Synthesis of 7-hydroperoxycholesterol and its separation, identification, and quantification in cholesterol heated model systems

Article abstract of DOI:10.1021/jf102252r

7-Hydroperoxycholesterol is considered to be an intermediate compound of the cholesterol oxidation path as the first product formed when cholesterol is oxidized by triplet oxygen. However, there is a limitation on cholesterol mechanism studies because of the lack of 7-hydroperoxycholesterol analytical standard due to its low stability. To verify the formation of hydroperoxides in cholesterol model systems heated at 140, 180, and 220 °C, 7α-hydroperoxycholesterol was synthesized by cholesterol photooxidation followed by rearrangement at room temperature in chloroform. Its structure was confirmed on the basis of ¹³C NMR and mass spectra obtained by APCI-LC-MS. The synthesized compound was also used as standard for the quantification of 7-hydroperoxycholesterol as the sum of 7α-and 7β-hydroperoxycholesterol. The results demonstrated that 7-hydroperoxycholesterol is the first compound formed when the temperature is lower (140 °C). However, the concentration of the 7-hydroperoxycholesterol depends on the temperature and time of exposure: the higher the time, the higher the amount of 7-hydroperoxycholesterol at lower temperatures, and the lower the time, the lower the amount of 7-hydroperoxycholesterol at higher temperatures (180 and 220 °C). By the formation of 7-hydroperoxycholesterol, the known cholesterol oxidation mechanism in three phases (initiation, propagation, and termination) could be confirmed; once at lower temperatures, the stage of cholesterol oxidation is at initiation, at which hydroperoxide formation predominates.

Full text of DOI:10.1021/jf102252r

10226 J. Agric. Food Chem. 2010, 58, 10226–10230
DOI:10.1021/jf102252r
Synthesis of 7-Hydroperoxycholesterol and Its Separation,
Identification, and Quantification in Cholesterol Heated
Model Systems
§
,
G
ISLAINE C. NOGUEIRA,† BRUNA Z. COSTA,‡ ANTONIO E. M. CROTTI
,†
AND
N
EURA
B
RAGAGNOLO
*
†Department of Food Science, Faculty of Food Engineering, and ‡Department of Organic Chemistry,
Chemistry Institute, University of Campinas, Campinas, SP, Brazil, and §Center of Research on Exact and
Technologic Sciences, University of Franca, Franca, SP, Brazil
7-Hydroperoxycholesterol is considered to be an intermediate compound of the cholesterol oxidation
path as the first product formed when cholesterol is oxidized by triplet oxygen. However, there is a limi-
tation on cholesterol mechanism studies because of the lack of 7-hydroperoxycholesterol analytical
standard due to its low stability. To verify the formation of hydroperoxides in cholesterol model sys-
tems heated at 140, 180, and 220 °C, 7R-hydroperoxycholesterol was synthesized by cholesterol photo-
oxidation followed by rearrangement at room temperature in chloroform. Its structure was confirmed
on the basis of 13C NMR and mass spectra obtained by APCI-LC-MS. The synthesized compound
was also used as standard for the quantification of 7-hydroperoxycholesterol as the sum of 7R- and
7β-hydroperoxycholesterol. The results demonstrated that 7-hydroperoxycholesterol is the first com-
pound formed when the temperature is lower (140 °C). However, the concentration of the 7-hydro-
peroxycholesterol depends on the temperature and time of exposure: the higher the time, the higher
the amount of 7-hydroperoxycholesterol at lower temperatures, and the lower the time, the lower the
amount of 7-hydroperoxycholesterol at higher temperatures (180 and 220 °C). By the formation of
7-hydroperoxycholesterol, the known cholesterol oxidation mechanism in three phases (initiation,
propagation, and termination) could be confirmed; once at lower temperatures, the stage of chole-
sterol oxidation is at initiation, at which hydroperoxide formation predominates.
