Journal of Chromatography A, 1364 (2014) 214–222

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Analysis of oxysterols and vitamin D metabolites in mouse brain and cell line samples by ultra-high-performance liquid chromatography-atmospheric pressure photoionization–mass spectrometry Linda Ahonen a , Florian B.R. Maire b , Mari Savolainen c , Jaakko Kopra c , Rob J. Vreeken b,d , Thomas Hankemeier b,d , Timo Myöhänen c , Petri Kylli a,∗∗ , Risto Kostiainen a,∗ a

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014, Finland Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands c Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014, Finland d Netherlands Metabolomics Centre, Leiden University, P.O. Box 9502, 22300 RA Leiden, The Netherlands b

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Article history: Received 25 June 2014 Received in revised form 22 August 2014 Accepted 26 August 2014 Available online 2 September 2014 Keywords: Liquid chromatography–mass spectrometry Atmospheric pressure photoionization Vitamin D Oxysterols Cell line samples Mouse brain samples

a b s t r a c t We have developed an ultra-high-performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometric (UHPLC-APPI–MS/MS) method for the simultaneous quantitative analyses of several oxysterols and vitamin D metabolites in mouse brain and cell line samples. An UHPLC-APPI-high resolution mass spectrometric (UHPLC-APPI-HRMS) method that uses a quadrupoletime of flight mass spectrometer was also developed for confirmatory analysis and for the identification of non-targeted oxysterols. Both methods showed good quantitative performance. Furthermore, APPI provides high ionization efficiency for determining oxysterols and vitamin D related compounds without the time consuming derivatization step needed in the conventionally used electrospray ionization method to achieve acceptable sensitivity. Several oxysterols were quantified in mouse brain and cell line samples. Additionally, 25-hydroxyvitamin D3 was detected in mouse brain samples for the first time. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Vitamin D is well known for its importance in various functions in the human body and vitamin D deficiency or insufficiency has long been known to be a risk factor for bone metabolic diseases, such as rickets, osteomalacia, and osteoporosis [1,2]. More recently, it was shown, that vitamin D deficiency is also involved in other diseases such as certain cancers (for example leukemia), autoimmune diseases and neurodegenerative diseases [1,3,4]. Vitamin D exists naturally either as vitamin D2 or as vitamin D3 . The

∗ Corresponding author at: Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland. Tel.: +358 294159134; fax: +358 294159566. ∗∗ Corresponding author at: Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland. Tel.: +358 294159453; fax: +358 294159566. E-mail addresses: linda.ahonen@helsinki.fi (L. Ahonen), [email protected] (F.B.R. Maire), mari.savolainen@helsinki.fi (M. Savolainen), jaakko.kopra@helsinki.fi (J. Kopra), [email protected] (R.J. Vreeken), [email protected] (T. Hankemeier), timo.myohanen@helsinki.fi (T. Myöhänen), petri.kylli@helsinki.fi (P. Kylli), risto.kostiainen@helsinki.fi (R. Kostiainen). http://dx.doi.org/10.1016/j.chroma.2014.08.088 0021-9673/© 2014 Elsevier B.V. All rights reserved.

main dietary sources of D2 and D3 are mushrooms and fish, respectively [1,5]. Vitamin D3 is additionally biosynthesized in the skin from its precursor 7-dehydrocholesterol after exposure to ultraviolet light. Vitamin D2 , on the other hand, is supplied only via dietary sources or from supplements [6]. Vitamin D as such is biologically inactive but it is hydroxylated in the liver to 25-hydroxyvitamin D (25-OH-D), which is the major circulating form in the body [1,2,5]. 25-OH-D is then further metabolized in the kidney to the biologically active form, 1␣,25-dihydroxyvitamin D (1␣,25-OH-D). Additionally numerous other metabolites of vitamin D have been well known for decades and the metabolism route has previously been presented thoroughly [2,7]. Cholesterol is the precursor of hormonal steroids and bile acids, and it can be detected at high levels in the blood, brain and other steroidogenic tissues [8,9]. Cholesterol is also the starting material for the synthesis of oxysterols. The oxysterols can be formed enzymatically in phase I metabolism of cholesterol [8,10] and directly from cholesterol by reactive oxygen species [8,11]. Recently, oxysterols were shown to be biologically active molecules [8,12,13]. Structurally different oxysterols have specific characteristic biological activities whereas the same oxysterols might have different

