HPLC-MS and MSMS analysis of 2-methoxy alkylglycerols isolated from shark liver oil. Aðalheiður Dóra Albertsdóttir

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1 HPLC-MS and MSMS analysis of 2-methoxy alkylglycerols isolated from shark liver oil Aðalheiður Dóra Albertsdóttir Faculty of Physical Sciences University of Iceland 2011

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3 HPLC-MS and MSMS analysis of 2-methoxy alkylglycerols isolated from shark liver oil Aðalheiður Dóra Albertsdóttir 90 ECTS thesis submitted in partial fulfillment of a Magister Scientiarum degree in Chemistry Advisors Dr. Sigurður V. Smárason Prof. Guðmundur G. Haraldsson Faculty Representative Prof. Ingvar H. Árnason Faculty of Physical Sciences School of Engineering and Natural Sciences University of Iceland Reykjavik, October 2011

4 HPLC-MS and MSMS analysis of 2-methoxy alkylglycerols isolated from shark liver oil HPLC-MS and MSMS analysis of 2-methoxy alkylglycerols 90 ECTS thesis submitted in partial fulfillment of a Magister Scientiarum degree in Chemistry Copyright 2011 Aðalheiður Dóra Albertsdóttir All rights reserved Faculty of Physical Sciences School of Engineering and Natural Sciences University of Iceland Hjarðarhaga , Reykjavik Iceland Telephone: Bibliographic information: Aðalheiður Dóra Albertsdóttir, 2011, HPLC-MS and MSMS analysis of 2-methoxy alkylglycerols isolated from shark liver oil, M.Sc. thesis, Faculty of Physical Sciences, University of Iceland. Printing: Háskólaprent, Fálkagata 2, 107 Reykjavík Reykjavik, Iceland, October 2011

5 Abstract The 1-O-alkyl-sn-glycerols, also known as glyceryl ethers, occur widely in Nature, but in particularly high amounts in the liver oil of various cartilaginous fish including shark species. Shark liver oil has been used in Scandinavia for centuries for its various remedial effects that have been attributed to the glyceryl ethers. Minor parts (2-4 %) of the glyceryl ether fraction of such oils are substituted with a methoxyl group located at the 2-position of the alkyl moiety. Such methoxylated ether lipids (1-O-(2 -methoxyalkyl)-sn-glycerols) are interesting compounds that have been claimed to offer various bioactivities including antifungal, antibacterial and antitumor properties. The objective of this work was to develop a HPLC- MS analytical method to investigate a mixture of such 2-methoxy alkylglycerols (MAGs) isolated from the liver oil of deep sea sharks originating in the North Atlantic Ocean. The work was focused on the ammonium and lithium adducts of the MAGs. Seven MAG adducts were found to account for the majority of the signals in the HPLC-MS analyses and accurate mass values were acquired for all of them. These compounds constitute two saturated (C16:0 and C18:0), three monounsaturated (C16:1 and two C18:1 isomers) and two polyunsaturated (C18:3 and C22:6) MAG derivatives. Confirmation of the MAGs general framework was achieved by analyzing the ammonium adduct MSMS spectra while the lithium MSMS spectra provided information regarding the location of the double bonds in the mono- and polyunsaturated MAGs.

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7 Útdráttur 1-O-alkýl-sn-glyseról, einnig þekkt sem glycerýl eterar, koma víða fyrir í náttúrunni, en þessi efni finnast í sérstaklega miklu magni í lýsi mismunandi brjóskfiska, þar á meðal í hákörlum. Hákarlalýsi hefur verið notað í Skandinavíu í margar aldir vegna ýmiskonar jákvæðra áhrifa sem hafa verið rakin til glycerýl etera. Lítill hluti (2-4%) glycerýl etera í olíunni eru með metoxýl hóp staðsettan á kolefni 2 á alkýlkeðjunni. Þessi metoxýleruðu eter lípíð (1-O-(2 - metoxýalkýl)-sn-glýseról) eru athyglisverð efni sem hafa verið tengd við hina ýmsu lífvirkni meðal annars sem sveppaeyðandi, bakteríueyðandi og æxliseyðandi eiginleikar. Markmiðið með verkefninu var að þróa HPLC-MS greiningaraðferð til þess að greina blöndu af þessum 2-metoxýleruðu alkýlglýserólum (MAGs) sem voru einangruð úr lýsi er unnið var úr hákörlum sem veiddust í norður Atlantshafinu. Eingöngu voru rannsakaðar ammoniumog liþíumjónir þessara efna. Sjö MAG efni stóðu fyrir meginhluta merkjanna í HPLC- MS greiningunni og var nákvæmum massagildum náð fyrir þau öll. Þessi efni samanstóðu af tveimur mettuðum (C16:0 og C18:0), þremur einómettuðum (C16:1 og tveimur C18:1 ísómerum) og tveimur fjölómettuðum (C18:3 og C22:6) MAG afleiðum. Staðfesting almennu MAG byggingarinnar fékkst með því að greina ammóníum MSMS rófin en liþíum rófin gáfu upplýsingar um staðsetningu tvítengja í ein-og fjölómettuðu MAG afleiðunum.

