Mass spectrometry characterization of the 5α-, 7α-, and 7β-hydroxy derivatives of β-sitosterol, campesterol, stigmasterol, and brassicasterol

Article abstract of DOI:10.1021/jf9812580

The 5α-hydroperoxides of β-sitosterol, campesterol, stigmasterol, and brassicasterol were obtained by photooxidation of the respective sterols in pyridine in the presence of hematoporphyrine as sensitizer. The reduction of the hydroperoxides gives the corresponding 5α-hydroxy derivatives. The 7α- and 7β-hydroperoxides of the sterols were obtained by allowing an aliquot of the 5α-hydroperoxides to isomerize to 7α-hydroperoxides, which in turn epimerize to 7β-hydroperoxides. The reduction gave the corresponding 7α- and 7β-hydroxy derivatives. The 5α-, 7α-, and 7β-hydroxy derivatives of β-sitosterol, campesterol, stigmasterol, and brassicasterol were identified by comparing thin-layer chromatography mobilities, specific color reactions, and mass spectral data with those of the corresponding hydroxy derivatives of cholesterol, which were synthesized in the same manner. The phytosterols had the same behavior to photooxidation as cholesterol and, moreover, the different phytosterols photooxidized at about the same rate. The mass spectra of the trimethylsilyl ethers of the hydroxy derivatives of the phytosterols investigated and of the corresponding hydroxy derivatives of cholesterol have the same fragmentation patterns and similar relative ion abundances.

Full text of DOI:10.1021/jf9812580

J. Agric. Food Chem. 1999, 47, 3069−3074
3069
Ma ss Sp ectr om etr y Ch a r a cter iza tion of th e 5r-, 7r-, a n d 7â-Hyd r oxy
Der iva tives of â-Sitoster ol, Ca m p ester ol, Stigm a ster ol, a n d
Br a ssica ster ol
Renzo Bortolomeazzi,* Michela De Zan, Lorena Pizzale, and Lanfranco S. Conte
Food Science Department, Udine University, via Marangoni 97, 33100 Udine, Italy
The 5R-hydroperoxides of â-sitosterol, campesterol, stigmasterol, and brassicasterol were obtained
by photooxidation of the respective sterols in pyridine in the presence of hematoporphyrine as
sensitizer. The reduction of the hydroperoxides gives the corresponding 5R-hydroxy derivatives.
The 7R- and 7â-hydroperoxides of the sterols were obtained by allowing an aliquot of the
5R-hydroperoxides to isomerize to 7R-hydroperoxides, which in turn epimerize to 7â-hydroperoxides.
The reduction gave the corresponding 7R- and 7â-hydroxy derivatives. The 5R-, 7R-, and 7â-hydroxy
derivatives of â-sitosterol, campesterol, stigmasterol, and brassicasterol were identified by comparing
thin-layer chromatography mobilities, specific color reactions, and mass spectral data with those of
the corresponding hydroxy derivatives of cholesterol, which were synthesized in the same manner.
The phytosterols had the same behavior to photooxidation as cholesterol and, moreover, the different
phytosterols photooxidized at about the same rate. The mass spectra of the trimethylsilyl ethers of
the hydroxy derivatives of the phytosterols investigated and of the corresponding hydroxy derivatives
of cholesterol have the same fragmentation patterns and similar relative ion abundances.
Keyw or d s: Sterols; phytosterols; hydroxysterols; photooxidation; gas chromatography/ mass
spectrometry; GC/ MS; mass spectra; trimethylsilyl ether
INTRODUCTION
Because of the structural similarity, phytosterols also
can oxidize in a similar way to cholesterol (Smith, 1981).
Among the vegetable sterols, some oxidation products
of â-sitosterol and stigmasterol have been studied to
verify their presence in vegetable oils and other plant-
based foods because of their possible toxicity like the
corresponding oxidation products of cholesterol (Lee et
al., 1985; Blekas et al., 1989; Nourooz-Zadeh et al., 1992;
Finocchiaro et al., 1983; Dutta et al., 1996).
