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ORIGINAL PAPER
Development of a novel method for quantification of sterols
and oxysterols by UPLC-ESI-HRMS: application
to a neuroinflammation rat model
Sophie Ayciriex &Anne Regazzetti &Mathieu Gaudin &
Elise Prost &Delphine Dargère &France Massicot &
Nicolas Auzeil &Olivier Laprévote
Received: 21 June 2012 /Revised: 24 August 2012 /Accepted: 29 August 2012
#Springer-Verlag 2012
Abstract Cholesterol and oxysterols are involved as key
compounds in the development of severe neurodegenerative
diseases and in neuroinflammation processes. Monitoring
their concentration changes under pathological conditions
is of interest to get insights into the role of lipids in diseases.
For numerous years, liquid chromatography coupled to
mass spectrometry has been the method of choice for metab-
olites identification and quantification in biological samples.
However, sterols and oxysterols are relatively apolar mole-
cules poorly adapted to electrospray ionization (ESI). To
circumvent this drawback, we developed a novel and robust
analytical method involving derivatization of these analytes
in cholesteryl N-4-(N,N-dimethylamino)phenyl carbamates
under alkaline conditions followed by ultra-performance
liquid chromatography–high resolution mass spectrometry
analysis (UPLC-HRMS). Optimized UPLC conditions led
to the separation of a mixture of 11 derivatized sterols and
oxysterols especially side chain substituted derivatives after
6 min of chromatographic run. High sensitivity time-of-
flight mass analysis allowed analytes to be detected in the
nanomolar range. The method was validated for the analysis
of oxysterols and sterols in mice brain in respect of linearity,
limits of quantification, accuracy, precision, analyte stabili-
ty, and recovery according to the Food and Drug Adminis-
tration (FDA) guidelines. The developed method was
successfully applied to investigate the impact of lipopoly-
saccharide (LPS) treatment on the rat cerebral steroidome.
Keywords UPLC/ESI/HRMS .Cholesterol .Oxysterols .
Derivatization .Quantification
Sophie Ayciriex and Anne Regazzetti contributed equally to this work.
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-012-6396-6) contains supplementary material,
which is available to authorized users.
S. Ayciriex :A. Regazzetti :M. Gaudin :D. Dargère :
F. Massicot :N. Auzeil (*):O. Laprévote
Chimie-Toxicologie Analytique et Cellulaire, EA 4463,
Université Paris Descartes, Sorbonne Paris Cité,
Faculté des Sciences Pharmaceutiques et Biologiques,
75006 Paris, France
e-mail: nicolas.auzeil@parisdescartes.fr
M. Gaudin
Division métabolisme, Technologie Servier,
45000 Orléans, France
M. Gaudin
Centre de Recherche de Gif,
Institut de Chimie des Substances Naturelles, CNRS,
Avenue de la Terrasse,
91198 Gif-sur-Yvette Cedex, France
E. Prost
UMR 8638 CNRS, Université Paris Descartes,
Sorbonne Paris Cité,
Faculté des Sciences Pharmaceutiques et Biologiques,
75006 Paris, France
O. Laprévote
Service de Toxicologie Biologique, Hôpital Lariboisière,
4 rue Ambroise Paré,
75475 Paris cedex 10, France
Present Address:
M. Gaudin
Biomolecular Medicine, Department of Surgery and Cancer,
Faculty of Medicine, Imperial College London,
SW7 2AZ London, UK
Anal Bioanal Chem
DOI 10.1007/s00216-012-6396-6
Introduction
Cholesterol is an important component of membrane lipids,
which regulates membrane fluidity, influencing the structural
organization and activity of membrane proteins. It is highly
abundant in the central nervous system, and especially in
neuronal cell membranes, where it represents about 25 % of
the total body cholesterol. Since the blood–brain barrier strictly
limits cholesterol uptake from the circulation into the brain,
brain cholesterol is mainly synthesized de novo and cholesterol
levels are tightly regulated [1].
To maintain brain cholesterol homeostasis, cholesterol is
converted into an oxygenated metabolite 24(S)-hydroxycho-
lesterol (24-OHC) by CYP46A1, a cytochrome P-450 en-
zyme, expressed in neurons. In contrast to cholesterol, 24-
OHC is able to cross the blood–brain barrier [2–4] and this
flux is an important part of the cholesterol turnover in the
brain [5]. Oxidized cholesterol metabolites, best known as
oxysterols, do not only play a role in cholesterol metabolism
but act as biologically active molecules [6]. They are indeed
involved in atherosclerosis, neurodegeneration, and inflam-
mation process [7–12].