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9 Table of contents List of Figures List of Tables Abbreviations Acknowledgements xi xiii xv xvii 1 Introduction Brief introduction into equipments and methods High performance liquid chromatography Mass spectrometry Nomenclature and additional concepts Experimental Lipid samples Chemicals High pressure liquid chromatography (HPLC) Mass spectrometry The MAG mixture from shark liver oil The synthesized MAG and its precursor Development and evaluation of HPLC-MS method The development process General results of the HPLC-MS runs Summary Synthetic MAG MSMS spectrum of C16:1 synth MSMS spectrum of C16:1 isop Summary NH 4 HPLC-MS/MSMS data of MAGs in mixture HPLC-MS data Visual comparison of standard and sample MSMS spectra of [M + NH 4 ] +, [M + H] + and [M CH 3 O] Change in fragmentation of [M + NH 4 ] + with increased collision energy Fragments in the upper mass range of [M + NH 4 ] + MSMS spectra Saturated MAGs Monounsaturated MAGs Polyunsaturated MAGs Comparison of fragments ix

10 5.6 Fragments in the lower mass range of [M + NH 4 ] + MSMS spectra Saturated MAGs Monounsaturated MAGs Polyunsaturated MAGs O 3 fragments Summary Li HPLC-MS/MSMS data of MAGs in mixture HPLC-MS data Change in fragmentation of [M + Li] + with increased collision energy Analysis of the major fragments in [M + Li] + MSMS spectra Saturated MAGs Monounsaturated MAGs Polyunsaturated MAGs Summary Conclusions 85 Bibliography 87 Appendices 89 Appendix A: Accurate mass values for natural MAGs Appendix B: [M + NH 4 ] + MSMS spectra of natural MAGs at various collision energies Appendix C: [M + NH 4 ] +, [M + H] + and [M CH 3 O] + MSMS spectra of natural MAGs Appendix D: [M+Li] + MSMS spectra of natural MAGs at various collision energies101 Appendix E: Sets of ammonium adduct MSMS spectra Appendix F: Sets of lithium adduct MSMS spectra Appendix G: Mass lists from the ammonium adduct MSMS spectra Appendix H: Mass lists from the lithium adduct MSMS spectra x

