Novel synthesis strategy for the preparation of individual phytosterol oxides

Article abstract of DOI:10.1021/jf304622s

Sterols (cholesterol and phytosterols) are important structural components of cell membranes and major constituents of lipid metabolism. Research on their oxides, such as the factors affecting oxidation, oxides' structures, and qualitative and quantitative analysis, aroused more attention in this decade. However, the biological roles of individual phytosterol oxides are still unclear because no commercial individual phytosterol oxide standards are available. Different from the traditional chemical synthesis, in the present study, chemical synthesis from a starting phytosterol mixture followed with a semipreparative HPLC separation produced individual oxides. TLC and analytical HPLC were used here to not only monitor the reaction process but also specifically analyze the synthetic intermediates and oxides. The chromatographic results exhibited strict rules and similar characteristics. Finally, for the first time, four individual phytosterol oxides were successfully separated and collected by a semipreparative HPLC system, thus providing a novel strategy for the preparation of individual phytosterol oxides.

Full text of DOI:10.1021/jf304622s

Article
pubs.acs.org/JAFC
Novel Synthesis Strategy for the Preparation of Individual
Phytosterol Oxides
Junlan Gao,† Qiulin Yue,† Yishun Ji,‡ Beijiu Cheng,† and Xin Zhang*,†
†School of Life Sciences, Anhui Agricultural University, 130 Changjiang West Road, Hefei 230036, People’s Republic of China
‡Grain and Oil Products Quality Supervision and Inspection Station of Anhui Province, 432 Qianshan Road, Hefei 230031,
People’s Republic of China
ABSTRACT: Sterols (cholesterol and phytosterols) are important structural components of cell membranes and major con-
stituents of lipid metabolism. Research on their oxides, such as the factors affecting oxidation, oxides’ structures, and qualitative
and quantitative analysis, aroused more attention in this decade. However, the biological roles of individual phytosterol oxides are
still unclear because no commercial individual phytosterol oxide standards are available. Different from the traditional chemical
synthesis, in the present study, chemical synthesis from a starting phytosterol mixture followed with a semipreparative HPLC
separation produced individual oxides. TLC and analytical HPLC were used here to not only monitor the reaction process but
also specifically analyze the synthetic intermediates and oxides. The chromatographic results exhibited strict rules and similar
characteristics. Finally, for the first time, four individual phytosterol oxides were successfully separated and collected by a
semipreparative HPLC system, thus providing a novel strategy for the preparation of individual phytosterol oxides.
KEYWORDS: phytosterols, individual phytosterol, chemical synthesis, semipreparative HPLC, isolation
Another is to chemically synthesize the high-purity target oxides
from a commercial sterol at specific oxidation sites.18,19 Because
no phytosterols are available except stigmasterol and β-sitosterol,
this also limits the formation of other phytosterol oxides.
Most papers available so far on phytosterol oxides were
focused on their qualitative and quantitative analysis in a food
matrix or in human plasma by gas or liquid chromatography in
combination with mass spectrometry.20,21 However, to consider
the preparation and purification purpose, a milder and non-
destructive process, such as crystallization or liquid chromatog-
raphy, should be better than other techniques.
The main aim of the present study was to develop a simple
and effective method for the preparation of the individual
phytosterol oxides. Different from the traditional chemical
synthesis mentioned above, in the present study, a phytosterol
mixture was used as the starting material to synthesize two
series of phytosterol oxide mixtures (7-keto- and 7-hydrox-
yphytosterols). Thin-layer chromatography (TLC) and ana-
lytical high-performance liquid chromatography (HPLC) were
used to analyze the reaction process. On the basis of the results
in analytical HPLC, a semipreparative HPLC system was first
applied to the separation of individual oxides.
