Simultaneous analysis of free phytosterols/phytostanols and intact phytosteryl/phytostanyl fatty acid and phenolic acid esters in cereals

Article abstract of DOI:10.1021/jf300878h

An approach based on solid-phase extraction for the effective separation of free phytosterols/phytostanols and phytosteryl/phytostanyl fatty acid and phenolic acid esters from cereal lipids was developed. The ester conjugates were analyzed in their intact

Full text of DOI:10.1021/jf300878h

Article
pubs.acs.org/JAFC
Simultaneous Analysis of Free Phytosterols/Phytostanols and Intact
Phytosteryl/Phytostanyl Fatty Acid and Phenolic Acid Esters in
Cereals
Rebecca Esche, Andreas Barnsteiner, Birgit Scholz, and Karl-Heinz Engel*
Lehrstuhl fur Allgemeine Lebensmitteltechnologie, Technische Universitat Munchen, Maximus-von-Imhof-Forum 2,
̈
̈
̈
D-85350 Freising-Weihenstephan, Germany
ABSTRACT: An approach based on solid-phase extraction for the effective separation of free phytosterols/phytostanols and
phytosteryl/phytostanyl fatty acid and phenolic acid esters from cereal lipids was developed. The ester conjugates were analyzed
in their intact form by means of capillary gas chromatography. Besides free sterols and stanols, up to 33 different fatty acid and
phenolic acid esters were identified in four different cereal grains via gas chromatography−mass spectrometry. The majority (52−
57%) of the sterols and stanols were present as fatty acid esters. The highest levels of all three sterol and stanol classes based on
dry matter of ground kernels were determined in corn, whereas the oil extract of rye was 1.7 and 1.6 times richer in fatty acid
esters and free sterols/stanols than the corn oil. The results showed that there are considerable differences in the sterols/stanols
and their ester profiles and contents obtained from corn compared to rye, wheat, and spelt. The proposed method is useful for
the quantification of a wide range of free phytosterols/phytostanols and intact phytosteryl/phytostanyl esters to characterize
different types of grain.
KEYWORDS: solid-phase extraction, sterols, steryl fatty acid ester, steryl phenolic acid ester, cereals
several classes of neutral lipids, for example, steryl esters, free
fatty acids, and free sterols, in rye and corn flour as well as in
sourdough and bread.19 Other approaches are based on the
isolation of steryl/stanyl fatty acid esters from corn oil by
column chromatography (CC) or thin layer chromatography
(TLC) and saponification of the ester fraction for the
subsequent analysis of liberated sterols by GC and flame
ionization detection (FID).20,21 Thereby, information concern-
ing the distribution and composition of individual steryl/stanyl
fatty acid esters gets lost.
Chromatographic methods for the investigation of intact
steryl and stanyl fatty acid esters in cereals are rare. Gordon et
al. analyzed intact esters in corn oil by means of GC-FID after
extraction by preparative NP-HPLC; however, the resolution of
individual esters was insufficient, and the esters were only
characterized on the basis of relative retention times without
identification of individual peaks.22 In wheat and spelt, the
fraction of steryl/stanyl fatty acid esters was extracted from the
lipids by TLC, and intact esters were analyzed via GC-FID and
RP-HPLC-ELSD.23 Using the RP-HPLC-ELSD method,
several peaks belonging to different steryl series were
overlapping. To distinguish between several esters, the analysis
had to be done with the use of mass detection by extraction of
single ions. In turn, the suggested GC-FID method resulted in a
better sensitivity and resolution compared to HPLC-ELSD.
However, no resolution between the steryl esters of oleic and
linoleic acid could be achieved; moreover, no stanyl esters were
included.23
INTRODUCTION
■
Phytosterols and phytostanols are cholesterol-like molecules
that play important roles in the structure and function of cell
membranes.1 The current interest in phytosterols/phytostanols
is mainly due to their potency to decrease low-density
lipoprotein (LDL) cholesterol levels in plasma, a potential
risk factor for cardiovascular diseases.2−4 This led to the
development of functional foods enriched in sterols/stanols and
their fatty acid esters (e.g., margarines and drinking yogurts). In
addition to the cholesterol-lowering effect, steryl and stanyl
ferulates are thought to possess other health-promoting
properties, for example, antioxidative and anti-inflammatory
effects.5−7 Thus, there is growing interest in increasing the
intake of these compounds in natural diets. The main natural
sources of phytosterols are cereals, seeds, and vegetable oils.
Cereals and cereal-based foods are of particular importance;
depending on the populations and the dietary patterns, they
contribute approximately 25−42% to the total daily intake of
phytosterols.8−10 As in many other foods, in cereal grains
sterols occur as free sterols, as esters of fatty acids and phenolic
acids, as steryl glycosides, and as acylated steryl glycosides.1
Currently, the analysis of phytosterols and phytostanols in
cereals is mainly based on the determination of total contents
after alkaline hydrolysis or after a combination of acidic and
alkaline hydrolysis.11−16 The liberated sterols and stanols can
be extracted as part of the unsaponifiable matter, commonly
followed by a purification step and final analysis via gas
chromatography (GC) or reversed phase high-performance
liquid chromatography (RP-HPLC). In whole corn grains, total
contents of steryl fatty acid esters were determined by means of
normal phase HPLC (NP-HPLC) equipped with an evapo-
rative light scattering detector (ELSD).17,18 A recently
published NP-HPLC-ELSD method was also used to analyze
Received: February 29, 2012
Revised: April 19, 2012
Accepted: April 23, 2012
Published: April 23, 2012
© 2012 American Chemical Society
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(BSTFA) + 1% trimethylchlorosilane (TMCS), thionyl chloride,
triethylamine (>99.5%), acetic anhydride (98%), hydrochloric acid
(25%), sodium sulfate (anhydrous), magnesium sulfate (anhydrous),
piperazine (99%), potassium hydroxide (85%), sodium hydroxide, and
4-dimethylaminopyridine (DMAP, 99%) were obtained from Sigma-
Aldrich (Steinheim, Germany). n-Hexane (AnalR Normapure), diethyl
ether (DEE) (extra pure), and ammonium chloride (Rectapur) were
purchased from VWR International (Darmstadt, Germany). Ethyl
acetate (EtOAc) and tert-butyl methyl ether (MTBE) were supplied by
Oxeno Olefinchemie (Marl, Germany) and were distilled prior to use.