11 List of Figures 1.1 Reported molecular structure of C16: Reported molecular structure of C16: Reported molecular structure of C18: Reported molecular structure of C22: [M + Na] + EIC of MAG mixture from a B. Sc. project BPC of HPLC-MS runs of MAG mixture using two different additives BPC and EIC of Li-Ac HPLC-MS run [M + Li] + EIC of Li-Ac HPLC-MS run Molecular structure of C16:1 synth [M + NH 4 ] + MSMS spectrum of C16:1 synth Lower end [M + NH 4 ] + MSMS spectrum of C16:1 synth Molecular structure of C16:1 isop [M + NH 4 ] + MSMS spectrum of C16:1 isop EIC of [M + NH 4 ] EIC of [M + NH 4 ] +, [M + H] +, [M CH 3 O] + and [m H] [M + NH 4 ] + MSMS spectrum of C16:1 synth [M + NH 4 ] + MSMS spectrum of C16: Lower mass range of [M + NH 4 ] + MSMS spectrum of C16:1 synth Lower mass range of [M + NH 4 ] + MSMS spectrum of C16: [M + NH 4 ] +, [M + H] + and [M CH 3 O] + MSMS spectra for C16: [M + NH 4 ] +, [M + H] + and [M CH 3 O] + MSMS spectra for C16: [M + NH 4 ] + MSMS spectra of C16:0 at various collision energy levels [M + NH 4 ] + MSMS spectra of C16:1 at various collision energy levels [M + NH 4 ] + MSMS spectra of C18:3 at various collision energy levels [M + NH 4 ] + MSMS spectra of C22:6 at various collision energy levels Upper mass range [M + NH 4 ] + MSMS spectrum of C16:0 at 15 ev Upper mass range [M + NH 4 ] + MSMS spectrum of C18:0 at 15 ev Upper mass range [M + NH 4 ] + MSMS spectrum of C16:1 at 15 ev Upper mass range [M + NH 4 ] + MSMS spectrum of C18:1 A and B at 15 ev Upper mass range [M + NH 4 ] + MSMS spectrum of C18:3 at 10 ev Upper mass range [M + NH 4 ] + MSMS spectrum of C22:6 at 10 ev Lower mass range [M + NH 4 ] + MSMS spectrum of C16:0 at 20 ev Lower mass range [M + NH 4 ] + MSMS spectrum of C18:0 at 20 ev Lower mass range [M + NH 4 ] + MSMS spectra of C16:1, C18:1 A and B at 20 ev Lower mass range [M + NH 4 ] + MSMS spectrum of C18:3 at 20 ev Lower mass range [M + NH 4 ] + MSMS spectrum of C22:6 at 20 ev EIC of [M + Li] xi

12 6.2 EIC of [M 2 + Li] [M + Li] + MSMS spectra of C16:0 at various collision energy levels [M + Li] + MSMS spectra of C16:1 at various collision energy levels [M + Li] + MSMS spectra of C18:3 at various collision energy levels [M + Li] + MSMS spectra of C22:6 at various collision energy levels [M + Li] + MSMS spectrum of C16:0 at 47 ev [M + Li] + MSMS spectrum of C18:0 at 50 ev Center mass range of [M + Li] + MSMS spectrum of C16:0 at 47 ev Center mass range of [M + Li] + MSMS spectrum of C18:0 at 50 ev [M + Li] + MSMS spectra of C16:1 (at 45 ev), C18:1 A and B (at 47 ev) Center mass range of [M + Li] + MSMS spectrum of C16:1 at 45 ev Center mass range of [M + Li] + MSMS spectrum of C18:1 B at 47 ev Center mass range of [M + Li] + MSMS spectrum of C18:1 A at 47 ev [M + Li] + MSMS spectra of C18:3 (at 40 ev) and C22:6 (at 43 ev) [M + Li] + MSMS spectrum of C18:3 at 40 ev, mass range m/z [M + Li] + MSMS spectrum of C18:3 at 40 ev, mass range m/z Center mass range of [M + Li] + MSMS spectrum of C22:6 at 43 ev xii

13 List of Tables 2.1 The solvent gradient used in HPLC-MS runs The main settings of the ESI source, quadrupole and collision cell used in the MS measurements of MAG mixture isolated from shark liver oil The settings used in the MSMS (MRM) measurements of MAG mixture isolated from shark liver oil The main settings of the ESI source, quadrupole and collision cell used in the MS measurements of the synthetic MAG and its precursor The settings used in the MSMS (MRM) measurements of the synthetic MAG and its precursor [M + NH 4 ] + MSMS spectral data for C16:1 synth Signals in lower mass region of [M + NH 4 ] + MSMS of C161 synth [M + NH 4 ] + MSMS spectral data for C16:1 isop HPLC-MS data for the EIC of [M + NH 4 ] Comparison of specific AF for [M + NH 4 ] +, [M + H] +, [M CH 3 O] + and [m H] Comparison of AF for [M + NH 4 ] +, [M + H] +, [M CH 3 O] + and [m H] Major fragments of upper mass range [M + NH 4 ] + MSMS spectrum of C16: Major fragments of upper mass range [M + NH 4 ] + MSMS spectrum of C18: Major fragments of upper mass range [M + NH 4 ] + MSMS spectrum of C16: Major fragments of upper mass range [M+NH 4 ] + MSMS spectrum of C18:1 A Major fragments of upper mass range [M+NH 4 ] + MSMS spectrum of C18:1 B Major fragments of upper mass range [M + NH 4 ] + MSMS spectrum of C18: Major fragments of upper mass range [M + NH 4 ] + MSMS spectrum of C22: Comparison of fragments present in the upper mass range [M+NH 4 ] + MSMS spectra of the natural MAGs and C16:1 synth Major fragments of lower mass range [M + NH 4 ] + MSMS spectrum of C16: Major fragments of lower mass range [M + NH 4 ] + MSMS spectrum of C18: Major fragments of lower mass range [M + NH 4 ] + MSMS spectra of C16: Major fragments of lower mass range [M + NH 4 ] + MSMS spectra of C18:1 A and B Major fragments of lower mass range [M + NH 4 ] + MSMS spectrum of C18: Major fragments of lower mass range [M + NH 4 ] + MSMS spectrum of C22: O 3 fragments present in the lower mass range of [M + NH 4 ] + MSMS spectra HPLC-MS data of [M + Li] + EIC HPLC-MS data of [M + Li] + EIC Comparison of specific ion AF for [M + Li] + and [M 2 + Li] Major fragments of [M + Li] + MSMS spectra of the sample MAGs O 4 containing fragments from [M + Li] + MSMS spectra of C16:0 and C18:0 70 xiii

