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Spectroscopic characterisation of dimeric oxidation products of phytosterols
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Ewa Sosińska1*, Roman Przybylski2, Felix Aladedunye3, Paul Hazendonk2
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Institute of Biochemistry and Biophysics Polish Academy of Sciences, 02-106 Warsaw, Poland
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Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4,
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Canada
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Max Rubner-Institut (MRI), Federal Research Institute for Nutrition and Food, D-32756 Detmold,
Germany
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* Corresponding author:
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E.Sosińska
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e-mail: esosinska@ibb.waw.pl
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mailing address: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Department of
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Lipid Biochemistry, Pawinskiego 5a, 02-106 Warsaw
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telephone number: +48 22 592 35 01
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fax number: +48 22 592 21 90
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Abstract
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Sterol dimers are the main oxidation products formed during sterols degradation at elevated temperatures.
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An investigation was carried out to decipher the structure of dimers differing in polarity, formed during β-
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sitosterol thermo-oxidation. The oxidation products were fractionated using silica gel into non-polar (NP),
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mid-polar (MP) and polar fractions (P). Oligomers were further separated by size-exclusion
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chromatography (SEC). Tentative chemical structures of non-polar, mid-polar and polar dimers were
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identified using Ag+/CIS-MS and APCI-MS procedures after on-line RP-HPLC separation. Further
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structures were verified by NMR and FT-IR spectroscopies.
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Key words
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β-Sitosterol; thermo-oxidation; dimer; disteryl ether; CIS-MS; APCI-MS; NMR; FT-IR
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Abbreviations
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SEC, size-exclusion chromatography; APCI-MS, atmospheric pressure chemical ionisation-mass
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spectrometry; CIS-MS, coordination-ion spray - mass spectrometry; FT-IR, Fourier transform infrared;
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ELSD, evaporative light scattering detector; DEPT, distortionless enhancement by polarization transfer;
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COSY, two-dimensional proton correlation spectroscopy; NOESY, nuclear Overhauser enhancement
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spectroscopy; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond
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correlation; BDE, bond dissociation enthalpy
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1. Introduction
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β-sitosterol is the most abundant plant sterol found in food products such as plant oils, nuts, seeds or fruits
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(Moreau, Whitaker & Hicks, 2002). Due to the ability of plant sterols and stanols to decrease amounts of
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low density lipoprotein-cholesterol in blood, the variety of phytosterol-enriched foods is still growing (de
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Jong, Ros, Ocke &Verhagen, 2008). Phytosterols are susceptible to auto- and/or enzymatic oxidation, and
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their oxidative stability is affected by the presence of unsaturation sites, conditions of oxidation (i.e.
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temperature, time, oxygen availability) and the matrix composition (Dutta & Savage, 2002; Rudzińska,
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Uchman &Wąsowicz, 2005; Winkler, Warner & Glynn, 2007). Recently, Rudzinska, Przybylski and
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Wąsowicz (2009) published the holistic approach to phytosterols’ thermo-oxidation, reporting the
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percentage of all groups of compounds formed during thermo-oxidative degradation of β-sitosterol
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standard at different temperatures (at 60ºC, 120ºC and 180ºC), including volatiles, fragmented sterols,
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oxysterols and oligomers. Amongst sterol oligomers, dimers are the most abundant, however higher-order
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oligomers are also formed (Rudzinska et al., 2009; Lampi, Kemmo, Mäkelä, Heikkinen & Piironen, 2009;
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Rudzinska, Przybylski, Zhao & Curtis, 2010). Recently, formation of oligomers during fatty acids
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cholesteryl esters oxidation at 100ºC and 140ºC was reported (Lehtonen, Lampi, Agalga, Struijs &
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Piironen, 2011).
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During oxidation, sterols are expected to form similar dimers as those observed for fatty acids, which are
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bound by ether (C-O-C) and peroxy (C-O-O-C) when oxygen is in excess, or direct carbon-carbon (C-C)
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linkages in oxygen starvation conditions (Christopoulou & Perkins, 1989; Muizebelt & Nielen, 1996). In
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fact our previous studies proved formation of sterol dimers during thermo-oxidation through ether bridge,
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as 3β,3β'-sitosteryl ether (Fig. 3a) was identified as the predominant non-polar dimer formed during β-
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sitosterol oxidative degradation at 180ºC for 24 h (Sosińska, Przybylski, Hazendonk, Zhao & Curtis,
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2013). Disteryl ethers were mainly investigated as by-products of industrial bleaching and were found in
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vegetables oils and table margarine (Kaufman, Vennekel & Hamza, 1970; Schulte & Weber, 1987). At
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frying conditions polymerization of lipids occurs resulting in changes of properties of the frying oil,
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moreover digestibility of frying fats has been suggested to decrease along with the polymerization (Billek,
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2000). Polymerization of sterols could affect their health-promoting properties, especially in case of the
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cholesterol-lowering plant sterol-fortified food products. No adverse effects of disteryl ethers were
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observed towards mice and rats (Kaufman et al., 1970; Weber, Benning & Schulte, 1988), however this
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dimer has to be considered a dimer of two non-oxidized units. Further consideration has to be addressed
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for possible adverse effects of sterol polymers when they consist of oxidized monomer units.
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During thermooxidation a wide variety of higher order molecules are formed, this works focuses on the
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structural identification of dimeric phytosterol oxidation products with differing polarity, applying
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different techniques such as Ag+/CIS-MS and APCI-MS after on-line RP-HPLC separation; along with
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NMR and FT-IR spectroscopies.
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2. Materials and methods
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2.1 Chemicals
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β-sitosterol (the supplier declared 78.3% purity, containing campesterol and β-sitostanol), deuterated
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chloroform (CDCl3, 99.8% D), tetramethylsilane (TMS) and AgBF4 were purchased from Sigma-Aldrich
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(St. Louis, MO, USA). The HPLC and LC-MS grade solvents, along with ACS grade silica gel (60Å, 70-
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230 mesh) were purchased from Fisher Scientific Company (Ottawa, ON, Canada) and VWR
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(Mississauga, ON, Canada).