Tetrahydrofuran (THF) was purchased from BASF (Ludwigshafen,
Germany). Sitosterol (75%) was obtained from Acros Organics
(Morris Plains, NJ, USA). Mixtures of phytosteryl/phytostanyl fatty
acid esters (Vegapure 95E) and of plant stanols (Reducol Stanol
Powder) were provided by Cognis GmbH (Illertissen, Germany).
Synthesis of Steryl/Stanyl Fatty Acid Esters. In accordance
with the procedure described in a patent (WO2007101580),34 steryl/
stanyl fatty acid esters were synthesized directly without a catalyst
under nitrogen atmosphere. Briefly, 0.25 mmol of stigmasterol or
sitostanol, respectively, and a 2−3-fold amount of fatty acid were
mixed and flushed with nitrogen. The reaction vial was sealed and
heated to 180 °C in a heating block for 25 h. Purification was achieved
by alkaline liquid−liquid extraction: 2.5 mL of potassium hydroxide
solution (1 M) was added to the residue, and the synthesized fatty acid
ester was extracted three times with 2.5 mL of n-hexane/MTBE (3:2,
v/v). After evaporation of the solvent by a gentle stream of nitrogen,
the residue was dried at 103 °C for 30 min. The obtained GC purities
for stigmasteryl palmitate and sitostanyl linoleate were 90 and 84 area
%, respectively.
Analytical methods for the investigation of free sterols and
stanols in cereals are also lacking. Pelillo et al. determined free
sterols and stanols in tetraploid and hexaploid wheat after TLC
and GC-FID.24 In whole corn kernels and corn oil, free sterols/
stanols were analyzed in total by NP-HPLC-ELSD or after
isolation by TLC or solid-phase extraction (SPE), subsequent
saponification, and final analysis via GC-FID.17,18,20,21,25
A
method for analysis of free sterols/stanols besides steryl/stanyl
fatty acid esters in several tetraploid and hexaploid wheats was
reported by Iafelice et al.26 The sterol classes were separated by
TLC and further saponified for analysis of the sterol moieties
by means of GC-FID. Neither qualitative nor quantitative data
on the distribution of free sterols and stanols in rye kernels are
available.
Investigations on intact steryl and stanyl phenolic acid esters
in cereals are commonly based on the isolation of this type of
esters by a base−acid cleanup and analysis by means of RP-
HPLC. Using this approach, intact phenolic acid esters were
determined in wheat and rye grains.27−29 Nevertheless,
drawbacks of this method are an insufficient resolution of
main compounds, for example, sitosteryl and campestanyl
ferulate, as well as missing information on naturally occurring
cis isomers of phenolic acid esters. Seitz30 and Norton31
analyzed several intact steryl ferulates and coumarates in corn
and corn oil via RP-HPLC, but with complex sample
preparation steps, for example, liquid−liquid extraction, CC,
TLC, and/or base−acid cleanup. Total contents of steryl
ferulates in corn grains were determined by means of a NP-
HPLC method, which was also used to analyze total amounts of
free sterols and steryl fatty acid esters, but without any
information on the distribution of individual compounds.17,18
Data on the contents of total or intact steryl and stanyl phenolic
acid esters in spelt grains are not available.
In conclusion, methods for the quantitative analysis of free
phytosterols/stanols as well as of intact phytosteryl/stanyl
esters in cereals are missing. Therefore, the aim of the present
study was the development of an SPE-based method for the
separation of free sterols/stanols and steryl/stanyl fatty acid
and phenolic acid esters isolated from cereals. GC-FID
equipped with a medium polar capillary column should be
used for the analysis of the SPE fractions as this chromato-
graphic method has been shown to be suitable for the
investigation of individual steryl/stanyl ferulic acid esters in
brown rice and of stanyl fatty acid esters in enriched foods.32,33
The developed methodology should be applied to demonstrate
the variability in composition and contents of individual steryl/
stanyl conjugates in different types of grain.
Synthesis of Steryl/Stanyl Phenolic Acid Esters. On the basis
of previously published methods,35−38 the synthesis of steryl/stanyl
phenolic acid esters was performed via (i) acetylation of the phenolic
acid, (ii) activation with thionyl chloride, (iii) esterification, (iv)
deprotection, and (v) purification. Briefly, 2.5 g of phenolic acid
dissolved in 4.5 mL of pyridine and 4.0 mL of acetic anhydride were
stirred at room temperature for 4 h. The mixture was transferred into
100 mL of ice−water; the resulting precipitate was isolated by filtration
via a Buchner funnel, washed with 20 mL of bidistillated water, and
̈
recrystallized from 30 mL of methanol. The acid chloride was prepared
by refluxing a mixture of the prepared O-acetylphenolic acid (1.2 g)
with thionyl chloride at 70−75 °C for 1.5 h. The reagents were
removed by rotary evaporation, and the residue was dried by a gentle
stream of nitrogen. For esterification, 300 mg of the O-acetyl acid
chloride and 100 mg of sterol/stanol were dissolved in 5 mL of dry
DMAP solution (2.5 mg/mL in DCM) and 50 μL of triethylamine.
