Direct analysis of sterols by derivatization matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and tandem mass spectrometry

Article abstract of DOI:10.1002/rcm.6766

RATIONALE Free sterols are neutral molecules that are difficult to analyze by MALDI or ESI and their molecular ions easily fragment. In order to increase their ionization efficiency and selectivity, sterols were derivatized by different reagents. METHODS Selected sterols were converted into their corresponding picolinyl esters, N-methylpyridyl ethers and sulphated esters. The derivatives were optimized for MALDI-TOFMS analysis through proper selection of the matrix. MALDI-TOF/TOF experiments were carried out to study the fragmentation pathways of the derivatives and their use in structural elucidation. Lipid extracts from mussels were used as test samples for MALDI analysis of sterols in biological samples also analyzed by GC/MS for comparison. RESULTS Sterol picolinyl esters were identified as sodiated adducts [M+Na] ⁺ and the signal significantly enhanced after addition of sodium acetate (20 mM). Sterol N-methylpyridyl ethers were easily detected as [M] ⁺ while sulphated sterols were best detected as [M-H]^-. The ester bonds of picolinyl and sulphated esters easily cleaved in MS/MS resulting in diagnostic derivative fragments at m/z 146.03 and 96.89, respectively. Cleavage of the ether bond of N-methylpyridyl ethers gave a diagnostic fragment ion at m/z 110.04. Sterol profiles in mussels obtained by MALDI-TOFMS were in close agreement with those obtained by GC/MS. Two sterols (cholesterol and β-sitosterol) were selected for quantification as their sulphated and picolinyl esters. Calibration curves gave excellent correlation coefficients. CONCLUSIONS Suitable matrices for picolinyl esters are DHB and THAP, for N-methylpyridyl ethers THAP, and for sulphated esters p-nitroaniline and dithranol. Using cholesterol, the limits of detection (LODs) for sulphated esters were 0.2 μg/mL and for picolinyl esters, 1.5 μg/mL. N-Methylpyridyl ethers were found unsuitable for sterol quantitation. Copyright 2013 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/rcm.6766

Research Article
Received: 21 May 2013
Revised: 8 October 2013
Accepted: 14 October 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 149–158
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6766
Direct analysis of sterols by derivatization matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry and
tandem mass spectrometry
*
Iyad Hailat and Robert J. Helleur
Department of Chemistry, Memorial University of Newfoundland, St. John’s, NL, Canada, A1B 3X7
RATIONALE: Free sterols are neutral molecules that are difficult to analyze by MALDI or ESI and their molecular ions easily
fragment. In order to increase their ionization efficiency and selectivity, sterols were derivatized by different reagents.
METHODS: Selected sterols were converted into their corresponding picolinyl esters, N-methylpyridyl ethers and
sulphated esters. The derivatives were optimized for MALDI-TOFMS analysis through proper selection of the matrix.
MALDI-TOF/TOF experiments were carried out to study the fragmentation pathways of the derivatives and their use
in structural elucidation. Lipid extracts from mussels were used as test samples for MALDI analysis of sterols in
biological samples also analyzed by GC/MS for comparison.
RESULTS: Sterol picolinyl esters were identified as sodiated adducts [M+Na]+ and the signal significantly enhanced after
addition of sodium acetate (20 mM). Sterol N-methylpyridyl ethers were easily detected as [M]+ while sulphated sterols
were best detected as [M–H]–. The ester bonds of picolinyl and sulphated esters easily cleaved in MS/MS resulting in
diagnostic derivative fragments at m/z 146.03 and 96.89, respectively. Cleavage of the ether bond of N-methylpyridyl
ethers gave a diagnostic fragment ion at m/z 110.04. Sterol profiles in mussels obtained by MALDI-TOFMS were in close
agreement with those obtained by GC/MS. Two sterols (cholesterol and β-sitosterol) were selected for quantification as
their sulphated and picolinyl esters. Calibration curves gave excellent correlation coefficients.
CONCLUSIONS: Suitable matrices for picolinyl esters are DHB and THAP, for N-methylpyridyl ethers THAP, and for
sulphated esters p-nitroaniline and dithranol. Using cholesterol, the limits of detection (LODs) for sulphated esters were
0.2 μg/mL and for picolinyl esters, 1.5 μg/mL. N-Methylpyridyl ethers were found unsuitable for sterol quantitation.