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2.2 Heating and pre-fractionation
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Heating procedure was as described before in Sosińska et al. (2013). Briefly, sitosterol standard was
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heated in the presence of oxygen at 180ºC for 24 h. Heated, dissolved in toluene sample (25 mg) was
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placed on silica gel column (1.3 g), and the non-polar (NP), mid-polar (MP) and polar (P) fractions were
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eluted with hexanes/diisopropyl ether (88:12, v/v), diisopropyl ether, and acetone, respectively. After
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evaporation of solvents, collected fractions were dissolved in dichloromethane prior to separation on SEC
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columns. The heating experiments and the pre-cleaning procedure were repeated in order to obtain enough
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material for the separation of dimers.
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2.3 Isolation of dimers on HPSEC columns
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Dimer fractions were isolated using high-performance SEC (HPSEC) on a Finnigan Surveyor liquid
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chromatograph (Thermo Electron, Waltham, MA, USA). The non-polar, mid-polar and polar fractions
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were injected on two Phenogel columns connected in series (500Å and 100Å, 5 μm, 300 x 7.80 mm with
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guard column; Phenomenex, Torrance, CA, USA) and kept at 25ºC. Dichloromethane was used as mobile
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phase at a flow rate of 1.0 mL/min. The non-polar, mid-polar and polar dimer fractions were collected
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using a Gilson FC 203B Fraction Collector (Middleton, WI, USA). Components were detected by an
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evaporative light scattering detector ELSD (Sedex 75; Sedere, Alfortville, France) operated at 30ºC with
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purified air at a pressure of 2.5 bar.
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2.4 RP-HPLC/ELSD
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Dimers fractions collected from HPSEC were separated on a Kinetex core-shell C18 column (2.6 µm; 150
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 3 mm; Phenomenex, MA, USA), using Finnigan Surveyor liquid chromatograph (Thermo Electron,
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Waltham, MA, USA) with an evaporative light scattering detector ELSD (Sedex 75; Sedere, Alfortville,
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France) operated at 30ºC with purified air at a pressure of 2.5 bar. Analyses were performed at 35oC with
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gradient elution of acetonitrile and dichloromethane (B) at a flow rate 0.4 mL/min (0-1 min isocratic at
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15% B, 1-6 min linear gradient to 35% B, 6-22 min linear gradient to 40% B, 22-26 min linear gradient to
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50% B, 26-30 min linear gradient to 100% B, after 10 min isocratic at 100% B column was equilibrated at
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initial conditions).
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2.5 RP-HPLC/UV and RP-HPLC/APCI-MS
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The analyses were performed on an Accela HPLC coupled to a Accela PDA detector and a Exactive
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Orbitrap MS (Thermo Fischer Scientific, West Palm Beach, FL, USA) using the same column and
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gradient elution as described above (2.4). Components were monitored at 243 nm and 280 nm. The mass
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spectrometer was equipped with an APCI ion source, and following conditions were used: positive ion
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mode; spray current 4.8 μA; capillary and heater temperature 300°C and 350°C; capillary, tube lens and
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skimmer voltage 50 V, 150 V and 25 V. Nitrogen was used as a sheath and auxillary gas with values set at
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35 and 5 arbitrary unit, respectively. Spectra of all-ions fragmentation preformed in HCD cell (higher
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energy collisional dissociation) at 25 eV were also recorded. Mass spectra were recorded within range
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from m/z 150 to 2400. Xcalibur 2.1.0 software was used for data acquisition and analysis. Mass
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spectrometer was calibrated within the range 195–1822 Da using caffeine, MRFA and Ultramark 1621
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(Sigma-Aldrich, St. Louis, MO, USA).
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2.6 RP-HPLC/Ag+ CIS-MS
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The analyses were performed on an Accela HPLC coupled to a Exactive Orbitrap MS (Thermo Fischer
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Scientific, West Palm Beach, FL, USA) using the same column and gradient elution as described above.
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50μM AgBF4 in isopropanol, delivered by Mighty Mini pump (Scientific Systems Inc., SSI, State College,
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PA, USA) at rate of 0.08 mL/min, was mixed post-column via a tee-piece with HPLC flow. The mass
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spectrometer was equipped with an ESI ion source, and following conditions were used: positive ion
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mode; spray voltage 5.2 kV; capillary temperature 400°C; capillary, tube lens and skimmer voltage 140 V,
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250 V and 50 V. Nitrogen was used as a sheath and auxillary gas with values set at 45 and 5 arbitrary
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unit, respectively. Mass spectra were recorded within range from m/z 300 to 1100. Xcalibur 2.1.0 software
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was used for data acquisition and analysis. Mass spectrometer was calibrated within the range 195–1822
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Da using caffeine, MRFA and Ultramark 1621 (Sigma-Aldrich, St. Louis, MO, USA).
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2.7 NMR and IR spectroscopy
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Solution state NMR spectra in CDCl3 were recorded on a Bruker Avance II 300 spectrometer, using 5 mm
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HX BB probe. The spectrometer was operated at 300.13 MHz for ¹H and 75.47 MHz for ¹³C. ¹H, ¹³C,
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DEPT 135, ¹H-¹H COSY, ¹H-¹H NOESY, ¹H-¹³C HSQC and ¹H-¹³C HMBC NMR spectra were recorded
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using tetramethylsilane (TMS) as an internal standard and chemical shifts are given in ppm (δ). Applied
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acquisition and processing parameters were as described before in Sosińska et al. (2013). IR spectra were
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recorded on a Bruker ALPHA-S equipped with DTGS detector, and Platinum ATR module with Diamond
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crystal plate and KBr as a beam splitter.
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IR-FT and 1H NMR spectral data
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NP2 fraction: IR (KBr) νmax [cm-1]: 2955, 2935, 2869, 1732, 1722, 1684, 1681, 1654, 1633, 1464, 1378,
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1340, 1253, 1173, 1137, 1092, 1069, 1015, 959. 1H NMR (CDCl3) δ values [ppm]: 0.65 (s), 0.68 (s), 0.81
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(d), 0.83 (d), 0.84 (t), 0.92 (d), 1.01 (m), 1.08-1.37, 1.43-1.56, 1.66 (m), 1.82-2.03, 2.21 (m), 2.30 (m),
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3.28 (dddd), 3.5 (br), 4.3 (br), 4.63 (br), 5.33 (ddd), 5.7 (br).
