chemical derivatization in combination with liquid …171930/fulltext01.pdf · chemical...

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 72

Chemical Derivatization inCombination with LiquidChromatography Tandem MassSpectrometry for Detection andStructural Investigation ofGlucuronides

MATILDA LAMPINEN SALOMONSSON

ISSN 1651-6192ISBN 978-91-554-7178-1urn:nbn:se:uu:diva-8670

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Papers discussed

This thesis is based on the following papers, which will be referred to in the text according to their Roman numerals:

I. Detection of altrenogest and its metabolites in post administration horse urine using liquid chromatography tandem mass spectrometry-increased sensitivity by chemical derivatization of the glucuronic acid conjugate. Matilda Lampinen-Salomonsson, Elin Beckman, Ulf Bondesson, Mikael Hedeland J. Chromatogr. B, Volume 833, Issue 2, 3 April 2006, 245-256. II. Differentiation of estriol glucuronide isomers by chemical derivatiza-tion and electrospray tandem mass spectrometry. Matilda Lampinen-Salomonsson, Ulf Bondesson, Carl Petersson, Mikael Hedeland. Rapid Commun. Mass Spectrom., Volume 20, Issue 9, 15 May 2006, 1429-1440. III. Structural evaluation of the glucuronides of morphine and for-moterol using chemical derivatization with 1,2-dimethylimidazole-4-sulfonyl chloride and LC-MSn. Matilda Lampinen Salomonsson, Ulf Bondesson, Mikael Hedeland. Submitted. IV. In vitro formation of phase I and II metabolites of propranolol and determination of their structures using chemical derivatization and liq-uid chromatography tandem mass spectrometry. Matilda Lampinen Salomonsson, Ulf Bondesson, Mikael Hedeland. In manuscript. Reprints were made with kind permission from the publishers.

Additional paper not included in the thesis

Validation of a method for quantification of ketobemidone in human plasma with liquid chromatography tandem mass spectrometry. Matilda Lampinen, Ulf Bondesson, Elisabeth Fredriksson, Mikael Hedeland J. Chromatogr. B, Volume 789, Issue 2, 15 June 2003, 347-354.

Contents

Introduction...................................................................................................11�Aims .........................................................................................................14�

Liquid chromatography- Mass spectrometry ................................................15�Liquid chromatography (LC) ...................................................................15�Mass Spectrometry (MS) .........................................................................15�

History .................................................................................................16�Ionization .............................................................................................17�Mass analyzer ......................................................................................18�

Drug metabolism...........................................................................................23�Substances investigated............................................................................24�

In vitro production of glucuronides ..............................................................26�

Structural investigation of metabolites .........................................................29�

Chemical derivatization ................................................................................37�Increased sensitivity in LC-MS................................................................37�Structural investigation with LC-MS .......................................................40�

Conclusions...................................................................................................57�

Future aspects ...............................................................................................59�

Populärvetenskaplig sammanfattning ...........................................................60�Kemiska aspekter för läkemedelsmetabolism ..........................................60�Individers olika metabolism .....................................................................61�Analysmetoder .........................................................................................62�

Acknowledgements.......................................................................................64�

References.....................................................................................................67�

Abbreviations

C18 Saturated carbon chain of 18 carbon atoms CID Collision-induced dissociation CMPI 2-chloro-1-methylpyridinium iodide dc Direct current DMISC 1,2-Dimethylimidazole-4-sulfonyl chloride E3G Estriol 3�-D-glucuronide E16G Estriol 16�-(�-D-glucuronide) E17G Estriol 17�-(�-D-glucuronide) EDC 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide EI Electron ionization ESI Electrospray ionization GC Gas chromatography LC Liquid chromatography M3G Morphine-3�-D-glucuronide M6G Morphine-6�-D-glucuronide MS Mass spectrometry MS/MS Tandem mass spectrometry MSn General designation of mass spectrometry to the nth degree m/z Mass-to-charge ratio NADPH Nicotinamide adenine dinucleotide phosphate PA 2-Picolylamine Q Quadrupole mass analyzer QQQ Triple quadrupole mass analyzer rf Radio frequency RP Reversed phase SRM Selected reaction monitoring TEA Triethylamine TOF Time-of-flight tR Retention time u Unified atomic mass unit UDPGA Uridine 5´-diphosphoglucuronic acid UDPGT Uridine 5´-diphosophoglucuronyl transferase UV Ultraviolet

11

Introduction

Once a xenobiotic (foreign) molecule such as a drug enters a living organ-ism, different enzyme systems take action to remove it through different metabolic reactions. Metabolism is a conversion of one chemical entity to another, that is, a change of characteristics, which makes it easier for a xenobiotic substance to be eliminated as it obtains more hy-drophilic properties.1,2 A drug’s biological effect and activity in the body depends on its interaction with the target structures, which in turn depends on its characteristics. Prop-erties such as lipophilicity, hydrophilicity, acid-base ionization, and chemi-cal reactivity affect the characteristics and hence the concentration of the drug at the site of action in the body through absorption, distribution, me-tabolism, and elimination.2 Metabolic modifications can occur as phase I and phase II reactions. In a phase I reaction, a part of the molecule is modified or removed, or a func-tional group is added, e.g., oxidation, reduction or hydrolysis. This conver-sion is often a preparation of the drug for a phase II reaction, which involves conjugation with a more water-soluble molecule. Three examples of phase II reactions are glutathione conjugation, glucuronidation, and sulfation. Conju-gated drug metabolites can easily be excreted in the urine, and thus be elimi-nated from the body.1,2 Metabolism generally involves deactivation of a drug’s activity. However, some studies have demonstrated situations where a drug metabolite is more active then the drug itself, for example the antidepressant drug imipramine and its phase I metabolite desipramine, or the drug diazepam (used for treatment of anxiolytic) and its more active phase I metabolite oxazepam. Both of these latter forms could therefore be used in medicinal treatment. Today, in development of a drug, a so called pro-drug can be purposefully created. A pro-drug is a drug which is pharmacologically inactive in the form in which it is administered, but which is converted/metabolized in the body to a pharmacologically active substance. Prontosil (used as a antibiotic in the 1930s) is a classic example of a pro-drug, as it is converted to its ac-tive metabolite sulfanilamide through azo reduction.1

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Metabolic reactions can also convert a drug to a toxic entity. An example of this is the metabolism of paracetamol, in which a reactive imine metabolite is formed in a phase I reaction. This metabolite is normally further detoxified through glutathione conjugation.1 In humans, approximately 40-70 % of xenobiotics are metabolized to form glucuronides, and thus it is essential to investigate this class of metabolites. It is important to determine the exact position of the conjugation during drug development, as the glucuronidation can produce substances which are more or less pharmacologically active2. The main metabolites of morphine — morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) — serve as examples of this phenomenon, as M6G is more pharmacologically active than morphine itself, and it has been claimed that the analgesic effect is antagonized by the M3G2,3. Other examples of drug glucuronides with direct therapeutic effects are the 16´-glucuronides of digitoxin and digoxin4, one of the O-glucuronides of ezetimibe5, and the O-glucuronide of thiocol-chicoside6. In other cases, conjugated metabolites (glucuronides) have shown to exert indirect effects on the pharmacokinetics of the parent drug. An example of this is the enterohepatic recycling (hydrolysis of the biliarly excreted glu-curonide conjugate in the intestine giving the aglycone) of ezetimibe, which results in a long plasma half-life7. It has also been proposed that glucuron-ides may be directly toxic, due to their direct interferences with cell func-tions8. An example of this is the acyl glucuronide, which is formed by con-jugation with a drug’s carboxylic acid; that is, an esterfication occurs. The NSAID drug diclofenac serves as an example of this, as it is partly metabo-lized through this reaction9. The N-O-glucuronides of hydroxamic acids comprise another example of potentially toxic metabolites3. Today, the number of new chemical entities — potential drugs — has in-creased rapidly with the use of combinatorial chemistry and modern high throughput bioassays. Due to this, there is a growing need for novel bioana-lytical systems which allow fast determination of the metabolites of these substances10. “In development, elucidation of biotransformation pathways of a drug can-didate by identifying its circulatory and excretory metabolites is vitally im-portant to understand its physiological effects.” -Chandra Parkash 2007 10 A number of regulatory bodies have emphasized the need to understand the metabolism of drugs. The U.S. Food and Drug Administration (FDA), which is responsible for regulating drug safety testing, recommends that the meta-bolic pathway of the drug should been identified using in vivo or in vitro

