# Characterization of Chemical Composition of Pericarpium Citri Reticulatae Volatile Oil by Comprehensive Two-Dimensional Gas Chromatography with High-Resolution Time-of-Flight Mass Spectrometry

Authors: Kunming Qin; Lijuan Zheng; Hao Cai; Gang Cao; Yajing Lou; Tulin Lu; Yachun Shu; Wei Zhou; Baochang Cai
Journal: Evidence-Based Complementary and Alternative Medicine
 (2013)

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## Abstract

Pericarpium Citri Reticulatae (Chenpi in Chinese) has been widely used as an herbal medicine in Korea, China, and Japan. Chenpi extracts are used to treat indigestion and inflammatory syndromes of the respiratory tract such as bronchitis and asthma. This thesis will analyze chemical compositions of Chenpi volatile oil, which was performed by comprehensive two-dimensional gas chromatography with high-resolution time-of-flight mass spectrometry (GC × GC-HR-TOFMS). One hundred and sixty-seven components were tentatively identified, and terpene compounds are the main components of Chenpi volatile oil, a significant larger number than in previous studies. The majority of the eluted compounds, which were identified, were well separated as a result of high-resolution capability of the GC × GC method, which significantly reduces, the coelution.β-Elemene is tentatively qualified by means of GC × GC in tandem with high-resolution TOFMS detection, which plays an important role in enhancing the effects of many anticancer drugs and in reducing the side effects of chemotherapy. This study suggests that GC × GC-HR-TOFMS is suitable for routine characterization of chemical composition of volatile oil in herbal medicines.


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## Body

## 1. Introduction

Pericarpium Citri Reticulatae (Chenpi in Chinese) has been widely used as an herbal medicine for a long time in China, Korea, and Japan, for its pharmacologic activity, rich resources, low toxicity, and costs. Chenpi is the dried ripe fruit peel ofCitrus reticulata Blanco and its cultivars, gathered from September to December [1]. Their main cultivars are Citrus reticulata “Chachi,” Citrus Reticulata “Dahongpao,” and Citrus erythrosa Tanaka. In Chinese people’s traditional use, Chenpi is mostly utilized to eliminate phlegm and strengthen spleen [2]. Moreover, Chenpi is extensively added to food as a condiment.It is well known that Chenpi contains various bioactive compounds, such as flavonoids, phenolic acids, and limonoids [2, 3]. In the present study, most reports on Chenpi focus on phenolic compounds and flavonoids [3–7], but few focus on volatile compounds which also have strong pharmacologic bioactivities. For example, high-performance liquid chromatography (HPLC), high-speed countercurrent chromatography (HSCCC), and capillary electrophoresis (CE) have been applied for the determination of phenolic compounds and flavonoids of Chenpi [8–10]. However, reviewing the literature, it seems that the chemical composition of the volatile oil of Chenpi has been little investigated [11]. Furthermore, the volatile compounds of Chenpi may contribute to pharmacological effects of Chenpi extracts reported above [12, 13]. Therefore, a method able to rapidly identify the volatile compounds of Chenpi could be a useful tool for the purpose of a complete phytochemical analysis.Gas chromatography-mass spectroscopy (GC-MS) has been used for the qualitative analysis of the volatile constituents in Chenpi [11]. But it is difficult to achieve the complete separation of minor volatile components and many coelution volatile constituents. To solve these problems, it is necessary to use multidimensional gas chromatography. Comprehensive two-dimensional gas chromatography with high-resolution time-of-flight mass spectrometry (GC × GC-HR-TOFMS) is a new developed powerful and versatile analytical tool, which combines two powerful analytical technologies with complementary attributes [14, 15]. GC × GC separates chemical species with two capillary columns interfaced by a modulator that traps and concentrates eluents from the first column, and it then introduces them into the second column, producing a full secondary chromatogram for each single data point of a traditional one-dimensional separation [16, 17]. HR-TOFMS provides mass precision that is fine enough to distinguish elemental compositions, providing a more definitive basis for molecular identification. GC × GC is important for HR-TOFMS because the better separations significantly reduce the coelution and the problems of mass spectral mixing. And, HR-TOFMS is important for GC × GC because the structural and compositional information available with HR-TOFMS aids in the interpretation of the rich, complex data from GC × GC separations [18]. GC × GC-TOFMS has been successfully applied in the volatile oil study and greatly improves the result of component separation and identification [19, 20]. In this study, the volatile oil of Chenpi was firstly separated and detected with GC × GC-HR-TOFMS (Figure 1).Figure 1
Flow chart of the chemical composition study of Chenpi volatile oil by GC × GC-HR-TOFMS.


## 2. Materials and Methods

### 2.1. Samples

Chenpi sample (fruit peels ofCitrus reticulate “Dahongpao”) was collected from Zigong in Sichuan province, China. The sample was authenticated by Professor Chen Jianwei from Nanjing University of Traditional Chinese Medicine, China.


### 2.2. Extraction of Volatile Oil

After the sample was dried for 2 h at 45°C and smashed, 50 g of sample was swollen with 600 mL of distilled water in a standard extractor for extracting volatile oil for 3 h. Then, the volatile oil was dried over anhydrous sodium sulphate until all the water was dried and then stored in the dark glass bottle at 4°C prior to GC × GC-HR-TOFMS analysis.


### 2.3. GC-MS System and GC × GC-HR-TOFMS Apparatus

GC × GC separations were performed by Tofwerk AG (Thun, Switzerland) on an Agilent 7890 A GC and 7693 autosampler with: 1μL splitless injection; column one DB-XLB (Agilent), 15 m × 0.25 mm, 0.25 μm film thickness; column two BPX-50 (SGE), 1 m × 0.1 mm, 0.1 μm film thickness; oven temperature from 50 to 230°C at 2.0°C min−1 ramp; inlet pressure from 35 PSI to 61.5 PSI at 0.28 PSI min−1; injection temperature 250°C; transfer line temperature 300°C; Zoex ZX2 thermal modulator with a 7 s modulation period, 300 ms modulation duration, 375°C hot jet temperature, 18 L min−1 cold jet nitrogen flow rate, and 40 PSI hot jet nitrogen pressure. The Zoex FasTOF time-of-flight- (TOF-)  HRMS system used 70 eV EI ion source, 280°C ion source temperature, a mass range of m/z 50–450 with 4000 FWHM resolution, and 100 spectra per second acquisition rate.


### 2.4. Data Conversion and Peak Table Generation

The final data for each chromatogram is an array of 1000 × 600 data points, each data point with a HRMS vector of 40 K intensities. Thus, each chromatogram has 24 billion values requiring 96 gigabytes for representing for single-precision floating point numbers without compression. The set of 18 chromatograms has more than 1.7 terabytes of uncompressed data. The data were compressed and stored by the Zoex FasTOF system to HDF5-format files and were processed with GC Image GC × GC Software R2.1. In order to manage such large files on computers with limited random access memory (RAM), GC Image Software maintains a chromatogram with integer mass or centroid-resampled spectra in RAM and accesses the HR-MS data from disk as needed. GC Image can export raw data and computed results to nonproprietary file formats for processing with external software. The components can be quantified by Zoex software (Zoex Corp, Lincoln, NE, USA).All peaks with signal-to-noise ratio higher than 100 were found in the raw GC × GC chromatogram. The workstation can automatically give the parameters such as similarity, reverse, and probability of peaks via comparing them with the compounds in the library. The results were combined in a peak table. The NIST/EPA/NIH Mass Spectral Library Version 2.0 was used in this work.


## 2.1. Samples

Chenpi sample (fruit peels ofCitrus reticulate “Dahongpao”) was collected from Zigong in Sichuan province, China. The sample was authenticated by Professor Chen Jianwei from Nanjing University of Traditional Chinese Medicine, China.


## 2.2. Extraction of Volatile Oil

After the sample was dried for 2 h at 45°C and smashed, 50 g of sample was swollen with 600 mL of distilled water in a standard extractor for extracting volatile oil for 3 h. Then, the volatile oil was dried over anhydrous sodium sulphate until all the water was dried and then stored in the dark glass bottle at 4°C prior to GC × GC-HR-TOFMS analysis.