KEYWORDS: APCI-LC-MS; cholesterol oxides; thermal degradation; NMR
INTRODUCTION
alcohol as mobile phase, allowing the separation of cholesterol and
11 COP (6, 9, 10); however, the quantification of 7-hydroperoxy-
cholesterol by this method has not been previously reported. Quan-
tification of 7-hydroperoxycholesterol has been currently based on
Cholesterol can undergo oxidation when exposed to high tempe-
ratures, light, dehydration, storage, radiation, and combinations
of these conditions (1-3). The resulting cholesterol oxidation
products (COP) have been reported to exhibit a wide range of
adverse biological effects on animals, such as atherogenesis, cyto-
toxicity, mutagenesis, and carcinogenesis (4).
The mechanism of cholesterol oxidation is similar to that
known for unsaturated lipids, which is divided in three phases:
initiation, propagation, and termination (3). 7-Hydroperoxycholes-
terol is considered to be the first intermediate compound in the
cholesterol oxidation path, as it is formed directly from choles-
terol by oxidation promoted by triplet oxygen (3, 5). Thus, the
identification of this intermediate plays a key role in quantitative
studies of cholesterol and COP by high-performance liquid chro-
matography (HPLC). However, such studies have been limited
because of the lack of a 7-hydroperoxycholesterol analytical
standard due to its low stability (6-8).
€
chromophoric formation by the Wurster dye method (6, 7) after
thin layer chromatography (TLC) or preparative HPLC separa-
tion; however, these methods present low sensitivity or accuracy.
In the past decade, liquid chromatography-mass spectro-
metry (LC-MS) using mostly atmospheric pressure ionization
(APCI) has emerged as a powerful analytical technique for the
identification and quantification of several COP (11-15). How-
ever, the use of this technique for the identification and quanti-
fication of 7-hydroperoxycholesterol has also been limited due to
its low stability, so that APCI-MS data for such compounds has
not been previously reported.
In this work the synthesis and characterization of 7R-hydro-
peroxycholesterol by 13C nuclear magnetic resonance (NMR)
and atmospheric pressure chemical ionization mass spectrometry
(APCI-MS) are described. The synthesized compound was also
used as standard for the quantification of 7-hydroperoxycholes-
terol as the sum of 7R- and 7β-hydroperoxycholesterol in cho-
lesterol heated model systems by HPLC to verify the formation of
this compound under high-temperature conditions.
The most commonly used HPLC method for COP quantifica-
tion employs CN column and mixture of n-hexane and isopropyl
*Author to whom correspondence should be addressed (phone 55
19 3521 2160; fax 55 19 3521 2153; e-mail neura@fea.unicamp.br).
pubs.acs.org/JAFC
Published on Web 08/27/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 18, 2010 10227
MATERIALS AND METHODS
The HPLC conditions were the same as described above. The MS para-
meters were set as follows: positive ion mode of analysis; source tempera-
ture, 400 °C; corona, 4000 nA; dry gas (N2) 300 °C, 5 L/min flow, and 65 psi
nebulizer gas; scan range from m/z 80 to 450. The MS spectrum of each
peak was compared with the MS spectrum of the COP standards at the
corresponding retention time. The MS/MS spectrum of the synthesized
7R-hydroperoxycholesterol was obtained using the ion m/z 401 as precursor.
The following conditions were used: Collision-induced dissociation was
produced with helium 99.999% (White Martins, RJ, Brazil) at a pressure
of 30 psi in the ion trap. Capillary voltage was 3500 V; end-plate offset,
500 V; skimmer (I), 10.0 V; and skimmer (II), 6.0 V. The octopole was at
2.5 V, octopole D 2.5, and octopole RF 100.0 (Vpp). The acquisition MS/
MS fragmentation amplitude was 1.4 V.
