Journal of Chromatographic Science, 2016, Vol. 54, No.

6, 879–887
doi: 10.1093/chromsci/bmw034
Advance Access Publication Date: 18 March 2016
Article

Article

GC–MS Analysis and Volatile Profile Comparison

for the Characteristic Smell from Liang-wai Gan

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Cao (Glycyrrhiza uralensis) and Honey-Roasting
Products
Min He1,*, Zhi-Yu Yang1, Wen-Na Guan2, Carlos M. Vicente Gonçalves3,
Juan Nie1, and Hai Wu4
1
Department of Pharmaceutical Engineering, School of Chemical Engineering, Xiangtan University, Xiangtan 411105,
People’s Republic of China, 2Qingdao Zhenzheng Analytical Instruments Co., Ltd, Qingdao 266000, People’s Republic
of China, 3Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Bergen, Bergen 5020,
Norway, and 4Institute of Forensic Science, Hunan Provincial Public Security Bureau, Changsha 410001, People’s
Republic of China
*Author to whom correspondence should be addressed. Email: dahai8214813@gmail.com
Received 22 September 2015; Revised 31 January 2016

Abstract
The characteristic smell of Liang-wai Gan Cao (Glycyrrhiza uralensis) and honey-roasting products
was comprehensively analyzed using gas chromatography–mass spectrometry (GC–MS). Steam dis-
tillation and headspace solid-phase microextraction (HS-SPME) were used to extract volatile organic
compounds (VOCs). Multiple fibers of SPME may reflect the samples’ comprehensive information to
the greatest extent, depending on their chemical characters. After chemometric resolution and spec-
tra interpretation, many aroma compounds could be identified from GC–MS data. As a result, prin-
cipal component analysis was set for the differentiation of several G. uralensis samples in different
regions, and some important peaks could be found. Next, VOCs’ profiles of honey-roasting products
suggested that the flavors could be influenced by honey and pharmaceutical technologies.

Introduction
Glycyrrhiza uralensis is one of the authenticated medicinal species of
licorice (1) with known health benefits for a variety of illnesses (2).
Since ancient China, “Liang-wai Gan Cao” in the Ordos region
(106°42′E–111°27′E, 37°35′N–40° 51′N) is an important source of
G. uralensis, which is known as “Dao Di medicinal material” (3) in
specific geographic regions with particular attention to cultivation
and processing techniques. It has a similar reputation as species such
as “Wang-ye Gan Cao” in the Alxa Left Banner region (97°10′E–106°
52′E, 37°21′N–42°47′N) and “Yan-chi Gan Cao” in the Yanchi
region (107°15′E–107°19′E, 37°32′N–37°36′N).
Subtle and characteristic smell is an important property of G. uralen-
sis. Presently, the chemical composition and aroma profiling of Glycyr-
rhiza species (G. glabra, G. inflata and G. echinata) have been
investigated (4, 5), and particularly for G. uralensis, some variations
of essential oils were found after irradiation (6). However, the volatile
organic compounds (VOCs) of Liang-wai Gan Cao and the honey-
roasting products, as well as of other “Dao Di medicinal material” of
G. uralensis, still need to be systematically and accurately studied. In
VOCs’ mixture research, extraction procedures using hydrodistillation
or headspace solid-phase microextraction (HS-SPME) (7–9), followed
by gas chromatography–mass spectrometry (GC–MS), have been widely
used (10, 11). To obtain accurate results, peak alignment, baseline
correction, smoothing, “mass accuracy” or determination of mass
centroids, computational analysis of fragmentation and structure eluci-
dation have been an essential part of problem solving (12, 13). Further-
more, many problems should be discussed in light of the current
challenges in hyphenated and multidimensional chromatographic
analysis (14), allowing an accurate identification of VOCs in natural
products or herbs.