## 2.3. GC-MS System and GC × GC-HR-TOFMS Apparatus

GC × GC separations were performed by Tofwerk AG (Thun, Switzerland) on an Agilent 7890 A GC and 7693 autosampler with: 1μL splitless injection; column one DB-XLB (Agilent), 15 m × 0.25 mm, 0.25 μm film thickness; column two BPX-50 (SGE), 1 m × 0.1 mm, 0.1 μm film thickness; oven temperature from 50 to 230°C at 2.0°C min−1 ramp; inlet pressure from 35 PSI to 61.5 PSI at 0.28 PSI min−1; injection temperature 250°C; transfer line temperature 300°C; Zoex ZX2 thermal modulator with a 7 s modulation period, 300 ms modulation duration, 375°C hot jet temperature, 18 L min−1 cold jet nitrogen flow rate, and 40 PSI hot jet nitrogen pressure. The Zoex FasTOF time-of-flight- (TOF-)  HRMS system used 70 eV EI ion source, 280°C ion source temperature, a mass range of m/z 50–450 with 4000 FWHM resolution, and 100 spectra per second acquisition rate.


## 2.4. Data Conversion and Peak Table Generation

The final data for each chromatogram is an array of 1000 × 600 data points, each data point with a HRMS vector of 40 K intensities. Thus, each chromatogram has 24 billion values requiring 96 gigabytes for representing for single-precision floating point numbers without compression. The set of 18 chromatograms has more than 1.7 terabytes of uncompressed data. The data were compressed and stored by the Zoex FasTOF system to HDF5-format files and were processed with GC Image GC × GC Software R2.1. In order to manage such large files on computers with limited random access memory (RAM), GC Image Software maintains a chromatogram with integer mass or centroid-resampled spectra in RAM and accesses the HR-MS data from disk as needed. GC Image can export raw data and computed results to nonproprietary file formats for processing with external software. The components can be quantified by Zoex software (Zoex Corp, Lincoln, NE, USA).All peaks with signal-to-noise ratio higher than 100 were found in the raw GC × GC chromatogram. The workstation can automatically give the parameters such as similarity, reverse, and probability of peaks via comparing them with the compounds in the library. The results were combined in a peak table. The NIST/EPA/NIH Mass Spectral Library Version 2.0 was used in this work.