The mixture was heated to 50 °C for 16 h. The solution was washed
three times with 3 mL of hydrochloric acid (0.1 M), twice with 3 mL
of a saturated ammonium chloride solution, and finally with 3 mL of
bidistillated water. The organic phase was dried with sodium sulfate.
After filtration, the solvent was removed by a gentle stream of
nitrogen. For deprotection, about 200 mg of the synthesized steryl/
stanyl O-acetylphenolic acid esters were dissolved in 20 mL of dry
THF, and 40 mL of piperazine solution (70 mg/mL in dry THF) was
added dropwise at room temperature under argon. The mixture was
stirred for 2 h at room temperature, diluted with 50 mL of ethyl
acetate, and washed 10 times with 50 mL of saturated ammonium
chloride solution. After drying with magnesium sulfate and filtration,
the solvent was removed by rotary evaporation. The purification was
achieved by an acid−base extraction and subsequent solid-phase
extraction. About 100 mg of the residue was dissolved in 20 mL of n-
hexane, and the solution was mixed with 20 mL of sodium hydroxide
(1 M). The aqueous solution was washed twice with 10 mL of n-
hexane, and the organic layer was discarded. After acidification with
hydrochloric acid, steryl/stanyl phenolic acid esters and remaining free
phenolic acids were extracted twice with 20 mL of MTBE. The
combined extracts were evaporated to dryness by rotary evaporation,
and the residue was used for SPE. The cartridge (Chromabond C18ec,
45 μm, 500 mg, Macherey-Nagel GmbH & Co. KG, Germany) was
conditioned with 4 mL of methanol. After transfer of the solid onto
the cartridge, the first fraction of 6 mL of methanol was discarded.
MATERIALS AND METHODS
■
Samples. Corn (Zea mays) was provided by Cornexo GmbH &
Co. KG (Freimersheim, Germany). Approximately six to eight cobs of
the genotype Starsky were harvested in 2010; the kernels
(approximately 1 kg) were manually removed and subsequently
dried in a cabinet dryer at 40 °C for 3 days. Wheat (Triticum aestivum
L.) and rye (Secale cereale) kernels produced by Alnatura GmbH,
Germany, and spelt kernels (Triticum spelta) produced by Neuform
International, Germany, were purchased in local stores. According to
the label, the wheat and rye material had been grown organically; no
other botanical or agronomic information was available.
Chemicals and Materials. Cholesteryl palmitate (≥98%), 5-α-
cholestan-3β-ol (∼95%), stigmasterol (∼95%), sitostanol (∼95%),
trans-p-coumaric acid (CA, >99%), trans-ferulic acid (99%), palmitic
acid (98%), linoleic acid (99%), pyridine (99.8%), dichloromethane
(DCM, 95%), methanol, N,O-bis(trimethylsilyl)trifluoroacetamide
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Elution of steryl/stanyl esters was carried out with 8 mL of MTBE.
The solvent was removed by a gentle stream of nitrogen. Fifty
micrograms of the synthesized ester was silylated with 100 μL of
BSTFA plus 1% TMCS (100 μL) and 20 μL of pyridine at 80 °C for
20 min. The obtained GC purities for cholestanyl-p-coumarate,
sitostanyl-p-coumarate, and sitostanyl ferulate were 95, 84, and 80 area
%, respectively.
Milling and Lipid Extraction. About 200 g of the self-dried corn
kernels and the commercially obtained wheat, rye, and spelt kernels,
respectively, were frozen in liquid nitrogen and subsequently ground
using a cyclone mill (1093 Cyclotec, Foss, Germany) equipped with a
500 μm sieve. The resulting flour was freeze-dried (Alpha 1-4 LSC,
Christ, Osterode, Germany) for 48 h and stored in plastic bags at −18
°C.
To avoid cis−trans isomerization of phenolic acid esters, all used
devices were wrapped with aluminum foil. Four grams of corn flour
and 8 g of rye, wheat, or spelt flour were weighed into an extraction
vessel. Five hundred microliters of cholesteryl palmitate (1.0 mg/mL
in n-hexane/MTBE (3:2, v/v)), 500 μL of 5-α-cholestan-3β-ol (1.0
mg/mL in MTBE), and 80 μL of cholestanyl-p-coumarate (1.0 mg/
mL in MTBE) were used as internal standards (IS). After the addition
of a magnetic stir bar, the oil was extracted with 40 mL of a mixture of
n-hexane/dichloromethane (1:1, v/v) under stirring for 1 h at room
temperature. After filtration, the extraction vessel and the filter were
washed, and the combined filtrates were evaporated to dryness by
rotary evaporation. The residue was dried at 103 2 °C to constant
weight. One hundred milligrams of the obtained oil was dissolved in
10 mL of n-hexane and was used for the SPE.
Solid-Phase Extraction. One milliliter of the oil solution was
loaded onto the SPE cartridge (Strata NH2, 55 μm, 70 Å, 1 g/6 mL,
Phenomenex, Germany), previously conditioned with 2 × 5 mL of n-
hexane. Steryl/stanyl fatty acid esters (fraction A) were eluted with 2 ×
5 mL of n-hexane/diethyl ether (98:2, v/v). Interfering triglycerides
were removed with 4 × 5 mL of n-hexane/ethyl acetate (96:4, v/v).