INTRODUCTION
■
Phytosterol is a general name for more than 100 plant sterols
with similar structures related to cholesterol.1 Phytosterol-
enriched foods (vegetable oils, margarine, yogurt, etc.) have
experienced a great increase in the market, due to the asserted
cholesterol-lowering effect of plant sterols.2−4 Owing to the
existence of double bonds in their chemical structures, phyto-
sterols may through the same process as cholesterol, including
nonenzymatic (such as heating treatment, UV radiation, or
catalytic reactions etc.) and enzymatic pathways, produce a
wide variety of oxides2,3,5,6 with possible controversial biological
effects. This becomes an important issue for human health.
It was known that cholesterol oxides had shown cytotoxic,
apoptotic, and pro-inflammatory effects.7−9 However, in the
case of phytosterol oxides, research is still limited and their bio-
logical roles are unclear,10,11 because no commercial phytoster-
ol oxide standards are available. In 2003, Maguire12 first pointed
out that it would be extremely interesting to extend the toxi-
cological analysis to individual phytosterol oxides rather than
mixtures of these compounds. To better understand and define
the single contribution of each phytosterol oxide on consumers’
health,13 it is valuable and urgent to develop some efficient
methods to prepare the individual oxides.
Phytosterol oxides are a quite complex mixture, which may
involve dozens to hundreds of different structural compounds
in natural existence or produced by artificial generation.2,3,5,6
Therefore, it is a challenge to perform all individual phytosterol
oxides. Two strategies are mainly explored in this area so far.
One is to thermo-oxidize or phyto-oxidize the sterols to produce
the mixture of sterol oxides and then separate the blend to
different structural compounds.14−16 However, although this
method opens the door to obtaining oxides, the formation of a
variety of different oxides together, even dimeric and polymeric
oxides,17 made the separation and isolation difficult and challenging.
MATERIALS AND METHODS
■
Materials and Reagents. Generol 95R, a commercial industrial
phytosterol mixture extracted from soybean oil, was provided by Cognis
Co. (Saint-Fargeau-Ponthierry, France). The blend of Generol 95R
contained 3% unknown sterol, 9% avenasterol, 10% brassicasterol, 32%
Received: November 5, 2012
Revised: December 31, 2012
Accepted: January 1, 2013
Published: January 1, 2013
© 2013 American Chemical Society
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Scheme 1. Chemical Synthesis Pathway of Cholesterol Oxides (4 and 6), Stigmasterol Oxides (4′ and 6′), and Phytosterol
a
Oxides (4″ and 6″)
a
(i) CH3COCl, Py, CH2Cl2; (ii) PCC, Celite, toluene; (iii) CH3OH/H2O, Na2CO3, 25°C; (iv) CeCl3·7H2O, NaBH4, CH3OH/THF, 25 °C.
campesterol, and 45% β-sitosterol, along with minor yellow industrial
impurities as their trimethylsilyl (TMS) ether derivates by GC analysis.
Standard compounds, including stigmasterol (95%), cholesterol
(99%), 7-ketocholesterol, and 7β-hydroxycholesterol, were purchased
from Sigma-Aldrich (St. Louis, MO, USA).
Acetonitrile, isopropanol, and methanol (Chromasolv, for HPLC,
≥99.9%) used for HPLC analysis were obtained from Sigma-Aldrich.
The chemicals and the organic reagents (analytical grade) used for
synthesis were obtained from commercial suppliers and were used
without further purification unless otherwise noted. Pyridine was
distilled from KOH prior to use. Toluene was dried with Al2O3. THF
was dried by refluxing over sodium/benzophenone immediately prior
to use. All chemical reactions were carried out under an argon
atmosphere under anhydrous conditions.
Chemical Synthesis of Cholesterol Oxides. Besides commercial
purchase from Sigma-Aldrich, two kinds of cholesterol oxides,
7-ketocholesterol 4 and 7β-hydroxycholesterol 6, were also chemically
synthesized in the laboratory from cholesterol 1 (in Scheme 1)
according to the description of a series of β-sitosterol oxides in the
literature.18 After the synthesis, they were characterized completely,
and the results were consistent with the commercial standards.