Copyright © 2013 John Wiley & Sons, Ltd.
Sterols are natural substances present in plants, animals and
fungi. β-Sitosterol, stigmasterol and 24-methylenecholesterol
occur abundantly in plants,[1] in animals it is cholesterol, and
in fungi ergosterol.[2] Plant-derived sterols are often called
phytosterols,[3,4] and are important constituents of plant
membranes where they assist in maintaining the phospholipid
bilayers in a stable form.[5] This function is similar to that of
cholesterol in animal cell membranes.[3,5] The chemical
structures of phytosterols are similar to that of cholesterol, but
differ in that phytosterols contain an additional methyl or ethyl
group at C-24 of the side chain.
Phytosterols have many positive impacts on human health.
It has been found that their consumption can reduce levels of
cholesterol in blood[6,7] via inhibition of the absorption of
cholesterol in the small intestine,[8] and protect against
cardiovascular and heart disease.[9] Therefore, phytosterols
have recently gained an important role in the nutraceutical
and pharmaceutical industries where they are extracted from
plants and added to prepared food as a health ingredient.[9]
Mussels are considered a good source of dietary
phytosterols. The diversity in content and composition of
phytosterols depends mainly on their diets which usually
consist of microscopic marine plants having an array of
phytosterols.[10] In addition, the season and the life cycle of
mussels affect the levels and composition of phytosterols.
Although the percentage of lipids in mussels is low (2% wet
weight), they contain a considerable proportion of beneficial
phytosterols.[10]
Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOFMS) has been used for
analysis of proteins,[11,12] oligosaccharides,[13] synthetic
polymers,[14,15] lipids,[16–18] and unstable or non-volatile
compounds.[19] One of the most important characteristic of
this technique is the fact that sample preparation can be done
easily and quickly. In addition, MALDI-TOF spectra are not
as adversely affected by the presence of small amounts of
buffer nor contaminants from sample workup.[20]
A variety of different techniques have been used for
analysis of free sterols including gas chromatography mass
spectrometry (GC/MS),[21,22] GC-flame ionization detection
(FID),[23] liquid chromatography/ultraviolet (LC-UV),[24]
LC/atmospheric pressure chemical ionization (APCI)-
MS,[24,25] LC/APCI-MS/MS,[26] LC/atmospheric pressure
photoionization (APPI)-MS/MS,[27] and high-performance
* Correspondence to: R. J. Helleur, Department of Chemistry,
Memorial University of Newfoundland, St. John’s, NL,
Canada, A1B 3X7.
E-mail: rhelleur@mun.ca
Rapid Commun. Mass Spectrom. 2014, 28, 149–158
Copyright © 2013 John Wiley & Sons, Ltd.

I. Hailat and R. J. Helleur
liquid chromatography/electrospray ionization (HPLC/ESI)-
MS.[28] These techniques usually require extensive sample
handling and chromatography.
the mussel was placed in a test tube and 12 mL of a mixture of
chloroform/methanol 2:1 (v/v) was added. The content of
the tube was homogenized using a polytron PCU-2-110
homogenizer. Chloroform-extracted water was added to
obtain a final ratio of 8:4:3 of chloroform/methanol/water
(v/v/v) followed by sonication for 10 min. Samples were
then centrifuged at a speed of 5000 g for 5 min. The lower
organic layer which contained the sterol extracts was then
drawn into a vial and then 12 mL of chloroform were added
and the extraction procedure was repeated three times.
Samples were kept at –20°C until analysis.
Another approach to sterol analysis which is suitable for
rapid analyte profiling is MALDI-TOFMS. Recently, it has been
used for the analysis of lipids.[29] Lipids and most matrices used
in MALDI easily dissolve in common solvents resulting in a
homogeneous mixture with a high degree of crystallization.
