ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1698

Mass spectrometry based analysisof endogenous sterols andhormones

NEIL DE KOCK

ISSN 1651-6214ISBN 978-91-513-0396-3urn:nbn:se:uu:diva-356767

Dissertation presented at Uppsala University to be publicly examined in B7:101a, BMC,Husargatan 3, Uppsala, Wednesday, 19 September 2018 at 09:15 for the degree of Doctorof Philosophy. The examination will be conducted in English. Faculty examiner: ProfessorWilliam J. Griffiths (Swansea University).

Abstractde Kock, N. 2018. Mass spectrometry based analysis of endogenous sterols and hormones.Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 1698. 62 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0396-3.

Bioanalytical applications using supercritical fluid chromatography (SFC) as analyticaltechnique are of increasing interest. In essence, bioanalysis involves measurement of bioactiveor endogenous compounds in biological matrices. SFC has emerged as an excellent choice forbioanalytical analysis, attributable to its speed, selectivity and efficiency compared with highperformance liquid chromatography. Moreover, coupling of SFC with mass spectrometry (MS)provides the additional benefits of specificity and sensitivity.

The aim of this thesis was to exploit these features by developing methods for the analysisof endogenous steroids, cholesterol oxidation products (COPs) and thyroid hormones (THs)by using ultra-performance supercritical fluid chromatography–tandem mass spectrometry(UPSFC–MS/MS) as analytical technique.

Endogenous steroids control many physiological processes, including reproduction,maturation, gene expression and neurological functions in humans and animals. In the firststudy, three steroids were measured in domesticated White Leghorn (WL) chickens andancestral Red Junglefowl (RJF) birds. Restraining stress caused a significantly larger increasein corticosterone levels in RJF than in WL, indicating a blunted hypothalamic–pituitary–adrenal(HPA) axis activity in domesticated chickens. The second study was a continuation of thefirst study and corticosterone levels from the F12 generation of an intercross between WL andRJF birds were measured before and after physical restraint stress. The expression levels ofthe glucocorticoid receptor (GR) in the hypothalamus and several genes in the adrenal glandswere correlated with the post-stress levels of corticosterone in plasma. In the third study, themeasurement of steroids was extended to assess more endogenous steroids from the four majorclasses, i.e. estrogens, androgens, progestogens and corticosterone.

Endogenous COPs are of interest in pathophysiology. COPs are more readily disposedby cells than cholesterol. Therefore, cholesterol is oxidised to the more polar COPs and aregenerally more bioactive than cholesterol. Moreover, if their production in cells and tissues and/or their introduction with dietary animal fat are excessive, COPs could indeed contribute to thepathogenesis of various disease processes. Fourteen COPs were included in the fourth study anda novel method for their separation was developed.

The last study in this thesis, involved the analysis of five THs. These hormones are vital forgrowth, developmental and metabolic processes of vertebrate life and play an important role inenergy homeostasis. Measurements of circulating thyroid hormone levels are used in thyroiddisorder diagnoses or treatment status monitoring. Two rapid methods for the separation of fiveTHs were developed.

In summary, the work in this thesis demonstrates the applicability of UPSFC–MS/MS as ananalytical technique in bioanalysis of endogenous compounds.

Keywords: mass spectrometry, supercritical fluid chromatography, UPSFC–MS/MS,bioanalysis, steroids, oxysterols, thyroid hormones

Neil de Kock, Department of Chemistry - BMC, Analytical Chemistry, Box 599, UppsalaUniversity, SE-75124 Uppsala, Sweden.

© Neil de Kock 2018

ISSN 1651-6214ISBN 978-91-513-0396-3urn:nbn:se:uu:diva-356767 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-356767)

Aan my gesin

To my family Education is the most powerful

weapon which you can use to change the world.

Nelson Mandela

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Fallahsharoudi, A., de Kock, N., Johnsson, M., Ubhayasekera, S.J.K.A., Bergquist, J., Wright, D., Jensen, P. (2015) Domesti-cation effects on stress induced steroid secretion and adrenal gene expression in chickens. Scientific Reports, 5, 15345. (DOI: 10.1038/srep15345)

II Fallahsharoudi, A.1, de Kock, N.1, Johnsson, M., Ubhayasekera,

S.J.K.A., Bergquist, J., Wright, D., Jensen, P. (2017) QTL mapping of stress related gene expression in a cross between domesticated chickens and ancestral red junglefowl. Molecular and Cellular Endocrinology, 446, 52–58. (DOI: 10.1016/j.mce.2017.02.010) 1 Equal contribution

III de Kock, N., Acharya, S.R., Ubhayasekera, S.J.K.A.,

Bergquist, J. (2018) A novel targeted analysis of peripheral steroids by ultra-performance supercritical fluid chromatog-raphy hyphenated to tandem mass spectrometry. Accepted in Scientific Reports with revision

IV de Kock, N., Bergquist, J., Ubhayasekera, S.J.K.A. (2018) A

novel ultra-performance supercritical fluid chromatography–tandem mass spectrometry method for separation of fourteen cholesterol oxidation products. Manuscript

V de Kock, N., Ubhayasekera, S.J.K.A., Bergquist, J. (2018) Rap-

id mass spectrometric methods for separation of five thyroid hormones by ultra-performance supercritical fluid chromatog-raphy and ultra-performance liquid chromatography. Manu-script

Reprints were made with permission from the respective publishers.

Contribution report

The author wishes to clarify his contribution to the research presented in papers I-V.

I Performed the hormonal analysis and wrote part of the paper.

II Performed the hormonal analysis and wrote part of the paper.

III Planning of the research project with the co-authors, execution

of analytical work, evaluation of the results and writing of the manuscript.

IV Planning of the research project with the co-authors, execution of analytical work, evaluation of the results and writing of the manuscript.

V Planning of the research project with the co-authors, execution of analytical work, evaluation of the results and writing of the manuscript.

The author also wishes to note that parts of this thesis are based on his licen-tiate thesis from 2016. The content has been updated, expanded and adapted to a doctoral thesis. de Kock, N. (2016) Targeted analysis of bioactive steroids and oxycholes-terols. Method development and application. Fil. Lic. Thesis, Acta Uni-versitatis Upsaliensis.

Papers not included in this thesis

Fallahsharoudi, A., de Kock, N., Johnsson, M., Bektic, L., Ubhayasekera, S.J.K.A., Bergquist, J., Wright, D., Jensen, P. (2017) Genetic and targeted eQTL mapping reveals strong candidate genes modulating the stress re-sponse during chicken domestication. G3-Genes Genomes Genetics, 7(2), 497–504. (DOI: 10.1534/g3.116.037721)

Contents

Aims .............................................................................................................. 13

Introduction ................................................................................................... 15

Mass spectrometry ........................................................................................ 17 Electrospray ionisation ............................................................................. 17 Mass analyser ........................................................................................... 19 Detector .................................................................................................... 20

Supercritical fluid chromatography .............................................................. 21

Endogenous steroids ..................................................................................... 23 Biosynthesis of endogenous steroids ........................................................ 23 Analysis of endogenous steroids .............................................................. 23

Endogenous cholesterol oxidation products (COPs) .................................... 26 Formation of COPs ................................................................................... 26 Analysis of endogenous COPs ................................................................. 28

Endogenous thyroid hormones ..................................................................... 32 Thyroid hormone production.................................................................... 32 Analysis of endogenous thyroid hormones .............................................. 33

Method development .................................................................................... 35 Materials ................................................................................................... 35 Sample preparation procedures ................................................................ 36

Preparation of standards and blank matrix .......................................... 37 Protein precipitation............................................................................. 38 Saponification ...................................................................................... 38 Liquid-liquid extraction ....................................................................... 39 Derivatisation ....................................................................................... 39

Mass spectrometric parameters ................................................................ 41 Chromatographic separation..................................................................... 44

Supercritical fluid chromatography ..................................................... 44 Liquid chromatography ....................................................................... 46

Method validation ......................................................................................... 47 Linearity and working range ................................................................ 47 Selectivity ............................................................................................ 47 Carryover ............................................................................................. 47 Sensitivity (limits of quantification, LOQ) .......................................... 47 Precision .............................................................................................. 48 Accuracy .............................................................................................. 48 Recovery .............................................................................................. 48

Summary ....................................................................................................... 49

Svensk sammanfattning ................................................................................ 51

Afrikaanse samevatting ................................................................................. 53

Acknowledgements ....................................................................................... 55

References ..................................................................................................... 56

Abbreviations

CO2 carbon dioxide COPs cholesterol oxidation products ESI electrospray ionisation GC gas chromatography IS internal standard LC liquid chromatography LLE liquid-liquid extraction LOQ limit of quantification MO methoxyamine/methoxime MRM multiple reaction monitoring MS mass spectrometry MS/MS tandem mass spectrometry MTBE tert-butyl methyl ether m/z mass-to-charge ratio R2 correlation coefficient RJF Red Junglefowl SFC supercritical fluid chromatography THs thyroid hormones UPLC ultra-performance liquid chromatography UPSFC ultra-performance supercritical fluid chromatography WL White Leghorn

13

Aims

The overall aim of the thesis was to develop methods for the analysis of en-dogenous compounds by using ultra-performance supercritical fluid chroma-tography–tandem mass spectrometry (UPSFC–MS/MS) as analytical tech-nique. The specific aims of the included papers were:

I To develop and apply a method in order to measure plasma lev-els of pregnenolone, dehydroepiandrosterone and corticosterone in White Leghorn and Red Junglefowl birds.

II To apply the developed method from Paper I to measure plasma levels of corticosterone from the F12 generation of an intercross between domesticated White Leghorn and ancestral Red Jungle-fowl birds.

III To develop a method for the simultaneous analysis of steroids from the four major classes (estrogens, androgens, progesto-gens, and corticosteroids).

IV To develop and validate a method for the separation of a mix-ture of more than ten cholesterol oxidation products.

V To develop a rapid method for separation of five thyroid hor-mones and compare with an ultra-performance liquid chroma-tography–tandem mass spectrometry method.