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methods during its development. Furthermore, any metabolites formed to a greater extent than 10% of the parent drug (systemic exposure at steady state) should be of safety concern. The safety concern leads to a further non-clinical study of the drug metabolite.11 These regulatory recommendations and requirements suggest that studies of drug metabolites require analytical methods which can determine both the compound’s concentration and its structure. There are a number of tech-niques used for elucidation of metabolites, including radiolabeling with ei-ther C14 or H3 in combination with liquid chromatography (LC) and a radio-chemical flow detector or a accurate radioisotope counting detector system, along with spectrometry and wet chemistry methods10. These are comple-mentary methods often used in combination with the dominant techniques mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectrome-try. For structural characterization and quantification of a substance in meta-bolic projects, MS has played an invaluable role as a detection method. The use of MS in combination with LC has increased enormously over the last ten years12, and it is now a well-known approach for structural determination of drugs and their metabolites, due to its selectivity and high sensitivity13. Although NMR is still the most commonly used technique for determining molecular structure, MS is the superior method for analysis of biological samples as it requires smaller sample amounts than NMR. Metabolic investigations mainly involve the identification of unknown sub-stances. When using MS, structural conclusions are drawn from measure-ments of the mass-to-charge ratios of the protonated/deprotonated molecules and their fragments. One analytical challenge in the structural determination of glucuronides (phase II metabolites) using tandem mass spectrometry (MS/MS) is the loss of monodehydrated glucuronic acid due to a break in the glucuronide bonding14. Thus, the product ion spectra from these types of analyses only give the protonated/deprotonated aglycone and its fragments, which results in no information about the position of the conjugation. If the information from the MS analyses is not enough for the structural evaluation, different types of chemical derivatization can be used to change the fragmentation pattern, and also to enhance the detection. Chemical deri-vatization in combination with LC-MS is a relatively unexploited technique, but its use has increased significantly in the last five years15. MS is indeed a powerful technique, but several drugs lack functional groups that can be easily ionized, using LC-ESI-MS, and at this point chemical derivatization has been most useful to enhance the detection15-49. Derivatization has also been applied to facilitate the structural evaluation of drug metabolites using LC-MS50-57.

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Aims This thesis focuses on enhancement of detection and structure evaluation of glucuronides (phase II metabolites) using chemical derivatization and LC-MS/MS or LC-MSn analyses. The specific aims were:

� to investigate the metabolism of altrenogest in horses, with the inten-tion of developing an LC-MS method for identification of the drug and its main metabolites in biological samples, specifically urine (Paper I);

� to differentiate isomeric glucuronides using chemical derivatization

as a tool together with LC-MS/MS or LC-MSn (Papers II and III);

� to apply the methods developed in Papers II and III for structural investigation of propranolol metabolites produced in-house in two in vitro systems (Paper IV).

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Liquid chromatography- Mass spectrometry

Liquid chromatography (LC) LC is one of the most widely used separation techniques in the field of anal-ysis of drugs in biological samples. Today, a simple LC system contains a mobile phase reservoir, a pump, an injection valve, and a column (often stainless steel). The separation takes place under high pressure in the column containing a stationary phase, and separation of the analytes is achieved by means of their different distribution between the mobile and stationary phas-es. The most common stationary phase is a non-polar material, often silica with covalently bonded carbon chains e.g., C18. This type of stationary phase is referred to as a reversed phase, and when it is used in combination with a mobile phase (delivered as a gradient or isocratically) mostly contain-ing water, often as a buffer or with a small proportion of an acid such as formic acid or acetic acid, the technique is known as reversed-phase chroma-tography58.

The separation technique used throughout the works in this thesis was LC using a reversed-phase system with a C8, C18, or Polar RP column and a mobile phase containing methanol or acetonitrile in combination with 0.1% acid (formic or acetic) in water. The separation system was coupled to dif-ferent types of mass spectrometers used as detectors for investigation of drugs and their metabolites.

Mass Spectrometry (MS) MS is a technique for measuring the mass-to-charge ratio of molecules; it has been in use for approximately one century13. Molecules are ionized with different types of techniques, before entering the mass spectrometer, where they travel as ions in the gas phase under low pressure and are separated in a mass analyzer before detection takes place (Figure 1).

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Figure 1. Simplified block diagram of a LC–MS system.

History In 1897 the electron was discovered and its mass-to-charge ratio was deter-mined by Thomson, who was awarded the 1906 Nobel Prize in Physics for this work13. Thomson also built the first mass spectrometer, in 1912, which he analyzed multiplied, negative, and metastable ions13. In 1948, Cameron et al.59 reported a design as well as mass spectra for a linear time-of-flight (TOF) mass analyzer; however, the first commercial instrument was intro-duced two decades later13. The first quadrupole mass analyzer was described in 1953 by Paul et al.60, who also explained the ion trap some years later in a patent61. Paul’s description and development of these instruments were re-warded many years later (1989) with the Nobel Prize in Physics. The colli-sion-induced dissociation (CID) procedure was introduced by McLafferty et al.62 and Jennings63 during 1967-1968, and the first commercial quadrupole mass spectrometer became available shortly after13. In the same period, the initial electrospray based mass analysis system was proposed by Dole et al.64. In 1973, Cooks made a landmark in MS/MS with the book Metastable Ions, 13 and a year later Arpino, Baldwin, and McLafferty presented the first LC-MS system13. The first triple quadrupole instrument was built by Yost et al. in the late 1980s, and the first commercial implementation became avail-able four years later13. The use of LC in combination with MS needed an improved ionization technique with high sensitivity which converted the molecule in the liquid to ions in a gas phase. The development of the elec-trospray to electrospray ionization (ESI), that is, integration of ESI with MS for small molecule analysis, was reported in 1984 by Yamasita and Fenn65,66 and simultaneously by Aleksandrov13,67. Later the same decade, the tech-nique was applied for analysis of multiply charged ions from proteins68. Fenn was awarded the 2002 Nobel Prize in Chemistry for his work. The very high sensitivity of the ESI has revolutionized the use of MS, and the coupling of different separation systems to MS, such as LC and capillary

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electrophoresis, also offers the possibility to gain more information from compounds in complex mixtures. New classes of instruments and applica-tions have been developed, and the technique has progressed extremely rap-idly over the last decade13. Today, LC-ESI-MS is one of the most commonly used methods for chemical analysis of both small polar molecules and larger complex biomolecules13,69.

Ionization As mentioned above, one problem with coupling separation systems like LC to a mass spectrometer is the requirement to transform molecules in a liquid phase to ions in a gas phase. When LC is used as a separation technique, the main ionization methods used are ESI and atmospheric pressure chemical ionization (APCI), both of which are atmospheric pressure ionization (API) methods13. In ESI, an aerosol is formed by the liquid exiting the LC, due to the electric charge set on the liquid surface. This formation also depends on the gas flow, which assists the evaporization of the liquid in the ion source (Figure 2). Droplets of the liquid in the aerosol are reduced in size, owing to evapo-ration closer to the inlet of the mass spectrometer. The highly charged drop-lets undergo coulombic explosions when the repulsion overrides the surface tension. Finally, gas-phase ions are produced and protonated/deprotonated molecules enter the MS due to the electriostatic attraction and the low pres-sure inside the instrument. The exact mechanisms for the formation of the gas-phase ions are not fully understood but there are two theories; the charged-residue model and the ion-evaporation model, both of which were discussed by Kebarle and Ho69,70.

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Figure 2. Schematic figure of the principle of ESI based on the charged-residue model.

ESI was the ionization technique used in the work described in this thesis.

Mass analyzer In a mass analyzer, the ions are separated according to their mass-to-charge ratios. The majority of mass analyzers are scanning devices like quadrupoles but there are also a small number of analyzers which can simultaneously transmit all ions. Two examples of this are ion trap and TOF mass analyzers. These mass analyzers are often combined with quadrupoles, resulting in an instrument with both characteristics e.g., quadrupole time-of-flight (Q-TOF) instrument. The choice of mass analyzer depends on the applications and on the type of information that is needed. The main characteristics differentiat-ing the analyzers are the resolution, the transmission, and the upper mass limit.13

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Quadrupole A quadrupole contains four rods of stainless steel with a circular hyperbolic section. The rods are placed perfectly parallel, and an electric field is applied in the form of direct current (dc) voltage and radio frequency (rf) voltage. The opposing rods have equal charges; that is, two are positively charged and two are negatively charged. The ions entering the quadrupole are sub-jected to the electric field and drawn against the oppositely charged rod, and as the potentials are changed the ions will travel through the quadrupole section. Depending on the supplied potential (controlling the rf-to-dc volt-age), different ions will be selected, and will either be transmitted or be dis-charged and not analyzed further. The ions are separated depending on their mass-to-charge ratio.13 A triple quadrupole system (QQQ) (Figure 3) contains three sections, with the middle one being a so-called collision cell. The collision cell is filled with an inert gas under a certain pressure. Using this gas, the kinetic energy of the ions is converted to internal energy and they undergo collision.13 This process is known as a CID procedure. Using all three sections QQQ in the analysis is known as tandem mass spectrometry (MS/MS) and in modern instruments the collision cells often contain a hexapole instead of a quadru-pole, but the form varies by manufacturer.

This type of mass analyzer was used in Papers I, II, and IV.

Figure 3. Schematic figure of a QQQ instrument. The picture was provided by Da-niel Salomonsson.

This type of mass analyzer offers the possibility of using a number of modes besides conventional full MS scan and product ion scan (MS/MS). The mod-es precursor ion scan (MS/MS), selected reaction monitoring (SRM) and neutral loss (NL) can also be performed which are very valuable tools to facilitate the evaluation in metabolism studies (Figure 4).

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Figure 4. Scan modes using a QQQ instrument A) product ion scan, B) precursor ion scan, C) neutral loss scan, and D) selected reaction monitoring.

