Simultaneous Determination of Four Major Components in Essential Oil from Litsea cubeba / Asian Journal of Traditional
Medicines, 2007, 2 ( 2 )
Simultaneous Determination of the 6-Methyl-5-hepten-2-one,
Limonene, Linalool and Citral in Essential Oil from Litsea cubeba
(Lour.) Pers by Capillary Gas Chromatography
Shuanghui Yu, Xiaohui Chen, Ling Tong, Kaishun Bi *
School of Pharmacy, Shengyang Pharmaceutical University, Shenyang 110016, China
A simple and fast Capillary Gas Chromatography (CGC) method was established for the simultaneous determination of four major
components, namely 6-methyl-5-hepten-2-one, limonene, linalool and citral in essential oil from Litsea cubeba (Lour.) Pers. The CGC
analysis was performed on a 30 m × 0.50 mm i.d. SE-54 capillary column, and was carried out with temperature programs by using
nitrogen as carrier gas. The good linear regression relationship was obtained over the range of 0.030–1.502 mg·mL-1 (r = 0.9999) for
6-methyl-5-hepten-2-one, 0.018–0.894 mg·mL-1 (r = 0.9997) for Limonene, 0.016–0.809 mg·mL-1 (r = 0.9997) for Linalool , 0.079–3.962
mg·mL-1 (r = 0.9999) for citral, respectively. The method is accurate with overall intra-day variation and repeatability less than 5 %
and the recovery more than 95 %. The contents of sample from different regions varied greatly. CGC was proved to be a quick and
informative tool for analysis of essential oil from Litsea cubeba (Lour.) Pers. The method was successfully applied to analyze four
major components.
Key words: Litsea cubeba（Lour.）Pers; 6-Methyl-5-hepten-2-one; limonene; linalool; citral; CGC
Litsea cubeba (Lour.) Pers is well-known
traditional Chinese medicine (TCM) that has been
used for warming and dispersing coldness as well
as relieving pain for more than thousands of years.
According to Pharmacopoeia of china, Litsea cubeba
(Lour.) Pers was derived from the ripe and dried
fruitage. It is widespread in the southern regions of
China, and mainly produced in Guangxi, Zhejinang,
Sichuan Provinces and so on. Photochemical and
pharmacological studies proved that essential oil
and fatty acid were the bioactive constituents,
and the former ones have the functions of antiasthma, eliminating phlegm, anti-bacteria, anticongregation of platelet, anti-myocardial infarction,
and anti-hypersusceptibility [1]. 6-Methyl-5-hepten2-one, Limonene, Linalool and Citral are the main
constituents of essential oil from Litsea cubeba (Lour.)
Pers. In the past documents, although the methods for
characterization of active compositions in essential
oil extracted from the Litsea cubeba (Lour.) Pers have
well been reported using GC-MS, there is no reports
on simultaneous determination of compositions in
essential oil. Therefore, it is necessary to develop a
new approach for the quality control of essential oil
from Litsea cubeba (Lour.) Pers.
The aim of the present study is to develop a direct
and rapid CGC with the internal standard method
for simultaneous determination of the four major
components, 6-methyl-5-hepten-2-one, limonene,
linalool and citral, and to produce a suitable method of
quality control.
Materials and Methods
Chemicals and materials
n-Hexane and ether of analytical grade were
purchased from Yu-Wang Chemical Company
(Shangdong, China). 6-Methyl-5-hepten-2-one
and limonene were supplied from J & K Chemical
(Beijing, China). Linalool was obtained from National
Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). Citral was
purchased from Sigma Chemical Company (St. Louis,
MO, USA). Drug samples of Litsea cubeba (Lour.)
Pers were collected from different regions in China and
authenticated as Litsea cubeba (Lour.) Pers by Prof.
Qishi Sun (Shenyang Pharmaceutical University).
Apparatus and conditions
* Author to whom correspondence should be addressed. Tel.:
+86-24-23928487; E-mail: bikaishun@yahoo.com
The analyses were performed on a Shimadzu 2010
66
Simultaneous Determination of Four Major Components in Essential Oil from Litsea cubeba / Asian Journal of Traditional
Medicines, 2007, 2 ( 2 )
Gas Chromatograghy (GC) system equipped with
FID detector. Data was processed by Lab solution 2.1
workstation. Separation was carried out on a 30 m×
0.50 mm i.d.× 0.32 µm film SE-54 capillary column
(Danlian, Zhonghuida, China). The non-polar was
75 ˚C programmed to 100 ˚C at 5 ˚C·min -1, then to
140 ˚C at 2 ˚C·min-1, and then to 230 ˚C at 6 ˚C·min-1;
Split injection was conducted with split radio of
1:1; Flow rate was 5.5 mL·min-1; Nitrogen was used
as carrier gas; injector temperature, 250 ˚C; flame
ioinization detection temperature, 250 ˚C; and
injected volume, 1 µL. Phenylethylketone was used
as internal standard. Some individual components
could be identified by co-injection of pure compounds
and comparison of the retention time.
obtained, and was added into 10 mL volumetric flasket,
then 0.5 mL internal standard solution (2.060 mg·mL-1)
was added, and was dilute with n-hexane, then shaken.