It is therefore crucial to develop fast, robust, and
sensitive methods for the quantification of oxysterols
and sterols. Several analytical tools were developed for
their analysis and quantification in biological samples
such as gas chromatography (GC) with flame ionization
detection or coupled with mass spectrometry (MS) and
liquid chromatography (LC) with ultraviolet detection
(UV) [13]. However, these techniques have limitations
attributable to the lack of specificity of the UV detection
system or the loss of derivatizing groups in the ion
source making molecular weight determination difficult
during GC-MS experiments. Thus, metabolite identifica-
tion remains difficult [14]. To date, LC-MS is the most
frequently used technique for the identification and quan-
tification of sterols and oxysterols in complex biological
samples including brain tissue sample, serum, and cere-
brospinal fluid. Atmospheric pressure chemical ionization
and atmospheric pressure photoionization are the only
ionization modes enabling a sensitive direct analysis of
these compounds without derivatization [15–19]. Howev-
er, these techniques lead to dehydrated protonated mole-
cules making the determination of the molecular weight
of the analyte sometimes difficult [19,20]. On the con-
trary, electrospray ionization (ESI) is a soft ionization
technique known to produce quasi-molecular ions in most
cases. However, owing to their non-polar character and
low gas-phase basicity, oxysterols and sterols are not
efficiently ionized in ESI leading to insufficient sensitiv-
ity for bioanalysis. To circumvent this drawback, a
derivatization step is required. Several chemical tags
for use with oxysterols in combination with ESI-HRMS have
been reported such as (2-hydrazinyl-2-oxoethyl)trimethylaza-
nium chloride (Girard reagent), N,N-dimethylglycyl, picoli-
noyl or danzyl derivatizations [20–26]. Depending on the
tagging, these derivatization procedures involve toxic reagents
or are complex and time consuming. Indeed, Griffiths et al.
developed a two-step elegant but complex derivatization pro-
cedure using enzymatic conversion of the 3′-hydroxyl moiety
into a ketone beforereacting with Girard P reagent [20]. Jiang
et al. proposed a simple method involving esterification of
hydroxyls by dimethylglycine [24]. Nevertheless, this one-step
protocol requires overnight heating of the sample. Re-
cently, Honda et al. proposed a novel derivatization
method that involves toxic reagents such as pyridine
as reaction solvent and three different reagents, namely
2-methyl-6-nitrobenzoic anhydride, picolinic acid, and 4-
dimethylaminopyridine [22,23]. This must be taken into
account in terms of safe handling.
Herein, we report a novel and robust analytical method for
sterols and oxysterols involving carbamate formation with 4-
(dimethylamino)phenyl isocyanate under alkaline conditions.
Our proposed protocol is a one-step fast derivatization method
leading to stable derivatives. The 11 cholesteryl N-4-(N,N-
dimethylamino)phenyl carbamates obtained are chromato-
graphically resolved after 6 min of chromatographic run and
readily ionized by ESI with high efficiency. In particular, side
chain hydroxylated derivatives 22(R)-hydroxycholesterol, 27-
hydroxycholesterol, 25-hydroxycholesterol, and 24(S)-
hydroxycholesterol were efficiently separated and exclu-
sively detected as quasi-molecular ions by high resolution
mass spectrometry (HRMS). The method was rigorously val-
idated according to the Food and drug Administration (FDA)
guidelines and was further tested on a neuroinflammation
model.
Materials and methods
Chemicals and reagents
22(R)-Hydroxycholesterol [5-cholestene-3β,22-diol], 27-
hydroxycholesterol [cholest-(25R)-5-ene-3β,27-diol], 25-
hydroxycholesterol [cholest-5-ene-3β,25-diol], 24(S)-
hydroxycholesterol [5-cholestene-3β,24-diol], 24(R/S)-
hydroxycholesterol (d
6
) [26,26,26,27,27,27-hexadeutero-
cholest-5-ene-3β,24-diol], 5α,6α-epoxycholestanol [cho-
lestanol, 5α,6α-epoxy], 7β-hydroxycholesterol [cholest-5-
en-3β,7β-diol], desmosterol [3β-hydroxy-5,24-cholesta-
diene], 7-dehydrocholesterol [Δ5,7-cholesterol], lathosterol
[5α-cholest-7-en-3β-ol], cholestanol [5α-cholestan-3β-ol],
cholesterol (d
7
) [cholest-5-en-3β-ol(d7)] were purchased
from Avanti Polar Lipids (Alabaster, AL, USA) (Electronic
Supplementary Material, Fig. S1). Cholesterol, triethyl-
amine, 4-(dimethylamino)phenyl isocyanate (DMAPI),
S. Ayciriex et al.
formic acid, lipopolysaccharide (LPS) from Salmonella enter-
ica serotype typhimurium were purchased from Sigma-
Aldrich (Saint-Quentin Fallavier, France). Hexane and
dichloromethane were obtained from Carlo Erba Reactifs
SDS (Val-de-Reuil, France). Acetonitrile, methanol, and iso-
propanol were of LC-MS grade (J.T. Baker, Phillipsburg, NJ,
USA). Leucine enkephalin was used as the lockmass solution
(Sigma, Saint-Quentin Fallavier, France).
Standard solution and quality control sample preparation
Stock solutions of 1 mg/mL 22(R)-hydroxycholesterol, 27-
hydroxycholesterol, 25-hydroxycholesterol, 24(S)-hydroxy-
cholesterol, 24(R/S)-hydroxycholesterol (d
6
) (oxysterols IS),
7β-hydroxycholesterol, 5α,6α-epoxycholestanol, desmos-
terol, 7-dehydrocholesterol, lathosterol, cholesterol, choles-
terol (d
7
) (sterols internal standards), and cholestanol were
prepared in methanol. Standard working solutions at
100 μM were prepared by diluting the stock solutions in
methanol. These working solutions were diluted and spiked
into mouse brain tissue C57BL/6 homogenates to assess the
effect of matrix on precision and recovery. The quality
control (QC) working solution was prepared in the same
way as the standard working solutions. Two concentrations
ranges (0.0017–0.17–1.25 μM; 0.017–0.33–5μM) for QC
samples were prepared by diluting the working solution
with methanol to calculate the precision and accuracy
experiments. The working IS solutions were prepared by
mixing the stock solutions at a final concentration of 10 μM.