## 3. Results and Discussion

### 3.1. Qualitative Analysis of Chenpi Volatile Oil

The column system is nearly orthogonal and provides a structured separation. A typical two-dimensional separation/total ion chromatogram (TIC) and three-dimensional chromatogram are shown in Figure2. In the GC × GC system, compounds are separated by volatility difference on the first dimension nonpolar column and by polarity on the second medium-polar column. The GC × GC system accomplishes the true orthogonal separation on account for both the change of the polarity of two fixed phases and the linear temperature programming.GC × GC-HR-TOFMS chromatogram (a) and three-dimensional chromatogram (b) of Chenpi volatile oil.
(a)
(b)Using GC × GC-HR-TOFMS, the quantity of the detected components was up to 834. Compared to the traditional identification method such as GC-MS, the analysis from GC × GC-HR-TOFMS becomes more reliable, relying on the combined identification information including retention times, similarity, reverse match factor, and probability. The similarity and reverse match factors indicate how well a mass spectrum matches the library spectrum, but the isomers have similar mass spectra. In this case, the probability is used to determine whether the peaks with the same name belong to one compound or several compounds. The GC × GC-HR-TOF/MS software was used to find all the peaks in the raw GC × GC chromatogram. A library search was carried out for all the peaks using the NIST/EPA/NIH version 2.0, and the results were combined in a single peak table. A similarity and reverse match factor above 583 and 612, respectively, indicates that an acquired mass spectrum usually shows a good match with the library spectrum. Because of the numerous isomers present in volatile oils, especially within monoterpenes and sesquiterpenes, more attention should be paid for identification using mass spectra. In order to enhance the reliability of the identification by MS, both similarity and reverse match factor should be used. According to our experience and the literature data [18–20], 167 compounds with good match were tentatively identified including 50 monoterpenes, 36 sesquiterpenes, 31 esters and acids, 9 aldehydes and ketones, 6 alcohols, 3 ethers, 12 phenyl compounds, and 20 other components. Compounds have lower search probabilities than these counted as unknowns, and were disqualified for Kovats index comparison. Table 1 listed 167 components identified in Chenpi volatile oil. The volatile fraction is characterized by high percentages of monoterpenes, sesquiterpenes, and esters, including β-elemene, p-mentha-1(7),8(10)-dien-9-ol, and limonene. In this study, many components have also been tentatively identified, which were found in Chenpi volatile oil for the first time such as globulol and  isoledene. There is high possibility that they will be literally useful for further pharmaceutical research of Chenpi volatile oil.Table 1
167 main volatile components identified in the Chenpi volatile oil.
No.
Compound name
Peak I/min
Peak II/s
Volume
Library formula
Library probability
Library CAS no.
(1)
Nonanal
28.62
1.6
2367.153
C9H18O
50.1
124-19-6
(2)
Pyrrolizidine-3-one-5-ol, ethyl ether
53.49
2.74
53.1709
C9H15NO2
17.7
0-00-0
(3)
2-Methoxy-4-vinylphenol
42.38
2.73
2959.926
C9H10O2
59.39
7786-61-0
(4)
Styrene
14.14
1.65
731.272
C8H8
36.4
100-42-5
(5)
Octanal
21.38
1.58
2235.185
C8H16O
62.19
124-13-0
(6)
Ethylbenzene
12.57
1.39
484.4996
C8H10
64.86
100-41-4
(7)
Pterin-6-carboxylic acid
11.24
0.82
50.3913
C7H5N5O3
40.94
948-60-7
(8)
Hexadecane, 1,1-bis(dodecyloxy)-
77.27
1.2
102.3988
C40H82O2
9.24
56554-64-4
(9)
1-Heptatriacotanol
75.09
2.76
169.2274
C37H76O
54.56
105794-58-9
(10)
Cholestan-3-ol, 2-methylene-, (3β,5α)-
64.35
2.11
268.4351
C28H48O
13.09
22599-96-8
(11)
[5,9-Dimethyl-1-(3-phenyl-oxiran-2-yl)-deca-4,8-dienylidene]-(2-phenyl-aziridin-1-yl)-amine
66.4
2.23
151.311
C28H34N2O
44.04
0-00-0
(12)
1,1′-(4-Methyl-1,3-phenylene)bis[3-(5-benzyl-1,3,4-thiadiazol-2-yl)urea]
23.55
2.15
106.0574
C27H24N8O2S2
24.95
0-00-0
(13)
Morphinan-4,5-epoxy-3,6-di-ol, 6-[7-nitrobenzofurazan-4-yl]amino-
46.49
0.79
105.5334
C26H27N5O6
9.48
0-00-0
(14)
Benzene, 1,1′-[3-(3-cyclopentylpropyl)-1,5-pentanediyl]bis-
7.74
1.18
149.2865
C25H34
15.18
55191-62-3
(15)
2-(2-Azepan-1-yl-2-oxoethyl)-1-hydroxy-1-phenyl-octahydro-pyrido[1,2-a]azepin-4-one
76.42
2.77
142.1083
C24H34N2O3
52.15
0-00-0
(16)
6,9,12,15-Docosatetraenoic acid, methyl ester
59.4
1.77
55.943
C23H38O2
19.28
17364-34-0
(17)
2-[4-Methyl-6-(2,6,6-trimethylcyclohex-1-enyl)hexa-1,3,5-trienyl]cyclohex-1-en-1-carboxaldehyde
53.49
2.15
64.4497
C23H32O
25.57
0-00-0
(18)
Naphthalen-2-yl-acetic acid, 6-hydroxy-6-methyl-cyclodecyl ester
41.54
2.63
65.5915
C23H30O3
27.3
0-00-0
(19)
Z-5-Methyl-6-heneicosen-11-one
80.89
1.11
115.1736
C22H42O
8.27
0-00-0
(20)
2H-Pyran, 2-(7-heptadecynyloxy)tetrahydro-
61.45
1.91
133.6559
C22H40O2
14.4
56599-50-9
(21)
Doconexent
17.88
1.63
127.9331
C22H32O2
40.98
6217-54-5
(22)
9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)-
31.16
2.22
105.1463
C21H36O4
19.78
18465-99-1
(23)
8,11,14-Eicosatrienoic acid, methyl ester, (Z,Z,Z)-
42.75
2.02
216.8234
C21H36O2
8.42
21061-10-9
(24)
5,8,11-Eicosatrienoic acid, methyl ester
57.59
2.03
96.1455
C21H30O2
43.95
0-00-0
(25)
cis-5,8,11,14,17-Eicosapentaenoic acid
24.52
4.25
53.7632
C20H30O2
13.99
10417-94-4
(26)
5,8,11,14-Eicosatetraynoic acid
58.8
3.17
144.9613
C20H24O2
29.78
1191-85-1
(27)
1,16-Cyclocorynan-17-oic acid, 19,20-didehydro-, methyl ester, (16S,19E)-
12.21
0.64
88.5548
C20H22N2O2
53.45
6393-66-4
(28)
Octadecane, 6-methyl-
82.09
1.25
184.9235
C19H40
18.41
10544-96-4
(29)
2-Methyl-E,E-3,13-octadecadien-1-ol
64.83
2.01
124.0941
C19H36O
9.74
0-00-0
(30)
Z,Z,Z-4,6,9-Nonadecatriene
26.81
1.7
109.996
C19H34
21.05
0-00-0
(31)
6,9,12-Octadecatrienoic acid, methyl ester
55.78
2.92
63.7153
C19H32O2
28.52
2676-41-7
(32)
Z,Z,Z-1,4,6,9-Nonadecatetraene
21.5
1.8
65.4446
C19H32
14.64
0-00-0
(33)
12,15-Octadecadienoic acid, methyl ester
49.14
2.35
158.5734
C19H30O2
19.33
57156-95-3
(34)
10,13-Octadecadienoic acid, methyl ester
50.47
2.47
161.0184
C19H30O2
15.45
18202-24-9
(35)
2,5-Octadecadienoic acid, methyl ester
59.52
1.99
254.7258
C19H30O2
10.15
57156-91-9
(36)
2,2,4,4-Tetramethyl-6-(1-oxo-3-phenylprop-2-enyl)-cyclohexane-1,3,5-trione
70.27
0.49
83.2401
C19H20O4
21.98
0-00-0
(37)
Gentamicina
63.63
3.21
62.6103
C18H36N4O10
27.05
13291-74-2
(38)
Z-10-Methyl-11-tetradecen-1-ol propionate
40.33
2
70.3719
C18H34O2
10.53
0-00-0
(39)
10-Heptadecen-8-ynoic acid, methyl ester, (E)-
34.18
2.06
63.2456
C18H30O2
14.6
16714-85-5
(40)
α-L-Fucopyranose 1,2:3,4-bis(benzeneboronate)
67.13
2.28
180.5156
C18H18B2O5
25.89
102281-26-5
(41)
3-(O-Anisidinomethyl)-5-(3-fluorobenzylidene)-2,4-thiazolidinedione
34.66
2.46
82.9072
C18H15FN2O3S
9.43
302954-96-7
(42)
1-Hexadecanol, 2-methyl-
44.19
1.03
65.1528
C17H36O
11.82
2490-48-4
(43)
10-Methyl-E-11-tridecen-1-ol propionate
33.33
1.56
164.2353
C17H32O2
6.69
0-00-0
(44)
13-Heptadecyn-1-ol
40.09
2.25
78.5129
C17H32O
12.04
56554-77-9
(45)
4,7,10-Hexadecatrienoic acid, methyl ester
32.97
2.05
83.5894
C17H28O2
7.98
17364-31-7
(46)
Methyl 5,7-hexadecadienoate
53.61
1.7
58.765
C17H26O2
14.34
0-00-0
(47)
3-(5-Benzyloxy-3-methylpent-3-enyl)-2,2-dimethyloxirane
35.02
2.61
96.359
C17H24O2
8.95
0-00-0
(48)
Falcarinol
37.07
1.57
55.0178
C17H24O
35.51
21852-80-2
(49)
tert-Hexadecanethiol
70.27
1.03
151.5898
C16H34S
13.87
25360-09-2
(50)
n-Hexadecanoic acid
79.08
1.74
705.7247
C16H32O2
43.05
10-3-1957
(51)
Cyclopentaneundecanoic acid
46.49
1.62
94.519
C16H30O2
13.97
6053-49-2
(52)
9-Hexadecenoic acid
65.56
1.91
89.144
C16H30O2
13.29
2091-29-4
(53)
Formic acid, 3,7,11-trimethyl-1,6,10-dodecatrien-3-yl ester
51.92
1.71
99.1647
C16H26O2
23.72
0-00-0
(54)
1,3-Dioxolane, 2-heptyl-4-phenyl-
7.74
0.67
137.1547
C16H24O2
20.64
55668-40-1
(55)
Peyonine
24.64
0.58
54.9972
C16H19NO5
27.28
19717-25-0
(56)
12,14,14-Trimethyl-3,6,9-trioxapentadecan-1-ol
66.52
1.18
58.5099
C15H32O4
16.94
55489-54-8
(57)
Isocalamendiol
73.65
2.76
52.3348
C15H26O2
24.27
0-00-0
(58)
Geranyl isovalerate
53.37
1.03
77.0183
C15H26O2
11.72
109-20-6
(59)
Cubeduel
63.26
1.88
203.2965
C15H26O
24.8
0-00-0
(60)
α-Cadinol
63.63
2.07
649.3081
C15H26O
24.36
481-34-5
(61)
Cubenol
62.42