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880
In this work, steam distillation and HS-SPME were used to prepare
the VOCs’ samples for GC–MS determination. Besides several chemo-
metric resolution methods, different spectral libraries together with
search algorithms were used for VOCs’ identification, and the results
were assembled to increase credibility. On the basis of the above
approaches, the aromatic character of “Liang-wai Gan Cao” was
compared with those of licorice medicinal materials grown in other
regions. Next, the aroma profiling of honey-roasting products was
investigated using different pharmaceutical technologies.
Experimental
Materials and the identification of crude drug
In the spring of 2015, the crude drugs of Liang-wai Gan Cao (G. ura-
lensis) were collected in the city of Ordos in Inner Mongolia, China. The
crude drugs were air-dried indoors prior to the isolation of essential oils.
Alkane standard solutions of C8–C20 (mixture no. 04070) and C21–C40
(mixture no. 04071) were purchased from Fluka Chemika (Buchs,
Switzerland). Glycyrrhizic acid and liquiritin reference substances
were purchased from the Chinese National Institute for the Control
of Pharmaceutical and Biological Products (Beijing, China).
The botanical origins of the roots of Liang-wai Gan Cao (G. ura-
lensis) were identified by Inner Mongolia Elion Energy Company
Limited, PR China. The chemical identification (glycyrrhizic acid
and liquiritin) was accomplished using HPLC-DAD.
Preparation of honey-roasting product
Traditional scheme (TS): each 100 g of Liang-wai Gan Cao slice was
soaked with 25 g of refined honey in 90 mL of water and then cooked
to deep yellow (China Pharmacopoeia Committee, 2010).
Drying at constant temperature (DT): Liang-wai Gan Cao slices
were soaked with refined honey and then air blast dried at 60°C for 6 h.
Ultra-ultrared drying (UD): Liang-wai Gan Cao slices were soaked
with refined honey and then ultra-ultrared dried at 250 W for 1 h.
Collection of VOCs by steam distillation
After air-drying the smashed G. uralensis, 50 g of samples were pre-
cisely weighed and mixed with 400 mL of distilled water. After keep-
ing it for 6 h at room temperature, different samples were subjected to
hydrodistillation for 8 h in the light of the standard extraction method
described in the Chinese pharmacopoeia (15). The volatile oils were
recovered with n-hexane, dehydrated with anhydrous sodium sulfate
and finally preserved in a sealed vial at 4°C for immediate analysis.
Enrichment of VOCs by SPME
One gram of G. uralensis samples was added into a 20-mL glass vial
and incubated for 30 min at 70°C. Then, enrichment of VOCs was
performed by fibers combined with a manual SPME device, which
was equilibrated at 70°C for 60 min on a heating and agitation plat-
form. After extraction, fibers were immediately conditioned in the GC
injection port for 10 min before being used for analyses. Fibers with
the following coatings were obtained from Zhenzheng (Qingdao,
China): 30 µm polydimethylsiloxane (PDMS); 65 µm silica/polydime-
thylsiloxane (SiO2/PDMS); 65 µm polydimethylsiloxane/divinylbenzene
(PDMS/DVB); 65 µm activated carbon/polydimethylsiloxane (ACAR/
PDMS); 40/60 µm activated carbon/polydimethylsiloxane/civinylben-
zene (ACAR/PDMS/DVB) and poly ( phthalazine ether sulfone ke-
tone)/activated carbon (ESK/ACAR).
He et al.
GC–MS and GC–FID analysis
