MOLECULAR CHARACTERISATION OF IN THE MAMMALIAN BRAIN

Martin Johannes Ebner

University College London

A thesis submitted for the degree of Doctor of Philosophy from the University of London, 2001. ProQuest Number: 10013862

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Thesis abstract

Steroid hormones from endocrine glands are known to regulate mammalian brain development and function. They act not only by regulation of gene transcription, but also via modulation of neurotransmitter receptors. More recently, steroids have been found to be synthesised within the brain, in addition to entry from the circulation. However, there is little information on the full complement of molecules present in the mammalian brain. In order to characterise brain steroid content, an improved procedure for the extraction and fractionation of steroids from nervous tissue was developed and used to generate samples from adult male rat brain suitable for analysis by gas capillary chromatography-electron impact mass spectrometry (GC-EIMS).

After extraction of free steroids and sulphate conjugates with acetic acid/ethanol, samples were purified by solvent partitioning followed by hydrophilic-lipophilic balance extraction on polymer based sorbents. Free steroids and sulphate ester fractions could then be separated using mixed mode anion exchange chromatography, which can also be used to separate steroid glucuronides.

For GC-EIMS, steroids were derivatised with methoxyamine (MO) and trimethylsilyl- (TMSI), heptafluorobutyric acid anhydride (HFBA) or N-methyl-N-(fer?-butyl- dimethylsilyl)trifluoroacetamide. Diagnostic ion screening procedures were developed for a wide range of free and sulphate conjugated steroids likely to be encountered in mammalian tissue. In initial screening, two diagnostic ions were monitored for the MO/ TMSI derivatives. Further possible generic steroids were screened with 32 ion monitoring. Confirmation employed three ion monitoring of MO/ TMSI and/or two ion monitoring of HFBA derivatives. The results unequivocally confirm the free steroids , , , 20a-dihydropregnenolone, 20(3- dihydropregnenolone, 3 a, 5a-tetrahydroprogesterone, 3 a, 5a-tetrahydrodeoxy- , corticosterone, and 5a-pregnan-3a,17-diol-20-one as present in male rat brain. Further evidence was obtained for the unconjugated steroids

5a-dihydroprogesterone, 2 0 a-dihydroprogesterone, 5a,20a-tetrahydroprogesterone, 5a- -3a,20a-diol, 3p,5a-tetrahydroprogesterone, 3p,5a-tetrahydrodeoxy- corticosterone and 5a-pregnan-3a,l ip-diol-20-one. In the sulphate fraction, dehydro­ was identified but not the previously described pregnenolone sulphate.

The results provide further implications for steroids in brain function. Acknowledgements

I would like to thank;

Drs. Jonathan Fry and John Honour, my supervisors for their guidance and help, their time and experience was invaluable.

Dr. David Allan for his role as subsidiary supervisor and many useful discussions that helped develop the thesis.

All people in the laboratories of Dr. Fry and Dr. Honour, especially Elvira Conway for introducing me to the practical side of mass spectrometry, Rachel Tilley for experimental assistance. Dr. Jayne Woodside and Richard Hodkinson for many fruitful discussions.

Dr. Mart Mojet for the photometric measurements of the haemoglobin samples.

The European Commission and the Department of Physiology, UCL for supporting me with grants, that enabled me to do this project.

My family, especially my parents, for their encouragement and help throughout, my sister and my grandmother for their support in many ways. Most of all Gill, for her endless support and patience.

This thesis is my own account of investigations carried out by myself under the supervision of Drs. Jonathan Fry and John Honour. Table of contents

Th e sis a bstr a ct 2

Acknowledgements 3

List o f ta bles 8

LIST OF FIGURES 10

Abbreviations 12

CHAPTER 1 INTRODUCTION AND BACKGROUND REVIEW______

1.1 INTRODUCTION 15

1.2 AIMS AND OBJECTIVES 16

1.3 B a c k g r o u n d r ev iew 17

1.3.1 O r ig in o f s t e r o id s in t h e n e r v o u s s y s t e m 17

1.3 .1.1 Steroid supply to the nervous system from peripheral sources 17

1.3.1.2 Neurosteroidogenesis 18

1.3 .1.3 Regulation of brain steroids 19

1.3 .1.4 Molecular regulation of neurosteroidogenesis 20

1.3 .1.5 Steroid metabolising enzymes in nervous tissue 21

1.3 .1.5 .1 Cytochrome P450 dependent enzymes 21

1.3.1.5.2 Non cytochrome P450 enzymes 27

1.3.2 MODES OF ACTION OF STEROIDS IN THE NERVOUS SYSTEM 3 3

1.3.2.1 GAB A A-receptor 3 3

1.3.2.2 N-methyl-D-aspartate receptor 37

1.3.2.3 Voltage-gated calcium channels 38

1 .3.2.4 Other steroid effects on neuronal membrane receptors 38

1.3.3 PHYSIOLOGICAL ROLES OF STEROIDS IN THE NERVOUS SYSTEM 3 9

1.3.4 BRAIN STEROIDS IN DISEASE MECHANISMS 43

1.4 C o n c lu sio n 46 CHAPTER 2 MATERIALS AND METHODS

2.1 C h em ic a ls 49

2.2 Gen era l Pr o c ed u res 50

2.3 E x tr a c t io n and fractionation o f ster o id s f r o m b r a in tissue 51

2.3.1 T is s u e s a m pl e s 51

2.3.2 T is s u e e x t r a c t io n 52

2.3.3 So l v e n t partitioning 54

2.3.4 So l id p h a s e e x t r a c t io n chromatography 55

2.3.5 CELITE chromatography 5 5

2.3.6 SOLVOLYSIS 56

2.3.7 Fin a l e x t r a c t io n a n d fractionation p r o c e d u r e f o r s t e r o id s f r o m m a m m a l ia n BRAIN TISSUE 56

2.4 G as chromatography - m ass spectrometry 58

2.4.1 Sa m p l e DERivATisATioN 58

2.4.2 Ga s CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS 5 9

CHAPTER 3 DEVELOPMENT OF A METHOD FOR THE ANALYSIS OF STEROIDS FROM MAMMALIAN BRAIN TISSUE BY GAS CHROMATOGRAPHY - ELECTRON IMPACT IONISATION MASS SPECTROMETRY (GC-EIMS)______

3.1 INTRODUCTION 76

3.1.1 G a s chromatography 77

3.1.2 H ig h performance l iq u id chromatography 79

3.1.3 M a s s spectrometry 80

3.1.4 Mass spectrometric identification 85

3.1.5 Q uantitative m a s s spectrometry 86

3.2 RESULTS AND DISCUSSION 88

3.2.1 DEVELOPMENT OF A METHOD FOR DERIVATISATION OF STEROIDS TO METHYLOXIME TRIMETHYLSILYL ETHERS FOR GAS CHROMATOGRAPHY-MASS SPECTROMETRY IN THE ELECTRON IMPACT MODE 8 8

3.2.2 O ptimisation o f g a s chromatographic a n d m a ss spectrometric c o n d it io n s f o r ANALYSIS OF STEROID MO- AND TMS- DERIVATIVES 89

3.2.3 C haracterisation o f MO-TMS- derivatives o f st e r o id s b y GC-EIMS 94

3.2.4 DERIVATISATION OF CONJUGATED STEROIDS BY MO AND TMSI 95 3.2.5 D e v e l o p m e n t o f t w o io n se l e c t e d io n m o n it o r in g m e t h o d s f o r a n a l y sis of STEROID MO-TMS- derivatives BY GC-EIMS 98

3.2.6 M u l t ipl e io n sc r e e n in g m e t h o d s f o r identification o f n o v e l c o m p o u n d s in MAMMALIAN BRAIN 100

3.2.7 M e t h o d s f o r confirmahon o f b r a in st e r o id s 10 1

3.2.7 .1 Derivatisation of steroids for GC-EIMS using perfluoroacylation 101

3.2 .7 .2 Derivatisation of steroids for GC-EIMS using rer/-butyldimethylsilylation 102

3.2 .7.3 Optimisation of HFBA derivatisation of steroids 104

3.2.7.4 Alternative derivatisation methods for conjugated steroids 105

3.2.7.5 Three ion selected ion monitoring for MO-TMS- derivatives of steroids 107

3.2.8 M e t h o d f o r quantitahon o f b r a in s t e r o id s 107

3.3 C o n c lu sio n 109

CHAPTER 4 AN IMPROVED PROCEDURE FOR THE EXTRACTION AND FRACTIONATION OF BRAIN STEROIDS______

4.1 INTRODUCTION 134

4.1.1 E x t r a c h o n 134

4.1.2 PURff ICAHON procedures 13 6

4.1.3 F r a c h o n a h o n o f f r e e a n d c o n ju g a t e d s t e r o id s 13 8

4.2 Re su lts an d d isc u ssio n 141

4.2.1 O r ig in a l p r o c e d u r e fo r e x t r a c h o n , frachonahon a n d purificahon o f STEROIDS FROM MAMMALIAN BRAIN HSSUE 141

4.2 .1.1 Steroid extraction and solvent partitioning of extracts 141

4.2.1.2 Solid phase extraction 141

4.2.1.3 Partition chromatography on celite 143

4.2 .1.4 Overall performance of original procedure for extraction and fractionation of steroids from brain tissue 144

4.2.2 Im p r o v e d a n d sim pl ifie d p r o c e d u r e fo r t h e e x t r a c h o n , purificahon a n d FRACHONAHON OF STEROIDS AND THEIR SULPHATE ESTERS FROM MAMMALIAN BRAIN HSSUE 145

4.2.2.1 Extraction of steroids from brain tissue in ethanol or ethanol/acetic acid 146

4.2.2 .2 Oasis HLB® solid phase extraction 146

4.2 .2 .3 Solvent partitioning 148 4.2.2.4 Separation of free and sulphate conjugated steroids by hydrophilic/hydrophobic balance and/or ion exchange chromatography 148

4.2.2.5 Purification of HFBA-derivatives of steroids extracted and fractionated from mammalian brain tissue 154

4.2.2.6 Overall recovery of improved procedure for extraction and fractionation of steroids from brain tissue 155

4.3 C o n c lu sio n 155

CHAPTER 5 A SURVEY OF STEROIDS IN THE MAMMALIAN BRAIN______

5.1 Introduction 172

5.2 Resu lts a nd d isc u ssio n 173

5.2.1 Two ION SELECTED ION MONITORING OF STEROIDS IN RAT BRAIN 173

5.2.2 M u l t ip l e io n sc r e e n in g o f st e r o id s in r a t b r a in 173

5.2.3 C onfirmation a n d quantitation o f b r a in s t e r o id s 174

CHAPTER 6 CONCLUSION 233

REFERENCES 236

APPENDICES

APPENDIX 1 Gas chromatographic-mass SPECTRAL DATA OF STEROIDS DERIVATISED BY MO/TMSI, TBDMS- AND HFBA AND SELECTED ION MONITORING METHODS 250

APPENDIX 2 Application of method for separation of free steroids and THEIR SULPHATE AND GLUCURONIDE CONJUGATES TO HUMAN URINE SAMPLES 265 List of Tables

Tables are located at the end of each Chapter on pages as listed below.

Table 1-1. Systematic and trivial names for P450 steroidogenic enzymes identified in rat brain ...... 47

Table 2-1. Systematic and trivial names of standard steroids and their abbreviations used here, sources and relative molecular weights ...... 64

Table 2-2. GC and scan MS parameter settings in methods used for steroid analysis. 6 8

Table 2-3. SIM MS settings of methods used in steroid analysis ...... 69

Table 3-1. GC-conditions employed in optimisation of separation of MO-TMS-steroids.

