Development of a novel method for quantification of

Anal Bioanal Chem DOI 10.1007/s00216-012-6396-6

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 metabolites identification and quantification in biological samples. However, sterols and oxysterols are relatively apolar molecules 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-offlight 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 stability, and recovery according to the Food and Drug Administration (FDA) guidelines. The developed method was successfully applied to investigate the impact of lipopolysaccharide (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: [email protected]

E. Prost UMR 8638 CNRS, Université Paris Descartes, Sorbonne Paris Cité, Faculté des Sciences Pharmaceutiques et Biologiques, 75006 Paris, France

M. Gaudin Division métabolisme, Technologie Servier, 45000 Orléans, France

O. Laprévote Service de Toxicologie Biologique, Hôpital Lariboisière, 4 rue Ambroise Paré, 75475 Paris cedex 10, 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

Present Address: M. Gaudin Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, SW7 2AZ London, UK

S. Ayciriex et al.

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)-hydroxycholesterol (24-OHC) by CYP46A1, a cytochrome P-450 enzyme, expressed in neurons. In contrast to cholesterol, 24OHC 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 inflammation 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 identification remains difficult [14]. To date, LC-MS is the most frequently used technique for the identification and quantification of sterols and oxysterols in complex biological samples including brain tissue sample, serum, and cerebrospinal 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]. However, these techniques lead to dehydrated protonated molecules making the determination of the molecular weight of the analyte sometimes difficult [19, 20]. On the contrary, 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 sensitivity 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)trimethylazanium chloride (Girard reagent), N,N-dimethylglycyl, picolinoyl 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 procedure using enzymatic conversion of the 3′-hydroxyl moiety into a ketone before reacting 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. Recently, 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 4dimethylaminopyridine [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,Ndimethylamino)phenyl carbamates obtained are chromatographically resolved after 6 min of chromatographic run and readily ionized by ESI with high efficiency. In particular, side chain hydroxylated derivatives 22(R)-hydroxycholesterol, 27hydroxycholesterol, 25-hydroxycholesterol, and 24(S)hydroxycholesterol were efficiently separated and exclusively detected as quasi-molecular ions by high resolution mass spectrometry (HRMS). The method was rigorously validated 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], 27hydroxycholesterol [cholest-(25R)-5-ene-3β,27-diol], 25hydroxycholesterol [cholest-5-ene-3β,25-diol], 24(S)hydroxycholesterol [5-cholestene-3β,24-diol], 24(R/S)hydroxycholesterol (d6) [26,26,26,27,27,27-hexadeuterocholest-5-ene-3β,24-diol], 5α,6α-epoxycholestanol [cholestanol, 5α,6α-epoxy], 7β-hydroxycholesterol [cholest-5en-3β,7β-diol], desmosterol [3β-hydroxy-5,24-cholestadiene], 7-dehydrocholesterol [Δ5,7-cholesterol], lathosterol [5α-cholest-7-en-3β-ol], cholestanol [5α-cholestan-3β-ol], cholesterol (d7) [cholest-5-en-3β-ol(d7)] were purchased from Avanti Polar Lipids (Alabaster, AL, USA) (Electronic Supplementary Material, Fig. S1). Cholesterol, triethylamine, 4-(dimethylamino)phenyl isocyanate (DMAPI),

Quantification of sterols and oxysterols by UPLC-ESI-HRMS

formic acid, lipopolysaccharide (LPS) from Salmonella enterica serotype typhimurium were purchased from SigmaAldrich (Saint-Quentin Fallavier, France). Hexane and dichloromethane were obtained from Carlo Erba Reactifs SDS (Val-de-Reuil, France). Acetonitrile, methanol, and isopropanol 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, 27hydroxycholesterol, 25-hydroxycholesterol, 24(S)-hydroxycholesterol, 24(R/S)-hydroxycholesterol (d6) (oxysterols IS), 7β-hydroxycholesterol, 5α,6α-epoxycholestanol, desmosterol, 7-dehydrocholesterol, lathosterol, cholesterol, cholesterol (d7) (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; 0.017–0.33–5 μM) 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.

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 compounds 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 μL was injected into the UPLC-ESI-HRMS system. UPLC-ESI-HRMS analysis

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.

The separation of sterols and oxysterols derivatives was achieved using an Acquity UPLC system (Waters, Milford, MA, USA) equipped with an Acquity UPLC CSH™ C18 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). Electrospray positive ion mode was used. The ESI source conditions 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/z 565.436 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 enkephalin (2 ng/μL in acetonitrile/water, 50:50, v/v).

Sample preparation: derivatization procedure

NMR spectra

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 homogeneization was performed for 15 s at 5,000 rpm (Precellys®24-Dual apparatus). Sterols were extracted with hexane/methanol mixture (7:1, v/v).

