Food Chemistry 117 (2009) 1519

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Isolation and identication of steroidal hydrocarbons in soybean oil

deodorizer distillate
Novy S. Kasim, Setiyo Gunawan, Yi-Hsu Ju *
Department of Chemical Engineering, National Taiwan University of Science and Technology, 43, Sec. 4, Keelung Road, Taipei 106-07, Taiwan

a r t i c l e

i n f o

Article history:
Received 3 October 2008
Accepted 17 March 2009

Keywords:
Soybean oil deodorizer distillate
Steroidal hydrocarbons
Modied Soxhlet extraction
Modied silica gel column chromatography

a b s t r a c t
In the isolation of squalene from soybean oil deodorizer distillate (SODD), ca. 30% of nonpolar lipid fraction (NPLF) is hydrocarbons (HCs), which can be divided into three main groups, namely aliphatic HCs,
steroidal HCs and terpene HCs. The steroidal HCs content of vegetable oil can be used as a means of identifying the oil and therefore, for detecting adulteration. The objective of this study was to isolate the steroidal HCs contained in SODD and identify the individual steroidal HCs. Isolation of steroidal HCs can be
achieved in three steps: modied Soxhlet extraction, modied silica gel column chromatography, and
distillation. Identication of individual steroidal HCs was accomplished by TLC, GC and GCMS analyses.
The GC chromatogram of steroidal HCs showed ve major peaks. Most of the major peaks were identied
as derivatives of free sterols. Special attention was needed in order to identify isomers which differ in the
position of double bonds and compounds which have three double bonds in the ring system.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Steroidal hydrocarbons (HCs) are formed in vegetable oils as the
dehydration products of D5-phytosterols, because of bleaching
with acidic earth and deodorization at high temperature during
the rening process. Steradienes and steratrienes are steroidal
HCs that have two double bonds and three double bonds, respectively. Their contents in vegetable oils can be used as a means for
identifying the oil and therefore, for detecting adulteration (Wretensj & Karlberg, 2002). The application limit of the steroidal
HCs in the virgin olive oil is between 0.01 and 4 mg/kg and that
is regulated by the International Olive Oil Council. The limit of
3,5-stigmastadiene and the total steroidal HCs content in virgin olive oil have been xed at 0.15 and 0.3 mg/kg, respectively and have
become a European Economic Community regulation (Toschi, Bendini, & Lercker, 1996).
Soybean oil deodorizer distillate (SODD) is a byproduct of the
rening process in obtaining rened soybean oil. Depending on
the sources, SODD usually has signicantly different characteristics, uses and value. It can be a good raw material for the production of tocopherols, phytosterols and fatty acids, (Nagao et al.,
2005; Torres, Torrelo, Senorans, & Reglero, 2007; Watanabe, Nagao,
Hirota, Kitano, & Shimada, 2004; Winters, 1986) and squalene
(Gunawan, Kasim, & Ju, 2008). Little information about detailed
steroidal HCs in SODD is available in the literature. Cert, Lanzn,
Carelli, and Albi (1994) and Verleyen et al. (2002) reported the
* Corresponding author. Tel.: +886 2 27376612; fax: +886 2 27376644.
E-mail address: yhju@mail.ntust.edu.tw (Y.-H. Ju).
0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.03.066

presence of 3,5-stigmastadiene in olive oil and compared its quantitative analyses using GC and HPLC. Bortolomeazzi, Pizzale, Novelli, and Conte (1996) and Bortolomeazzi, Zan, Pizzale, and Conte
(2000) detected the existence of steradienes and steratrienes in
olive oil from the occurrence of ions at m/z 255 and 213 and at
m/z 253 and 211 in the gas chromatographymass spectrometry
(GCMS) chromatogram, respectively. Mennie, Moffat, and McGill
(1994) detected the presence of steroidal HCs at different stages
in the rening process of vegetable oils (maize oil, sunower oil,
rapeseed oil, palm oil and soybean oil) and succeeded in identifying 3,5-, 2,5-, 4,6-, 3,5,22-, and -4,6,22- steroidal HCs using
thin-layer chromatography (TLC), gas chromatography (GC), and
GCMS. Gunawan et al. (2008) reported that, in the isolation of
squalene from SODD, ca. 3040% of nonpolar lipid fraction (NPLF)
is HCs. Their preliminary results suggest that the HCs contained
in NPLF can be divided into four main groups, which are aliphatic,
steroidal, sesquiterpene and triterpene (squalene) HCs.
A better knowledge of the separation, purication, and chemical
composition of steroidal hydrocarbons in SODD will help researchers nding a better utilization of this byproduct. The American Oil
Chemists Society (AOCS) method Cd27-96 (1997) and the International Union of Pure and Applied Chemistry (IUPAC) method pp.
M7M11 (1994) are used to analyze the steradienes content in a
vegetable oil by GC. Steradienes were isolated from the unsaponiable matters by silica gel column chromatography (Toschi et al.,
1996). The weak point of these methods is the interferences of
steradienes with other hydrocarbons in the chromatogram and this
makes it difcult to quantify the exact steradiene amount. In order
to determine the exact content and composition of steroidal HCs in

