Vol.1, No.3, 203-206 (2009)
doi:10.4236/health.2009.13034
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
Isolation and identification of an isomer of β-sitosterol
by HPLC and GC-MS
Yi Sheng, Xiao-Bin Chen*
BS.Luh Food Safety Research Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, China;
Received 5 June 2009; revised 10 July 2009; accepted 12 July 2009.
ABSTRACT
Phytosterols are a group of steroids alcohols
which had been regarded as a functional factor.
An unknown compound in phytosterol samples
and phytosterol standard samples was detected
by HPLC using symmetry C18 column. The quan-
tity of the compound was increased with the
enrichment of β-sitosterol. After being collected
and analyzed by GC-MS and compared with
standard diagram from Wiley and Nist standard
chart library, it proved to be γ-sitosterol, a 24β
epimer of β-sitosterol.
Keywords: β-Sitosterol; γ-Sitosterol; Isomer;
Stereochemistry; HPLC; GC-MS
1. INTRODUCTION
Plant sterols are steroid alcohols. Phytosterols regulate
the membrane properties of the plant cells and participate
in the control of membrane-associated metabolic proc-
esses. Sterols also play an important role in cellular and
developmental processes in plants as precursors of brassi-
nosteroids. They also act as substrates for a wide variety
of secondary metabolites such as the glycoalkaloids,
saponins. A major function of phytosterols in diet is the
inhibition of absorption and subsequent compensatory
stimulation of the synthesis of cholesterol. They are
generally regarded as a kind of functional factor which
could lower serum cholesterol and LDL-C level. Among
different kinds of phytosterols, β-sitosterol has the most
powerful serum cholesterol-lowering effect.
Phytosterols are made up of a tetracyclic cyclopenta [α]
phenanthrene ring and a long flexible side chain at the C-
17 carbon atom. The four rings (A, B, C, D, from left to
right) have trans ring junctures, forming a flat α system.
The most common representatives are sitosterol, stigmas-
terol and campesterol (4-desmethyl sterol). Campesterol
and sitosterol have a △5 double bond and an additional
one-carbon or two-carbon substituent in the side chain at
C-24. Brassicasterol and stigmasterol have △5 and △22
double bonds, also an additional methyl or ethyl sub-
stituent at C-24 (Figure 1). These substituents are intro-
duced by trans-methylation reactions. Methyl and ethyl
substituents may have α or β chirality. Most 24-ethyl
sterols are 24α-epimers, whereas 24-methyl sterols occur
as mixtures of 24α-epimers and 24β-epimers. Alkylation
of C-24 is a reaction specific to plants.
Sitosterol is the principal sterol in plant materials. Gen-
erally it refers to β-sitosterol which has △5 double bond
and α-ethyl at C-24 [1]. The structure of β-sitosterol is
definite. But little has been reported concerning γ-sitos-
terol, an epimer of β-sitosterol, which has been described
as a △5 sterol and β-ethyl at C-24. Thompson (1963) [2]
and Nishioka (1965) [3] respectively reported on γ-sitos-
terol in early years, but little chromatography information
about how to separate these two epimers could be found.
In the course of an experiment aimed to enrich β-sitos-
terol, it was found that an unknown compound of small
quantity also had been enriched. This compound had been
neglected in the previous research and determination. It is
important to define what this compound is.
2. EXPERIMENTAL
2.1. Chemicals
All chemical solvents are HPLC-grade, were purchased
from Shanghai Chemical solvents company.
2.2. Samples
Four samples were used for determination by HPLC.
Sample A was phytosterol raw material and its purity was
87.1%. Sample B was a β-sitosterol enriched phytosterol
product prepared in the lab. Sample C was a blended phy-
tosterol standard sample (total phytosterols purity was
95.7%) containing brassicasterol, stigmasterol, campes-
terol and β-sitosterol. Sample D was a stigmasterol stan-
dard sample with the purity of 97.3%. All the standard
samples were from Sigma.

Y. Sheng et al. / HEALTH 1 (2009) 203-206
SciRes Copyright © 2009 http://www.scirp.org/journal/HEALTH/
204
Figure 1. The structures of phytosterols.