In the present study, three hydroxy derivatives,
namely, the 5R-, 7R-, and 7â-hydroxy of cholesterol,
â-sitosterol, campesterol, stigmasterol, and brassicast-
erol, have been synthesized and characterized by mass
spectral data of their trimethylsilyl (TMS) ether deriva-
tives as a necessary preliminary step to analyze their
degradation products during the refining process of
vegetable oils, the last part being the subject of an other
paper.
Phytosterols are among the most important compo-
nents of the unsaponifiable matter of vegetable oils.
Their composition is characteristic of the botanical
species from which the oil is obtained, and the analysis
of sterols in vegetable oils is used for identity control.
Among phytosterols, â-sitosterol (Ib) is the most
representative and widely distributed in vegetable oils
and other products of plant origin, together with other
minor sterols such as campesterol (Ic), stigmasterol (Id ),
and brassicasterol (Ie) (Itoh et al., 1973).
Phytosterols can undergo transformation during the
refining process of oils with the formation of isomers
and dehydrated products (Niewiadomski et al., 1966;
Kaufmann and Hamza, 1970; Grob et al., 1994; Mennie
et al., 1994), which in turn can be analyzed to evaluate
the quality of the oil and of other products (Lanzon et
al., 1989; Cert et al.,1994; Grob et al., 1995; Biedermann
et al., 1996; Crews et al., 1997). Little is known of the
fate of sterol oxides eventually present in the oil during
the refining processes (Niewiadomski et al., 1964;
Kaufmann et al., 1970; Grob et al., 1994; Bortolomeazzi
et al., 1996).
The molecular structures of phytosterols are strictly
related to the structure of cholesterol as represented in
Scheme 1. Cholesterol can oxidize by thermal (autoxi-
dation) as well by photosensitized (photooxidation)
processes, and the oxidation products have been thor-
oughly studied due to their biological activity (Smith,
1981).
EXPERIMENTAL PROCEDURES
Ma ter ia ls. All solvents were of analytical grade. Hexam-
ethyldisilazane (HMDS), trimethylchlorosilane (TMCS), the
silylating mixture Fluka II according to Horning, and 7-de-
hydrocholesterol were from Fluka (Buchs, Switzerland). Cholest-
5-en-3â-ol (Ia ), cholest-5-ene-3â,7R-diol (7R-OH-cholesterol)
(VIa ), cholest-5-ene-3â,7â-diol (7â-OH-cholesterol) (VIIa ), and
cholest-4,6-dien-3â-ol were supplied by Nu-Chek-Prep, Inc.
(Elysian, MI); [24R]-24-ethylcholest-5-en-3â-ol (Ib) and [24S]-
24-ethylcholest-5,22-dien-3â-ol (Id ) by were provided by Merck
(Bracco, Milan, Italy).
The gas chromatography (GC) and GC/mass spectrometry
(MS) analyses of â-sitosterol standard as TMS ether revealed
the following composition: â-sitosterol, 76%; [24R]-24-meth-
ylcholest-5-en-3â-ol (campesterol), 8%; [24R]-24-methylcholest-
3â-ol (campestanol), 1.4%; [24R]-24-ethylcholest-3â-ol (sito-
stanol), 14.6%.
* Author to whom correspondence should be addressed (fax
+39-0432590719; e-mail Renzo.Bortolomeazzi@DSA.uniud.it).
10.1021/jf9812580 CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/29/1999

3070 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Bortolomeazzi et al.
Sch em e 1
injection was in split mode (split ratio ) 1:40) with helium as
carrier gas at a flow rate of 1 mL/min. The injector, transfer
line, and ion trap temperatures were 300, 300, and 170 °C,
respectively. The electron impact (70 eV) spectra were recorded
at 1 s/scan with a filament emission current of 10 µA.