14 6.6 Two heaviest O 3 containing fragments from [M + Li] + MSMS spectra of C16:0 and C18: Other O 3 containing fragments from [M + Li] + MSMS spectra of C16:0 and C18: O 4 containing fragments from [M + Li] + MSMS spectra of C16:1 and C18:1 B Two largest O 3 containing fragments from [M+Li] + MSMS spectra of C16:1, C18:1 A and B Other O 3 containing fragments from [M + Li] + MSMS spectra of C16:1 and C18:1 B O 4 containing fragments from [M + Li] + MSMS spectrum of C18:1 A Other O 3 containing fragments from [M + Li] + MSMS spectrum of C18:1 A The three small signals of C18:3 in [M + Li] + MSMS spectrum O 4 containing fragments from [M + Li] + MSMS spectrum of C18: O 3 containing fragments from [M + Li] + MSMS spectrum of C18: O 4 containing fragments from [M + Li] + MSMS spectrum of C22: Three largest O 3 containing fragments from [M + Li] + MSMS spectrum of C22: Other O 3 containing fragments from [M + Li] + MSMS spectrum of C22:6. 82 xiv

15 Abbreviations ACN AF APCI BPC DAcG DHA EIC ESI HPLC LC Li-Ac MAcG MAG min MRM MS MSMS NH 4 -Ac RT TAcG TOF Acetonitrile Area fraction Atmospheric pressure chemical ionization Base peak chromatogram Diacyl alkylglycerol Docosahexaenoic acid Extracted ion chromatogram Electrospray ionization High pressure liquid hromatography Liquid chromatography Lithium acetate Monoacyl alkylglycerol 2 -Methoxy alkylglycerol Minute Multiple reaction monitoring Mass spectrometry Tandem mass spectrometry Ammonium acetate Retention time Triacyl alkylglycerol Time of flight xv

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17 Acknowledgements First and foremost I would like to thank my two advisors, Dr. Sigurður V. Smárason and Prof. Guðmundur G. Haraldsson for their guidance and excellent advice. Additionally, I like to thank Prof. Ingvar H. Árnason, a member of my M.Sc. committee, for his support throughout my studies. I would like to thank Dr. Carlos D. Magnusson and Dr. Anna Valborg Guðmundsdóttir for their help and Edda Katrín Rögnvaldsdóttir is acknowledged for providing me with the sample. I would like to thank Ísak Sigurjón Bragason for his immense help on LaTex related matters. The Icelandic Research Fund is acknowledged for financial support. At last but certainly not the least, I would like to thank my family and my friends for their endless support during my studies. xvii