14

15

Introduction

Bioanalytical applications using supercritical fluid chromatography (SFC) as analytical technique are of increasing interest. In essence, bioanalysis in-volves the qualitative determination and quantitative measurement of bioac-tive or endogenous compounds in biological matrices. SFC has emerged as an excellent choice for bioanalytical analysis, attributable to its enhanced performance compared with high performance liquid chromatography. These advantages are speed, selectivity and efficiency.1 Moreover, coupling of SFC with mass spectrometry (MS) provides the additional benefits of specificity, sensitivity and versatility in determination of compound structures.2

Endogenous steroids, cholesterol oxidation products (COPs) and thyroid hormones are present in both normal and pathological conditions of the body. Endogenous steroids control many physiological processes, including reproduction, maturation, gene expression and neurological functions. Dur-ing the last two decades, there has been an increased focus on the application of steroids as biomarkers in healthcare practice.3-9 Steroids have been impli-cated in the development and/or progression of many diseases, such as breast cancer, ovarian cancer, prostate cancer, endometrial cancer, osteoporosis, cardiovascular disease, obesity, and neurodegenerative disorders.10 The analysis of steroids in biological samples such as plasma, serum, and urine is routinely used in clinical diagnosis as an essential source of information on endocrine and metabolic disorders,3-6,9 and in neurodegenerative disorders.7,8 Therefore, an accurate analysis of steroids in biological tissues has become important for contemporary medicine – even if troublesome, especially due to the low concentration levels in biological samples.5 Steroid profiles, gen-erated by the simultaneous determination of steroids from the four major classes (estrogens, androgens, progestogens and corticosteroids), could pro-vide useful data in the clinical environment.6

Cells more readily dispose of endogenous COPs than cholesterol. There-fore, an oxygen function, such as a hydroxyl, epoxide or ketone group, is introduced to the sterol ring or side chain of cholesterol to make it more po-lar. These COPs are generally more bioactive than cholesterol and are of interest in pathophysiology. Moreover, if their production in cells and tissues and/or their introduction with dietary animal fat are excessive, COPs could indeed contribute to the pathogenesis of various disease processes.11

Thyroid hormones are vital for growth, developmental and metabolic pro-cesses of vertebrate life and play an important role in energy homeo-

16

stasis.12,13 Measurements of circulating thyroid hormone levels are used in thyroid disorder diagnoses or treatment status monitoring.14 Some of the most common thyroid disorders include hyperthyroidism and hypothyroid-ism.15 High reverse-triiodothyronine, the inactive hormone, and low triiodo-thyronine, the active hormone, levels in circulating blood have been associ-ated with the prognosis of critical illness and can be an important biomarker in health assessment.16,17

In this context, ultra-performance supercritical fluid chromatography–tandem mass spectrometry (UPSFC–MS/MS) was employed as analytical technique to develop methods for the measurement of endogenous steroids, COPs and thyroid hormones.

17

Mass spectrometry

Mass spectrometry is a technique which generates ionised chemical species and separate these ions based on their mass-to-charge ratio (m/z). Mass spec-trometry has unique characteristics among analytical methods. It provides (1) unsurpassed molecular specificity because of its unique ability to meas-ure accurate molecular mass, (2) unparalleled versatility to determine the structures of most classes of compounds and individual elements, (3) ultra-high detection sensitivity, (4) coupling with different chromatographic sepa-ration techniques, and (5) flexibility in its application to a large variety of sample types: volatile, non-volatile, polar and non-polar.2

A mass spectrometer consists of three essential components: ion source, mass analyser, and detector (Figure 1). The ion source converts analytes into gas phase ions. The ions are transferred to the mass analyser which uses a magnetic and/or electric field to separate these ions based on their m/z. The ions are detected by the detector which generates a signal corresponding to the electrical current of each ion.2 Generally, a data processing system is used for collection and analysis of data.

Figure 1. Schematic diagram of a mass spectrometer with an ionisation source at atmospheric pressure.

Electrospray ionisation Different types of ion sources exist and can be operated under vacuum or in atmospheric pressure, depending on the technique. Electrospray ionisation (ESI) is an atmospheric pressure technique and was used in Papers I–V. ESI is a soft ionisation technique which transfers analytes in solution into gas phase ions with little or no fragmentation.2

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The mechanism of ESI can be described in three main steps: formation of charged droplets, evaporation of solvent from the droplets, and formation of gas phase ions. The first step entails the generation of a potential difference between the capillary and the mass spectrometer by application of a high voltage (3–5 kV) to the spray capillary and a counter electrode. A charge accumulates at the tip of the capillary due to the electrostatic field generated and an enrichment of ions will occur with the formation of a Taylor cone. The surface tension of the liquid is overcome by the electrostatic repulsion forces and highly charged droplets are emitted (Figure 2).2

Figure 2. Schematic illustration of the electrospray ionisation process (positive mode).

The second step involves the evaporation of solvent from the droplets. As solvent evaporates from the charged droplets, the droplets’ radii decrease and an increase in surface charge density occurs. At a certain point, called the Rayleigh limit, the Coulombic force overcomes the surface tension of the liquid and the droplets undergo irregular fission into several smaller droplets. This process is repeated several times, leading to very small, highly charged second-generation droplets. The final step is the formation of gas phase ions when the droplets become small enough.2

The mechanism for the transfer from solvated ions to gas phase ions is not fully understood. The two main theories are the charged residue model (CRM) and the ion desorption model (IDM). The charge residue model pro-poses the production of small droplets containing only a single analyte mole-cule with a number of charges, for example protons in positive mode. As the last liquid molecules on each droplet evaporate, analyte molecules are dis-persed into the ambient gas, retaining the charge of the droplets. The ion evaporation model suggests the expulsion of the solvated ions into the gas phase at some intermediate droplet size when the electric field, due to the

19

surface charge density, is sufficiently high but less than the Rayleigh insta-bility limit.2

The eruption of charged droplets into naked ions is strongly influenced by the solvent physico-chemical properties (viscosity, surface tension, pKa), the concentration and chemical nature of analytes, as well as ionising agents and the voltage applied between the inlet capillary and the counter electrode.2

ESI can be operated in either positive or negative ion mode. In Papers I–V, positive ionisation was used with the most typical adduct ions generated being [M+H]+ or [M+Na]+.

Mass analyser The instrument used in Papers I–V was a Waters TQ-S triple quadrupole. The mass analyser is the heart of a mass spectrometer. Ionic species are sep-arated and mass-analysed based on their mass-to-charge (m/z) ratio and all mass-resolved ions are focused at a single focal point. By definition, the m/z is the mass of an ion (m) divided by the number of charges (z) the ion car-ries. Static or dynamic magnetic and electric fields can be used alone or in combination to control the motion of the ions. Some of the most common mass analysers include the quadrupole, time-of-flight, magnetic sector, or-bitrap, quadrupole ion trap, and Fourier transport ion cyclotron resonance instruments. A mass analyser is best defined according to its mass range limit, scan speed, ion transmission, mass accuracy and resolving power (or resolution). The limit of m/z over which the mass analyser can measure ions, is determined by the mass range. The scan speed is the rate at which the mass analyser can measure ions over a particular mass range. Ion transmis-sion efficiency involves the ability of a mass analyser to deliver ions to the detector and is a reflection of the sensitivity of the instrument. Mass accura-cy is the difference observed between the true m/z and the measured m/z. By definition, resolving power is the analyser’s capability to distinguish be-tween two neighbouring signals of ions that differ only slightly in their mass (m/Δm).2

A quadrupole mass analyser consists of four cylindrical rods that are per-fectly parallel to one another. A potential of +(U–Vcos(ωt)) is applied to one pair of two opposite rods and a potential of –(U–Vcos(ωt)) is applied to the other pair of opposite rods. The value of U represents a constant direct po-tetial (DC). Vcos(ωt) represents an oscillating alternating potential (AC) with V as the ‘zero-to-peak’ amplitude of the radio-frequency (RF) voltage. The ions accelerated along the z-axis enter the space between the rods and maintain their velocity along this axis. Due to the forces induced by the elec-tric fields, these ions are also accelerated in the x- and y-directions. Ions with different m/z ratios will have different ion trajectories as a consequence of the oscillating electric fields. Ions with a specific m/z value will have a stable

20

trajectory and pass through the geometry of the rods to the detector when a set of defined DC and AC potentials are applied. Ions with unstable trajecto-ries will be discharged and not reach the detector.2

Figure 3 shows a typical diagram of an instrument with three quadru-poles. The first and third quadrupoles (Q1 and Q3) are mass spectrometers and the second quadrupole (q2) is a collision cell using RF only. A precursor ion passing through Q1 will collide with an inert gas in q2 and fragment into product ions which will be analysed by Q3.

The low cost, mechanical simplicity, high scan speeds, high transmission, increased sensitivity, independence from the initial energy distribution of ions, and linear mass range are advantageous attributes of a quadrupole mass analyser.

Figure 3. Schematic diagram of a triple quadrupole instrument. Q1: first quadrupole; q2: second quadrupole; Q3: third quadrupole.

Detector The detector is responsible for converting the ion current into signals. The MS used in Papers I–V was equipped with a photomultiplier detector. The ions from the mass analyser are accelerated to a high velocity in order to enhance detection efficiency by holding the conversion dynode at a high potential (from ±3 to ±30 kV). A positive ion striking the conversion dynode causes the emission of several electrons. These electrons then strike a phos-phorous screen which in turn releases a burst of photons. The photons pass into a multiplier and are amplified by a cascade effect to produce an ampli-fied electric current which is proportional to the abundance of the incident ions.2

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Supercritical fluid chromatography

Supercritical fluid chromatography (SFC) was invented as an alternative to gas chromatography (GC) over fifty years ago and was described as a tech-nique with “liquid-like solvation power and gas-like viscosity”.18 The role of the mobile phase was the main difference between GC and SFC, which was near neutral in GC versus the active role it played in compound elution in SFC. Development of the technique has gone through several stages to met-amorphose into the current state. Initially, SFC was more gas-like, using open tubular capillary columns and GC type detectors.18 SFC uses a super-critical fluid as mobile phase, which behaves like a compressible fluid of very low viscosity, where mass transfer operates very rapidly, allowing the use of high linear velocities and, therefore, very short analysis times without any loss of efficiency. Early mobile phases consisted of neat solvents like carbon dioxide (CO2), ammonia, and nitrous oxide. However the use of CO2 has prevailed as the mobile phase of choice owing to several advantageous characteristics: availability, low cost, low critical point (Pc = 7.3 MPa, Tc = 31 ºC), non-flammability, inertness to most compounds, low viscosity and surface tension, low toxicity, and miscibility with various organic solvents, like methanol.19

In most analyses at the moment, a modifier is mixed with CO2 to prevent precipitation of a sample at the column inlet when introduced, and to im-prove analyte solubility.19 Moreover, small volumes of water and additives (acid or base) at low concentration can be added to polar organic modifiers resulting in increased solubility of analytes and more symmetric peak shapes.19,20

Choice of stationary phase has a strong impact on selective separation of analytes using SFC.21,22 Both polar and non-polar stationary phases can be used, resulting in normal-phase, reverse-phase or ion-pairing elution pro-files.23,24 Furthermore, the system allows for high flow rates, due to the strong elution power of supercritical mobile phases. Small particle sizes (2–3 µm) are necessary to achieve high efficiency at these high flow rates as a result of low pressure drops induced by the low fluid viscosity.23 Recent advances in SFC instrumentation and the coupling to mass spectrometry (MS) have enabled the development of analytical methods for the analysis of a wide range of compounds, mostly non-volatile compounds.19 SFC has proven to be especially suited for the separation of isomers within a very short chromatographic run time.25-27 Thus, coupling of SFC with MS/MS

22

provides several advantages related to sensitivity and specificity.20,28 SFC–MS/MS) was explored as a more promising technique for the targeted analy-sis of endogenous compounds in biological matrices.