' 110

Table 3-2. Responses of steroid sulphate esters after different derivatisation methods or after microsolvolysis and derivatisation relative to tetracosane ...... 1 1 0

Table 3-3. Predicted EIMS -ions (m/z) of MO-TMS-derivatives of steroids for multiple ion screening of brain extracts for new compounds ...... I ll

Table 3-4. Relative response ratios of free steroids (10 ng) derivatised with HFBA in different reaction conditions as shown and tetracosane (10 ng) in GC-EIMS... 113

Table 3-5. Calibration curve equations, R-values and accuracy (as % of added amount) of two ion SIM analysis of steroids in GC-EIMS ...... 114

Table 3-6. Intra- and inter-assay reproducibility as well as detection limits of two ion SIM analysis of steroids in GC-EIMS ...... 116

Table 4-1. Recovery (%) of steroids in 90% or 80% in H 2O after solvent partitioning against isooctane ...... 157

Table 4-2. Recovery of steroids from Sep Pak Cl 8 ® chromatography ...... 157

Table 4-3. Recovery of selected steroids from celite chromatography with

glycol stationary phase ( 1 :1 , w/v)...... 158

Table 4-4. Recoveries (%) of ^H-CORT and ^H-5a-DHPR0G from ethylene and propylene glycol stationary phases on celite ( 1 g)...... 158

Table 4-5. Recoveries (%) of reference steroids from celite (1 g) columns with ethylene and propylene glycol (4:1:1, w/v/v) stationary phases ...... 159

Table 4-6. Recoveries of free steroids added to and concentrations of endogenous free steroids in human cerebral cortex (approx. 5 g each) extracted by original procedure ...... 159 Table 4-7. Responses of DHEA or DHEAS and PREG or PREGS after derivatisation with MO and TMSI to the internal standard ME-17-OH-PROG after different pre­ treatments on GC-MS ...... 160

Table 5-1. Typical concentrations of steroids in adult male rat brain as characterised by GC-MS in reports by other authors ...... 185

Table 5-2. Results of screening in male rat whole brain extracts in GC-MS two ion selected ion monitoring after MO-TMS-derivatisation ...... 186

Table 5-3. Comparison of relative retention times (to ME-17-OH-PROG) given by the diagnostic ions of putative free steroids from rat whole brain extracts in comparison to known standard steroids ...... 187

Table 5-4. Examples of identification of free and sulphate conjugated (as indicated in Table) steroids in adult male rat brain ...... 190

Table 5-5. Examples of identification of free and sulphate conjugated (as indicated in Table) steroids in adult male rat brain ...... 193

Table 5-6. Summary of identification of free and sulphate conjugated steroids in adult male rat whole brain ...... 197

Table 5-7. Quantitation of steroids in adult male rat whole brain ...... 200

Table A-1. Retention index after Kovats (RI), major ions, target and qualifier diagnostic ions (m/z) for two-ion SIM of MO-TMS-derivatives of steroids in GC-EIMS .252

Table A-2. Two ion SIM methods for the analysis of MO-TMS-derivatives of steroids...... 254

Table A-3. Target (T) and two qualifier ions (Ql, 2), ion set end times, retention indices (RI) and detection limits for three ion SIM methods used to confirm identities of a) unconjugated and b) sulphate conjugated brain steroids as well as indications of IS used to calculate RRT ...... 256

Table A-4. Retention index after Kovats (RI), major ions, diagnostic (target, T and qualifier, Q) ions (m/z) for SIM of TBDMS-derivatives of steroids in GC-EIMS 260

Table A-5. Retention index after Kovats (RI) and major ions of HFB-derivatives of steroids in GC-EIMS ...... 261

Table A- 6 . Two ion SIM methods for the analysis of HFB-derivatives of free (a) and sulphate conjugated (b) steroids ...... 263 List of Figures

Figures are located at the end of each Chapter on pages as listed below.

Figure 1-1. Ring numbering of basic steroid structure ...... 47

Figure 1-2. Biosynthetic pathways of steroid formation ...... 48

Figure 3-1. Quadrupole mass separator ...... 119

Figure 3-2. Total ion chromatogram of TMS-20a-DHPREG as analysed by GC-MS.. 119

Figure 3-3. Elution of MO and TMSI derivatised reference steroids from Lipidex 5000® chromatography with cy do hexane : pyridine : HMDS (98:1:1, v/v/v) ...... 120

Figure 3-4. Signal to noise ratios for 3a -DHPROG ion 417 m/z (50pg) analysed by GC- MS...... 124

Figure 3-5. GC-separation of MO-TMS-derivatives of reference steroids on a 25 m, 0.25 mm ID, 0.12 |im film thickness CP SIL 5 CB column ...... 125

Figure 3-6. GC-separation of MO-TMS-derivatives of reference steroids on a 30 m, 0.25 mm inner diameter, 0.25 pm film thickness ZBl column ...... 127

Figure 3-7. El- mass spectra (99-800 m/z) of a) PREG, b) 5a-pregnan-3a,17-diol-20- one derivatised with MO and TMSI and proposed fragmentation pathways. .. 129

Figure 3-8. El-mass spectrum (50-800m/z) of HFB -PROG and possible fragmentations...... 131

Figure 3-9. Total ion chromatogram and El- mass spectra (3 -TBDMS-PROG, 3-,20-di- TBDMS-PROG, 50-800 m/z) for PROG derivatised with MTBSTFA/ TBDMSCl/ NH4I/ pyridine/ acetonitrile together with their proposed fragmentation pathways...... 132

Figure 3-10. Calibration curve for MO-TMS-DHEA, ion 358 m/z to IS MO-TMS-ME- 17-OH-PROG ion 443 m/z from 0.5-10 ng, IS amount 50 ng ...... 133

Figure 4-1. Stationary phases of solid phase extraction cartridges ...... 161

Figure 4-2. Removal of from rat brain extracts by Sep Pak C l 8 ® solid phase extraction ...... 162

Figure 4-3. Recovery (%) of ^H-PROG and ^^C-CHOL from Oasis HLB® cartridges...... 163

Figure 4-4. Elution profiles of ^H-5a-DHPR0G (5P), ^H-PROG (P) and ^H-CORT (C) from celite with ethylene glycol stationary phase (1:1, w/v) ...... 164

Figure 4-5. Elution of ^H-5a-DHPR0G from celite (1 g): ethylene glycol : propylene glycol (circles: 2:1:0, squares: 4:1:1) stationary phases ...... 164

10 Figure 4-6. Original procedure for extraction and separation of free steroids and their sulphate esters from nervous tissue ...... 165

Figure 4-7. Selected ion chromatograms of MO-TMS-DHEA from human cerebral cortex extracted by the original procedure and analysed by GC-MS in two ion SIM. 166

Figure 4-8. Recoveries of ^H-PROG in rat brain extracts from Oasis HLB® cartridges with (a) 60 % (circles) and (b) 80% (squares) ethanol in potassium phosphate buffer (5mM, pH 7.4, v/v) ...... 166

Figure 4-9. Elution of a) steroid sulphate esters and b) free steroids from Oasis HLB® cartridges...... 167

Figure 4-10. Elution of ^H-DHEA from Oasis MAX in different load solvents ...... 168

Figure 4-11. Elution of HFB-derivatised reference steroids from Lipidex 5000® chromatography columns ...... 168

Figure 4-12. Final procedure for extraction and separation of free steroids and their sulphate esters from nervous tissue ...... 171

Figure 5-1. Selected ion chromatograms of male rat whole brain extract free steroid fractions derivatised by MO/TMSI and analysed in two ion SIM ...... 204

Figure 5-2. Selected ion chromatograms of male rat whole brain extract steroid sulphate fractions derivatised by MO/TMSI and analysed in two ion SIM ...... 206

Figure 5-3. Typical selected ion chromatograms of the free steroid fraction of rat brain extracts analysed in GC-MS by multiple ion screening ...... 207

Figure 5-4. Typical selected ion chromatograms of rat brain extracts analysed in GC-MS by two or three ion monitoring of MO-TMS-derivatives for identification ...... 209

Figure 5-5. Typical selected ion chromatograms of rat brain extracts analysed in GC-MS by two ion monitoring of HFB-derivatives for identification ...... 220

Figure 5-6. Possible pathways of steroid synthesis and metabolism in rat brain ...... 232

Figure A-1. Total ion chromatograms of urine steroids ...... 267

11 Abbreviations

Systematic, trivial names and abbreviations of steroids are listed in Table 2-1.

AMPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ANC adenine nucleotide carrier APCI atmospheric pressure chemical ionisation AR receptor BBB blood-brain barrier BSA sulphonic acid Cl chemical ionisation CNS central nervous system CPH cyclohexane:pyridine:hexamethyldisilazane (98; 1:1,v/v/v) CRH corticotropin-releasing hormone CV coefficient of variation DBI binding inhibitor DRG dorsal root ganglia El electron impact ionisation ER oestrogen receptor ESI electro spray ionisation FAB fast atom bombardment FID flame ionisation detector GABA y-aminobutyric acid GABA a "R GABA type A-receptor/ion channel complex GC-MS gas chromatography-mass spectrometry GR glucocorticosteroid receptor HFBA heptafluorobutyric acid anhydride HLB hydrophilic-lipophilic balance HMDS hexamethyldisilazane HMG-CoA hydroxymethylglutaryl-CoA HOR hydroxysteroid oxidoreductase HPLC high performance liquid chromatography HRMS high resolution mass spectrometry HSD hydroxysteroid dehydrogenase HST Hydroxysteroid sulphotransferase ip intraperitoneal IS internal standard i.v. intravenous LTD long term depression LTP long term potentiation

12 M molecular ion MALDI matrix-assisted laser desorption ionisation MAX mixed mode anion exchange MR mineralocorticosteroid receptor

Mr relative molecular weight MTBSTFA N-methyl-N-(^^r^-butyl-dimethylsilyl)- trifluoroacetamide nAch-R nicotinic acetylcholine receptor NICI negative ion chemical ionisation NMDA N-methyl-D-aspartate NMDA-R NMDA-receptor P450aro aromatase P450c 11 AS synthase P450cl Ip 11 P-hydroxylase P450c 17 17a-hydroxylase/17,20-lyase P450c21 21-hydroxylase P450c7b 7a-hydroxylase P450scc P450 side chain cleavage PER peripheral receptor PFTBA perfluorotributylamine PICI positive ion chemical ionisation PKA protein kinase A PMS pre-menstrual syndrome PNS peripheral nervous system PR Q qualifier ion Q/T qualifier-target ion area ratio RI retention index RRT relative retention time RT retention time RT-PCR reverse transcriptase-polymerase chain reaction S/N signal-to-noise ratio SAPK stress activated protein kinase SAX strong anion exchange SD standard deviation SDN-PO A sexually dimorphic nucleus of the hypothalamic preoptic area S .E.M. standard error of the mean SIM selected ion monitoring SPE solid phase extraction SSRI selective serotonin reuptake inhibitor StAR steroidogenic acute regulatory protein

13 STS steroid sulphatase T target ion TBDMSCl /er/-butyl-dimethylsilylchloride TFAA trifluoroacetic acid anhydride TMCS trimethylchlorosilane TMSI trimethylsilylimidazole TOP time of flight VDAC voltage dependent anion channel VGCC voltage gated calcium channel VGKC voltage gated potassium channel WCOT wall coated open tubular

14 Chapter 1 Introduction and background review

1.1 Introduction

Steroid hormones are produced in a controlled manner in the adrenal cortex, gonads and placenta. They are well known to enter the nervous system and exert important regulatory functions. The influence of certain steroids on excitability of the central nervous system was observed long ago in the ground-breaking work of Selye when injection of these compounds was found to induce anaesthesia [168]. In addition, it has more recently been found that the nervous system can synthesise steroids de novo and independently from peripheral steroidogenic glands. The latter compounds were termed (see 1.3.1.2). Furthermore, several enzymes of steroid metabolism are present in both central and peripheral nervous systems (CNS and PNS). In addition to their action on gene transcription mediated by cytosolic and/or nuclear receptors, steroids can modulate neuronal activity in a fast manner via allosteric regulation of neurotransmitter and voltage gated ion flux through membrane channels (see 1.3.2).