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19 1 Introduction The majority of the Earths biomass stems from marine sources where, for example, phytoplankton accounts for half of the organic matter production. However of all species currently existing, less than 13 % are found in the ocean. This can largely be attributed to the great difference between marine and terrestrial environments where among other factors dehydration and greater effect of gravity impact land based life 1 while marine creatures face e.g. salinity, low oxygen availability 2 and connectivity of habitat. 1 In order to deal with the salinity the oceans inhabitants have come up with two methods, intracellular isosmotic and extracellular anisosmotic regulation. The former is chosen by most invertebrates and is essentially having the same osmolarity (osmotic pressure) as the surrounding seawater. The latter, adapted by e.g. most vertebrates, revolves around regulating both the amount of fluid and cell osmolarity but battling loss of water by constantly drinking sea water but discharging the salts through the gills. The gills also serve the part of respiratory organ but sea water, on average, contains 30 times less oxygen than air. Despite names such as the Atlantic and Indian Ocean, most of the hydrosphere is a continuos global body. Therefore, at least in theory, the marine organisms are free to migrate. Terrestrial animals are often isolated by landmark boundaries such as sea and mountain ranges and thus the feasibility of greater speciation is created. 1 An example of this is the critically endangered kakapo (Strigops habroptilus), the world s largest parrot in New Zealand (NZ). Because NZ was isolated for 70 million years and there were no mammal predators the parrots grew big and lost their ability to fly. Therefore when human settlement began, their numbers decreased drastically as habitat destruction and predation took its toll. 3 Due to the connectivity of the ocean such a case of self-handicapping evolution could not be possible. Therefore the marine environment is highly competitive and aggressive where a high emphasis is put on viability and thus of producing effective and distinct potent molecules. These molecules or compounds show great diversity in structure and biological activities. 4 Algae is an example of organism whose adaptation to a variety of habitats, ranging from freshwater to open ocean, has resulted in co-evolution of immense range of bioactive compounds with diverse uses for humans such as pigments and biodiesel. 5 Furthermore, research is underway towards identifying compounds extracted from marine by-products to improve human health, including vitamins, antioxidants, enzymes, proteins and lipids. 4,6 An example of such by-product is shark liver oil but it has been used in Scandinavia for 1

20 centuries for its remedial effects on injuries. 7 The shark liver oil contains high quantities of 1-O-alkylglycerols which are bio-active ether lipids that take part in the function and structure of membranes of particular cells e.g. white blood cells. Studies have shown that they can reduce injuries caused by radiotherapy and haematopoiesis stimulation. 8 The focus of this study are 2-methoxyl substituted alkylglycerols (MAGs) or more precisely (2 -R)-1-O-(2 -methoxyalkyl)-sn- glycerols which are a subcategory of 1-O-alkylsn-glycerols, 7 whose bioactivity include antifungal, antibacterial and antitumor properties. 9 They were first discovered and isolated by Hallgren and Stallberg in 1967 from the liver oil of Greenland shark (Somniousus microcephalus) 10 and accounted for 4 % of the glycerol ethers. 11 In addition to Greenland shark liver oil the 2 -methoxylated alkylglycerols can be found in cartilaginous fish, marine animals and land mammals in varying concentrations. The highest percentages are present in deep sea sharks in the North Atlantic Ocean. The published molecular structures of these compounds are 1-O-(2 -methoxyhexadecyl)- sn-glycerols (C16:0) shown in figure 1.1, (Z)-1-O-(2 -methoxyhexadec-4 -enyl)-sn-glycerol (C16:1) shown in figure 1.2, (Z)-1-O-(2 -methoxyoctadec-4 -enyl)-sn-glycerol (C18:1) shown in figure 1.3 and 1-O-(2 -methoxydocosa-4, 7, 10, 13, 16, 19 -hexaenyl)-sn-glycerol (C22:6) shown in figure 1.4 where the letter M is used to represent the entire compound while m stands for the alkyl chain excluding the methoxylgroup. The configuration of all of the double bonds is 4 cis. 7 O OCH 3 m OH OH Figure 1.1: Reported molecular structure of C16:0. The molecular formula is C 20 H 42 O 4 (M) while the molecular formula of the alkyl chain, excluding the methoxylgroup is C 16 H 32 (m). M O OCH 3 m OH OH Figure 1.2: Reported molecular structure of C16:1. The molecular formula is C 20 H 40 O 4 (M) while the molecular formula of the alkyl chain, excluding the methoxylgroup is C 16 H 30 (m). M 2