Besides the mentioned benefits, SFC is considered as a more environmen-tally friendly chromatographic technique compared to LC.29 A typical SFC setup consists of several major components (Figure 4): binary pump, mixer, injector, column oven, splitter, make-up pump, backpressure regulator, and detector(s).

Figure 4. Schematic illustration of the major components in a typical SFC system coupled to a MS detector.

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Endogenous steroids

Endogenous steroids are derived from cholesterol which is absorbed through the diet or synthesised de novo in various tissues and cells including the brain, adrenal glands, gonads, and placenta.30 Endogenous steroids such as estrogens, androgens, progestogens, corticosteroids, and their metabolites are naturally occurring physiologically important compounds controlling differ-ent functions in the human body.10

Biosynthesis of endogenous steroids Steroids are formed during steroidogenesis through a series of enzyme con-trolled reactions (Figure 5). These enzymes belong to two major classes of protein: the cytochrome P450 proteins (CYP11, CYP17, CYP19, CYP21, etc.) and the hydroxysteroid dehydrogenases (3βHSD, 11βHSD, 17βHSD, etc.).30 The cytochrome P450 enzymes are products of a single gene, while the HSDs are products of distinct genes. Steroids have different primary production sites due to variation in distribution of these enzymes between tissues, including the brain, adrenal glands, gonads, and placenta, resulting in biosynthesis of many of the steroids in more than one type of tissue.30

Conversion of cholesterol to pregnenolone is the rate-limiting step in the biosynthetic pathway. Pregnenolone is formed on the inner membrane of mitochondria. Conversions of pregnenolone to other steroids occur through further enzymatic reactions during back and forth transfers between the mi-tochondria and endoplasmic reticulum.30

Steroids bind to steroid receptors on the surface of target cells to initiate their physiological effect. Steroids are divided into four major classes or groups, namely estrogens, androgens, progestogens, and corticosteroids, depending on the type of receptor to which they bind.30

Analysis of endogenous steroids Several techniques are used for the quantification of steroids. The most common methods of steroid quantification in clinical practice include immu-noassays such as radioimmunoassay or enzyme immunoassay. The main disadvantages of immunoassay techniques are the cross reactivity of the

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Figure 5. Steroid hormone biosynthesis pathway with some steroid metabolites in the human body. The four steroid classes are progestogens (yellow), corticosteroids (green), androgens (blue), and estrogens (pink).

antibodies used in the assay with the related steroids, and being prone to matrix effects.5,6 Separation methods like liquid and gas chromatography (LC and GC) rely on modern mass spectrometry (MS) techniques. These high-tech methods are practical and offer tremendous value in obtaining useful structural information on individual steroids and their metabolites.4 These methods have been compared and evaluated for factors such as sensi-tivity, specificity, limits of detection and quantification, etc.6 Analysis of steroids and their metabolites in biological samples with GC–MS is usually accompanied by different chemical derivatisation methods.31 The derivatisa-tion of steroids for GC–MS analysis aids in the enhancement of volatility, stability, ionisation properties, and fragmentation behaviour of these analytes in the electron ionisation (EI) mode.31 With the recent developments in MS, GC has been hyphenated with many different types of mass spectrometers, including triple quadrupole (TQ, tandem MS),32 quadrupole ion trap (QIT–MS),33,34 and time-of-flight (TOF–MS),35 in order to improve the sensitivity of the steroid analysis. Likewise, LC has been coupled to different MS sys-tems with electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) as the most common ionisation techniques.6 Yet, the ap-plication of ESI–MS in steroid analysis is limited. This is due to low proton affinity of the carbonyl and hydroxyl groups.31 Therefore, chemical derivati-sation of steroid analytes is also a useful step prior to ESI–MS analysis. The chemical addition to the corresponding steroid (carbonyl and/or hydroxyl groups) allows formation of derivatives with enhanced sensitivity compared to the underivatised form of steroids.5 However, analysis of steroids without derivatisation by LC–MS/MS is well documented and is also widely used in

25

clinical practice.5,6 The advantages of LC–MS/MS are less sample prepara-tion and shorter analytical time in comparison to GC–MS/MS, with the latter providing superior chromatographic resolution.4 Furthermore, GC–MS/MS or LC–MS/MS methods for steroids analysis are commonly focused on the determination of only a few steroids within one or two classes, as was evi-dent in a review by Abdel-Khalik et al.6

More recently, supercritical fluid chromatography MS (SFC–MS/MS) has been used in a few reported studies of analysis of steroids and their metabo-lites.21,36-38 Xu et al. analysed standards of the estrogenic class and its metab-olites. The steroids were derivatised with dansyl chloride prior to analysis.36 In a more recent publication, Quanson et al. described the development of a high-throughput analysis of underivatised androgenic steroids,37 and which was subsequently applied in a study by du Toit et al. to analyse eleven dif-ferent oxygenated steroids in 4 min.39 A new SFC–MS/MS method was also reported by Doué et al. for the analysis of eight glucuronide and ten sulphate steroids from the estrogenic and androgenic classes in urine.21 In the most recent publication, Parr et al. reported the analysis of 32 underivatised ster-oids.38

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Endogenous cholesterol oxidation products (COPs)

Cholesterol oxidation products (COPs) are bioactive lipids, which are oxy-genated derivatives of cholesterol. The major routes for formation of these metabolites in biological systems are through enzymatic or autoxidation processes. Enzymatic processes occur mainly by cytochrome P450s, which are a class of enzymes that can add a hydroxyl group either to the isooctyl side chain or the steroid nucleus of the parent cholesterol molecule, giving rise to 24S-hydroxycholesterol (24S-HC), 26-hydroxycholesterol (26-HC), or 7α-hydroxycholesterol (7α-HC). 25-Hydroxycholesterolgenerated by cho-lesterol 25 hydrolase (CH25H) is an exception. 7β-Hydroxycholesterol (7β-HC) and 7-ketocholesterol (7-KC) are the autoxidation products generated by reactive oxygen species’ (ROS) attack on the particularly susceptible C7 on the sterol B-ring. Pathways involving formation of some of the key COPs discussed in this thesis are illustrated in Figure 6. Due to the additional hy-droxyl group, this family of compounds is more polar compared to choles-terol. This change in physical property makes COPs biologically potent mol-ecules involved in cell signalling.

COPs have been shown to play a role in development and are potentially involved in the pathogenesis of numerous diseases and conditions, such as cardiovascular disease,40 various types of cancers,11,41 atherosclerosis,11,42 neurodegenerative disorders,11,42,43 and inflammatory bowel disease.11,42

The COPs analysed in Paper IV were sterol ring and side chain oxygen-ated cholesterols, epimeric epoxycholesterols and cholestanetriol (Figure 6). The separation and identification were conducted by UPSFC–MS/MS. Sam-ple preparation included saponification, extraction and derivatisation prior to analysis by UPSFC–MS/MS. COP standards were used for method devel-opment.

Formation of COPs COPs are formed by enzymatic and non-enzymatic oxidation of cholesterol. Non-enzymatic oxidation includes autoxidation and photoxidation. The ster-ol ring of cholesterol is most often oxidised by non-enzymatic mechanisms and leads to products such as 7-ketocholesterol, 7β-hydroxycholesterol,

27

Figure 6. Chemical structures of selected cholesterol oxidation products (COPs).

5α,6α-epoxycholesterol and 5β,6β-epoxycholesterol, while 7α-hydroxy-cholesterol is formed by enzymatic oxidation. All of the side chain oxida-tions of cholesterol follow an enzymatic mechanism and produce COPs like 24S-hydroxycholesterol, 25-hydroxycholesterol and 26-hydroxychol-esterol.11 Free radicals, or triplet oxygen, initiate autoxidation of lipids, gen-erating a series of autocatalytic free radical reactions (Figure 7). Oxidation products are formed during the breakdown of lipids by the autoxidation reac-tions. Cholesterol, an unsaturated lipid (RH), is subjected to the free radical (R•) chain reaction, which includes three processes: initiation, propagation and termination. A peroxy radical reaction with another sterol molecule yields a sterol hydroperoxide and a sterol radical, thus altering the number of sterol radicals in the reaction sequence.44-46 Cholesterol autoxidation usually starts at the C-7 position by the abstraction of a hydrogen atom following the addition of an oxygen atom forming primary COPs, isomers of 7-hydroperoxycholesterols (Figure 8). These 7-hydroperoxycholesterols can further convert into 7α-hydroxycholesterol and 7β-hydroxycholesterol. In addition, 7-ketocholesterol can be formed by the dehydration of isomeric 7-hydroperoxycholesterols (Figure 8). The side chain oxidation occurs at C-20,

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C-22, C-24, C-25 and C-26 with free radical attacks at these positions result-ing in the production of relevant hydroperoxides, which can be further con-verted into 20α-hydroxycholesterol, 22S-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol and 26-hydroxycholesterol.44

The formation of isomeric epoxycholesterols occur due to interaction be-tween cholesterol molecules and hydroxy radicals (Figure 9) and these epoxy compounds can be further hydrolysed in an acidic medium converting them into cholestanetriol.44 Conversely, 24S,25-epoxycholesterol is pro-duced de novo from acetyl-CoA.

Initiation: RH → R• + H•

Propagation: R• + O2 → ROO•

ROO• + RH → ROOH + L•

Termination: ROO• + H• → ROOH R• + H• → RH

Figure 7. Lipid autoxidation pathway.