The sterols and their hydroxy derivatives were derivatized
to TMS ethers prior to GC and GC/MS analyses. The deriva-
tization was carried out with a mixture of pyridine, HMDS,
and TMCS (5:2:1 v/v/v) (Sweeley et al., 1963) for ∼1 h at room
temperature. In these conditions the 5-hydroxy sterols were
only partially silanized, probably due to steric hindrance, with
the formation of different products. For these compounds the
silanization was obtained by using a more powerful silylating
mixture formed by N,O-bis(trimethylsilyl)acetamide, 1-(trim-
ethylsilyl)imidazole, and trimethylchlorosilane (3:3:2 v/v/v)
(silylating mixture Fluka II) for ∼2 h at room temperature.
Syn th esis of 5-r-OH-, 7r-OH-, a n d 7â-OH-ch olester ol.
Cholesterol (1.0795 g) and hematoporphyrin (15 mg) were
dissolved in 20 mL of pyridine in a three-neck, double-wall
glass container equipped with a bulb condenser. The container
was then placed in the center of four cross-disposed 100 W
tungsten lamps, at ∼2 cm from each lamp. A flow of ∼300 mL/
min of air, supplied by a gas cylinder, was bubbled into the
solution, ensuring a vigorous stirring, and the temperature of
the mixture was maintained at 10 °C by external circulation
with a water cooling bath.
The reaction was carried out for ∼5 h, and then the pyridine
was removed in a Rotavapor at 35 °C. The residue, dissolved
in chloroform, was mixed with silica and then absorbed by
removing the chloroform under reduced pressure at 35 °C. The
sample was then loaded into a silica chromatography column
(14 cm length × 3 cm i. d.) and eluted with diethyl ether/n-
hexane (6:4 v/v). The separation was monitored by spotting
the eluate from the column on a thin-layer chromatography
(TLC) silica plate sprayed with 80% sulfuric acid, and warming
with hot air cholesterol gave a red color while the hydroper-
oxides gave a blue-green color and were, moreover, positive
to the KI test, producing a yellow-brown spot on a silica plate
impregnated with a KI solution. The hydroperoxides fraction,
evaporated to dryness under reduced pressure, gave 850 mg
of product. The hydroperoxides were dissolved in 50 mL of
chloroform and then separated in two aliquots of, respectively,
10 and 40 mL.
The 10 mL aliquot was evaporated to dryness, dissolved in
methanol/diethyl ether, and immediately reduced with NaBH4
to yield 170 mg of the corresponding hydroxy compounds. A
TLC silica analysis (eluent diethyl ether) of the reduced
product revealed four spots with Rf 0.42 (predominant), blue
color with 80% sulfuric acid; Rf 0.27, blue color; Rf 0.23, light
blue-gray color; Rf 0.21, yellow color.
The mixture was absorbed on silica and then loaded into a
silica flash chromatography column (25 cm length × 2 cm i.d.)
and eluted with a mixture of diethyl ether/n-hexane starting
from a 7:3 ratio to 100% ether and then methanol. The main
fraction, 84 mg, gave after crystallization from aqueous
methanol 68 mg of pure cholest-6-ene-3â,5R-diol (5-R-OH-
cholesterol) (Va ), Rf 0.42 (silica gel, eluent diethyl ether),
immediate blue color with 80% H2SO4, mp 176-179 °C [lit.
mp 148-149, 147-150, 170-175, 166-171, 134-135, 181 °C)
(Teng et al., 1973; Smith et al., 1978)]. Calcd for C27H46O2: C,
80.54; H, 11.52. Found: C, 79.51; H, 11.51.