In analysis modes product ion scan, precursor ion scan, NL scan or SRM the ions undergo CID in the collision cell. In product ion scan the first quadru-pole (Q1) is fixed on a selected ion, whereas the third quadrupole (Q3) is used to scan for fragment ions, while using precursor ion scan the Q1 is used in scan mode whereas the Q3 is fixed. In NL scan both the Q1 and Q3 are used in scan modes but with the certain mass offset between, and in SRM both the Q1 and Q3 are fixed for specific ions.13

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Ion trap An ion trap (Figure 5) consists of one ring electrode and the two end-cap electrodes, one at the electron inlet and one at the electron outlet, which traps ions through a change in the potential69. This type of system is very useful in structural analysis, as it can perform repeated collision and trapping (i.e. MSn) of selected ions. Inside the ion trap, all the ions can be present at the same time13. This mass analyzer was used in Papers II, III, and IV.

Figure 5. Schematic figure of a Q-TOF trap instrument. The picture was provided by Daniel Salomonsson.

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Time-of-flight In a TOF instrument (Figure 6), the ions travel in a field-free region and their different flight times are measured. The flight time depends on the ion’s m/z; ions of lower mass have a higher velocity, and thus travel faster than ions of higher mass13. From the source, the ions are delayed and accelerated with a voltage pulse that separates the ions according to their velocity69. The ions fly towards an electrostatic reflector, whereupon a deflection occurs and the ions change direction against the detector. The flight times are measured and the ions’ mass-to-charge ratios are determined. This instrument provides very exact measurements and is therefore often used to accurately determine the masses of ions. This type of mass analyzer was used in Paper II for determination of the elemental compositions of fragments.

Figure 6. Schematic figure of a TOF instrument.

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Drug metabolism

The main parts of metabolic reactions take place in the liver and are cata-lyzed by microsomal enzymes. Different enzymes produce different types of metabolites. For the phase I reactions, the cytochrome P450 family is the main actor and the reactions require the presence of the cofactor nicotina-mide adenine dinucleotide phosphate (NADPH). The cytochrome P450 is the major enzyme system in the body which contains many isoforms.1 In this thesis, two phase II reactions were studied; glucuronidation and sul-fation.

The main enzyme responsible for the formation of a glucuronide of a drug (phase II reaction) is uridine 5’diphosophoglucuronyl transferase (UDPGT). This metabolite is formed through a conjugation between a phase I metabo-lite of a drug (or its native form) and the cofactor uridine 5´-diphosphoglucuronic acid (UDPGA)1. The reaction is catalyzed by UDPGT, which is mainly found in the liver but can also occur in other tissues1. Glu-curonidation can take place with different functional groups, such as alco-hols, phenols, hydroxylamines, carboxylic acids, amines, sulfonamides, or thiols1. Depending on the binding position to the drug, these reactions can result in four types of glucuronides; O-, N-, S-, or C-glucuronides1,15. The most common glucuronides are O- and N-glucuronides1. Sulfation is another phase II reaction; it occurs mainly between a drug’s phenol group and 3’-phosphoadenosine-5’-phosphosulfate (PAPS). This reaction is catalyzed by the cytosolic enzyme sulfotransferase1. The characterization of a substance’s metabolism in the body is very impor-tant during the development of a new drug. As discussed in the introduction, a metabolite can be more or less pharmacologically active or toxic, and so knowledge of the metabolite’s effect can change the use of a drug. The knowledge of the metabolic rate and elimination will also partly determine the dose of a drug in a treatment.

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Substances investigated In this thesis, five model compounds and their metabolites were structurally investigated. In Paper I, the metabolism of the steroid altrenogest (Figure 7), used in breeding for control of estrus in horses71 was studied as the bio-transformation of altrenogest was previously unknown. Using LC-ESI-MS this substance has earlier been qualitatively studied, in doping control pur-poses72,73. Furthermore a quantitative method for determination of altreno-gest in horse urine was published last year74.

Figure 7. Structure of altrenogest.

Paper II comprised an investigation of another steroid; estriol, which is a natural female estrogen hormone formed via 16� hydroxylation of estrone in the ovaries and through 16� hydroxydehydroepiandrosterone in the placenta of pregnant women2. One of the main metabolic pathways for elimination of estriol is through glucuronidation2. The conjugated metabolites have previ-ously been qualitatively detected and quantitatively determined using LC-MS methods75-77. In this study, the conjugated glucuronides of estriol (Figure 8) were investigated structurally with the aim of being able to differentiate them from each other when all are present in the same sample.

Figure 8. Structures of estriol and its metabolites estriol-3-glucuronide, estriol-16-glucuronide, and estriol-17-glucuruonide.

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In Paper III, morphine, morphine-3-glucuronide, morphine-6-glucuronide, formoterol (Figure 9), and in-house produced formoterol glucuronides were investigated. Morphine is a well-known analgesic drug which has been used for centuries to relieve severe pain. The metabolism of this substance is well documented, and in 2007 Bosch et al.78 discussed in a review article the analysis methods used the last three decades for determination of morphine and its main metabolites in various matrix. Among other analysis methods described, LC-ESI-MS was used for quantification of M3G and M6G in biological samples79-84.

Figure 9. Structures of the model compounds formoterol, morphine, and two of its phase II metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G).

Conversely, studies of the metabolism of formoterol, which is used in man-agement of asthma, are few. The studies of this substance using MS have mostly been qualitative85-88 but one quantification have also been reported89.

Finally, Paper IV describes an investigation of the model compounds pro-pranolol and its phase I metabolite 4’-hydroxypropranolol, and their glu-curonide metabolites. The metabolism of propranolol using LC-MS has pre-viously been reported90-93.

Figure 10. Structures of the model compounds propranolol and 4’-hydroxypropranolol. The asterisk point out the asymmetric centra in the molecule.

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In vitro production of glucuronides

Reference substances for studies of phase II metabolites are not particularly widely available. There are a few ways to obtain these metabolites for a study; buying commercially available substances, obtaining substances in biological material such plasma or urine, chemical synthesis94, or production using an in vitro enzyme system95 such as specific UDPGT96,97 or micro-somes93,98 and incubation with UDPGA. The choice of approach will depend on the purpose of the study and the availability of the substance in question.

The difficulty of obtaining biological samples varies with the type of sub-stance. Horse urine samples were obtained for the study in Paper I. Com-mercially-available reference substances are often very expensive, and very few glucuronides are produced due to the low demand and the high produc-tion costs; however, in Paper II (the glucuronides of estriol) and Paper III (the glucuronides of morphine) some or all reference substances were avail-able, and therefore bought and used. In these types of studies, structural in-vestigation of glucuronide isomers, characterized reference substances are preferred in the method development before it can be applied on a biological sample. Another approach to be able to study glucuronides are to produce them using chemical synthesis or an in vitro system. In Paper III and Paper IV, pro-duction of glucuronides was described with a specific human UDPGT; UGT2B7, which is the enzyme mainly responsible for the production of glucuronides of opioids. This enzyme has also shown the ability to produce glucuronides with aliphatic alcohols and phenols. Interestingly, the produc-tion of glucuronides using UGT2B7 and UDPGA gave two chromatographic peaks with m/z agreeing well with that of glucuronides for each model com-pound; two formoterol glucuronides (Paper III) and two 4´-hydroxypropranolol glucuronides (Paper IV). For Paper IV, an in vitro system with rat liver microsomes was set up to evaluate the metabolites of propranolol. The main metabolites studied were the glucuronides formed from the incubation of S-propranolol, R-propranolol, rac-propranolol, and rac-4´-hydroxypropranolol with cofactors NADPH and UDPGA together with microsomes. For the production of phase II metabolites, UDPGA was added together with the microsomes. The

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results from the incubations showed one peak for each enantiomer, with an m/z agreeing with that of a glucuronide metabolite of propranolol, (Figures 11A-11B). The incubation of the rac-propranolol resulted in two peaks which agreed well (regarding retention time (tR)) with the two peaks ob-tained for the enantiomers; thus, two diastereoisomeric glucuronides were formed (Figure 11C). The incubation of rac-4´-hydroxypropranolol with microsomes and UDPGA gave only one peak for a glucuronide (data not shown). The production of metabolites (through phase I and phase II reac-tions of S-propranolol, R-propranolol, and rac-propranolol) gave five phase I metabolites with m/z agreeing with the following proposed metabolites: three monohydroxypropranolol isomers and two dihydroxypropranolol iso-mers, and three phase II monohydroxypropranolol glucuronides, but the amount of the formed metabolites varied between the enantiomers (Figures 12A-12D).