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N.S. Kasim et al. / Food Chemistry 117 (2009) 1519

SODD, enrichment, followed by separation of these lipid classes

prior to analysis, is important. The objective of this study was to
isolate the steroidal HCs contained in SODD and identify the individual steroidal HC.
2. Materials and methods
2.1. Materials and reagents
SODD was donated by Taiwan Sugar Company (Tainan, Taiwan).
Stigmasterol (95%) and cholesta-3,5-diene (approx. 95%) standards
were obtained from Sigma Chemicals Company (St. Louis, MO). Reagents used in the isolation of steroidal HCs were of industrial
grade and those used in the analyses (GC, TLC, GCMS) were of
analytical grade. Silica gel (pore size: 60 , 70230 mesh) was purchased from Silicycle (Quebec, Canada). Analytical TLC silica gel
plates (0.25 mm thickness) were obtained from Merck KGaA
(Darmstadt, Germany). The silica gel was rst activated by heating
in the oven at 150 C for 12 h prior to use.
2.2. Isolation of steroidal hydrocarbons from SODD
Fig. 1 shows the ow chart for the isolation of steroidal HCs
from SODD. About 20 g of SODD were subjected to a modied
Soxhlet extraction (Gunawan et al., 2008) to obtain a nonpolar lipid
fraction (NPLF) which is rich in fatty acid steryl esters (FASEs),
squalene and other HCs. The NPLF was then subjected to a modied silica gel column chromatography (MSC) to obtain the HCs
fraction. Detail of the MSC was described by Gunawan, Ismadji,
and Ju (2008). During MSC elution, the HCs fraction was obtained
in the rst 4 h. Squalene was obtained in the next two and half
hours. It is important to obtain the rst HCs fraction that is free
of any squalene, because squalene and steroidal HCs have similar
molecular weight and boiling points. If present together, it is difcult to separate them by distillation. The HCs fraction was distilled
at 270280 C and 35 mTorr to obtain pure steroidal HCs in the
residue. The distillate contained aliphatic, sesquiterpene, and a
small amount of steroidal HCs. The steroidal HCs obtained in the
residue were analyzed by GC, TLC, and GCMS to identify the individual steroidal HC.
2.3. Gas chromatography (GC) and GCmass spectrometry (GCMS)
studies
A Shimadzu GC-17A equipped with a splitsplit injector and a
ame ionization detector (FID) was employed for analyzing steroidal HC. The fused silica column used was a DB-5HT, 15 m 

SODD

Modified Soxhlet Extraction

PLF

NPLF

Modified Column Chromatography

HCs

Squalene, FFAs, FASEs

Distillation

Aliphatic, terpene, steroidal

HCs

Steroidal HCs

Fig. 1. Schematic diagram of steroidal HCs isolation.