2.3. Sample Preparation
The four samples were dissolved in absolute alcohol.
The concentrations of sample A, B, C, and D were
0.7563mg/ml, 0.8322mg/ml, 1.360mg/ml and 0.7520mg/
ml respectively. Preparation of well-dissolved samples
were achieved by ultra-sonic treatment. Undissolved
particulate matter was removed by membrane filtration
before the HPLC analysis.
Openly accessible at
3. CHROMATOGRAPHY METHODS [4]
3.1. HPLC Conditions
Waters 600 HPLC equipment combined with UV detector
at UV 210nm. Symmetry C18 Column (5μm, 3.9×150mm)
from Waters. Column temperature was 30. Mobile ℃
phase: MeOH (HPLC grade). The flow rate was 1.0ml/
min. Sample Loading was 30μl.
3.2. GC-MS Conditions
TRAC-MS (Finnigan company), OV-1701 column (30m×
0.25mm×0.25μm) from Vertical Chromatography Co.,
Ltd. Mobile phase: He gas (99.99% purity), flow speed
was 0.8ml/min, split ratio was 10:1. Sample temperature:
280oC. Column temperature: from 240oC and rose up to
265 oC at the rising speed of 10oC/min. Remained at 265
oC for 40 minutes. Ionization mode: EI+. Electron energy:
70eV. Interface temperature: 250oC. Ion source tempera-
ture: 200oC. Detection voltage: 350V. Sample loading:
0.5μl.
3.3. Separation of Four Samples by HPLC
HPLC was applied to separate phytosterol samples for
quantity determination. The RT (retention time) and
quantity of each kind of sterol in phytosterol standard
samples (sample C and D) were already known (Table 1).
Sample B was prepared for the purpose of concentrating
β-sitosterol. The unknown compound was also enriched
while the β-sitosterol enriched. This phenomenon caused
our interest. Usually the unknown compound we dis-
cussed in this report was too little to be noticed, though it
existed in almost all the samples. Their HPLC separation
results are shown in Figures 2-5 (Sample A, B, C and D).
The information about the name and RT of each phy-
tosterol in Figures 2-5 was listed in Table 1.
The first small peak is a compound with low concen-
tration which was detected around 15min to 15.3min in
sample A, B and C. It was a long chain alkane with SiO-
group. After collection and determination by GC-MS, this
small compound was judged to be material lost from the
C18 column.
The content of β-sitosterol in sample B was more than
in sample A which was due to the enriching course of β-
sitosterol. The content of β-sitosterol in raw material was
37.21% and in concentrated phytosterol sample was
56.46%. Simultaneously, the unknown compound was
also enriched in the experiment which showed the same
concentrated tendency as β-sitosterol. Its content was
raised from 4.92% in raw material to 6.55% in treated
sample. It must be a kind of phytosterol for it has a similar
chemical reaction ability and solvent dissolved property
as β-sitosterol. This unknown compound was collected
and analyzed by GC-MS for further study. Repeated col-
lecting the unknown compound manually until 5ml was
gathered when the unknown peak shown up from the
HPLC UV detector. Dry the gathered sample by high
purity of nitrogen gas, then dissolve it in 0.2ml cyclo-
hexanone before GC-MS analysis.
3.4. Analysis of the Unknown Compound by
GC-MS
The unknown compound was collected and analyzed by
GC-MS. The information of MS was given from the
Wiley and Nist standard chart library. The MS informa-
tion is listed in Figure 6 and Figure 7.
The unknown compound (Figure 6) probably was an
isomer of β-sitosterol. The name stigmast-5-en-3-ol was
given by IUPAC (International Union of Pure and Ap-
plied Chemistry). The only difference from β-sitosterol
was the stereochemistry in the position of C-24 ethyl.

Y. Sheng et al. / HEALTH 1 (2009) 203-206
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205
Figure 2. HPLC of phytosterol raw material. Figure 3. HPLC of phytosterol product.
Figure 4. HPLC of β-sitosterol standard sample. Figure 5. HPLC of stigmasterol standard sample.