1.9
408.4446
C15H26O
16.17
21284-22-0
(62)
.tau.-Cadinol
63.02
2
707.983
C15H26O
11.7
11-1-5937
(63)
α-Acorenol
63.63
2.17
1949.884
C15H26O
9.72
0-00-0
(64)
Globulol
60.13
1.88
278.3377
C15H26O
6.4
51371-47-2
(65)
7-Epi-cis-sesquisabinene hydrate
54.57
1.3
81.2625
C15H26O
6.4
0-00-0
(66)
Limonen-6-ol, pivalate
44.56
1.85
78.8648
C15H24O2
24.04
0-00-0
(67)
Aromadendrene oxide-(2)
62.78
2.09
96.7312
C15H24O
15.84
0-00-0
(68)
Caryophyllene oxide
59.88
2
161.5517
C15H24O
13.15
1139-30-6
(69)
Spiro[4.5]dec-6-en-8-one, 1,7-dimethyl-4-(1-methylethyl)-
38.16
2.27
73.2272
C15H24O
7.02
39510-36-6
(70)
Humulene
53
1.61
5204.711
C15H24
44.49
6753-98-6
(71)
(Z,E)-α-farnesene
55.78
1.52
31067.6
C15H24
42.94
26560-14-5
(72)
α-Copaene
48.42
1.4
7508.707
C15H24
42.37
0-00-0
(73)
δ-Elemene
45.88
1.33
5692.139
C15H24
38.01
20307-84-0
(74)
Naphthalene, 1,2,3,5,6,8a-hexahydro- 4,7-dimethyl-1-(1-methylethyl)-, (1S-cis)-
56.75
1.68
12956.64
C15H24
31
483-76-1
(75)
1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-, (Z,E)-
54.94
1.51
196.9983
C15H24
29.01
26560-14-5
(76)
Caryophyllene
50.95
1.54
3006.936
C15H24
17.9
87-44-5
(77)
β-Elemene
49.14
1.45
20347.87
C15H24
17.11
515-13-9
(78)
α-Guaiene
52.16
1.47
556.5085
C15H24
16.63
12-1-3691
(79)
Cyclohexane, 1-ethenyl-1-methyl-2,4-bis (1-methylethenyl)-
52.76
1.23
160.6527
C15H24
16.42
110823-68-2
(80)
1,6-Cyclodecadiene, 1-Methyl-5-methylene-8-(1-methylethyl)-, [S-(E,E)]-
54.45
1.65
14112
C15H24
15.25
23986-74-5
(81)
γ-Elemene
51.68
1.51
742.7373
C15H24
15.14
29873-99-2
(82)
Naphthalene, 1,2,3,4,4a,7- hexahydro-1,6-dimethyl-4-(1-methylethyl)-
57.35
1.71
200.7951
C15H24
14.55
16728-99-7
(83)
Cyclohexane, 1-ethenyl-1-methyl- 2,4-bis(1-methylethenyl)-, [1S-(1α,2β,4β)]-
48.66
1.47
549.6758
C15H24
10.66
515-13-9
(84)
β-Copaene
54.45
1.55
561.4817
C15H24
10.53
0-00-0
(85)
γ-Elemene
58.8
1.82
1698.139
C15H24
8.31
29873-99-2
(86)
β-Guaiene
57.59
1.75
60.1417
C15H24
7.16
88-84-6
(87)
Guaia-1(10),11-diene
54.82
1.67
1093.799
C15H24
6.6
0-00-0
(88)
Isoledene
54.21
1.58
405.0377
C15H24
6.27
0-00-0
(89)
4,5-Di-epi-aristolochene
47.81
1.43
90.0937
C15H24
4.2
0-00-0
(90)
trans-calamenene
56.51
1.85
84.299
C15H22
38.35
0-00-0
(91)
β-Vatirenene
73.65
2.34
437.4147
C15H22
25.71
0-00-0
(92)
4,4-Dimethyl-3-(3-methylbut-3-enylidene)-2-methylenebicyclo[4.1.0]heptane
72.68
2.32
284.3733
C15H22
11.28
79718-83-5
(93)
7-Hydroxy-6,9a-dimethyl-3-methylene-decahydro-azuleno[4,5-b]furan-2,9-dione
75.21
3.61
61.6952
C15H20O4
11.61
0-00-0
(94)
Propanoic acid, 3-(2,3,6-trimethyl- 1,4-dioxaspiro[4.4]non-7-yl)-, methyl ester
33.93
2.7
84.8793
C14H24O4
19.92
0-00-0
(95)
2,5-Furandione, 3-(2-decenyl)dihydro-
40.33
2.76
154.1135
C14H22O3
11.38
62568-81-4
(96)
trans-(2-Decenyl)succinic anhydride
72.08
1.6
67.8793
C14H22O3
10.39
81949-64-6
(97)
1,4-Benzenediol, 2,6-bis(1,1-dimethylethyl)-
77.51
0.5
70.9573
C14H22O2
7.13
2444-28-2
(98)
Tetraacetyl-d-xylonic nitrile
58.32
1.62
100.275
C14H17NO9
18.48
0-00-0
(99)
α-Ionol
30.19
2.18
277.9312
C13H22O
15.16
25312-34-9
(100)
1-(2-Acetoxyethyl)-3,6-diazahomoadamantan-9-one oxime
73.28
0.5
55.2116
C13H21N3O3
8.78
0-00-0
(101)
1b,5,5,6a-Tetramethyl-octahydro-1-oxa-cyclopropa[a]inden-6-one
33.21
1.89
138.6104
C13H20O2
6.45
0-00-0
(102)
Pyrimidin-2-one, 4-[N-methylureido]-1-[4-methylaminocarbonyloxymethyl
68.94
0.38
54.3663
C13H19N5O5
23.12
0-00-0
(103)
2H-Indeno[1,2-b]furan-2-one, 3,3a,4,5,6,7,8,8b-octahydro-8,8-dimethyl
55.3
1.93
203.2335
C13H18O2
28.93
0-00-0
(104)
Dodecanal
49.26
1.58
1282.104
C12H24O
6.6
112-54-9
(105)
2,6-Octadien-1-ol, 3,7-dimethyl-, acetate, (Z)-
46.37
1.74
3059.872
C12H20O2
37.64
141-12-8
(106)
Geranyl acetate
47.45
1.79
3772.587
C12H20O2
28.7
105-87-3
(107)
(R)-Lavandulyl acetate
46.37
2.25
81.7472
C12H20O2
14.19
0-00-0
(108)
Geranyl vinyl ether
36.83
1.77
76.0262
C12H20O
14.31
0-00-0
(109)
3′-Hydroxyquinalbarbitone
39.61
1.5
51.4505
C12H18N2O4
10.72
839-21-4
(110)
Non-1-yn-5-en-9-aldehyde, 4-carbethoxy-
44.31
2.11
115.1319
C12H16O3
17.87
0-00-0
(111)
Undecanal
42.75
1.59
704.5031
C11H22O
33.86
112-44-7
(112)
Cyclohexane, 2-ethenyl-1,1-dimethyl-3-methylene-
30.31
1.34
135.2002
C11H18
23.83
95452-08-7
(113)
Cyclohexene, 2-ethenyl-1,3,3-trimethyl-
42.02
2.22
591.2578
C11H18
14.36
5293-90-3
(114)
Acetic acid, octyl ester
36.47
1.49
323.6557
C10H20O2
17.84
112-14-1
(115)
Decanal
35.74
1.61
7031.098
C10H20O
61.62
112-31-2
(116)
Cephrol
37.55
1.66
328.2375
C10H20O
12.34
40607-48-5
(117)
Linalol
28.74
1.52
3645.373
C10H18O
81.59
78-70-6
(118)
α-Terpineol
34.9
1.89
7440.985
C10H18O
67.75
98-55-5
(119)
Terpinen-4-ol
34.18
1.79
3293.372
C10H18O
50.03
562-74-3
(120)
Citronellal
32.12
1.69
615.3934
C10H18O
42.59
106-23-0
(121)
2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, cis-
30.31
1.67
159.2231
C10H18O
39.11
29803-82-5
(122)
Cyclohexanol, 1-methyl-4-(1-methylethenyl)-, cis-
31.64
1.8
726.8632
C10H18O
10.59
7299-41-4
(123)
2-Cyclohexen-1-ol, 2-methyl-5-(1-methylethyl)-, (1S-cis)-
36.11
1.85
101.2405
C10H18O
10.53
536-30-1
(124)
exo-2,7,7-trimethylbicyclo[2.2.1]heptan-2-ol
32.97
1.89
169.9902
C10H18O
8.15
0-00-0
(125)
5-Hepten-1-ol, 2-ethenyl-6-methyl-
45.76
1.68
397.1537
C10H18O
7.75
18479-48-6
(126)
Bicyclo[4.1.0]heptane, 3,7,7-trimethyl-, [1S-(1α,3β,6α)]-
45.76
1.58
368.8305
C10H18
9.72
2778-68-9
(127)
Desulphosinigrin
66.64
1.06
73.3518
C10H17NO6S
8.92
5115-81-1
(128)
R-Limonene
39.37
2.73
97.0311
C10H16O3
40.37
0-00-0
(129)
Limonene oxide, trans-
31.28
1.78
570.8168
C10H16O
54.24
4959-35-7
(130)
Limonene oxide, cis-
30.92
1.78
1068.476
C10H16O
40.43
13837-75-7
(131)
trans-p-Mentha-2,8-dienol
30.07
1.78
448.7691
C10H16O
34.35
0-00-0
(132)
3-Cyclohexene-1-acetaldehyde,α,4-dimethyl-
36.35
2.09
181.7561
C10H16O
31.38
29548-14-9
(133)
cis-p-Mentha-1(7),8-dien-2-ol
37.43
2.05
398.5489
C10H16O
26.42
0-00-0
(134)
(Z)-Carveol
35.5
1.97
452.2402
C10H16O
25.48
1197-06-4
(135)
trans-Carveol
36.71
2
1591.657
C10H16O
22.66
1197-07-5
(136)
cis-p-Mentha-1(7),8-dien-2-ol
33.93
1.91
336.4337
C10H16O
21.5
0-00-0
(137)
1-Cyclohexene-1-methanol, 4-(1-methylethenyl)-
35.62
1.89
195.9113
C10H16O
20.76
536-59-4
(138)
p-Mentha-1(7),8(10)-dien-9-ol
49.14
2.02
1162.984
C10H16O
20.12
29548-13-8
(139)
cis-p-Mentha-2,8-dien-1-ol
43.47
1.91
99.0278
C10H16O
7.83
3886-78-0
(140)
2,6-Dimethyl-3,5,7-octatriene-2-ol,  E,E-
31.88
1.98
187.0291
C10H16O
7.6
0-00-0
(141)
(S)-(-)-(4-Isopropenyl-1-cyclohexenyl)methanol
35.38
1.89
172.7702
C10H16O
5.86
18457-55-1
(142)
Bicyclo[3.1.0]hex-2-ene, 4-methyl-1-(1-methylethyl)-
17.4
1.11
7918.412
C10H16
65.04
28634-89-1
(143)
β-Pinene
20.66
1.31
19964.34
C10H16
40.8
127-91-3
(144)
β-Ocimene
25.48
1.34
4519.345
C10H16
37.56
13877-91-3
(145)
α-Phellandrene
22.47
1.32
2085.008
C10H16
33.42
99-83-2
(146)
β-Myrcene
21.5
1.27
69260.99
C10H16
31.3
123-35-3
(147)
α-Pinene
17.88
1.16
48376.96
C10H16
23.82
80-56-8
(148)
Camphene
18.85
1.22
406.4708
C10H16
19.93
79-92-5
(149)
β-Phellandrene
20.29
1.27
4173.575
C10H16
19.68
555-10-2
(150)
D-Limonene
24.52
1.69
1784450
C10H16
19.59
5989-27-5
(151)
α-Terpinene
23.43
1.33
4905.547
C10H16
17.58
99-86-5
(152)
Isoterpinene
28.38
1.46
14607.02
C10H16
16.01
586-62-9
(153)
γ-Terpinene
26.33
1.5
189808.1
C10H16
15.28
99-85-4
(154)
1,5,5-Trimethyl-6-methylene-cyclohexene
26.33
1.86
305.9981
C10H16
9.81
514-95-4
(155)
Cyclohexene, 1-methyl-4-(1-methylethenyl)-, (S)-
24.52
3.31
51.7426
C10H16
9.58
5989-54-8
(156)
γ-Pyronene
23.07
1.27
60.3834
C10H16
5.33
514-95-4
(157)
5-Isopropenyl-2-methylcyclopent-1-enecarboxaldehyde
36.11
2.3
711.2013
C10H14O
34.31
0-00-0
(158)
(-)-Carvone
37.8
2.35
909.8454
C10H14O
32.76
6485-40-1
(159)
1-Cyclohexene-1-carboxaldehyde, 4-(1-methylethenyl)-
39.85
2.43
1404.104
C10H14O
31.76
2111-75-3
(160)
3,5-Heptadienal, 2-ethylidene-6-methyl-
36.95
2.28
238.0085
C10H14O
28.28
99172-18-6
(161)
Benzenemethanol,α,α,4-trimethyl-
34.05
2.28
288.3869
C10H14O
16.59
1197-01-9
(162)
Cyclohexanone, 2-(2-butynyl)-
42.38
3.18
59.1028
C10H14O
15.02
54166-48-2
(163)
D-Verbenone
41.42
2.16
475.2141
C10H14O
11.63
18309-32-5
(164)
3,5-Heptadienal, 2-ethylidene-6-methyl-
36.11
2.5
184.88
C10H14O
8.97
99172-18-6
(165)
o-Cymene
23.55
1.55
35991.09
C10H14
47.51
527-84-4
(166)
2,6-Dimethyl-1,3,5,7-octatetraene, E,E-
31.64
1.71
164.8089
C10H14
20.19
460-01-5
(167)
Benzene, 1-methyl-4-(1-methylethenyl)-
27.9
1.8
271.6784
C10H12
20.42
1195-32-0