0.32 mm i.d., 0.1 lm lm thickness (Agilent Tech. Palo Alto, California, USA). The initial column temperature was 80 C, which then
was raised to 365 C at 15 C/min and held for 8 min. The temperature of the injector and detector was 370 C. For GCMS analysis,
a GC Shimadzu model GC-2010 coupled with MS model QP2010
was used. The fused silica column was a DB-5 ms, 30 m  0.25 mm
i.d., 1 lm lm thickness (Agilent Tech. Palo Alto, California, USA).
The initial column temperature was 80 C, which was then raised
to 320 C at 15 C/min and held for 14 min. The injection was in
split mode (split ratio was 1:50) with helium as the carrier gas
and with a linear velocity of 30.6 cm/s. The temperature of injector,
interface, and ion source were, 250, 250, and 200 C, respectively.
Electron ionization was carried out at 70 eV. The mass spectra were
recorded at 1250 scans/s.
2.4. Thin-layer chromatography (TLC)
The specic reagent used to detect the steroidal HCs spot was
made by dissolving 50 mg FeCl3.6H2O in 90 ml H2O, along with
5 ml acetic acid and 5 ml sulfuric acid. The desired spot yields a
redviolet colour (Fried, 2003). The mobile phase used in the TLC
analyses was HPLC grade n-hexane (95% purity). The spot was observed at 366 nm for analytical TLC. The reagent was sprayed onto
the TLC plate, and the plate was heated at 90100 C until the desired spot appeared.
3. Results and discussion
3.1. Isolation of steroidal HCs
Based on IUPAC and IUBMB recommendations of steroid
nomenclature (1989), steroidal HCs and free sterols have similar
structures. For free sterols, there is an OH group at position 3.
For steroidal HCs, there is no OH group on the skeleton; however,
there are double bonds in the ring system. In the bleaching process,
activated (acid) earth is added to rened oil to remove colour,
odour, other impurities, and residual soap. Deodorization is a high
temperature, high vacuum steam-distillation process that is necessary for the removal of volatile avour and odour compounds to
transform the oil into a bland-tasting clear liquid desirable to consumers (Pryde, 1995). These processes may cause the free sterol in
soybean oil to undergo isomerization reactions, such as the shifting
of the double bond position and also the dehydration of sterol, to
produce steroidal HCs (Biedermann, Grob, Mariani, & Schmid,
1996). The reaction scheme is shown in Fig. 2. The dominant products of this dehydration reaction are products B. This is because pbonding in products B are the strongest bond among other
products.
SODD in Taiwan typically contains about 40% FFAs and 17% triacylglycerols (TAGs) (Gunawan, 2008). The elimination of these
compounds is crucial for enriching steroidal HCs due to their high
contents in SODD. By using modied Soxhlet extraction, we were
able to isolate all HCs in the NPLF (5 g NPLF from 20 g of SODD).
TLC and HTGC analyses of the polar lipid fraction (PLF) showed
the absence of HCs. More than 90% of TAGs and other polar components were concentrated in the PLF after modied Soxhlet extraction. NPLF (3 g), which contains 43.7% HCs, was subjected to
modied silica gel column chromatography, using hexane as the
mobile phase. All aliphatic, steroidal and sesquiterpene HCs
(1.3 g) were obtained in the rst fraction after eluting the column
with hexane for 4 h. Analysis of this fraction showed that it contained only HCs. The rst fraction had a steroidal HCs content of
11.9% (100% recovery). Modied Soxhlet extraction and modied
silica gel column chromatography were efcient for the extraction
of HCs using hexane as the solvent. Advantages of the current
method are: a large sample to silica gel ratio, easy recovery and

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N.S. Kasim et al. / Food Chemistry 117 (2009) 1519

B1, B2, B3
- H2O

OH

A1, A2, A3
C1, C2, C3

Fig. 2. Reaction scheme for isomerization during the dehydration of free sterol to become steroidal HCs, where 1: R = CH3; 2: R i = C2H5; 3: R = C2H5 and one hydrogen
removed from carbon 22 and another hydrogen from carbon 23.

reuse of solvents, and simple operational procedure. Distillation

was employed for the purication steroidal HCs due to the large
differences in the boiling point of steroidal HCs and other HCs. Steroidal HCs (purity 90.8%, recovery 81.3%) were obtained as residue.
The quantication of the steroidal HCs is based on external calibration of cholesta-3,5-diene. The chromatogram of the steroidal HCs
obtained from distillation showed that there was a small hump, as

3.2. Identication of steroidal HCs

b
4

FID response

2 3
5

10
15
20
Retention time (minute)

25

Fig. 3. Results of HTGC and TLC analyses: (a) GC chromatogram of residue

containing mainly steroidal HCs; (b) TLC plate of cholesta-3,5-diene standard (lane
I) and the residue (lane II).