GC–MS analysis (hydrodistillation samples) was performed on a
GC-2010 gas chromatograph (Shimadzu, Kyoto, Japan) equipped
with a QP2010 mass spectrometer (Shimadzu); GC–MS analysis
(HS-SPME samples) was performed on a 7890A gas chromatograph
(Agilent, Santa Clara, CA, USA) equipped with a 5975C mass spec-
trometer (Agilent). An HP-5 capillary column (30,006D × 0.25 mm
i.d., film thickness of 0.25 µm, Agilent) was used for separation and a
DB-5MS capillary column (50 m × 0.25 mm i.d., film thickness of
0.25 µm, Agilent) was also used for the preliminary study. The follow-
ing oven temperature program was applied, initiated at 40°C for 5 min,
increased to 90°C at the rate of 8°C min−1, then increased to 190°C at
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the rate of 4°C min−1 and finally increased to 300°C at the rate of 10°
C min−1 for 3 min. The spectrometers were operated in electron impact
mode; the scan range was 20–500 amu; the ionization energy was 70 eV
and the scan rate was 0.50 s per scan. Injector, interface and ion source
were kept at 280, 250 and 200°C, respectively. For hydrodistillation
samples, split injection was 1 µL conducted with a split ratio of
1 : 40, whereas for HS–SPME samples, unsplit stream sampling
(2 min) was selected, and the probe was removed after 10 min.
In gas chromatography (GC–FID) analysis, the VOC samples were
collected using the hydrodistillation method, and the analysis was
performed on a 7890A gas chromatograph (Agilent) with the same
chromatographic condition described above, and a split ratio of 1 : 5
was conducted.
Data processing
The identification of VOCs was made by comparisons of their retention
indices (RIs), mass spectrum from the Nist 08, Nist 2011 and Wiley 8
data. All data analyses were carried out on a Pentium IV (Intel) personal
computer. Heuristic evolving latent projections (HELP) and baseline
correction methods were implemented in C++ and MFC with a nice
graphic user interface. Alternative moving window factor analysis
(AMWFA), selective ion analysis (SIA) and principal component anal-
ysis (PCA) programs were coded in MATLAB 2010 (a) for windows.
Results
In this study, the characteristic smell of Liang-wai Gan Cao was mere-
ly described by the qualitative and quantitative values of VOCs. After
extraction using hydrodistillation and HS–SPME methods, VOCs of
Liang-wai Gan Cao were analyzed, using GC separation and MS
detection in a short run time. The analysis results of different
extraction methods or fibers are presented in Table I, and the detailed
resolution process is discussed in the following section.
The characteristic smell of Liang-wai Gan Cao can be influenced
by multiple factors. First, PCA results for GC–FID chromatograms in-
dicated that cultivation regions or technologies play an important role.
Moreover, GC–MS was used to determine the samples prepared by
hydrodistillation, as illustrated by the results in Table II. In addition,
HS-SPME/GC–MS results also clearly revealed some differences be-
tween the chromatograms of Liang-wai Gan Cao and those of honey-
roasting products by different pharmaceutical technologies.
Discussion
Pure mass spectrum extraction and resolution
from “black” GC–MS data
In terms of complex matrix analysis, pure mass spectra are a guarantee
for the researcher to accurately identify the unknown compounds, or
GC–MS Analysis and Volatile Profile Comparison 881