Table 1. RT(min) of each sterol in sample A-D.
Compound name Sample A
RT(min)
Sample B
RT(min)
Sample C
RT(min)
Sample D
RT(min)
1 Column loss 15.245 15.080 －
2 Unknown compound 16.187 16.669 16.282 －
3 brassicasterol 17.201 17.605 17.330 17.308
4 stigmasterol 19.635 19.780 19.775 19.745
5 campesterol 20.440 20.580 20.574 －
6 β-sitosterol 21.743 21.879 21.880 －
Hit Spectrum Compound Structure
100 200 300 400
m/z
0
20
40
60
80
100
Rela
tive
Abu
nda
nce
43 81
95 145
147 159 213 396
255
163 381 414
303 329
231 354 416
OH
RT Name SI RSI Probability
34.97 STIGMAST-5-EN-3-OL, (3.BETA. 24S)- (CAS) 878 890 54.40
Figure 6. MS information of unknown compound.

Y. Sheng et al. / HEALTH 1 (2009) 203-206
SciRes Copyright © 2009 http://www.scirp.org/journal/HEALTH/
206
Hit Spectrum Compound Structure
100 200300 400
m/z
0
20
40
60
80
100
Relative Abundance
43
55
57 81 107 414
145 303213
161 396
329
255
173 330 41
6
OH
RT Name SI RSI Probability
37.93 STIGMAST-5-EN-3-OL, (3.BETA)- (CAS) 921 922 62.73
Figure 7. MS information of β-sitosterol.
4. CONCLUSIONS
β-sitosterol is one important kind of phytosterols that
commonly occurs in raw material which was extract from
deodorizer distillates. It is a △5 4-desmethyl sterol with
an additional ethyl substituent in the side chain at C-24.
This 24-ethyl substituent has α chirality which had been
already known. An unknown compound was found in the
sample prepared in lab. The unknown compound could be
separated from other phytosterols and collected by HPLC
under our chromatography conditions. In further study,
GC-MS information was given and shown that the un-
known compound was probably to be an epimer of β-
sitosterol, which was called γ-sitosterol by its trivial name.
The only difference of these two epimers was the 24-ethyl
substituent. The 24-ethyl in the side chain of γ-sitosterol
has beta chirality, which was indicated as 24s in the Wiley
and Nist standard chart library.
Openly accessible at
It must be emphasized most strongly that the α-, β- as-
signments for side chain stereochemistry bear no rela-
tionship to the use of α- and β- to define substituents
attached to the sterol rings. The two systems of α/β as-
signments are quite unrelated.
Although γ-sitosterol is an important epimer of β-si-
tosterol, it has been neglected in past research. Especially
the chromatography conditions for detecting and sepa-
rating γ-sitosterol from other phytosterols in raw material
samples. So, isolation and identification of γ-sitosterol by
HPLC and GC-MS in this study provided a new, precise
determination of phytosterol for further research.
5. ACKNOWLEDGMENTS
Thanks for the help from Professor Gu Wen-ying, Mr. Wang li-ping, Mr.
Tao Guan-jun, Mr. Dai jun, they did a lot of work in chromatography.
The project was sponsored by the funds of School of Agriculture and
Biology, Shanghai Jiaotong University.
REFERENCES
[1] V. Piironen, D. G. Lindsay, T. A. Miettinen, etc. (2000)
Plant sterols: Biosynthesis, biological function and their
importance to human nutrition. J. Sci. of Food and Agric.,
80, 939-966.
[2] M. J. Thompson, W. E. Robbins, G. L. Baker, etc. (1963) The
nonhomogeneity of soybean-Gamma-Sitosterol. [J]. Steroids.,
29(6), 407-418.
[3] I. Nishioka, N. Ikekawa, A. Yagi, ect. (1965) Studies on the
plant sterols and triterpenes. [J]. Chem. Pharm. Bull., 11(5),
579-584.
[4] L. J. Goad and T. Akihisa, (1997) Analysis of sterol. Pub-
lished by Blackie Academic & Professional, London.