(a)
m/z 41

m/z 43

m/z 57

(b)

m/z 255

m/z 213

(c)

m/z 253

seen in Fig. 4. The calculation of steroidal HCs content is based on

the summation of all resolvable steroidal HC peaks. Although the
ve resolvable peaks made up only 70.0% of the steroidal HCs,
the steroidal HCs obtained had very high purity. This was conrmed on TLC analysis (Fig. 3). Based on the steroidal HCs content
obtained in the distillation residue, we can calculate the steroidal
HCs content in SODD (Gunawan, 2008), which is (2.46 0.12)
wt% of SODD.

m/z 211

Fig. 4. Common fragmentations of steroidal HC compounds: (a) of its side chains,

(b) of its ring system with two double bonds, and (c) of its ring system with three
double bonds.

The identication of steroidal HCs was achieved by comparing

results from GC, TLC, and GCMS analyses. The separation between
peaks in the chromatograms of both GC and GCMS analyses is
based on differences in molecular weight and boiling point of the
compound that each peak represents. For GCMS, the fragmentation of certain functional groups can be detected. By applying a
standard, that has the same or similar structure as the target compound, on a TLC plate, together with the target compound, and
detecting them using a specic reagent; the target compound
structure or its functional group can be revealed. In this study,
we used these three methods of analysis to identify the individual
steroidal HCs in the mixture which was obtained from SODD.
The TLC chromatogram of the residue from distillation shows
absence of aliphatic and sesquiterpene HCs (Fig. 3b). The fact that
the peaks represent steroidal HCs was supported by the TLC chromatogram (Fig. 3b). TLC analysis showed that the Rf value of the
residue spot (lane 2) was 0.750.81, which has about the same
Rf value of the spot of cholesta-3,5-diene standard (lane 1). When
the TLC plate was treated with a ferric chloride reagent, both spots
gave a violet colour which is a specic character of compounds
with sterol ring structures. The relative retention times (RRT) of
the ve major peaks, as well as their weight percentages, which
are presented in parentheses, in Fig. 3a were 0.94 (5.21%), 0.97
(16.80%), 0.98 (16.05%), 1.00 (25.56%), and 1.01 (5.37%), for peak
numbers 1 until 5, respectively. The RRT of a peak was calculated,
based on the retention time of that peak divided by the retention
time of peak number 4.
From the results of GC and TLC analyses of the sample, as shown
in Fig. 3, there are several major peaks with different retention
times in the GC chromatogram with each peak representing one
particular steroidal HC. However, in the TLC chromatogram, there
is only one spot appeared, which means that all steroidal HCs in
the sample have almost the same polarity. Further isolation of individual steroidal HCs from the residue of distillation is extremely
difcult, if not impossible. The residue was applied for GCMS
analysis to extract information on the structure of individual steroidal HCs. Table 1 shows names of the ve major peaks, their

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N.S. Kasim et al. / Food Chemistry 117 (2009) 1519

Table 1
GCMS fragmentations of the ve major peaks in Fig. 3a.
HTGC Peak
No.

Name and MW

m/z (relative intensity, %)

24-ethylcholesta-2,5-diene
(C2); 396

24-methylcholesta-3,5-diene
(B1); 382
24-ethylcholesta-3,5,22triene (B3); 394

41(50%), 43(98.10%), 55a(100%), 57(35.71%), 67(47.62%), 69(35.71%), 71(28.57%), 79(47.62%), 81(71.43%), 83(56.19%),
91(63.33%), 93(46.67%), 95(47.62%), 105(64.76%), 121(21.43%), 135(22.86%), 147(26.19%), 161(13.81%), 213(14.29%),
255(29.52%), 382(40.48%), 396(14.29%).
41(34.29%), 43(76.19%), 55(41.90%), 57(33.33%), 67(34.29%), 81(71.43%), 91(47.62%), 105(52.38%), 121(26.19%), 133(23.81%),
147(57.14%), 159(14.29%), 213(16.67%), 255(19.05%), 261(18.57%), 274(23.81%), 367(28.57%), 382a(100%).
41(33.33%), 55(83.33%), 67(41.90%), 69(47.62%), 81a(100%), 91(40.48%), 105(42.86%), 119(19.05%), 133(28.57%), 145(28.57%),
159(19.05%), 213(11.90%), 255(38.10%), 267(0.45%), 282(0.93%), 295(0.40%), 309(0.10%), 323(<0.10%), 337(<0.10%),
351(0.95%), 365(0.10%), 379(0.95%), 394(95.24%).
41(40.94%), 43(86.55%), 55(52.63%), 57(49.71%), 67(40.94%), 81a(100%), 91(55.56%), 105(71.93%), 121(38.01%), 133(32.16%),
147(86.55%), 213(23.98%), 255(26.90%), 275(22.81%), 288(27.19%), 381(35.09%), 396 (87.13%).
41(30%), 55a(100%), 67(25.71%), 69(52.38%), 81(38.10%), 97(30%), 105(28.57%), 121(10%), 213(5.71%), 253(4.76%),
281(15.71%), 296(61.90%), 394(21.43%).