The 40 mL aliquot was allowed to isomerize for 5 days at
room temperature on the laboratory bench. After isomeriza-
tion, the 40 mL aliquot was reduced with NaBH4 in methanol/
diethyl ether to yield 608 mg of hydroxy compounds. A TLC
silica analysis revealed four principal bands with Rf 0.38, blue
color, with 80% sulfuric acid; Rf 0.27 (predominant), blue color;
Rf 0.23, light blue-gray color; and Rf 0.21, yellow color. The
mixture of hydroxides was absorbed on silica and then loaded
into a silica flash chromatography column (30 cm length × 2
cm i.d.) and eluted with diethyl ether/n-hexane from an initial
7:3 ratio to 100% diethyl ether followed by methanol. The first
fraction eluted, 71 mg, gave after crystallization from aqueous
methanol 40 mg of pure cholest-5-ene-3â,7â-diol (7â-OH-
cholesterol) (VIIa ), Rf 0.38 (silica gel, eluent diethyl ether),
The composition of the stigmasterol standard was as fol-
lows: stigmasterol, 91%; â-sitosterol, 4.8%; campesterol, 2.6%;
[24S]-24-methylcholest-5,22-en-3â-ol (brassicasterol), 1.5%.
The melting points were determined on a Gallen Kamp
MFB-595 (England) melting apparatus.
GC a n d GC/MS. A Carlo Erba Mega 5160 gas chromato-
graph equipped with a split-splitless injector and a flame
ionization detector was used. The fused silica column was an
SPB5, 30 m × 0.32 mm i.d., 0.25 µm film thickness (Supelco,
Bellefonte, PA). The column temperature was 280 °C isother-
mal, and the detector and injector temperatures were 300 °C.
The carrier gas (helium) flow rate was 1.3 mL/min and the
split ratio 1:40 (v/v).
For the GC/MS analysis a Varian 3400 gas chromatograph
coupled to a Varian Saturn ion trap detector was used. The
fused silica column was a DB-5 (J &W, Folsom, CA), 30 m ×
0.25 mm i.d., 0.25 µm film thickness, operating at 280 °C. The

5R-, 7R-, and 7â-Hydroxy Derivatives of Phytosterols
J. Agric. Food Chem., Vol. 47, No. 8, 1999 3071
Ta ble 1. Ma ss Sp ectr om etr ic Da ta of th e 7r- a n d 7â-Hyd r oxy TMS Der iva tives of Ch olester ol a n d P h ytoster ols
m/z (%)
ion
VIa a
VIIa a
VIba
VIIba
VIca
VIIca
VId a
VIId a
VIea
VIIea
M
546 (1)
531 (1)
546 (2)
531 (0.3) 559 (1)
574 (2)
574 (1)
559 (0.3) 545 (1)
560 (2)
560 (1)
545 (-)
572 (2)
557 (1)
572 (2)
558 (2)
558 (0.8)
M - CH3
M - (TMSOH)
557 (0.2) 543 (0.3) 543 (-)
456 (100) 456 (100) 484 (100) 484 (100) 470 (100) 470 (100) 482 (100) 482 (100) 468 (100) 468 (100)
M - (TMSOH + CH3) 441 (2)
441 (2)
-
366 (0.5) 394 (1)
469 (1)
-
469 (2)
-
394 (1)
379 (1)
455 (2)
-
380 (1)
365 (2)
455 (3)
-
380 (-)
365 (2)
343 (-)
253 (1)
211 (2)
467 (1)
467 (1)
453 (2)
453 (2)
M - (TMSOH + C3H7)
M - (2TMSOH)
-
439 (0.4) 439 (0.5) 425 (0.2) 425 (-)
392 (0.3) 392 (0.1) 378 (-)
377 (1)
343 (1)
253 (2)
211 (1)
366 (1)
378 (-)
363 (-)
343 (0.3)
253 (2)
211 (-)
M - (2TMSOH + CH3) 351 (2)
351 (2)
379 (1)
377 (0.5) 363 (1)
M - (Rb + TMSOH)
M - (Rb + 2TMSOH)
M - (2TMSOH +
Rb + C3H6)
343 (0.3) 343 (0.2) 343 (0.2) 343 (0.3) 343 (1)
253 (2)
211 (1)
343 (1)
253 (1)
211 (1)
343 (1)
253 (2)
211 (1)
253 (1)
211 (1)
253 (2)
211 (1)
253 (1)
211 (1)
253 (2)
211 (1)
a
b
Analyzed as TMS derivatives. R ) side chain.