All the stock, standard working, and QC working solutions
were stored at −80 °C.
LPS rat treatment
All experiments were performed according to protocols
approved by the Institutional Animal Care and Use
Committee. In addition, the number of animals used
and their suffering were minimized in all experiments
designed. Wistar rats were treated once a week by
intraperitoneal injections of either LPS or saline buffer
(0.9 % sodium chloride) for controls. After 8 weeks of
treatment, the rats were killed and cryostat sections
were performed in the cortex of the frontal lobe and
in the hippocampus.
Sample preparation: derivatization procedure
Weighed rat brain tissues were placed in 2-mL Precellys®
CK 14 lysing tubes pre-filled with 1.4-mm ceramic beads.
Six hundred microlitre of cold water was added and homo-
geneization was performed for 15 s at 5,000 rpm (Pre-
cellys®24-Dual apparatus). Sterols were extracted with
hexane/methanol mixture (7:1, v/v).
Dried sterols were resuspended with 200 μL of a solution
containing DMAPI in dichloromethane (10 mg/mL). Thirty
microlitre of triethylamine was added. The resulting mixture
was vortexed and subsequently shaken for 2 h at 65 °C and
150 rpm in an incubator shaker. To quench the reaction,
150 μL of phosphate buffer (pH 8) was added, followed by
3 mL of hexane. The mixture was vortexed for 30 s and
centrifuged. The upper layer containing the carbamate com-
pounds was withdrawn and the organic solvent was evaporated
under reduced pressure. The dry residues were reconstituted in
200 μL of acetonitrile/isopropanol (1:1, v/v) and 5 μLwas
injected into the UPLC-ESI-HRMS system.
UPLC-ESI-HRMS analysis
The separation of sterols and oxysterols derivatives was
achieved using an Acquity UPLC system (Waters, Milford,
MA, USA) equipped with an Acquity UPLC CSH™C
18
column (100×2.1 mm; 1.7 μm) heated at 70 °C. A binary
gradient system was used consisting of 0.01 % (v/v) formic
acid in water as eluent A and acetonitrile/methanol mixture
(70:30, v/v) as eluent B. The flow rate was 0.4 mL/min. The
sample analysis was carried out over a 13-min total run
time; initially, elution was performed isocratically for 3 min
at 85 % eluent B, following by an increase to 100 % in 4 min
(curve 3) and held at this composition for 3 min (curve 6).
Thereafter the system was switched back to 85 % B and 15 %
A (curve 1).
The UPLC system was coupled to a hybrid quadrupole
orthogonal time-of-flight mass spectrometer (SYNAPT G2
HDMS, Waters MS technologies, Manchester, UK). Elec-
trospray positive ion mode was used. The ESI source con-
ditions were as follows: 900 L/h for the desolvation gas
flow, 250 °C for the desolvation temperature, +2.50 kV for
the capillary voltage, and +40 V for the cone voltage. Data
were acquired in the mass range 100–1,000m/z. Enhanced
duty cycle (EDC) function was applied for the first 5 min of
run time centered on m/z565.436 and from 5 to 6 min on m/z
549.441 corresponding to the [M+H]
+
ion of carbamates
derivatives of oxysterols and cholesterol, respectively. The
ion source was equipped with a LockSpray unit from which
the acquisition software collects a reference scan every 20 s.
The LockSpray internal reference used was leucine enkepha-
lin (2 ng/μL in acetonitrile/water, 50:50, v/v).
NMR spectra
All NMR experiments were recorded on a Bruker AVANCE-
400MHzat400MHzand75MHzfor
1
Hand
13
C, respec-
tively, and equipped with an inverse broadband probe (BBI)
(Bruker Biospin). About 5 mg of 22(R)-hydroxycholesterol
carbamate derivative and 9 mg of cholesterol carbamate
derivative were dissolved in CDCl
3
. Assignments were
Quantification of sterols and oxysterols by UPLC-ESI-HRMS
performed by two-dimensional correlation spectroscopy
(COSY), heteronuclear single quantum correlation (HSQC),
and heteronuclear multiple bond correlation experiment
(HMBC) experiments.
Data processing
Data acquisition was carried out using MassLynx software
version 1.4 (Waters MS Technologies, Manchester, UK). Tar-
getLynx software was used to determine peak areas of com-
pounds of interest (Waters MS Technologies, Manchester, UK).
Method validation
Method validation was performed according to the recom-
mendations of the FDA guidance for industry [27].
Data analysis
All measurements and calculations were expressed as mean±
SD (standard deviation) except for the stability experiment
performed on the entire mice brain.
Linearity of the calibration curves was analyzed by a
simple linear regression. Accuracy and precision were de-
termined using six determinations per concentration.