Analysis of endogenous COPs Given the growing biological importance of COPs, precise and sensitive analysis in broad biological matrices has merited significant attention. Due to their isomeric nature and the greatly differing activities of COPs, individ-ual measurement has been required and therefore chromatographic tech-niques have been almost solely employed. Furthermore, due to the low en-dogenous concentration of COPs, highly sensitive mass spectrometric detec-tion has also been largely utilised.

A comprehensive review on the analysis of COPs was recently published by Griffiths et al.47 A short summary of the review pertaining to analysis of COPs is presented here.

Quantification of COPs has been performed by several analytical tech-niques. The most common methods use gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS). GC–MS based methods for the analysis of COPs48-52 have not changed much over the past two decades since Ulf Diczfalusy and co-workers described their GC–MS method for COP analysis.53 The method uses deuterated internal standards for quantification and is known as isotope dilution–MS. Sample preparation for COP analysis by GC–MS usually includes the saponification, extraction,

29

Figure 8. Autoxidation of cholesterol.

30

Figure 9. Formation of epoxycholesterols and cholestanetriol.

enrichment, and derivatisation of the COPs. Silylation reagents are mostly used to form trimethylsilyl ether (TMS-ether) derivatives of the COPs. De-tection by MS is mostly performed in selected ion monitoring (SIM) mode.47

LC–MS analysis of COPs has been performed with and without derivati-sation. Sample work-up usually includes extraction, hydrolysis and enrich-ment for most of the reviewed literature. McDonald, Russell and co-workers have analysed ten COPs, without derivatisation, together with sterols and

31

secosteroids.54 Ionisation was performed with ESI and acquisition was con-ducted in multiple reaction monitoring (MRM) mode.47

Methods using LC–tandem MS (LC–MS/MS) for analysis of native COPs have been reported.55-58 These methods require the availability of authentic standards and full chromatographic separation of isomeric COPs as a conse-quence of similar MS/MS spectra. Moreover, due to COPs being neither basic nor acidic, [M+H]+, [M+NH4]+ or [M−H]− ions do not readily form and do not give strong signals in electrospray ionisation (ESI), atmospheric pres-sure chemical ionisation (APCI), or other desorption ionisation methods.43 In contrast, derivatisation of COPs enhances ionisation efficiency and exhibit improved sensitivity when analysed by LC–MS/MS.

Different derivatisation reagents have been used to derivatise COPs to picolinyl esters,59 nicotinyl esters,60 N,N-dimethylglycine esters,61,62 oximes,63,64 and Girard hydrazones65,66 prior to LC–MS/MS analysis.43 The majority of the LC–MS/MS methods with derivatisation used ESI59-61,63-66 instead of APCI62 as ionisation source.43

Supercritical fluid chromatography (SFC) has remained fairly unexplored for the analysis of COPs.67 To our knowledge, McAllister and Wu reported the first and only separation of COPs using SFC in 2013.68 They evaluated a mixture of eight COPs without derivatisation and achieved full chromato-graphic resolution on an achiral column within 35 min using an evaporative light scattering detector for detection. However, the method was not validat-ed.

32

Endogenous thyroid hormones

Thyroid hormones (THs) are vital for growth, developmental and metabolic processes of vertebrate life and play an important role in energy homeosta-sis.12,13 The effect of THs under pathological conditions has increased during the last twenty years. Nevertheless, thyroid dysfunction is closely associated with significant morbidity and mortality. Measurements of circulating THs’ concentrations are used in thyroid disorder diagnoses or treatment status monitoring.14 Some of the most common thyroid disorders include hyperthy-roidism and hypothyroidism.15 High levels of reverse-triiodothyronine (rT3), the inactive hormone, and low levels of the active hormone, triiodothyronine (T3), have been associated with the prognosis of critical illness and can be an important biomarker in health assessment.16,17

Thyroid hormone production All the THs are derived from tyrosine and contain an amino group, a car-boxylic acid, and a hydroxyl group (Figure 10). The prohormone L-thyroxine (T4) and 20% of the circulating T3 are synthesised in the thyroid gland.69 The production of thyroid hormones is based on the organisation of thyroid epithelial cells in functional units. THs are regulated via a negative feedback mechanism involving the hypothalamus-pituitary-thyroid (HPT) axis.12,69 In the thyroid gland, T4 and T3 are bound to thyroglobulin and stored in large follicles.16 Secretion of T4 and T3 from the thyroid gland into the bloodstream is stimulated by thyroid stimulating hormone (TSH). In circulation, the majority of T4 (99.97%) and T3 (99.7%) are bound to thy-roxine-binding globulin, thyroxine-binding prealbumin and albumin, with the remaining part present in the free form.16 Free T4 and T3 are transported into the cytoplasm of peripheral tissue cells where these thyroid hormones are metabolised by three deiodinase enzymes. Type 1 deiodinase (D1) is present in the thyroid, pituitary, liver, and kidney. Type 2 deiodinase is ex-pressed in the central nervous system (CNS), brown adipose tissue, anterior pituitary, and placenta. Type 3 deiodinase is present in CNS, skin, placenta, and fetal tissue.69 D3 and D1 converts T4 into T3, while D1 and D2 trans-forms T3 into diiodothyronine (T2). T4 can also be deiodinated to rT3 by D1 and D2, which in turn can be metabolised to T2 by D3 and D1. Thyroid syn-thesis of T2 is currently still a topic of debate.69

33

Figure 10. Chemical structures of thyroid hormones.

Analysis of endogenous thyroid hormones Given that T4 (99.97%) and T3 (99.7%) are predominantly bound to plasma proteins in the blood circulation with only a small fraction in the free form, measurements of either total THs (free THs plus protein bound THs) or free THs can be performed.17

Several techniques are used for THs analysis. The chromatographic anal-ysis of THs is often a challenge due to their structural similarities (Figure 10). Radioimmunoassay (RIA)70 and non-radioimmunoassay techniques are widely used for T4 and T3 measurements whilst the determination of rT3 levels are only done by RIA.14 Despite the sensitivity obtained using immu-noassays, several hindrances exists, including measurement of only a single analyte at a time and lack of specificity and accuracy due to analytical inter-ferences.70-73 In the past decade, the analysis of THs has moved towards the use of LC–MS/MS applications which are superior in sensitivity, specificity, automation and gaining structural information.14 LC–MS/MS methods have been described for quantification of THs in humans,14,17,74,75 as well as in other vertebrates.12,14,71,75-79 Only a few of these methods include derivatisa-tion of the THs.74,79,80 A comparison of these methods are summarised in Table 1.

34

Tabl

e 1.

Ove

rvie

w o

f sel

ecte

d ar

ticle

s ana

lyzi

ng th

yroi

d ho

rmon

es.L

OD

, lim

it of

det

ectio

n; L

OQ

, lim

it of

qua

ntifi

catio

n; IM

, ion

izat

ion

mod

e; R

ef, r

efe-

renc

e. *

Valu

e ca

lcul

ated

bas

ed o

n th

e lo

wes

t cal

ibra

tor,

conc

entra

tion

fact

or a

nd in

ject

ion

volu

me.

Re

f.

Chen

et

al.80

Saka

i et

al.14

Álv

arez

et

al.17

Saba

et

al.74

Ngu

yen

et a

l.81

Wel

sh

et a

l.75

Wan

g et

al

.82

IM

+ + + + + + + /

–

Mas

s spe

ctro

met

er

Bruk

er E

VO

Q

Elite

MS/

MS

tripl

e qu

adru

pole

SCIE

X A

PI 5

000

tripl

e qu

adru

pole

QTO

F

SCIE

X A

PI 4

000

tripl

e qu

adru

pole

SCIE

X A

PI 5

000

tripl

e qu

adru

pole

Agi

lent

646

0 tri

ple

quad

rupo

le

Agi

lent

641

0 tri

ple

quad

rupo

le

Chro

m.

time

(min

)

10.5

12.5

4

13

14

11

11

25

Coul

mn

Acq

uity

BEH

C18

(2.1

×

100

mm

, 1.7

µm

)

Syne

rgi P

olar

-RP

80A

(50

mm

× 4

m, 2

m

m)

ACE

Exc

el C

18-

pent

aflu

orop

heny

l (2

.1 ×

50

mm

, 2 µ

m)

Gem

ini C

18 (2

× 5

0 m

m, 3

µm

)

Zorb

ax S

B-C 1

8 (2.

1 ×

30 m

m, 1

.8 µ

m)

Eclip

se X

BD-C

8 (2.

1 ×

100

mm

, 1.7

µm

)

Syne

rgi P

olar

-RP

100A

(2 ×

50

mm

, 2.

5 µm

)

Ana

lytic

al

met

hod

UPL

C–ES

I–M

S/M

S

HPL

C–ES

I–M

S/M

S

LC–E

SI–

QTO

F–M

S

HPL

C–ES

I–M

S/M

S

HPL

C–ES

I–M

S/M

S

LC–E

SI–

MS/

MS

LC–E

SI–

MS/

MS

Inje

ctio

n vo

lum

e (µ

L)

5 70

30

30

300

200

5

Der

ivat

is-at

ion

Buty

latio

n

No

No

Buty

latio

n

No

No

No

Sam

ple

pr

epar

atio

n

Hyd

rolis

atio

n +

LLE

+ SP

E

LLE

+ on

line

SPE

PP +

mix

ed

mod

e ca

tion

exch

ange

SPE

Hom

ogen

isat-

ion

+ PP

+

SPE

PP +

onl

ine

SPE

PP +

onl

ine

SPE

PP +

SPE

Sam

ple

type

an

d vo

lum

e

Zabr

afish

: 15

0 la

rvae

Seru

m: 2

0

Seru

m: 3

00

Hea

rt tis

sue

Se-

rum

/pla

sma:

10

0

Se-

rum

/pla

sma:

10

0Se

rum

: 500

LOQ

(p

g on

col

umn)

(0.2

ng/

mL)

1* fo

r all

anal

y-te

s

(0.0

5 ng

/mL)

3.

5* fo

r all

anal

ytes

30–4

2 fo

r all

anal

ytes

(0.2

ng/

mL)

6* fo

r all

anal

y-te

s

Not

stat

ed

Not

stat

ed

Not

stat

ed

LOD

(pg

on c

olum

n)

0.5

0.

6

0.5

0.