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activities in different cells [8]. There is also evidence of changes in the quantities of oxysterols (higher or lower amounts depending on the compound) as a result of Alzheimer’s disease compared to healthy subjects [14,15]. Numerous methods for analyzing vitamin D (and its metabolites) and oxysterols in biological samples have been described previously [16–19]. Many of the conventional methods are based on gas chromatography (GC) [8,20] or gas chromatography–mass spectrometry (GC–MS) [16,21], high performance liquid chromatography (HPLC) [16,19,21] or immunological methods [19,21,22]. The selectivity or quantitative performance of HPLC and immunological methods may, however, be limited in the analyses of complex biological samples [19,22,23]. GC–MS provides high selectivity and sensitivity, but requires time-consuming derivatization of the analytes [16,20,21]. LC–MS using electrospray ionization (ESI) [16,17] and atmospheric pressure chemical ionization (APCI) [16,17,24] are increasingly used in the analysis of vitamin D and oxysterols. ESI based methods also often require time-consuming derivatization procedures, because the ionization efficiency of the non-polar vitamin D and oxysterols as such is poor [16,17]. Derivatization is nowadays also often used in APCI based methods in order to enhance the sensitivity. Atmospheric pressure photoionization (APPI), on the other hand, provides high sensitivity for both classes of compounds without any additional derivatization steps [18,25,26]. This makes the LC-APPI–MS methods faster and easier to implement compared to ESI or APCI methods using derivatization for achieving the required sensitivity. In this work we describe an ultra-high-performance liquid chromatography (UHPLC)-APPI–MS/MS and an UHPLC-APPI-HRMS method for the simultaneous analyses of several oxysterols and vitamin D derived compounds in biological samples. Both methods provide simple, fast, and sensitive analyses of these compounds without recourse to time-consuming derivatization procedures. As oxysterols and vitamin D may have a role in neurodegenerative diseases, the UHPLC-APPI–MS/MS method was developed for the analysis of these compounds in mouse brain samples. In order to study the role of oxidation of cholesterol in neurodegenerative diseases non-stressed cell line samples were compared to cell line samples that had been exposed to oxidative stress and thus were overexpressing alpha-synuclein (␣-syn) aggregates (Parkinson’s disease aggregates). This comparison is expected to provide information on the role of oxysterols in the formation of the aggregates. Furthermore, the performances of UHPLC-APPI–MS/MS and UHPLC-APPI-HRMS are compared in the analysis of cell line samples.

2. Materials and methods 2.1. Chemicals The water was purified using a Milli-Q purification system (Millipore, Molsheim, France). LC–MS grade methanol (MeOH), isopropanol (IPA), and acetonitrile (ACN), and HPLC grade dichloromethane (DCM) and toluene were purchased from Sigma-Aldrich (Steinheim, Germany). The following compounds were also purchased from Sigma-Aldrich: progesterone (PROG, 4-pregnene-3,20-dione), deuterated vitamin D3 (d3 D3 , 6,19,19-deuterated cholecalciferol), 7-dehydrocholesterol (7-DHYD, 3␤-hydroxy-5,7-cholestadiene), vitamin D2 (D2 , ergocalciferol), 1␣,25-dihydroxyvitamin D3 (1␣,25-OH-D3 , 1␣,25dihydroxycholecalciferol), and 25-hydroxyvitamin D3 (25-OH-D3 , 25-hydroxycholecalciferol). Vitamin D3 (D3 , cholecalciferol) was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA), 27-hydroxycholesterol (27-OH-Chl, cholest-5-ene-3␤,26diol) from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany),