Three QC concentrations level samples were used: low,
middle, and high QC samples according to the concen-
tration range. Precision is expressed as the relative
coefficient of variation (CV%) according to the follow-
ing formula: CV%¼standard deviation mean
=
ðÞ100
and should be lower than 15 %. Accuracy is defined
as the relative error (RE) between the determined mean
value and the theoretical value, which is calculated as
RE %ðÞ¼measured value theoretical valueðÞ=theoretical
value 100 and should be within ±15 %. The same set of
samples was used to estimate the extraction yield.
The stability of native oxysterols and sterols solution
and their respective carbamates was assessed at three
concentrations (1, 0.17, and 0.03 μM) after short-term
(24 h and 72 h) and long-term (7 days and 30 days)
storage at different temperatures (room temperature, +10 °C,
−20 °C, and −80 °C). Repeated measures analysis of variance
(rANOVA) was used to evaluate stability condition parame-
ters (storage time and temperatures). The concentrations de-
termined immediately after solubilisation and derivatization,
respectively, for native and derivatized oxysterol and sterol
(time 0) were assigned as C
0
. Stability was expressed as
percentage change in mean concentration from C
0
and the
95 % confidence interval for the percentage change was
calculated on the basis of six replicates at each concentration
point.
To assess the stability of oxysterols and sterols
contained in brain tissue matrix, three storage conditions
of C57BL/6 mice brain tissue were evaluated. First,
brain tissue homogenate (ca. 10 mg/mL protein assay)
was kept for short-time storage (4 h) at room tempera-
ture and on ice. Second, intact mice brain tissue was
stored for 1 month at −80 °C.
For each oxysterol and sterol detected in the brain tissue,
the peak area ratio of the carbamate derivatives normalized
to the deuterated internal standard was calculated (R
x
) and
compared to the one obtained without brain storage (R
0
).
Results were expressed as RxR0
=
ðÞ100:
For the precision in matrix experiments, each oxysterol
and sterol found in the mice brain tissue was quantified.
Four samples were prepared, analyzed in triplicate, and the
precision measured. Recovery experiments were performed
on mice brain tissues (40 mg) spiked with different concen-
trations close to the expected endogenous concentrations.
Recovery was calculated as amount found after spiking ð
endogenous amountÞamount added 100
=
Biological data were analyzed with Student’s two-tailed
unpaired ttest to assess differences between rats treated with
LPS and rats treated with saline buffer. For all analyses, p<
0.05 was considered statistically significant. All the data
were analyzed using GraphPad Prism vs 5.0.
Fig. 1 Reaction scheme for cholesterol derivatization with DMAPI
S. Ayciriex et al.
Results and discussion
Method development
Derivatization method
Because of their neutral character, oxysterols and sterols are
not efficiently detected in ESI. The use of tertiary amine
groups is a popular method for analyte tagging and improves
the ionization yield in positive ion mode under ESI conditions.
Although esterification has been extensively used to
derivatize the hydroxyl function of sterols and oxysterols
[22–24], carbamate chemistry has not yet been applied to
these analytes. Since aromatic isocyanates offer a better reac-
tivity than alkyl ones, the derivatization reagent chosen was 4-
(dimethylamino)phenyl isocyanate (DMAPI). In the presence
of triethylamine, it readily reacts with oxysterols and sterols to
afford a stable carbamate derivative (Fig. 1)[28]. Whereas for
oxysterols the two hydroxyl groups could theoretically react,
only carbamates corresponding to monoderivatives were gen-
erated. The question arises which hydroxyl group, on ring A
or on the side chain, was involved in the reaction. In order to
answer this question, a sufficient amount of 22(R)-hydroxy-
cholesterol and cholesterol carbamate was synthesized and
NMR experiments (COSY, HSQC, and HMBC) were per-
formed on the carbamate derivative and on 22(R)-hydroxy-
cholesterol and cholesterol [29]. The NMR study revealed that
the derivatization occurred only on position 3 of ring A
(Electronic Supplementary Material, Table S1). The total
and selective mono-addition of the tagging moiety is a benefit
of this method because it avoids possible mixtures of mono
and di-addition products, thus improving robustness.
Different derivatization parameters, including reaction sol-
vents (dioxane, dichloromethane, pyridine, tetrahydrofuran,
dimethylformamide), concentration of DMAPI reagent (10,
20, 30 mg/mL), amount of triethylamine (10, 20, and 30 μL),
and reaction times (1 h, 2, and 3 h), were optimized with a
mixture of oxysterols and sterols frequently present in biolog-
ical samples (Electronic Supplementary Material, Fig. S2). As
a result, the derivatization was performed by adding 200 μLof
a 10 mg/mL DMAPI solution in dichloromethane and
30 μL of triethylamine heated for 2 h at 65 °C under gentle
shaking. The amount of reagents used per sterol extract is
2 mg, i.e., ten times lower than the total amount of reagents
used in the picolinic ester procedure [22,23].