However, the number of known pharmacological actions of steroid hormones and possible metabolic conversions is in contrast to the information available on steroid metabolites present in the nervous system and the regulation of their formation. The reasons for this lack of information are largely analytical difficulties. Firstly, due to their lipophilic nature, free steroids need to be extracted and extensively purified from the lipid rich milieu of nervous tissues prior to analysis. On the other hand, the more hydrophilic sulphate esters need also to be extracted and furthermore separated from other conjugated and unconjugated steroids. They usually have to be cleaved to release the free steroids before analysis. Secondly, steroids are present at low levels in this tissue (low ng/g tissue range) and thus require highly sensitive analysis methods. Finally, the diversity of structures of steroid activity and possible molecules formed in or entering this organ with high chemical similarity necessitates highly specific assay techniques.

Previously, analysis of steroids in nervous tissue has been mainly carried out using radioimmunoassay (RIA) (e.g. [22,213]). Although this technique possesses excellent sensitivity, unequivocal identification cannot usually be achieved owing to cross­ reactivities of the antisera between chemically very similar steroids. Specificity for

15 unequivocal identification of trace amounts of steroids in complex biological mixtures is usually only achieved by gas chromatography-mass spectrometry (GC-MS), with specificity being inferred by unique mass spectral fragmentation patterns obtained for each compound and by the high efficiency separation of mixtures by gas chromatography.

Knowledge of the identities and quantities of steroids in nervous tissues is an important factor in order to establish their physiological relevance. Further, it is possible that metabolites are present for which the pharmacological actions have not yet been investigated. Brain steroids are thought to be implicated in clinical conditions such as some forms of depression, anxiety and epilepsy as well as deficits of cognitive performance. The possible physiological and pathological roles of steroids in the nervous system are discussed in more detail in Sections 1.3.3 and 1.3.4 below. An understanding of the involvement of certain steroid metabolites in physiological and pathological processes could aid in the development of possible cures for those conditions.

Investigations of the physiological regulation of synthesis in CNS/PNS and their regional distribution usually require analysis of metabolites in small tissue sample sizes. This requires highly sensitive assay methods, which often do not meet the highest specificity standards. Quantitations with such methods can however be carried out, when they are first validated by specific methods.

1.2 Aims and objectives

The main aim of this study was to develop procedures for the extraction, fractionation and unequivocal identification and quantitation of a range of free steroids and steroid conjugates in the mammalian brain and to study the steroid content of rat brain under normal physiological conditions. In detail, the objectives can be summarised as follows;

• To evaluate and further develop, if necessary, techniques of steroid extraction, purification and fractionation from mammalian nervous tissue.

• To evaluate derivatisation methods for GC-MS of a wide range of different steroids, to optimise separation of those steroids by GC and the sensitivity of the analysis in order to allow screening of steroids at the low levels that they are present at in brain tissue.

16 • To apply selected ion monitoring GC-MS methods for screening of free steroids and steroid sulphate esters in rat brain and confirmation and quantitation of preliminarily identified compounds by specific methods, such as three ion monitoring and/or selected ion monitoring of compounds derivatised by different methods.

In order to provide a background to the present study knowledge about the supply to and synthesis and metabolism of steroids in the mammalian nervous system, their modes of action and possible physiological and pathological roles is briefly reviewed.

1.3 Background review

1.3.1 Origin of steroids in the nervous system

Steroid molecules are derived from the basic four ring cyclopentanoperhydrophenanthrene system. The precursor compound for all steroid hormones, cholesterol (CHOL), is derived from the diet or biosynthesised from acetate. It can be made in the nervous system of mammals, as in all other body organs. The synthesis proceeds via mevalonate (C6) to the basic isoprene units (C5), famesyl pyrophosphate (Cl 5), squalene (C30) and lanosterol (C30). Loss of three methyl groups and rearrangements of the structure finally leads to CHOL. The rate-limiting step in this biosynthetic pathway is the formation of mevalonate by hydroxymethylglutaryl-CoA (HMG-CoA) -reductase. Modification of CHOL gives rise to the three classes of basic pregnane (C21), androstane (C l9) and oestrane (C l8) structures from which a wide variety of active hormone metabolites can be derived. The convention on the numbering of the carbon atoms in the steroid nucleus is shown in Figure l-I. The main known steroidogenic pathways existing in peripheral organs, mainly gonads, adrenals and placenta are shown in Figure 1-2.

1.3.1.1 Steroid supply to the nervous system from peripheral sources

Permeability of the blood-brain barrier (BBB) to unconjugated endogenous steroids is determined by their lipid solubility. The radiolabelled (^H) sex steroids testosterone (TESTO), oestradiol (OESTR) and progesterone (PROG) can cross the BBB more or less freely, the glucocorticosteroid corticosterone (CORT) is about 40% permeable in

17 the rat relative to ^'^C-butanol which is considered to be freely diffusible in the assay [138]. The crossing of the BBB by steroids is however reduced if bound to plasma steroid binding globulins. Rat serum does not prevent partitioning of the free steroids PROG, TESTO and OESTR into the brain. Neonatal rat serum, in contrast, almost completely prevents OESTR from crossing into the brain, but not TESTO or PROG [138,140], consistent with the existence of a specific binding protein a-fetoprotein [110]. Albumins, carrying the majority of most steroids in blood of rat through low affinity binding, do not significantly prevent steroids from transport through the BBB. CORT is retained in rat plasma to about 70%, indicating existence of a -binding globulin (CBG)[138,139]. Evidence exists for brain uptake from blood of sulphate conjugated steroids. PREGS is distributed in brain following i.v. injection of rats [202]. As with other organic anions, specific transporter proteins might play a role for transport of those conjugates.

1.3.1.2 Neurosteroidogenesis

The first evidence of steroid synthesis in nervous tissue independently of peripheral steroidogenic glands was the discovery in 1981 by the group of Robel and Baulieu of higher concentrations of DHEAS in rat brain than plasma. The brain concentrations did not decrease significantly even several weeks after adrenalectomy and orchidectomy [48]. Later studies also identified PREG and its sulphate ester PREGS in brains of rats under these experimental conditions [49]. Subsequently, the presence of the enzyme responsible for the formation of PREG from CHOL, P450 side chain cleavage (see) or desmolase was demonstrated at the protein level by immunocytochemistry throughout the white matter of rat brain [102]. Several enzymes active in peripheral steroidogenic glands, adrenals, gonads and placenta have since been shown to be present in the CNS and/or PNS, as described below.

Neurosteroids have been thus defined as steroids that are formed in nervous tissue, at least in part, independently from peripheral steroidogenic glands, but not including compounds that are produced in the nervous system from precursors derived mainly from blood [12]. Thus far DHEA, PREG, their sulphate and lipid esters, PROG and 3a,5a-THPROG fulfil the above criteria for neurosteroids [48-50,153]. Steroids exerting

18 effects on neural cells can be derived from blood or synthesised locally and are referred to as neuroactive [12].

1.3.1.3 Regulation of brain steroids

Measurements by RIA have shown that steroid levels in brain undergo variations according to the physiological situation of the animals. Time of day, exposure to light, food, exposure to animals of the other sex and stress appear to be factors influencing brain steroid concentrations. Diurnal variations of brain and plasma PREG and DHEA in male rats have been shown [190], with the brain PREG and plasma DHEA rhythms peaking at the beginning of the dark phase. Independent regulatory mechanisms for the synthesis of the different steroids are thus indicated. Large variations of brain PROG, 5a-DHPR0G, 3a,5a-THPROG during the mouse oestrous cycle following a 24 hour rhythm with peaks at the beginning of the dark phase were also shown [47]. In contrast, plasma PROG showed a different rhythm with only two major peaks during the oestrous cycle. The plasma PROG variations were small compared to the variations of brain PROG, 5a-DHPR0G, 3a,5a-THPROG. Brain PREGS content was also analysed and was surprisingly found to correlate with plasma PROG levels. Strong correlations were also found between brain PROG and both 5a-DHPR0G, 3a,5a-THPROG. Neither brain PROG, 5a-DHPR0G nor 3a,5a-THPROG correlated with plasma PROG These findings suggest regulation of the brain PROG metabolites independent from plasma steroids and different to the regulation of PREGS in brain. Furthermore, the major regulating factor for brain 5a-DHPR0G, 3a,5a-THPROG seems to be the supply of PROG from within brain.

RIA measurements of brain 3a,5a-THPROG and 3a,5a-THDOC also suggest a rise within around 10 minutes after swim stress in rats. The 3a,5a-THPROG increase was also observed in adrenalectomised rats, whereas no such change was observed for 3a,5a-THDOC. The 3a,5a-THPROG peak level in brain precedes the peak level in plasma, suggesting an independent regulation [153].

More detailed studies with specific assay methods including measurement of several further metabolites are necessary to establish the physiological relevance and regulation of neurosteroids.

19 1.3.1.4 Molecular regulation of neurosteroidogenesis

In classical steroidogenic tissues, the side chain cleavage of CHOL to PREG is considered to be the rate limiting step. The limiting factor in this reaction is the supply of substrate to the enzyme on the inner side of the inner mitochondrial membrane. Steroidogenic acute regulatory protein (StAR) is considered to be involved in inducing steroidogenesis in adrenals and gonads. Its function is assumed to be in aiding CHOL transport from the cytosol to the inner mitochondrial membrane. Recently StAR was shown by RT-PCR to be expressed in a region specific manner in brain [61]. StAR transcripts were found in hippocampus, dentate gyrus, cerebellum, where also P450 side chain cleavage (P450scc) and 3 p-hydroxysteroid dehydrogenase (3P-HSD) are found. Furthermore StAR was expressed in the cerebral cortex. The levels of expression, however were 100-1000 fold lower than those in the adrenal. The transport of CHOL through the mitochondrial membranes occurs through the so-called peripheral benzodiazepine receptor complex (PER). PER is a specific benzodiazepine binding site distinct from the GAEAA/benzodiazepine-receptor complex that occurs also outside the CNS. The PER complex in the mitochondria contains also the proteins voltage dependent anion channel (VDAC) and adenine nucleotide carrier (ANC). Together these proteins are thought to form pores and through conformational changes bringing the two membranes into contact to translocate CHOL to P450scc at the inner mitochondrial membrane. PER is abundant in peripheral steroidogenic cells and has also been described in brain [121,209]. Endogenous ligands of the PER are the polypeptide diazepam binding inhibitor (DEI) and its fragments. In traditional steroidogenic models, signalling is mediated by an increase in cAMP levels and activation of protein kinase A (PKA) upon binding of a G-protein coupled receptor by a steroidogenic hormone. It has been found that cAMP changes PER conformation and topography to states of higher affinity for ligands. This could lead to increased binding of abundant DBI and consequently increased mitochondrial CHOL transport. Exposure to the lipid soluble cAMP analogue, dibutyrilic cAMP was found to increase PREG or ^H-PREG formation in a dose dependent manner in slices from rat brain cortex [11,162] and in glial cell cultures [78]. Other messengers like (NO) and signal transduction proteins such as protein kinase C could also be involved in acute steroidogenesis in certain situations. In the long term steroidogenesis is presumed to be regulated through transcription of the steroidogenic enzymes.

20 1.3.15 Steroid metabolising enzymes in nervous tissue

Since the initial discovery of apparent independent synthesis of DHEA(S) in the brain (see above), the enzymatic apparatus with a multitude of steroidogenic capabilities has been demonstrated in the nervous system. These enzymes will be indicated below. Steroidogenic enzymes can be classified into cytochrome P450 and non-cytochrome P450 dependent. These enzymes vary in their subcellular location. Figure 1-2 summarises known steroidogenic conversions regardless of tissue. Enzyme activities not demonstrated in the mammalian nervous system to date are indicated by dotted arrows.

1.3.1.5.1 Cytochrome P450 dependent enzymes

Cytochrome P450 enzymes are a group of monoxygenases that are involved in the metabolism of a wide range of biological molecules such as steroids, fatty acids, prostaglandins, biogenic amines, etc. They are often responsible for metabolism of substances foreign to the body (xenobiotics). They are present at high levels in adrenals, gonads and liver, but low levels are found in the brain [205]. The underlying structure is a protoporphyrin haemprotein of the cytochrome b5 type [116]. Steroid converting P450 enzymes have a somewhat different functionality to others as they show relatively high substrate specificity. Molecular oxygen is reduced via electrons from the haem function of the enzymes. For mitochondrial P450s, the electrons are received from NADPH via the non-haem iron protein adrenodoxin, through a reaction catalysed by the flavoprotein adrenodoxin reductase. By contrast, microsomal P450s receive electrons from NADPH via cytochrome bS and P450 reductase [116].