Figure 11. Extracted ion chromatograms of m/z 436 from full scan MS analyses (triple quadrupole). A) S-propranolol glucuronide, B) R-propranolol glucuronide and C) rac-propranolol glucuronides. The glucuronides were produced with microsomes and UDPGA. (Paper IV)

28

Figure 12. Full MS scan analyses (triple quadrupole) and extracted ion chroma-tograms of S-propranolol and R-propranolol (microsome incubated with NADP and UDPGA). A) [M + H]+ m/z 276 S-hydroxypropranolol, B) [M + H]+ m/z 452 S-hydroxypropranolol glucuronides, C) [M + H]+ m/z 276 R-hydroxypropranolol, D) [M + H]+ m/z 452 R-hydroxypropranolol glucuronides. (Paper IV)

29

Structural investigation of metabolites

As explained earlier, it is very important to determine the identities and structures of a new drug’s metabolites early in the development process, since the body’s reaction to a foreign substance may result in different levels of pharmacological effects or toxicity. Most metabolic studies performed today make use of LC-MS/MS, as this technique offers high selectivity, sensitivity, and as it also can give rapid and accurate structural information. It is also important to study drug metabolism and pharmacological effects of drugs used in veterinary medicine, in order to ensure animal welfare. Paper I concerns the metabolism of the steroid altrenogest (Figure 7), which is used in horse breeding to control estrus71. Estrus can be a problem in horse-racing, as it can have a negative effect on the horse’s performance. The pharmacological effects of altrenogest are necessary in breeding, but it may not be used under competition conditions as the rules of horseracing do not allow a horse to compete if it is under the influence of any kind of medica-tion (rules in many European countries)99. The purpose of this rule is to maintain animal welfare, as well as to ensure fairness of competition and gambling. When seeking to detect prohibited drugs, it is of great importance to have a safe and efficient control system. Identification of a positive dop-ing sample from a horse is defined as a finding of either the drug itself in a biological fluid, or a metabolite of the drug, or an isomer of the substance99. The samples in this study consisted of urine collected from three different horses which had been given orally administered Regu-Mate® (altrenogest 0.044 mg/kg bodyweight). In investigations of metabolism of a substance, it is crucial to analysis the parent compound, and so altrenogest was evaluated using full MS and MS/MS scan mode, in addition to do SRM for the detec-tion of low levels of analyte. The full MS/MS analyses (performed in both positive and negative mode) gave fragments as shown in Figures 13A-13B. The predominant fragment shown in positive mode was m/z 227, which was observed earlier by Thevis et al.100. The full MS/MS analysis in negative mode showed similarities as well as differences compared with positive mode; see the proposed cleavage sites in Figures 13A-13B.

30

Figure 13. A) Positive product ion spectrum and suggested cleavage sites of altreno-gest, precursor [M+H]+ m/z 311, collision energy 29 V. B) Negative product ion spectrum and suggested cleavage sites of altrenogest, precursor [M-H]- m/z 309, collision energy 20 V. (QQQ instrument) (Paper I)

In the search of metabolites in the urine samples the result indicated that no phase I metabolites of altrenogest were found. However, both sulfate conju-gate [M - H]- m/z 389 and glucuronic acid conjugate [M + H]+ m/z 487 were detected by doing full MS/MS analyses (Figures 14 and 15), though the de-tection signals were low. Their CID patterns agreed well with those of the protonated aglycone fragmentation pattern (c.f. Figures 13A-13B). These results are analogous with previous results regarding metabolism of other steroids, where the main metabolites formed were phase II metabolites such as glucuronides and sulfates101.

31

Figure 14. (A) Extracted ion chromatograms of a directly injected urine sample taken on the 8th day of Regu-Mate® administration analyzed in positive mode. Full scan product ion, precursor m/z 487, collision energy 35 V. (B) Full scan product ion spectrum of the peak eluting at 3.37 min. (QQQ-instrument) (Paper I)

32

Figure 15. (A) Extracted ion chromatograms of significant peaks of altrenogest in negative mode. Full scan product ion, precursor m/z 389 corresponding to altreno-gest sulfate collision energy 40 V. (B) Full scan product ion spectrum of the peak eluting at 1.3 min. (QQQ-instrument). (Paper I)

33

Paper I presents a useful qualitative analytical method for determination of altrenogest and its metabolites in horse urine. With this method, 0.042 nM of altrenogest in horse urine can be determined using LC-MS/MS in the SRM mode. The MS/MS analysis of altrenogest glucuronide (Figur 14B) shows the pre-dominant ion of the protonated algycone i.e., a neutral loss of 176 u. In the LC-MS/MS analyses, the most commonly observed collision-induced frag-ments from the glucuronide conjugated drugs are [M + H – 176]+, i.e., a neutral loss of monodehydrated glucuronic acid14. The loss of water (- 18 u) could also be observed, but this does not provide any structural information on the binding site of the glucuronyl moiety. As discussed earlier, conjugation can occur with different functional group of a drug, thus isomers of the metabolites are commonly formed. The iso-meric glucuronides have the same m/z, and their full scan MS/MS spectra are often identical; it is thus impossible to differentiate the metabolites from each other using only an MS/MS instrument. This problem has been noted for decades in structural evaluations of phase II metabolites such as glu-curonides14, but possible solutions have received much less attention. Levsen et al.14 published a review article of structural elucidation of phase II me-tabolites in 2005, in which it was discussed that the use of MS alone usually could not provide enough information to determine the glucuronidation site. For this exact determination, NMR was claimed to be the only technique which could be used. Studies of different isomeric glucuronides are described in Papers II, III, and IV. Figure 16 (E16G and E17G), Figures 17A-17B, Figures 18A-18B, and Figures 19A-19B show the full MS/MS analyses of the isomeric glu-curonides. These analyses did not give any structural information regarding the binding site for the glucuronide, as the fragmentation patterns of the iso-mers were too similar. These results are analogous with earlier results from studies14, and demonstrate the above-mentioned difficulty of establishing the position of glucuronidation by MS/MS alone.

34

Figure 16. Mass spectra from the positive product ion mode analysis, using the Q-TOF instrument, with precursor ion m/z 465 [M+H] + for E3G, E16G and E17G, and with a collision energy of -10 eV. (Paper II)

35

Figure 17. Positive product ion spectrum from full MS/MS analyses (ion trap in-strument) of precursor [M+H]+ m/z 462 of A) M3G and B) M6G, collision energies 23 and 26, respectively. (Paper III)

36

Figure 18. Mass spectra from a positive full scan MS/MS analysis (ion trap instru-ment) of synthesized formoterol glucuronides (precursor [M + H] + m/z 521) colli-sion energy 23 %. A) Spectrum of the peak with a retention time of 12.94 min and B) spectrum of the peak with a retention time of 14.00 min. (Paper III)

Figure 19. Mass spectra from a full scan MS/MS analysis (triple quadrupole, colli-sion energy 30 V) of UDPGT produced 4’-hydroxypropranolol glucuronides for the peaks at A) 7.2 min and B) 8.8 min. (Paper IV)

37

Chemical derivatization

Derivatization is a modification of a molecule which has been used in drug analysis for many years. The main reasons to use derivatization in combina-tion with LC are to increase the analyte stability, the separation of the ana-lyte from the matrix, the differences between isomers/enantiomers, or the detector sensitivity. Many derivatization reagents used today in LC-MS were formerly used as fluorescent and/or UV tagging agents in conventional LC. Two examples of this are the reagents 2-chloro-1-methylpyridinium iodide (CMPI)102,103 and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)104,105, which have mainly been used for the derivatization of carboxyl acids. Other reagents which have been used to improve the detection, using common fluorescent and UV detectors, are sulfonyl chlorides106 (e.g. dansyl chloride107-111) for derivatization of primary amines, secondary amines, and phenols.

Increased sensitivity in LC-MS Chemical modifications for enhancing detection in LC-ESI-MS, e.g., by introduction of a easily ionizable group or a permanent charge, have been described in a number of publications. Quirke et al.39 used chemical derivati-zation to make alkyl halides, alcohols, phenols, thiols, and amines more eas-ily ionizable, while Barry et al.18,19 used it to enhance detection of low mo-lecular weight and polar metabolites. Barry et al.19 derivatized substances to increase the molecular weight and thereby the detection to a higher m/z in-terval where the background ions are relative low in intensity.

In 2004, Higashi24 reviewed the area of chemical derivatization for enhanced detector sensitivity for neutral steroids in LC-MS. Neutral steroids lack a functional group which can be easily ionized, which is necessary for detec-tion with MS. Hence derivatization has revolutionized analyses of these ana-lytes. Many new reagents for LC-MS analysis have been reported in recent years, for example 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ)38, 4-(4-methyl-1-piperazyl)-3-nitrobenzoyl azide (APZ)38, 12-(Difluoro-1,3,5-triazinyl)-benz[f]isoindolo-[1,2b][1,3]benzothiazolidine36, dansyl chloride22,41, 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC)46, and other sulfonyl chlorides47. Xu et al.46 used DMISC, a phenol selective

38

reagent, to enhance sensitivity for determination of estrogens. In a later study by the same group, looking at enhancement of ESI properties, DMISC was compared with three other sulfonyl chlorides47. As previously mentioned above Paper I investigated the metabolism of the steroid altrenogest. As this substance has a steroid skeleton with (conju-gated) double bonds, that is, pi-electrons (Figure 7), it was fairly well de-tected in positive ion mode. Conversely, its metabolite altrenogest glucuron-ide had low detectability, due to the introduction of the glucuronyl moiety, but as described earlier in the section on structural investigation of metabo-lites, detection was accomplished using full product ion mode or SRM mode and direct injection of the urine sample. To increase the ionization in ESI and thus the detection response of the metabolite, derivatization with hy-droxyammonium chloride, was applied to form a basic oxime (Figure 20).