21 O OCH 3 m OH OH Figure 1.3: Reported molecular structure of C18:1. The molecular formula is C 22 H 44 O 4 (M) while the molecular formula of the alkyl chain, excluding the methoxylgroup is C 18 H 34 (m). M O OCH 3 m OH OH Figure 1.4: Reported molecular structure of C22:6. The molecular formula is C 26 H 42 O 4 (M) while the molecular formula of the alkyl chain, excluding the methoxylgroup is C 22 H 32 (m). M These compounds were analyzed by isolating each from the mixture and subjected to nuclear magnetic resonance (NMR) spectroscopy, infrared spectroscopy, gas chromatography-mass spectroscopy (GC-MS) and GC-liquid chromatography (LC) analysis. Furthermore the same compounds had to be synthesized and subjected to the same analysis in order to prove the proposed structure. 10,12 However the main drawback of using GC-MS to separate and identify glycerolipids is that it requires that derivatives of the compounds in question to be made before using GC-MS which is time consuming and may produce unexpected results thus considerable efforts have been made to develop alternative methods. 13 In recent years various MS ionization techniques have been utilized to analyze glycerolipids. Among those are fast atom bombardment (FAB), matrix assisted laser desorption/ionization (MALDI), electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). 13 In order to gain structural details tandem mass spectrometry (MS/MS) has been utilized as it allows focus on one particular ion and all signals that appear are the result of the fragmentation of that ion. Extensive research has been done to locate the position of double bonds in free fatty acids (FA), mono-, di- and triacylglycerols (MAcG, DAcG and TAcG) but also, in the case of diand triacylglycerols, to determine the location of each fatty acid on the glycerol backbone. 14 Multiresidue analysis of low erucic acid rapeseed oil lipids coupled normal phase liquid 3

22 chromatography with positive electrospray ionization (ESI) mass spectrometry (MS) where the ammonia adduct was utilized. A vast quantity of standards was analyzed and comparison between the spectra of the standards and the sample was used to confirm the structure. In spite of the effort, the researchers were unable to determine the location of the double bonds in the case of the unsaturated free fatty acids based on the obtained data. 15 In an ESI tandem MS (MSMS) analysis of MAcG, DAcG and TAcG present in tear samples, characteristic product ions facilitating the determination of the position of the double bonds were identified when the lithium adduct was used. 16 As previously mentioned, FAB has also been used as an ionization source for glycerolipids and in one such study MS and MSMS spectra of the sodium adducts were used to analyze MAcGs extracted from marine sponge where the spectra yielded characteristic fragments and location of double bonds was deduced. 13 However results from an ESI-MS and MSMS structural study of TAcGs utilizing the sodium, ammonium and lithium adducts, showed that although all three adducts produced data where the location of the fatty acids on the glycerol backbone could be established the double bond elucidation on the fatty acid chain was not successful when using sodium and ammonium adducts. 14 When analyzing free fatty acids the lithium adducts ([M H + 2 Li] + ) provide simplified spectra as the two lithium ions stabilize the oxygens completely and thus the only fragments visible are due to the chain. These studies utilized ESI 17,18 and MALDI. 19 As the charge is positioned on the polar end of the FA the fragmentations occur without the involvement of the charge or by so called charge remote fragmentation (CRF) These often result in neutral fragment losses of C n H 2n+2 where n is an integer. 17,20 Multidimentional MS experiments, where ESI has been coupled together with ion mobility spectrometry and mass spectrometry, have been applied to structural analysis of phospholipids and related structures. 21 A B. Sc. project performed at the University of Iceland utilizing HPLC-MS to analyze and quantify methoxy glycerols isolated by same procedure as presented here, identified the major compounds as C16:0, C16:1, C18:0, C18:3, C22:6 and C18:1. The chromatogram suggested that the C18:1 consisted of two different isomers where the position of the double bond varied between the two. 22 Therefore the purpose of this work consists of developing and improving the chromatographic separation of the compounds present in the sample with focus on separating compounds that have similar masses. Furthermore, to develop a MSMS method which produces quality spectra in a reproducible fashion and explore the feasibility of structural analysis based on the data obtained. 4