All NMR experiments were recorded on a Bruker AVANCE400 MHz at 400 MHz and 75 MHz for 1H and 13C, respectively, 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 CDCl3. Assignments were

LPS rat treatment

S. Ayciriex et al.

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). TargetLynx software was used to determine peak areas of compounds of interest (Waters MS Technologies, Manchester, UK). Method validation Method validation was performed according to the recommendations 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 determined using six determinations per concentration. Three QC concentrations level samples were used: low, middle, and high QC samples according to the concentration range. Precision is expressed as the relative coefficient of variation (CV%) according to the following formula: CV % ¼ ðstandard deviation=meanÞ 100 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 ð%Þ ¼ ðmeasured value theoretical valueÞ=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

Fig. 1 Reaction scheme for cholesterol derivatization with DMAPI

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 parameters (storage time and temperatures). The concentrations determined immediately after solubilisation and derivatization, respectively, for native and derivatized oxysterol and sterol (time 0) were assigned as C0. Stability was expressed as percentage change in mean concentration from C0 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 temperature 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 (Rx) and compared to the one obtained without brain storage (R0). Results were expressed as ðRx =R0 Þ 100: 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 concentrations close to the expected endogenous concentrations. Recovery was calculated as ðamount found after spiking endogenous amountÞ= amount added 100 Biological data were analyzed with Student’s two-tailed unpaired t test 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. 2 Chromatographic separation of derivatized sterols and oxysterols mixture (1 μM) in a 6-min run (A): a 22(R)-hydroxycholesterol (tR 0 2.62 min; m/z 565.436); b 27-hydroxycholesterol (tR 02.85 min; m/z 565.436); c 25-hydroxycholesterol (tR 02.99 min; m/z 565.436); d 24(S)hydroxycholesterol (tR 03.13 min; m/z 565.436); e 7β-hydroxycholesterol (tR 03.72 min; m/z 565.436); f 5β,6β-epoxycholestanol (tR 02.62 min; m/z 565.436); g 5α,6α-epoxycholestanol (tR 04.88 min; m/z 565.436); h desmosterol (tR 05.28 min; m/z 547.426); i 7-dehydrocholesterol (tR 0 5.41 min; m/z 547.426); j lathosterol (tR 05.66 min; m/z 549.441); k cholesterol (tR 05.72 min; m/z 549.441); l cholestanol (tR 05.94 min; m/z 551.457)]

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 reactivity 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 generated. 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)-hydroxycholesterol and cholesterol carbamate was synthesized and NMR experiments (COSY, HSQC, and HMBC) were performed on the carbamate derivative and on 22(R)-hydroxycholesterol 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 solvents (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 biological samples (Electronic Supplementary Material, Fig. S2). As a result, the derivatization was performed by adding 200 μL of 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 selected 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

Linear regression equation

Correlation coefficient (R2)

LOQ (μM)

Linear dynamic range (μM)

22(R)-Hydroxycholesterol

1.7×10−3

7.35×102

y=20.696x+0.021

0.998

27-Hydroxycholesterol 25-Hydroxycholesterol 24(S)-Hydroxycholesterol 7β-Hydroxycholesterol 5α,6α-Epoxycholestanol Desmosterol 7-Dehydrocholesterol Lathosterol Cholesterol Cholestanol

1.7×10−3 1.7×10−3 1.7×10−3 3×10−4 3.3×10−3 3.3×10−3 1.7×10−2 3.3×10−3 3.3×10−3 3.3×10−3

7.35×102 7.35×102 5.57×103 1.51×103 1.51×103 1.51×103 2.99×102 1.51×103 1.51×103 1.51×103

y=10.448x+0.001 y=12.947x+0.002 y=8.907x+0.004 y=3.175x+0.006 y=2.894x+0.059 y=5.144x+0.003 y=2.656x−0.001 y=9.756x+0.021 y=4.48x+0.04 y=2.989x+0.014

0.998 0.999 0.997 0.995 0.999 0.998 0.995 0.999 0.999 0.996

S. Ayciriex et al.

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 QTOF 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 signalto-noise ratio by a factor of 10 [31]. We applied this function centered on m/z 565.4 during the first 5 min of run time and on Table 2 Repeatability of the quantification of oxysterols and sterols in QC samples

Oxysterols and sterols

22(R)-Hydroxycholesterol

27-Hydroxycholesterol


24(S)-Hydroxycholesterol

7β-Hydroxycholesterol

5α,6α-Epoxycholesterol

Desmosterol

7-Dehydrocholesterol

Lathosterol

Cholesterol

Cholestanol

m/z 549.4 from 5 to 6 min corresponding to the carbamate quasi-molecular ion [M+H]+ of oxysterols and sterols, respectively. On average we obtained a 7-fold increase of the signalto-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 fragmentation. In contrast, the picolinate esters derivatives lead to the loss of picolinoyl in our ESI source conditions [22, 23].