Figure 20. Derivatization to a basic oxime of altrenogest glucuronide (Paper I).

This derivatization method had previously been used for analysis of oxoster-oids by nano-ESI-MS35. Figure 21 shows the full scan MS analyses of al-trenogest glucuronide with and without derivatization. The sensitivity was significantly increased, and it was possible to detect the metabolite even in a full scan MS analysis. In conclusion, the derivatization used in Paper I enhanced detection sensi-tivity and made it possible to determine the altrenogest glucuronide during a full scan MS analysis.

39

Fi

gure

21.

Ful

l MS

scan

ana

lyse

s (us

ing

a Q

QQ

inst

rum

ent)

of a

ltren

oges

t glu

curo

nide

with

out (

A) a

nd w

ith (B

) oxi

me

deriv

atiz

atio

n. (P

aper

I)

40

Structural investigation with LC-MS Structural evaluation with LC-ESI-MS/MS in combination with chemical derivatization for has only been reported in a few studies. Liu et al.15 dis-cussed in a review 2005 derivatization for structural investigation of metabo-lites. In one study of phase I metabolites, aimed at structural determination of isomers, chemical derivatization was applied as the fragmentation path-ways were identical when CID was used in isolation50. Chemical derivatization has also been useful in structural determination of conjugated metabolites53-57,112. The discrimination of isomeric phase II me-tabolites such as glucuronides has been studied in the context of determining the site of glucuronidation. As described earlier, the metabolic pathway in-volving conjugation of glucuronic acid is a common reaction for excretion of a xenobiotic15. As the glucuronidation can occur on many different func-tional groups, structural isomers are common1. Structural determination of the glucuronides is most important, as the pharmacological effect or toxicity can vary by structure3. Acetic anhydride was used by Schaefer et al.53 for acetylation of carvedilol and carvedilol glucuronides, for structural determination. Cui et al.57 used chemical modification with 3-pyridylcarbinol in combination with LC-MS/MS to determine the site of glucuronidation of an N-(3,5-dichlorophenyl)succinimide metabolite. Glucuronide isomers was also eluci-dated by preparing dimethylated glucuronides, with diazomethane, of a an-giotensin II receptor antagonist56.

In the work described in this thesis (Papers II, III, and IV), structural elu-cidation of glucuronides was studied using chemical derivatization to modify specific functional groups and thereby change the CID of the isomers. Paper II described derivatization of estriol glucuronides. As many estrogens have low detection respons in LC-ESI-MS, the choice of derivatization re-agent was based on two principles. Firstly, the reagent should help the ioni-zation and thereby increase the detection of the compound. Secondly it should be able to be attached to the carboxylic acid in the glucuronyl moiety and induce a change of characteristics which could lead to a changed CID fragmentation pattern. The 2-picolylamine (PA) reagent was chosen due to its possibility to be easily ionized in positive ESI-MS, as it contains a free pyridine, and its ability to affect the fragmentation of the molecule.

41

First, an activation of the carboxylic acid on the glucuronide was performed with the well-known carboxyl acid activators 2-chloro-1-methylpyridium iodide (CMPI) (Figure 22A) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (Figure 22B) in the presence of triethylamine. Then nucleophilic substitution was carried out with 2-picolylamine. None of these derivatives gave different fragmentation for E16G and E17G. The E3G was distinguished from the other two isomers, as its reaction with the CMPI was different. Derivatization was also separately performed with CMPI and the phenol, for E16G and E17G (Figure 22C), and this resulted in a very stable derivative but no mass spectral differences were found between the deriva-tives. Using a combination of two reactions forming a double reaction, that is, reacting with both the carboxylic acid and the phenol resulted in different CIDs patterns for the glucuronides E16G and E17G (Figure 23).

Figure 22. A) Reaction scheme for activation with CMP and PA substitution. B) Reaction scheme for activation with EDC and PA substitution. C) Reaction scheme for CMP binding to the free phenolate for E16G and E17G (for explanation of R2 and R3 see Figure 8). (Paper II)

42

Figure 23. Derivatization reaction scheme for the double reaction of E16G. The same procedure was applied for E17G. (Paper II)

The derivative from the double reaction was analyzed with three different MS instruments: a QQQ, a Q-TOF, and an ion trap. The full MS/MS analy-ses gave a selective fragmentation for the E16G-(EDC)-PA-CMP (Figure 24 and 25) which was not seen for the E17G-(EDC)-PA-CMP derivative. All three instruments gave approximately the same results for these derivatives. In order to determine the structure of the characteristic fragment m/z 306, accurate mass measurements and MS3 experiments were performed on the Q-TOF and ion trap, respectively. To gain more information, estriol was also derivatized with CMPI. The estriol-CMP was analyzed with the TOF and accurate mass determination of some fragments was also performed in order to assist the evaluation of the glucuronides derivatives. The predominant ion m/z 380 from the full MS/MS analyses of the glucuronides derivative (Fig-ures 24 and 25) was probably the result of a loss of monodehydrated glu-curonic acid and PA, yielding estriol-CMP. Furthermore, an MS3 analysis (m/z 646�380�scan) demonstrated that the selective fragment m/z 306 was not formed from m/z 380; however, it was concluded that the fragment m/z 362 did come from m/z 380.

43

Figure 24. Mass spectra from product ion analysis of precursor ion m/z 646 (M+) with a collision energy of 55% (ion trap mass spectrometer LCQ) for the E16G-(EDC)-PA-CMP and E17G-(EDC)-PA-CMP derivatives. (Paper II)

Figure 25. Mass spectra from product ion analysis of precursor ion m/z 646 (M+) with a collision energy of -55 eV (Q-TOF instrument) for the E16G-(EDC)-PA-CMP and E17G-(EDC)-PA-CMP derivatives. (Paper II)

44

Table 1. Accurate mass determination of the E16G-(EDC)-PA-CMP fragments in positive ESI with collision energy -55 eV. Internal calibration substance clemastine [M+H] + m/z 344.1781. (Paper II)

The results of the accurate mass determinations of some fragments, using the TOF instrument, are shown in Table 1. Calculation of possible elemental compositions based on the masses determined, assuming that the remaining number of nitrogens was one, did not yield any realistic formula other than C21H24NO for the fragment m/z 306. This conclusion was drawn using a tolerated m/z difference of ± 10 mTh. A proposed structure for the m/z 306 fragment is also shown in Table 1.

Paper II describes a method of using chemical derivatization and LC-ESI-MS/MS to structurally differentiate between E16G and E17G for the first time. The fragment m/z 306 was only formed for the E16G-(EDC)-PA-CMP derivative, giving a selective fragmentation pattern.

In Paper III, the two main phase II metabolites of morphine, i.e., M3G and M6G, were investigated as model compounds for the determination of the conjugation site. M6G is an aliphatically hydroxylated glucuronide whereas M3G is a phenolic one. As described earlier, these glucuronides could not be discriminated from each other using MS/MS only. Morphine, M3G and M6G were subjected to reaction with DMISC to improve their fragmentation (Figure 26). The derivatives were analyzed using the ion trap in full MS, MS/MS, MS3, and MS4 modes. This reagent has earlier been described as phenol-selective in the presence of aliphatic alcohols46.

45

Figure 26. Schematic reaction of 1,2-dimethylimidazole sulfonyl chloride (DMISC) with phenol46. (Paper III)

The analyses showed that the derivatives were successfully formed for mor-phine-DMIS (m/z 444) and M6G-DMIS (m/z 620), but no derivative corre-sponding to M3G-DMIS could be detected. The MS/MS analysis of M6G-DMIS gave the predominant fragment at m/z 444, corresponding to the agly-cone with the DMIS still attached, implying a loss of monodehydrated glu-curonic acid. The full MS/MS analysis (Figure 27A) of precursor [M + H]+ m/z 444 gave the predominant fragment m/z 285, which probably corresponds to a radical cation of morphine, as protonated morphine itself has an m/z of 286 (cf. Figure 28). The CID of the DMIS derivatives gave a fragmentation pathway similar to that earlier obtained in GC-EI-MS113. Further fragmentation of m/z 285 in a MS3 gave several more radical ions as m/z 226 and 198 (Figure 27B). These fragments are analogous with those of protonated morphine m/z 229 and 201 (c.f. Figure 28), structures have been proposed by Raith et al.114. The conclusions drawn from the derivatization of morphine, M3G and M6G, were that selective reaction was performed on M6G. The M3G gave no product, probably because the phenol group was blocked. Furthermore, the fragmentation pattern for the M6G-DMIS suggests that the derivative was attached to the phenol. DMISC was proven to be a very useful tool for the isomer that contained a free phenol.