immediate blue color with 80% H2SO4 (Smith et al., 1967), mp
172-175 °C [lit. mp 175-178, 174-178 °C) (Smith et al., 1973,
1978]. Calcd for C27H46O2: C, 80.54; H, 11.52. Found: C, 79.64;
H, 11.48. GC retention time and MS spectrum of the TMS
derivative were in agreement with those of an authentic
sample. The second fraction eluted, 428 mg, gave after
crystallization from aqueous methanol 364 mg of pure cholest-
5-ene-3â,7R-diol (7R-OH-cholesterol) (VIa ), Rf 0.27 (silica gel,
eluent diethyl ether), immediate blue color with 80% H2SO4
(Smith et al., 1967), mp 181-184 °C [lit. mp 181-183, 183-
186 °C) (Smith et al., 1973, 1978]. Calcd for C27H46O2: C, 80.54;
H, 11.52. Found: C, 79.80; H, 11.43. GC retention time and
MS spectrum of the TMS derivative were in agreement with
those of an authentic sample.
The third fraction, 42 mg, consisted of a mixture of VIa and
of the two epimers cholest-4-ene-3â,6â-diol (6â-OH-cholesterol),
Rf 0.23, light blue-gray color with 80% H2SO4, and cholest-4-
ene-3â,6R-diol (6R-OH-cholesterol), Rf 0.21, yellow color. The
two latter compounds were identified on the basis of the Rf
values, which are lower than those of the other diols, on the
color developed with H2SO4, (Kulig and Smith, 1973), and by
computer matching of the mass spectra with the NIST library.
The two 6R and 6â diols derived from the photooxidation of
cholesterol (Kulig and Smith, 1973) and were present also in
the aliquot of hydroperoxides reduced immediately after the
photooxidation. The presence also in this aliquot of the 7R-
diol derivative was probably due to isomerization during
reduction with NaBH4.
to 7R-hydroperoxide (IIIa ), which in turn isomerizes to
7â-hydroperoxide (IVa ) (Beckwith et al., 1989); in this
way, the three hydroperoxides and their corresponding
hydroxides Va , VIa , and VIIa , by reduction, can be
prepared in a relatively simple and clean way as shown
in Scheme 1.
Identification of the hydroxy derivatives of â-sitosterol
and campesterol present as impurities of the â-sito-
sterol, stigmasterol, and brassicasterol present as im-
purities of stigmasterol was carried out by comparing
them with the corresponding oxidation products of
cholesterol. The latter, contrary to the oxidation prod-
ucts of phytosterols, have been, in fact, widely studied
and well characterized.
The presence of campesterol as a relatively abundant
impurity (8%) in the â-sitosterol standard (ratio â-sito-
sterol/campesterol ) 9:1) allowed the characterization
of the oxidation products of the campesterol itself. The
same was valid also for the hydroxides of brassicasterol,
which was present as 1.5% impurity in the stigmasterol
standard.
The GC analyses showed that the ratio between the
corresponding hydroxy derivatives was about the same
as between â-sitosterol and campesterol, suggesting that
the two sterols oxidized at about the same rate.
Syn th esis of th e Hyd r oxy Der iva tives of P h ytoster ols.
The synthesis of the 5R-, 7R-, and 7â-hydroxy derivatives of
â-sitosterol and stigmasterol was carried out under the same
experimental conditions used for the hydroxy derivatives of
cholesterol.
The same was observed also for the hydroxides of
stigmasterol and the other sterols present as impurities
in the stigmasterol standard.