Chemical Synthesis of Stigmasterol Oxides. From stigmasterol
1′, 7-ketostigmasterol 4′ and 7β-hydroxystigmasterol 6′ were also
chemically synthesized in the laboratory via the same pathway
(in Scheme 1) as above.
where A means the percentage of each sterol in the total amount of
sterols (4% Δ5-avenasterol, 9% brassicasterol, 44% campesterol, and
40% β-sitosterol, which was different from the content of the original
Generol 95R).
Phytosterol Oxidation Intermediates 2″. 1″ (7.50 g, 18.84 mmol)
and pyridine (4.47 g, 4.39 mL, 56.52 mmol) were dissolved in 120 mL
of CH2Cl2; acetyl chloride (4.36 mg, 4.02 mL, 56.52 mmol) was then
added dropwise at 4 °C. The reaction mixture was stirred under
nitrogen at room temperature for 3 h. The residue was washed with
water (50 mL) and extracted with CH2Cl2 (5 × 50 mL). The organic
phase was washed sequentially with a 10% HCl solution (50 mL) and
a saturated NaCl aqueous solution (50 mL) and then dried over
MgSO4. After filtration, the solvent was evaporated under reduced
pressure. The crude product was recrystallized from methanol to give
compound 2″ as a white solid (7.46 g, 16.96 mmol, 90% yield).
Phytosterol Oxidation Intermediates 3″. Celite (40 g) and PCC
(20.58 g, 95.4 mmol) were added to a solution of compound 2″
(3.50 g, 7.95 mmol) in toluene (300 mL). After being refluxed for 30 h
using a Dean−Stark apparatus, the reaction mixture was allowed to
cool to room temperature. The residue was filtered and washed with
Et2O, distilled under reduced pressure. The crude product was eluted
with a stepwise gradient of ethyl acetate in n-hexane (50 mL each of
10:90, 20:80, and 40:60, v/v) in a silica gel column to give compound
3″ as a white solid (1.98 g, 4.37 mmol, 55% yield).
7-Ketophytosterols 4″. Compound 3″ (1.50 g, 2.20 mmol) in
CH3OH (100 mL) was treated with sodium carbonate (1.17 g,
11.01 mmol) and H2O (20 mL). After being stirred under nitrogen at
room temperature for 15 h, the solvent was removed under reduced
pressure. Water (60 mL) was added, and the mixture was extracted
with CH2Cl2 (5 × 50 mL). The organic phase was dried over MgSO4,
filtered, and concentrated under reduced pressure. The crude product
was recrystallized from methanol to give compound 4″ as a white solid
(862 mg, 2.09 mmol, 95% yield).
Chemical Synthesis of Phytosterol Oxides. As shown in
Scheme 1, from the phytosterol mixture 1″ (crystallization from Generol
95R by acetone), the oxides were chemically synthesized as above. The
average molecular weight (M ) of the starting compound was
̅
w
calculated as
M
̅
= A1 × Mw(avenasterol) + A2 × Mw(brassicasterol)
+ A3 × Mw(campesterol) + A4 × Mw(β‐sitosterol)
w
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Table 1. Rf Values of Sterols (Cholesterol 1, Stigmasterol 1′,
and Phytosterols 1″) and Their Synthetic Intermediates and
a
Oxides on TLC Plate
compound
Rf (n-hexane/ethyl acetate = 2:1)
cholesterol 1
0.66
stigmasterol 1′
phytosterols 1″
cholest-5-en-3β-ol acetate 2
0.97
0.84
0.30
0.69
0.17
stigmast-5-en-3β-ol acetate 2′
phytosterol oxidation intermediates 2″
3β-acetoxycholest-5-en-7-one 3
3β-acetoxystigmast-5-en-7-one 3′
phytosterol oxidation intermediates 3″
7-ketocholesterol 4
7-ketostigmasterol 4′
7-ketophytosterols 4″
3β-acetoxycholest-5-en-7α-ol 5
3β-acetoxystigmast-5-en-7α-ol 5′
phytosterol oxidation intermediates 5″
7β-hydroxycholesterol 6
7β-hydroxystigmasterol 6′
7β-hydroxyphytosterols 6″
a
TLC analyses were performed on silica gel 60 F254 precoated
aluminum plates (Merck, Darmstadt, Germany).