### 3.2. Group Separation of Chenpi Volatile Components

In GC × GC-HR-TOFMS analysis, the 167 identified volatile components in Chenpi volatile oil were mainly classified into two groups that can be seen in Figure3. Based on GC × GC-HR-TOFMS, it can be found that the peaks in areas A and B are monoterpenes and sesquiterpenes, respectively. These monoterpenes and sesquiterpenes are mainly alkenes, alcohols, and ethers. It was also found that a lot of saturated and unsaturated fatty acid esters and phenyl compounds constitute the Chenpi volatile oil. This study demonstrates that GC × GC-HR-TOFMS is a powerful separation and identification tool that allows for the identification and group separation of a much larger number of complex volatile oil components.Figure 3
The GC × GC contour plot of Chenpi volatile oil group separation result. Regions marked by squares (A) and (B) were identified mainly as monoterpenes and sesquiterpenes, respectively.


### 3.3. Identification of Three Coelution Volatile Components in Chenpi Volatile Oil

The high-resolution mass spectra in the TIC can be used for accurate identification of volatile compounds in Chenpi volatile oil, and these identified compounds will be significant to the further pharmaceutical research. For example, Figure4 compares the high resolution mass spectrum of the blob (peak) marked with 138 (49.14 min, 2.02 s), 104 (49.26 min, 1.58 s), and 77 (49.14 min, 1.45 s), head-to-tail with the mass spectrum of p-mentha-1(7),8(10)-dien-9-ol, dodecanal, and β-elemene TMS from the NIST/EPA/NIH library mass spectra. For p-mentha-1(7),8(10)-dien-9-ol, the forward match factor is 806; reverse match factor is 855; and probability is 20.12%. For dodecanal, the forward match factor is 763; reverse match factor is 773; and probability is 7.16%. For β-elemene, the forward match factor is 911; reverse match factor is 914; and probability is 17.11%. The above three volatile components cannot be clearly separated or identified by traditional one-dimensional gas chromatography or GC-MS method, because they are coelution volatile components, which have very similar chemical properties including volatility and polarity. In this study, the three coelution volatile components in Chenpi volatile oil were well separated and identified by GC × GC-HR-TOFMS, which have not been reported in other studies (Figure 4).Details of three coelution volatile components (peak 77, 104, 138) in GC × GC chromatogram. The spectra ofβ-elemene (peak 77), dodecanal (peak 104), and p-mentha-1(7),8(10)-dien-9-ol (peak 138) in sample and in NIST library, respectively.
(a)
(b)
(c)
(d)This study showed that GC × GC-HR-TOFMS represents a powerful separation and analysis tool for the analysis of complex volatile oils of herbal medicines. GC × GC-HR-TOFMS can give the information about the formula and structures, can provide the opportunity for differentiating different volatile oils, can give the subtle differences of the oils from different areas, and can find new compounds that have the possible pharmaceutical effect on some diseases.