Model Systems. To verify COP formation during heating, pure
cholesterol 99% (Sigma-Aldrich, St. Louis, MO) was submitted to a heat
process in a heating block (Marconi, Piracicaba, Brazil) under constant oxy-
gen 99.9999% (White Martins) flow (ca. 10 mL/min). The tubes contain-
ing 1 mL of a 1 mg/mL cholesterol solution had the solvent (isopropyl
alcohol) evaporated under a gentle nitrogen stream and then were placed
into the holes of the heating block set at 140, 180, or 220 °C. The tubes were
heated until cholesterol concentration reached at least 25% of its initial
content. To determine cholesterol, 7-hydroperoxycholesterol, and COP
concentrations during the heating process, 12 tubes were sampled at
different times. After the heating time, the tubes were immediately chilled
to stop any reactions, and their contents were diluted with 1 mL of mobile
phase and injected into the HPLC to quantify the remaining cholesterol
and COP. The initial amount of cholesterol was measured by the quanti-
fication of cholesterol in the test tube without heating.
Synthesis of 7r-Hydroperoxycholesterol. Cholesterol, 95% pure
(Sigma-Aldrich, Steinheim, Germany), and 1% Rose Bengal (Sigma Chemi-
cal, St. Louis, MO) were mixed with 10 mL of analytical grade pyridine
(Merck, Darmstadt, Germany) and submitted to photooxidation by
sodium light exposure (400 W) and oxygen insufflation under continuous
stirring and controlled temperature (5 °C), as described by Beckwith and
co-workers (16).
The synthesized compound was purified by flash column chromato-
graphy (silica gel, 4:1 and 1:1 n-hexane/ethyl acetate v/v). Twenty fractions
of 15 mL each were collected and analyzed by TLC using silica gel
G/aluminum precoated plates with 250 μm (Whatman, Freiburg, Germany).
The development of TLC was carried out in a glass tank lined with filter
paper and equilibrated for 30 min with 5 mL of n-hexane/ethyl acetate
(4:1 v/v). Approximately 10 μL of each extract was spotted on the plate.
The chromatogram was developed over a distance of 3 cm at room tem-
perature, after which the plate was dried and the color development of
cholesterol, Rose Bengal, and the reaction product was carried out with an
acid solution of anisaldehyde. Reaction product presented three bands,
one at the same Rf of cholesterol, another at the same Rf of Rose Bengal,
and a third one, which should be 5R-hydroperoxycholesterol. Fractions
11-17 (n-hexane/ethyl acetate 1:1 v/v), which presented a single band at
the same Rf supposed to be 5R-hydroperoxycholesterol, were grouped and
concentrated under a gentle nitrogen stream to afford pure 5R-hydroper-
oxycholesterol, which was dissolved in chloroform and kept at room
temperature for 68 h to rearrange and form 7R-hydroperoxycholesterol.
To obtain pure 7R-hydroperoxycholesterol, the solvent was evaporated
under a gentle nitrogen stream. The purity of the synthesized compound
was determined on the basis of peak area percentages obtained by UV
(210 nm) detector in the same chromatographic conditions used to quan-
tify COP. All solvents were of analytical grade.
RESULTS AND DISCUSSION
Synthesis and Identification of 7-Hydroperoxycholesterol. The
synthesis produced 7R-hydroperoxycholesterol (39% yield) with
68% of purity. To increase the purity, recrystallization was carried
out as described by Beckwith and co-workers (16); however, such
procedure led to the decomposition of 7R-hydroperoxycholes-
terol into 7-ketocholesterol and 7R-hydroxycholesterol, which
were already identified as part of the impurity of the synthesized
7R-hydroperoxycholesterol. On the other hand, Geiger and co-
workers (17) reported the formation of 7R- and 7β-hydroperoxy-
cholesterols as the most abundant peroxide species in photooxidized
cholesterol liposomes. In the present work, the epimerization of
7R-hydroperoxycholesterol into 7β-hydroperoxycholesterol was
not observed.
The identification of 7R-hydroperoxycholesterol was made by
comparison of 13C NMR data (Figure 1) with previously pub-
lished data for cholesterol (18) and other very closely related
structures (19). The main difference between data of 7R-hydro-
peroxycholesterol and 7R-hydroxycholesterol is in the chemical
shift of C-7, which is 12.1 ppm deshielded in comparison with the
corresponding carbon of 7R-hydroperoxycholesterol. This che-
mical shift is very similar to that reported for 7-hydroperoxy-
stigmasterol (77.9 ppm) (19).