The names for the steroidogenic P450 enzymes found in mammalian brain are shown in Table 1-1, listed by their systematic and trivial names. The names for the genes are the same as the systematic names for the proteins, however italicised. The trivial names are still more commonly used and are thus employed throughout the remainder of this text [134].

Cholesterol side chain cleavage (P450scc)

Nervous system cells can make the main precursor of steroid hormones, PREG. The first and rate limiting step in steroidogenesis in peripheral endocrine tissues is the cleavage of

21 the side chain of CHOL to form PREG mediated by the mitochondrial cytochrome P450 side chain cleavage (P450scc, see Figure 1-2). In humans and rodents, this enzyme is, as far as is known, encoded by a single gene. The complete reaction involves two hydroxylations at C-20 and C-22 to 20a,22-dihydroxycholesterol and subsequent scission of the C-20-C-22 bond, leading to the reaction products PREG and isocaproic aldehyde [125]. As mentioned above, the presence of the P450scc protein has been demonstrated in rat brain by immunocytochemistry using antibodies against bovine adrenal P450scc. It exists predominantly in the white matter, with only few cell clusters labelled elsewhere [102]. This suggested that the enzyme could be located in myelin, which is formed by oligodendrocytes in the CNS. Subsequent studies with mitochondrial incubations found the enzyme activity indeed to be present in primary cultures of oligodendrocytes from rat [78]. Primary mixed glial cell cultures from rat brain cortex could also produce ^H-PREG and ^H-20a-DHPREG after incubation with ^H- mevalonolactone. Immunostaining for P450scc and cell markers indicated its expression in oligodendrocytes, but not astrocytes in these cultures [79,84].

Further studies using reverse transcription of mRNA and polymerase chain reaction for cDNA amplification (RT-PCR) [125,165] and in situ hybridisation with cRNA probes [165] found highest expression of P450scc in subcortical and cerebellar white matter structures. In the former, corpus callosum, fimbria and internal capsule fibres were stained by cRNA probes. Further expression was seen in grey matter of cerebral cortex, in amygdala, hippocampus, midbrain and spinal cord. Low levels of expression were seen throughout the brain. The differing findings from protein and mRNA studies could be due to different sensitivities of the assays. When P450scc protein was detected, much more protein than mRNA was found when compared to adrenal levels. The protein abundance in rat brain was estimated to be 1% of adrenal, but mRNA only 0.001-0.01% of adrenal levels [125]. This difference in relative mRNA and protein concentrations suggests that the enzyme protein and mRNA are more stable in brain and /or transcription and translation are different in these tissues. The possibly higher stability of the P450SCC protein could be an indicator that there is no regulation of steroidogenesis at this level in the nervous system. Alternatively, it was speculated that the dissimilarity of the protein/mRNA ratio in brain and peripheral endocrine glands is caused by the existence of another CHOL side chain cleavage activity that has similar immunoreactive

22 properties to adrenal P450scc [125]. However, evidence for any such mechanism is still outstanding.

Although the above findings of highest expression levels in white matter structures and in oligodendrocytes in culture of new-born rat brain cortex, but not in astrocytes, suggested the localisation of the enzyme expression to be mainly in the former in brain, subsequent investigations have demonstrated presence in cortical neurones, astrocytes, cerebellar granule neurones and Purkinje cells as well as in oligodendrocytes at the protein, mRNA and activity levels [125,165,193,217]. Neurosteroidogenic activity as shown by the formation of PREG was found in each cell type of the cortical cultures. However, these results carry with them the caution that cells in culture have possibly different biochemical profiles due to the lack of the appropriate humoral and extracellular signals.

P450SCC is expressed during embryogenesis in rodents, with higher mRNA levels found in the PNS than in CNS. Expression was found mainly in trigeminal and dorsal root ganglia, retina, neocortex, diencephalon, hippocampus and spinal cord using in situ hybridisation. Expression was detected from gestational day (GD) 9.5 onwards in rat. Analysis of cell types expressing P450scc using co-immunolabelling with cell specific markers showed sensory neurones in dorsal root ganglia to contain the protein [43].

Adrenodoxin is involved in electron transport to the cytochrome P 4 5 0 enzymes.

Adrenodoxin was detected throughout the brain by RT-PCR [125]. Immunohistochemistry with specific antibodies detected adrenodoxin in the same areas as P450SCC [102].

17a-hydroxylase/l7,20-lyase (P450c 17)

This enzyme converts PREG into DHEA or PROG into . The enzyme located in the endoplasmic reticulum carries out two successive steps, 17-hydroxylation precedes the side chain cleavage. Although both these activities are catalysed by the same enzyme, they are physiologically separately regulated. It is a major control point in steroidogenesis as its action determines whether mineralocorticosteroids, glucocorticosteroids or /oestrogens are eventually produced (see Figure 1-2). Thus, when 17-hydroxylation is predominant, the steroidogenic pathway goes towards the glucocorticosteroids. When 17-hydroxylase and 17,20-lyase are both active, the pathway leads to sex steroid formation. When the enzyme activity is not present.

23 mineralocorticosteroids are formed. The enzyme activities are regulated by presence of cofactors on the one hand and phosphorylation on the other hand, where the latter increases 17,20-lyase over 17-hydroxylase activity [129]. Expression of P450cl7 mRNA could initially not be detected in the rat brain [125], although DHEA and DHEAS were the first neurosteroids discovered (see 1.3.1.2). However, the enzyme has now been detected at the mRNA and protein level during embryogenesis in the rodent CNS and PNS. Its expression remains in the PNS into adulthood, but not in the CNS. In the embryonic CNS, specific expression was seen in neurites leading to the cerebral cortex and also other structures in hindbrain and mesencephalon [42]. The potential fiinctional significance of the regulated P450cl7 expression in the developing CNS was demonstrated by the findings of Compagnone and Mellon [44] on the specific neurotrophic actions of DHEA and DHEAS. These findings are fiarther described below in 1.3.3.

The above findings led to the hypothesis that although P450cl7 probably plays an important role in DHEA synthesis in the CNS during development, another pathway might be existing in adulthood. It could be considered that the PNS is the provider of locally synthesised DHEA for the CNS, as P450cl7 expression continues in PNS through to adult life. A further possibility is discussed below (1.3.1.5.2, p. 31).

Aromatase (P450aro)

The endoplasmic reticulum enzyme P450aro or aromatase is responsible for the conversion of androgens into oestrogens, e.g. TESTO to OESTR and androstenedione to oestrone. The enzyme carries out two hydroxylations at C-19 and one at C-2. The C- 19 methyl group is subsequently cleaved off and the A-ring aromatised [116]. This catalytic activity is important for the mediation of most androgen effects in the brain, as TESTO has to be converted to OESTR for its action in several areas. Sexual differentiation of the CNS is thought to be mediated mainly by this mechanism [110,189].

The aromatase activity is thought to be mainly located in preoptic, hypothalamic and limbic structures in most animals [10]. mRNA expression of the enzyme is first observed from GDI 6 in the CNS of the developing rat, in medial preoptic and sexually dimorphic nucleus of the preoptic area. The expression in these areas decreases after birth. Other areas with high mRNA expression throughout development and into adulthood are the

24 bed nucleus of stria terminalis and medial amygdala [99]. Virtually no aromatase was seen in rat cerebral cortex at the enzyme activity level [201]. Substantial enzyme activity was however measured in several cortical areas of the early postnatal female and male rhesus monkey brain [109]. In the rat brain overall, adult females have lower p450aro expression than males [201]. The cellular location of aromatase is probably mainly in neurones [104].

21-hydroxyIase (P450c21)

Adrenal P450c21 hydroxylates PROG and 17-OH-PROG to 11-deoxycorticosterone (DOC) and 11-deoxycortisol. The enzyme activity has been found in several extra­ adrenal tissues including the brain. However, mRNA expression could not be demonstrated and thus it is believed that the activity is mediated by a different enzyme in those tissues [60,126].

lip-hydroxylase (P450cllp)

This mitochondrial enzyme catalyses 11-hydroxylation and is the final step in CORT and formation. RT-PCR detected the enzyme expression at high levels in amygdala and cortex, and also in cerebellum and hippocampus in rat. Females had considerably higher levels in hippocampus than males. No P450cllp could be detected in mixed glial cell cultures or C6 glioma cells, indicating that the enzyme is expressed in neurones only [125]. Further, the enzyme was detected at the activity level in rat whole brain homogenates incubated with ^H-DOC [137].

Aldosterone synthase (P450cllAS)

The last step in synthesis is catalysed by P450cllAS. This step uses a single enzyme for 11-hydroxylation, 18-hydroxylation and 18-oxidoreduction located in the mitochondria to form aldosterone (ALDO). This enzyme is different to P450clip. Initially, RNAse protection assays and RT-PCR failed to detect P450cllAS in the rat brain [125]. Recently, however, one study identified mRNA of the enzyme in rat hypothalamus, hippocampus, amygdala, cerebrum and cerebellum by RT-PCR and Southern blotting. Furthermore, the enzyme was found biologically active, with minces of various brain regions showing ALDO production from endogenous precursors and

25 also synthesis of ^H-ALDO and the following tritiated intermediates CORT, 11- dehydrocorticosterone, 18-OH-DOC after incubation with ^H-DOC [64].

7a-hydroxyIase (P450c7b)

This microsomal enzyme is abundant in the liver where it is involved in metabolism [116]. The 7-hydroxylated DHEA metabolites have been described as beneficiary in counteracting effects on the immune system by high levels of [70]. In rat brain microsomes, DHEA and PREG are converted to their 7a-0H-derivatives [2]. An enzyme has been found in the brain, CYP7b, that shares 39% sequence homology with the liver enzyme. This enzyme is predominantly expressed in the brain in rat, mainly in hippocampus [186]. It has been found to be more specifically hydroxylating PREG and DHEA than the liver enzyme [163]. DHEA 7-hydroxylation was increased in astrocytic cultures under high cell density. This is a situation resembling inflammation and could indicate the possible functional significance of 7-hydroxylated metabolites [3].

3p,5a-diol-hydroxylase

The prostate enzyme 5a-androstane-3p,17p-diol hydroxylase was found throughout the rat CNS [204]. This protein is the most abundant P450-steroid hydroxylase in the brain by far as identified so far, however its cDNA has not been cloned to date. Its function is to hydroxylate 3p,5a-reduced steroids such as 5a-androstane-3P,17P-diol and 3p,5a- THPROG at positions C-6 and C-7. It is evenly distributed throughout the brain and its activity not dependent on adrenals or gonads.

The hydroxylation of 3p,5a-reduced steroids could be an important pathway of steroid catabolism. Hydroxylation by 3p,5a-diol-hydroxylase at C-6 or C-7 renders the steroids more polar ready for excretion. Thus the 3p-reduction of 5a-DHPR0G could act to reduce levels of 3a,5a-THPROG [188].

26 L 3.1.5.2 Non cytochrome P450 enzymes

Sp-hydroxysteroid oxidoreductase (SP-HSD)

This microsomal enzyme converts 3p-hydroxy-A5 steroids to 3-keto-A4-steroids, thus having two separate catalytic activities: a 3-dehydrogenase and a A5-A4 isomerase. The enzyme is important in all steroidogenic pathways, converting PREG, 17-OH-PREG and DHEA into PROG, 17-OH-PROG and androstenedione, respectively. In situ hybridisation studies revealed the expression of 3p-HSD mRNA in neurones in many areas of the rat brain, including olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, hippocampus and cerebellum, in which levels were highest. The protein also could be detected by immunocytochemistry with specific antibodies [68]. Another study found 3p-HSD mRNA co-expressed with P450scc mRNA in cortex, cerebellum and spinal cord [165]. The mRNA levels in rat brain were again very low compared to adrenals and gonads (see section on P450scc). Subsequently, Schwann glial cells of dorsal root ganglia (DRG) were shown to express 3p-HSD by in situ hybridisation [93]. The enzyme is active as these cells produce PROG from PREG in cultures [69]. The possible significance of the expression of this enzyme in peripheral nervous system glia is in regulation of myelination (see 1.3.3).