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MP2: IR (KBr): 3450, 2954, 2934, 2869, 1715, 1673, 1657, 1629, 1463, 1379, 1259, 1234, 1179, 1136,
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1089, 1069, 1017, 960. 1H NMR (CDCl3): 0.61 (s), 0.64 (s), 0.67 (s), 0.81 (d), 0.83 (d), 0.84 (t), 0.92 (d),
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1.01 (m), 1.10-1.33, 1.49-1.56, 1.66 (m), 1.83-2.03, 2.13 (m), 2.29 (m), 2.42 (m), 2.62 (br), 3.53 (m), 4.63
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(br), 5.36 (br), 5.7 (br).
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P2: IR (KBr): 3465, 2955, 2935, 2870, 1716, 1691, 1633, 1462, 1379, 1260, 1188, 1049. 1H NMR
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(CDCl3): 0.61 (s), 0.65 (s), 0.68 (s), 0.81 (d), 0.83 (d), 0.84 (t), 0.92 (m), 1.01 (br), 1.12-1.31, 1.47-1.56,
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1.66 (m), 1.83 (br), 1.99 (br), 2.29 (br), 2.42 (br), 3.52 (br), 4.06 (br), 4.65 (br), 5.36 (br).
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P1: IR (KBr): 3448, 2954, 2933, 2869, 1715, 1688, 1462, 1378, 1239, 1180, 1082, 1057. 1H NMR
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(CDCl3): 0.61 (s), 0.64 (s), 0.67 (s), 0.81 (d), 0.83 (d), 0.84 (t), 0.92 (br), 1.00, 1.05-1.33, 1.48-1.56, 1.66
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(m), 1.83 (br), 2.00 (br), 2,13 (br), 2.30 (br), 2.42 (br), 2.60 (br), 3.52 (br), 4.07 (br), 4.66 (br), 5.36 (br),
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5.72 (br).
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P0: IR (KBr): NMR (CDCl3): 3440, 2955, 2934, 2869, 1710, 1689, 1462, 1378, 1228, 1179, 1069, 1042.
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1.83 (br), 2.00-2.10, 2.14 (m), 2.24 (br), 2.29-2.37, 2.56 (br), 3.53 (m), 4.07 (br), 4.69 (br), 5.04 (br), 5.35
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(br), 5.52 (br).
H NMR (CDCl3): 0.66-0.72, .81 (d), 0.83 (d), 0.84 (t), 0.93 (br), 1.01 (br), 1.12-1.35, 1.49-1.54, 1.65 (m),
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3. Results and discussion
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3.1. Thermo-oxidation of sitosterol standard and dimer fractions collection
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HPSEC/ELSD profiles of non-polar (NP), mid-polar (MP) and polar (P) silica-gel-fractions of thermo-
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oxidized sitosterol standard are presented in Fig. 1C, D and E, respectively. For comparison
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chromatograms of un-oxidized sitosterol standard (Fig. 1A) and disteryl ether (Fig. 1B) are shown.
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Disteryl ether standard was synthesised from sitosterol standard as described previously in Sosińska et al.
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(2013). Phenogel columns, connected in series with pore sizes 500Å and 100Å with dichloromethane as a
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mobile phase, gave separation of disteryl ether and monomer standard by 2.48 min (Fig. 1A and B),
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however, three fractions had profiles far more complex containing not only higher oligomers (on the left
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from RT=18.06 min of disteryl ether), dimers (differing in number of oxygen atoms in the molecule, thus
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in polarity, mass and spatial size), and un-oxidized monomers, but also oxidized monomers and
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fragmented molecules. For each of the NP, MP and P silica gel-fractions three SEC-fractions were
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collected within the window time collection marked in Fig. 1C-E.
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3.2. Separation of dimer fractions on RP-HPLC with ELSD and UV detection
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Kinetex core-shell C18 column with gradient elution of acetonitrile and dichloromethane was used for
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separation of individual dimeric compounds present in the non-polar (NP2, NP1, NP0), mid-polar (MP2,
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MP1, MP0), and polar (P2, P1, P0) SEC-fractions. RP-HPLC/ELSD chromatograms of these fractions are
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shown in Fig. 2A, 2B and 2C. Compound profiles on C18 column were strongly influenced by the polarity
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of the fraction (MP, NP or P) and collection window (2, 1 or 0). Taking into consideration that un-
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oxidized standards and synthesized disteryl ethers were eluting at RT ca. 10 min and ca. 54 min,
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respectively, on C18 column, it can be assumed that almost exclusively monomers (oxidized or un-
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oxidized) were present in the NP0, NP1, MP0 and MP1 fractions (Fig. 2A, 2B). All three polar fractions
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i.e. P2, P1 and P0 contained monomer compounds in addition to oligomeric compounds (Fig. 2C),
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whereas fractions NP2 and MP2 contained almost exclusively oligomers. These assumptions were
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confirmed by MS analysis (see below). In this paper we focused on dimers, therefore, only results of SEC-
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fraction containing oligomers i.e. NP2, MP2, P2, P1 and P0 will be presented and discussed. There were
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three collection windows 2, 1 and 0 (Fig. 1C-E) applied, which was dictated by the difference in elution
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times of dimers disparate in polarity (thus in mass and spatial size) on the SEC columns. Chromatograms
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in Fig. 2A-C indicate a high degree of complexity in each of the NP, MP and P SEC-fractions. In non-
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polar fraction dimers bearing one or two oxygen atoms are the most expected, whereas in the polar
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fraction compounds with four or more oxygen atoms could be present. Moreover oxygen atoms can be
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involved in the linkage between two molecules (i.e. ether or peroxy), or be present in the ring or side chain
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of the sterols molecules as hydroxyl, keto, epoxy or peroxy group. A number of possible structures of
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dimers should increase exponentially with the increase of oxygen atoms in the dimer structure, therefore
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in the NP faction we should observe less diversity in dimer structures than in MP, whereas in P fraction
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the diversity should be greatest. This is observed in the lack of baseline separation among oligomeric
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compounds in collected SEC-fractions. Moreover, sterol standard used for heating experiment, besides β-
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sitosterol, contained also other phytosterols with ratio β-sitosterol: sitostanol : campesterol : stigmasterol
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as 100: 4.6 : 3.9 : 0.2 (calculated on basis of RP-HPLC/ELSD) (data not-shown), therefore involvement of
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four sterol in dimers formation further increased the number of possible structures.