Table I. GC–MS Results for Steam Distillation and HS-SPME Samples from Liang-wai Gan Cao

Compounds Odor type/odor strengtha Peak no. Formula Identify Hydrodistillation SPME

Terpenes
α-Thujene Woody/medium 11 C10H16 RI, MS – √1,5,7
2,4(10)-Thujadiene – 13 C10H14 RI, MS √ √5,7
α-Terpinene Woody/medium 20 C10H16 RI, MS √ √5,7
β-Cymene – 22 C10H14 RI, MS √ √1,3,5,7
γ-Terpinene Terpenic/medium 25 C10H16 RI, MS √ √5,7
Umbellulon – 34 C10H14O RI, MS – √1,3,4,5,7
Terpinen-4-ol Spicy/medium 35 C10H18O RI, MS √ √1,3,5,7
Alcohols

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1-Hexanolb Herbal/medium 3 C6H14O RI, MS √ √1,3,5,6,7
p-Cymen-8-ol Fruity/medium 36 C10H14O RI, MS √ √1,3
6,9-Pentadecadien-1-ol – 61 C15H28O MS √ –
Aldehydes
Hexanalb Green/high 1 C6H12O RI, MS √ √1,3,4,5,6,7
2-Hexenal Green/high 2 C6H10O RI, MS √ √1,5,7
Heptanalb Green/high 9 C7H14O RI, MS √ √1,3,5,6,7
Benzaldehyde Fruity/high 14 C7H6O RI, MS √ √1,5
2,4-Heptadienal, (E,E)- Fatty/high 21 C7H10O RI, MS √ –
2-Octenal, (E)-b Fatty/high 26 C8H14O RI, MS √ √1,3,6
Nonanalb,c Aldehydic/high 30 C9H18O RI, MS √ √1,3,4,5,6,7
2,6-Nonadienal, (E,Z)- Green/high 32 C9H14O RI, MS √ –
2-Nonenal, (E)-b,c Green/high 33 C9H16O RI, MS √ √1,3,4,5,6,7
Decanalb Aldehydic/high 37 C10H20O RI, MS √ √1,3,4,5,6,7
2,4-Nonadienal Fatty/high 38 C9H14O RI, MS √ –
2-Decenal, (E)- Waxy/high 39 C10H18O RI, MS √ –
(E,Z)-2,4-Decadienalc Fried/high 43 C10H16O RI, MS √ –
2,4-Decadienal, (E,E)-c Fatty/high 44 C10H16O RI, MS √ –
Tetradecanal Waxy/medium 53 C14H28O RI, MS √ √1,3,4,6,7
Pentadecanal (E-2-Tetradecen-1-ol) Waxy/medium 56 C15H30O RI, MS √ √1,3,4,5,6,7
cis,cis,cis-7,10,13-Hexadecatrienal – 62 C16H26O MS √ –
cis,cis-7,10,-Hexadecadienal – 63 C16H28O MS – √1,3,4,6,7
Ketones
2-Heptanone Cheesy/high 8 C7H14O RI, MS √ –
2,3-Octanedione Dill/medium 15 C8H14O2 RI, MS √ √1,5
5-Hepten-2-one, 6-methyl- Citrus/medium 17 C8H14O RI, MS √ –
3-Octen-2-one Earthy/high 23 C8H14O RI, MS – √1,3,5,6,7
5,9-Undecadien-2-one, 6,10-dimethyl- – 50 C13H22O RI, MS √ –
Acids
Hexanoic acidb Fatty/medium 18 C6H12O2 RI, MS – √1,3,4,5,6,7
Pentadecanoic acid Waxy/medium 59 C15H30O2 RI, MS √ –
n-Hexadecanoic acidc Waxy/low 64 C16H32O2 RI, MS √ √1,4
Heptadecanoic acidc – 67 C17H34O2 RI, MS √ –
9,12,15-Octadecatrienoic acid, (Z,Z,Z)- None 68 C18H30O2 RI, MS √ –
9,12-Octadecadienoic acid (Z,Z)-c – 69 C18H32O2 RI, MS √ –
Linoelaidic acid – 70 C18H32O2 MS √ –
Octadecanoic acid Odorless/none 72 C18H36O2 RI, MS √ –
Esters
2-Propenoic acid, butyl ester – 6 C7H12O2 RI, MS – √1,5
Hexanoic acid, methyl ester Fruity/medium 10 C7H14O2 RI, MS – √1,5
Hexanoic acid, 2-propenyl ester Fruity/high 27 C9H16O2 RI, MS – √1,3,6
Hexanoic acid, hexyl ester Green/high 47 C12H24O2 RI, MS – √1,3,4,5,6,7
Phthalic acid, diisobutyl ester Bland/low 60 C16H22O4 RI, MS – √3,4,5,6,7
Hexadecanoic acid, ethyl ester Waxy/low 65 C18H36O2 MS √ √1,3,4,7
Linoleic acid ethyl ester – 71 C20H36O2 RI, MS √ –
Phenols
Phenol, 2-methyl-5-(1-methylethyl)- – 40 C10H14O RI, MS – √1,3,5,6
Furanic
2-n-Butyl furan Spicy/medium 4 C8H12O RI, MS √ √1,3,5,7
Furan, 2-pentyl-b,c Fruity/medium 16 C9H14O RI, MS √ √1,3,4,5,6,7
trans-2-(2-Pentenyl)furan – 19 C9H12O RI, MS √ √1,5
2(3H)-Furanone, dihydro-5-pentyl- – 46 C9H16O2 RI, MS √ –

Table continues
882 He et al.

Table I. Continued

Compounds Odor type/odor strengtha Peak no. Formula Identify Hydrodistillation SPME

Alkanes
Nonane Gasoline 7 C9H20 RI, MS √ –
Tridecane – 41 C13H28 RI, MS – √1,3,5,6
Tetradecane Waxy/low 49 C14H30 RI, MS – √1,2,3,4,5,6,7
Pentadecane Waxy/low 51 C15H32 RI, MS – √1,3,4,5,6,7
Hexadecane – 52 C16H34 RI, MS – √1,3,4,5,6,7
Heptadecane – 54 C17H36 RI, MS – √1,3,4,5,6,7
Pentadecane, 2,6,10,14-tetramethyl- – 55 C19H40 RI, MS – √
Hexadecane,7,9-dimethyl- – 57 C18H38 MS – √1,3,4,5,6,7