24-ethylcholesta-3,5-diene
(B2); 396
24-ethylcholesta-2,4,6-triene;
394

5
a

The base peak.

molecular weight (MW), and mass fragmentations with their relative intensities.
Steroidal HCs have a strong p-bond in the ring system. As ions
in mass spectrometry bombard steroidal HCs, side chains will be
cleft rst, followed by cleavage of the ring system. The mass spectrum of the cholesta-3,5-diene standard can be seen in the NIST
database (2007). From this mass spectrum we were able to analyze
some common fragmentations. The fragmentation of steroidal HC
side chain results in groups, such as CH3 at m/z 15, C3H5+, at m/z
41, C3H7+, at m/z 43, C4H9+, at m/z 57, Fig. 4a (Johnsson & Dutta,
2003). The breakdown of the ring system in the steroidal HCs results either in groups such as fragmentations at m/z 255 and at
m/z 213 (Fig. 4b), which show that the compounds have two double bonds in the ring system or in groups such as fragmentations at
m/z 253 and at m/z 211 (Fig. 4c), which show that the compounds
have three double bonds in the ring system (Bortolomeazzi, Zan,
Pizzale, & Conte, 2000).
HTGC peak 2 shows a base peak at its molecular ion peak (Table
1). This indicates that the structure of the corresponding compound is very stable. Fragmentations at m/z 255 and 213 show that
there are two pairs of double bonds inside the ring system. The
compound of peak 2 has a MW of 382; it is derived from the dehydration of campesterol (A1 in Fig. 2, MW = 400). Based on its fragmentations at m/z 55, 67, and 81 (Fig. 5), this compound may
have double bonds at positions 3 and 5 or 2 and 5. The amount
of isomer with double bonds at positions 3 and 5 is much larger
than that with double bonds at positions 2 and 5, due to the fact
that double bonds at positions 3 and 5 are more stable that those
at 2 and 5, (Bortolomeazzi, Pizzale, Novelli, & Conte, 1996; Grob,
Biedermann, Artho, & Schmid, 1994; Mennie et al., 1994). The double bonds located at positions 3 and 5 are in a conjugated system,
which is why this isomer is more stable than other isomers. There
are unique fragmentations at m/z 261 and 274, which are present
because of side chain effect (Mennie et al., 1994). Hence, this compound is identied as B1 in Fig. 2.
The compound of peak 4 has a molecular ion peak at m/z 396,
fragmentations at m/z 255 and 213, and high relative intensities

m/z 55

m/z 67

of fragmentations at m/z 55, 67, and 81 (Table 1). These show that
the compound of peak 4 is B2 (Fig. 2), which is derived from bsitosterol (A2 in Fig. 2, MW = 414). There are unique fragmentations
at m/z 275 and 288, which are present because of the side chain effect (Mennie et al., 1994).
The compound of peak 3 has a molecular ion peak at m/z 394.
Fragmentation patterns of its side chain occur at m/z 255, 267,
282, 295, 309, 323, 337, 351 and 365. These fragmentations exist
because of one double bond at the side chain that easily moves
along the side chain and has a tendency to move to the end of
the side chain. High relative intensities occur at fragmentations
of m/z 55, 67 and 81 (Table 1). The fragmentations of the side chain
appeared in reduced intensity because the side chain in this compound is not stable, due to the double bond effect in the side chain.
These facts suggest that the compound of peak 3 is B3 (Fig. 2),
which is derived from stigmasterol (A3, MW = 412).
The compound of peak 1 has a mass spectrum very similar to
that of B2 (Table 1). Their most favourable fragmentations are
the same, namely at m/z 43, 55, 67, 81, 213 and 255. The double
bond positions of compound of peak 1 and B2 are in the same ring
system. They differ at the positions inside the ring system. The
compound of peak 1 is suggested as C2 (Fig. 2). The fragmentations
at m/z 67 and 81 show that the double bond is at position 5. From
the fragmentation at m/z 55, the rst cyclohexane ring has only
one double bond, but its position has yet to be determined. Based
on the area percentage ratio of C2 and B2, and the isomerization
scheme (Fig. 2), B2 is more abundant than its isomer C2. The fragmentation patterns of C2 will be quite similar to those of B2 because the only difference between the structures of C2 and B2
are that C2 and B2 have double bonds at positions 2 and 3, respectively. The fragmentation of the compound of peak 1 at m/z 55 is
the base peak. This fragmentation is the most favoured one because it will result in a stable butadiene fragmentation (Roderbourg & Kuzdal-Savoie, 1979). Isomers with double bounds at
positions 2 and 5 may occur, but their percentages are much smaller than those of the 3,5 isomers. Hence GCMS analyses of minor
peaks in Fig. 3a were not carried out in this study.

