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

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.

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 of Citrus 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 of Citrus 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: 1 μL 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 Figure 2. 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.

Figure 2

GC × GC-HR-TOFMS chromatogram (a) and three-dimensional chromatogram (b) of Chenpi volatile oil.

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 Figure 3. 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, Figure 4 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).

Figure 4

Details of three coelution volatile components (peak 77, 104, 138) in GC × GC chromatogram. The spectra of β-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.

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.