Free sterols and stanols (fraction B) were eluted with 2 × 5 mL of n-
hexane/ethyl acetate (5:95, v/v), and finally steryl/stanyl phenolic acid
esters (fraction C) were eluted with 2 × 5 mL n-hexane/ethyl acetate
(5:95, v/v) followed by 5 mL of MTBE. After evaporation of the
solvents by rotary evaporation, the residues of fraction A and B were
dissolved in 500 and 1000 μL of n-hexane, respectively. Fraction C was
silylated with 20 μL of pyridine and 100 μL of BSTFA:TCMS (99:1)
at 80 °C for 20 min. The silylation reagents were removed by a gentle
stream of nitrogen, and the residue was dissolved in 50 μL of n-hexane.
GC-FID Analysis. Analysis was performed using a gas chromato-
graph equipped with a flame ionization detector (Agilent Technologies
Identification by Gas Chromatography−Mass Spectrometry
(GC-MS). Identification of the steryl/stanyl esters and the free sterols/
stanols was performed on a Finnigan Trace GC ultra (Thermo Electro
Corp., Austin, TX, USA) coupled with a Finnigan Trace DSQ mass
spectrometer (Thermo Electro Corp.). Mass spectra were obtained by
electron impact ionization at 70 eV in the full scan mode at unit
resolution from 40 to 750 Da. Helium was used as carrier gas with
constant flow rate (1.0 mL/min). The interface was heated to 330 °C
and the ion source to 250 °C. Gas chromatographic separations were
performed on an Rtx-200MS (30 m × 0.25 mm i.d., 0.1 μm film)
fused-silica capillary column (Restek), and conditions were as
described for GC-FID analysis. Data acquisition was performed by
Xcalibur software.
Validation of the Method. Recoveries were determined by
spiking corn flour (4 g) with defined amounts of internal standards
and of two selected plant steryl/stanyl derivatives representative for
each SPE fraction: 0.5 mg of cholesteryl palmitate, stigmasteryl
palmitate, and sitostanyl linoleate, respectively, as reference com-
pounds for steryl/stanyl fatty acid esters; 0.5 mg of cholestanol,
stigmastanol, and sitostanol, respectively, as reference compounds for
free sterols/stanols; 80 μg of cholestanyl-p-coumarate, sitostanyl-p-
coumarate, and sitostanyl ferulate as reference compounds for steryl/
stanyl phenolic acid esters. The repeatability of the method was
confirmed by working up a control sample (corn flour) once on each
day of analysis (in total, five replicates). The limits of detection (LOD)
and the limits of quantification (LOQ) were determined according to
standard procedures and criteria.39
RESULTS AND DISCUSSION
■
SPE Separation and GC Analysis. On the basis of SPE, a
facile method for the separation of free sterols/stanols, steryl/
stanyl fatty acid, and phenolic acid esters from corn oil was
developed (Figure 1). Fatty acid esters (fraction A) were eluted
Instrument 6890N, Boblingen, Germany). Separations of 1 μL of the
̈
Figure 1. Elution sequence employed to separate sterols/stanols and
their esters isolated from cereals via SPE.
sample solutions were carried out on a 30 m × 0.25 mm i.d. fused-
silica capillary column coated with a film of 0.1 μm trifluoropro-
pylmethyl polysiloxane (Rtx-200MS, Restek, Bad Homburg, Ger-
many). The temperature of the injector was set to 280 °C, and
hydrogen was used as carrier gas with a constant flow rate of 1.5 mL/
min. Split flow was set to 11.2 mL/min, resulting in a split ratio of
1:7.5. Nitrogen was used as makeup gas with a flow of 25 mL/min.
The oven temperature program was as follows: initial temperature, 100
°C; programmed at 15 °C/min to 310 °C (2 min), then at 1.5 °C/min
to 315 °C, and at 15 °C/min to 340 °C (2 min). The detector
temperature was set to 360 °C. Data acquisition was performed by
ChemStation software.
Quantification by GC-FID. Steryl/stanyl fatty acid esters were
quantified by generating three-point calibration functions with 0.1, 0.3,
and 0.5 μg of total esters (Vegapure 95E) per microliter of injection.33
Each calibration point was done in triplicate. Linear regression was
confirmed in ratio of areas (area steryl or stanyl ester/area IS) and
amounts (amount steryl or stanyl ester/amount IS). Free sterols/
stanols were quantified using cholestanol as internal standard with a
response factor of 1.00. Steryl/stanyl phenolic acid esters were
determined as their trimethylsilyl derivatives with the following
response factors related to cholestanyl-p-coumarate (IS): stanyl-p-
coumarates, 1.05; steryl ferulates, 1.05; and stanyl ferulates, 1.15.
with a mixture of n-hexane/diethyl ether (98:2, v/v) and were
separated from triglycerides, which were subsequently washed
out. With aminopropyl-modified silica cartridges a better
removal of interfering triglycerides could be achieved compared
to normal silica. The elution of free sterols and stanols (fraction
B) was performed with a mixture of n-hexane/ethyl acetate
(5:95, v/v) followed by elution of the steryl/stanyl phenolic
acid esters (fraction C). The separation of the lipids from corn
into the three classes was very efficient (Figure 2). In fraction
C, containing the phenolic acid esters, several other
constituents were detected. Preliminary investigations by GC-
MS indicated the presence of free fatty acids and
monoglycerides. However, a full structural elucidation has not
been performed.
The GC separation was achieved using a medium polar
trifluoropropylmethyl polysiloxane capillary column, which has
been shown to be suitable for the separation of steryl/stanyl
ferulic acid and stanyl fatty acid esters.32,33 Under the employed
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Figure 2. Capillary gas chromatographic analysis of (A) steryl/stanyl fatty acid esters, (B) free sterols and stanols, and (C) steryl/stanyl phenolic acid
esters obtained from ground corn kernels. Peak numbering correlates to Tables 3−5. (IS) internal standard: (A) cholesteryl palmitate; (B)
cholestanol; (C) cholestanyl-p-coumarate.