5

0.5

Not

stat

ed

12 fo

r all

anal

y-te

s

Not

stat

ed

4500

30

30

0.75

4500

30

2.5/

68.5

7/68

.5

1.5/

73

3.5/

24.5

4.5/

71.5

Ana

lyte

T4

T3

rT3

3,5-

T2

3,3’

-T2

T4

T3

rT3

T4

T3

rT3

T4

T3

T4

T3

rT3

3,3’

-T2

T4

T3

T4

T3

rT3

3,5-

T2

3,3’

-T2

35

Method development

In this thesis, ultra-performance supercritical fluid chromatography–tandem mass spectrometry (UPSFC–MS/MS) was employed as analytical technique to develop methods for the separation of endogenous compounds of interest. In general, analytical method development requires consideration of the chemical properties and concentration levels of the compound, type of sam-ple matrix, type of measurement i.e. qualitative and quantitative, necessary accuracy and precision, analytical time, equipment needed and experimental cost.

Materials All analytes included in Papers I–V are listed in Tables 2–4. All chemicals used were of analytical or chromatographic grade. Details are provided in Papers I–V.

Table 2. Classes, names and abbreviations of steroids included in Paper I–III.

Class Compound Abbreviation

Estrogens Estrone E1 Androgens Dehydroepiandrosterone DHEA Androsterone AN Etiocholanolone ECN Androstenedione AE Testosterone T Dihydrotestosterone DHT Progestogens Pregnenolone Preg 17α-Hydroxypregnenolone 17OHPreg Progesterone P 17α-Hydroxyprogesterone 17OHP Pregnanolone PONE Allopregnanolone Allo Corticosteroids Cortisone E Cortisol F Corticosterone B 11-Deoxycortisol S 11-Deoxycorticosterone DOC Aldosterone A

36

Table 3. Nomenclature and abbreviations of cholesterol oxidation products (COPs) included in Paper IV.

Trivial name Abbreviation Systemic name

4β-Hydroxycholesterol 4β-HC cholest-5-en-3β,4β-diol 7α-Hydroxycholesterol 7α-HC cholest-5-en-3β,7α-diol 7β-Hydroxycholesterol 7β-HC cholest-5-en-3β,7β-diol 7-Ketocholesterol 7-KC cholest-5-en-3β-ol-7-one 20α-Hydroxycholesterol 20α-HC cholest-5-en-3β,20α-diol 22S-Hydroxycholesterol 22S-HC cholest-5-en-3β,22S-diol 22R-Hydroxycholesterol 22R-HC cholest-5-en-3β,22R-diol 24S-Hydroxycholesterol 24S-HC cholest-5-en-3β,24S-diol 25-Hydroxycholesterol 25-HC cholest-5-en-3β,25-diol 26-Hydroxycholesterol 26-HC cholest-5-en-3β,26-diol Cholestanetriol CT cholestane-3β,5α,6β-triol Α-Epoxycholesterol α-EC 5α,6α-epoxycholestan-3β-ol Β-Epoxycholesterol β-EC 5β,6β-epoxycholestan-3β-ol 24S,25-Epoxycholesterol 24S,25-EC 24S,25-epoxycholest-5-en-3β-ol

Table 4. Nomenclature and abbreviations of thyroid hormones (THs) included in Paper V.

Trivial name Abbreviation Systemic name

Thyroxine T4 3,3′,5,5′-tetraiodo-L-thyronine

Triiodothyronine T3 3,3′,5-triiodo-L-thyronine

Reverse triiodothyronine rT3 3,3′,5′-triiodo-L-thyronine

– 3,5-T2 3,5-diiodo-L-thyronine

– 3,3’-T2 3,3′-diiodo-L-thyronine

Sample preparation procedures Suitable and appropriate sample preparation procedures can ensure success-ful analytical results. Usually, these procedures determine the overall analyt-ical time required for analysis and may influence analytical method perfor-mance parameters such as limits of quantification, accuracy and precision. The main objectives of sample preparation are to decrease the presence of possible interfering substances from the matrix, to increase the concentration of the compound of interest and to ensure that the compound of interest is dissolved in a solvent suitable injection into the chromatographic system. Biological samples are complex mixtures containing a wide range of sub-stances that can interfere with the analysis and the chromatographic column. Furthermore, these substances can affect the mass spectrometric detection of the compound of interest through ion enhancement or suppression during the ionisation process. The most common interfering substances include pro-teins, salts, metabolites and other endogenous analytes such as phospholip-

37

ids. In general, sample preparation procedures can include dilution followed by injection, filtration, protein precipitation, and sample clean-up such as liquid-liquid extraction, solid-phase extraction or desalting.

The sample preparation procedures performed for the analysis of steroids (Papers I–III), COPs (Paper IV) and THs (Paper V) are presented as a flow chart in Figure 11.

Figure 11. Flow chart of the sample workup performed for analysis of (A) steroids, (B) COPs, and (C) thyroid hormones.

Preparation of standards and blank matrix Stock and working solutions were prepared for all analytes using the appro-priate solvents. Steroid standards were prepared in methanol-acetonitrile (1:1, v/v) (Papers I–II), except acetone was used for estrone and methanol-acetonitrile-chloroform (1:1:1, v/v/v) was used for 17OHPreg (Paper III). Standards of COPs were prepared in chloroform (Paper IV) and 0.1 M NH4 in methanol was used for preparation of TH standards (Paper V). All stock and working solutions were stored at –80 ºC. In Papers I–V, plasma free of sterols and hormones was prepared as described by Aburuz et al.83 in order to have a matrix similar to the true samples. In brief, 400 µg of activated charcoal were added to 10 mL of normal human plasma and gently mixed for 4 h at room temperature on a programmable rotator mixer, followed by centrifugation at 3000 rpm for 3 h at 4 ºC. The supernatant plasma was fil-tered using a PVDF syringe filter and stored at –80 ºC.

38

Protein precipitation Proteins often interfere with the analysis procedure and protein precipitation (PP) is routinely used to remove proteins from the sample matrix. Further-more, many compounds of interest are protein bound and PP is applied to liberate these compounds from the protein by disrupting the binding interac-tions. Protein solubility can be influences by changing the pH of the matrix or by the addition of a salt or an organic modifier, resulting in precipitation of the protein. In general, precipitation is followed by centrifugation and the supernatant is transferred to a clean vesicle. The supernatant can either be directly injected into the chromatographic system or dried by evaporation and dissolved in a suitable solvent. The use of protein precipitation does present some benefits compared to extraction methods. Precipitation is less labour intensive and thus less time consuming, as well as less organic modi-fier or other solvents are used. Despite these advantages, there is a risk that endogenous compounds or drugs might interfere with the analysis, since protein precipitation is a non-selective sample clean-up technique. In order to overcome this drawback, protein precipitation is often used in combina-tion with an extraction method such as liquid-liquid or solid-phase extraction to produce a clean extract.

Cold acidified methanol was used for protein precipitation in Papers I–III, and cold acidified acetonitrile was used in Paper V for the same pur-pose. In brief, internal standards and 300 µL of cold acidified methanol or acetonitrile was added to either 200 µL (Papers I–II), 50 µL (Paper III) or 100 µL (Paper V) of plasma, vortex-mixed and kept at room temperature for 30 min before extraction was performed.

Saponification Saponification is the most common purification method for COPs. The method involves the hydrolysis of acyl lipids and sterol esters to produce water soluble fatty acid salts and an organic solvent soluble unsaponifiable fraction. The unsaponifiable fraction contains COPs, sterols, lipid soluble vitamins, etc. The reaction is shown below:

R-CO-OR’ + KOH → R’-OH (sterol or COPs) + R-CO-OK+

Saponification with alcoholic KOH can be performed at cold (20–40 ºC) or hot (60–100 ºC) conditions; however degradation of COPs leads to artefact formation during hot saponification. Cold saponification was performed in Paper IV with the addition of 500 µL of 1 M ethanolic KOH to 100 µL of plasma and kept at 37 ºC. After 1 h, the reaction was stopped by the addition of 500 µL of saturated NaCl solution and liquid-liquid extraction was per-formed.

39

Liquid-liquid extraction Liquid-liquid extraction (LLE) is a common sample preparation choice in bioanalysis. The use of LLE presents several benefits such as high analyte recoveries, generation of clean extracts, removal of inorganic salts with ease, short method development time, and low cost. LLE does have some draw-backs. Generally, LLE requires the use of organic solvents in large quanti-ties, it is difficult to automate, it is often prone to extract phospholipids, and it can be time consuming and labour intensive. Choice of extraction solvent is very important with the concept of like dissolves like very much applica-ble. Analyte partitioning into one of two immiscible solvents determine the extraction efficiency and depending on the density of the two immiscible solvents, the analyte may preferentially reside in the upper or lower layer.

For analyte extraction, different solvents and solvent combinations were tested as summarised in Table 5, with the best solvent for each type of ana-lyte highlighted in bold. The hydrolysis process and extraction procedure were controlled by thin-layer chromatography as described by Ubhayasekera et al.84 (data not shown).

Table 5. Organic solvents tested for liquid-liquid extraction of analytes.

Steroids (Paper I–III) COPs (Paper IV) THs (Paper V)

tert-Butyl methyl ether Chloroform-methanol Chloroform-methanol Diethyl ether Dichloromethane Diethyl ether Dichloromethane Dichloromethane-methanol tert-Butyl methyl ether Hexane-diethyl ether Hexane Hexane-ethyl acetate – Hexane-isopropanol Ethyl acetate

Extraction of steroids from plasma was performed by the addition of 2 mL of MTBE (Papers I–III), COPs were extracted twice from plasma into 1 mL of n-hexane (Paper IV), and 2 mL of ethyl acetate was used for THs extraction from plasma (Paper V). The samples were vortexed for 10 min at room temperature and centrifuged at 3000 rpm for 5–10 min at 4 ºC. The upper layer supernatant was collected and evaporated to dryness under a gentle stream of nitrogen gas, followed by derivatisation (Papers I–V) or for UPLC analysis of THs in Paper V, the dried residue was dissolved in ace-tonitrile.

Derivatisation The use of an analytical technique is often incompatible with the chemical properties of an analyte, such as solubility, polarity, volatility, stability and ionisability. Thus, chemical modification of an analyte is often necessary in order to change the chemical properties of an analyte. In general, the derivat-isation reaction targets a specific functional group of the analyte and gener-

40

ates a derivative. Several derivatisation reagents are available and choice of reagent depends on the type of functional groups present in the chemical structure of the analyte and the desired change of the chemical property or properties needed.