## 3.1. Qualitative Analysis of Chenpi Volatile Oil

The column system is nearly orthogonal and provides a structured separation. A typical two-dimensional separation/total ion chromatogram (TIC) and three-dimensional chromatogram are shown in Figure2. In the GC × GC system, compounds are separated by volatility difference on the first dimension nonpolar column and by polarity on the second medium-polar column. The GC × GC system accomplishes the true orthogonal separation on account for both the change of the polarity of two fixed phases and the linear temperature programming.GC × GC-HR-TOFMS chromatogram (a) and three-dimensional chromatogram (b) of Chenpi volatile oil.
(a)
(b)Using GC × GC-HR-TOFMS, the quantity of the detected components was up to 834. Compared to the traditional identification method such as GC-MS, the analysis from GC × GC-HR-TOFMS becomes more reliable, relying on the combined identification information including retention times, similarity, reverse match factor, and probability. The similarity and reverse match factors indicate how well a mass spectrum matches the library spectrum, but the isomers have similar mass spectra. In this case, the probability is used to determine whether the peaks with the same name belong to one compound or several compounds. The GC × GC-HR-TOF/MS software was used to find all the peaks in the raw GC × GC chromatogram. A library search was carried out for all the peaks using the NIST/EPA/NIH version 2.0, and the results were combined in a single peak table. A similarity and reverse match factor above 583 and 612, respectively, indicates that an acquired mass spectrum usually shows a good match with the library spectrum. Because of the numerous isomers present in volatile oils, especially within monoterpenes and sesquiterpenes, more attention should be paid for identification using mass spectra. In order to enhance the reliability of the identification by MS, both similarity and reverse match factor should be used. According to our experience and the literature data [18–20], 167 compounds with good match were tentatively identified including 50 monoterpenes, 36 sesquiterpenes, 31 esters and acids, 9 aldehydes and ketones, 6 alcohols, 3 ethers, 12 phenyl compounds, and 20 other components. Compounds have lower search probabilities than these counted as unknowns, and were disqualified for Kovats index comparison. Table 1 listed 167 components identified in Chenpi volatile oil. The volatile fraction is characterized by high percentages of monoterpenes, sesquiterpenes, and esters, including β-elemene, p-mentha-1(7),8(10)-dien-9-ol, and limonene. In this study, many components have also been tentatively identified, which were found in Chenpi volatile oil for the first time such as globulol and  isoledene. There is high possibility that they will be literally useful for further pharmaceutical research of Chenpi volatile oil.Table 1
167 main volatile components identified in the Chenpi volatile oil.
No.
Compound name
Peak I/min
Peak II/s
Volume
Library formula
Library probability
Library CAS no.
(1)
Nonanal
28.62
1.6
2367.153
C9H18O
50.1
124-19-6
(2)
Pyrrolizidine-3-one-5-ol, ethyl ether
53.49
2.74
53.1709
C9H15NO2
17.7
0-00-0
(3)
2-Methoxy-4-vinylphenol
42.38
2.73
2959.926
C9H10O2
59.39
7786-61-0
(4)
Styrene
14.14
1.65
731.272
C8H8
36.4
100-42-5
(5)
Octanal
21.38
1.58
2235.185
C8H16O
62.19
124-13-0
(6)
Ethylbenzene
12.57
1.39
484.4996
C8H10
64.86
100-41-4
(7)
Pterin-6-carboxylic acid
11.24
0.82
50.3913
C7H5N5O3
40.94
948-60-7
(8)
Hexadecane, 1,1-bis(dodecyloxy)-
77.27
1.2
102.3988
C40H82O2
9.24
56554-64-4
(9)
1-Heptatriacotanol
75.09
2.76
169.2274
C37H76O
54.56
105794-58-9
(10)
Cholestan-3-ol, 2-methylene-, (3β,5α)-
64.35
2.11
268.4351
C28H48O
13.09
22599-96-8
(11)
[5,9-Dimethyl-1-(3-phenyl-oxiran-2-yl)-deca-4,8-dienylidene]-(2-phenyl-aziridin-1-yl)-amine
66.4
2.23
151.311
C28H34N2O
44.04
0-00-0
(12)
1,1′-(4-Methyl-1,3-phenylene)bis[3-(5-benzyl-1,3,4-thiadiazol-2-yl)urea]
23.55
2.15
106.0574
C27H24N8O2S2
24.95
0-00-0
(13)
Morphinan-4,5-epoxy-3,6-di-ol, 6-[7-nitrobenzofurazan-4-yl]amino-
46.49
0.79
105.5334
C26H27N5O6
9.48
0-00-0
(14)
Benzene, 1,1′-[3-(3-cyclopentylpropyl)-1,5-pentanediyl]bis-
7.74
1.18
149.2865
C25H34
15.18
55191-62-3
(15)
2-(2-Azepan-1-yl-2-oxoethyl)-1-hydroxy-1-phenyl-octahydro-pyrido[1,2-a]azepin-4-one
76.42
2.77
142.1083
C24H34N2O3
52.15
0-00-0
(16)
6,9,12,15-Docosatetraenoic acid, methyl ester
59.4
1.77
55.943
C23H38O2
19.28
17364-34-0
(17)
2-[4-Methyl-6-(2,6,6-trimethylcyclohex-1-enyl)hexa-1,3,5-trienyl]cyclohex-1-en-1-carboxaldehyde
53.49
2.15
64.4497
C23H32O
25.57
0-00-0
(18)
Naphthalen-2-yl-acetic acid, 6-hydroxy-6-methyl-cyclodecyl ester
41.54
2.63
65.5915
C23H30O3
27.3
0-00-0
(19)
Z-5-Methyl-6-heneicosen-11-one
80.89
1.11
115.1736
C22H42O
8.27
0-00-0
(20)
2H-Pyran, 2-(7-heptadecynyloxy)tetrahydro-
61.45
1.91
133.6559
C22H40O2
14.4
56599-50-9
(21)
Doconexent
17.88
1.63
127.9331
C22H32O2
40.98
6217-54-5
(22)
9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)-
31.16
2.22
105.1463
C21H36O4
19.78
18465-99-1
(23)
8,11,14-Eicosatrienoic acid, methyl ester, (Z,Z,Z)-
42.75
2.02
216.8234
C21H36O2
8.42
21061-10-9
(24)
5,8,11-Eicosatrienoic acid, methyl ester
57.59
2.03
96.1455
C21H30O2
43.95
0-00-0
(25)
cis-5,8,11,14,17-Eicosapentaenoic acid
24.52
4.25
53.7632
C20H30O2
13.99
10417-94-4
(26)
5,8,11,14-Eicosatetraynoic acid
58.8
3.17
144.9613
C20H24O2
29.78
1191-85-1
(27)
1,16-Cyclocorynan-17-oic acid, 19,20-didehydro-, methyl ester, (16S,19E)-
12.21
0.64
88.5548
C20H22N2O2
53.45
6393-66-4
(28)
Octadecane, 6-methyl-
82.09
1.25
184.9235
C19H40
18.41
10544-96-4
(29)
2-Methyl-E,E-3,13-octadecadien-1-ol
64.83
2.01
124.0941
C19H36O
9.74
0-00-0
(30)
Z,Z,Z-4,6,9-Nonadecatriene
26.81
1.7
109.996
C19H34
21.05
0-00-0
(31)
6,9,12-Octadecatrienoic acid, methyl ester
55.78
2.92
63.7153
C19H32O2
28.52
2676-41-7
(32)
Z,Z,Z-1,4,6,9-Nonadecatetraene
21.5
1.8
65.4446
C19H32
14.64
0-00-0
(33)
12,15-Octadecadienoic acid, methyl ester
49.14
2.35
158.5734
C19H30O2
19.33
57156-95-3
(34)
10,13-Octadecadienoic acid, methyl ester
50.47
2.47
161.0184
C19H30O2
15.45
18202-24-9
(35)
2,5-Octadecadienoic acid, methyl ester
59.52
1.99
254.7258
C19H30O2
10.15
57156-91-9
(36)
2,2,4,4-Tetramethyl-6-(1-oxo-3-phenylprop-2-enyl)-cyclohexane-1,3,5-trione
70.27
0.49
83.2401
C19H20O4
21.98
0-00-0
(37)
Gentamicina
63.63
3.21
62.6103
C18H36N4O10
27.05
13291-74-2
(38)
Z-10-Methyl-11-tetradecen-1-ol propionate
40.33
2
70.3719
C18H34O2
10.53
0-00-0
(39)
10-Heptadecen-8-ynoic acid, methyl ester, (E)-
34.18
2.06
63.2456
C18H30O2
14.6
16714-85-5
(40)
α-L-Fucopyranose 1,2:3,4-bis(benzeneboronate)
67.13
2.28
180.5156
C18H18B2O5
25.89
102281-26-5
(41)
3-(O-Anisidinomethyl)-5-(3-fluorobenzylidene)-2,4-thiazolidinedione
34.66
2.46
82.9072
C18H15FN2O3S
9.43
302954-96-7
(42)
1-Hexadecanol, 2-methyl-
44.19
1.03
65.1528
C17H36O
11.82
2490-48-4
(43)
10-Methyl-E-11-tridecen-1-ol propionate
33.33
1.56
164.2353
C17H32O2
6.69
0-00-0
(44)
13-Heptadecyn-1-ol
40.09
2.25
78.5129
C17H32O
12.04
56554-77-9
(45)
4,7,10-Hexadecatrienoic acid, methyl ester
32.97
2.05
83.5894
C17H28O2
7.98
17364-31-7
(46)
Methyl 5,7-hexadecadienoate
53.61
1.7
58.765
C17H26O2
14.34
0-00-0
(47)
3-(5-Benzyloxy-3-methylpent-3-enyl)-2,2-dimethyloxirane
35.02
2.61
96.359
C17H24O2
8.95
0-00-0
(48)
Falcarinol
37.07
1.57
55.0178
C17H24O
35.51
21852-80-2
(49)
tert-Hexadecanethiol
70.27
1.03
151.5898
C16H34S
13.87
25360-09-2
(50)
n-Hexadecanoic acid
79.08
1.74
705.7247
C16H32O2
43.05
10-3-1957
(51)
Cyclopentaneundecanoic acid
46.49
1.62
94.519
C16H30O2
13.97
6053-49-2
(52)
9-Hexadecenoic acid
65.56
1.91
89.144
C16H30O2
13.29
2091-29-4
(53)
Formic acid, 3,7,11-trimethyl-1,6,10-dodecatrien-3-yl ester
51.92
1.71
99.1647
C16H26O2
23.72
0-00-0
(54)
1,3-Dioxolane, 2-heptyl-4-phenyl-
7.74
0.67
137.1547
C16H24O2
20.64
55668-40-1
(55)
Peyonine
24.64
0.58
54.9972
C16H19NO5
27.28
19717-25-0
(56)
12,14,14-Trimethyl-3,6,9-trioxapentadecan-1-ol
66.52
1.18
58.5099
C15H32O4
16.94
55489-54-8
(57)
Isocalamendiol
73.65
2.76
52.3348
C15H26O2
24.27
0-00-0
(58)
Geranyl isovalerate
53.37
1.03
77.0183
C15H26O2
11.72
109-20-6
(59)
Cubeduel
63.26
1.88
203.2965
C15H26O
24.8
0-00-0
(60)
α-Cadinol
63.63
2.07
649.3081
C15H26O
24.36
481-34-5
(61)
Cubenol
62.42
1.9
408.4446