This leads to improved reproducibility of the method and
better signal-to-noise (S/N) ratio for the analysis of conjugated
sterols.[20] However, free sterols are neutral molecules that are
difficult to ionize by ESI and MALDI. In order to increase their
ionization efficiency and selectivity, sterols can be converted
into more polar derivatives.[30] Studies include the analysis of
sterols in human serum by LC/ESI-MS/MS as their picolinyl
esters,[30] determination of cholesterol by nano-ESI-MS/MS
after derivatization to cholesterol sulfate,[31] and determination
of cholesterol by ESI-MS as its N-methylpyridyl ether.[32] These
techniques require sample cleanup. However, analysis of these
sterol derivatives using MALDI-TOFMS should not require this
since the method is more tolerant to sample complexity and
contaminants. For example, Wang et al.[33] described a
MALDI-TOF/TOF method for direct analysis of oxosteroids
after their derivatization using Girard P (GP) hydrazine to give
GP hydrazones without the need for sample cleanup.
Lipid samples are complex mixtures ranging from nonpolar
compounds like sterols and TAGs to very polar compounds like
phospholipid. Analysis of sterols by GC/MS and LC/MS
requires preparative chromatography (i.e., solid-phase extraction
(SPE) and thin-layer chromatography (TLC)) that takes a long
time. However, the present study will show that the analysis
of sterols by derivatization MALDI-TOFMS will not require
sample cleanup, one reason being that derivatization targets
only the sterol alcohols thus lessening interference from other
lipid classes. This study describes for the first time the
analysis of sterols by MALDI-TOF and MALDI-TOF/TOF
and their quantitation after derivatization to picolinyl esters,
N-methylpyridyl ethers and sulphated esters. The influence
of the derivative on sensitivity, the effect of additives and the
selection of proper matrix will be discussed. The derivatization
MALDI-TOF approach was further investigated for analysis of
sterols extracted from mussels.
Analysis of free sterols using GC/MS
Free sterols were isolated from total lipid extracts using column
chromatography. The bottom of a Pasteur pipette was plugged
with glass wool. It was then placed in an oven at 250°C
overnight, cooled and filled with activated silica gel which
had been heated at 150°C for 1 h. The column was washed with
2 mL of diethyl ether then with 4 mL of hexane. Lipid extracts
were loaded to the top of the column. The free sterol fraction
was eluted with 15 mL of a mixture of 1:1 diethyl ether/hexane.
The samples were evaporated until dryness under nitrogen
flow. The trimethylsilyl ether derivatives (TMS) of sterols were
obtained by adding 100 μL of N,O-bis(trimethylsilyl)
trifluoroacetamide (BSTFA) containing 1% of trimethylchloro-
silane (TMCS) and heating at 85°C for 15 min. Samples were
then dried under nitrogen flow and 500 μL of hexane/
dichloromethane 1:1 (v/v) were added to the dried sample
followed by analysis using GC/MS.
Sterols were analyzed using an Agilent 6890 gas
chromatograph with an Agilent 5973 mass-selective detector.
The column was a DB-5 (5%-phenylmethyl polysiloxane; 30
m long, 0.25 μm film thickness and 0.25 mm i.d.; J& W
Scientific USA). The carrier gas was helium with a column
pressure of 14 psi. The injected volume was 1 μL in
splitless mode with an inlet temperature of 290°C. The
temperature program was 80°C (1 min) ramping to 200°C at
50°C/min, then to 280°C at 4°C/min with a 5 min hold.
The detector transfer line was set at 280°C. The mass
detector was operated in EI mode (70 eV) with mass range
of 50–550 m/z and source temperature of 160°C. Sterols
were identified using the NIST MS library and by
comparing retention times with those of standards.
Calibration curves of sterol standards were constructed
for sterol quantification.
EXPERIMENTAL
Chemicals and reagents
2,5-Dihydroxybenzoic acid (DHB), 2,4,6-trihydrooxyaceto-
phenone monohydrate (THAP), α-cyano-4-hydroxycinnamic
acid (CHCA), dithranol, p-nitroaniline, picolinic acid,
2-methyl-6-nitrobenzoic anhydride, 4-dimethylaminopyridine,
triethylamine, pyridine, sodium acetate, 2-fluoro-1-methylpyri-
dinium p-toulenesulfonate, sulfur trioxide pyridine and barium
acetate were obtained from Sigma Chemical Co. (St. Louis, MO,
USA). Cholesterol, β-sitosterol, ergosterol and cholestanol were
obtained from Steraloids, Inc. (New Port, RI, USA). All other
chemicals and solvents were of analytical grade.