HPLC analysis was performed on an Agilent 1200 series instru-
ment (Agilent, Santa Clara, CA, USA) equipped with a manual 7725i
injector, a quaternary pump system, a VWD UV−vis detector, and
Agilent Chemstation software.
With a standard circulation valve and a 20 μL injection loop, in anal-
ytical system, an Agilent Eclipse XDB-C8 column (150 × 4.6 mm i.d.,
5 μm) was used to analyze phytosterols, their reaction intermediates,
and oxides at ambient temperature with an isocratic mobile phase
(acetonitrile/isopropanol = 95:5, v/v) at a flow rate of 1.0 mL/min.
The chromatograms were monitored by UV detection at 208 nm, 0.1
AUFS.
Figure 1. Scan of TLC aluminum plate (silica gel 60 F254) with sterols
(cholesterol 1, stigmasterol 1′, and phytosterols 1″) and their synthetic
intermediates and oxides developed with mobile phase n-hexane/ethyl
acetate = 2:1 (v/v). It was observed (A) directly by UV detector at
254 nm and (B) then visualized after spraying with 20% phosphomolybdic
acid reagent in ethanol followed by heating at 120 °C. The compounds are
described in Table 1.
The synthesized phytosterol oxides, 7-ketophytosterols 4″ or
7β-hydroxyphytosterols 6″, were separated by semipreparative HPLC
system with a manual injector, a 2 mL injection loop, and an Agilent
Eclipse XDB-C8 column (250 × 9.4 mm i.d., 5 μm). After filtration
with a 0.45 μm filter membrane, 1 mL of saturated acetonitrile solution
of oxides 4″ or 6″ was injected successively into the semipreparative
HPLC system with acetonitrile/isopropanol = 10:1 (v/v) as mobile
phase at a flow rate of 1.5 mL/min. The major fractions were manually
collected into different collectors. After several injections and runs,
the organic solvents in collected fractions were removed by rotary
evaporation under reduced pressure at 40 °C. The pure individual
compounds as white powder were obtained and stored at 4 °C under
nitrogen gas. They were characterized by NMR, MS, specific rota-
tion, etc. Until now, 350 mg of 7-ketocampesterol, 300 mg of
7-ketositosterol, 320 mg of 7β-hydroxycampesterol, and 300 mg of
7β-hydroxysitosterol were isolated by semipreparative HPLC.
Spectrometric Characterization. Melting points (mp) were
measured with a Mettler Toledo FP62 (Sweden) melting apparatus.
NMR spectra were taken in a Bruker 400 MHz Ultrashield spec-
trometer (400 and 100 MHz for 1H and 13C, respectively) with CDCl3
as the solvent at room temperature. Optical rotations were measured
with a Perkin-Elmer 343 polarimeter at 20 °C in a 1 dm length cell.
Phytosterol Oxidation Intermediates 5″. Compound 3″ (1.60 g,
3.52 mmol) and CeCl3·7H2O (3.95 g, 10.56 mmol) were dissolved in a
mixture of THF/CH3OH (5:1, 90 mL), followed by portionwise
addition of NaBH4 (399 mg, 10.56 mmol) at room temperature. The
reaction mixture was stirred under nitrogen at room temperature for
1 h and, then, hydrolyzed with a 10% HCl solution (30 mL) and
extracted with CH2Cl2 (4 × 50 mL). The organic phase was dried over
MgSO4, filtered, and concentrated under reduced pressure. The crude
product was recrystallized from methanol to give compound 5″ as a
white solid (1.36 g, 2.99 mmol, 85% yield).