5a-reductase

5a-reductase converts a variety of steroidal substrates with the 3-keto-A4-conformation to 3-keto, 5a-dihydrosteroids [34]. Known natural steroidal substrates are TESTO, PROG, 20a-DHPROG, DOC, CORT and cortisol. The enzymatic reaction is considered to proceed only in the reductive direction and thought to be irreversible, using NADPH as cofactor. This enzyme activity is responsible for converting some steroids into active metabolites in both peripheral endocrine structures and the nervous system. For example, TESTO is converted into 5a- (5a-DHT) in target tissues, which binds to the androgen receptor (AR) with much higher affinity than its precursor and is mainly responsible for induction of androgenic effects in the periphery. On the other hand, PROG and DOC are converted into 5a-DHPR0G and 5a-DHD0C, which are substrates for the enzyme 3a-H0R. The products of this reaction are 3a,5a-THPROG

and 3a,5a-THDOC, both potent GABA a -R enhancers (see 1.3.2.1). There are also

27 indications that 5a-reductase serves a catabolic function. This could be the case with 5a- reduction of TESTO to remove substrate from aromatase enzyme in order to prevent more oestrogen formation in certain areas [136]. Support for this notion also comes from studies of 5a-reductase type I knockout mice. The foetuses have only 50% viability, which was attributed to the effects of excess oestrogens [112]. Two isoforms of the enzyme in rat have been demonstrated I [100] and II [136], with low and high KmS for PROG. Both isoforms have higher affinity for PROG as substrate than for TESTO, CORT and cortisol. 20a-DHPROG was found to be more reactive than PROG and much more than TESTO when production rates of 5a-reduced metabolites were compared in incubations of rat medial basal hypothalamus homogenates [39]. The enzyme expression was detected in several CNS regions at protein, mRNA and enzyme activity levels in rats [71,100,123,144,161]. The expression is highly regulated in the CNS, both developmentally and regionally.

Type I isozyme mRNA expression in CNS is high from fetal stages through to adulthood, whereas the type II enzyme is only transiently highly expressed during late fetal and early postnatal life [146]. In later postnatal stages and adulthood, expression of the type I isozyme is widespread throughout the CNS, with highest expression in white matter structures [100]. Type II isoform is highest on postnatal day 2 and then decreases to overall low levels in whole brain in adulthood. Nevertheless, selective high expression in hypothalamus was seen in adults brains. It is also expressed after stress in the hippocampus [146]. Enzyme activity measurements were in line with these findings, with higher activities in the subcortical white matter than in cerebral cortex and hypothalamus in male and dioestrous female rats and mice [123]. Subsequent studies also found high levels of 5a-reductase activity in the myelin rich structures of the peripheral nervous system, such as the sciatic nerve [122].

The differential expression of the isozymes in different regions and stages during development suggests an involvement in differentiation of the CNS. It is not clear, however, how this is mediated. The early ontogeny of 5a-reductase type I in proliferating neuroepithelia of cerebral cortex, basal telencephalon and spinal cord could indicate a role in guidance and/or development of cells in those regions. It is not known what are the functions of the expression of type I isozyme in more differentiated regions in the early postnatal period as compared to the transient expression of type II in late fetal and early postnatal life in several regions of the CNS. A possible implication of

28 these findings could be for 5a-reduced progestins being involved in fetal brain development. With the reduced progestin 3a,5a-THPROG being a modulator of

GABA a -R function, it could influence trophic actions of GABA in early brain development [167].

The significance of high levels of 5a-reductase enzyme expression and activity in white matter structures and the demonstration of the enzyme in myelin sheaths is not yet known. Implications for myelination were suggested [145]. However the time course of the enzyme expression in developing CNS does not coincide with the time course of myelination [1 0 0 ] and thus a role in myelination is unlikely. Alternatively, as this enzyme is often closely associated with 3a-hydroxysteroid oxidoreductase (3a-H0R), the high expression in myelin could mean that these structures are part of the neuroactive producing structures of the nervous system. This is further supported by the fact that P450SCC is also found highly expressed in white matter, producing the steroid precursor PREG (see above).

Another implication for 5a-reductase type II activity could be the modulation of stress responses in the CNS. This is supported by the finding that the enzyme expression is induced after stress in hippocampus [146], a structure where stress induced in excess can cause neuronal death. Formation of the reduced 3a,5a- THPROG could lead to a modulation of the stress response [153].

5p-reductase

3a,5P-reduced are equally potent as the 3a,5a-reduced compounds as

GABA a -R modulators in some systems investigated [143]. 5p-reduction is however considered to be mainly a hepatic pathway for elimination of steroids. The enzyme activity has not been detected in the rodent or primate CNS.

3a-hydroxysteroid oxidoreductase (3a-HOR)

Mammalian 3a-HORs belong to the aldoketo reductase family of enzymes [142]. In the nervous system this enzyme is involved in the conversion of 5a-reduced steroids to the potent neuroactive 3a,5a-reduced compounds. It is thus an important regulator of steroid activity (see in description of 5a-reductase). The enzyme expression was found in several areas co-located with 5a-reductase (cerebral cortex, hippocampus.

29 hypothalamus, thalamus, cerebellum) at protein and mRNA levels in rat. However, the highest protein levels were found in the olfactory bulb. There are two different 3a-H0R activities in brain with different substrate affinities, subcellular localisation and co-factor requirements [29,95,96], The hypothalamic enzymes are both NADPH- and NADH- dependent. The cytosolic isoform requires NADPH and has lower Km for 5a-DHPR0G than 3a,5a-THPROG. A plasma membrane associated isoform requires NADH and has a lower Km for 3a,5a-THPROG than 5a-DHPR0G. This suggests that the two activities operate in opposite directions in vivo.

20a-hydroxysteroid oxidoreductase (20a-HOR)

PREG and PROG are converted into their 20a-reduced metabolites by this enzyme. The enzyme from rodents belongs to the aldoketo reductase family, the human isoform is a member of the short-chain dehydrogenase family. The enzyme mRNA was detected in human brain [214]. Enzyme activity was observed by formation of 20a- dihydropregnenolone (20a-DHPREG) in primary glial cell cultures of rat brain cortex after incubation with ^H- mevalonolactone as precursor [79,84]. In the mouse brain, 20- reduction was the main activity found upon incubation with PROG, whereas the main product in the rat brain incubation was 5a-DHPR0G [31].

20p-hydroxysteroid oxidoreductase (20P~HOR)

The enzyme for 20p-reduction of PREG and PROG has so far only been identified in brain of mouse and neonatal pig [80,92]. The mouse enzyme was detected at mRNA and protein, the pig enzyme at protein level.

17P-hydroxysteroid oxidoreductase (17P-HOR)

17P-H0R is an enzyme catalysing a step in the formation of androgens, converting e.g. androstenedione to TESTO. Its expression was investigated by RT-PCR in brain cell cultures from rat cortex [217]. Only astrocytes, not oligodendrocytes nor neurones were found to express the mRNA for the enzyme. Enzyme activity was demonstrated in hypothalamus, cerebellum and cerebral cortex of the rat [161].

30 llp-hydroxysteroid oxidoreductase (lip-HOR)

In ALDO target tissues this microsomal enzyme converts CORT or cortisol to 11 - dehydrocorticosterone or 11 -dehydrocortisol, respectively, which are inactive at the mineralocorticoid receptor (MR) [131]. Thus this enzyme prevents activation of the MR by these glucocorticoids. In situ hybridisations showed lip-HO R mRNA expression to be high in cerebral cortex and hippocampus, and lower expression in hypothalamus. MRs are also mainly expressed in those regions. Enzyme activity, measured by conversion of ^H-CORT to ^H-11-dehydrocorticosterone was also demonstrated in these areas as well as brain stem and spinal cord [131].

Novel pathways of steroid formation

Research by Lieberman and co-workers [147] suggested a pathway for the formation of PREG and DHEA different to the classical one mediated by P450scc and P450cl7. Such a pathway could provide an explanation for the presence of DHEA in adult brain despite the lack of P450cl7 expression (see Section 1.3.1.5.1). Extracts of brain in organic solvent treated with organic base, HCl and Fe^^ yield higher amounts of PREG than without treatment. Increased yield after treatment with Fe^^ suggests that these extracts contain oxidised species as steroid precursors. PREG is then formed from those not by the known steroidogenic enzymes, but by other unknown enzymes or chemical transformations. DHEA could be formed by a similar mechanism. Potential precursors for PREG could be a steroid 20-hydroperoxide or 20,22-cycloperoxide, for DHEA 17- hydroperoxide or 17,20-cycloperoxide. By contrast extracts of adrenals and testes, tissues which do express P450cl7, do not yield higher amounts of PREG and DHEA upon treatment with the above mentioned reagents [147]. Furthermore, rat C6 glioma cells produced higher amounts of PREG and DHEA in the presence of FeS 0 4 than without. P450cl7 was not detected in the same cells [33]. The synthesis could be mediated by Fe^^, although the concentration employed in the above studies (10 mM) is generally too high for physiological conditions. It could be that cells locally accumulate such high concentrations, however. Another possible factor which could be relevant in this pathway is the presence of reactive oxygen species found in oxidative stress or P- amyloid, a peptide that occurs in plaques in tissues of subjects with Alzheimer’s disease (also increasing reactive oxygen species) [26].

31 Hydroxysteroid sulphotransferase

Hydroxysteroid sulphotransferases (HST) transfer inorganic sulphates to steroids with a A-5, 3- or 17- hydroxy- configuration. Whereas this enzyme action is considered a major catabolic pathway in the periphery, sulphation of steroids leads to a change of their pharmacological properties in the NS (see 1.3.2). Little information has been obtained about the presence of the enzyme in the nervous system. For a long time attempts at identification were negative. Recently low HST activity was detected in the rat brain [156]. The activity was found to be highest in the fetal stages, starting to decline after birth. It is not known whether this low enzyme activity is responsible for the levels of PREGS and DHEAS in the adult rat brain previously determined (see 5.1). This is counter-indicated by experiments where supply of DHEA to the brain after peripheral injection failed to increase DHEAS levels [12].

Steroid sulphatase

Steroid sulphatase (STS) hydrolyses sulphate esters of A-5, 3-hydroxysteroids. Its expression (mRNA) was found in murine brain in the cortex, hippocampus and thalamus and in the PNS. The expression was observed during late embryogenesis until 9 days after birth, then declined to low levels in adulthood [45,132].

Acyl transferase

Steroids are conjugated with fatty acids by this enzyme, of which the activity has been demonstrated in rat brain [200]. The enzyme has 3P-hydroxy-A5-steroids as preferred substrates, but also esterifies 17p-hydroxysteroids (e.g. TESTO), and is distinct to CHOL acyl transferase. The activity is located mainly in the microsomal fi*action. Several endogenous fatty acids are being conjugated to ^H-PREG after addition to rat brain microsomal incubations, with the main esters being palmitate, oleate, linoleate and stearate.

Steroid glucuronosyl transferase

Steroids are conjugated to glucuronic acid mainly in the liver for excretion. The enzyme mRNA has also been demonstrated in brain of monkeys [13]. Expression of the cloned cDNA revealed glucuronylisation of steroid 3a-hydroxyl and 17p-hydroxyl groups.

32 TESTO, and 5p-androstane-3a,17p-diol were substrates for this enzyme.

1.3.2 Modes of action of steroids in the nervous system

The nervous system contains intracellular receptors for all classes of steroid hormones, glucocorticoids, , androgens, oestrogens and progesterone with varying prevalence in different regions. The transcription regulating properties of steroids are well characterised (see e.g. [86,111]), thus only their non-genomic effects are discussed here.