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24(S)-hydroxycholesterol (24-OH-Chl, cholest-5-ene-3␤,24␣-diol) from AH diagnostics Oy (Helsinki, Finland), desmosterol (DESMO, 3␤-hydroxy-5,24-cholestadiene), 7-ketocholesterol (7-OXO, 3␤-hydroxy-5-cholesten-7-one) and 27-hydroxycholesterol-d6 (d6 -27-OH-Chl, 25,26,26,26,27,27-hexadeuterocholest-5-ene3␤,27-diol) from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and 7␣-hydroxycholesterol (7␣-OH-Chl, 5-cholesten-3␤,7␣-diol), 7␤-hydroxycholesterol (7␤-OH-Chl, 5-cholesten-3␤,7␤-diol) and 22(S)-hydroxycholesterol (22-OH-Chl, 5-cholesten-3␤,22(S)-diol) from Fountain Limited (Naxxar, Malta). The structures of the compounds studied appear in Fig. 1. 2.2. Samples 2.2.1. Animals Three months old male NMRI-mice were used in this study. The mice were kept in a 12/12 light/dark cycle at ambient temperature (20–22 ◦ C), and between 2 and 5 animals per cage. All mice were given ad libitum access to standard mouse chow and to tap water. The protocol was accepted by the Animal Experiment Board of Finland and experiment was performed in accordance with Finnish legislation. The mice were anaesthetized using sodium pentobarbital (100 mg kg−1 , i.p.) and then transcardially perfused with phosphate buffered saline (PBS) solution for 4 min to purge all the blood out of the brain. Afterwards the brains were removed from the skull, frozen on dry ice and stored in −80 ◦ C until assayed. 2.2.2. Cell line samples Wild type (WT) SH-SY5Y human neuroblastoma cell line was purchased from ATCC (LGC Standards; Product # CRL-2266, Middlesex, UK) and cultured as described by Myöhänen et al. [27]. Stable cell lines expressing A30P and A53 T ␣-syn were generated using a lentiviral vector as described by Gerard et al. [28], and transfected cells were selected by their resistance to puromycin. The ␣-syn overexpressing cells were cultured as described by Myöhänen et al. [27]. Cell lines were used at passages 3–15 and grown at 37 ◦ C and 5% CO2 in a humidified atmosphere. In the oxidative stress treatment, 1 × 106 cells were seeded in T25-flasks and allowed to grow overnight. Thereafter, the aggregation process of ␣-syn was induced by adding 100 ␮M H2 O2 and 10 mM FeCl2 in the cell culturing medium for 3 days as previously described [27,28]. All cells (non-stressed and stressed) were homogenized as described by Myöhänen et al. using a 0.1 M Na–K-phosphate buffer at pH 7.0 [27]. The homogenates were centrifuged at 16,000 × g, at 4 ◦ C, for 20 min, and thereafter the supernatant (soluble fraction) and pellet were separated and stored at −80 ◦ C for further analysis. 2.3. Sample preparation Stock solutions (1.0 mg mL−1 ) of the analytes were prepared by dissolving the analytes in MeOH. The working standard solutions were prepared by diluting the stock solutions in MeOH to the appropriate concentrations. The standard solutions were used to prepare a calibration curve with the following concentration levels: 0.5, 1.0, 2.5, 5.0, 7.5, 10, 15, 25, 35, 50, 100, 250, 500 and 1000 ng mL−1 . 22 ␮L of a 1 ␮g mL−1 internal standard working solution containing d3 D3 , and d6 -27-OH-Chl was added to 198 ␮L of each concentration level. Intra- and inter-day repeatabilities were measured using a freshly prepared working standard solution at a concentration level of 100 ng mL−1 . 2.3.1. Brain samples The intact mouse brains were weighed and cut into four equal parts. A 2.5 ␮L volume of the 1 ␮g mL−1 internal standard working solution was added to each brain part. The samples

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Fig. 1. Structures of the studied compounds. For abbreviations, see Section 2.1.

were then homogenized and extracted by liquid-liquid extraction (LLE). The procedure was as follows: 0.5 mL of a DCM:MeOH mixture (1:1, v/v) was added to the samples, which were homogenized using ultrasonication by a Branson Sonifier 450 (Branson

Ultrasonics Corporation, Danbury, CT, USA) in an ice bath for 1 min. The ultrasonication had an amplitude and efficiency of 90% and 2, respectively. The samples were subsequently centrifuged (at 13,200 rpm for 5 min), the supernatants were removed and the