7β-Hydroxyphytosterols 6″. The same procedure as for com-
pound 4″ was used: compound 5″ (1.20 g, 2.63 mmol), CH3OH/H2O
(4:1, 100 mL), Na2CO3 (951 mg, 8.97 mmol). After workup, the
reaction produced compound 6″ as a white solid (870 mg, 2.10 mmol,
80% yield).
TLC and HPLC. TLC was performed on silica gel 60 F254 precoated
aluminum plates (Merck, Darmstadt, Germany) and observed by UV
detector at 254 nm or visualized after spraying with 20% phos-
phomolybdic acid reagent in ethanol followed by heating at 120 °C.
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Figure 2. Analytical HPLC chromatograms of phytosterols 1″ (A), synthetic intermediates 2″, 3″, and 5″ (B, C, and E) and oxides
7-ketophytosterols 4″ (D), 7β-hydroxyphytosterols 6″ (F), and semipreparative HPLC chromatograms of 4″ (G) and 6″ (H). HPLC analysis was
performed on an Agilent 1200 series instrument (Santa Clara, CA, USA). Analytical HPLC conditions: Agilent Eclipse XDB-C8 column (150 × 4.6
mm i.d., 5 μm); mobile phase, acetonitrile/isopropanol = 95:5 (v/v) at a flow rate of 1.0 mL/min; UV detection at 208 nm. Semipreparative HPLC
conditions: Agilent Eclipse XDB-C8 column (250 × 9.6 mm i.d., 5 μm); mobile phase, acetonitrile/isopropanol = 10:1 (v/v) at a flow rate of 1.5
mL/min; UV detection at 208 nm. Injection volume = 1 mL of saturated acetonitrile solution of 4″ or 6″.
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Table 2. Characterization of 7-Ketocampesterol, 7-Ketositosterol, 7β-Hydroxycampesterol, and 7β-Hydroxysitosterol
1H NMR (400 MHz, CDCl3), 13C NMR (100
compound
property
mp (°C)
δ
MHz, CDCl3), δ
[α]2D0 (deg)
(ESI+)-MS m/z
7-ketocampesterol
white powder,
purity ≥ 95%
172.5
5.69 (1H, br s, H-6),
202.25, 165.10,
126.11, 70.52,
54.81, 49.97,
45.43, 43.12,
41.83, 38.84,
38.28, 36.37,
36.10, 35.82,
33.73, 32.41,
31.21, 30.34,
28.53, 26.32,
21.23, 20.19,
18.85, 18.25,
17.31, 15.39,
11.97.
−88.5 (c 1, CHCl3)
415.4 (32),
397.4 (100)
3.70−3.63 (1H, m, H-3),
2.42−2.37 (3H, m), 2.24
(1H, br t, J = 9.0 Hz),
2.05−1.12 (25H, m,
including 1.20 (3H, s, CH3-
19)), 0.92 (3H, d, J = 6.5 Hz,
H-21), 0.86−0.77 (9H, m,
H-28, H-27, and H-26),
0.69 (3H, s, H-18)
7-ketositosterol
white powder,
purity ≥ 95%
123.8 (lit. value
5.69 (1H, br s, H-6),
202.27, 165.11,
126.11, 70.52,
54.74, 49.97,
45.86, 45.43,
43.12, 41.83,
38.72, 38.29,
36.37, 36.08,
33.97, 31.21,
29.18, 28.54,
26.33, 26.16,
23.08, 21.23,
19.78, 19.05,
18.93, 17.31,
11.97.