The Rf values of the 5R-, 7R-, and 7â-hydroxy deriva-
tives of phytosterols Vb-VIb-VIIb, Vc-VIc-VIIc,
Vd -VId -VIId , and Ve-VIe-VIIe were 0.42, 0.27,
and 0.38, respectively, on TLC (silica gel, eluent diethyl
ether). In the case of the derivatives of â-sitosterol and
campesterol, a single spot was visible and only the GC/
MS analysis revealed the presence of the hydroxides of
the two sterols. In a similar way, the hydroxides of
stigmasterol presented a single spot, with the GC/MS
analysis revealing, instead, the presence also of the
corresponding hydroxides of the other sterols present
as impurities. The same Rf values were obtained for the
corresponding hydroxy derivatives of cholesterol; more-
over, all of the hydroxy derivatives of phytosterols as
well as those of cholesterol developed an intense blue
color after spraying with 80% H2SO4. The order of
migration and the color developed in the case of the
7-OH diols VIb and VIIb were in agreement with
literature data (Daly et al., 1983; Yanishlieva et al.,
1980, 1983).
From â-sitosterol: 5R-OH-â-sitosterol (Vb), Rf 0.42, blue
color with 80% H2SO4; 7R-OH-â-sitosterol (VIb), Rf 0.27, blue
color; 7â-OH-â-sitosterol (VIIb), Rf 0.38, blue color.
The GC/MS analysis of the â-sitosterol hydroxides as TMS
ethers revealed also the presence of the corresponding hydroxy
derivatives of the campesterol, which were not separated on
TLC.
From stigmasterol: 5R-OH-stigmasterol (Vd ), Rf 0.42, blue
color with H2SO4; 7R-OH-stigmasterol (VId ), Rf 0.27, blue
color; 7â-OH-stigmasterol (VIId ), Rf 0.38, blue color.
The GC/MS analysis of the stigmasterol hydroxides as TMS
ethers revealed also the presence of the corresponding hydroxy
derivatives of campesterol, â-sitosterol, and brassicasterol,
which were not separated on TLC.
RESULTS AND DISCUSSION
Photooxidation was used as a synthetic route for the
preparation of the hydroxy derivatives of sterols because
of the lower number of oxidation products formed with
respect to autoxidation. The 5R-hydroperoxide (IIa ) is
the main product of photooxidation of cholesterol,
together with small amounts of 6R- and 6â-hydroper-
oxides (Kulig and Smith, 1973). IIa can then isomerize
The mass spectra of VIa , VIIa , and Va as TMS
derivatives are represented in Figure 1. The mass
spectra of the two 7-OH-diols VIa and VIIa show the

3072 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Bortolomeazzi et al.
Ta ble 2. Ma ss Sp ectr om etr ic Da ta of th e 5r-Hyd r oxy TMS Der iva tives of Ch olester ol a n d P h ytoster ols
m/z (%)
ion
Va a
Vba
Vca
Vd a
Vea
M
546 (0.5)
531 (5)
456 (100)
441 (17)
-
366 (13)
351 (25)
343 (3)
253 (7)
211 (5)
574 (1)
559 (3)
484 (100)
469 (15)
-
394 (12)
379 (22)
343 (3)
253 (7)
211 (5)
560 (-)
545 (2)
470 (100)
455 (17)
-
380 (12)
365 (25)
343 (5)
253 (9)
211 (7)
572 (1)
557 (4)
558 (-)
543 (1)
M - CH3
M - (TMSOH)
M - (TMSOH + CH3)
M - (TMSOH + C3H7)
M - (2TMSOH)
M - (2TMSOH + CH3)
M - (Rb + TMSOH)
M - (Rb + 2TMSOH)
M - (2TMSOH + Rb + C3H6)
482 (100)
467 (12)
439 (1)
392 (10)
377 (17)
343 (8)
468 (100)
453 (10)
425 (-)
378 (11)
363 (19)
343 (3)
253 (11)
211 (5)
253 (12)
211 (2)
a
b
Analyzed as TMS derivatives. R ) side chain.
fragments, m/z values, and relative abundances of all
7-OH-diol TMS derivatives are given in Table 1.