Picolinyl esters
Picolinyl ester derivatization was carried out using
a
procedure reported by Yamashita et al.[35] with minor
modifications. A 200 μL aliquot of a reagent mixture
consisting of picolinic acid (80 mg), 4-dimethylaminopyridine
(30 mg), 2-methyl-6-nitrobenzoic anhydride (100 mg),
pyridine (1.5 mL), and triethylamine (250 μL) was added
immediately to 2.0 mg of sterol standard or dried mussel
extract. The reaction mixture was heated at 60°C for 20 min
followed by addition of 750 μL of hexane and vortexed for
1 min. The reaction mixture was then centrifuged for 5 min.
The supernatant was transferred to a vial and evaporated to
dryness under a nitrogen flow. The residue was re-dissolved
in 2 mL of dichloromethane and analyzed immediately.
Sterol extraction from mussels
Free sterols and other lipid components were extracted from the
meat of whole blue mussel (average 3.7 g; Newfoundland-
cultured) using a modified Folch procedure.[34,41] The meat of
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Analysis of sterols by MALDI-TOFMS and MALDI-TOF/TOFMS
N-Methylpyridyl ethers
10 mg/mL followed by addition of trifluoroacetic acid to a final
concentration of 0.1%. Each sterol standard was mixed with the
matrix at a ratio of 1:1.
N-Methylpyridyl ether derivatives were prepared according
to Mukiyama et al.[36] with minor modifications: 9.4 mg of
2-fluoro-1 methylpyridinium p-toluenesulphonate was added
to a solution of 13.0 mg of sterol standard in 1 mL of
dichloromethane and 10 μL of triethylamine were then added
to the reaction. The solution was allowed to stir at room
temperature for 1 h. The derivative was diluted with
dichloromethane to 100 μg/mL for analysis.
Picolinyl esters of sterols
Picolinyl esters dissolved in dichloromethane were mixed with
THAP (10 mg/mL in acetonitrile) at a ratio of 1:1 followed by
addition of sodium acetate to a final concentration of 20 mM.
N-Methylpyridyl ethers of sterols
Sulphated esters
N-methylpyridyl ethers dissolved in dichloromethane were
mixed with THAP (10 mg/mL in dichloromethane/acetone
(1:1)) at a ratio of 1:1.
The sulphated ester derivatization was performed using a
modified Duff procedure:[37] 12.5 mg of sulphur trioxide
pyridine complex were dissolved in 2.5 mL of pyridine and
the solution sonicated for 30 s and 200 μL of sulphur trioxide
pyridine solution were immediately added to 2.0 mg of sterol
standard or to dried mussel extract. The vials were sonicated
for 30 s then left to sit at room temperature for 10 min,
followed by addition of 20 μL of barium acetate solution
(314.0 mM). The sample was vortexed for 30 s and sonicated
for 1 min, then left for 10 min at room temperature followed
by 1 h at 4°C in order to allow hydrolysis and precipitation
of barium sulphate. Dichloromethane (500 μL) was added
and the mixture was centrifuged for 10 min. The supernatant
was stored in the freezer until analyzed. Figure 1 shows the
chemical structures of each type of derivative based on
cholesterol.
Sterol sulphates
Sterol sulphates in dichloromethane were mixed with
dithranol (2.5 mg/mL in dichloromethane) at a ratio of 1:1.
Quantification of derivatized sterols
Cholesterol and β-sitosterol were measured in mussel extracts
by MALDI-TOFMS as their picolinyl and sterol sulphate
esters. Calibration curves for cholesterol and β-sitosterol
picolinyl esters, and cholesterol and β-sitosterol sulphates,
were constructed using cholestanol picolinyl and cholestanol
sulphate ester as internal standards, respectively. A 100 μg
sample of cholestanol picolinyl and cholestanol sulphate ester
was added to 500 μL of picolinyl and sulphated ester
standards prepared at different concentrations (1000, 500,
250, 125, 62.5 μg/mL), respectively. The same amount of
internal standards was added to dried mussel samples after
sample derivatization.