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The most abundant constituent (ca. 38% estimated on basis of RP-HPLC/ELSD profile) of the NP2
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fraction was eluted at RT=54.1 min (NP2-H) with several other compounds that are well resolved from
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the mixture and marked with letter from NP2-A to NP2-I (Fig. 2A). In MP2 faction compounds were less
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effectively separated; however, some of them can be easily distinguished and were labelled as MP2-A to
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MP2-G (Fig. 2B). In oligomeric region the P2 fraction has only few peaks that are well separated (P2-A,
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P2-B, P2-C), whereas other compounds form very broad band, however in next two fractions: P1 and P0
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other oligomeric compounds were effectively separated (peaks P1-A to P1-C, and P0-1 to P0-D) (Fig.
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2C).
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Depending on the presence of: diene (homo- or heteroannular) and enone system; additional conjugation
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and exocyclic characters of double bonds; ring-residue C-C bonds etc. in sterol molecule, it’s UV
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absorption spectrum can show a maximum at 235-245 nm (e.g. 234 nm calc. for 3,5-diene and 244 nm for
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4-en-3-one), 280-285 nm (e.g. 280 nm calc. for 4,6-dien-3-one or 283 nm calc. for 5,7-diene) or 315 nm
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(e.g. 313 nm calc. for 3,5,7-triene) with strong end absorption at shorter wavelengths (Kasal, Budesinsky
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& Griffiths, 2010). Therefore conjugated diene structure features in dimers were monitored at 243 nm,
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above the dicholomethane cut-off wavelength, whereas diene conjugated with enone system was
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monitored at 280 nm. In Supplementary Material in Figure S1A ELSD chromatogram of NP2 fraction is
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presented with UV chromatograms at 243 nm (Fig. S1C) and 280 nm (Fig. S1D). Oligomeric compounds
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in NP fraction marked as NP2-B, NP2-C and NP2-D were detected at 243 nm and 280 nm, whereas NP2-
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A only at 243 nm (Figures 2A, S1C and S1D, Table 1). In MP2 fraction, peaks MP2-E, MP2-F, MP2-G
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and MP2-A were observed at both 243 and 280 nm, while MP2-B and MP2-D responded only at 243 nm
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(Fig. 2B). In polar fractions only compounds P2-A, P2-B and P2-C were detected at 243 nm, and none of
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oligomeric compounds found in fraction P0 or P1 (Fig. 2C).
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3.3. RP-HPLC/APCI-MS and RP-HPLC/CIS-MS of SEC-fractions
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In our previous paper we employed APCI ionisation to identify non-polar dimers, however, many dimers
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were unstable under these ionisation conditions, producing fragment monomer ions with low intense
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dimer ions (Sosińska et al., 2013). Consequently in this paper we employed CIS-MS with AgІ, as olefins
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and polyolefins form highly stable π or π-allyl complexes with Ag+. Moreover Ag+-CIS/MS method was
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previously employed for lipid peroxides and recently for stigmasterol dimers identification (Yin & Porter,
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2007; Struijs, Lampi, Ollilainen & Piironen, 2010).
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Un-oxidized sterols produce a mass spectrum under APCI-MS conditions that consists of a base peak
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[M+H-H2O]+ representing loss of the hydroxyl group, along with dehydrogenated sterol ions [M+H-4H]+,
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and [M+H-2H]+ and low intensity (if observed) [M+H]+ ions, whereas in case of oxysterols dehydration is
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closely related to a functional oxygen group and their structure (Rozenberg et al., 2003). For instance,
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under the APCI/MS applied in this study, 3-ol-5-en-7-one produced a mass spectrum with a base peak
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[M+H]+ ion, along with less intense [M+H-H2O]+ and [M+H-2xH2O]+ ions, whereas 3-ol-5-ens were
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represented by a base peak [M+H-H2O]+ and [M+H-4H]+ ions (data not shown). Ionization patterns of
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sterols are usually consistent among various APCI/MS systems; however, the relative ion abundances
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vary. Similarly, under these conditions dehydration and dehydrogenation of sterol oligomers is expected.
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Additionally we have observed solvent derived adducts such as [M+C3H4NCl+H]+ under APCI-MS
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(which may be a 3-chloropropionitrile formed by a substitution of one of chlorine atoms by the methyl
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carbon of acetonitrile). We have observed formation of other solvents adducts of disitosteryl ether under
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APCI conditions when tetrahydrofuran or acetonitrile/2-propanol were used (Sosińska et al., 2013).
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Similar phenomenon, in respect to other lipids including sterol oxides, was previously reported (Ma &
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Kim, 1997; Manini, Andreoli, Careri, Elviri & Musci, 1998). In addition we have observed formation of
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solvent derived adducts to both dimer and monomer molecules.
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In Table 1 list of tentatively assigned dimers and trimers found in non-polar NP2, mid-polar MP2 and
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polar P2, P1, P0 fractions is given, including information such as chemical formula, monoisotopic mass,
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UV and mass spectrometric CIS/MS data of identified compounds. In Supplementary Material in Table S1
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additional APCI/MS mass spectrometric data is given.