46

Figure 27. Positive product ion spectra, using a ion trap instrument. A) Full MS/MS analysis and suggested cleavage sites of morphine-DMIS, precursor [M+H]+ m/z 444, collision energy 35%. B) MS3 analysis of morphine-DMIS, precursor [M+H]+ m/z 444� 285�scan, collision energies 32% and 35%, respectively, and with pro-posed structures for two fragments. (Paper III)

47

Figure 28. Positive product ion spectrum from a full MS/MS analysis of morphine, precursor [M+H]+ 286, collision energy 38 %, using a ion trap instrument. (Paper III)

Paper III describes also an investigation of formoterol glucuronides using the same derivatization reagent, DMISC. The enzymatic reaction of for-moterol with UDPGT gave two chromatographic peaks with m/z 452 which corresponded to [M+H]+ of formoterol glucuronides (Figure 29C). This is discussed further in the section on in vitro production of glucuronides. The derivatization with DMISC also produced the fascinating result that one of the two formed glucuronides disappeared (Figure 29C c.f. Figure 29H) and the derivatization resulted in one peak for the m/z of a formoterol glucuron-ide-DMIS derivative.

48

Fi

gure

29.

Mas

s chr

omat

ogra

ms f

rom

pos

itive

ion

full

MS

anal

ysis

(QQ

Q in

stru

men

t) of

synt

hesi

zed

form

oter

ol g

lucu

roni

des b

efor

e (A

-E)

and

afte

r (F-

J) d

eriv

atiz

atio

n w

ith D

MIS

C. A

) and

F) t

otal

ion

chro

mat

ogra

ms,

B) a

nd G

) ext

ract

ed io

n ch

rom

atog

ram

s for

[M +

H] +

m/z

345

(f

orm

oter

ol),

C) a

nd H

) m/z

521

(for

mot

erol

glu

curo

nide

), D

) and

I) m

/z 5

03 (f

orm

oter

ol-D

MIS

der

ivat

ive)

and

E)

and

J) m

/z 6

79 (f

orm

oter

ol

gluc

uron

ide-

DM

IS d

eriv

ativ

e). (

Pape

r II

I)

49

The CID of formoterol-DMIS gave information that lead to a proposed fragmentation pathway for the formoterol-DMIS derivative (Figure 30). The full MSn analysis of the formoterol glucuronide-DMIS derivative gave a neutral loss of 176 u, and the subsequent fragmentation was the same as for formoterol-DMIS (Figure 31). These findings led to the structural determina-tion of the conjugation site for the two glucuronide metabolites of for-moterol. Thus the first eluted formoterol glucuronide (Figure 29C, tR 12.94 min), which remained unchanged after the derivatization, was most likely the phenol-bound isomer; as the phenol was blocked, no derivative could be formed. The second eluted formoterol glucuronide (Figure 29C, tR 14.00 min) was probably an aliphatically attached glucuronide with a free phenol which could react with the DMIS forming a derivative with the m/z 610. The full MS/MS analysis of formoterol glucuronides (see Figure 18A-B) gave a higher intensity for the fragment m/z 503 for one of the isomers, which indicates that this fragment is an aromatic glucuronide as it can easily lose water (18 u); this result is also in agreement with a previous study by Rosenborg et al.85. In conclusion, DMISC was used for the first time to differentiate an aliphatic and an aromatic glucuronidation in the work described in Paper III.

50

Fi

gure

30.

Pro

pose

d fr

agm

ent s

truct

ures

of t

he fo

rmot

erol

-DM

IS d

eriv

ativ

e (m

/z 5

03) f

rom

the

MS3 a

nd M

S4 ana

lyse

s (io

n tra

p). (

Pape

r II

I)

51

Figure 31. A) Mass spectrum from positive ion full MS/MS analysis (ion trap in-strument) of formoterol glucuronide-DMIS derivative m/z 679, collision energy 26 % and purposed structure of the formoterol glucuronide derivative. B) Mass spec-trum from positive ion MS3 analysis of formoterol glucuronide-DMIS derivative m/z 679� 503�scan, collision energies 27 %, and 25 %, respectively. C) Mass spec-trum from positive ion MS3 analysis of formoterol glucuronide-DMIS derivative m/z 679� 485�scan, collision energies 26 %, and 35 %, respectively. (Paper III)

52

In the study described in Paper IV, the information from the earlier studies was used to perform a structural investigation of in vitro produced metabo-lites, mainly glucuronides of propranolol and hydroxypropranolol. Both the in vitro produced glucuronides and the model compounds pro-pranolol and rac-4´-hydroxypropranolol were derivatized and analyzed for structure information. Full scan MS analysis of the DMIS derivative of phase II reacted propranolol (Figure 32) resulted in one chromatographic peak at tR 19.76 min for the propranolol-DMIS derivative [M + H]+ m/z 418 and one chromatographic peak with tR 14.57 min for a propranolol glucuron-ide-DMIS derivative [M + H]+ m/z 594.

Figure 32. Extracted ion chromatograms from a full scan MS analysis (QQQ in-strument) of microsome incubated S-propranolol. A) m/z 260 remaining S-propranolol, B) m/z 436 remaining S-propranolol glucuronide, C) m/z 418 S-propranolol DMIS derivative, D) m/z 594 S-propranolol glucuronide DMIS deriva-tive. (Paper IV)

Both these derivatives were also analyzed with full MS/MS and MS3, and the proposed structures for the fragments are shown in Figure 33. The frag-mentation suggested that DMIS was bound to the secondary amine (Figure 33).

53

Figure 33. Suggested structures of obtained fragments from MSn analyses (ion trap) of the S-propranolol-DMIS derivative. (Paper IV)

The derivative 4’-hydroxypropranolol-DMIS was also analyzed; the result from the full MS, MS/MS, and MS3 analyses showed a different fragmenta-tion pathway from that of propranolol-DMIS. The fragmentation of the 4’-hydroxypropranolol-DMIS derivative (Figure 34) implied that the DMIS was attached to the naphtol. These results suggest that the naphtol group was preferred for the reaction if both the naphtol and the secondary amine were

54

available. Previous studies using dansyl chloride have shown that both pri-mary and secondary amines and phenols are possible functional groups for derivatization with a sulfonyl chloride106.

Figure 34. Suggested structures of the obtained fragments from MSn analyses (ion trap) of 4’-hydroxypropranolol-DMIS derivative. (Paper IV)

The produced glucuronides of hydroxypropranolol were also derivatized and analyzed as the derivative of the model compounds. The full scan MS analy-sis of the 4’-hydroxypropranolol glucuronide-DMIS derivative (4’-hydroxypropranolol glucuronide from the incubation with UGT2B7 and UDPGA) gave three peaks (Figure 35H) with m/z agreeing with the theoreti-cal m/z for a 4’-hydroxypropranolol glucuronide-DMIS derivative. How-ever, two of the peaks corresponded to derivatized glucuronides and one (tR 14.69 min) to background noise, as this peak also was found in blank sam-ples containing no model compound. The two peaks of the 4’-hydroxypropranolol glucuronide-DMIS derivative (Figure 34H) were further analyzed using MS/MS, MS3, and MS4. Interest-ingly, the results showed that fragmentation using MS3 and MS4 gave differ-ent fragmentation pathways for the two peaks (Figures 36A-36B).

55

Fi

gure

35.

Ful

l MS

scan

ana

lyse

s (Q

QQ

inst

rum

ent)

and

extra

cted

ion

chro

mat

ogra

ms o

f 4’-

hydr

oxyp

ropr

anol

ol g

lucu

roni

de (i

ncub

ated

with

U

DPG

T an

d U

DPG

A) b

efor

e (A

-D) a

nd a

fter (

E-H

) the

der

ivat

izat

ion

with

DM

ISC

. A) a

nd E

) [M

+ H

]+ m/z

276

4’-

hydr

oxyp

ropr

anol

ol, B

) an

d F)

[M +

H]+ m

/z 4

52 4

’-hy

drox

ypro

pran

olol

glu

curo

nide

, C) a

nd G

) [M

+ H

]+ m/z

434

4’-

hydr

oxyp

ropr

anol

ol D

MIS

der

ivat

ive,

D) a

nd

H) [

M +

H]+ m

/z 6

10 4

’-hy

drox

ypro

pran

olol

glu

curo

nide

DM

IS d

eriv

ativ

e. (P

aper

IV)

RT:0

,00

- 23,

00SM

:3B

05

1015

20

Tim

e (m

in)

050100050100050100

Relative Abundance

050100

8,84

9,14

7,05

15,0

09,

8918

,31

4,18

21,5

111

,30

2,30

7,03

8,79

20,3

115

,07

10,9

85,

1012

,34

18,4

54,

060,

84

8,81

9,47

8,53

11,1

211

,68

8,11

6,48

14,1

819

,61

18,1

822

,31

4,83

0,46

3,10

11,1

19,

058,

29

7,96

6,13

14,0

815

,05

5,10

20,0

14,

5122

,66

19,5

21,

55

NL: 5

,73E

6

m/z

= 27

5,70

-276

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_1

6

NL: 8

,92E

6

m/z

= 45

1,70

-452

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_1

6

NL: 5

,45E

5

m/z

= 43

3,70

-434

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_1

6

NL: 2

,70E

4

m/z

= 60

9,70

-610

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_1

6

RT:0

,00

- 23,

00SM

:3B

05

1015

20

Tim

e (m

in)