Sample preparation
Free sterols
Cholesterol, cholestanol and β-sitosterol were separately
prepared in a mixture of chloroform/methanol (1:1). The matrix
(DHB) was prepared in the same solvent at a concentration of
MALDI-TOF instrumentation
A 1 μL sample of sterol derivative/matrix mixture was applied
to the MALDI target plate and dried slowly before insertion
into a 4800 MALDI TOF/TOF analyzer (AB Sciex). The
machine was equipped with a Nd:YAG laser of 355 nm with
a firing rate of 200 Hz and a camera to help direct the laser
beam. For MS/MS studies, 1 kV positive or negative ion mode
was used. Air at medium pressure was used for collision-
induced dissociation.
(a)
(b)
RESULTS AND DISCUSSION
Free sterols
(c)
(d)
Sterol standards, cholesterol, cholestanol and β-sitosterol
were analyzed by MALDI-TOFMS to observe their ionization
behavior. Figure 2(a) shows the MALDI-TOF mass spectrum
of cholesterol. The sterol fragment ion [M+H–H2O]+ at m/z
369.24 can only be observed with low signal intensity. Wang
et al.[33] and Schiller et al.[38] also reported that the molecular
ion of cholesterol cannot be detected by MALDI-TOFMS
and that free sterols readily lose a water molecule. Other
ions observed in the spectrum are related to matrix ions
of DHB. Cholestanol and β-sitosterol were also detected as
[M+H–H2O]+ ions at m/z 371.28 and 397.29, respectively
(spectra not shown).
(e)
Figure 1. Chemical structures of (a) cholesterol, (b)
cholesterol N-methylpyridyl ether, (c) cholesterol picolinyl
ester, (d) cholesterol sulphate ester, and (e) cholesterol
trimethylsilyl ether.
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I. Hailat and R. J. Helleur
Figure 2. MALDI-TOF mass spectra of (a) cholesterol detected at m/z 369.24 [M+H–H2O]+,
(b) cholesterol picolinyl ester at 514.36 [M+Na]+, and (c) cholesterol N-methylpyridyl ether
at 478.40 [M]+. * signifies the target analyte; $ = N-methylpyridyl fragment.
Picolinyl esters
analysis of serum sterols as their picolinyl esters; however,
the esters were analyzed by LC/ESI-MS/MS and observed as
In order to study the enhanced ionization efficiency of sterols
by derivatization, cholesterol, β-sitosterol, ergosterol and
cholestanol were derivatized to their picolinyl esters. The
picolinyl esters were easily detected as their sodiated adducts
[M+Na]+ and their ion intensities were significantly enhanced
after addition of sodium acetate (final concentration 20 mM).
The most suitable matrix was either THAP or DHB. By
illustration, the picolinyl ester of cholesterol is shown in
Fig. 2(b) showing a strong ion at m/z 514.36 ([M+Na]+)
using THAP. In additional experiments, the picolinyl esters
of cholestanol, β-sitosterol and ergosterol showed strong
[M+Na]+ signals at m/z 516.36, 542.37 and 524.30, respectively
(spectra not shown). When sodium acetate dopant was
added, the intensity of [M+Na]+ increased by 90%.
Dopants ammonium acetate (20 mM) and trifluoroacetic acid
(0.1%) were attempted but only weak [M+Na]+ signals were
observed. In another study, Honda et al.[30] described the
[M+Na+CH3CN]+.
N-Methylpyridyl ethers
Sterols were also converted into their N-methypyridyl ethers.
These derivatives were easily detected in positive mode (using
THAP) since the derivative is positively charged. Cholesterol
N-methylpyridyl ether was detected at m/z 478.40 [M]+
(Fig. 2(c)). N-Methylpyridyl ethers of cholestanol and β-sitosterol
were observed at m/z 480.41 and 506.41, respectively (spectra
not shown). Ions related to incomplete derivativation of the
sterol standard, i.e., [sterol+H–H2O]+, were observed at m/z
369.33 for cholesterol (Fig. 2(c)) and m/z 397.34 for β-sitosterol
(spectra not shown). N-Methylpyridyl ethers exhibited
significant fragmentation to give the ion at m/z 110.00
(N-methylpyridyl fragment). Attempts to improve the
conversion yield of this ether derivative were unsuccessful.