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Under CIS-MS conditions both AgІ adducts [M+107Ag]+ and [M+109Ag]+ were observed for disteryl ethers
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(synthesized as described in Sosińska et al. (2013)), and other oligomeric sterol derivatives found in
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collected SEC-fractions. Moreover acetonitrile/ silver (I) adducts [M+C2H3N+Ag]+ were found. On basis
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of CIS/MS mass spectra and comparison of retention times with synthesized disteryl ether the most
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abundant compound in the NP2 fraction at RT=54.1 min (NP2-H) was confirmed to be a disitosteryl ether
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(Fig. 3A) with ion at m/z 917.6656 assigned as [M+107Ag]+ where M= 810.7612 Da, and has elemental
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composition C58H98O (mass accuracy Δ= -0.8 ppm) (Table 1). In the APCI/MS mass spectrum the
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[M+H]+ molecular ion of the ether was present but at very low intensity, along with the more intense
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solvent adduct [M+C3H4NCl+H] + at m/z 900.7720 (Δ= -0.4 ppm) (Table S1). Moreover a few other
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disteryl ethers were found in NP2 fraction, i.e. sitosteryl-sitostanyl ether C58H100O (NP2-I) or sitosteryl-
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campesteryl C57H96O (NP2-G) (Table 1). All ethers were highly unstable under APCI ionization
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conditions forming monomer fragments i.e. sitosteryl (C29H49 at m/z 397.3828), sitostanyl (C29H51 at m/z
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399.3980), campesteryl (C28H47 at m/z 383.3673) and stigmasteryl (C29H47 at m/z 395.3676) (Table S1),
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but their [M+107Ag]+ ions were very abundant under CIS/MS conditions. Fragmentation of the ether
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molecule by cleavage of the adjacent bond to a heteroatom can be explained via a charge site-initiated
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reaction (inductive cleavage, i), similar to loss of water in sterol. However we observed both [R-CH+] and
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[+O-R'-2H] ions, which is explained invoking a special case of a σ bond cleavage with simultaneous
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dehydrogenation (de Hoffmann & Stroobant, 2007). For example, in case of disitosteryl ether (NP2-H) in
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APCI/MS spectrum ions at m/z 397.3828 (C29H49, [R-CH+]) and at m/z 411.3625 (C29H47O, [+O-R'-2H])
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were observed (Fig. 3A, Table S1). None of the disteryl ethers (NP2-G, NP2-H, NP2-I) were detected at
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243 and 280 nm (Fig. S1C, Fig. S1D, Table 1), indicating an absence of diene or enone conjugated
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features in the structure. On the basis of RP-HPLC/ELSD analysis, NP2 fraction consisted of 38% of
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disitosteryl ether (NP2-H) along with sitosteryl-campesteryl (NP2-G, 6%) and sitosteryl-sitostanyl (NP2-I,
289
5%) ethers. Dimers NP2-B, NP2-C and NP2-D were also present in MP2 fraction: MP2-E, MP2-F, MP2-
290
G, respectively, and will be discussed below, as they were main components of MP2 fraction. On the basis
12
291
of its MS spectra, dimer NP2-F was assigned as C58H98O2, most likely having an ether linkage and
292
hydroxyl or epoxide group on one of the steryl units, as the molecular ion has low intensity in the
293
APCI/MS spectrum coincident with the dehydrated [M+H-H2O]+ ion, and no absorption was observed at
294
243 or 280 nm (Table 1 and S1). Dimer NP2-E was assigned as a structural isomer of NP2-F.
295
296
In MP fraction the most abundant oligomer was detected at RT = 47.3 min (Fig. 2B). MP2-F was assigned
297
as C58H94O2 with a base peak at m/z 823.7329 ([M+H]+, Δ= 0.3 ppm) in the APCI/MS spectrum (Table
298
S1). This molecular ion was very stable and remained as the base peak even after HCD fragmentation at
299
25 eV (data not shown). Under CIS/MS conditions, silver adducts [M+107Ag]+ at m/z 929.6305 were
300
observed. The main monomer fragment ions at m/z 427.3573 (C29H47O2), and m/z 397.3830 (C29H49)
301
along with less intense ions fragment ions were found in the APCI spectrum (Table S1). The presence and
302
high abundance of the molecular ion of this dimer suggests a very stable dimer linkage such as direct a C-
303
C, or an ether linkage, with concomitant efficient protonation of the molecule, possibly owing to the
304
presence of a keto group. In general if sterol has a keto group, the APCI/MS spectrum has a [M+H]+ ions
305
as a base peak (i.e. 3-ol-7-one, 6-ol-3-one, 3,5-diene-7-one), whereas [M+H-H2O]+ is the most intense in
306
case of 3-ol-5,6-epoxy, 3-ol-6-ol, 3-ol-5-en-7-ol derivatives, and [M+H-2xH2O]+ is a base peak in mass
307
spectra of triols and 3-ol-5-en-7-peroxy sterols (Kemmo, Ollilainen, Lampi & Piironen, 2007; Nogueira,
308
Costa, Crotti & Bragagnolo, 2010). Taking into consideration the data presented, along with the absence
309
of the dehydrated dimer ion (which should be formed when hydroxyl group is present), it can be
310
concluded that dimer MP2-F has an ether linkage and a keto group in one of the units. The tentative
311
structure of MP2-F dimer is presented in Fig. 3B. Other similar dimers eluting before or after MP2-F were
312
observed, such as dimer MP2-G with chemical formula C58H96O2 with a base peak [M+107Ag]+ in the
313
CIS/MS spectrum at m/z 931.6467 and in the APCI/MS spectrum molecular ion [M+H]+ at m/z 825.7476;
314
or dimer MP2-E (C57H92O2) with monomer fragment ions: sitosteryl fragment C29H49 and campesteryl
315
fragment at m/z 383.3678 (C28H47) in the APCI/MS spectrum (Table 1, Table S1). All these dimers (MP213
316
E, MP2-F and MP2-G) were observed at 243 and 280 nm, indicating presence of conjugated enone and
317
diene systems in the molecule. Dimer MP2-A, having molecular formula C58H94O4, on basis of its mass
318
spectral and UV data was tentatively assigned as a dimer with a keto group, along with hydroxyl or epoxy
319
group and diene moiety conjugated in one unit. Dimer MP2-D with molecular formula, C58H94O3 is
320
proposed to be linked via direct C-C linkage, with keto group and hydroxyl or epoxy groups in one of the
321
units, and hydroxyl or epoxy group on the second molecule, as it had a base peak in the APCI/MS
322
spectrum at m/z 427.3577 (C29H47O2), and a medium intense molecular ion [M+H]+ at m/z 839.7289 along
323
with a dehydrated dimer ion [M+H-H2O]+ at m/z 821.7188 (Table 1 and S1).
324
325
All three compounds of the P2 fraction, marked with symbols on the ELSD chromatogram shown in Fig.