1). The fragmentation patterns observed for the ferulates were
in agreement with previously reported GC-MS data for silylated
stanyl ferulates.40 The steryl/stanyl-p-coumarates were resolved
and identified in intact and silylated form by GC-MS for the
first time. Mass spectra obtained for synthesized sitosteryl and
sitostanyl-p-coumarate as TMS ethers are shown in Figure 3;
characteristic fragments are listed in Table 1. Molecular ions
[M]+ were observed only for stanyl esters, whereas the
fragment [M − 15]+ was obtained for all steryl/stanyl-p-
coumarates. The mass spectra of steryl esters exhibited [M −
CA]+ and those of stanyl esters the fragment m/z 236 as base
ions. The fragments m/z 236, 219, and 191 are characteristic
for TMS derivatives of coumaric acid.
The recoveries, LODs, and LOQs of the used internal
standards and of representatives of the three substance classes
are presented in Table 2. The recoveries of the internal
Figure 3. EI mass spectra of TMS ethers of (A) sitosteryl-p-coumarate
standards and of selected plant sterols/stanols and their esters
and (B) sitostanyl-p-coumarate.
after lipid extraction, SPE, and GC analysis were >90%. The
total contents of fractions A, B, and C from five replicate
analyses of the control sample were 643.8 32.5, 351.1 15.5,
conditions, free sterols and stanols were better resolved than
their trimethylsilyl (TMS) derivatives, whereas the response
and 80.3
3.4 μg/g, respectively; the relative standard
and the resolution of steryl/stanyl phenolic acid esters could be
improved by TMS derivatization. Under the applied conditions,
besides the steryl and stanyl esters of ferulic acid, also small
peaks of coumaric acid esters were detected and quantified
(Figure 2C). The stanyl ferulates or coumarates eluted just after
the corresponding steryl esters. Additionally, occurring cis
derivatives of steryl and stanyl ferulic acid esters were
completely separated under the applied conditions. Owing to
the high temperature stability and the low bleeding, the
employed stationary phase is particularly suitable for mass
spectrometric investigations.
deviations of <5% indicate good repeatability. The reproduci-
bility in terms of interlaboratory precision of the approach has
not been assessed.
Comparison of Different Cereals. The developed
methodology was applied to the analysis of steryl/stanyl fatty
acid esters (A), free sterols/stanols (B), and steryl/stanyl
phenolic acid esters (C) in corn, rye, wheat, and spelt kernels.
Lipids were extracted from the ground and freeze-dried cereal
grains with a mixture of n-hexane/dichloromethane (1:1, v/v),
resulting in oil contents of 3.57 0.07, 1.46 0.02, 1.69
0.03, and 2.47
0.03% for corn, rye, wheat, and spelt,
Identification of individual compounds was performed on the
basis of relative retention times and mass spectral data (Table
respectively. Rye, wheat, and spelt revealed very similar
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Table 1. Mass Spectrometric and Chromatographic Data of Sterols/Stanols and Steryl/Stanyl Esters
a
RRT
molecular ion [M]+
characteristic fragments
steryl/stanyl fatty acid esters
campesteryl-16
stigmasteryl-16
campestanyl-16
Δ7-campesteryl-16
sitosteryl-16
1.040
1.044
1.049
1.060
1.076
1.083
1.096
1.118
1.124
1.125
1.129
1.135
1.135
1.150
1.159
1.167
1.173
1.176
1.176
1.195
638 (−)
650 (−)
640 (7)
638 (100)
652 (−)
654 (5)
652 (88)
664 (−)
676 (−)
662 (1)
666 (1)
664 (4)
674 (−)
662 (25)
678 (−)
676 (3)
380 (2)
678 (5)
674 (4)
676 (22)
383 (34), 382 (100), 367 (12), 255 (4), 213 (3), 81 (5)
395 (27), 394 (100), 379 (13), 255 (13), 213 (3), 81 (7)
385 (37), 384 (100), 369 (31), 257 (11), 215 (57), 81 (10)
383 (−), 382 (19), 367 (3), 255 (23), 213 (1), 81 (8)
397 (35), 396 (100), 381 (17), 255 (13), 213 (8), 81 (19)
399 (33), 398 (90), 383 (40), 257 (23), 215 (100), 81 (36)
397 (4), 396 (20), 381 (15), 255 (100), 213 (19), 81 (1)
383 (39), 382 (100), 367 (17), 255 (−), 213 (10), 81 (5)
395 (34), 394 (100), 379 (7), 255 (28), 213 (7), 81 (18)
383 (59), 382 (100), 367 (18), 255 (13), 213 (9), 81 (31)
385 (100), 384 (47), 369 (17), 257 (21), 215 (25), 81 (28)
385 (100), 384 (48), 369 (15), 257 (−), 215 (13), 81 (45)
395 (41), 394 (100), 379 (9), 255 (38), 213 (9), 81 (32)
383 (100), 382 (28), 367 (5), 255 (14), 213 (1), 81 (8)
397 (38), 396 (100), 381 (14), 255 (12), 213 (10), 81 (22)
397 (53), 396 (100), 381 (20), 255 (11), 213 (11), 81 (45)