QC concentrations (μM) Targeted concentration

Measured concentration

Precision (CV%)

Accuracy (RE%)

0.0017 0.17 1.25 0.0017 0.17

0.0017±0.000 0.171±0.005 1.253±0.009 0.0017±0.000 0.172±0.007

1.28 3.06 0.69 2.27 3.88

−1.36 0.48 0.20 1.47 1.07

1.25 0.0017 0.17 1.25 0.0017 0.17 1.25 0.017 0.33 5 0.017 0.33 5 0.017 0.33 5 0.017 0.33

1.248±0.020 0.0017±0.000 0.168±0.007 1.257±0.022 0.0017±0.000 0.171±0.005 1.257±0.020 0.017±0.000 0.332±0.011 5.012±0.067 0.017±0.000 0.334±0.008 5.048±0.064 0.017±0.000 0.3342±0.0148 5.0508±0.20 0.017±0.000 0.3320±0.0142

1.55 2.39 4.08 1.75 2.65 3.06 1.58 2.74 3.40 1.33 3.33 2.33 1.28 4.96 4.43 3.94 5.01 4.28

−0.16 0.32 −1.22 0.58 −0.22 0.48 0.55 0.34 0.67 0.23 −0.88 1.34 0.97 −0.49 1.26 1.02 −1.58 0.60

5 0.017 0.33 5 0.017 0.33 5 0.017 0.33 5

5.0314±0.1450 0.017±0.000 0.34±0.01 5.04±0.14 0.017±0.000 0.34±0.02 5.09±0.15 0.017±0.000 0.34±0.016 4.95±0.202

2.88 4.70 4.40 2.83 4.48 4.83 3.03 4.98 4.88 4.08

0.63 −0.38 2.46 0.74 −0.80 3.53 1.72 −2.08 1.52 −0.97


The specificity of the SRM method between two adducts is thus limited. In our source conditions, these picolinate derivatives are detected as a mixture of protonated and sodiumcationized molecules, thus reducing sensitivity. The dimethylglycine 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 separation of 22(R)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24(S)-hydroxycholesterol. The

Fig. 3 Stability of endogenous oxysterol and sterol compounds in mice brain 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%

S. Ayciriex et al.

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. S3 and 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-d6 and cholesterol-d7, respectively, derivatized and analyzed as described in the “Materials and methods” section. The peak area of the oxysterols and sterol carbamate derivatives normalized to the deuterated analogue was plotted against the corresponding oxysterol and sterol concentrations. The linearity of the standards curves, as determined by simple linear regression, exhibit an R2 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 7dehydrocholesterol. 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 concentration 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 precision ranged from 2.83 to 4.96 % and the accuracy 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 extraction 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 carbamate derivatives in the mixture were stable with no significant 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)

27-Hydroxycholesterol 25-Hydroxycholesterol 24(S)-Hydroxycholesterol 5α,6α-Epoxycholesterol Desmosterol Cholesterol Cholestanol

1.9×10−3 1.7×10−3 1.8 1.1×10−2 8.5 200 2.1×10−3

CV (%)

8.9 7.4 7.3 6.3 6.1 9.2 10.6

Quantification of sterols and oxysterols by UPLC-ESI-HRMS Table 4 Recovery of the major oxysterols and sterols from mice brain Oxysterols and sterols

Amount added (nmol/mg proteins)



24(S)-Hydroxycholesterol

5α,6α-Epoxycholestanol

Desmosterol

Cholesterol

Cholestanol

Recovery (%) (n012)

CV (%)

2×10−3 4×10−3 6×10−3 2×10−3 4×10−3 6×10−3 2.5 5 7.5 0.01 0.02 0.03 3.5

97 102 109 102 98 107 104 107 85 87 104 99 99

11 9 12 12 9 10 10 8 11 8 9 7 10

7 9 350 700 1,000 2×10−3

101 105 99 96 109

14 13 12 7 8

102 99 111

8 12 9

4×10−3 6×10−3

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-epoxycholestanol, cholesterol, and the two internal standards, 24 (R/S)-hydroxycholesterol-d6 and cholesterol-d7. No significant changes were observed except for the B-ring substituted oxysterol, 7β-hydroxycholesterol. This oxysterol is deteriorated at room temperature from 7 days. However, no degradation occurs during storage period at −20 °C or −80 °C (Electronic Supplementary Material, Table S6). Fig. 4 Effects of LPS treatment in 25-hydroxycholesterol contents in cortex tissue (A) and in 7-dehydrocholesterol, cholestanol, 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