326
2C, had low intensity ions in the APCI/MS spectra in the region characteristic for dimers i.e. m/z 795 -900
327
but also for trimers i.e. m/z 1190 – 1320, along with the expectedly intense fragment monomer ions. The
328
trimer P2-B (RT=52.3 min) was assigned as C87H142O6 with the molecular ion [M+H]+ at m/z 1284.0895
329
and more intense dehydrated ion [M+H-H2O]+ at m/z 1266.0788, along with the dimeric fragment ions at
330
m/z 869.7031 (C58H93O5) and at m/z 851.6921 (C58H91O4), and monomer fragment ions from C29H47 to
331
C29H47O3 (Table S1). All oxygen atoms in this trimer should be comprised in ether, as well as hydroxyl or
332
epoxy groups, and accompanied by some diene features in the structure, stemming from the characteristics
333
ions found in the MS spectrum including intensity and presence of UV absorption at 243 but not at 280
334
nm. In the APCI/MS spectrum of the trimer P2-A, assigned as C86H140O6 with one campesteryl unit, the
335
molecular ion at m/z 1270.0741 was found, along with the most intense amongst dimer fragment ions
336
[M+H]+ at m/z 853.7076 (C58H93O4), whereas the trimer P2-C was assigned as C87H144O6 (Table 1).
337
In the P1 fraction there were three dimeric products found, and the most abundant being P1-B (RT=43.0
338
min) (Fig. 2C). In the APCI/MS of compound P1-B, assigned as C58H100O3, the [M+H]+ was not found
339
but the solvent adduct [M+C3H4NCl+H]+ at m/z 934.7782 and dehydrated ions [M+H-H2O]+ and [M+H-
340
2xH2O]+ were detected at low intensity (Table S1). Furthermore diagnostic monomer fragments were
14
341
found, such as starting from C29H49 at m/z 397.3831 to C29H49O2 at m/z 429.3731. Taking into
342
consideration the MS spectrum ions, their intensity and absence of UV absorption at 243 or 280 nm, the
343
tentative structure of P1-B is proposed and presented in Fig. 3C. Dimeric compounds detected in the P1
344
fraction differ in the monomer building unit. Compound P1-A was assigned as C57H98O3 having one
345
campesteryl unit, whereas dimer P1-C was assigned as C58H102O3 having two sitostanyl units. In the P0
346
fraction four main dimeric compounds were found, moreover all three dimers found in the P1 fraction
347
within RT 41-45 min (Fig. 2C) were also detected at low abundances. The P0-D compound (RT=39.3
348
min), assigned as C58H98O3, is proposed to be an ether with two hydroxyl and/or epoxy groups in its units,
349
as its dehydrated dimer ions were found in the APCI/MS spectrum. Compounds P0-B and P0-C were both
350
assigned as C58H94O3. These structural isomers had different dehydrated ions; at m/z 809.7541 in case of
351
P0-B and at 807.7385 in P0-C, both ions being most prominent in the dimeric region of the spectrum.
352
None of these compounds were detected by UV at 243 nm or 280 nm. In the polar fraction also other
353
dimers with higher number of oxygen atoms were found, but at very low concentration. For example a
354
dimer in the P1 fraction at RT=33.1 min was assigned as C58H99O4 ([M+H]+, m/z 859.7556, Δ=1.8 ppm),
355
and in the P0 fraction a dimer detected at RT=30.4 min was assigned as C58H97O4 ([M+H]+, m/z 857.7403,
356
Δ=2.2 ppm) and one at RT=21.5 min was assigned as C58H93O5 ([M+H]+, m/z 869.7042, Δ=2.5 ppm).
357
358
3.4. NMR spectroscopy of SEC-fractions
359
Collected SEC-fractions are complex mixtures of dimeric and trimeric oxidation products, including
360
monomers of all phytosterols present in β-sitosterol standard. Therefore, in addition to HPLC and MS, we
361
employed NMR and IR techniques primarily to identify the functional groups and characteristic
362
connetivities within the molecules in collected fractions. Comparison of ¹H NMR spectra of NP2, MP2
363
and P2, P1 and P0 SEC-fractions with the β-sitosterol standard is shown in Fig. 4A-F (spectral data also
364
presented in Supplementary Material). Signals of all methyl protons of β-sitosterol (Fig. 4A) are mostly
365
preserved in the NP2 fraction (δH-18 0.67, s; δH-19 0.99, s; δH-21 0.92, d, J=6.2 Hz;, δH-26 0.81, d, J=6.6 Hz;
15
366
δH-27 0.83, d, J=6.6 Hz and δH-29 0.84, d, J=7.4 Hz) (Fig. 4B), but much less prominent in the P0 fraction
367
(Fig. 4F). Similarly all other signals present in β-sitosterol within range 1-2.6 ppm are the mostly
368
preserved in NP2, and least in the P2, P1 and P0 fractions. NMR data for NP2 fraction agrees with our
369
previous work, where non-polar dimers were separated on SEC column with tetrahydrofuran instead of
370
dichloromethane (Sosińska et al., 2013). In agreement with mass spectrometric data, the most dominant
371
constituent of NP2 fraction is the 3β,3β'-sitosterol ether and all its ¹H and ¹³C signals, along with
372
corresponding correlations were found in 1D and 2D NMR spectra. The most important correlations
373
validating structure of this ether are δC-3 76.34 with δH-3 3.28 (-HC-O-C'H') and δC-6 121.35 with δH-6 5.33
374
(-C=CH-) in HSQC spectrum where correlations between the aliphatic carbon and its attached protons are
375
shown, as well as δC-3' with δH-3 (-HC-O-C'H') and δC-3 with δH-3’ (-HC-O-C'H') in HMBC spectrum, which
376
proves that H-3α /H-3α' is two- or three-bonds away from the C-3' /C-3 in the dimer molecule.