399 (100), 398 (51), 383 (17), 257 (19), 215 (26), 81 (29)
399 (100), 398 (26), 383 (11), 257 (19), 215 (22), 81 (45)
397 (70), 396 (100), 381 (12), 255 (16), 213 (11), 81 (25)
397 (100), 396 (51), 381 (1), 255 (20), 213 (50), 81 (77)
sitostanyl-16
Δ7-sitosteryl-16
campesteryl-18:1
stigmasteryl-18:1
campesteryl-18:2
campestanyl-18:1
campestanyl-18:2
stigmasteryl-18:2
Δ7-campesteryl-18:2
sitosteryl-18:1
sitosteryl-18:2
sitostanyl-18:1
sitostanyl-18:2
sitosteryl-18:3
Δ7-sitosteryl-18:2
free sterols/stanols
cholesterol
1.011
1.041
1.047
1.050
1.072
1.081
386 (100)
400 (100)
412 (100)
402 (100)
414 (100)
416 (100)
371 (39), 368 (61), 353 (51), 255 (31), 213 (43), 129 (10)
385 (52), 382 (73), 367 (50), 255 (7), 213 (11), 129 (2)
397 (9), 394 (22), 379 (19), 255 (87), 213 (37), 129 (20)
387 (68), 384 (11), 369 (53), 257 (10), 215 (31), 129 (23)
399 (40), 396 (67), 381 (46), 255 (46), 213 (50), 129 (11)
401 (36), 398 (17), 383 (15), 257 (16), 215 (81), 129 (3)
campesterol
stigmasterol
campestanol
sitosterol
sitostanol
steryl/stanyl phenolic acid esters
cis-campesteryl ferulate
0.944
0.956
0.807
0.986
1.019
1.029
1.047
1.056
1.085
1.127
1.144
1.154
1.170
1.188
1.198
1.227
648 (2)
633 (2), 382 (10), 367 (5), 266 (100), 249 (5), 221 (1), 194 (−)
635 (1), 384 (2), 369 (2), 266 (9), 249 (12), 221 (3), 194 (1)
647 (1), 396 (10), 381 (5), 266 (100), 249 (−), 221 (1), 194 (1)
649 (3), 398 (3), 383 (1), 266 (3), 249 (16), 221 (1), 194 (1)
673 (5), 422 (37), 407 (39), 266 (4), 249 (100), 221 (1), 194 (−)
603 (1), 382 (100), 367 (21), 236 (28), 219 (29), 191 (5), 164 (2)
605 (1), 384 (13), 369 (4), 236 (100), 219 (35), 191 (21), 164 (2)
617 (1), 396 (100), 381 (15), 236 (34), 219 (20), 191 (5), 164 (1)
619 (1), 398 (4), 383 (3), 236 (100), 219 (22), 191 (3), 164 (1)
633 (3), 382 (14), 367 (4), 266 (100), 249 (4), 221 (1), 194 (−)
635 (6), 384 (2), 369 (1), 266 (14), 249 (14), 221 (2), 194 (1)
633 (7), 382 (7), 367 (4), 266 (5), 249 (17), 221 (1), 194 (−)
647 (2), 396 (13), 381 (4), 266 (100), 249 (4), 221 (1), 194 (−)
649 (6), 398 (2), 383 (1), 266 (13), 249 (13), 221 (2), 194 (−)
647 (3), 396 (8), 381 (4), 266 (6), 249 (8), 221 (1), 194 (−)
673 (6), 422 (15), 407 (16), 266 (1), 249 (100), 221 (−), 194 (−)
cis-campestanyl ferulate
650 (100)
662 (3)
cis-sitosteryl ferulate
cis-sitostanyl ferulate
664 (100)
688 (10)
618 (−)
cis-24-methylenecycloartanyl ferulate
trans-campesteryl-p-coumarate
trans-campestanyl-p-coumarate
trans-sitosteryl-p-coumarate
trans-sitostanyl-p-coumarate
trans-campesteryl ferulate
trans-campestanyl ferulate
trans-Δ7-campesteryl ferulate
trans-sitosteryl ferulate
620 (42)
632 (−)
634 (17)
648 (−)
650 (100)
648 (100)
662 (2)
trans-sitostanyl ferulate
664 (100)
662 (100)
688 (10)
trans-Δ7-sitosteryl ferulate
trans-24-methylenecycloartanyl ferulate
a
Retention times relative to cholesteryl palmitate for steryl/stanyl fatty acid esters, to cholestanol for free sterols/stanols, and to cholestanyl-p-
coumarate for steryl/stanyl phenolic acid esters (Rtx-200MS).
patterns; the capillary gas chromatograms of fractions A, B, and
C are exemplarily shown for rye in Figure 4.
mainly campesteryl and sitosteryl linoleate, made up 95.6% of
total esters. In contrast, in rye, wheat, and spelt approximately
50% of the sterols/stanols in fraction A were esterified to
palmitic acid. It is interesting to note that the level of fatty acid
esters based on micrograms per 100 mg of extracted oil in rye
was 1.7-fold above the level in corn oil (Table 3); however,
taking into account the low fat content of rye, the amounts
based on dry matter of ground kernels were highest in corn.
The determined total contents of steryl/stanyl fatty acid esters
in wheat and spelt were in line with reported results.23 To the
Plant Steryl/Stanyl Fatty Acid Esters (A). The majority
(51.5−56.5%) of the sterols and stanols in the investigated
cereal samples occurred as fatty acid esters. Amounts of
individual esters are shown in Table 3. The total contents of
fatty acid esters (A) ranged from 370 μg/g in wheat to 651 μg/
g in corn. The steryl and stanyl ester profile of corn was clearly
distinguishable from those obtained from rye, wheat, and spelt.
In corn, the steryl/stanyl esters of unsaturated fatty acids,
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amounts of campestanol were higher. Cholesterol was present
in all cereals and made up 0.7−1.2% of free sterols/stanols.