Mass spectrometry analysis
A triple quadrupole mass spectrometer operated in the select-
ed reaction monitoring (SRM) mode is the gold standard in
quantitative bioanalysis. Nevertheless, last-generation hybrid
quadrupole time-of-flight mass spectrometers are designed to
Table 1 Limit of quantification, linear dynamic range, linearity of the plot of area response ratio versus concentration, correlation coefficient
Oxysterols and sterols LOQ (μM) Linear dynamic range (μM) Linear regression equation Correlation coefficient (R
2
)
22(R)-Hydroxycholesterol 1.7× 10
−3
7.35× 10
2
y=20.696x+ 0.021 0.998
27-Hydroxycholesterol 1.7× 10
−3
7.35× 10
2
y=10.448x+ 0.001 0.998
25-Hydroxycholesterol 1.7× 10
−3
7.35× 10
2
y=12.947x+ 0.002 0.999
24(S)-Hydroxycholesterol 1.7× 10
−3
5.57× 10
3
y=8.907x+ 0.004 0.997
7β-Hydroxycholesterol 3× 10
−4
1.51× 10
3
y=3.175x+ 0.006 0.995
5α,6α-Epoxycholestanol 3.3× 10
−3
1.51× 10
3
y=2.894x+ 0.059 0.999
Desmosterol 3.3× 10
−3
1.51× 10
3
y=5.144 x+ 0.003 0.998
7-Dehydrocholesterol 1.7× 10
−2
2.99× 10
2
y=2.656x−0.001 0.995
Lathosterol 3.3× 10
−3
1.51× 10
3
y=9.756x+ 0.021 0.999
Cholesterol 3.3× 10
−3
1.51× 10
3
y=4.48x+ 0.04 0.999
Cholestanol 3.3× 10
−3
1.51× 10
3
y=2.989 x+ 0.014 0.996
Fig. 2 Chromatographic separation of derivatized sterols and oxysterols
mixture (1 μM) in a 6-min run (A): a22(R)-hydroxycholesterol (t
R
0
2.62 min; m/z565.436); b27-hydroxycholesterol (t
R
02.85 min; m/z
565.436); c25-hydroxycholesterol (t
R
02.99 min; m/z565.436); d24(S)-
hydroxycholesterol (t
R
03.13 min; m/z565.436); e7β-hydroxycholesterol
(t
R
03.72 min; m/z565.436); f5β,6β-epoxycholestanol (t
R
02.62 min; m/z
565.436); g5α,6α-epoxycholestanol (t
R
04.88 min; m/z565.436); hdes-
mosterol (t
R
05.28 min; m/z547.426); i7-dehydrocholesterol (t
R
0
5.41 min; m/z547.426); jlathosterol (t
R
05.66 min; m/z549.441); k
cholesterol (t
R
05.72 min; m/z549.441); lcholestanol (t
R
05.94 min;
m/z551.457)]
Quantification of sterols and oxysterols by UPLC-ESI-HRMS
perform both qualitative and quantitative analysis on the same
instrument [30]. Indeed, their linear dynamic range has been
increased and detectors are less prone to saturation. Owing to
its high resolution and high mass accuracy analyzer, our Q-
TOF instrument (SYNAPT G2) allowed analytes of interest to
be detected with a mass window as narrow as 4 ppm ensuring
specificity of the analysis, while at the same time monitoring
interfering matrix. This can be useful when working with
complex biological samples. Moreover, synchronization of
the release of ions from the transfer ion guide with the high
field pusher in the EDC mode led to an increase of the signal-
to-noise ratio by a factor of 10 [31]. We applied this function
centered on m/z565.4 during the first 5 min of run time and on
m/z549.4 from 5 to 6 min corresponding to the carbamate
quasi-molecular ion [M+H]
+
of oxysterols and sterols, respec-
tively. On average we obtained a 7-fold increase of the signal-
to-noise ratio (Electronic Supplementary Material, Table S2).
The carbamates derivatives obtained led exclusively to
quasi-molecular ions in ESI-TOF HRMS mode without any
alkali cation adduct. In contrast, the picolinic acid method led
to ion adducts of sterols such as [M+Na +ACN]
+
used as a
precursor ion for collision-induced dissociation experiments,
which provide a [M+Na]
+
fragment ion [23]. Moreover, the
sterol carbamate derivatives exhibit no in-source fragmenta-
tion. In contrast, the picolinate esters derivatives lead to the
loss of picolinoyl in our ESI source conditions [22,23].
Table 2 Repeatability of the
quantification of oxysterols and
sterols in QC samples
Oxysterols and sterols QC concentrations (μM)
Targeted
concentration
Measured
concentration
Precision (CV%) Accuracy (RE%)