1.3.2.1 GABAA-receptor

The rapid effects of the ring A reduced pregnane steroids on neuronal excitation were first observed through the increase in the time span of inhibitory post synaptic conductances [166]. This effect was seen of the anaesthetic alphaxalone, a synthetic progestin analogue of 3a,5a-THPROG (3a-hydroxy-5a-pregnane-l 1,20-dione), in slices of olfactory cortex of guinea pig. This steroid was found to selectively potentiate y-amino butyric acid (GABA) and evoked responses in rat cuneate slices [74], thus indicating that the basis of the increase of inhibition in neural tissue by the steroid is

due to modulation of the GABA type A-receptor/ion channel complex (GABA a -R). The muscimol evoked response potentiation by alphaxalone was dose dependent and occurred at lower concentrations than the effect observed with pentobarbitone. The naturally occurring steroids 3a,5a-THPROG and 3a,5a-THDOC were also shown to

modulate the GABA a -R in this fast manner [114]. Potentiation of ^H- binding, inhibition of binding of the convulsant ^^S-/-butylbicyclophosphorothionate (TBPS) and stimulation of uptake were all observed upon application of the steroids

in a dose dependent manner to synaptosomal fractions. Activation of the GABA a -R in cultured neurones was also found to be positively modulated as measured by action

potential inhibition, current potentiation and increase in decay time of GABA a-R mediated current.

The GABA a -R is the most widespread inhibitory neurotransmitter receptor in the CNS, with its activation leading to inhibition of the cell, manifested in the case of most CNS

33 neurones by a hyperpolarisation of the somatic membrane. The receptor consists usually of five subunits with a multitude of possible subunits [178], mainly of the a-, P-, y-, 5-,

8- or 0- type. Of the former three, several sub-types exist. The variation of GABA a -R subunit composition results in different electrophysiological properties and thus by differential expression within the brain, brain regions and even cells probably different functions are served.

The site of the steroid effects and their mechanism on the receptor are not clear yet. Experiments on recombinant receptors showed that 3a,5a-THPROG had similar effects

on alply2L and alp2y2L GABAa-R [113], thus suggesting the identity of the p- subunit has little consequence for potentiation by steroids. The y- subunit does not seem to be essential for steroid binding. However, a higher efficacy is displayed by 3a,5a- THPROG, but its effect is of lower potency in receptors without the y- subunit. Potentiation of flunitrazepam binding by 3a,5a-THPROG was more effective on recombinant receptors of a3piy2 than of a lp ly 2 or a2piy2 composition [97]. Receptors containing ô-subunits are insensitive to potentiation of GABA-gated currents by 3a,5a-THDOC, but not the direct effect on the current by the steroid [216]. The s- or 0-subunits do not seem to influence receptor sensitivity to potentiation by steroids [16]. Thus it seems that a, P and y subunits are important in mediating the potentiating effects of the steroids and there are different sites on GABAa-Rs influenced by the direct steroid and the agonist potentiating effects. The differential sensitivity of different GABAa-R subtypes to steroids thus possibly reflects the existence of different functions of the steroid effects in different locations. GABAa-R with changing steroid sensitivity have recently been described in rat hypothalamic synaptic neurotransmission (see 1.3.3, p. 42).

There is also evidence on dependence of the GABA a-R properties on membrane CHOL. Enrichment of synaptosomal membranes from cerebral cortex with CHOL led to a decreased sensitivity of ^H-flunitrazepam binding to 3a,5P-THPROG, and to an increased sensitivity for membranes from spinal cord [17]. Enrichment of hippocampal neurone membranes with CHOL decreased the potentiation of GABA induced currents by 3a,5a-THPROG and 3a,5p-THPROG [183]. Thus it is possible that CHOL

influences the GABA a -R function either by direct, low affinity binding to a steroid

34 recognition site or by changing the conformation of the receptor via an effect on membrane fluidity.

Investigation of the effects of the ring A reduced pregnane steroids on the GABA a-R showed that they increase the duration and the amplitude of the current evoked by GABA, thus resulting overall in an increased transfer of charge and subsequently higher strength of inhibition. The steroids were effective at an estimated concentration near the active sites of as low as 10 nM. At higher concentrations (1 piM), direct gating of the ion channel by 3a ,5-reduced pregnane steroids was observed [114,143], The modes of action of steroids are actually quite similar to those of , although with higher (100-1000 fold) potency. To see whether these steroids were actually active in synaptic neurotransmission, 3a,5a-THPROG was applied to preparations of hippocampal slices. The steroid prolonged inhibitory postsynaptic currents (IPSCs) evoked by presynaptic action potentials [73].

Closer investigations of the steroid effects on the GABA a -R in nucleated cerebellar granule cell patches looked at the modulation of GABA evoked currents in the presence of 3a,5a-THDOC. This showed that the steroid enhances the GABA induced current by interacting with the desensitisation of the receptor channel [215]. The GABA evoked current behaves in a biphasic manner after a fast rise, with first a fast and then a slow component of exponential decay. These characteristics of the current were found very similar to those seen in IPSCs. Application of 3a,5a-THDOC after a short, saturating pulse of GABA increases the time constant of the slow decay, but not the fast phase.

Desensitisation of the GABA a -R in these preparations is thought to be due to interconverting channel conformations after GABA binding. First, deactivation occurs followed by a reopening of unbound receptor. 3a,5a-THDOC is possibly interacting with the channel in the latter state. 3a,5a-THDOC had no effect, when GABA was applied for a long time, but an increase of the decay time due to GABA itself was seen. It was further found that the steroid largely increases the time needed for recovery of the current amplitude after desensitisation by GABA, thus actually decreasing efficacy of GABAergic transmission under high frequency stimulation. The significance of these effects is not clear yet and further investigations of the transmission conditions in functioning synaptic pathways are needed [215].

35 The above-described GABA a -R function potentiating steroids all had in common the structure of the steroid A-ring. Further studies gave more detailed insight into structural requirements. In a system using potentiation of muscimol stimulated ^^Cf-uptake in rat cerebral cortical synaptoneurosomes to test the potency of compounds, 5a-pregnan-3a- ol-20-one (3a,5a-THPROG), 5a-pregnan-3a,21-diol-20-one (3a,5a-THDOC) and 4- pregnen-3a-ol-20-one (3a-DHPR0G) were similarly effective [73]. On the other hand, 4-pregnen-3 p-ol-20-one (3P-DHPR0G) was ineffective. Compounds with 3 a - hydroxy,5a,17-one configuration also were potent stimulators. 5p-pregnan-3a,21-diol- 20-one (3a,5p-THDOC) was less potent than the 3a,5a-. Acetate conjugates of the 3a-hydroxyl-compounds, oximes or the oxidised 5a-pregnan-3,20-dione (5a- DHPROG), 5a-pregnan-21 -ol-3,20-dione (5a-DHD0C) and 4-pregnen-3,20-dione (PROG) were inactive, as were 20-conjugated compounds. Thus it seems that a 3a- hydroxyl-group is a requirement for activity, but the C-5 atom can be reduced as well as oxidised. The side chain at C-17 is not necessary, however a ketone at either C-17 or C- 20 seems to be important. Several compounds related to active compounds with a double bond between C-9 and C-11 or a hydroxyl-group at C-11 were inactive, indicating structural requirements in the C-ring. Interestingly, 3a-hydroxy steroids conjugated with a sulphate group (3a ,5a-THPROGS, 3a,5p-THPROGS and 5a-androstan-3a-ol-17-one sulphate ( S) also stimulated muscimol induced -uptake in synaptoneurosomes, albeit at somewhat lower potency than observed for 3a,5a- THPROG (1-10 p.M) [55]. This is contrary to the effects of the sulphate esters of 3p- hydroxy steroids (see below). Similar findings were obtained by measuring ^^S-TBPS displacement of steroids and potentiation of GABA-induced currents in hippocampal neurones [152]. In addition 5a-pregnan-3a-ol-l 1,20-dione (alphaxalone), 5p-pregnan- 3a-ol-l 1,20-dione, 3a,5p-THPROG, 3p,5a-THPROG and 3P,5P-THPROG were investigated in this assay. Alphaxalone and 3a,5p-THPROG were found equally potent as 3a,5a-THPROG, which is consistent with earlier findings of alphaxalone as a potent enhancer of GABAa-R function [74]. The 5P-isomer of alphaxalone, on the contrary, was much less potent. The 3p~compounds were inactive as were 5a-pregnane-3a,20a- diol, 5a-pregnane-3a,20p-diol, 5P-pregnane-3a,20a-diol and 5p-pregnane-3a,20p-diol. However, in a recent study on pharmacological interactions at a human recombinant GABAa-R expressed in Xenopus oocytes, the latter compounds were shown to be of limited, but significant efficacy in enhancing GABA evoked currents [15]. 5a-pregnane-

36 3a,20a-diol had similar potency as 3a,5a-THPROG, with an effective range of 10 nM-

3|liM. The maximum increase of the GABA-induced current was 40% compared to 69% for 3a,5a-THPROG. The other - were less potent, and the 5p- reduced compounds had lower efficacy. This would be consistent with action as partial

agonists of all these compounds at the 3a,5a-THPROG site on the GABA a -R.

Other steroids were found to negatively modulate GABA a -R function. The sulphate ester of 5-pregnen-3 p-ol-20-one (PREGS) in analyses of whole cell [115] and patch recordings [127] from rat cortical neurones showed antagonism of GABA mediated currents. The effect was non-competitive, similar to . Analysis of single channel recordings showed that PREGS acted by reduction of channel opening frequency [127]. Other steroids with sulphate esters on a 3 p-hydroxyl-function also inhibit

GABA a-R currents. Thus, the sulphate esters of 5-androsten-3 p-ol-17-one (DHEA), 5- androstene-3P,17p-diol () and 3p,5a-THPROG all showed this effect [55]. Therefore sulphate conjugated steroids exhibit both positive and negative

modulation of GABA a -R function. The potency of these steroids in antagonising

GABA a -R function is somewhat lower than of the most potent steroids in enhancing

GABA a -R, in the 1-100 pM range [55,127,210].

Steroids with a 3p-hydroxyl group were found to be mainly inactive in the above

described studies on the GABA a-R. Antagonistic effects of these steroids were however discovered on the positive modulation of the receptor by their 3a-isomers [148,150]. When 3a,5a-THPROG and 3a,5P-THPROG potentiation of ^H-flunitrazepam binding to rat brain membranes was measured in presence of 3p,5a-THPROG or 3P,5P- THPROG a significant shift of the dose-response curve to lower potencies was observed. The maximum enhancement of binding did not change significantly. This would indicate that these compounds are competitive antagonists.

1.3.2.2 N-methyl-D-aspartate receptor

The N-methyl-D-aspartate type excitatory glutamate receptor (NMDA-R) is important in excitatory CNS neurotransmission and associated with synaptic events occurring in long­ term potentiation and involved in excitotoxicity leading to neural damage after conditions such as stroke. The receptor is linked to an ion channel permeable to Ca^^, Na^ and

37 in the open state [210]. Several steroids modulate this receptor. PREGS potentiates the NMD A evoked current in concentrations between 10-100 |iM. PREGS also slightly inhibits kainate and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) types

of glutamate receptor responses. At 10 |liM, PREGS leads to 25% and 100 pM PREGS to 200% potentiation of the NMD A induced-current [210]. Single channel studies showed the steroid increased channel open frequency and channel open time, but not single channel conductance. Other steroids, 3a,5a-THPROGS, 3a,5p-THPROGS, 3p,5p-THPROGS and OESTR attenuate the response of the NMDA-R, whereas DHEAS has no effect [12]. Necessary structural elements for an NMDA-enhancing effect were found to be the side chain at C-17 and a double bond between C-5 and C-6. The necessary concentration for modulating function is, as for the above antagonistic

effects of steroids at the GABA a -R, higher than the positive GABA a -R modulatory effects of steroids (10-100 pM). DHEA was found to acutely enhance NMDA-R function in rat hippocampal neurones [18]. This effect was observed at low doses, however was indirect and mediated by the a-receptor.