The presence of the hydroperoxy group was easily confirmed by
APCI-MS through the loss of 34 mass units directly from the proto-
nated 7R-hydroperoxycholesterol (m/z 419), which is characteristic
for peroxide groups (20) (Figure 2). The formation of the product
ion (m/z 383) can be considered useful to distinguish 7-hydroper-
oxycholesterol from other COP, the mass spectra of which are very
similar due to water elimination resulting in isobaric ions (9).
The peak relative to 7R-hydroperoxycholesterol in the model
system samples was identified on the basis of comparison of its
retention time and mass spectrum with those of the synthesized
7R-hydroperoxycholesterol standard. The epimeric 7β-hydroper-
oxycholesterol was identified following the elution order of the
epimeric 7R-hydroxycholesterol (9), as shown in Figure 3.
Cholesterol Heating and Formation of Hydroperoxycholesterol.
The initial amounts of cholesterol were 1.5, 1.4, and 1.6 mg/mL at
Structure Confirmation by 13CNMR. The synthetic 7R-hydroperoxy-
cholesterol was solubilized in deuterated chloroform (CDCl3) (Cambridge
Isotope Laboratories Inc., Andover, MA) and characterized by 13C nuclear
magnetic resonance (NMR) in an INOVA-500 spectrometer (B0 = 11T)
(Varian, Palo Alto, CA) operating at 125.7 MHz for 13C and equipped
with a 5 mm probe for direct and indirect detection, selective pulse, and
Sun workstation. Spectra were obtained at 24.6 °C with 45° pulses, using
broad band decoupling (WALTZ sequence), spectral width of 30 kHz,
acquisition time of 1.3 s, and delay time of 1.5 s. The chemical shifts were
obtained in parts per million using tetramethylsiloxane (TMS) and deute-
rated chloroform (CDCl3) as reference standards. The difference between
methylic, methylenic, methynic, and not bound carbons was established by
DEPT spectra.
Cholesterol and COP Quantification by HPLC-UV-RI. A liquid
chromatograph (Shimadzu, Kyoto, Japan), equipped with UV (SPD-10
AVVP) and RI (RID 10A) detectors was used. The analytical column used
was a 300 mm ꢀ 3.9 mm i.d., 4 μm, Nova Pack CN HP (Waters, Milford,
MA); the injection loop was 20 μL, and the oven temperature was 32 °C. A
mixture of hexanes (minimum 63% of n-hexane) and 2-propanol was used
as mobile phase (97:3 v/v) at a flow rate of 1 mL/min (9). Cholesterol,
7R- and 7β-hydroxycholesterol, 7-ketocholesterol, and 7-hydroperoxy-
cholesterol (sum of 7R- and 7β-hydroperoxycholesterol) were quantified
using the UV detector at 210 nm. R- and β-epoxycholesterols were
quantified using an RI detector at 32 °C. Identification of cholesterol
and COP was made by comparison of the retention times of peaks in sam-
ples with those of reference standards and spiking. The compound identi-
ties were further confirmed by LC-APCI-MS.
7-Hydroperoxycholesterol was quantified as the sum of 7R- and 7β-
hydroperoxycholesterols using a calibration curve plotted with six points
with a concentration range from 0.7 to 182 μg/mL of the synthesized
7R-hydroperoxycholesterol. Cholesterol, 7R- and 7β-hydroxycholesterols,
7-ketocholesterol, and R- and β-epoxycholesterols were quantified by
external calibration, with curves ranging from 0.2 to 6 mg/mL for choles-
terol and from 0.5 to 100 μg/mL for each COP.
Confirmation of Compound Identities by LC-APCI-MS. A liquid
chromatograph (Shimadzu) equipped with quaternary pumps (LC-20AD)
and a degasser unit (DGU-20A5) connected inseriesto a photodiode array
detector (PDA) (SPD-M20A) and to an Esquire 4000 mass spectrometer
(Bruker Daltonics, Bremen, Germany), fitted with an atmospheric pres-
sure chemical ionization source (APCI) and an ion-trap analyzer was used.