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Analysis of sterols by MALDI-TOFMS and MALDI-TOF/TOFMS
Sterol sulphates
Product ion scanning was carried out on the picolinyl
ester of cholesterol with the precursor ion m/z 514.34
(Fig. 4(a)). A strong product ion was observed at m/z
146.03 representing [picolinate+Na]+ and indicates facile
ester bond cleavage. No product ion associated with the
steryl moiety was observed. By subtracting the mass of
picolinyl ion from the precursor ion the molar mass of
steryl moiety can be determined, i.e., 369 Da for
cholesterol’s carbon skeleton.
The MALDI-TOF/TOF mass spectrum of cholesterol
N-methylpyridyl ether (precursor ion m/z 478.40) is
shown in Fig. 4(b). CID results in ether cleavage to give the
charged N-methylpyridyl moiety at m/z 110.04. The MALDI-
TOF/TOF mass spectrum of cholesterol sulphated ester
(precursor ion at m/z 465.32) is given in Fig. 4(c). The ion
[HSO4]– at m/z 96.89 is the result of ester bond cleavage. An
unexplained ion at m/z 458.95 is observed, likely an artifact
from the derivative preparation.
Sterol standards were derivatized to their sulphated esters
and analyzed by MALDI-TOFMS. Finding the best MALDI
matrix proved difficult and DHB, CHCA, THAP, dithranol
and p-nitroaniline were tested. p-Nitroaniline or dithranol
was the most suitable resulting in strong analyte ion intensity
with lowest matrix ion interference. As expected sulphated
esters were observed best in negative mode [M–H]– and the
use of base additives was not required to observe strong ion
signals. The MALDI-TOF mass spectrum of cholesterol
sulphate is shown in Fig. 3(a) with [M–H]– at m/z 465.32.
[M–H]– signals for other sterol sulphate esters were as
follows: cholestanol (m/z 467.32), β-sitosterol (m/z 493.33)
and ergosterol (m/z 475.28) (spectra not shown).
Fragmentation for sterol sulphates was observed by the
presence of ions HSO–3 (m/z 79.95) and HSO–4 (m/z 96.92) in
Figs. 3(a) and 3(b). A higher abundance of these fragment ions
was noted using p-nitroaniline (Fig. 3(b)) than with dithranol
matrix (Fig. 3(a)). Neto et al.[39] demonstrated the usefulness of
sulphate esters for sterol analysis in tissue using LC/ESI-MS.
It was found that steryl sulphates were best detected as
[M–H]– which is in agreement with the present method. Sandhoff
et al.[31] reported that sulphate ester derivatives easily lose a
proton and thus result in high ionization efficiency.
Reproducibility and sensitivity
The picolinyl and sulphate ester derivatives were selected for
statistical analysis. Cholesterol at a concentration of 200 μg/mL
was derivatized as its picolinyl and sulphated esters in replicates
(n = 3) and their molecular ion intensities measured by MALDI-
TOFMS. Sources of variability include the preparation of
derivatives and MALDI-TOFMS measurements which can be
expressed as relative standard deviation (RSD %). Variability
resulting from derivative preparation was found to be 8.3% for
MALDI-TOF/TOF analysis
MALDI-TOF/TOF experiments were performed on the
derivativized sterols in order to confirm the identity of
these derivatives and to study their fragmentation.
Figure 3. MALDI-TOF mass spectra of cholesterol sulphated ester detected at m/z 465.3
[M–H]– using (a) dithranol and (b) p-nitroaniline. * signifies the target analyte; $ = fragments.
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I. Hailat and R. J. Helleur
Figure 4. MALDI-TOF/TOF product ion spectra of (a) cholesterol picolinyl ester,
precursor ion of m/z 514.36; (b) cholesterol N-methylpyridyl ether, precursor ion of m/
z 478.40; and (c) cholesterol sulphated ester, precursor ion of m/z 465.32.
cholesterol picolinyl and 8.5% cholesterol sulphated esters
within spots was calculated as 3.7% and 5.9% for cholesterol
picolinyl and cholesterol sulphated esters, respectively. Variability
among sample spots was 5.3% (picolinyl esters) and 6.6%
(sulphated esters) (Table 1).