377
Additionally in HMBC spectrum correlation of δC-5 141.34 with δH-4α 2.27 and δH-4β 2.21 (-HHC-C=C-)
378
was present. Moreover, additional correlations for other ethers were found: campesteryl ether (δH-28 0.76
379
with δC-28 15.37 in HSQC spectrum), sitostanyl (δH-18 0.64 with δC-18 12.02 in HSQC spectrum). Detailed
380
assignment of 3β,3β'-sitosterol ether present in NP2 fraction has already been published by our group
381
(Sosińska et al., 2013). Besides signals and correlations mentioned above, other important signals,
382
although of weak intensity and broad, were detected in NP2 fraction. In HSQC spectrum, correlations
383
proving that proton at δH 2.31 is attached to δC 38.1; δH 4.3 to δC 77.1; δH 4.63 to δC 73.8 and δH 5.33 to δC
384
122.7 were found, whereas in HMBC spectrum correlation of δH 2.29 with δC 173.3 was observed. COSY
385
spectrum showed strong through-bond couplings between δH 4.63 and δH 2.3 (data not shown). Additional
386
signals at δH 4.6 and δC 73.8 suggested the presence of steryl ester, and are assigned as: H-3 and C-3
387
(numeration in steryl ester molecule, where ester group is on C-3 atom); δC 173.3 as C-1' (-CH-COO-
388
steryl unit); δH 2.29 as H-2' (-CH-COO-steryl unit); including δH 2.31 and δC 38.13 as H-4 and C-4; and δH
389
5.33 and δC 122.7 as H-6α and C-5 (Wilson, Sumpter, Warren, Rogers, Ruan & Schroepfer, 1996; Lou,
390
Liu, Qi, Wu & Zhang, 2006). Moreover correlation of δH 3.5 ppm (H-3α in β-sitosterol) and δC at 71.7 (C16
391
3 in β-sitosterol) found in HSQC spectrum, indicates presence of compounds with hydroxyl group
392
preserved e.g. some dimers or monomers in NP2 fraction.
393
In the mid-polar and polar SEC-fractions, typical signals of β-sitosterol were present in proton NMR
394
spectra (Fig. 4C-F) along with δH 3.5 ppm and δH 5.3 ppm, indicating the presence of hydroxyl in the ring
395
A and ethene functionality in the ring B, respectively. Signal at δH 5.7 would typically arise from protons
396
near keto groups e.g. in the ring B, whereas signals at δH 4.3, 5.3 and 5.9 suggest the presence of ethene
397
function in rings A and B (Ning, 2005). Those signals can indicate presence of dimer with structure
398
tentatively assigned on basis of mass spectra and shown in Fig. 3B. In the polar fractions, beyond
399
correlations and signals described above, an additional correlation on HSQC spectrum was found
400
indicating the presence of hydroxyl group next to the double bond (δH 4.1 with δC 67.9) (Wilson et al.,
401
1996). Another important feature of 1H NMR spectra of mid-polar fraction; MP2 (Fig.4C) and polar
402
fractions; P2, P1 and P0 (Fig. 4D-F) is lack of a signal at 3.28 ppm present in NP2 fraction (Fig. 4B) due
403
to the proton neighbouring ether linkage -HC-O-C'H'. However chemical shift of proton next to the ether
404
bond is influenced also by the presence and distance of additional oxygenated groups in the dimer,
405
therefore signals of protons next to the ether linkage in such dimers as shown in Fig. 3B and 3C will have
406
a chemical shift above 3.3 ppm.
407
408
3.5. FT-IR spectroscopy of SEC-fractions
409
In the IR spectra of the MP2, P2, P1 and P0 fractions (data presented in Supplementary Material) a very
410
broad band with maximum intensity at 3450-3465 cm-1 typical of O-H-stretching modes were clearly
411
visible, whereas in NP2 fraction a very low intensity band at 3450 cm-1 was barely discerned. This broad
412
band confirms presence of sterol derivatives with a hydroxyl group involving in an intermolecular
413
hydrogen bonding, or a hydroperoxide (-O-O-H) group in the collected fractions. In the β-sitosterol IR
414
spectrum the intensity of O-H-stretching mode at 3450 cm-1 represents 26% (1:4) of the very intense,
415
asymmetrical -CH3 stretching band at 2955cm-1 (Sosińska et al., 2013), whereas in the P2, P1, P0, MP2
17
416
and NP2 fractions it represents 11%, 12%, 17%, 5% and less than 1%, respectively. All of the fractions
417
showed broad bands covering the range 1800 -1550 cm-1 characteristic for C=C and C=O-stretching
418
vibrations in carbonyl compounds (Ning, 2005). In the NP2 fraction these bands were of moderate
419
intensity with maximum at 1732 cm-1 along with other well resolved local peaks i.e. at 1684 or 1633 cm-1,
420
whereas in the MP2 and the P2 fractions these band were more intense with a maximum at 1715 cm-1 with
421
others having less well resolved local maxima. C=C-stretching bands in IR spectra are typically weak in
422
absence of conjugation (eg. 3-ol-5-en) due to high psuedosymmetry, whereas in diene systems or enone
423
systems, conjugation with one or more double bonds can be very intense, appearing in range from 1650 to
424
1580 cm-1. For example, 4,6-dien-3-one gives two C=C bands at 1620 cm-1 and a weaker band at 1587 cm-
425
1
426
bands in the range 1720–1700 cm-1, whereas carbonyl stretching modes for ketones in the five-membered
427
ring D occur in the region of 1750–1740 cm-1. However, conjugation of the C=O group with a C=C bond
428
shifts the absorption maximum in IR spectrum to a lower frequency by ca. 30–40 cm-1 for α, β-unsaturated
429
ketones, such as 4-en-3-one with C=O stretching band at 1680 (Ning, 2005). Beyond C=C and ketone
430
C=O bands, the aldehydic C=O-stretching band is observed in the region 1730–1720 cm-1; however, the
431
weak, but characteristic C–H-stretching band for CHO group at ~2750 cm-1 was not present in any of the
432
collected fractions.
433
On the basis of the MP2 and polar fractions' IR spectra, the presence of some carboxylic acids cannot be
434
ruled out as some broadness is observed in range 3600 to 2400 cm-1, which would be associated with O-H
435
stretching vibrations for hydroxyl group in sterols (broad band in range 3550–3200 cm-1 for
436
intermolecularly bonded hydrogen), but also with hydrogen bonding in carboxylic acids with very broad
437
band in the region 3330-2500 cm-1. The C=O stretching mode of free carboxylic acid is strong, narrow
438
and usually found at 1710–1700 cm-1 (Ning, 2005). Carboxylic esters of hydroxyl steroids are
439
characterized by three strong bands: one for C=O stretching vibrations at frequency dependent on the acyl
440
component (i.e. band at ~1740 cm-1 in acetates, at ~1730 cm-1 in formates); and two C-O stretching
(Ning, 2005). Ketones in unsaturated six-membered rings or in the side chains show C=O-stretching
18
441
vibrations in the range 1270-1160 cm-1 (O=C-O-C band) and 1100-1000 cm-1 (O=C-O-C band). All
442
fractions have absorptions (very crowded and overlaid) in these two last ranges, but only NP fraction has
443
additional band at 1732 cm-1. Thus, the intensity and spread of the broad band at ca. 1750-1570 cm-1 can
444
be explained by the presence of a variety of compounds with enone and diene functionality. The spectra
445
are very crowded in the range 1400 – 900 cm-1; however, a few important bands can be distinguished in
446
the IR spectra: at ca. 1050 cm-1 due to C-O-stretching vibrations present in the polar fractions; and at ca.