m/z 81

Fig. 5. Unique fragmentations which show double bond at positions 3 and 5, where (. . .) indicate locations where bonds can be cleft.

N.S. Kasim et al. / Food Chemistry 117 (2009) 1519

The compound of peak 5 has the same molecular ion as that of

B3, at m/z 394 (Table 1). However, the compound of peak 5 has
fragmentations at m/z 253 and 211 while B3 has fragmentations
at m/z 255 and 213. This compound has three double bonds inside
the ring system. From the molecular ion and the fragmentations,
this compound is derived from the hydroxy derivative of A2, (Bortolomeazzi, Zan, Pizzale, & Conte, 1999), not derived from A3. The
fragmentations at m/z 55, 67, 81, 93, and 105 suggest that the double bonds are located at positions either 2,4,6, or 3,5,7. Fragmentations at m/z 55, 95, 107, 119, 131 and 145 suggest that the double
bonds are located at positions 3, 8, and 11. This may happen because the impact energy of EI is high enough to cleave bonds in
the compound and give enough energy to the compound to undergo isomerization. This is why intramolecular isomerization reactions can occur. Based on the relative intensities of
fragmentations mentioned above, the 2,4,6 or 3,5,7-isomer is more
abundant than is the 3,8,11-isomer. The 2,4,6- or 3,5,7-isomer
forms a conjugated system, so it will result in a more preferable
isomer product. Hence, the compound of peak 5 is identied as
24-ethylcholesta-2,4,6-triene.
4. Conclusion
From the analyses of steroidal HC mass spectra, it can be concluded that there are ve major steroidal HCs in SODD, which
are 24-ethylcholesta-3,5-diene, 24-ethylcholesta-3,5,22-triene,
24-methylcholesta-3,5-diene, 24-ethylcholesta-2,5-diene, and 24ethylcholesta-2,4,6-triene. Their content in steroidal HCs are
25.6%, 16.0%, 16.8%, 5.21% and 5.37%, respectively, based on their
weight percentages in the GC chromatogram. Most of these compounds are derived from the dehydration of free sterol in SODD.
There are several minor peaks in the GC chromatogram insert of
Fig. 4a. Because the amount of each minor peak is very small, it
is more difcult to identify these minor peaks by GCMS. These
minor peaks are believed to be the products of unfavourable dehydration reaction of free sterol or hydroxy derivatives of free sterol.
Acknowledgements
The authors wish to thank Dr. Jiang, Jyh-Chiang for technical
assistance and to the National Science Council of Taiwan for funding the project (NSC94-2214-E011-004).
References
Biedermann, M., Grob, K., Mariani, C., & Schmid, J. P. (1996). Detection of
desterolized sunower oil in olive oil through isomerized D7-sterols.
Zeitschrift fr Lebensmitteluntersuchung und-Forschung A, 202, 199204.