22(R)-Hydroxycholesterol 0.0017 0.0017±0.000 1.28 −1.36
0.17 0.171± 0.005 3.06 0.48
1.25 1.253± 0.009 0.69 0.20
27-Hydroxycholesterol 0.0017 0.0017±0.000 2.27 1.47
0.17 0.172± 0.007 3.88 1.07
1.25 1.248± 0.020 1.55 −0.16
25-Hydroxycholesterol 0.0017 0.0017±0.000 2.39 0.32
0.17 0.168± 0.007 4.08 −1.22
1.25 1.257± 0.022 1.75 0.58
24(S)-Hydroxycholesterol 0.0017 0.0017±0.000 2.65 −0.22
0.17 0.171± 0.005 3.06 0.48
1.25 1.257± 0.020 1.58 0.55
7β-Hydroxycholesterol 0.017 0.017± 0.000 2.74 0.34
0.33 0.332± 0.011 3.40 0.67
5 5.012± 0.067 1.33 0.23
5α,6α-Epoxycholesterol 0.017 0.017 ± 0.000 3.33 −0.88
0.33 0.334± 0.008 2.33 1.34
5 5.048± 0.064 1.28 0.97
Desmosterol 0.017 0.017± 0.000 4.96 −0.49
0.33 0.3342±0.0148 4.43 1.26
5 5.0508±0.20 3.94 1.02
7-Dehydrocholesterol 0.017 0.017 ±0.000 5.01 −1.58
0.33 0.3320±0.0142 4.28 0.60
5 5.0314±0.1450 2.88 0.63
Lathosterol 0.017 0.017± 0.000 4.70 −0.38
0.33 0.34± 0.01 4.40 2.46
5 5.04± 0.14 2.83 0.74
Cholesterol 0.017 0.017±0.000 4.48 −0.80
0.33 0.34± 0.02 4.83 3.53
5 5.09± 0.15 3.03 1.72
Cholestanol 0.017 0.017± 0.000 4.98 −2.08
0.33 0.34± 0.016 4.88 1.52
5 4.95± 0.202 4.08 −0.97
S. Ayciriex et al.
The specificity of the SRM method between two adducts is
thus limited. In our source conditions, these picolinate deriv-
atives are detected as a mixture of protonated and sodium-
cationized molecules, thus reducing sensitivity. The dimethyl-
glycine derivatization of oxysterols produces di-derivatives
detected as doubly charged ions in the positive ion mode,
leading to complex MS/MS spectra [24].
The UPLC-ESI-HRMS method proposed in this work
provides adequate and reproducible separation of sterols and
oxysterols and a selective and sensitive detection thanks to
improved behavior of the analytes in the ion source of the
mass spectrometer (Fig. 2).
Chromatographic separation of oxysterols isomers
We optimized the liquid chromatography conditions in order to
allow the quantification of oxysterols isomers. Indeed, the
main chromatographic challenge was to achieve adequate sep-
aration of 22(R)-hydroxycholesterol, 25-hydroxycholesterol,
27-hydroxycholesterol, and 24(S)-hydroxycholesterol. The
Fig. 3 Stability of endogenous oxysterol and sterol compounds in micebrain cell lysate stored at room temperature for 4 h (A) and at 4 °C for 4 h (B)and
at −80 °C for 1 month (C). The results are expressed as the mean of a triplicate of six tissue samples. Errors bars CV%
Quantification of sterols and oxysterols by UPLC-ESI-HRMS
mobile phase composition (variation of acetonitrile percentage
in methanol) and the column temperature were optimized
and provided good results for the separation of side
chain substituted oxysterols with a mean asymmetry factor
for the four aforementioned analytes of 1.13, in agreement
with FDA recommendations for chromatography (Electronic
Supplementary Material, Fig. S3and S4)[27]. The separation
of isomeric oxysterols was finally achieved with a mixture of
acetonitrile/methanol (70:30, v/v) as eluent B and a column
temperature of 70 °C after a 3-min chromatographic run.
Method validation
Validation of the proposed analytical method was performed
according to the FDA guidelines on general principles of
process validation in term of linear range, precision, accuracy,
stability, and recovery [27].
Linearity of calibration curve
A calibration plot was established for each oxysterol and sterol
present together in a mixture. Different amounts of oxysterols
and sterol standards were mixed with deuterated internal
standard, 24(R/S)-hydroxycholesterol-d
6
and cholesterol-d
7
,
respectively, derivatized and analyzed as described in the
“Materials and methods”section. The peak area of the oxy-
sterols and sterol carbamate derivatives normalized to the
deuterated analogue was plotted against the corresponding
oxysterol and sterol concentrations. The linearity of the stand-
ards curves, as determined by simple linear regression, exhibit
an R
2
above 0.995 (Table 1).
Limits of quantification (LOQ)
The LOQ was defined as the lowest concentration on the
calibration curve at which the analyte can be measured with a
precision and accuracy better than 20 %. The calculated LOQ
values for each oxysterol and sterol are shown in Table 1.The
lowest value of the LOQ calculated corresponds to 0.0003 μM
for the 7β-hydroxycholesterol (2 fg injected on the column)
and the highest one to 0.017 μM (96.4 fg injected) for 7-
dehydrocholesterol. For the other oxysterols and sterols the
LOQ values range from 0.0017 to 0.0033 μM (10.1–19.1 fg
injected). Our method enabled us to determine the concentra-
tion of sterols and oxysterols with a higher sensitivity than
earlier published methods [22,23].
Extraction yield, precision, and accuracy of the present
method
The extraction yield was determined in six replicates by
comparing in the extracted QC sample the peak area ratio of
the analyte to the corresponding deuterated analogue at the
lower LOQ (LLOQ), medium and high concentrations with
those obtained without the hexane/methanol extraction step.
The extraction yield of oxysterols and sterols varied from 92
to 105 %, except for two oxysterols, 27-hydroxycholesterol
and 7β-hydroxycholesterol, whose extraction yields are
around 79–80 % and 62–82 %, respectively. These data are
summarized in Table S3(Electronic Supplementary Material).
The extraction yield of oxysterols and sterols was consistent,
precise, and reproducible.
