acetate
Durenol
Carvacrol
Thymol
Thymolacetate
-Caryophyllene
-Elemene
Aromadendrene
-Elemene
-Humulene
Ledene
-Muurolene

954
959
985
980
954
955
947
953
951
929
953
910

1276
1281
1292
1298
1346
1407
1413
1420
1425
1459
1474
1491

0.72
17.23
60.12
4.38
3.12

0.64

0.27

9.48
3.96
2.81
4.14
5.19

5.36

11.90

0.21
3.56
23.95
48.24
0.75
0.89
0.32
1.07
0.25
0.25

0.21

2.23
4.97
2.74
3.10
5.74
6.22
3.76
3.64
4.75
8.88

6.11

43

-Cadinene

936

1502

0.87

7.53

Food Anal. Methods

Table 1 (continued)
No.

44
45
46
47
a

Chemicala

Spathulenol
Caryophyllene oxide
Guaiol
-Eudesmol

RMFb

926
948
917
946

RIc

1566
1570
1579
1585

Percentage
Garden thyme

RSDd

Wild thyme

RSD

0.41
0.39

9.31
10.53

0.21
0.56
0.07
0.15

10.05
2.20
13.46
6.48

Identification was based on comparing the resolved MCR-ALS mass spectra with those of standards using MS library database and RI

Reverse match factor for the resolved mass spectrum MS library database

Kovats retention indices in HP-5MS column

Relative standard deviation on relative peak area for three times replication (n=3). The RSD on peak area for all components was less than 12 %

As Fig. 4c, d reveals, the results of both morphological scores

and OPA-SIMPLISMA are in agreement with each other. It
should be noted that the results of local rank analysis using
FSMW-EFA also confirms the chemical rank determination
results. In the next step, MCR-ALS was implemented to resolve the peak clusters A and B into chromatographic profiles
and mass spectra of related components. Besides, initial estimates using SIMPLISMA method and some constraints such
as non-negativity, unimodality, and selectivity were used in
MCR-ALS algorithm. Figure 4e, f displayed the resolved
chromatogram of the peak clusters A and B, respectively.
The peak cluster A has components of -terpineol, linalool, and terpinen-4-ol. The components in the peak cluster B
are -caryophyllene, -elemene, aromadendrene, and elemene. It should be noted that the reverse match factor
(RMF) for identified components are acceptable. Moreover,
reliability of the resolution results was demonstrated by similarity between the mass spectra of resolved components with
those of their counterpart standards. As examples, resolved
a n d s t a n d a r d m a s s s p e c t r a o f t e r p i n e n -4 - o l a n d

aromadendrene in the peak clusters A and B are presented in

Fig. 5. As this figure shows, there is a good agreement between resolved and standard mass spectra. Other GC-MS peak
clusters are also resolved in a similar way as described above.
As Table 1 shows, 32 and 43 components have been identified
using GC-MS combined with chemometrics for the volatiles
of garden and wild thyme, respectively. The amount of each
component is proportional to the overall volume of its
two-way response. This response is equal to the outer
product of the concentration and spectrum vectors.
Overall volume integration (OVI) (Jalali-Heravi et al.
2014) was applied for calculating the quantity of each
component implementing chemometric resolution techniques. Despite general peak area integration, all mass
spectra absorbing points are taken into consideration in
this method and subsequently more accurate results can
be reached. In addition, it avoids the disadvantage of
peak splitting in general peak area approximately. It
should be noted that it is not possible to perform quantitative
analysis for co-eluted peaks without using chemometric

Fig. 6 a Score plot and b loading plot in PCA plot of the garden and wild thyme

Food Anal. Methods

resolution techniques. In the peak cluster A, the relative percentages of -terpineol, linalool, and terpinen-4-ol are 0.34,
0.48, and 0.64, respectively. In the peak cluster B, the relative
percentages of -caryophyllene, -elemene, aromadendrene,
and -elemene are 0.89, 0.32, 1.07, and 0.25, respectively.
For comparative analysis between garden and wild thyme,
PCA was used to recognize intrinsic patterns in the GC-MS
data in an unbiased way. This technique helps determine hidden structure and cluster the garden and wild thyme into two
different classes. The relative percentages of the resolved
peaks were used to perform PCA. Figure 6a, b illustrates the
score and loading plots using ten samples (five samples of
each garden and wild thyme). The PC1 and PC2 explained
67.84 and 25.92 % of the total variance, respectively. As
Fig. 6a indicates, the garden and wild thyme can be easily
classified in two distinct species with acceptable variation between and within two classes. PCA also helps determine those
volatiles which were the most differentiating within the entire
data. The loading plot (Fig. 6b) reveals those volatiles which
were the most differentiating within the volatiles of garden
and wild thyme. The 14 components which account for the
most of the variance in the dataset are given more weight in
the loading plot. These results show that garden and wild
thyme are different enough in terms of chemical composition
to put them in two distinct classes. Table 1 shows the volatiles
and the relative percentages of each component in the garden
and wild thyme. The reproducibility of analysis was explored
with three replicate experiments (n=3) for both garden and
wild thyme samples. The Kovats retention index (RI) as a
complementary identification tool in HP-5MS was calculated
for each component. The RI, RMF, and relative standard deviations (RSD) are presented in Table 1 for all components in
the both samples. The results revealed that thymol and carvacrol are major components in the both samples. As shown in
this table, thymol (60.12 %), carvacrol (17.23 %),
thymolacetate (4.38 %), p-cymene (3.3 %), and caryophyllene (3.12 %) are the main components of garden
thyme. Also, thymol (48.24 %), carvacrol (23.95 %), terpinene (3.95 %), thymol methyl ether (2.28 %), and camphene (1.64 %) are the major components of wild thyme.

Discussion
Both garden and wild thyme volatiles were analyzed by
SPME with polyamide nanofibers together with GC-MS and
chemometric resolution techniques. Moreover, different problems of baseline drift, noisy data, and co-elution of components in GC-MS have been addressed using chemometrics. In
addition, polyamide nanofibers were superior compared to
typical PDMS in terms of extraction performance.
Therefore, the described combined strategy was utilized for
the first time to analyze the volatile components of the garden

and wild thyme extracts. Highly overlapped and embedded

peak clusters can be resolved and analyzed using different
chemometrics techniques such as morphological score, subspace comparison, and MCR-ALS techniques. A total of 32
and 43 components of garden and wild thyme extracts were
identified using proposed strategy. Finally, PCA was used to
identify the components responsible for differentiating the
garden and wild thyme from MCR-ALS results. The results
of this study demonstrate the ability of chemometrics as an
essential step for comprehensive analysis of real complex
samples to achieve more reliable results.

Conflict of Interest M. Asadollahi-Baboli declares that he has no conflict of interest. A. Aghakhani declares that he has no conflict of interest.
V. Bikdeloo declares that he has no conflict of interest. This article does
not contain any studies with human or animal subjects.

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