In Papers I–III methoxyamine (MO) was used which reacts with the car-bonyl groups of the steroids to form the corresponding oximes. The optimum incubation condition for the derivatisation reaction to occur was established to be 45 min at 60 ºC.

COPs were chemically modified with picolinic acid in Paper IV to pico-linyl ester derivatives in the presence of 2-methyl-6-nitrobenzoic anhydride, 4-dimethylaminopyridene, pyridine and triethylamine. The picolinic acid reacts with the hydroxyl functional group(s) present on the sterol ring and/or the side chain of the molecules. The reaction was allowed to occur at 80 ºC for 60 min.

In Paper V, the THs were found to not dissolve appreciably in supercriti-cal CO2. Three strategies to increase the solubility of the analytes were con-sidered: (1) increasing the solvating power of the mobile phase with the ad-dition of a polar organic solvent (e.g. acetonitrile, isopropanol or methanol) as modifier,85 (2) increasing the solvating power of supercritical CO2, deac-tivating the stationary phase, or both, with the addition of a highly polar or ionic component to the modifier as additive,86,87 and (3) chemical modifica-tion of one or more of the functional groups (amino group, carboxylic acid, and hydroxyl group) to increase the analyte hydrophobicity. The addition of a polar organic solvent with or without an additive did not substantially in-crease the solubility of the THs. Recently, butylation of the carboxylic acid of the THs have been used in analysing THs with LC–MS/MS74,79,80 and was subsequently selected to increase the analyte hydrophobicity. Thus, 3 M hydrochloric acid in n-butanol was used and the reaction was allowed to occur for 2 h at 60 ºC.

41

Mass spectrometric parameters Electrospray ionisation (ESI) can be operated in either positive or negative ion mode. Ionisability of analytes was assessed using both modes. In Papers I–V, standard solutions of the individual analytes at a concentration between 1 and 10 µg/mL were introduced into the ion source at a flow rate of 10 µL/min using the Waters Xevo TQ-S mass spectrometer IntelliStartTM pro-gramme in infusion mode. Mass spectra for each analyte were acquired in MS and MS/MS mode. All analytes in Papers I–IV lack easily ionisable moieties in the molecule structures. Steroids and COPs are neither basic nor acidic and have hydroxyl, carbonyl and/or epoxyl groups in their structures, which are low proton affinity functional groups. Chemical modification of these functional groups was performed as discussed in the derivatisation section above.

Infusion of individual standard solutions of each analyte derivative were performed and resulted in improved ionisation efficiency for all analytes. In contrast, THs (Paper V) have an amino group, a carboxylic acid and a hy-droxyl group and the amino group and carboxylic acid readily ionise in ESI. For LC analysis, ESI and mass spectrometric settings were optimised for underivatised THs, whilst for SFC analysis, the THs were derivatised due to low solubility in supercritical CO2 as discussed in the previous section. The molecular mass of the individual derivatised steroids, COPs and THs (Pa-pers I–V) as well as the individual underivatised THs (Paper V) is denoted by M. The [M+H]+ ion was selected as precursor for all the steroids and THs, whilst the [M+Na]+ ion was selected as precursor for all COPs. The most abundant product ions observed were selected to construct the multiple reaction monitoring (MRM) acquisition methods. The collision energy and cone voltage parameters were optimised for each precursor to product ion transition. Furthermore, the capillary voltage, desolvation temperature, desolvation gas flow and collision gas flow settings were optimised for each analytical method in Papers I–V. The optimised ionisation and mass spec-trometric settings are presented in Tables 6–8.

42

Table 6. Optimised ionisation and mass spectrometric parameters used in Papers I–III. Capillary voltage, 2.8 kV; cone voltage, 30 V; desolvation temperature, 500 ºC; source temperature, 150 ºC; desolvation gas flow, 750 L/h; cone gas flow, 150 L/h; collision gas flow, 0.15 mL/min; nebuliser gas flow pressure, 7.0 bar. Paper no. Analyte

Precursor ion (m/z) Product ion (m/z) Dt (s) [M+H]+ Quantifier CE (eV) Qualifier CE (eV)

1 DHEA-MO 318.3 286.2 18 110.2/253.2 25/17 0.025 Preg-MO 346.2 300.1 26 100.1 23 0.028 B-diMO 405.1 343.1 28 – – 0.112 d2-DHEA-MO 320.2 288.2 18 112.2/255.2 27/17 0.025 13C2-d2-Preg-MO 350.3 304.3 21 104.3 27 0.028 d4-F-diMO 425.2 363.0 29 288.0 28 0.112 2 CORT-diMO 405.1 343.1 28 – – 0.112 d4-F-diMO 425.2 363.0 29 288.0 28 0.112 3 E1-MO 300.1 253.2 15 157.0 25 0.004 DHEA-MO 318.3 286.2 18 110.2/253.2 25/17 0.004 AN-MO 320.3 288.2 18 255.1 18 0.004 ECN-MO 320.1 288.2 18 255.1 18 0.004 AE-diMO 345.3 283.2 26 260.2 27 0.019 T-MO 318.1 138.0 30 126.1 29 0.004 DHT-MO 320.2 140.2 30 128.3 29 0.004 Preg-MO 346.2 300.1 26 100.1 23 0.004 17OHPreg-MO 362.3 344.1 5 – – 0.033 P-diMO 373.1 286.2 28 327.2 28 0.032 17OHP-diMO 389.1 268.1 25 228.1/286.2 34/25 0.004 PONE-MO 348.1 100.0 29 – – 0.004 Allo-MO 348.2 100.0 29 – – 0.003 E-diMO 419.2 357.1 29 300.1/316.1 26/30 0.004 F-diMO 421.2 284.1 28 359.2 27 0.033 B-diMO 405.1 343.1 28 – – 0.004 S-diMO 405.2 286.2 25 343.2 25 0.004 DOC-diMO 389.1 327.2 28 126.0/138.0 40/40 0.004 A-diMO 419.2 357.1 29 – – 0.033 d4-E1-MO 304.2 257.0 15 159.0 25 0.004 d2-AN-MO 322.3 290.2 18 257.1 18 0.004 13C3-T-MO 321.3 141.1 30 129.2 29 0.004 13C2-d2-Preg-MO 350.3 304.3 21 104.3 27 0.004 d2-Allo-MO 353.2 105.0 29 – – 0.003 d9-P-diMO 382.3 292.2 28 333.3 28 0.032 d8-17OHP-diMO 397.2 273.1 25 129.0/291.1 25/25 0.004 d4-F-diMO 425.2 363.0 29 288.0 28 0.033 d8-B-diMO 413.3 349.3 29 – – 0.004

CE, collision energy; Dt, dwell time.

43

Table 7. Optimised ionisation and mass spectrometric parameters used in Paper IV. Capillary voltage, 3.6 kV; cone voltage, 30 V; desolvation temperature, 500 ºC; source temperature, 150 ºC; desolvation gas flow, 750 L/h; cone gas flow, 150 L/h; collision gas flow, 0.16 mL/min; nebuliser gas flow pressure, 7.0 bar.

Analyte Precursor ion (m/z) Product ion (m/z)

Dt (s) [M+Na]+ Quantifier CE (eV) Qualifier CE (eV) 4β-HC 635 146 22 512 20 0.163 7α-HC 635 146 15 – – 0.108 7β-HC 635 146 15 – – 0.108 7-KC 528 146 22 – – 0.163 20α-HC 530 146 22 – – 0.163 22S-HC 635 146 26 512 22 0.081 22R-HC 635 146 26 512 22 0.038 24S-HC 635 512 22 146 31 0.038 25-HC 635 512 19 146 28 0.038 26-HC 635 512 12 146 31 0.038 CT 635 143 28 – – 0.330 Α-EC 530 146 22 – – 0.108 Β-EC 530 146 22 – – 0.108 24S,25-EC 528 146 20 – – 0.108 19-HC (IS) 635 146 28 – – 0.108

CE, collision energy; Dt, dwell time.

Table 8. Optimised ionisation and mass spectrometric parameters used in Paper V. Capillary voltage, 3–4.5 kV; cone voltage, 35–40 V; desolvation temperature, 450–500 ºC; source temperature, 150 ºC; desolvation gas flow, 750 L/h; cone gas flow, 150 L/h; collision gas flow, 0.15–0.17 mL/min; nebuliser gas flow pressure, 7.0 bar. Chromatographic technique Analyte

Precursor ion (m/z) Product ion (m/z) Dt (s) [M+Na]+ Quantifier CE (eV) Qualifier CE (eV)

UPSFC T4 833.6 731.3 32 604.5/777.4 49/20 0.010 T3 707.7 605.4 26 478.6/197.9 42/63 0.010 rT3 707.7 605.4 26 478.6/507.6 45/27 0.010 3,5-T2 581.8 479.7 22 352.7/381.8 38/24 0.010 3,3’-T2 581.8 479.7 22 352.7/381.8 38/24 0.010 13C6-T4 839.6 737.4 33 610.4/783.4 48/20 0.010 13C6-rT3 713.8 611.6 28 484.7/657.6 47/19 0.010 UPLC T4 777.8 731.5 25 – – 0.033 T3 651.8 605.9 20 – – 0.033 rT3 651.8 605.8 20 – – 0.033 3,5-T2 525.6 479.6 20 – – 0.033 3,3’-T2 525.6 479.6 20 – – 0.033 13C6-T4 783.8 737.8 25 – – 0.033 13C6-rT3 657.8 611.9 20 – – 0.033

CE, collision energy; Dt, dwell time.

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Chromatographic separation Chromatography is an analytical technique for separation of compounds in a mixture. The compounds separate based on different partitioning rates be-tween a stationary and a mobile phase. The most common chromatographic separation methods are gas chromatography (GC), liquid chromatography (LC) and supercritical chromatography (SFC). Separation of compounds of interest can be optimised by consideration of one or several parameters de-pending on the type of system. The most common parameters include the type of stationary phase, the type of modifier(s) and additives used as mobile phase, utilisation of an isocratic or gradient elution programme, flow rates, column temperatures and backpressures. In this study SFC was used.