Precision and accuracy were assessed by analyzing
QC samples (LLOQ, middle and high concentrations
levels) during intraday assay. The intraday precision
(n06) ranged from 0.69 to 5.01 % and the accuracy
from −2.08 to 1.52 % at the three concentrations levels
(QC) of the oxysterols carbamates derivatives. The pre-
cisionrangedfrom2.83to4.96%andtheaccuracy
from −0.80 to 3.53 % at the three concentrations levels
of the three sterol carbamate derivatives. The results
obtained for precision and accuracy are summarized in
Table 2. The data indicated that the proposed method has
satisfactory precision, accuracy, and reproducibility. No drift
in retention time was detected and peak area variation
exhibited a CV% lower than 15 % (Electronic Supplementary
Material, Table S4).
Stability
The stability of the carbamate derivatives after long-term
storage at −80 °C and short-term storage at +10 °C (in the
autosampler) and at room temperature was investigated
using UPLC-ESI-HRMS. For this purpose, peak area of
the carbamate derivative was normalized to the internal
standard freshly prepared and added just before the extrac-
tion step. All the stability studies were conducted at three
different concentrations levels with six replicates each. The
stability results are summarized in Table S5(Electronic
Supplementary Material). All the oxysterol and sterol car-
bamate derivatives in the mixture were stable with no sig-
nificant variation over the storage period whatever the
temperatures and the durations tested.
Table 3 Precision for oxysterol and sterol quantification in mice brain
Oxysterols and sterols Amount added
(nmol/mg proteins)
CV (%)
27-Hydroxycholesterol 1.9× 10
−3
8.9
25-Hydroxycholesterol 1.7× 10
−3
7.4
24(S)-Hydroxycholesterol 1.8 7.3
5α,6α-Epoxycholesterol 1.1× 10
−2
6.3
Desmosterol 8.5 6.1
Cholesterol 200 9.2
Cholestanol 2.1× 10
−3
10.6
S. Ayciriex et al.
As indicated by the FDA, we have also investigated the
stability of some analytes in stock solution including 24(S)-
hydroxycholesterol, 7β-hydroxycholesterol, 5α,6α-epoxy-
cholestanol, cholesterol, and the two internal standards, 24
(R/S)-hydroxycholesterol-d
6
and cholesterol-d
7
. No signifi-
cant changes were observed except for the B-ring substituted
oxysterol, 7β-hydroxycholesterol. This oxysterol is deterio-
rated at room temperature from 7 days. However, no degra-
dation occurs during storage period at −20 °C or −80 °C
(Electronic Supplementary Material, Table S6).
We have also evaluated the stability of the endogenous
oxysterols and sterols in mice brain tissue samples stored at
room temperature, 4 °C for 4 h, and −80 °C for 30 days. The
selected conditions are those frequently encountered in the
laboratory.
In mice brain, besides cholesterol, the major oxysterols
and sterols identified according to our analysis conditions
were 22(R)-hydroxycholesterol, 27-hydroxycholesterol, 24
(S)-hydroxycholesterol, 7β-hydroxycholesterol, 5α,6α-
epoxycholestanol, desmosterol, 7-dehydrocholesterol, and
cholestanol. The results for the stability experiments are
shown in Fig. 3. After 4 h at room temperature, we observed
degradation of cholesterol and an increase in 22(R)-hydrox-
ycholesterol, 7β-hydroxycholesterol, and 5α,6α-epoxycho-
lestanol content.
A stability study was conducted on mice brain tissue ho-
mogenate stored at 4 °C for 4 h and at room temperature for
the same duration. Among the nine oxysterols and sterols
detected in the brain, three different behaviors were observed
(Fig. 3). At 4 °C, a first set of compounds including 24(S)-
hydroxycholesterol, 27-hydroxycholesterol, desmosterol, and
7-dehydrocholesterol was found to be stable. A second group
of analytes (5α,6α-epoxycholestanol, 22(R)-hydroxycholes-
terol, 7β-hydroxycholesterol, and cholestanol) was increased
indicating an oxidative process involving cholesterol and
mediated enzymatically and/or chemically [4,32–36]. The
strong decrease of the cholesterol abundance was in favor of
such a hypothesis. At room temperature, the observed oxida-
tive phenomena are magnified.
We also performed a second stability study on intact
mice brain tissue stored at −80 °C for 1 month. 27-
Hydroxycholesterol, 24(S)-hydroxycholesterol, and choles-
tanol were found to be stable under such conditions. 7β-
Hydroxycholesterol, 22(R)-hydroxycholesterol, and 5α,6α-
epoxycholestanol are increased, whereas cholesterol, desmos-
terol, and 7-dehydrocholesterol are decreased. In order to
minimize oxysterol and sterol concentration change, it is
Fig. 4 Effects of LPS treatment
in 25-hydroxycholesterol con-
tents in cortex tissue (A) and in
7-dehydrocholesterol, cholesta-
nol, and desmosterol contents in
hippocampus tissue (B). The
results are expressed as the
mean of a triplicate of six tissue
samples. Errors bars SD. (n06;
*p<0.05; ***p< 0.001)
Table 4 Recovery of the major oxysterols and sterols from mice brain
Oxysterols and sterols Amount added
(nmol/mg proteins)
Recovery (%)
(n012)
CV
(%)
27-Hydroxycholesterol 2×10
−3
97 11
4×10
−3
102 9
6×10
−3
109 12
25-Hydroxycholesterol 2×10
−3
102 12
4×10
−3
98 9
6×10
−3
107 10
24(S)-Hydroxycholesterol 2.5 104 10
5 107 8
7.5 85 11
5α,6α-Epoxycholestanol 0.01 87 8
0.02 104 9
0.03 99 7
Desmosterol 3.5 99 10
7 101 14
9 105 13
Cholesterol 350 99 12
700 96 7
1,000 109 8
Cholestanol 2×10
−3
102 8
4×10
−3
99 12
6×10
−3
111 9
Quantification of sterols and oxysterols by UPLC-ESI-HRMS
thus recommended to treat samples immediately after tissue
withdrawal.