(Table 1). The same two derivatives were tested for reproducibility
in measurement by MALDI-TOFMS where variability can be
divided into within-spot and among-spot analyses. Variability
Table 1. Comparison of figures of merit among derivatized sterols (cholesterol as standard) versus free sterols by
MALDI-TOFMS
Reproducibility (RSD %)
LOD
Preparation (n=3)
Measurements
S/N
(μg/mL)
LOQ
At 60 μg/mL at S/N=3 (μg/mL)
within spot (n=3) among spots (n=3)
Free sterols
Cholesterol picolinyl ester
Cholesterol sulphated ester
54
780
4028
12.0
1.5
0.2
40.0
5.0
0.67
-
-
-
8.3%
8.5%
3.7%
5.9%
5.3%
6.6%
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Analysis of sterols by MALDI-TOFMS and MALDI-TOF/TOFMS
Figure 5. Total ion chromatogram of sterols found in cultivated mussels using
GC/MS. Peaks: 1. 24-nor-dehydrocholesterol, 2. occelasterol, 3. trans-22-
dehydrocholesterol, 4. cholesterol, 5. desmosterol, 6. brassicasterol, 7. ergosterol,
8. 24-methylenecholesterol, 9. campesterol, 10. stigmasterol, 11. β-sitosterol.
Figure 6. MALDI-TOF mass spectra of sterol profile in mussels as (a) sulphated esters
and (b) picolinyl esters. * signifies derivatized sterols, $ signifies sterol isomers, IS:
internal standard.
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I. Hailat and R. J. Helleur
The limits of detection for cholesterol was 12.0 μg/mL, lower
for the picolinyl ester at 1.5 μg/mL and lowest for the sulphate
ester at 0.2 μg/mL (Table 1). The limit of quantitation values are
also given. The derivatization MALDI-TOFMS approach using
the sterol picolinyl ester (LOD 1.5 μg/mL) shows similar a LOD
to that reported for cholesterol trimethylsilyl ether by GC/MS
single ion monitoring (1.19 μg/mL).[40] The use of sulphated
ester derivatives with MALDI-TOF analysis gives significantly
better LODs than GC/MS.
Sensitivity was calculated based on the S/N ratio at an
analyte concentration of 60 μg/mL. Sulphated ester derivatives
had the highest S/N ratios (4028). MALDI-TOFMS analyses of
sterols as their sulphated esters were 5 times more sensitive than
picolinyl esters and 75 times more sensitive than free sterols.
method. Mussel sterols were converted into their sulphated
and picolinyl esters for MALDI-TOFMS sterol profiling and
quantification. Figure
5 shows the GC/MS total ion
chromatogram (TIC) of the TMS sterols of mussels while
Fig. 6 shows the resulting MALDI-TOF mass spectra of mussel
sterols, as their sulphated esters using dithranol (Fig. 6(a)) and
as their picolinyl esters using DHB (Fig. 6(b)). Confirmation of
the sterols labeled in Fig. 6 was performed by MS/MS
experiments whereby sulphate esters were screened for
fragment ion m/z 97 and sterol picolinyl esters using fragment
ion m/z 146.
Sterols identified in mussels are listed in Table 2 based on
MALDI-TOFMS data and confirmed by GC/MS analyses. The
chemical structures of these sterols are given in Fig. 7. The GC/
MS sterol data revealed that two groups of sterol isomers could
not be resolved by MALDI-TOFMS. The three sterol isomers
occelasterol, trans-22-dehydrocholesterol and desmosterol
(Table 2) for example show up as m/z 512.30 as their picolinyl
ester derivatives while two other sterols isomers, brassicasterol
and 24-methylenecholesterol, as picolinyl esters at m/z 526.31.
Identification of sterols in blue mussels
Application of MALDI-TOFMS of sterol derivatives was
applied to sterols extracted from cultivated mussels. GC/MS
analysis was used alongside MALDI-TOFMS, as a comparative
Table 2. Calibration curve equations for derivatized sterol standards
Reproducibility (RSD %)
(n=3)
Calibration curve equation
R2
cholesterol picolinyl ester
cholesterol sulphated ester
β-sitosterol picolinyl ester
β-sitosterol sulphated ester
y = 0.0027x À 0.0183
y = 0.0031x À 0.0317
y = 0.0019x À 0.0385
y = 0.0025x À 0.0609
0.9995
0.9973
0.9987
0.9993
6.3
7.2
7.5
8.6
Figure 7. Chemical structures of sterols found in mussels.