Results and Discussion
Calibration curves and quantification limits
The reference standards of the target compounds,
namely mehty heptenone, limonene, linalool and citral
were accurately weighed and dissolved in n-hexane to
produce the stock mixed standard solution, then diluted
to a series of appropriate concentration for construction
of calibration curves and determination of the limit
of quantification (LOQ). A 0.5 mL (2.060 mg·mL-1)
volume of the internal standard solution was added
in each concentration solution. Calibration curves
were calculated with six different concentrations by
plotting the peak area rations of analyte to internal
standard versus analyte concentration. All the target
compounds showed good linearity over a relatively
wide concentration range. The results are expressed as
the values of the correlation coefficient (r) in Table 1.
and the chromatograms are shown in Fig. 1.
Sample pretreatment
10 g of Litsea cubeba (Lour.) Pers was weighed
accurately. A ten fold mass of distilled water was added
and refluxed for 6 hours with essential oil’s extractor
after being soaked for 5 hours according to appendix
XD in Chinese Pharmacopoeia (2005). When cooled
down to the room temperature, extracted for three
times by ether, and evaporated to dry, then re-dried
with sodium sulfate anhydrous, essential oil was
Table 1 Calibration curves of four compounds and limits of quantitation­­
Calibration curve
r
Linear range (mg·mL-1)
LOQ (mg·mL-1)
6-Methyl-5-hepten-2-one
Y =10.878 X­­­­­­­−0.0638
0.9999
0.030–1.502
0.030
Limonene
Y =15.158 X−0.133
0.9997
0.019–0.894
0.019
Linalool
Y =11.530 X−0.0919
0.9997
0.016–0.809
0.016
Citral
Y =11.288 X−0.132
0.9999
0.079–3.962
0.079
Compound
X denotes the concentration and Y denotes peak area rations
Precision, repeatability and accuracy
linalool and 4.5 % for citral. The recovery was determined
by spiking with the mixed standard solution at high,
medium and low concentration levels in samples. The
ratios of measured and added amounts were calculated
to reflect the recovery. The results are shown in Table
2. and demonstrate that the developed method is
reproducable and accurate.
The intral-day variability of the content was used
for assessing the precision of the developed method by
performing six duplicated injection of the same solution in
a single day. The relative standard derivation (RSD) were
2.6 %, 2.8 %, 2.7 %, and 1.8 % for 6-methyl-5-hepten2-one, limonene, linalool and citral, respectively. The
repeatability of the total procedure was tested using
six processed samples of the essential oil from Litsea
cubeba (Lour.) Pers, and the RSD (n = 6) were 1.9 % for
6-Methyl-5-hepten-2-one, 2.6 % for limonene, 4.5 % for
Application
Sample solution was obtained according to
the sample pretreatment method, then 1 μL of each
67
Simultaneous Determination of Four Major Components in Essential Oil from Litsea cubeba / Asian Journal of Traditional
Medicines, 2007, 2 ( 2 )
Table 2 Method recoveries (n=3)
Compound
Basic value
(mg)
6-Methyl-5-hepten-2-one
0.542
Limonene
0.299
Linalool
0.357
Citral
0.465
Amount (mg)
Added
0.300
0.601
0.901
0.179
0.358
0.536
0.162
0.324
0.485
0.792
1.585
2.377
Recovery (%)
Found
0.844
1.153
1.464
0.478
0.644
0.806
0.516
0.679
0.842
2.221
3.044
3.850
Average recovery (%)
RSD (%)
101.5
2.1
96.9
3.0
99.6
3.6
98.6
4.3
100.7
101.0
102.7
99.9
95.9
95.0
99.9
98.5
100.3
96.5
98.7
100.6
Table 3 Results of the determination in 6 samples (mg·g-1 )
Region
6-Methyl-5-hepten-2-one
Limonene
Linalool
Citral
Sichuan
Guangdong
Jiangxi
Zhejiang
Hubei
Guangxi
0.092
0.187
0.053
0.317
0.159
0.108
0.039
1.363
0.225
2.129
0.731
0.060
0.040
0.222
0.097
0.446
0.127
0.071
0.296
0.289
0.354
1.463
0.750
0.294
listed as follows: 75 ˚C programmed to 100 ˚C at 5 ˚C
·min-1, then to 140 ˚C at 2 ˚C·min-1, and then to 230 ˚C
at 6 ˚C·min-1; Split injection was conducted with split
radio of 1:1; Flow rate was 5.5 mL·min-1; Nitrogen
was used as carrier gas; injector temperature, 250 ˚C;
flame ionization detection temperature, 250 ˚C; and
injected volume, 1 µL. The results revealed that four
components could be separated successfully on the
SE-54 capillary columns with temperature programs.