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Octadecane – 58 C18H38 RI, MS – √3,4,5,6,7
Eicosane Waxy/low 66 C20H42 MS – √1,3,4
Docosane Waxy/low 73 C22H46 RI, MS – √1,7
Olefin
1-Undecene – 28 C11H22 RI, MS √ –
4-Undecene, (Z)- – 29 C11H22 RI, MS √ –
1-Tetradecene – 48 C14H28 RI, MS – √3,5,6
1,3,6-Octatriene, 3,7-dimethyl-, (Z)- Floral/medium 24 C10H16 RI, MS √ –
Acetals
Benzaldehyde dimethyl acetal – 31 C9H12O2 MS – √1,3,5
Others
Naphthalene, 1-methyl- Naphthyl/medium 42 C11H10 RI, MS √ √1,3,5,6
Naphthalene, 2-methyl- Floral/medium 45 C11H10 RI, MS – √ 1,3,5,6
Oxime-, methoxy-phenyl- – 5 C8H9NO2 MS – √1,3,7
Oxepine, 2,7-dimethyl- – 12 C8H10O RI, MS √ √5

Peak no: number assigned to each compound.

1, ACAR–PDMS–DVB (Zhenzheng); 2, PDMS (Zhenzheng); 3, PDMS–DVB (Zhenzheng); 4, SiO2–PDMS (Zhenzheng); 5, ACAR–PDMS (Zhenzheng); 6,
ESK–ACAR (Zhenzheng); 7, PDMS–DVB–CAR (Supelco).
RI, compounds identified on the basis of the retention index from the literature; MS, compounds identified by comparison of their mass spectra with the
corresponding pure standards.
a
The data came from the good scents company information system (http://www.thegoodscentscompany.com/search2.html).
b
The main VOCs in SPME samples.
c
The main VOCs in hydrodistillation samples.

at least, narrow the possibilities down to a few compounds from a lot
of potential chemicals. “Pure” represents a spectrum with less noise or
impurities. In this study, multiple chemometric resolution methods
(16, 17), including HELP, AMWFA and SIA methods, have been ap-
plied to resolve the two-way data into pure chromatograms and pure
spectra, which made “blind” action against some overlapped peaks
and embedded peaks clearer, as the specific examples that can be
seen in the references (18, 19). For the segment of 9.33–9.55 min
from the hydrodistillation sample, 2,4-heptadienal, (E,E)- and
β-cymene could be resolved by SIA after baseline correction and
smoothing (20, 21), which could be seen in Figure 1.
Currently, pure mass spectra can provide the researchers with some
structural information after searching in some libraries of reference
spectra, including NIST and Wiley libraries. However, a single spectral
library is not complete to contain various fragmentation spectra from a
reference compound measured on a given instrument condition. For the
sake of reliability of the identification results, two or more spectral
libraries combined with search algorithms (workstations) should be re-
peatedly used to identify the unknown mass spectra (22), especially for
some certain kinds of compounds with minimal differences (structurally
related compounds). On the basis of these methods, the erroneous de-
terminations sparked by only one reference library could be excluded to
the utmost. For the pure mass spectrum extracted from the segment of
9.18 min, several candidate compounds (similarity >93%) were found
in different libraries, including α-terpinene, 2-carene and terpinolen,
which can be seen in Figure 2. After the discrimination of RI to leave
out/confirm the ambiguous results (red symbol “RI” in Figure 2), the
characteristic ratios of m/z 77 (C6H+5) and m/z 79 (C6H+7) were also
used as a powerful tool. Often the molecular ion’ isotopes can be
used in deriving the composition of unknown samples, and fragmental
intensities can also be used for the identification due to the different
structure or bond energy of corresponding molecules. The problem is
focused on the fragmental intensities’ uncertainty due to various instru-
mental conditions, as the solution multiple spectra in libraries were uti-
lized to distinguish their characteristic ratios of m/z 77 and m/z 79. As
seen from Figure 3 (α-terpinene’s data from the different libraries), frag-
mental intensities of m/z 77 are entirely stronger than those of m/z 79. In
this way, the structural property has resulted in a unique and reproduc-
ible spectrum or fragmentation of compounds. Finally, α-terpinene was
identified in a quick and inexpensive fashion.
Mass spectrum analysis for natural VOCs in
Liang-wai Gan Cao
Overall investigation for VOCs in steam distillation and
SPME samples
Compared with steam distillation, SPME is a convenient method that
requires minimal sample preparation and time. Attributable to a vari-
ety of factors (23, 24), the amount of analytes extracted from G. ura-
lensis samples strongly depends on the properties of the fiber coatings
and the aromatic substance, after the fiber coating is directly exposed
in the headspace with a long-time thermal equilibration. In the test, the
amount and strength of adsorbed compounds were represented in the
chromatograms derived from six SPME fibers, in which ACAR/
GC–MS Analysis and Volatile Profile Comparison 883