Supercritical fluid chromatography Generally with supercritical fluid chromatography (SFC), packed columns with different stationary phases, such as illustrated in Figure 12, can be screened with the same mobile phase composition to assess selectivity and retention of analytes.23,88

SFC stationary phase columns are most commonly packed with silica-based bonded phases. SFC columns can be classified as containing non-polar, polar alkyl, aromatic or polar phases.19 Consideration of the packing material, particle size and column dimensions are important for stationary phase selection. Parameters such as retention, resolution, backpressure, sol-vent consumption and chromatographic run time are influenced by choice of column. Typically, SFC columns are 50–150 mm in length, have inner di-ameters of 2–5 mm and have particle sizes of 1.7–5 µm. Mobile phase flow rates of between 0.2 and 4 mL/min can be used, depending on the physical properties of the column.

In papers I–III, separation of steroid analytes was assessed on three dif-ferent stationary phases, namely BEH, BEH 2EP and CSH FP (3 mm × 100 mm, 1.7 µm). Seven columns with different stationary phases were screened for their ability to separate fourteen COPs (Paper IV) or five THs (Paper V): BEH (3 mm × 150 mm, 1.7 µm), BEH 2EP (3 mm × 100 mm, 1.7 µm), CSH FP (2.1 mm × 100 mm, 1.7 µm), HSS C18 SB (3 mm × 100 mm, 1.8 µm), 2-PIC (3 mm × 150 mm, 1.7 µm), 1-AA (2.1 mm × 100 mm, 1.7 µm), and DIOL (3 mm × 150 mm, 1.7 µm). Selection of the stationary phase was based on chromatographic resolution of the target analytes and the mass spectrometric sensitivity obtained.

Composition of the modifier, especially polar ones such as methanol or isopropanol, also affects retention of the analytes. These polar solvents can reduce negative interactions between the stationary phase and the analytes and deactivate the active sites in the stationary phase.24,88

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Figure 12. Chemical structure of the tested stationary phases on ultra-performance supercritical fluid chromatography (UHSFC).

Different modifiers and modifier compositions were used in optimisation of the mobile phase in Papers I–V. Screening of the columns for analyte sepa-ration was performed with methanol, isopropanol, acetonitrile, and combina-tions of the three solvents. Furthermore, solubility of the analytes, mass spectrometric sensitivity and peak shape can be enhanced with the addition of additives (acids or bases) to the modifier.88 Ammonium hydroxide (NH4OH) and formic acid (FA) was used as additives for screening in Paper III, FA and ammonium acetate (CH3COONH4) in Paper IV, whilst the use of FA, trifluoroacetic acid (TFA) and acetic acid (CH3COOH) was investi-gated in Paper IV.

Additional chromatographic parameters were assessed after stationary phase selection as well as establishment of the modifier and additive compo-sition for each method in Papers I–V. These parameters included the flow rate, column temperature, backpressure and mobile phase elution pro-gramme. In general, the largest effect on the separation of the analytes was observed by using either a gradient or isocratic mobile phase elution pro-gram in combination with varying flow rates. Lower temperatures resulted in faster elution of the analytes due to an increase in the density of the mobile phase. In contrast, a decrease in the backpressure lowered the density of the mobile phase resulting in an increased retention of all the analytes. These observed effects are in agreement with previously reported observations.24,89

In Papers I–II an Acquity UPC2 BEH column (3 mm × 100 mm, 1.7 µm) at 40 ºC with a flow rate of 3 mL/min was used for separation of the steroid analytes. The injection volume was 2 µL and a gradient elution was per-formed with the mobile phase consisting of (A) CO2 and (B) methanol as follows: linear gradient from 2–17% B in 2.01 min followed by a linear gra-dient to 2% B for 1 min and held for 1 min at 2% B. The make-up solvent

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contained 0.1% NH4OH in methanol-water (97.5:2.5, v/v) with a flow rate of 0.4 mL/min and the backpressure was set to 1800 psi (124.1 bar).

In Paper III the number of steroid analytes increased to nineteen from three steroids and one steroid analysed in Papers I and II, respectively. An Acquity UPC2 BEH column with an increased length was used (3 mm × 150 mm, 1.7 µm) to achieve full separation of the analytes. The column tempera-ture was 40 ºC. The flow rate and back pressure was decreased to 2 mL/min and 1500 psi (103.4 bar) in order to compensate for the increased pressure generated as a result of the increased column length. The volume of injection was 1 µL and the optimum mobile phase composition was (A) CO2 and (B) 0.1% FA in methanol-isopropanol (1:1, v/v). The linear gradient was adjust-ed to 2–17% B over 3 min, held at 17% B for 0.5 min, linearly decreased to 2% B in 0.5 min and maintained at 2% B for 1 min. The make-up solvent was introduced post-separation at a flow rate of 0.2 mL/min and consisted of 0.1% FA in methanol.

Separation of the COPs in Paper IV was performed on an Acquity UPC2 BEH column (3.0 × 150 mm, 1.7 µm) at 30 °C. The mobile phase consisted of (A) CO2 and (B) 10 mM ammonium acetate in methanol-isopropanol (1:1, v/v). The following programmed gradient was used at a flow rate of 1.5 mL/min: 0–5 min, 5% B; 5–15 min, a linear gradient from 5–15% B, 15–16 min, 15–5% B and kept constant for 1.0 min at 5% B. The back pressure was set at 1600 psi (110.3 bar) and a make-up solvent of 0.1% formic acid in methanol was introduced post-column at a flow rate of 0.2 mL/min.

THs were separated on a Viridis HSS C18 SB column (3.0 mm × 100 mm, 1.8 µm) maintained at 35 °C in Paper V. The injection volume was 1 µL. The mobile phase consisted of (A) CO2 and (B) 0.1% acetic acid in acetoni-trile-isopropanol-water (47.5:47.5:5, v/v/v). A 3 min isocratic program at 20% B at a constant flow rate of 1.5 mL/min was used. The backpressure was set to 2200 psi (151.7 bar) and a make-up pump introduced 0.1% acetic acid in methanol into the mixer prior to the MS line at a flow rate of 0.2 mL/min.

Liquid chromatography In Paper V, THs were also separated using an UPLC system fitted with an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) and maintained at 30 °C. The injection volume was 5 µL. The mobile phase consisted of (A) 0.1% acetic acid and 5% acetonitrile in water, and (B) 0.1% acetic acid and 95% acetonitrile in water. A 4 min gradient elution program at a constant flow rate of 0.4 mL/min was used. Eluent B was increased from 10–35% in 1 min, kept at 35% for 1 min, increased to 100% in 1 min, decreased to 10% in 0.5 min and kept at 10% for 0.5 min.

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Method validation

Method validation is an important requirement to confirm that an analytical method is fit for purpose. In general, validation includes the assessment of linearity and working range, selectivity, carryover, sensitivity (limits of quantification), accuracy, precision, and recovery. Slightly modified guide-lines of the EU Commission Decision/657/EEC were used in Papers III–V.

Linearity and working range A calibration curve is the relationship between instrument response and known concentration of the analyte. A calibration curve should be construct-ed for each individual analyte in the same biological matrix as the samples. The curve should at least contain five points by spiking matrix-matched samples at five different concentration levels in triplicate. The deviation should be within 20% at the lowest calibrator concentration and within 15% for the other calibrators. Matrix-matched blank (sample spiked with internal standard) and double blank (without internal standard) samples should be included as well. The working range of the curve is the expected concentra-tion interval of the study and usually determines the calibrator concentra-tions.

Selectivity The selectivity of an analytical method is the ability to determine the analyte without interference from other components in a complex mixture. Blank samples should be processed and evaluated for interferences from the sample matrix.

Carryover Carryover is assessed by injection of a double blank sample following injec-tion of the highest concentration calibrator.

Sensitivity (limits of quantification, LOQ) The sensitivity of an analytical method is expressed as the limit of quantifi-cation (LOQ) with the lowest standard on the calibration curve providing an

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analyte response of at least five times higher than the blank response. Preci-sion should be below 20% and accuarcy should be within ±20%.

Precision The precision of an analytical method is a representation of the closeness of a series of replicate measurements to each other of at least five matrix-matched samples spiked at the same analyte concertation level. The preci-sion is expressed as the coefficient of variance (CV%) of the series of meas-urements. Intraday precision is performed on the same day and interday pre-cision is performed on at least two different days. The acceptance criteria for precision is defined as a CV% within 15% for each concentration level and below 20% for the LOQ.

% = standard deviationmean measured concentration × 100

Accuracy The accuracy of an analytical method is a representation of the closeness of the measured analyte concentration to the nominal analyte concentration added to the sample. At least five replicate analysis of spiked samples with known concentrations should be performed. The replicate samples used for precision determinations can be used for accuracy determinations with the same acceptance criteria (±15% except LOQ ±20%).

% = measured concentration − nominal concentrationnominal concentration × 100

Recovery Analyte recovery from sample matrix is assessed by comparing the response from extracted samples with the response from non-extracted standards with the same nominal concentration as the extracted samples.

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Summary

Supercritical fluid chromatography with tandem mass spectrometry (SFC–MS/MS) was explored as a promising technique for the targeted analysis of endogenous compounds in biological matrices. In this study, simple, rapid and specific SFC–MS/MS analytical methods were developed for the target-ed analysis of steroids, cholesterol oxidation products (COPs) and thyroid hormones (THs) present in biological samples.

As part of the study in Paper I, the levels of three central steroids in plasma with adrenal origin were compared at baseline and after restraint stress (ten minutes of physical restraint in a hanging net) in six weeks old domesticated White Leghorn (WL) and ancestral Red Junglefowl (RJF) birds in order to evaluate the effects of domestication on acute stress sensitivity of chickens at hormonal levels. The results indicated that the basal levels of pregnenolone and dehydroepiandrosterone were significantly higher in RJF, but there was no significant difference between WL and RJF in baseline levels of corticosterone. Ten minutes of physical restraint led to significant elevation of the corticosterone levels in both WL and RJF, with the levels after restraint being higher in RJF. With the exception of basal corti-costerone, there was no significant effect of sex on the levels of the meas-ured hormones. The results supported previous observations that endocrino-logical stress responses, and the associated breed levels of steroid hormones, have been modified by domestication in chickens.

The same method used in Paper I was applied in Paper II to measure corticosterone levels from the F12 generation of an intercross between do-mesticated WL and ancestral RJF birds before and after physical restraint stress. The expression levels of the glucocorticoid receptor (GR) in the hypo-thalamus and several genes in the adrenal glands were correlated with the post-stress levels of corticosterone in plasma. The presence of eQTL for GR in the hypothalamus combined with the negative correlation between GR expression and corticosterone response suggests GR as a candidate for fur-ther functional studies regarding modification of stress response during chicken domestication.