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Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 149–158

Analysis of sterols by MALDI-TOFMS and MALDI-TOF/TOFMS
Table 3. Sterols identified in mussels as their picolinyl and sulphated ester derivatives, and their characteristic MS/MS
fragmentation
Picolinyl esters
Sulphated esters
Sterols
m/z
m/z
[M+Na]+
MS/MS data
[M–H]–
MS/MS data
24-nor-dehydrocholesterol
498.28
512.30
514.31
524.30
526.31
528.29
540.30
542.30
498.28 → 145.98
512.30 → 145.97
514.31 → 146.01
524.30 → 146.01
526.31 → 145.97
528.29 → 145.99
540.30 → 146.01
542.30 → 146.01
449.27
463.29
465.30
475.28
477.30
479.30
491.32
493.33
449.27 → 96.91
463.29 → 96.89
465.30 → 96.90
475.28 → 96.90
477.30 → 96.91
479.30 → 96.89
491.32 → 96.90
493.33 → 96.91
occelasterol or trans-22-dehydrocholesterol or desmosterol
cholesterol
ergosterol
brassicasterol or 24-methylenecholesterol
campesterol
stigmasterol
β-sitosterol
and its signal enhanced after addition of sodium acetate using
either DHB or THAP. N-Methylpyridyl ethers were detected
as [M]+ using THAP. Sulphated esters were detected as
[M–H]– using either p-nitroaniline or dithranol. The ester
bonds of picolinyl and sulphated esters showed facile
cleavage during CID MS/MS resulting in diagnostic fragments
at m/z 146 and 97, respectively. Based on MALDI-TOFMS and
GC/MS data, the most abundant sterols in mussels were
cholesterol, desmosterol, brassicasterol, 24-nor-dehydro-
cholesterol, 24-methylenecholesterol and trans-22-dehydro-
cholesterol. The sterol profile of mussels obtained by
MALDI-TOF is in good agreement with those obtained by
GC/MS. Cholesterol and β-sitosterol were successfully
quantified using MALDI-TOF. Sulphated esters showed
highest sensitivity, 5 times more sensitive than picolinyl esters
and with a better detection limit for sterols than GC/MS.
Calibration curves exhibited excellent linear regression which
indicates that MALDI-TOF can be used for quantitation of
sterols. Finally, preliminary research on the analysis of these
sterol derivatives using direct injection ESI-QIT MSn shows
promise. ESI-MS3 should have the ability to distinguish among
sterol isomers and allow for a more comprehensive MS
approach to sterol analysis in biological samples.
Table 4. Sterol amounts (mg/kg SD wet wt) in mussel
samples obtained by MALDI-TOFMS and GC/MS (n = 3)
MALDI-TOFMS
picolinyl sulphated
GC/MS
trimethylsilyl
ether
ester
ester
cholesterol 157.8 8.4 232.9 12.3
β-sitosterol 32.7 1.8 35.9 1.9
211.0 10.2
38.2 2.1
Comparing the MALDI-TOFMS spectra of the two derivatives
for sterol analysis (Fig. 6) the sulphate ester derivative profile
showed significantly better S/N ratio and has lower matrix
ion interference.
Calibration for derivatives and sterol quantification in mussels
As a trial, two sterols (cholesterol and β-sitosterol) were
quantified in the mussel extract. Calibration curves for cholesterol
and β-sitosterol picolinyl esters, and cholesterol and β-sitosterol
sulphate esters, were constructed using cholestanol picolinyl
and cholestanol sulphate ester as internal standards, respectively.
The derivatized sterol standard curves gave excellent correlation
coefficients (R2), and good reproducibility (Table 3).
The concentrations of cholesterol and β-sitosterol in
mussels measured by MALDI-TOFMS as their picolinyl and
sulphated esters and by GC/MS as their TMS derivatives
are given in Table 4. The results of the two methods are
reasonably comparable noting that the comparison was not
between the same mussel extract, but from different mussel
extracts from the same mussel supplier. Although these
results represent only one biological sample the results are
very promising for the use of derivativation MALDI-TOFMS
for rapid analysis of sterols extracted from biological samples.
Acknowledgements
We would like to express our thanks to the Chemistry Depart-
ment and the School of Graduate Studies at Memorial
University of Newfoundland and NSERC for funding. We would
also like to acknowledge the Center for Chemical Analysis and
Research Training for assistance in chemical analysis.
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