Precision in biological matrix and recovery
The precision of the quantification method was investigated
on mice brain samples by analyzing four samples in tripli-
cate by UPLC-ESI-HRMS. The precision for each oxysterol
and sterol was calculated and the CV % was lower than
15 % (Table 3).
For the recovery experiment, known amounts of oxysterols
and sterols were spiked into the same amount of mice brain
homogenate before sample preparation. The recoveries of the
known spiked amounts of the oxysterols ranged from 85 to
111 % and from 96 to 109 % for sterols with a CV less than
15 % (Table 4). No matrix effects were found.
Application of the method to a neuroinflammation model
The proposed methodology was applied to a neuroinflamma-
tion model consisting in rats treated with LPS. We investigat-
ed the oxysterol and sterol profiles from brain hippocampus
and cortex of rats treated with LPS and compared them to
controls (saline buffer injection) (Electronic Supplementary
Material, Fig. S5).
Briefly, oxysterols and sterols were extracted from tissues
with an optimized lipid extraction procedure and diluted
prior to derivatization. Subsequent to derivatization, oxy-
sterols and sterols detected in the brain tissue samples were
identified and quantified as their carbamate derivatives by
comparison with commercial standard using UPLC combined
with HRMS.
Rats treated with LPS exhibit a significant decrease in
25-hydroxycholesterol in cortex tissue compared to rats
treated with saline buffer (3.88±0.43 ng/mg of tissue vs
6.04±0.62 ng/mg) (Fig. 4A). Hippocampus samples of rats
treated with LPS exhibit an increase in 7-dehydrocholesterol
(0.34±0.04 μg/mg vs 0.19±0.04 μg/mg), desmosterol (3.74±
0.21 μg/mg vs 1.90±0.22 μg/mg), and cholestanol (39.99±
1.77 ng/mg vs 22.45±1.74 ng/mg) content compared to con-
trol (Fig. 4B). No other significant differences were observed
between treated and control rat brain tissue (Electronic Sup-
plementary Material, Table S7). In this work we showed that
LPS treatment induces an increased abundance of three cho-
lesterol precursors in hippocampus (7-dehydrocholesterol,
cholestanol, and desmosterol) which are considered as
markers of the synthesis rate of cholesterol. In contrast, 25-
hydroxycholesterol was decreased in the cortex after LPS
treatment. It has been shown previously that an increased
production of 25-hydroxycholesterol by macrophages oc-
curred in healthy volunteers receiving an intravenous injection
of LPS [37]. 25-Hydroxycholesterol is known to stimulate the
release of pro-inflammatory cytokines in several cellular
systems [38,39] and is a well-known bioactive oxysterol
involved in lipid metabolism regulation and inflammatory
processes [37]. The reduced level of 25-hydroxycholesterol
content observed in the cortex may suggest that 25-
hydroxycholesterol is able to pass through the blood–brain
barrier. This hypothesis was supported by our preliminary
results showing reduced blood–brain barrier integrity in these
LPS-treated rats.
Conclusion
We developed a novel, simple, and robust method for the
quantitative analysis of oxysterols and sterols. It involves the
easy and selective conversion of C3-OH in a carbamate deriv-
ative by DMAPI. The aromatic amino group introduced pre-
vents in-source fragmentation and leads exclusively to
protonated molecules under electrospray conditions. The pro-
posed method enables the simultaneous detection and quanti-
fication of 11 oxysterols and sterols in a mixture by UPLC-
ESI-HRMS. The method was successfully validated according
to the FDA recommendations and exhibited good sensitivity,
stability, and repeatability for all the oxysterols and sterols
analyzed. Moreover, it illustrates the use of quadrupole time-
of-flight instruments in quantitative analysis of low abundance
bioactive lipids, demonstrating the advantageous use of the
enhanced duty cycle mode to significantly increase the sensi-
tivity of bioanalytical methods over a defined mass range.
This method can be widely used for the quantification in
biological samples (brain tissue, plasma, cerebrospinal flu-
id) of oxysterol and sterol biomarkers involved in neurode-
generative diseases.
Acknowledgment The post-doctoral position of S.A. was funded by
ANR Chol AD (French-Canadian Cooperation-2010-MALZ-10303)
and the PhD position of M.G. by Technologie Servier (Orléans,
France). O.L. is indebted to Fondation pour la Recherche Médicale,
Région-Île-de-France and Centre National de la Recherche Scientifique
for their financial support. We thank Fathia Djelti (INSERM U745) for
providing the C57BL/6 mice brain tissue.
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Quantification of sterols and oxysterols by UPLC-ESI-HRMS