−96.8 (c 1, CHCl3)
429.2 (33),
411.3 (100)
121−12418
)
3.71−3.63 (1H, m, H-3),
2.53−2.37 (3H, m), 2.24
(1H, br t, J = 9.0 Hz),
2.05−1.12 (27H, m,
(lit. value −9818
)
including 1.25 (3H, s, H-
19)), 0.93 (3H, d, J = 6.5 Hz,
H-21), 0.87−0.81 (9H, m,
H-29, H-27, and H-26),
0.69 (3H, s, H-18)
7β-hydroxycampesterol
white powder,
purity ≥ 95%
182
5.29 (1H, br s, H-6), 3.84
(1H, d, J = 7.5 Hz,, H-7),
3.57−3.51 (1H, m, H-3),
2.36−1.29 (30H, m,
143.48, 125.47,
73.36, 71.43,
55.99, 55.46,
48.30, 42.91,
41.74, 40.94,
39.59, 38.87,
36.96, 36.45,
36.13, 35.84,
33.76, 32.42,
31.59, 30.33,
26.38, 21.09,
20.19, 19.15,
18.95, 18.76,
18.26, 11.83.
+5.8 (c 1, CHCl3)
399.4 (100),
381.3 (60),
349.0 (25),
302.2 (53)
including 1.05 (3H, s, H-
19)), 0.92 (3H, d, J = 6.5 Hz,
H-21), 0.87−0.81 (9H, m,
H-28, H-27, and H-26),
0.70 (3H, s, H-18)
7β-hydroxysitosterol
white powder,
purity ≥ 95%
219.7 (lit. value
218−22018
5.29 (1H, br s, H-6), 3.84
(1H, d, J = 7.5 Hz, H-7),
3.58−3.51 (1H, m, H-3),
2.40−1.30 (32H, m,
143.48, 125.48,
73.36, 71.44,
55.99, 55.42,
48.30, 45.88,
42.95, 41.75,
40.94, 39.58,
36.96, 36.46,
36.10, 34.01,
31.60, 29.19,
28.54, 26.39,
26.17, 23.09,
21.09, 19.80,
19.15, 19.04,
18.84.
+6 (c 1, CHCl3) (lit.
413.4 (100),
359.2 (20),
320.6 (78)
)
value +618
)
including 1.04 (3H, s, H-
19)), 0.92 (3H, d, J = 6.5 Hz,
H-21), 0.87−0.80 (9H, m,
H-29, H-27, and H-26),
0.70 (3H, s, H-18)
Electrospray ionization tandem mass spectrometry (ESI-MS) data
were measured on a Finnigan LCQ Advantage Max ion trap mass
spectrometer (Thermo, USA), which was operated in positive ion
mode using the following settings: spray voltage, 4.5 kV; spray current,
0.24; capillary voltage, 45.5 V; capillary temperature, 270 °C; nitrogen
of pure quality as carrier gas and sheath gas flow, 24 units; scan range,
m/z 50−600. One percent of acetic acid was added into the sample to
aid protonation of the sample molecules before detection.
possible interconversion in food, their adsorption in plasma,
and their biological activities.18 Chemical synthesis is an ideal
method to obtain the products with specific structures. How-
ever, traditional chemical synthesis is generally from a pure
commercially available compound or natural precursor. But so
far it is a challenge to isolate or purify some major individual
phytosterols with gram scale to serve the synthesis need.
Thus, in the present study, a novel synthetic strategy was
employed. It provides a chemical synthesis of the oxides mix-
ture from a sterols mixture as starting material followed by a
semipreparative HPLC separation.
First, two cholesterol oxides (7-ketocholesterol 4 and
7β-hydroxycholesterol 6) and two stigmasterol oxides
(7-ketostigmasterol 4′ and 7β-hydroxystigmasterol 6′) were
laboratory-synthesized from cholesterol and stigmasterol
separately as control experiments. With the same chemical
RESULTS AND DISCUSSION
■
Until now, the biological research of phytosterol oxides was
scarce.11 Most of these research works investigated the cyto-
toxicity of the mixture effects on different cell lines10−13,22
because no commercial phytosterol oxide standards were
available. It is important to obtain individual standards of
oxides and, consequently, to research their concentration or
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1
synthetic pathway as shown in Scheme 1, the phytosterol oxides
with the same oxidation sites were also synthesized from phyto-
sterol mixture 1″. Phytosterol acetate 2″ was synthesized and
underwent allylic oxidation using PCC and toluene to produce
7-ketophytosterols 4″. From the reaction intermediate 3″,
stereoselective reduction by NaBH4 and CeCl3 with a deprotec-
tion reaction might produce 7β-hydroxyphytosterols 6″.