C15H26O
16.17
21284-22-0
(62)
.tau.-Cadinol
63.02
2
707.983
C15H26O
11.7
11-1-5937
(63)
α-Acorenol
63.63
2.17
1949.884
C15H26O
9.72
0-00-0
(64)
Globulol
60.13
1.88
278.3377
C15H26O
6.4
51371-47-2
(65)
7-Epi-cis-sesquisabinene hydrate
54.57
1.3
81.2625
C15H26O
6.4
0-00-0
(66)
Limonen-6-ol, pivalate
44.56
1.85
78.8648
C15H24O2
24.04
0-00-0
(67)
Aromadendrene oxide-(2)
62.78
2.09
96.7312
C15H24O
15.84
0-00-0
(68)
Caryophyllene oxide
59.88
2
161.5517
C15H24O
13.15
1139-30-6
(69)
Spiro[4.5]dec-6-en-8-one, 1,7-dimethyl-4-(1-methylethyl)-
38.16
2.27
73.2272
C15H24O
7.02
39510-36-6
(70)
Humulene
53
1.61
5204.711
C15H24
44.49
6753-98-6
(71)
(Z,E)-α-farnesene
55.78
1.52
31067.6
C15H24
42.94
26560-14-5
(72)
α-Copaene
48.42
1.4
7508.707
C15H24
42.37
0-00-0
(73)
δ-Elemene
45.88
1.33
5692.139
C15H24
38.01
20307-84-0
(74)
Naphthalene, 1,2,3,5,6,8a-hexahydro- 4,7-dimethyl-1-(1-methylethyl)-, (1S-cis)-
56.75
1.68
12956.64
C15H24
31
483-76-1
(75)
1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-, (Z,E)-
54.94
1.51
196.9983
C15H24
29.01
26560-14-5
(76)
Caryophyllene
50.95
1.54
3006.936
C15H24
17.9
87-44-5
(77)
β-Elemene
49.14
1.45
20347.87
C15H24
17.11
515-13-9
(78)
α-Guaiene
52.16
1.47
556.5085
C15H24
16.63
12-1-3691
(79)
Cyclohexane, 1-ethenyl-1-methyl-2,4-bis (1-methylethenyl)-
52.76
1.23
160.6527
C15H24
16.42
110823-68-2
(80)
1,6-Cyclodecadiene, 1-Methyl-5-methylene-8-(1-methylethyl)-, [S-(E,E)]-
54.45
1.65
14112
C15H24
15.25
23986-74-5
(81)
γ-Elemene
51.68
1.51
742.7373
C15H24
15.14
29873-99-2
(82)
Naphthalene, 1,2,3,4,4a,7- hexahydro-1,6-dimethyl-4-(1-methylethyl)-
57.35
1.71
200.7951
C15H24
14.55
16728-99-7
(83)
Cyclohexane, 1-ethenyl-1-methyl- 2,4-bis(1-methylethenyl)-, [1S-(1α,2β,4β)]-
48.66
1.47
549.6758
C15H24
10.66
515-13-9
(84)
β-Copaene
54.45
1.55
561.4817
C15H24
10.53
0-00-0
(85)
γ-Elemene
58.8
1.82
1698.139
C15H24
8.31
29873-99-2
(86)
β-Guaiene
57.59
1.75
60.1417
C15H24
7.16
88-84-6
(87)
Guaia-1(10),11-diene
54.82
1.67
1093.799
C15H24
6.6
0-00-0
(88)
Isoledene
54.21
1.58
405.0377
C15H24
6.27
0-00-0
(89)
4,5-Di-epi-aristolochene
47.81
1.43
90.0937
C15H24
4.2
0-00-0
(90)
trans-calamenene
56.51
1.85
84.299
C15H22
38.35
0-00-0
(91)
β-Vatirenene
73.65
2.34
437.4147
C15H22
25.71
0-00-0
(92)
4,4-Dimethyl-3-(3-methylbut-3-enylidene)-2-methylenebicyclo[4.1.0]heptane
72.68
2.32
284.3733
C15H22
11.28
79718-83-5
(93)
7-Hydroxy-6,9a-dimethyl-3-methylene-decahydro-azuleno[4,5-b]furan-2,9-dione
75.21
3.61
61.6952
C15H20O4
11.61
0-00-0
(94)
Propanoic acid, 3-(2,3,6-trimethyl- 1,4-dioxaspiro[4.4]non-7-yl)-, methyl ester
33.93
2.7
84.8793
C14H24O4
19.92
0-00-0
(95)
2,5-Furandione, 3-(2-decenyl)dihydro-
40.33
2.76
154.1135
C14H22O3
11.38
62568-81-4
(96)
trans-(2-Decenyl)succinic anhydride
72.08
1.6
67.8793
C14H22O3
10.39
81949-64-6
(97)
1,4-Benzenediol, 2,6-bis(1,1-dimethylethyl)-
77.51
0.5
70.9573
C14H22O2
7.13
2444-28-2
(98)
Tetraacetyl-d-xylonic nitrile
58.32
1.62
100.275
C14H17NO9
18.48
0-00-0
(99)
α-Ionol
30.19
2.18
277.9312
C13H22O
15.16
25312-34-9
(100)
1-(2-Acetoxyethyl)-3,6-diazahomoadamantan-9-one oxime
73.28
0.5
55.2116
C13H21N3O3
8.78
0-00-0
(101)
1b,5,5,6a-Tetramethyl-octahydro-1-oxa-cyclopropa[a]inden-6-one
33.21
1.89
138.6104
C13H20O2
6.45
0-00-0
(102)
Pyrimidin-2-one, 4-[N-methylureido]-1-[4-methylaminocarbonyloxymethyl
68.94
0.38
54.3663
C13H19N5O5
23.12
0-00-0
(103)
2H-Indeno[1,2-b]furan-2-one, 3,3a,4,5,6,7,8,8b-octahydro-8,8-dimethyl
55.3
1.93
203.2335
C13H18O2
28.93
0-00-0
(104)
Dodecanal
49.26
1.58
1282.104
C12H24O
6.6
112-54-9
(105)
2,6-Octadien-1-ol, 3,7-dimethyl-, acetate, (Z)-
46.37
1.74
3059.872
C12H20O2
37.64
141-12-8
(106)
Geranyl acetate
47.45
1.79
3772.587
C12H20O2
28.7
105-87-3
(107)
(R)-Lavandulyl acetate
46.37
2.25
81.7472
C12H20O2
14.19
0-00-0
(108)
Geranyl vinyl ether
36.83
1.77
76.0262
C12H20O
14.31
0-00-0
(109)
3′-Hydroxyquinalbarbitone
39.61
1.5
51.4505
C12H18N2O4
10.72
839-21-4
(110)
Non-1-yn-5-en-9-aldehyde, 4-carbethoxy-
44.31
2.11
115.1319
C12H16O3
17.87
0-00-0
(111)
Undecanal
42.75
1.59
704.5031
C11H22O
33.86
112-44-7
(112)
Cyclohexane, 2-ethenyl-1,1-dimethyl-3-methylene-
30.31
1.34
135.2002
C11H18
23.83
95452-08-7
(113)
Cyclohexene, 2-ethenyl-1,3,3-trimethyl-
42.02
2.22
591.2578
C11H18
14.36
5293-90-3
(114)
Acetic acid, octyl ester
36.47
1.49
323.6557
C10H20O2
17.84
112-14-1
(115)
Decanal
35.74
1.61
7031.098
C10H20O
61.62
112-31-2
(116)
Cephrol
37.55
1.66
328.2375
C10H20O
12.34
40607-48-5
(117)
Linalol
28.74
1.52
3645.373
C10H18O
81.59
78-70-6
(118)
α-Terpineol
34.9
1.89
7440.985
C10H18O
67.75
98-55-5
(119)
Terpinen-4-ol
34.18
1.79
3293.372
C10H18O
50.03
562-74-3
(120)
Citronellal
32.12
1.69
615.3934
C10H18O
42.59
106-23-0
(121)
2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, cis-
30.31
1.67
159.2231
C10H18O
39.11
29803-82-5
(122)
Cyclohexanol, 1-methyl-4-(1-methylethenyl)-, cis-
31.64
1.8
726.8632
C10H18O
10.59
7299-41-4
(123)
2-Cyclohexen-1-ol, 2-methyl-5-(1-methylethyl)-, (1S-cis)-
36.11
1.85
101.2405
C10H18O
10.53
536-30-1
(124)
exo-2,7,7-trimethylbicyclo[2.2.1]heptan-2-ol
32.97
1.89
169.9902
C10H18O
8.15
0-00-0
(125)
5-Hepten-1-ol, 2-ethenyl-6-methyl-
45.76
1.68
397.1537
C10H18O
7.75
18479-48-6
(126)
Bicyclo[4.1.0]heptane, 3,7,7-trimethyl-, [1S-(1α,3β,6α)]-
45.76
1.58
368.8305
C10H18
9.72
2778-68-9
(127)
Desulphosinigrin
66.64
1.06
73.3518
C10H17NO6S
8.92
5115-81-1
(128)
R-Limonene
39.37
2.73
97.0311
C10H16O3
40.37
0-00-0
(129)
Limonene oxide, trans-
31.28
1.78
570.8168
C10H16O
54.24
4959-35-7
(130)
Limonene oxide, cis-
30.92
1.78
1068.476
C10H16O
40.43
13837-75-7
(131)
trans-p-Mentha-2,8-dienol
30.07
1.78
448.7691
C10H16O
34.35
0-00-0
(132)
3-Cyclohexene-1-acetaldehyde,α,4-dimethyl-
36.35
2.09
181.7561
C10H16O
31.38
29548-14-9
(133)
cis-p-Mentha-1(7),8-dien-2-ol
37.43
2.05
398.5489
C10H16O
26.42
0-00-0
(134)
(Z)-Carveol
35.5
1.97
452.2402
C10H16O
25.48
1197-06-4
(135)
trans-Carveol
36.71
2
1591.657
C10H16O
22.66
1197-07-5
(136)
cis-p-Mentha-1(7),8-dien-2-ol
33.93
1.91
336.4337
C10H16O
21.5
0-00-0
(137)
1-Cyclohexene-1-methanol, 4-(1-methylethenyl)-
35.62
1.89
195.9113
C10H16O
20.76
536-59-4
(138)
p-Mentha-1(7),8(10)-dien-9-ol
49.14
2.02
1162.984
C10H16O
20.12
29548-13-8
(139)
cis-p-Mentha-2,8-dien-1-ol
43.47
1.91
99.0278
C10H16O
7.83
3886-78-0
(140)
2,6-Dimethyl-3,5,7-octatriene-2-ol,  E,E-
31.88
1.98
187.0291
C10H16O
7.6
0-00-0
(141)
(S)-(-)-(4-Isopropenyl-1-cyclohexenyl)methanol
35.38
1.89
172.7702
C10H16O
5.86
18457-55-1
(142)
Bicyclo[3.1.0]hex-2-ene, 4-methyl-1-(1-methylethyl)-
17.4
1.11
7918.412
C10H16
65.04
28634-89-1
(143)
β-Pinene
20.66
1.31
19964.34
C10H16
40.8
127-91-3
(144)
β-Ocimene
25.48
1.34
4519.345
C10H16
37.56
13877-91-3
(145)
α-Phellandrene
22.47
1.32
2085.008
C10H16
33.42
99-83-2
(146)
β-Myrcene
21.5
1.27
69260.99
C10H16
31.3
123-35-3
(147)
α-Pinene
17.88
1.16
48376.96
C10H16
23.82
80-56-8
(148)
Camphene
18.85
1.22
406.4708
C10H16
19.93
79-92-5
(149)
β-Phellandrene
20.29
1.27
4173.575
C10H16
19.68
555-10-2
(150)
D-Limonene
24.52
1.69
1784450
C10H16
19.59
5989-27-5
(151)
α-Terpinene
23.43
1.33
4905.547
C10H16
17.58
99-86-5
(152)
Isoterpinene
28.38
1.46
14607.02
C10H16
16.01
586-62-9
(153)
γ-Terpinene
26.33
1.5
189808.1
C10H16
15.28
99-85-4
(154)
1,5,5-Trimethyl-6-methylene-cyclohexene
26.33
1.86
305.9981
C10H16
9.81
514-95-4
(155)
Cyclohexene, 1-methyl-4-(1-methylethenyl)-, (S)-
24.52
3.31
51.7426
C10H16
9.58
5989-54-8
(156)
γ-Pyronene
23.07
1.27
60.3834
C10H16
5.33
514-95-4
(157)
5-Isopropenyl-2-methylcyclopent-1-enecarboxaldehyde
36.11
2.3
711.2013
C10H14O
34.31
0-00-0
(158)
(-)-Carvone
37.8
2.35
909.8454
C10H14O
32.76
6485-40-1
(159)
1-Cyclohexene-1-carboxaldehyde, 4-(1-methylethenyl)-
39.85
2.43
1404.104
C10H14O
31.76
2111-75-3
(160)
3,5-Heptadienal, 2-ethylidene-6-methyl-
36.95
2.28
238.0085
C10H14O
28.28
99172-18-6
(161)
Benzenemethanol,α,α,4-trimethyl-
34.05
2.28
288.3869
C10H14O
16.59
1197-01-9
(162)
Cyclohexanone, 2-(2-butynyl)-
42.38
3.18
59.1028
C10H14O
15.02
54166-48-2
(163)
D-Verbenone
41.42
2.16
475.2141
C10H14O
11.63
18309-32-5
(164)
3,5-Heptadienal, 2-ethylidene-6-methyl-
36.11
2.5
184.88
C10H14O
8.97
99172-18-6
(165)
o-Cymene
23.55
1.55
35991.09
C10H14
47.51
527-84-4
(166)
2,6-Dimethyl-1,3,5,7-octatetraene, E,E-
31.64
1.71
164.8089
C10H14
20.19
460-01-5
(167)
Benzene, 1-methyl-4-(1-methylethenyl)-
27.9
1.8
271.6784
C10H12
20.42
1195-32-0