Table II. GC–MS Results for Steam Distillation Samples from Liang-wai Gan Cao, Wang-ye Gan Cao and Yan-chi Gan Cao

RI Compounds Formula Identify/similarity Liang-wai Wang-ye Yan-chi Gan

Gan Cao (%) Gan Cao (%) Cao (%)

800 Hexanala C6H12O GC–FID Undected Undected Undected

851 2-Hexenala C6H10O RI, MS/95% 2.57 0.52 2.42
882 1-Hexanola C6H14O RI, MS/93% 1.37 3.2 2.28
892 2-n-Butyl furan C8H12O RI, MS/91% 0.52 0.27 0.86
900 Nonane C9H20 RI, MS/97% 0.21 0.06 0.54
908 2-Heptanone C7H14O RI, MS/92% 0.2 0.42 0.58
911 Heptanal C7H14O RI, MS/95% 0.68 0.43 1.43
928 α-Thujene C10H16 RI, MS/93% – 0.1 0.11
933 α-Pinene C10H16 RI, MS/85% – 0.1 0.12

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941 Oxepine, 2,7-dimethyl- C8H10O RI, MS/80% 0.04 0.01 0.2
947 2,4(10)-Thujadiene C10H14 RI, MS/87% 0.04 0.05 0.26
971 Benzaldehyde C7H6O RI, MS/95% 0.54 0.1 0.4
988 Heptane, 2,2,4,6,6-pentamethyl- C12H26 RI, MS/95% – – 0.21
988 2,3-Octanedione C8H14O2 RI, MS/90% 0.01 1.41 –
993 Furan, 2-pentyl-a C9H14O RI, MS/96% 11.13 2.75 15.92
1000 5-Hepten-2-one, 6-methyl- C8H14O RI, MS/92% 0.01 – 0.65
1004 trans-2-(2-Pentenyl)furan C9H12O RI, MS/89% 0.52 – 0.74
1012 Octanal C8H16O RI, MS/93% – 0.25 –
1015 α-Terpinene C10H16 RI, MS/96% 0.25 0.23 0.71
1020 Hexanoic acid C6H12O2 RI, MS/95% – 0.19 –
1023 2,4-Heptadienal, (E,E)- C7H10O RI, MS/89% 0.13 – 0.28
1025 β-Cymene C10H14 RI, MS/93% 0.51 0.35 5
1052 1,3,6-Octatriene, 3,7-dimethyl-, (Z)- C10H16 RI, MS/95% 0.1 – 0.36
1052 3-Octen-2-one C8H14O RI, MS/90% – 0.24 0.14
1060 γ-Terpinenea C10H16 RI, MS/90% 0.27 0.31 0.65
1067 2-Octenal, (E)- C8H14O RI, MS/93% 0.81 0.48 1.74
1100 1-Undecene C11H22 RI, MS/88% 0.03 – –
1103 4-Undecene, (Z)- C11H22 RI, MS/86% 0.05 – –
1106 Linalool C10H18O RI, MS/90% – 0.22 0.68
1108 Nonanal C9H18O RI, MS/95% 0.54 0.34 1.31
1158 2,6-Nonadienal, (E,Z)- C9H14O RI, MS/90% 0.14 0.69 0.24
1162 2-Nonenal, (E)-a C9H16O RI, MS/90% 1.25 0.1 2.43
1179 Terpinen-4-ola C10H18O RI, MS/95% 0.49 0.76 1.38
1191 p-Cymen-8-ol C10H14O RI, MS/89% 0.01 0.29 0.53
1194 α-Terpineol C10H18O RI, MS/92% – – 0.36
1209 Decanal C10H20O RI, MS/92% 0.23 0.13 0.64
1220 2,4-Nonadienal C9H14O RI, MS/91% 0.07 0.05 0.2
1265 2-Decenal, (E)- C10H18O RI, MS/90% 0.05 0.82 0.11
1269 Bicyclo[3.2.0]heptan-2-one C7H10O RI, MS/90% 0.24 0.28 0.23
1276 2H-Pyran-4-carboxylic acid, 3,4-dihydro-5-methyl-, ethyl ester C9H14O3 MS/86% 0.16 – 0.45
1289 Naphthalene, 1-methyl- C11H10 RI, MS/80% 0.04 0.29 0.16
1298 (E,Z)-2,4-Decadienala C10H16O RI, MS/95% 1.95 0.22 2.75
1319 2,4-Decadienal, (E,E)-a C10H16O RI, MS/96% 8.27 0.84 16.88
1368 2(3H)-Furanone, dihydro-5-pentyl- C9H16O2 RI, MS/88% 0.06 0.1 –
1371 2-Octenal, 2-butyl- C12H22O RI, MS/94% – 0.51 –
1456 5,9-Undecadien-2-one, 6,10-dimethyl- C13H22O RI, MS/91% 0.11 0.11 1.33
1498 2,5-Etheno[4.2.2]propella-3,7,9-triene C12H10 MS/91% 0.02 0.06 –
1614 Tetradecanal C14H28O RI, MS/90% 0.17 0.1 0.45
1679 1-Tetradecanol C14H30O RI, MS/94% – 0.49 –
1716 Pentadecanal-a C15H30O RI, MS/89% 2.08 0.88 3.22
1771 Tetradecanoic acid C14H28O2 RI, MS/94% – 0.4 –
1871 Pentadecanoic acid C15H30O2 RI, MS/88% 0.33 1.52 1.15
1888 6,9-Pentadecadien-1-ol C15H28O MS/90% 4.11 1.14 5.14
1894 cis,cis,cis-7,10,13-Hexadecatrienal C16H26O MS/90% 1.06 0.26 1.11
1978 n-Hexadecanoic acid C16H32O2 RI, MS/94% 20.97 42.34 0.78
1998 Hexadecanoic acid, ethyl ester C18H36O2 RI, MS/90% 0.21 0.62 0.65
2073 Heptadecanoic acid C17H34O2 RI, MS/90% 0.3 0.52 –
2076 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- C18H30O2 RI, MS/85% 0.57 0.08 0.3
2149 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 RI, MS/96% 14.36 23.2 1.13
2153 Linoelaidic acid C18H32O2 MS/89% 4.49 4.71 –
2167 Linoleic acid ethyl ester C20H36O2 RI, MS/88% 0.83 0.4 1.1
2173 Octadecanoic acid C18H36O2 RI, MS/85% 0.9 0.68 0.23