10228 J. Agric. Food Chem., Vol. 58, No. 18, 2010
Nogueira et al.
Figure 1. 13C NMR chemical shifts (ppm) of 7R-hydroperoxycholesterol.
Figure 4. Concentration of 7-hydroperoxycholesterol during heating at
140, 180, and 220 °C.
Table 1. Cholesterol Degradation and Formation of 7-Hydroperoxychol-
esterol and COP at the Heating Time at Which Maximum 7-Hydroperoxy-
cholesterol Formation Occurred
140 °C
180 °C
220 °C
cholesterol degradation (%)
7-hydroperoxycholesterol (μg/mL)
timea (s)
42
21
52
87
92
31
83
59
1494
79
45
110
total COP (μg/mL)
a Time at which 7-hydroperoxycholesterol presented the maximum formation.
Figure 2. MS spectrum of 7R-hydroperoxycholesterol. Peaks: (a) [M -
H2O]þ; (b) [M - 2H2O]þ; (c) [M - H2O - H2O2]þ; (d) [M - 3H2O]þ.
7-hydroperoxycholesterol formed, possibly because of differences
in the cholesterol oxidation rates (21).
A direct relationship was verified between 7-hydroperoxy-
cholesterol formation and cholesterol degradation, where the lowest
temperature (140 °C) presented higher values than the other
temperatures (Table 1). At 180 °C smaller amounts of 7-hydro-
peroxycholesterol and cholesterol degradation percentages were
found, whereas at higher temperatures the amount of 7-hydro-
peroxycholesterol increased again, as well as the cholesterol degra-
dation, suggesting that cholesterol degradation can be correlated
to 7-hydroperoxycholesterol formation, independently of tempe-
rature. At 140 °C the formation of 7-hydroperoxycholesterol was
slower than at the higher temperatures, and probably its degradation
was slower, too, explaining the higher concentration of 7-hydro-
peroxycholesterol when compared to the higher temperatures.
The maximum amounts of 7-hydroperoxycholesterol detected
represented 5, 5, and 10% of the remaining cholesterol and 6, 4,
and 4% of the initial content cholesterol at 140, 180, and 220 °C,
respectively. Nawar and co-workers (12) reported the amount of
COP as representing 22% of initial cholesterol; however, the
authors heated the cholesterol at 180 °C for 1 h.
Figure 3. Chromatogram of cholesterol and COP from model system
containing pure cholesterol submitted to 220 °C for 45 s obtained by HPLC
(UV at 210 nm). Retention times: cholesterol, 3.5 min; 7-ketocholesterol,
14.4 min; 7β-hydroperoxycholesterol, 15.5 min; 7R-hydroperoxycholesterol,
16.1 min; 7β-hydroxycholesterol, 21.3 min; 7R-hydroxycholesterol, 22.7 min.
Chien and co-workers (6) used much higher quantities of
cholesterol (100 mg) in a heated model system and detected
ca. 0.35 μg of 7-hydroperoxycholesterol at 150 °C for 90 min and
ca. 8 μg of 7-hydroperoxycholesterol for each gram of cholesterol
at 140 °C for 120 min (7). However, the authors quantified the
€
7-hydroperoxycholesterol by using the Wurster dye method,
140, 180, and 220 °C, respectively, which were at least 75% degra-
ded at the end of the heating process.
which uses preparative HPLC analysis followed by color reaction
and UV detection.
Cholesterol Oxidation and 7-Hydroperoxycholesterol Forma-
tion. Cholesterol oxidation is known to occur by two different
paths: C-7 oxidation and epoxidation (6-8). The C-7 oxida-
tion path is observed by the formation of 7-ketocholesterol and
During heating, the amount of 7-hydroperoxycholesterol (sum
of7R- and 7β-hydroperoxycholesterols) reached a maximum con-
centration, after which time degradation occurred (Figure 4). The
time to reach the maximum concentration of 7-hydroperoxy-
cholesterol varied at each temperature as well as the amount of

Article
J. Agric. Food Chem., Vol. 58, No. 18, 2010 10229
Table 2. Content (Micrograms per Milliliter) of Cholesterol Oxides Formed at
the Heating Time at Which Maximum Formation of 7-Hydroperoxycholesterol
Occurred
Moreover, degradation of cholesterol at 140 °C occurs slowly, as
this temperature is lower than its melting point. These data
indicate that a greater part of degraded cholesterol did not form
7-hydroperoxycholesterol or other COP, but other polymers or
volatile compounds, which were not identified in this work.