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procedure was repeated once. After the second extraction process, the supernatants of the four equal parts of the mouse brain were combined into one sample, which was evaporated to dryness (N2 , 40 ◦ C). The samples were reconstituted in 100 ␮L of MeOH, then centrifuged (at 13,200 rpm for 5 min) and the supernatants were pipetted into a vial with a glass insert immediately before analysis. 2.3.2. Cell line samples 10 ␮L of the 1 ␮g mL−1 internal standard working solution was added to an eppendorf tube that included the cell pellet and the samples were homogenized and extracted using 0.5 mL of a DCM:MeOH mixture (2:1, v/v) following the same procedure as for the brain samples. Finally, the supernatants were combined, evaporated to dryness (N2 , 40 ◦ C) and reconstituted in 100 ␮L of MeOH before analysis. 2.4. Ultra performance liquid chromatography (UPLC) Two different UPLC systems were used in this work. The system used with the triple quadrupole mass spectrometer was a Waters ACQUITY UPLC® I Class (Waters, Milford, MA, USA) and the system used with the quadrupole-time of flight mass spectrometer was a Waters ACQUITY UPLC® (Waters). Both UPLC systems were equipped with a sample manager (maintained at 8 ◦ C), a binary solvent manager and a column thermostat (maintained at 25 ◦ C). Separations were performed in 2.1 mm × 100 mm C18-columns. The column used for the triple quadrupole system was packed with 1.7 ␮m particles (ACQUITY UPLC® BEH C18, Waters) and the column used for the quadrupole-time of flight system was packed with 1.8 ␮m particles (ACQUITY UPLC® HSS C18, Waters). The flow rate was 0.3 mL min−1 and the injection volume was 5 ␮L (triple quadrupole system experiments) or 7.5 ␮L (quadrupole-time of flight system experiments). Water (A) and MeOH (B) were used as the mobile phases for the gradient elution. The gradient was as follows: from 0 to 14 min 80–100% B and from 14 to 17 min 100% B. Each run was followed by a five min washout period, whereby the column was flushed with 10% DCM in MeOH. After this, the column was re-equilibrated for 6 min under initial conditions (80% B). 2.5. Mass spectrometry Two different mass spectrometers were also used in this work. One was a triple quadrupole system, Waters® XevoTM TQ-S MS, interfaced with a ZsprayTM Atmospheric Pressure Photoionization/Atmospheric Pressure Chemical Ionization (APPI/APCI) dual source (Waters). The analytes were ionized by APPI in positive ion mode with toluene as the dopant. The toluene flow was kept constant at 20 ␮L min−1 and it was introduced into the mass spectrometer via a Agilent series 1100 capillary pump with a degasser (Agilent Technologies, Waldbronn, Germany). Nitrogen was used as the nebulizing gas at a pressure of 4.0 bar and as the desolvation gas at a flow rate of 1000 L h−1 . Argon was used as the collision gas at a flow rate of 0.15 mL min−1 . The probe temperature was maintained at 450 ◦ C and the APPI lamp repeller at 0.5 kV. Cone gas was set at 150 L h−1 and cone voltage at 6 V. MS-spectra (scan range m/z 200–500) and MS/MS-spectra (scan range m/z 50–500) were acquired for each analyte in order to select the best precursor and product ions for selected reaction monitoring (SRM) experiments. The collision energies (CE) were separately optimized for each ion transition (Table S1 in Supplementary material). The other mass spectrometer was a SYNAPT G2-S HDMS (Waters, Wilmslow, UK) interfaced with a similar dual source as the triple quadrupole. The analytes were ionized by APPI in positive ion mode using 0.1 ␮g mL−1 progesterone in toluene, at a flow rate of 30 ␮L min−1 , as the lock mass and dopant, respectively. Nitrogen was used as the nebulizing gas at a pressure of 6.0 bar and as

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the desolvation gas at a flow rate of 800 L h−1 . The probe temperature was 450 ◦ C and the APPI lamp repeller voltage was 0.5 kV. Cone voltage was set at 10 V and the acquisition mass range was m/z 50–1200. The instrument was run in sensitivity mode, i.e. resolution of 10,000 FWHM measured on the [M+6H]6+ isotope cluster from bovine insulin (m/z 956). The CE used for the MSe experiments were 10 eV and 30 eV for the low and high energy function, respectively. MassLynx 4.1 (Waters) was used for all data acquisition and processing. 3. Results and discussion 3.1. LC–MS The APPI–MS spectra show abundant protonated molecules ([M+H]+ ) and/or fragment ions formed by the loss of one ([M+H–H2 O]+ ), two ([M+H–2H2 O]+ ) or three ([M+H–3H2 O]+ ) water molecule(s) (Table S2 in Supplementary material). The spectra of the analytes having only one hydroxyl group and conjugated double bonds in their structures (7-OXO, D2 , D3 , and d3 -D3 ) showed intense [M+H]+ as the main ion, whereas the fragments [M+H–H2 O]+ or [M+H–2H2 O]+ were the main ions for all the other analytes. The number of fragmented water molecules correlated with the number of hydroxyl groups present in the analyte. Based on the MS spectra, the most abundant precursor ions for the SRM experiments were chosen in order to maximize the sensitivity. The MS/MS spectra (Fig. S1 in Supplementary material) show product ions formed by the loss of one or two water molecules ([M+H–H2 O]+ , [M+H–2H2 O]+ ) and a number of product ions at lower m/z values formed by the dissociation of the ring structures of the oxysterols and vitamin D based compounds. Product ions for the SRM experiments were chosen according to highest intensity and optimal selectivity and the collision energies were separately optimized for each ion transition (Table S1 in Supplementary material). The UHPLC method shows good chromatographic performance (Fig. 2). The plate numbers (N) and tailing factors (As ) were within acceptable values (∼40,000 and

Analysis of oxysterols and vitamin D metabolites in mouse brain and cell line samples by ultra-high-performance liquid chromatography-atmospheric pressure photoionization-mass spectrometry.

We have developed an ultra-high-performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometric (UHPLC-APPI-MS/MS) me...
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