Peak no: number assigned to each compound.

RI, compounds identified on the basis of the retention index from the literature; MS, compounds identified by comparison of their mass spectra with the
corresponding pure standards.
a
The important peaks deduced from the PCA analysis for GC–FID data.
884
Figure 1. After baseline correction and smoothing, 2,4-heptadienal, (E,E)- and β-cymene could be resolved by SIA. This figure is available in black and white in print
and in color at JCS online.
PDMS/DVB fiber coating (bipolar) had much better extraction effi-
ciencies than the others. It is noteworthy that each fiber coating has
a respective sensitivity and selectivity for different compounds based
on the adsorption–absorption theory, such as PDMS (non-polar),
SiO2/PDMS ( polar) and PDMS/DVB (semi-polar).
For steam distillation, the largest drawback is the prolonged heating
period affecting the extract composition, as well as the yield, because the
high temperatures led to significant losses of volatile compounds (25).
To achieve a comprehensive understanding of VOCs in Liang-wai
Gan Cao, all GC–MS data should be combined for analysis. As seen
from Table I, peak number, identified compounds, odor type (odor
strength), formula and diversity could be found. First, terpene com-
pounds are irreplaceable to provide the aroma of herbs, such as
α-thujene, α-terpinene, β-cymene, γ-terpinene, umbellulon, 2,4
(10)-thujadiene and terpinen-4-ol identified, extracted by both hydro-
distillation and SPME methods. Volatile aldehydes, especially many
unsaturated aldehydes, also contribute greatly to the desirable flavors
of many foods/herbs. In our test, volatile aldehydes could be easily
observed in hydrodistillation samples, due to the prolonged heating
period effects (the auto-oxidation process of unsaturated fatty acids
or others). For example, 2-hexenal is formed by the oxidation of
He et al.
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linoleic acid (26, 27); decanal, nonanal, 2-decenal and octenal are
formed by the oxidation of oleate (28); 2,4-decadienal and hexanal
are formed by the oxidation of linoleate (28) and2,4-heptadienal
and 2,6-nonadienal are formed by the oxidation of linolenate (28)
and so on. All these lipid-derived aldehydes are known to be the im-
portant flavor contributors. In addition, esters, alkanes and olefin
might contribute largely to the characteristic aroma of G. uralensis.
Mass spectrum analysis and GC–FID comparison for Liang-wai
Gan Cao and other G. uralensis in different regions
Except for gene, maturity stages, post-harvest treatments and so on,
geographical location and cultivar differences play an important role
in the concentration and composition of herb VOCs. Thus, reporting
the aroma characters of “Liang-wai Gan Cao”, “Wang-ye Gan Cao”
and “Yan-chi Gan Cao” would provide valuable knowledge for future
research focused on the G. uralensis. After GC–MS analysis (hydrodis-
tillation samples), the results indicated that some differences existed in
three varieties. As seen from Table II, the total concentration and some
aroma characters, such as terpenes, alcohol and ketones, varied signif-
icantly due to different cultivation regions or technologies. In
GC–MS Analysis and Volatile Profile Comparison 885