The mass spectra of the hydroxides of phytosterols
VIb-VIIb, VIc-VIIc, VId -VIId , and VIe-VIIe as
TMS derivatives present the same fragmentation pat-
terns and have similar relative ion abundances of the
corresponding hydroxides of cholesterol with the base
peaks at, respectively, m/z 484, 470, 482, and 468. The
differences of 28, 14, 26, and 12 amu with respect to
the base peak (m/z 456) of the 7-OH-diols VIa and VIIa
reflect the differences of the side-chain structure in the
original sterols (Scheme 1). The ions at m/z 343, 253,
and 211 do not contain the side chain and are common
to all of the hydroxy derivatives of phytosterols as well
of cholesterol. These ions originate, respectively, from
the loss of one TMSOH group plus the side chain for
m/z 343, the loss of two TMSOH groups, and the side
chain for m/z 253 and the loss of two TMSOH groups
and the side chain plus ring D for m/z 211. These
fragmentations are characteristic of sterolic structures
(Dumazer et al., 1986).
The mass spectral data of the two epimeric 7-OH-diols
VIb-VIIb are in accord with the data reported by
Nourooz-Zadeh and Appelqvist (1992).
The ion at m/z 439 in the spectra of the 7-OH-diols
VId -VIId and the ion at m/z 425 in the spectra of the
7-OH-diols VIe-VIIe derived from the loss of one
TMSOH plus isopropyl (C25, C27) group at the end of
the side chain and are characteristic of a ∆22 double
bond in the side chain, although with a much lower
relative abundance with respect to the corresponding
sterols TMS (Dumazer et al., 1986; Pizzoferrato et al.,
1993). The presence of a second hydroxy group at C7
results in a very close similarity of the mass spectra of
all the hydroxy sterols analyzed, keeping into account
the different molecular weights.
The fragments, m/z, and relative abundances of the
5R-OH-diols Va , Vb, Vc, Vd , and Ve as TMS derivatives
are shown in Table 2. The mass spectra of the 5-hydroxy
derivatives of phytosterols present the same fragmenta-
tion patterns and similar relative ions abundances of
the corresponding 5R-OH-cholesterol TMS. The consid-
erations made previously about the ions with differences
of 28, 14, 26, and 12 amu with respect to the corre-
sponding fragments of the hydroxy derivative of cho-
lesterol are valid also in the analysis of the spectra of
the hydroxy derivatives of phytosterols.
The mass spectrum of the 5R-OH Va has a quite
different appearance with respect to the spectrum of the
7-OH-diols VIa -VIIa , although the fragmentation pat-
terns are pratically the same. In all spectra the base
peak is the m/z 456 ion due to the loss of one TMSOH
group, but in the case of Va , all of the other high-mass
F igu r e 1. Mass spectra of (A) 7R-OH-cholesterol TMS, (B)
7â-OH-cholesterol TMS, and (C) 5R-OH-cholesterol TMS.
same fragmentation patterns, which are characterized
by an intense fragment at m/z 456 (M - TMSOH) with
all of the other high-mass ions lower than 5%. The

5R-, 7R-, and 7â-Hydroxy Derivatives of Phytosterols
J. Agric. Food Chem., Vol. 47, No. 8, 1999 3073
C3 should not lead to the formation of structures
stabilized by double-bond conjugation.
In the case of the spectra of the two diol epimers VIa
and VIIa it seems there is no formation of the ion at
m/z 456 with a structure like that of 7-dehydrocholes-
terol TMS. Among the possible losses of the TMSO
group, the 1,2 elimination between the TMSO group at
C3 and H at C4 with the formation of a structure like
3,5-cholestadien-7-ol TMS, stabilized by double-bond
conjugation, seems the more probable.