1.3.2.3 Voltage-gated calcium channels

Voltage gated Ca^^-channels (VGCC) are universally important in the nervous system, for instance, in neurotransmitter release. There are several types of VGCC differing in electrophysiological and pharmacological properties. Steroid inhibition of these ion channels is seen partially at very low concentrations (from 10 nM). Active steroids are PREG, PREGS, DHEAS, 3a,5a-THDOC as well as some synthetic steroids. They only inhibit a percentage of the voltage gated current, but at higher concentrations strong inhibitions are observed, e.g. DHEAS inhibits 80% of the current at 100 pM in CAl hippocampal neurone whole cell recordings. The steroid effects were found to be G- protein dependent. Further, protein kinase C signalling is involved [57,58].

1.3.2.4 Other steroid effects on neuronal membrane receptors

Other known steroid effects on neuronal membrane receptors are the negative modulation of PROG on nicotinic acetylcholine (nAch) R [12,90], positive modulation of the a-R by DHEA and DHEAS [12,18], of the voltage gated K^-channel (VGKC) by

38 PREGS [203] and positive modulation of the glycine-R by 20a-dihydrocortisol, a- cortol and hydrocortisone [149].

1.3.3 Physiological roles of steroids in the nervous system

Steroids play a multitude of important roles in the nervous system. The wide range of modes of action and multitude of steroids possibly formed within the nervous system as described above suggests that there are wider implications for steroids than previously thought.

Neurosteroids are possibly autocrine/paracrine regulators of signalling in the nervous system, as opposed to and in addition to their endocrine actions. Oligodendrocytes are the main site of PREG formation in the CNS and contain several steroid metabolic enzymes (see 1.3.1.5). Neurones and astrocytes also contain several steroid forming enzymes. In 1.3.2 it was described that steroids are modulators of several kinds of mechanisms of neurotransmission. Oligodendrocytes (and Schwann cells in the PNS) and astrocytes are in close anatomic proximity to neuronal cell bodies, axons and synaptic structures in the nervous system. Thus steroids formed in oligodendrocytes, Schwann cells or astrocytes by diffusion to neighbouring neurones can exert influences on the neuronal signals, or act in an autocrine function after further metabolism by the neurones. Furthermore, it has been shown that some glial cells in certain developmental stages contain neurotransmitter receptors and voltage gated ion channels, including the

GABA a -R [199]. Thus steroids produced locally could also influence neuronal signals received by glial cells.

Until very recently, it was assumed that oestrogens produced in the nervous system through aromatase action mainly had effects mediated via the classical oestrogen receptor a (ER a). However, lack of co-localisation of this receptor and aromatase activity in certain brain areas suggests other mechanisms are in place in these areas. The discovery of the subcellular location of aromatase immunoreactive material in axon terminals indicates paracrine action via other ways than the ER might be existing [10], such as modulation of neurotransmitter receptors and/or ion channels. The localisation of the newly discovered oestrogen receptor p (ER p) has not yet been fully elucidated. This could also be a target of locally produced oestrogen.

39 However, as most steroids are more or less freely diffusible through cell membranes, the signal would spread probably quickly through neighbouring regions. There are enzymes for inactivation of neuroactive steroids (see 1.3.1.5). Thus it is possible that production rates of the metabolites and regulation of their inactivation enzymes determine how the signal spreads.

Physiological responses probably depend on endocrine actions of steroids entering from peripheral sources and local actions upon confined cellular synthesis both on neurotransmission in a fast manner and on transcription in a slower fashion in varying degrees of interaction.

Sexual differentiation

One of the earliest functions discovered of steroids in the nervous system was their influence on its differentiation. This is derived from several studies. Several CNS functions in mammals, including reproductive behaviour, regulation of gonadotropin release and non-reproductive behaviours such as aggression are sexually dimorphic. They have been found to depend on exposure to gonadal hormones in early life in studies in many mammals. Exposure of rats to TESTO during gestation and/or early postnatal life leads to a decrease in lordosis and increase in mounting behaviour in adulthood. There are morphological differences in the CNS between sexes, the most prominent of which is the size and the cell number of the sexually dimorphic nucleus of the hypothalamic preoptic area (SDN-POA). This area is generally larger in size and cell number in males. Other regions are larger in the female, e.g. areas of the bed nucleus of the stria terminalis. Gonadal hormones seem to induce these differences. Gonadectomy in male rats in early postnatal life changes the morphology to a female pattern. On the contrary, exposure of females to TESTO early on induces male patterns.

Sex differentiation affects the nervous system at several levels. Besides the size of certain nuclei, differences are also seen in the synaptic organisation and cellular/synaptic organelles. The effects of TESTO are presumed to be mediated by aromatisation to OESTR, as they can be mimicked by exposure of appropriate doses of this steroid. Also, the TESTO effects on female brain are not observed upon administration of 5a-DHT. Furthermore, aromatase inhibitor treatment of male rats results in a decrease in size of SDN-POA and a change in their sexual preferences. The sex differences are considered

40 to be induced mainly via genomic effects of steroids. However, recently experiments indicated possible non-genomic influences of oestrogens might play part as well. Furthermore, it cannot be excluded that ring A- reduced pregnanes are involved in differential sexual organisation of the brain. GABÂA-R-activation in early postnatal stages prevents masculinisation of the SDN-POA. GABA a -R action is often depolarising in early life stages [167,189].

Neurone development

Steroids are probably more generally involved in the development of the CNS. Other potential steroidal effects on CNS development are those of DHEA and DHEAS. Studies on neurone groAvth and differentiation in selected CNS areas showed trophic actions of DHEA and DHEAS [44]. Their effects have been shown in areas where P450cl7 is expressed in the embryonic brain. Neurite outgrowth in mouse embryo neocortical neurones in culture was found increased, when grown in the presence of DHEA and DHEAS. The neurites with increased growth in the presence of DHEA contained specific markers of axons, those with DHEAS of dendrites. Hydroxysteroid sulphotransferase (HST) and steroid sulphatase (STS) are also expressed in CNS of rodents, in an increased manner in early developmental stages. STS is expressed in the thalamus, a region where axon projections to the neocortex are initiated. The differential action of those enzymes could be a regulatory mechanism for the DHEAS/DHEA ratio and subsequently influence the axon/dendrite development and shape neuronal pathways. A possible mechanism for these effects was also shown by the finding that DHEA increases intracellular Ca^^ in neocortical neurones. This action involved the NMDA-R. DHEAS did not have this effect [44].

It is conceivable that other neurosteroids influence the development of the nervous system, as has been hypothesised for ring A-reduced pregnanes above. Their actions could be not restricted to sexual differentiation, as well as other steroids could be active. Neurosteroids and peripherally derived steroids may work in complex interactions to influence the wiring of the developing CNS and PNS.

Myelination

PROG promotes myelination of peripheral nerves. Peripheral nerves of the rat have high production rates of PROG that are persistent long after removal of peripheral endocrine

41 glands [93]. They also have been shown to remyelinate after degeneration of the myelin sheath following axonal lesion. The remyelination was reported to be inhibited when incubation was done in presence of trilostane, an inhibitor of 3|3-HSD activity, the enzyme converting PREG to PROG [93]. This could signify a possible signalling role of PROG in plasticity of myelin structures. The steroid involvement in this process is most likely compartmentalised due to a signalling cascade. As glial cells are thought to be the main locations of P450scc activity (see 1.3.1.5.1), PREG possibly diffuses into neighbouring axons after synthesis in the surrounding Schwann cells. After transformation into PROG in the neurones, it could then be supplied back to the Schwann cells. There it could serve as signal for remyelination, which is supported by the above described experiments. Further support to the myelination promoting role of PROG comes from the finding that its addition to dorsal root ganglion cultures increased myelination [93].

Regulation of synaptic plasticity

PROG and 3a,5a-THPROG are involved in regulating synaptic plasticity and long term potentiation of GABAergic neurotransmission. The subunit composition of the GABAa- R in hypothalamic oxytocin neurones changes during late pregnancy in rat, with a l subunit levels decreasing and levels of a2 subunits remaining constant at the same time. This subunit switch renders the receptors less sensitive to the neurosteroid 3a,5a- THPROG. The physiological implication of this finding is that GABAergic neurotransmission inhibits the oxytocinergic neurones during pregnancy. When PROG and consequently 3a,5a-THPROG are reduced towards the time of parturition this inhibition is lifted and allows the cells to fire and release oxytocin. This hormone is important in enabling delivery and lactation [27]. The structure change of the GABAa-R is thoughtto be mediated by PROG [56].

Involvement of 3a,5a-THPROG in GABA a-R plasticity was also shown in another context. A fall in 3a,5a-THPROG concentration similar to those observed in pregnancy

in a rat model was found to reduce the GABA a -R mediated current in hippocampal CAl neurones [182]. It was found that this effect depended on gene transcription, altering the

subunit composition of the GABA a -R. Thus it could be seen that 3a,5a-THPROG

suppressed expression of the a4-subunit of the GABA a -R. After the decrease in 3a,5a- THPROG, a4-subunit expression increased, leading to a decreased sensitivity to

42 neuroactive steroids and other GABA a -R modulatory drugs, accompanied by an apparent increased anxiety. Suppression of a4-subunit expression prevented such withdrawal effects [181].

In the context of the above findings it is interesting that brain PROG and 3a,5a- THPROG content undergoes large diurnal variations in the female mouse (larger than plasma PROG over the oestrus cycle) ([47], see 1.3.1.3). However, it is possible that during pregnancy these levels are “overridden” by PROG entering from plasma, which is at high concentration during pregnancy. It is not known whether the diurnal variations in

non-pregnant mice are sufficient to affect GABA a -R plasticity. It would be interesting to see whether the plasticity of GABAergic synapses mediated by PROG and its metabolites is a more widely distributed mechanism in the CNS.

1.3.4 Brain steroids in disease mechanisms

Aberrations in regulation of steroid concentrations can, after consideration of the possibly multiple influences on nervous system functions, be expected to result in more or less severe disorders. Several clinical studies have shown the possible involvement of abnormal steroid levels in diseases.

Mood

Steroids are likely to be involved in depression/anxiety disorders. Several studies have shown the anxiolytic, sedative and hypnotic properties of 3a,5a-THPROG and 3a,5a- THDOC in animal experiments [6,21,52,158]. Preliminary clinical studies showed that levels of 3a,5a-THPROG and 3a,5P-THPROG were significantly lower in depressed patients compared to control subjects in cerebrospinal fluid [195] and plasma [160]. The selective serotonin reuptake inhibitors (SSRI) used to treat depression, and fluvoxamine specifically increased 3a,5a-THPROG and 3a,5p-THPROG levels in those studies. Concentrations of other steroids, PREG and PROG were not affected through the treatment. Change in the 3a,5a/p-THPROG CSF levels was correlated with improvement of depression symptoms. It was previously shown that fluoxetine increases the brain content of 3a,5a-THPROG at similar rates in adrenalectomised/orchidectomised and sham operated rats [194].The possible underlying

43 mechanism has been recently shown to be that fluoxetine directly increases activity of 3a-H0R. 5a-reductase activity is not affected [65].

Pre menstrual syndrome

The depression like symptoms frequently experienced in women in the later stages of the luteal phase of the menstrual cycle (pre-menstrual syndrome, PMS) also possibly involve changes in 3a,5a-THPROG levels. Evidence for this comes from studies showing serum 3a,5a-THPROG concentrations to be significantly lower in the later stages of the menstrual cycle in subjects with PMS compared to control subjects [157]. The ratio of 3a,5a-THPROG to PROG was also lower, whereas PROG levels were not different.

The modulatory effects of 3a,5a-THPROG on GABA a-R subunit composition and responses described earlier could be a mechanism underlying post partum depression and PMS.

Stress

Steroids are possibly involved in stress induced homeostatic processes mediated through the brain. 3a,5a-THPROG and 3a,5a-THDOC are increased in the brain in stressed rats with a short delay after the stressful event (see 1.3.1.3). Activity of 5a-reductase is upregulated in stress in the hippocampus (1.3.1.5.2). The stress hormones, the glucocorticoids can increase levels of excitatory neurotransmitters and potentiate their effects [198]. In excess, this can eventually lead to neurotoxicity. A brain region particularly vulnerable to effects of excess glucocorticoids is the hippocampus. A way by which 3a,5a-THPROG and/or 3a,5a-THDOC could be involved in the regulation of

stress is by the increase in the inhibitory tone via potentiation of the GABA a -R. By increasing overall inhibition in cells, the increase in 3a,5a-THPROG and/or 3a,5a- THDOC could reduce the excitatory neurotransmitter effects. Furthermore, these steroids are possibly factors involved in the negative feedback control of glucocorticoids, as 3a,5a-THPROG has been shown to reduce corticotropin-releasing hormone (CRH) mRNA expression in rats upon adrenalectomy and CRH induced anxiety significantly [141].