447
1090 cm-1 assigned as a C-O-C-stretching modes present in the NP2, MP2 and P1 fractions in IR spectra.
448
449
450
Lipid polymers, observed in model reaction systems and in processed food, are proposed to be formed on
451
a route of peroxyl, alkoxyl and alkyl radical recombinations e.g. cross-chain addition to the C=C, as
452
shown in Fig. S2 (Supplementary Material). Due to higher concentration of oxygen and the relatively
453
lower dissociation energy of the O-O bond (~160 kJ/mol) compared to C-O and C-H, peroxy-linked
454
dimers are observed mostly at lower temperatures (Neff, Frankel & Fujimoto, 1988; Schaich, 2005),
455
whereas C-C linked non-polar dimers are believed to be formed in the absence of oxygen through
456
interaction of radicals R· (Fig. S2D) (Velasco, Marmesat & Dobarganes, 2008). Experimental data of
457
oxidation of sterols in a model and in products show that the primary oxidation site in sterol molecule is
458
on C7, as the most abundant derivatives formed during oxidation of sterols are 7-keto, 7α-hydroxy and 7β-
459
hydroxysterols (Dutta et al., 2002; Rudzińska et al., 2005; Lampi et al., 2009; Derewiaka & Obiedzinski,
460
2012). Recently Lengyel, Rimarčík, Vagánek, Fedor, Lukeš and Klein (2012) theoretically studied sterols
461
oxidation in terms of bond dissociation enthalpies (BDE). Their findings correlate with experimental data,
462
as they computed the lowest BDE for C7-H in cholesterol or sitosterol (328-329 kJ/mol), whereas for
463
other Δ5-sterols having double bond in the side chain lowest BDE were found for C20-H (315-320
464
kJ/mol). Therefore radical formation of dimers seems to favor the C7 of sitosterol, however in our study
465
the most abundant dimeric product formed during thermo-oxidation has configuration of 3,3'-disteryl
19
466
ether, based on detailed spectrometric data. Indeed, the profile of dimers found in thermo-oxidized
467
standard was influenced by various factors such as: (1) stability at high temperature, peroxy linked dimers
468
being very unstable; (2) availability of substrates, un-oxidized standards being the most abundant; and (3)
469
steric hindrance around the double bonds (i.e. position of addition reaction) or the bonds with the lowest
470
dissociation energies. Considering the last two factors, dimers formed directly from un-oxidized
471
molecules or by linkages between atom on ring A instead of ring B (Fig. 3A) should be more favored. As
472
shown in Fig. S2C and Fig. 5A dimer ethers can be formed on route of radical recombinations, however
473
other alternative mechanism, via nucleophilic substitution (Fig. 5B), should be considered. In the
474
presence of a strong acid, and at elevated temperature, ethers or alkenes are formed from alcohols by
475
nucleophilic substitution in case of ether, or elimination, in case of alkene. First step is the protonation of
476
the hydroxyl oxygen with formation of an oxonium ion, followed by the cleavage of the C-O bond to give
477
a carbocation intermediate (Fig. 5B). Subsequent E2 elimination of proton results in the creation of a
478
double bond as in steryl-3,5-diene. The secondary carbocation at position 3 (Fig. 5B) can also react with
479
second sterol molecule via the hydroxyl functional group, yielding a disteryl ether. This latter mechanism
480
was used to explain the formation of disteryl ether during oil bleaching with acid treated earth (Weber,
481
Bergenthal, Brühl & Schulte, 1992). Similarly ethers are formed in reaction catalyzed by copper sulphate
482
at high temperature, where the intermediate carbocation can be formed if transition metal draws off the
483
electron density from the hydroxyl group, leading to an electron defect on C3 and more favourable
484
cleavage of the C-O bond (Bergenthal, Schulte & Weber, 1990). Our study shows that high temperature
485
can be sufficient catalyser for formation of disteryl ethers, however, the prevailing mechanism
486
(carbocation vs radical) requires further study.
487
488
4. Conclusions
489
Thermo-oxidative degradation of sitosterol results in a complex mixture of dimeric and oligomeric
490
compounds with varying polarities. Our previous study (Sosińska et al., 2013) showed formation of
20
491
dimers consisting from two un-oxidized units such as disitosteryl ether – found in non-polar fraction of
492
thermo-oxidized sterols - on basis of data obtained by NMR, FT-IR and Raman spectroscopies, along with
493
HPSEC/APCI-MS analysis. In this study we have investigated not only non-polar but also mid-polar and
494
polar fraction of thermo-oxidized sitosterol standard, employing much better separation of the compounds
495
on RP-HPLC column prior to ionisation and detection by APCI/MS and also Ag+-CIS/MS techniques. In a
496
result, we have elucidated the chemical structures of major dimeric components in the mixture using a
497
combination of chromatographic and spectrometric methods. Available data supported 3,3’-disteryl ether
498
as the most abundant compound in the non-polar fraction while 7-keto disteryl ether was the major dimer
499
in the mid-polar. A 7-hydroxy disteryl ether was identified as a prominent dimeric component in the
500
complex polar phytosterol degradation products. Contrary to a C7 (B-ring) linked disteryl ether expected
501
on the basis of radical mediated mechanism, detailed NMR data from the present study supported a C3-
502
C3' linked (A-ring) disteryl ether, suggesting an alternative pathway for thermal dimerization of sterols.
503
Steric hindrance around the C7 compared to C3, coupled with high concentration of unoxidized sterol
504
substrate presumably inform the preference for a 3,3'-disteryl ether, however, further study is required to
505
better understand the prevailing mechanism of formation.
506
507
Acknowledgments
508
This research was funded by the Alberta Value Added Corporation and the Agriculture and Food Council
509
through the Alberta Agriculture Funding Consortium.
21
510
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511
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