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Figure 2. Examples of mass spectral identification of α-terpinene from the segment of 9.18 min, by comparison with literature mass spectra including α-terpinene,
γ-terpinene, 2-carene, terpinolen, cyclohexene, 4-methyl-3-(1-methylethylidene)- and 4-carene. Reference mass spectra plotted from values in the Willy or Nist
libraries. RI: compounds identified on the basis of the retention index from the literature. This figure is available in black and white in print and in color at JCS online.

882, 993, 1060, 1162, 1179, 1298, 1319 and 1716) seem to play
an important role.

Honey-roasting products influence on characteristic

Figure 3. For different reference spectra, the relative abundance of m/z 77 and
m/z 79 of α-terpinene was compared, and intensities of m/z 77 are stronger than
those of m/z 79.
smell of Liang-wai Gan Cao
Throughout Chinese history, honey-roasting licorice (29) is widely
used in clinical diseases and crude medicinal materials. In our re-
search, three methods were used to prepare honey-roasting products,
including TS, UD and DT. Characteristic smell could be obtained in
three kinds of products, due to the adjunction of honey and heat treat-
ment. There were some differences in three kinds of products, for ex-
ample, the samples (TS method) were hard to be mixed evenly,
accompanied with easily being burnt. Accordingly, the amount of
VOCs produced in the drying process using both UD and DT methods
is decreased. Furthermore, UD consumes less working time together
with less pollution. As a result, an intuitive vision was conducted to
compare the aroma VOCs’ differences due to different pharmaceutical
technologies, which could be seen in Figure 5.

addition, three groups of aromatic components were analyzed by GC–

FID, and 6–7 samples were included in each group. Finally, PCA re- Conclusion
sults indicated that Liang-wai Gan Cao is different from other In this work, VOCs of Liang-waiGan Cao and honey-roasting products
G. uralensis groups (Figure 4), and the peaks with RI (800, 851, were analyzed using GC–MS apparatus coupled with chemometric
886
Figure 4. The PCA results for three groups of aromatic components which were analyzed by GC–FID, and 6–7 samples were included in each group. 1—Liang-wai
Gan Cao; 2—Wang-ye Gan Cao; 3—Yan-chi Gan Cao. This figure is available in black and white in print and in color at JCS online.
Figure 5. GC–MS comparison for the different honey-roasting products
(Segment 0–35 min). Arabic numerals represent the different VOCs from
Liang-wai Gan Cao. S1: interference peak 1.
resolution, and the comprehensive information could be obtained in
spectral libraries. The samples were obtained using an overall way of
diversification; all these efforts could avoid untrustworthy results
He et al.
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about aromatic substance in samples, in which 75 compounds could
be identified, including terpenes, alcohols, aldehydes, ketones and es-
ters. In addition, PCA was set for the differentiation between Liang-
wai Gan Cao and other G. uralensis samples, and the geographical lo-
cation differences accompanied with some important peaks could be
found. That is to say, the overall flavors are mainly influenced by
gene, followed by geographical position, maturity stages, cultivar differ-
ences and so on. Beyond that, honey-roasting technologies also influ-
enced the smell of products due to the addition of honey and heat
treatment.
Funding
This work is financially supported by the general project of Hunan
Provincial Education Department (grant no. 14C1098), Hunan Prov-
ince Natural Science Fund (no. 2016JJ4085) and Xiang Tan Universi-
ty Research Fund (no. 13QDZ34). The studies meet with the approval
of the university’s review board.
Conflict of interest statement. None declared.
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