Comparable to the fatty acid esters, rye contained the highest
amounts of free sterols/stanols in the lipid extract and corn the
highest contents based on ground kernels. The levels of free
sterols/stanols in corn, wheat, and spelt were comparable to
those previously reported.17,24
Table 2. Recoveries, Limits of Detection (LOD), and Limits
of Quantification (LOQ) for Steryl/Stanyl Fatty Acid Esters
a
(A), Free Sterols/Stanols (B), and Stanyl Phenolic Acid
Esters (C)
b
c
c
recovery
(%)
LOD
LOQ
(μg/mL)
(μg/mL)
internal standards
Plant Steryl/Stanyl Phenolic Acid Esters. Up to 16 intact
phenolic acid esters were identified in the investigated cereal
samples. The contents of individual steryl and stanyl phenolic
acid esters are presented in Table 5. The highest total amounts
were determined in corn grains and were mainly composed of
steryl and stanyl ferulic acid esters, representing 97.6% of total
phenolic acid esters. Additionally, small amounts of coumarates
occurred in all samples. Steryl and stanyl coumarates have been
described in corn and corn bran as minor components, but to
the authors' knowledge not in other cereals.30,31,41 In this study,
campestanyl and sitostanyl-p-coumarate could also be identified
in rye, wheat, and spelt grains. The contents of coumarates
were very low; however, this is the first report on the
occurrence of these particular esters in cereals other than corn.
In accordance with data from previous studies, the contents
of sitosteryl/sitostanyl ferulate in corn were higher compared to
those of campesteryl/campestanyl ferulate; in the samples of
rye, wheat, and spelt the esters of campesterol and campestanol
are dominating.27,29,30 Wheat contained the highest total
amounts of phenolic acid esters in the oil extract and corn
the highest level based on ground kernels. The phenolic acids
occurred predominantly as esters of stanols (74.6−90.8%),
whereas in fraction A 79.0−85.5% of the fatty acids were
esterified to sterols. The employed GC analysis also allowed the
determination of cis derivatives of steryl and stanyl ferulates.
The cis/trans ratios were 0.64, 0.17, 0.05, and 0.13 for corn, rye,
(A) cholesteryl palmitate
(B) cholestanol
97.9 1.9
98.0 2.0
93.6 5.3
0.04
0.03
1.48
0.09
0.07
3.36
(C) cholestanyl-p-coumarate
plant sterols/stanols and esters
(A) stigmasteryl palmitate
(A) sitostanyl linoleate
(B) stigmasterol
96.7 1.9
102.9 1.4
99.1 0.6
95.2 0.3
96.9 4.5
92.8 3.7
0.13
0.16
0.03
0.03
1.10
3.60
0.34
0.52
0.07
0.07
3.30
7.01
(B) sitostanol
(C) sitostanyl-p-coumarate
(C) sitostanyl ferulate
a
b
Designations (A−C) according to the SPE fractions. Average of
three values standard deviation. Determined according to ref 39
and expressed on μg/mL of injection volume of fraction A, B, or C.
c
authors' knowledge, there are presently no data available for
fatty acid esters in rye. In corn kernels the fatty acid esters have
been analyzed by means of NP-HPLC.17 Thereby, the ranges of
total contents reported for ground kernels (0.76−3.09 wt % of
oil; n = 49) were in good agreement with the data determined
for the corn sample in this study (1.82 0.04 wt % of oil).
Free Plant Sterols/Stanols (B). In all cereal grains, sitosterol
was the dominating free sterol (55.4−61.5%, Table 4).
Sitostanol and campestanol represented 9.5% of total free
sterols/stanols in corn, 10.6% in rye, 16.3% in wheat, and 15.5%
in spelt. The amounts of stigmasterol in corn and rye were
higher compared to campestanol; in wheat and spelt the
Figure 4. Capillary gas chromatographic analysis of (A) steryl/stanyl fatty acid esters, (B) free sterols and stanols, and (C) steryl/stanyl phenolic acid
esters obtained from ground rye kernels. Peak numbers correlate to Tables 3−5. (IS) internal standard: (A) cholesteryl palmitate; (B) cholestanol;
(C) cholestanyl-p-coumarate.
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Table 4. Contents of Free Sterols and Stanols (B) Determined in the Investigated Cereals
corn
rye
wheat
spelt
a
free sterols/stanols
μg/g of dm
μg/100 mg of oil μg/g of dm μg/100 mg of oil μg/g of dm μg/100 mg of oil μg/g of dm μg/100 mg of oil
b
c
1
2
3
4
5
6
7
cholesterol
campesterol
stigmasterol
campestanol
sitosterol
2.7 0.7
69.8 2.9
25.2 1.1
11.5 0.5
238.7 9.5
25.5 0.6
15.0 1.7
7.5 1.9
195.3 4.3
70.4 1.8
32.3 0.7
668.1 13.8
71.3 1.1
41.8 4.1
1.8 0.2
54.1 0.4
14.4 0.3
8.5 0.2
12.3 1.8
370.5 7.3
98.3 2.3
2.7 0.4
38.1 0.7
6.8 0.1
16.1 2.2
225.2 3.9
40.5 0.1
80.0 3.6
808.7 16.4
136.8 3.5
21.6 1.0
2.3 0.2
37.0 0.2
6.9 0.0
9.1 0.9
149.4 2.3
28.0 0.5
46.9 1.1
532.4 12.0
85.4 4.1
15.1 2.2
57.8 0.5
13.5 0.4
136.8 3.7
23.1 0.8
3.7 0.2
11.6 0.3
131.7 1.5
21.1 0.8
3.7 0.5
145.1 1.8
19.4 0.9
18.4 0.3
992.7 12.9
132.7 6.0
126.0 2.9
sitostanol
d
others
total free sterols/
stanols
388.3 15.8
1086.7 23.9
261.6 3.7
1790.4 12.9
224.8 4.7
1328.8 18.7
214.3 2.4
866.3 20.9
a
b
c
Based on dry matter (dm) of ground kernels. Peak numbering according to Figures 2B and 4B. All contents are the average of three values
d
standard deviation. Tentatively identified as Δ7-sitosterol and unidentified sterol.