050100050100050100

Relative Abundance

050100

8,86

7,31

9,12

10,9

35,

124,

2515

,30

18,2

219

,32

22,0

30,

34

6,98

20,1

311

,37

8,71

14,9

919

,68

5,43

4,53

20,5

51,

10

9,98

9,18

11,1

98,

7811

,61

7,49

20,2

414

,67

18,4

34,

8322

,45

1,19

2,01

9,78

12,0

5

14,6

9

9,31

8,15

12,9

319

,06

5,43

21,7

317

,94

4,22

2,02

NL: 5

,65E

6

m/z

= 27

5,70

-276

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_2

3

NL: 3

,38E

6

m/z

= 45

1,70

-452

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_2

3

NL: 4

,41E

6

m/z

= 43

3,70

-434

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_2

3

NL: 4

,53E

5

m/z

= 60

9,70

-610

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_2

3

A

RT:0

,00

- 23,

00SM

:3B

05

1015

20

Tim

e (m

in)

050100050100050100

Relative Abundance

050100

8,84

9,14

7,05

15,0

09,

8918

,31

4,18

21,5

111

,30

2,30

7,03

8,79

20,3

115

,07

10,9

85,

1012

,34

18,4

54,

060,

84

8,81

9,47

8,53

11,1

211

,68

8,11

6,48

14,1

819

,61

18,1

822

,31

4,83

0,46

3,10

11,1

19,

058,

29

7,96

6,13

14,0

815

,05

5,10

20,0

14,

5122

,66

19,5

21,

55

NL: 5

,73E

6

m/z

= 27

5,70

-276

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_1

6

NL: 8

,92E

6

m/z

= 45

1,70

-452

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_1

6

NL: 5

,45E

5

m/z

= 43

3,70

-434

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_1

6

NL: 2

,70E

4

m/z

= 60

9,70

-610

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_1

6

RT:0

,00

- 23,

00SM

:3B

05

1015

20

Tim

e (m

in)

050100050100050100

Relative Abundance

050100

8,86

7,31

9,12

10,9

35,

124,

2515

,30

18,2

219

,32

22,0

30,

34

6,98

20,1

311

,37

8,71

14,9

919

,68

5,43

4,53

20,5

51,

10

9,98

9,18

11,1

98,

7811

,61

7,49

20,2

414

,67

18,4

34,

8322

,45

1,19

2,01

9,78

12,0

5

14,6

9

9,31

8,15

12,9

319

,06

5,43

21,7

317

,94

4,22

2,02

NL: 5

,65E

6

m/z

= 27

5,70

-276

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_2

3

NL: 3

,38E

6

m/z

= 45

1,70

-452

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_2

3

NL: 4

,41E

6

m/z

= 43

3,70

-434

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_2

3

NL: 4

,53E

5

m/z

= 60

9,70

-610

,70

F: +

c E

SI Q

1MS

[50,

000-

700,

000]

MS

0712

07_2

3

C

B

D

G

F

E

H

56

Figure 36. Mass spectra from a full MS3 analyses (ion trap) of UDPGT produced 4’-hydroxypropranolol glucuronides derivatized with DMISC A) corresponding to the peak at 9.78 min (c.f. Fig. 35 H) and B) corresponding to the peak at 12.05 min (c.f. Fig. 35H). (Paper IV)

The fragments of the first peak agreed well with those of propranolol-DMIS, which implies that this was probably an aromatic O-glucuronide, due to the fact that DMIS was attached to the secondary amine and not the naphtol. The fragments of the second peak agreed with those of the 4’-hydroxypropranolol-DMIS derivative, which indicated that this metabolite was an aliphatic O-glucuronide and that DMIS was attached to the naphtol.

The derivatization of the hydroxypropranolol glucuronides (produced by microsomes) resulted in the same fragmentation pathways as the pro-pranolol-DMIS, suggesting that all three glucuronides formed were aromatic glucuronides (Figures 12B and 12D). Paper IV provides important results which show the power of chemical derivatization and LC-MS/MS or MSn for structural discrimination between isomeric glucuronides. The derivatizations with DMISC changed the charac-teristics and fragmentation pathways of the isomeric glucuronides produced, resulting in the determination of the glucuronidation sites.

57

Conclusions

Chemical derivatization has proven to be a very powerful approach in analy-ses of phase II metabolites using LC-MS/MS. In this thesis, derivatization was used both to enhance detection sensitivity through improvement of ioni-zation in ESI and as a tool for structural evaluation of phase II metabolites, specifically isomeric glucuronides. The low levels of metabolites present in a complex matrix constitute a prob-lem for the quantification and qualitative detection of substances that do not contain a functional group that can be easily ionized in ESI. In Paper I, chemical derivatization was tested for enhancement of the detection of the steroid altrenogest and its metabolites. Hydroxyammonium chloride was used to form a basic oxime, a modification that significantly increased the detection sensitivity for the altrenogest glucuronide in horse urine. Furthermore, derivatization together with LC-MS/MS has been used to dif-ferentiate between isomeric glucuronides, as analyses using only MS/MS often do not give enough information for determination of the glucuronida-tion site, as the fragmentation pattern is almost identical for the isomers. In Paper II, three isomeric estriol glucuronides (E3G, E16G, and E17G) were evaluated using a combination of the three reagents CMPI, EDC, and PA. Interestingly, the derivatization gave a selective fragmentation pattern for a derivative of E16G (i.e. E16G-(EDC)-PA-CMP) that could be used to dis-tinguish between E16G and E17G. The third isomer, E3G, was differentiated due to its different type of reaction product when exposed to CMPI. In Papers III and IV chemical derivatization with another derivatization reagent, DMISC, was applied for the first time to aid structural determina-tion of isomeric glucuronides site of conjugation by LC-MS/MS. In Paper III, the substances morphine and formoterol, both possessing one phenolic and one aliphatic hydroxyl group where the glucuronidation could take place, were investigated. The results from these studies showed that the glu-curonidation site can be determined through selective binding of the reagent to free phenols as well as through selective fragmentation of the derivatives.

58

In Paper IV, the developed derivatization approach using DMISC was used to structurally determine phase II metabolites of in vitro produced (with mi-crosomes or UGT2B7) glucuronides of propranolol, that is, hydroxypro-pranolol glucuronides. This metabolite possesses one phenolic and one ali-phatic hydroxyl group where glucuronidation could take place. In this study, DMISC was proven to react with both the phenol and secondary amine, but the reaction with the phenol was preferred in the case where both groups were unconjugated. The fragmentation pattern was shown to be different depending on the binding site of DMISC, giving secondary information on the site of glucuronidation.

59

Future aspects

In terms of structural investigation, chemical derivatization in combination with LC-MS/MS analysis is a relatively unexploited area today, and only a few studies have been published. However, these reactions are more thor-oughly used and reported for enhancement of detection sensitivity in ESI. As chemical modification has been used for many decades (for GC and LC), there are probably many more useful derivatization reagents, both old and new, that could be helpful in the analysis of drugs and their metabolites with LC-MS. This thesis is an important contribution to the development of deri-vatization in combination with LC-MS. There are many more areas, i.e., compounds that could use this or a similar approach to differentiate between isomeric substances or to change the fragmentation pattern of a compound to facilitate the structural investigation.

Furthermore, these developed methods should be tested in biological samples for structural determination of isomeric glucuronides.

60

Populärvetenskaplig sammanfattning

Utredning av ett läkemedels omvandling i kroppen är av yttersta vikt för en ökad förståelse av ett läkemedels verkan, funktion och biverkningar. Det finns ett flertal exempel på fall där ett läkemedels omvandlingsprodukter både ger önskad verkan och oönskade biverkningar.

Ett läkemedel som tillförs kroppen utsätts för påverkan av olika slags en-zymsystem, vilket leder till kemiska förändringar i olika hög grad, dvs ämnet metaboliseras. De metabola förändringarna syftar i allmänhet till att om-vandla substansen till mer vattenlöslig form för att påskynda dess utsönd-ring. Läkemedelsmetabolism processen är således engagerad i att avlägsna främmande ämnen från kroppen2.

Kemiska aspekter för läkemedelsmetabolism Metabolism kan ske genom så kallade fas I- och fas II-reaktioner. Vid fas I-reaktioner modifieras eller tillförs kemiska funktionella grupper eller delar av molekylen avlägsnas. Fas II reaktioner innebär konjugering med vatten-lösliga molekyler, se figur 37. Vid konjugering kopplas en vattenlöslig kroppsegen substans till läkemedlet eller metaboliten. Komplexet blir där-igenom mer vattenlösligt och kan därmed lättare utsöndras i urinen1.

Figur 37. De två faserna för läkemedelsmetabolism. I första steget (fas I) avspjälkas COCH2 och OH lämnas kvar och i nästa steg (fas II) binds en vattenlöslig kropps-egen substans (glukuronsyra) till molekylen.