For the selection of internal standard compound,
eicosane, nonadecane and octadecane were once
considered, however, under the present conditions,
their retention time were lagged to 20 minutes, and
their peaks were tailing with bad symmetry. The
phenylethylketone mentioned in the previous document
was still selected as the internal standard. Good
reproducability and accuracy for the quantification of
four major bioactive terpenoid of the essential oil from
Litsea cubeba (Lour.) Pers were permitted.
solution was injected to GC-FID analysis (Fig. 1). The
content of each component was calculated from the
corresponding calibration curve. The results are shown
in Table 3. The contents of sample from different
regions vary greatly.
Chromatographic condition
To assay components of the essential oil from
Litsea cubeba (Lour.) Pers with good resolution
and a reasonable retention time, capillary columns
of different types and temperature programs were
investigated. Thus, we analyzed the performance of
DB-1, SE-54, DB-17, DB-WAX capillary column
at the same temperature, in the case of the DB-1,
6-methyl-5-hepten-2-one could not be completely
separated from impurities; in the case of DB-17 or
DB-WAX, citral could not be completely separated
from impurities. It was demonstrated that optimal
chromatographic condition were obtained by using
SE-54 column. Hence, the SE-54 column was applied
to analyze the essential oil from Litsea cubeba (Lour.)
Pers as shown in Fig. 1. The optimal conditions for
achieving a good chromatographic resolution were
Essential oil extraction condition
Vapor distilment was a common way of extracting
essential oil, which was introduced in the Chinese
68
Simultaneous Determination of Four Major Components in Essential Oil from Litsea cubeba / Asian Journal of Traditional
Medicines, 2007, 2 ( 2 )
Pharmacopoeia, and also applied in our study.
Consequently, we have acquired that granularity affects
greatly on the contents of limonene and linalool. When
traditional Chinese medicine (TCM) was reduced into
thin powder, the contents of limonene and linalool
from Litsea cubeba (Lour.) Pers were hardly detected.
It was likely that they were apt to volatilize. The
repeatability of the total procedure is satisfactory by
using thick powder.
The results indicated that there were great
differences in essential oil of Litsea cubeba (Lour.)
Pers from various areas. Compared with the previous
study[4], they made also sharp distinctions in citral
content. In addition, the content and component
of essential oil were greatly influenced by the
environment such as collecting time, ways of storage,
duration of storage and detecting apparatus and so on.
In conclusion, the established CGC-FID method
A
B
Fig. 1 (A) Chromatogram of a standard mixtuere containing 6-methyl-5-hepten-2-one (1), Limonene (2), I.S. (3) Linalool (4), Citral
(5, 6) and (B) chromatogram of essential oil from Litsea cubeba（Lour.）Pers
is reproducable with excellent resolution, recovery
and reproducability for simultaneously quantitative
analysis of terpenoid in Litsea cubeba (Lour.) Pers. It
provided a suitable quality control method for Litsea
cubeba (Lour.) Pers samples and could be readily
utilized for the determination of the major biologically
active ingredients in Litsea cubeba(Lour.)Pers.
Meanwhile，essential oil from Litsea cubeba (Lour.)
Pers namely 6-methyl-5-hepten-2-one, limonene,
linalool and citral were simultaneously determined by
this method for the first.
on Essential Oil from Litsea cubeba（Lour.）Pers in
Guizhou by GC-MS. Journal of Guizhou University
(Natural Sciences), 2001, 18 (1): 45-47
[4] Li BY, Li YZ, et al. Chromatographic Quantification of
Citral in Essential Oil from Litsea cubeba（Lour.）Pers.
Chemical World, 1998, 2: 99-100
References
[1] Song LR, Hong X, Ding XL, et al. Modern Dictionary of
Chinese Pharmacy. Beijing: People's Medical Publishing
House, 2000, 2397
[2] Zhou RH, Wang LS, Liu XM, et al. Analysis of Guangxi
Litsea cubeba（Lour.）Pers by GC-MS. Journal of
Chemical Industry of Forest Products, 2003, 37 (1): 19-21
[3] Zhou X, Mo BB, et al. Analysis of chemical components
69