19

Bortolomeazzi, R., Pizzale, L., Novelli, L., & Conte, L. S. (1996). Steroidal
hydrocarbons formed by dehydration of oxidized sterols in rened oils.
Rivista Italiana delle Sostanze Grasse, 73, 457460.
Bortolomeazzi, R., Zan, M. D., Pizzale, L., & Conte, L. S. (1999). Mass spectrometry
characterization of the 5a-, 7a-, and 7b-hydroxy derivatives of b-sitosterol,
campesterol, stigmasterol, and brassicasterol. Journal of Agricultural and Food
Chemistry, 47, 30693074.
Bortolomeazzi, R., Zan, M. D., Pizzale, L., & Conte, L. S. (2000). Identication of new
steroidal hydrocarbons in rened oils and the role of hydroxy sterols as possible
precursors. Journal of Agricultural and Food Chemistry, 48, 11011105.
Cert, A., Lanzn, A., Carelli, A. A., & Albi, T. (1994). Formation of stigmasta-3, 5-diene
in vegetable oils. Food Chemistry, 49, 287293.
Fried, B. (2003). Lipids. In J. Sherma, B. Fried (Eds.), Handbook of thin-layer
chromatography (3rd ed.). Chromatographic science series (Vol. 89, pp. 654
655); Marcel Dekker: New York
Grob, K., Biedermann, M., Artho, A., & Schmid, J. P. (1994). LC, GC, and MS of sterol
dehydration products in rened olive oil. Rivista Italiana delle Sostanze Grasse,
71, 533538.
Gunawan, S. (2008). Isolation and purication of squalene and fatty acid steryl esters
from soybean oil deodorizer distillate. Ph.D. dissertation, National Taiwan
University of Science and Technology, Taipei, Taiwan.
Gunawan, S., Ismadji, S., & Ju, Y.-H. (2008). Design and operation of a modied silica
gel column chromatography. Journal of the Chinese Institute of Chemical
Engineers, 39, 625633.
Gunawan, S., Kasim, N. S., & Ju, Y.-H. (2008). Separation and purication of squalene
from soybean oil deodorizer distillate. Separation and Purication Technology, 60,
128135.
Johnsson, L., & Dutta, P. C. (2003). Characterization of side-chain oxidation products
of sitosterol and campesterol by chromatographic and spectroscopic methods.
Journal of American Oil Chemists Society, 80, 767776.
Mennie, D., Moffat, C. F., & McGill, A. S. (1994). Identication of sterene compounds
produced during the processing of edible oils. Journal of High Resolution
Chromatography, 17, 831838.
Nagao, T., Kobayashi, T., Hirota, Y., Kitano, M., Kishimoto, N., Fujita, T., Watanabe, Y.,
& Shimada, Y. (2005). Improvement of a process for purication of tocopherols
and sterols from soybean oil deodorizer distillate. Journal of Molecular Catalysis
B: Enzymatic, 37, 5662.
Pryde, E. H. (1995). Composition of soybean oil. In D. R. Erickson (Ed.), Practical
handbook of soybean processing and utilization (pp. 1331). Champaign, IL: AOCS
Press.
Roderbourg, H., & Kuzdal-Savoie, S. (1979). The hydrocarbon of anhydrous
butterfat: Inuence of technological treatments. Journal of American Oil
Chemists Society, 56, 485488.
Torres, C. F., Torrelo, G., Senorans, F. J., & Reglero, G. (2007). A two steps enzymatic
procedure to obtain sterol esters, tocopherols and fatty acid ethyl esters from
soybean oil deodorizer distillate. Process Biochemistry, 42, 13351341.
Toschi, T. G., Bendini, A., & Lercker, G. (1996). Evaluation of 3, 5-stigmastadiene
content of edible oils: Comparison between the traditional capillary gas
chromatographic method and the on-line high performance liquid
chromatography-capillary gas chromatographic analysis. Chromatographia, 43,
195199.
Verleyen, T., Szulczewska, A., Verhe, R., Dewettinck, K., Huyghebaert, A., & Greyt, W.
D. (2002). Comparison of steradiene analysis between GC and HPLC. Food
Chemistry, 78, 267272.
Watanabe, Y., Nagao, T., Hirota, Y., Kitano, M., & Shimada, Y. (2004). Purication of
tocopherols and phytosterols by a two-step in situ enzymatic reaction. Journal
of American Oil Chemists Society, 81, 339345.
Winters, R. L. (1986). In A. R. Baldwin (Ed.), Proceedings of the world conference on
emerging technologies in the fats and oils industry (p. 186). Champaign, IL:
American Oil Chemists Society.
Wretensj, I., & Karlberg, B. (2002). Characterization of sterols in rened borage oil
by GCMS. Journal of American Oil Chemists Society, 79, 10691074.