detection. They were also characterized by their H and 13C
NMR profiles, optical rotations, and electrospray ionization
mass spectra (ESI-MS) (Table 2). The data of 7-ketositosterol
and 7β-hydroxysitosterol were consistent with the information
in the literature.18 7-Ketocampesterol and 7β-hydroxycampes-
terol were first isolated and characterized as pure compounds.
To the best of our knowledge, this is the first report to syn-
thesize the mixed oxides directly from a phytosterols mixture.
The facile RP-HPLC system used here not only monitored and
analyzed the reaction process but also isolated high-purity
individual oxides in sufficient amounts for the further sys-
tematic biological investigations in vitro or in vivo. On the basis
of the above results, we might predict that other individual
phytosterol oxides (19-hydroxy-, 25-hydroxy-, 7α-hydroxy-,
5α,6α-epxy-, 5β,6β-epoxy-, etc.) could be produced according
to this method. This strategy thus provided a simple and easy-
to-handle method to obtain the individual target sterol oxides
or other different structural sterol derivatives.
TLC or column chromatography (CC) was usually used
for the preliminary separation of phytosterol oxides on the basis
of their different oxidation sites.23,24 In the present study, as
shown in Figure 1 and Table 1, TLC Rf values of synthetic
phytosterol oxidation intermediates (2″, 3″, and 5″) and oxides
(4″ and 6″) were similar to the data of the corresponding struc-
tural derivatives of cholesterol (2−6) or stigmasterol (2′−6′).
The mobilities of these compounds were Rf(2″) > Rf(3″) > Rf(5″)
>
Rf(1″) > Rf(4″) > Rf(6″) using n-hexane/ethyl acetate = 2:1 (v/v)
as eluant. No separation could be achieved between individual
compounds in each kind of phytosterol derivate by TLC. This
result declared that Rf values strongly depended on their chem-
ical structures in sterol compounds, but little on their side-chain
structures.
AUTHOR INFORMATION
Corresponding Author
■
Indeed, because of its higher resolution, gas chromatography
(GC) has some obvious advantages over TLC and HPLC. It
was used in most previous research to separate complex phyto-
sterols and their oxides and analyze their content in foods and
biological samples.20,21 However, a time-consuming silylation
or derivatization step is necessary before the injections,23,25 so
the reference oxides could not be finally obtained after the
structural modification and the destructive testing. For the
preparative and collection purpose, HPLC should be an ideal
technique because it could be operated under milder column
temperature and nondestructive detection of sterols. In the
present study, with an Agilent Eclipse XDB-C8 column and
acetonitrile/isopropanol = 95:5 (v/v) as mobile phase, this
analytical HPLC system could separate not only individual
phytosterol peaks in the starting mixture, except avenasterol
with brassicasterol coelution (Figure 2A), but also the peaks in
the synthetic intermediates (Figure 2B,C,E) and the peaks in
target oxides (Figure 2D,F). All of these compounds could be
eluted out of the column in <10 min. No sample pretreatment
step was necessary before HPLC separation.
*Phone/fax: +86 551 65786021. E-mail: xinzhang@ahau.edu.
cn.
Funding
This work was financially supported by National Natural
Science Foundation of China (Grant 21072002) and the
Scientific Research Foundation for Returned Scholars, Ministry
of Education of China (2011-1568).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Yu-Peng Tian of Anhui University for help with the
NMR test. We also thank Dr. Paul K. Kienker of Albert Einstein
College of Medicine, USA for his valuable suggestion on this paper.
REFERENCES
■
(1) Moreau, R. A.; Whitaker, B. D.; Hicks, K. B. Phytosterols,
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