23 1.1 Brief introduction into equipments and methods In this section a very short presentation is provided of the techniques used and the nomenclature that was used in this work High performance liquid chromatography High performance liquid chromatography (HPLC) is a separation technique based on the interaction of a solid stationary phase and liquid mobile phase under pressurized flow. 24 Using reverse phase chromatography results in faster elution of more polar compounds than the less polar ones which is caused by the mobile phase being more polar than the stationary phase Mass spectrometry The mass spectrometer used in this work contains among other a hexapole, an analytical quadrupole, a collision cell and a time of flight (TOF) mass analyzer. Tandem spectra can be measured and in those cases the analytical quadrupole acts as a mass selector ensuring that only the wanted ions, one at a time, are allowed to enter the collision cell and then fragmented. In ESI the ionization takes place directly from the solution where it is pumped through a high voltage capillary resulting in the formation of highly charged droplets. These solvents are desolvated by a stream of drying gas leaving the analytes ionized. 27 In APCI it is the opposite where the solution is vaporized at high temperatures and the analytes are ionized by a gas phase ion-molecule reactions commenced by a corona discharge. 28 The mass analyzer is a high resolution and so can differentiate between different isotopes. Therefore only monoisotopic mass is shown and discussed. Furthermore, all the ions and fragments are singly charged and unless otherwise specified the unit of intensity of the MS and MSMS spectra is counts Nomenclature and additional concepts BPC vs. EIC The raw output of data from a HPLC-MS or MSMS measurement is a chromatogram. Base peak chromatogram (BPC) shows the intensity of the highest ion in every spectrum while in an extracted ion chromatogram that ion has been predefined and the resulting signal indicates (EIC) a mass has been defined and the chromatogram only shows the intensity of that particular mass. 30 AF vs. AF ion When running HPLC-MS experiment, sometimes more than one ion is visible for each compound. Therefore a comparison of area fractions (AF) and ion specific area fractions (AF ion ) 5

24 is feasible. For a compound X where the area (A) under its specific ion is A X ion these quantities would be calculated as follows: AF = (A X ion / S total ) 100% AF ion = (A X ion / S ion ) 100% where S total is the sum of all measured areas but S ion the sum of the signals that stem only from the particular ion. I Err vs. E Err Internal error (I Err ) is a measurement of precision or how well the measured mass values compare to each other where the average measured mass is compared to each and then the average calculated. External error (E Err ) is the evaluation of accuracy or how well the measured mass values compare to the correct value or in this case the theoretical mass. 6

25 2 Experimental 2.1 Lipid samples (Z)-(2 R)-1-O-(2 -methoxyhexadec-4 -enyl)-sn-glycerol (C16:1 synth ) and its precursor (Z)- (2 R)-1-O-(2 -methoxyhexadec-4 -enyl)-2,3-o-isopropylidene-sn-glycerol (C16:1 isop ) were synthesized and exact structure confirmed by NMR and accurate mass. 9 The synthetic C16:1 was diluted in a chloroform/acetonitrile mixture (1:1; v/v) prior to injection but its precursor diluted in n-hexane/acetonitrile/chloroform solution (1:1:1; v/v/v). The 2 -methoxylated alkylglycerols (MAGs) were isolated from the liver oil of deep sea shark originating from the North Atlantic Ocean. In short the isolation procedure consists of methanolysis of the squalene free liver oil, separation utilizing silica gel column chromatography and lastly, the fraction containing the desired compounds being further purified by prep. TLC. 31 To 46 mg of the 2 -methoxylated alkylglycerol mixture, 500 µl of chloroform were added in order to dissolve it. The resulting solution was used as a stock solution. Portions of it were diluted by factor 1/10 with chloroform and used in HPLC-MS and MSMS measurements. Both were stored in the dark at -25 C when not in use. 2.2 Chemicals Acetonitrile (ACN), chloroform, n-hexane and 2-propanol were all of HPLC grade and purchased from Sigma Aldrich. All aqueous solutions were prepared using deionized water purified by EasyPure RoDi system (Barnstead). Formic acid (Fluka) and acetic acid (Riedel dehaën) were both of puriss grade ( %). Lithium acetate dihydrate was purchased from Sigma Aldrich and was purum p.a. grade (crystalized). Lithium hydroxide (monohydrate, 56 %) was from Acros and ammonium salt of acetic acid (98+ %, anhydrous) from Acros organics. 2.3 High pressure liquid chromatography (HPLC) All HPLC measurements were carried out using Agilent Technologies 1200RR series HPLC consisting of a degasser (model no. G13798), a binary SL pump (model no. G1312B), an autosampler (model no. HiP-ALS SL G1367C) and a thermostatted column compartment (model no. G1316B). The HPLC system was coupled to a Bruker microtof-q spectrometer which was used as the detector. Chromatographic separation was achieved using a 3 µm particle size reverse phase C18 Gemini-NX, 110 Å, 150 x 2.00 mm column (Phenomenex). 7

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