As described in Paper III, we have developed and optimised a UPSFC–MS/MS method for targeted analysis of nineteen steroids within 5 min. The novelty of the study was a fast, sensitive and reliable method for simultane-ous quantification of endogenous steroids across the four major classes (es-trogens, androgens, progestogens and corticosteroids). Inclusion of a derivat-

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isation step prior to analysis improved sensitivity of detection and out-weighed the drawback of an increased analytical time. Different stationary phases, modifiers, additives, and gradient elutions were assessed after MS detection conditions were established. The validation data suggests that it is possible to identify and quantify these steroid analytes in small sample vol-umes.

A UPSFC–MS/MS method was developed and validated for measurement of fourteen COPs as described in Paper IV. Derivatisation of COPs into picolinyl esters allowed for sensitive and reliable determination. The method demonstrated excellent linearity, accuracy, precision, and selectivity for the intended purpose of using plasma. In addition to the ecological and econom-ic advantages of SFC, the UPSFC–MS/MS method represents a good alter-native to LC–MS/MS for the analysis of COPs in biological samples.

In Paper V, we reported the development of a rapid analytical UPSFC–MS/MS method for the separation of five THs (T4, T3, rT3, 3,5-T2 and 3,3’-T2) after derivatisation within a short chromatographic run time of 3 min. The separation profile was compared with a developed and validated UPLC–MS/MS method. The method was validated through evaluation of linearity and determination of carryover effects, limits of quantification, intraday and interday accuracy and precision, and recovery.

In conclusion, this work demonstrates the applicability of UPSFC–MS/MS as an analytical technique in bioanalysis of endogenous compounds. To the best of our knowledge, two of the studies in this thesis are the first reported methods using SFC in combination with MS to analyse COPs and THs, respectively.

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Svensk sammanfattning

Bioanalytiska tillämpningar som använder superkritisk vätskekromatografi (SFC) som analytisk teknik har på senare år kommit att få ökat intresse. I huvudsak innefattar bioanalys mätning av bioaktiva eller endogena (kropps-egna) föreningar i biologiska matriser (kroppsvätskor eller vävnader). SFC har utvecklats till en teknik som utgör ett utmärkt alternativ eller komple-ment vid bioanalys, framförallt på grund av teknikens hastighet, selektivitet och effektivitet jämfört med till exempel vätskekromatografi. Dessutom ger koppling av SFC till masspektrometri (MS) som detektor ytterligare förde-larna vad avser specificitet och känslighet.

Syftet med denna avhandling var att utnyttja dessa kombinerade egen-skaper genom att utveckla metoder för analys av endogena steroider, koleste-roloxidationsprodukter (COPs) och sköldkörtelhormoner (THs) genom an-vändning av superkritisk vätskekromatografi-tandem masspek-trometri (UPSFC–MS/MS ) som analytisk teknik.

Endogena steroider kontrollerar många fysiologiska processer, inklusive reproduktion, mognad, genuttryck och neurologiska funktioner hos männi-skor och andra djur. I den första studien mättes tre steroider i domesticerade (tama) White Leghorn (WL) kycklingar och jämfördes med individer från de mer vilda förfäderna Red Junglefowl (RJF) fåglar. Stressen hos dessa fåglar då de hölls i bur, orsakade en signifikant större ökning av kortikosteronnivå-erna i RJF än i WL, vilket indikerar en förändrad hypotalamus–hypofys–adrenal (HPA)-axelaktivitet hos tama kycklingar. Den andra studien var en fortsättning på den första studien och kortikosteronnivåerna från individer som var en korsning mellan WL och RJF-fåglar mättes före och efter stress induktion. Expressionsnivåerna för glukokortikoidreceptorn (GR) i hypota-lamus och flera gener i binjurarna var korrelerade med post-stressnivåerna av kortikosteron i plasma. I den tredje studien utvidgades mätningsmetoden av steroider för att kunna inkludera fler av de endogena steroiderna från alla de fyra huvudklasserna, dvs östrogener, androgener, progestogener och kor-tikosteron.

Analys av endogena kolesteroloxidationsprodukter (COPs) är av stort in-tresse för att i detalj kunna förstå patofysiologin vid flertalet sjukdomar. Dessa oxidationsprodukter är lättare för cellen att utsöndra än kolesterol. Därför oxideras kolesterol till de mer polära COP och dessa är i allmänhet mer bioaktiva än kolesterol själv. Om produktion av COPs i celler och väv-nader är förhöjd så kan de bidra till patogenesen vid olika sjukdomsprocesser

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så som neurodegeneration och cancer. Totalt fjorton COP studerades i den fjärde studien och en helt ny metod för separation och detektion utvecklades.

Den sista studien i denna avhandling beskriver utvecklingen och utvärde-ring av analysmetoder för fem sköldkörtelhormoner (THs). Dessa hormoner är bland annat viktiga för tillväxt, utveckling och metaboliska processer hos många djurslag. Mätningar av cirkulerande sköldkörtel-hormonnivåer an-vänds vid diagnos av sköldkörtelstörningar eller vid övervakning av behand-lingseffekter. Två snabba metoder för separation och detektion av fem THs utvecklades i denna studie.

Sammanfattningsvis demonstrerar arbetet i denna avhandling tillämplig-heten av framför allt UPSFC-MS/MS som en viktig bioanalytisk teknik vid studier av viktiga endogena föreningar i komplexa biologiska prover.

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Afrikaanse samevatting

Bioanalitiese toepassings wat superkritiese vloeistofchromatografie (SFC) as analitiese tegniek gebruik, het die afgelope jare toenemend belangstelling gelok. In wese behels bioanalise die bepaling van bioaktiewe of endogene (liggaamlike) molekulêre stowwe in biologiese matrikse (liggaams-vloeistowwe of -weefsels). SFC het tot 'n tegniek ontwikkel wat ‘n uitstekende alternatief of komplement tot bioanalise verteenwoordig, hoofsaaklik as gevolg van die spoedigheid, selektiwiteit en doeltreffendheid van die tegniek in vergelyking met byvoorbeeld vloeistofchromatografie. Daarbenewens bied die koppeling van SFC met massaspektrometrie (MS) as die detektor bykomende voordele met betrekking tot spesifisiteit en sensitiwiteit.

Die doel van hierdie verhandeling was om hierdie gekombineerde eienskappe op die proef te stel deur metodes vir die analise van endogene steroïede, cholesteroloksidasieprodukte (COP’s) en skildklierhormone (TH’s) te ontwikkel deur gebruik te maak van ultraprestasiesuperkritiese vloeistof-chromatografie–tandemmassaspektrometrie (UPSFC-MS/MS) as analitiese tegniek.

Endogene steroïdes beheer menige fisiologiese prosesse, insluitende reproduksie, volwassewording, geenuitdrukking en neurologiese funksies in mense en ander diere. Drie steroïede is in die eerste studie in gedomestikeerde (mak) White Leghorn (WL)-hoenders bepaal en met individue van die meer wilde voorouer Red Junglefowl (RJF)-voëls vergelyk. Die stres van hierdie voëls wanneer hulle in hokke gehou word, veroorsaak 'n aansienlik groter toename in kortikosteroonvlakke in RJF as in WL, wat 'n veranderde hipotalamus–pituïtêre–adrenale (HPA)-asaktiwiteit in mak hoenders aandui.

Die tweede studie was 'n voortsetting van die eerste studie en kortikosteroonvlakke van individue van ‘n kruising tussen die WL- en RJF-voëls is voor en na stresinduksie gemeet. Die uitdrukkingsvlakke van die glukokortikoïede reseptor (GR) in die hipotalamus en verskeie gene in die byniere het met post-stresvlakke van kortikosteroon in plasma gekorreleer. In die derde studie is die skeidingsmetode van steroïede uitgebrei om meer endogene steroïede vanuit al vier hoofklasse, dit wil sê die estrogeen-, androgeen-, progestogeen- en kortikosteroïedklas, in te sluit.

Analise van endogene cholesteroloksidasieprodukte (COP’s) is van groot belang om die patofisiologie van verskeie siektes in detail te verstaan.

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Hierdie oksidasieprodukte is makliker as cholesterol vir die sel om uit te skei. Derhalwe word cholesterol na die meer polêre COP’s, wat oor die algemeen meer bioaktief as cholesterol self is, geoksideer. As die produksie van COP’s in selle en weefsels verhoog word, kan hulle bydra tot patogenese in verskeie siekteprosesse soos neurodegenerasie en kanker. ‘n Totaal van veertien COP’s is in die vierde studie bestudeer en ‘n hele nuwe metode van skeiding en opsporing is ontwikkel.

Die laaste studie in hierdie verhandeling beskryf die ontwikkeling en evaluering van skeidingsmetodes vir vyf skildklierhormone (THs). Hierdie hormone is belangrik vir groei, ontwikkeling en metaboliese prosesse in baie diersoorte. Bepalings van sirkulerende skildklierhormoonvlakke word in die diagnose van skildklierstoornisse of in die monitering van behande-lingseffekte gebruik. Twee vinnige metodes vir skeiding en opsporing van vyf TH’s is in hierdie studie ontwikkel.

Samevattend toon die werk in hierdie verhandeling die toepaslikheid van veral UPSFC–MS/MS as 'n belangrike bioanalitiese tegniek in die studie van belangrike endogene molekulêre stowwe in komplekse biologiese monsters.

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Acknowledgements

What a journey it has been! At times, a PhD can be a lonely path to walk and at times you can be joined by great people.

Jonas B, you are a cool and collected person and a great supervisor. Thank you for always being extremely encouraging, positive and understanding throughout my PhD. Also, we had good times discussing my work, attending conferences and it was great to have you visit South Africa! I also appreciate your support with regards to my involvement as representative within the faculty and the university. We had good times!

Kumari, words cannot describe how grateful I am for having had you as a supervisor. Thank you for the encouragement, motivation and tremendous support you gave me. We had a rocky start, but we steadied the boat and commanded the winds! Thank you for sharing your knowledge, working in the lab with me, putting in long hours to help me get the work done, sharing great experiences at conferences, visiting in South Africa, and always looking out for my well-being.

Thanks to all my colleagues, old and new, for support and encouragement throughout my time at the department.

To all my friends in the department and not in the department, thanks for being there in the good and not so good times. I really appreciate the time we spent together.

My greatest thanks and appreciation I would like to express to my family. You are awesome! Thank you for your love and continuous encouragement.

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