61

Metabolism innebär i huvudsak omvandling av ämnen till mer overksamma och mindre giftiga former. Det finns dock ämnen som kan genom sin om-vandling överföras till ett aktivare ämne, dvs ha större effekt än ursprungs-ämnet. Ett exempel på en sådan substans är desipramin (läkemedel mot de-pression) som genom omvandling i kroppen bildas av imipramin (inaktivt)1. Ett annat exempel är kortison som i kroppen omvandlas till den aktiva sub-stansen hydrokortison. Ytterligare ett exempel är morfin och dess två konju-gerade fas II-metaboliter, morfin-3-glukuronid och morfin-6-glukuronid (Figur 38). Morfin-6-glukuronid ger mer smärtlindring än morfin själv, me-dan morfin-3-glukuronid påstås minska smärtlindringen av morfin2. Med tanke på detta är det mycket viktigt att veta vilka typer av metaboliter ett läkemedel bildar i kroppen eftersom dessa kan förändra ett läkemedels ver-kan och eventuella biverkningar.

Figur 38. Metabolism av morfin till dess fas II metaboliter morfin-3-glukuronid och morfin-6-glukuronid.

Individers olika metabolism Läkemedelsmetabolism sker i olika omfattning hos olika individer beroende på bland annat art, ålder, kön samt även genetiska faktorer1. Vid jämförelser av människans metabolism med den hos försöksdjur (t ex-hund, katt, kanin och råtta) har det konstaterats att det finns stora likheter men vissa viktiga skillnader också. Ett exempel är metabolism av paraceta-mol som är den aktiva substansen i bland annat läkemedlet Alvedon®. Hos människor och hundar utsöndras denna som ett glukuronsyra konjugat, lik-nade exempel kan ses i figur 37, medan sådana inte bildas hos katter efter-som de saknar det leverenzym som krävs för dess bildning.

62

Enzymaktiviteten och därmed förmågan att metabolisera läkemedel föränd-ras med åldern. Spädbarn och barn kan därför vara mycket känsliga för lä-kemedel som normalt inaktiveras genom metabolism, eftersom låg enzymak-tivitet bidrar till att sådana läkemedel finns i större mängder och får därmed längre verkan i kroppen än hos vuxna.

Analysmetoder Utveckling av nya metoder för bestämning av både fas I och fas II-metaboliter i biologiskt material (t ex urin och blod) behövs för att få mer information om ett läkemedels metabolism. Enkla och snabba analystekniker för upptäckt av nya metaboliter, samt identifiering och mängdbestämning av redan kända, krävs för ökad förståelse för kroppens omvandling av kemiska ämnen10. En teknik som i dag används för detta är masspektrometri tillsam-mans med olika typer av separationsmetoder.

Figur 39. Schematisk bild över en masspektrometer.

I en masspektrometer, se figur 39, sker schematiskt följande: det introduce-rade provet i vätska förångas. Molekylerna i gasen får en laddning (nega-tiv/positiv) och accelereras av ett elektriskt fält i vakuum varefter de separe-ras beroende på deras vikt och laddning. I masseparatorn kan de laddade molekylerna brytas ned i mindre delar såkallade fragment, som sedan regi-streras av en detektor. Den information som utvärderas är ett så kallat mass-spektrum där det procentuella förekomsten av delarna som träffar detektorn summeras13. Masspektrometri ger möjligheten till att bestämma bland annat metaboliter genom att jämföra data med referenser samt även genom att titta på specifika sönderfall som erhålls vid analysen. Detta kan liknas vid att varje läkeme-delssubstans har ett eget fingeravtryck som är de specifika fragmenten som uppkommer vid analysen. Metaboliternas förekomst verifieras sedan genom att jämföra metabolitens fingeravtryck med läkemedlets.

63

Ökad forskning om läkemedelsmetaboliternas verkan och biverkningar ger förhoppningsvis information som kan föra fram till nya och bättre läkemedel och att tidigt stoppa potentiella läkemedel med stora biverkningar. Nya enkla och snabba analystekniker är förutsättningen för att detta skall kunna ske. I denna avhandling har analysmetoder utvecklats för att undersöka läkeme-dels- och dess metaboliters förekomst, kemiska struktur och mängd.

I det första delarbetet (arbete I) undersöks metabolismen av läkemedelsub-stansen altrenogest (finns i djurläkemedlet Regu-Mate®) som används till häst för att dämpa brunst. Detta läkemedel kan påverka en hästs prestation varför det därför klassificeras som dopning, inom trav- och galoppsport i många europeiska länder99. För att upptäcka om en häst är dopad analyseras hästens urin- eller blodprov som tagits efter tävlingen. I detta prov söker man efter läkemedel eller en metabolit av läkemedel. I arbete I togs en analysme-tod fram för att bestämma små mängder av detta läkemedel i hästurin. I ar-bete II och III har nya principer för utredning av metaboliters strukturer tagits fram. De ämnen som studerats är metaboliter av estriol (kroppseget östrogen som bildas hos gravida kvinnor men som även kan ges som läke-medel), metaboliter av morfin (används för kraftig smärtlindring) och formo-terol (ingår bland annat i astmamedicinen Symbicort Forte®). I arbete IV användes de i tidigare framtagna metoderna från arbete II och III för att kemiskt strukturbestämma metaboliter av propranolol (ingår bland annat i läkemedlet Inderal®).

64

Acknowledgements

The work for this thesis was carried out at the Division of Analytical Phar-maceutical Chemistry, Department of Medicinal Chemistry, Uppsala Uni-versity; and at the Department of Chemistry, National Veterinary Institute (SVA), Uppsala. During the period in which this work was accomplished, financial help in the form of travel grants was received from Norrlands nation and the Swedish Pharmaceutical Society. Many people have contributed to this thesis in many different ways. I would like to thank all of them, and in particular the following:

My supervisors, Associate Professor Mikael Hedeland and Professor Ulf Bondesson. Mikael, my official supervisor, for your outstanding scientific guidance, your supervision, and for always taking the time to read my texts. Ulf, for your endless enthusiasm and positivity, for introducing me to mass spectrometry, and for convincing me to become a PhD student.

Professor Curt Pettersson, head of the Division of Analytical Pharmaceutical Chemistry, for your support and your belief in my ability. My co-authors, Dr Carl Petersson and Elin Beckman, for your contributions to this thesis; and my co-workers in various projects during these years: Dr Gunilla Åkersson Nilsson, Dr Birger Scholz, and Christian Johansson for nice collaboration. Professor Per Andrén at BMMS, for your kindness in lending me the TOF instrument. All personnel at SVA, for your pleasant company; special thanks go to Eli-sabeth Fredriksson, Gabor Sellei, Anders Ingvast, and Lotta Thorelius for your support with laboratory, instrumental, and administrative assistance. Kerstin Ståhlberg, Gunilla Eriksson, and Maj Blad at the Department of Me-dicinal Chemistry, for administrative support; and Sorin Srbu at the same department, for computer support.

65

All my present and former colleagues at or associated with the Division of Analytical Pharmaceutical Chemistry, for interesting analytical discussions, pleasant company, and making my years at the department so enjoyable; in particular Professor Douglas Westerlund (thank you for always taking the time to answer an analytical question), Associate Professor Emeritus Per Beronius, Dr Anders Sokolowski, Dr Birgit Hakkarainen, Dr Ingela Sund-ström (thank you for critically reading of Paper III), Dr Ylva Hedeland, Dr Sheila Mohabbati, Dr Johan Pierson, Dr Johan Lindholm, Anita Persson, Victoria Barclay, and Jakob Haglöf (without your help with understanding my old and tired computer I don’t think this thesis would ever have been finished). Henrik Lodén, my roommate at work; thank you for your support during these years! I wonder if I would ever have made it if it hadn’t been for your never-ending computer animations with low humor those days! Annica Tevell Åberg, my roommate at SVA, for your support, our long dis-cussions about everything from science to being a parent, and for critically reading Paper III, IV and this thesis. All my other friends at BMC; those from my time on the committee of the Pharmaceutical Postgraduate Students’ Council; those from my stay at BMMS while using the TOF instrument; and all “fika” friends from the divi-sion next door, for your pleasant company and for our funny and sometimes slightly strange discussions :o). My friends outside the university. Those from back home in Gällivare who moved to Uppsala at the same time as me (especially thanks to Helena Buhr for reading parts of this thesis); without all of you, I would probably not have stayed in Uppsala. My friends from “Ingenjörsprogrammen”, my friends from ski trips, and of course my “fika” friends at the “studentnation-erna” on Sundays (before all our little ones came along); thank you all for the funny times we have spent together — and I look forward to many more… My friends from the “dog world”, who have helped me focus on something other than this thesis by giving me, Zabbis and Fia, who have regularly lifted me up with long walks to refresh my mind. My husband’s family, for your interest in my work. My grandparents and my relatives, for being close and supportive. My mother Magnhild and father Håkan (and Dixy), for always being there for me, believing in me, and giving me much love and support. My sister Josefin and my niece Isabelle, and my brother Christoffer, and my sister in

66

law Ann, and my nieces Olivia and Amanda, for always being there for me and for your support. Most importantly of all, my son Elliot , You are the dearest thing in my life and your twinkling eyes warm my heart every day. Your energy and happi-ness have given me will power to conclude this thesis. My husband Daniel, for all your love and endless encouragement. I could not have finished this thesis without your outstanding support. You are the best ! Matilda, Uppsala 10/4 - 2008

67

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