Table 5. Contents of Steryl/Stanyl Phenolic Acid Esters (C) Determined in the Investigated Cereals
corn
rye
wheat
spelt
μg/g
of dm
μg/100 mg
of oil
μg/g
of dm
μg/100 mg
of oil
μg/g
of dm
μg/100 mg
of oil
μg/g
of dm
μg/100 mg
of oil
a
steryl/stanyl phenolic acid esters
b
c
d
6
7
8
9
1
trans-campesteryl-p-
<LOQ
−
−
−
−
−
−
coumarate
e
trans-campestanyl-p-
coumarate
1.0 0.1
2.7 0.2
2.9 0.3
0.5 0.0
−
3.5 0.2
−
1.7 0.2
−
10.3 1.3
−
<LOQ
<LOQ
trans-sitosteryl-p-
coumarate
<LOQ
<LOQ
−
−
trans-sitostanyl-p-
coumarate
7.7 0.8
0.3 0.0
1.8 0.2
0.3 0.0
2.0 0.2
cis-campesteryl ferulate
1.9 0.1
6.5 0.6
5.7 0.2
34.5 1.8
4.3 0.3
13.2 0.9
44.6 3.5
39.0 1.7
0.9 0.0
11.6 0.2
3.0 0.3
53.0 2.0
2.1 0.2
5.2 0.3
68.6 2.5
18.0 1.7
313.5 14.5
12.3 1.2
1.5 0.1
6.8 0.8
4.7 0.2
35.7 1.3
1.5 0.1
6.0 0.3
10 trans-campesteryl ferulate
cis-campestanyl ferulate
2.8 0.1
23.4 3.1
21.7 0.6
1.5 0.4
7.7 0.8
65.5 8.6
60.8 1.4
4.2 1.2
27.6 2.9
19.0 1.2
144.4 3.4
6.1 0.5
2
11 trans-campestanyl ferulate
12 trans-Δ7-campesteryl
236.2 9.4
29.3 1.6
ferulate
3
cis-sitosteryl ferulate
13 trans-sitosteryl ferulate
cis-sitostanyl ferulate
2.7 0.3
3.1 0.5
35.4 5.8
58.5 1.0
4.4 1.1
−
7.5 0.9
8.5 1.5
0.9 0.0
4.7 0.5
4.0 0.2
23.6 1.5
2.1 0.1
0.7 0.1
6.0 0.3
32.4 2.8
27.4 1.3
161.4 9.3
14.4 0.6
4.7 0.6
<LOQ
<LOQ
6.4 0.1
2.2 0.1
39.9 1.4
1.5 0.1
37.6 1.0
13.2 0.7
236.0 10.7
8.9 0.5
4.4 0.6
4.6 0.2
31.8 3.2
1.3 0.0
−
18.0 2.2
18.7 0.6
128.3 12.2
5.1 0.1
−
4
99.1 15.5
14 trans-sitostanyl ferulate
15 trans-Δ7-sitosteryl ferulate
5
164.5 2.2
12.3 3.2
−
cis-24-methylene-
cycloartanyl ferulate
<LOQ
16 trans-24-methylene-
<LOQ
2.2 0.2
15.4 1.8
1.5 0.1
8.6 0.8
<LOQ
cycloartanyl ferulate
total phenolic acid esters
157.5 13.3
441.0 35.8
92.0 5.1
629.2 29.0
124.2 4.1
734.3 31.0
92.4 5.9
373.2 21.5
a
b
c
d
Based on dry matter (dm) of ground kernels. Peak numbering according to Figures 2C and 4C. Content below LOQ (Table 2). (−) content
e
below LOD (Table 2). All contents are the average of three values standard deviation.
wheat, and spelt, respectively. Comparably high cis/trans ratios
in cereals have been reported earlier.42 It has been shown that
the trans forms of phenolic acids can be partially converted into
their cis forms by ultraviolet light and daylight.43,44 Therefore,
the sample preparation was performed protected from daylight,
and during the analysis no isomerization of the internal
standard cholestanyl-p-coumarate was observed. Steryl and
stanyl ferulates were determined, among others, by Nurmi et al.
via RP-HPLC.29 Thereby, the total amounts of steryl/stanyl
ferulates were 65−74 μg/g in rye and 74−114 μg/g in wheat
and were a little lower compared to the investigated samples.
The determined amounts of phenolic acid esters in corn were
in agreement with previous data.17
stanyl fatty acid esters, and steryl/stanyl phenolic acid esters
from corn, rye, wheat, and spelt grain oils. The analysis of intact
steryl/stanyl esters by means of capillary gas chromatography
provides detailed information on the profiles and contents of
individual esters. The methodology revealed quantitative
differences between the patterns of fatty acid and phenolic
acid esters in corn compared to rye, wheat, and spelt.
The approach should be a suitable tool for future
investigations of the natural variability of individual phytoster-
ol/phytostanol conjugates in various types of grain and could,
for example, be employed to investigate the phenotypic
plasticity of sterol patterns as a response to environmental
factors.
In conclusion, the described solid-phase extraction method
enables the effective isolation of free sterols/stanols, steryl/
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49 8161 714250. Fax: +49 8161 714259. E-mail:
k.h.engel@wzw.tum.de.
Notes
The authors declare no competing financial interest.
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