Heating cholesterol at too high temperatures (>200 °C) may
have caused thermal degradation rather than cholesterol oxida-
tion (22). Moreover, the amount of COP detected could be
reduced by thermal degradation, because the quantity observed
after a given time interval represents the net balance between
formation and degradation of each COP (21).
COP
140 °C
180 °C
220 °C
7-ketocholesterol
13
13
14
14
27
28
17
20
11
17
21
20
25
17
27
7R-hydroxycholesterol
7β-hydroxycholesterol
R-epoxycholesterol
β-epoxycholesterol
In summary, the method used takes advantage of mass spectro-
metry for the identification of 7-hydroperoxycholesterol. The
potential of this technique for the analysis of such compounds has
not been previously investigated in the literature. By applying this
method in cholesterol heated model systems it was possible to
verify that 7-hydroperoxycholesterol is the first compound
formed when the temperature is lower (140 °C). However, the
concentration of the 7-hydroperoxycholesterol is dependent on
the temperature and time exposure: the higher the time, the higher
the amount of 7-hydroperoxycholesterol at lower temperature,
and the lower the time, the lower the amount of 7-hydroperoxy-
cholesterol at higher temperature (180 and 220 °C).
These results corroborate the three phases of cholesterol
oxidation, being that at 140 °C the oxidation is still at initiation
phase at which the formation of hydroperoxides predominates,
whereas at higher temperatures the oxidation is already at
propagation phase at which the formation of other COP pre-
dominates.
Figure 5. Formation of 7-hydroperoxycholesterol (maximum) and COP
observed in pure cholesterol model system heated at 140, 180, and 220 °C
for 1494, 87, and 45 s, respectively.
7R- and 7β-hydroxycholesterols, which are formed by the decom-
position of 7R- and 7β-hydroperoxycholesterols, respectively.
The epoxidation path is observed by the formation of R- and
β-epoxycholesterols due to the presence of 7-hydroperoxycholes-
terol by a bimolecular reaction, at which 7-hydroperoxycholes-
terol probably attacks another cholesterol molecule to form
epoxycholesterol (3).
The COP detected in the present study were those known
to result from cholesterol oxidation such as 7-ketocholesterol,
7R- and 7β-hydroxycholesterols, and R- and β-epoxides. Side-chain
derivatives or 3β-,5R-,6β-cholestanetriol were not detected. At
140 °C, only 7-hydroperoxycholesterol was formed until 213 s;
between 213 and 593 s, 7-ketocholesterol and 7R- and 7β-hydroxy-
cholesterols were formed; and after that, the formation of R- and
β-epoxycholesterols began. However, this behavior was not
observed at higher temperatures, at either 180 and 220 °C, when
at the first sampling time, all five of these cholesterol oxides were
detected and quantified. COP formation presented a direct cor-
relation with temperature: the higher the temperature, the higher
the COP formation, which is consistent with previously published
data (21).
ACKNOWLEDGMENT
We thank Dr. Anita J. Marsaioli from the Department of
Organic Chemistry, Chemistry Institute, University of Campinas,
for her technical assistance on the synthesis and characterization
of 7R-hydroperoxycholesterol and Dr. Lilian R. B. Mariutti from
the Department of Food Science, Faculty of Food Engineer,
University of Campinas, for her valuable contribution to the
cholesterol oxidation discussion.
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August 13, 2010. Accepted August 14, 2010.
(16) Beckwith, A. L. J.; Davies, A. G.; Davison, I. G. E.; Maccoll, A.;
Mruzek, M. H. The mechanisms of the rearrangements of allylic