As reported under Experimental Procedures, all of the
hydroxy derivatives of sterols were analyzed by GC/MS
as TMS ethers. The silylation with the mixture of
HMDS and TMCS in pyridine at room temperature,
normally utilized for the sterols, works well also with
the 7-OH-sterols, whereas in the case of the 5R-OH-
sterols, the GC/MS analysis reveals the presence of
three principal products. The more abundant one has a
spectrum that is compatible with the structure of a
mono-TMS derivative with the TMS group bonded to
C(3), characterized by the base peak at m/z 456 (M -
18), the (M - 90) ion at m/z 384, and the rest of the
spectrum like that of 7-dehydrocholesterol TMS. The
second has the retention time and the spectrum in
agreement with the di-TMS derivative; the third, which
is characterized by a pronounced tail, has a retention
time and the spectrum in agreement with that of 4,6-
cholestadien-3â-ol TMS. This last compound is likely to
be the decomposition product of the mono-TMS deriva-
tive, formed by the thermal loss of a molecule of water
from C5 in the injection port of the gas chromatograph.
This behavior reflects the difficulty, probably due to
steric hindrance, of introducing the second TMS group
at C5. A higher temperature would probably lead the
reaction to completness but with a high risk of decom-
position by dehydration. The 5R-OH derivatives of
sterols have been successfully silylated at room tem-
perature by using a more powerful silanizing mixture
as reported under Experimental Procedures.
F igu r e 2. Mass spectra of (A) 7-dehydrocholesterol TMS and
(B) 4,6-cholestadien-3â-ol TMS.
fragments have a much higher relative abundance.
Particularly interesting is the ion at m/z 325, the
formation of which can be explained by the loss of a 131
amu fragment, which comprises the TMS group and
C1-C3, from the m/z 456 ion. This elimination is
characteristic of ∆5,7 di-unsatered sterols as reported by
Brooks et al. (1968) for the TMS derivatives of 7-dehy-
drocholesterol and ergosterol. The spectrum of Va
(Figure 1C) is, in fact, similar to that of 7-dehydrocho-
lesterol TMS in the m/z 200-500 range (Figure 2A),
with the difference that in the first the m/z 456 ion is
the base peak. In Va the 1,4 elimination between the
TMSO group at C5 and the H at C8 with rearrangement
of double bonds can lead to the formation of a structure,
stabilized by double-bond conjugation, like that of the
molecular ion of 7-dehydrocholesterol TMS. Another
more probable fragmentation pattern with the formation
of a species stabilized by double-bond conjugation is the
1,2 elimination between the TMSO group at C5 and the
H at C4 with the formation of a structure like that of
4,6-cholestadien-3â-ol TMS, the spectrum of which
(Figure 2B) is characterized by the base peak at m/z 456
and all of the other fragments of low intensity. This can
explain the base peak at m/z 456 and the low intensity
of the ions under 200 amu in the spectrum of Va . On
the bases of these considerations and by comparison
with the spectra of Figure 2, we can hypothesize that
in the Va spectrum, the ion at m/z 456 is due mainly to
two species, one with a structure like the molecular ion
of 7-dehydrocholesterol TMS and one, probably pre-
dominant, because of the m/z 456 ion as base peak, with
a structure like the molecular ion of 4,6-cholestadien-
3â-ol TMS. The possible losses of the TMSO group at
CONCLUSIONS
â-Sitosterol, campesterol, stigmasterol, and brassi-
casterol have the same behavior to photooxidation as
cholesterol, resembling in this the similarity already
seen in the case of autoxidation. Moreover, the different
phytosterols photooxidized at about the same rate as
evidenced by the similar percent composition of the
sterols and of the corresponding sterol oxides. The mass
spectra of the corresponding hydroxy derivatives of the
phytosterols investigated and of the cholesterol have the
same fragmentation patterns and similar relative ion
abundances. In particular, the two 7-OH epimers of each
sterol have practically the same spectra, which are
instead quite different from the spectra of the 5R-OH
derivative.
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Received for review November 16, 1998. Revised manuscript
received J une 2, 1999. Accepted J une 9, 1999.
J F9812580