## 3.2. Group Separation of Chenpi Volatile Components

In GC × GC-HR-TOFMS analysis, the 167 identified volatile components in Chenpi volatile oil were mainly classified into two groups that can be seen in Figure3. Based on GC × GC-HR-TOFMS, it can be found that the peaks in areas A and B are monoterpenes and sesquiterpenes, respectively. These monoterpenes and sesquiterpenes are mainly alkenes, alcohols, and ethers. It was also found that a lot of saturated and unsaturated fatty acid esters and phenyl compounds constitute the Chenpi volatile oil. This study demonstrates that GC × GC-HR-TOFMS is a powerful separation and identification tool that allows for the identification and group separation of a much larger number of complex volatile oil components.Figure 3
The GC × GC contour plot of Chenpi volatile oil group separation result. Regions marked by squares (A) and (B) were identified mainly as monoterpenes and sesquiterpenes, respectively.


## 3.3. Identification of Three Coelution Volatile Components in Chenpi Volatile Oil

The high-resolution mass spectra in the TIC can be used for accurate identification of volatile compounds in Chenpi volatile oil, and these identified compounds will be significant to the further pharmaceutical research. For example, Figure4 compares the high resolution mass spectrum of the blob (peak) marked with 138 (49.14 min, 2.02 s), 104 (49.26 min, 1.58 s), and 77 (49.14 min, 1.45 s), head-to-tail with the mass spectrum of p-mentha-1(7),8(10)-dien-9-ol, dodecanal, and β-elemene TMS from the NIST/EPA/NIH library mass spectra. For p-mentha-1(7),8(10)-dien-9-ol, the forward match factor is 806; reverse match factor is 855; and probability is 20.12%. For dodecanal, the forward match factor is 763; reverse match factor is 773; and probability is 7.16%. For β-elemene, the forward match factor is 911; reverse match factor is 914; and probability is 17.11%. The above three volatile components cannot be clearly separated or identified by traditional one-dimensional gas chromatography or GC-MS method, because they are coelution volatile components, which have very similar chemical properties including volatility and polarity. In this study, the three coelution volatile components in Chenpi volatile oil were well separated and identified by GC × GC-HR-TOFMS, which have not been reported in other studies (Figure 4).Details of three coelution volatile components (peak 77, 104, 138) in GC × GC chromatogram. The spectra ofβ-elemene (peak 77), dodecanal (peak 104), and p-mentha-1(7),8(10)-dien-9-ol (peak 138) in sample and in NIST library, respectively.
(a)
(b)
(c)
(d)This study showed that GC × GC-HR-TOFMS represents a powerful separation and analysis tool for the analysis of complex volatile oils of herbal medicines. GC × GC-HR-TOFMS can give the information about the formula and structures, can provide the opportunity for differentiating different volatile oils, can give the subtle differences of the oils from different areas, and can find new compounds that have the possible pharmaceutical effect on some diseases.


## 4. Conclusions

In this study, GC × GC-HR-TOFMS not only tentatively identified 167 volatile components in Chenpi volatile oil, but also provided several kinds of identification information that make the result more reliable. Among 167 components, there are 50 monoterpenes, 36 sesquiterpenes, 31 esters and acids, 9 aldehydes and ketones, 6 alcohols, 3 ethers, 12 phenyl compounds, and 20 other components. Monoterpenes and sesquiterpenes are the main components of Chenpi volatile oil. This study demonstrates a dependable method for the qualitative analysis of volatiles, which can achieve an accurate and comprehensive chromatographic profile with a low contamination risk and cost, as well as shortened sample preparation time. GC × GC-HR-TOFMS will play an important role in the analysis of volatile oils of herbal medicines in the future.



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*Source: 237541-2013-04-28.xml*