Cognition

44 Glucocorticosteroids have complex actions on behaviour, cognition and emotional states [75]. Glucocorticosteroids in the brain can act on mineralocorticoid (MR) and (GR) receptors, with MRs having about ~10 fold higher affinity to CORT (in rat). Whereas low levels (mainly MRs occupied) are important for hippocampal cell function and increase long term potentiation (LTP), a process believed to be underlying some forms of memory formation, at excessive levels of glucocorticoids, LTP is decreased or prevented and long term depression (LTD) was found to set in [53] (see also above). Glucocorticoids also have been found to potentiate neuronal damage induced by glutamate analogues. Poor performance in memory tasks has been correlated with high blood levels of cortisol [75].

Stress, depressive disorders and cognitive dysfunction are probably connected [75]. Atrophies of specific brain structures including the hippocampus during depressive illnesses are often observed [172]. Glucocorticoids in excess have deleterious effects on hippocampus cells and cognitive functions. High levels of glucocorticoids occur in depressive subjects compared to controls [133]. It is not known, whether this is an accompanying phenomenon or causally linked to the depressive state. However, administration of high doses of glucocorticoids has been shown to lead to depression. So how could GABA-potentiating steroids be involved in these phenomena? As described above, abnormalities in levels of 3a,5a-THPROG were observed in clinical studies of depression. In such circumstances a disturbance in feedback control of CRH release would be expected, leading to higher glucocorticoid secretion, which exerts the damaging effects on hippocampus and maybe other brain structures.

The steroid DHEA can protect the hippocampus against the neurotoxic effects of corticoids and glutamate analogues. CORT added to embryonic rat hippocampal cell cultures resulted in a substantial loss of neurones [89]. This effect did not take place, when DHEA was added together with CORT . DHEA also prevented hippocampal neurone death induced by NMDA both in vitro and in vivo after infusion [88]. The cell viability increase was neurone specific in the rat embryonic hippocampal cell cultures and substantial even at very low levels of the hormone (10 nM). DHEAS also had a protective effect, but only at much higher levels. The underlying mechanism of these DHEA effects is not known. DHEA and DHEAS have several effects on neurotransmitter receptors and voltage gated channels (see 1.3.2). Thus it is possible that one of those effects plays a role in the inhibition of corticosteroid or NMDA induced

45 neurotoxicity, but is not known how this influence would lead to the effects. However, it was found that DHEA can prevent induction of stress induced protein kinases (SAPK) in culture, that follow CORT administration [89]. Thus this could be a possible mechanism of DHEA prevention of the toxic effects of CORT and NMDA.

However, it is not known whether DHEA plays a role in vivo. There is evidence that a decreased DHEA/cortisol ratio occurs in depressed subjects, and as deleterious effects of glucocorticoids on nerve cells are often related, this could indicate that DHEA plays a natural role in these conditions.

DHEA, however could be a cellular protectant. Cellular stress, such as free radical formation is also known to induce SAPKs [63]. As mentioned above, DHEA can prevent induction of SAPKs. A possible factor in the DHEA formation via the peroxide pathway are reactive oxygen species. Thus this could be a natural protection mechanism against increased cellular oxidative stress.

1.4 Conclusion

As has been described in the above sections, the mammalian nervous system is able to produce several steroid metabolites de novo, in addition to entry of metabolites synthesised in adrenals, gonads, placenta, etc. They have been shown to possibly be involved in a multitude of signalling roles in the nervous system. The above hypotheses of the genomic and non-genomic effects on neurotransmission lead to the conclusion that steroids represent a general mechanism to regulate the excitation state of brain amongst other roles. Evidence suggests that disturbances in the regulation of levels of certain steroids can result in mild neurodegenerative and/or emotional disorders.

The following Chapters show the development of gas chromatographic-mass spectrometric methods for steroid analysis, together with improved procedures for the extraction and fractionation of steroids from nervous tissue. An overview of previous steroid identification, extraction and fractionation procedures is given in the introductions to these Chapters. The main outcome of the present work has been to apply new and/or improved procedures to a survey of free steroids and steroid conjugates in male rat brain.

46 Table 1-1. Systematic and trivial names for P450 steroidogenic enzymes identified in rat brain. Names of mRNA and protein are the same, the names for the genes are the systematic names written italicised. *not demonstrated in rat brain yet.

Systematic name Trivial name

Mitochondrial

CY PllA P450SCC

C Y PllB l P450clip

CYP11B2 P450cllAS

Microsomal

CYP17 P450cl7

CYP19 P450aro

CYP7b P450c7b

CYP21A* P450c21

Figure 1-1. Ring numbering of basic steroid structure.

47 Figure 1-2. Biosynthetic pathways of steroid formation. Included are enzymatic conversions known to be present in “classical” peripheral steroidogenic glands gonads, adrenal and placenta. Enzymes not identified in the mammalian nervous system to date are indicated by dotted arrows. Modified from [12,116,124]. Dehydroepi- Fatty acid ester of Cholesterol Pregnenolone sulphate androsterone Dehydroepiandrosterone Fatty acid ester sulphate of Pregnenolone

CH3 ,O H 20p-HOR c H3^.oh Acyl- transferase CH3_^0 OH

Dehydroepi­ 20p-Dihydro- 20a-Dihydro- HD' Pregnenolone androsterone pregnenolone pregnenolone 17a-OH-pregnenolone 17,20-lyase 17a-OHIase 3p-HSD 20a-HOR 3P-HSD 3P-HSD CHspO CH3s ^O CH3pP o C--OH CH3^.0H

HO' Androstenedione Progesterone 17a-OH-progesterone 5a-pregnan-3a,17- 20a-Dihydro- diol-20-one progesterone — 21-OHIase 17P-H0R 11p-OHIase 17a-OHIase CH3^0 CH3yD JNS.O H 3 C H 2 .^ 0 OH

5a-Reductase H I HO- Testosterone 5a-Dihydro- 11-Deoxycorticosterone 11-Deoxycortisol 3p, 5a-T etrahydro- 3a, 5a-T etrahydro- progesterone 11 p-OHIase progesterone progesterone 3a-HOR Aromatase Sp-HOR HOCHz 's^O HDCH2 | HDCHz^O

HDCHzyfP HDCH;^ OH

HD- 3p,5a-T etrahydrodeoxy- 3a,5a-Tetrahydrodeoxy- 5a-Dihydrodeoxy- HO- corticosterone corticosterone corticosterone Corticosterone Cortisol Oestradiol

48 Chapter 2 Materials and methods

2.1 Chemicals

All chemicals used were analytical grade, unless otherwise stated. The systematic and trivial names of the steroids used here as well as their abbreviations to be used throughout the text, source and molecular weight are listed in Table 2-1. Free steroids and steroid fatty acid esters were made up at 100 p.g/ml in ethanol, steroid sulphate esters at this concentration in ethanol containing 0.5% NH3 (v/v). All were stored at - 20°C. ^H-pregnenolone (PREG, 777.0 GBq/mmol), ^H-dehydroepiandrosterone (DHEA, 3422.5 GBq/mmol), ^H-progesterone (PROG, 5106.0 GBq/mmol), and corticosterone (CORT, 3256.0 GBq/mmol) were all obtained from NEN Life Science Products and stored at -20°C in ethanol. ^'^C-cholesterol (CHOL, specific activity not available) was a kind gift by Dr. David Allan, University College London. ^H-5a- dihydroprogesterone (5a-DHPR0G) was prepared in the laboratory by incubation of ^H- PROG with rat brain homogenate, purification by thin layer chromatography (TLC) and storage at -20°C. The sulphate esters of ^H-PREG and ^H-DHEA were synthesised from their respective free steroids by Dr. Fry in this laboratory after a method modified from [54] and then purified by procedures given in [51] with purities tested to be greater than 99%. As for the unlabelled sulphate esters, these ^H-labels were stored at -20°C in ammoniated ethanol.

The solvents ethanol (96%), methanol, ethyl acetate, isooctane (trimethylpentane), , cyclohexane, (diethyl) ether and benzene were redistilled before use from analytical grade. Deactivated charcoal was added at 1 g/1 and the solvent left for approximately 12 h at room temperature. The charcoal was removed by filtration through filter paper (Whatman) and the solvent then distilled over a Vigreux type fractionating column. Aliquots were checked for purity by gas chromatography-mass spectrometry (GC-MS) prior to use for experiments. Pyridine was analytical grade according to American Chemical Society specifications and stored over NaOH pellets. Ether was washed with 5% ferrous sulphate solution in a separating funnel to remove peroxides prior to distillation. Glacial acetic acid was recrystallised and washed with

49 ethanol before use. The scintillation fluid was Ecoscint H (National Diagnostics) and used unmodified except for counting samples of Soluene 350 (Packard) solubilised brain tissue (see 4.2.2.1). The derivatisation reagents for GC-MS methoxyamine hydrochloride (MO), trifluoroacetic acid anhydride (TFAA), heptafluorobutyric acid anhydride (HFBA), N-methyl-N-(/gr?-butyl-dimethyIsilyl)- trifluoroacetamide (MTBSTFA) and /gr/-butyl-dimethylsilylchloride (TBDMSCl) were from Sigma, trimethylsilylimidazole (TMSI) and hexamethyldisilazane (HMDS) from Pierce. TFAA and HMDS were stored at room temperature, HFBA and TMSI at 4 °C and MTBSTFA and TBDMSCl at -20°C, all in desiccators. MO was made up at 2% (w/v) in pyridine and stored at room temperature. Lipidex 5000® gel (Packard) was prepared by removing the solvent methanol under vacuum, rinsing three times with cyclohexane and once with cyclohexane ; pyridine : hexamethyldisilazane 98:1:1 (v/v/v) (CPH). It was then stored at 4 °C in this solvent until use. All water was double glass-distilled.

Buffers and miscellaneous solutions

Acidified ethyl acetate was prepared by saturating ethyl acetate with 2 M H2SO4. Constitutions of other buffers and solutions used are given where appropriate. All solutions were prepared from fresh frequently and stored at 4°C, except acidified ethyl acetate, benzene sulphonic acid (BSA)Zacidified ethyl acetate (over Na 2 S0 4 ), which all were stored at room temperature.

2.2 General Procedures

Preparation o f glassware

All glassware was cleaned by soaking in dilute Decon 90® alkaline detergent then nitric acid followed by extensive washes with tap and then double glass-distilled water. Glassware in contact with standard steroids or radio-labelled sources was decontaminated by soaking in Decon 90® for 12 h followed by continued rinsing in tap water for 3 days prior to the above cleaning procedure. All glassware that would come into contact with steroids was deactivated by silanisation using 2% dimethyldichlorosilane in or cyclohexane. The glassware was thoroughly rinsed with the above reagent. Remaining reagent was removed by thorough rinsing with cyclohexane followed by H 2 O. Excess silanising reagent was removed by baking the

50 treated glassware at 220 “C for 2 hours. All apparatus used for processing of the human brain samples was decontaminated using Virkon®.

Scintillation counting

Samples in polypropylene counting vials (Sarstedt) were dried down under vacuum and 3 ml Ecoscint H scintillation fluid was added. All samples were taken in triplicate and counted for 10 minutes unless otherwise stated on a Packard Tri-Carb 2200 CA liquid scintillation analyser. The counter had been calibrated with ^H-hexadecane standards in the same scintillation fluid and counting vials. Background samples with scintillation fluid only taken before and after the samples were included in each determination. Counts per minute (CPM) were transformed into decays per minute (DPM) using counting efficiency for each sample vial assessed from the transformed spectral index obtained with an external radiation source (^^^Ba).

Thin layer chromatography

Aluminium backed, silica coated plates from Merck, were activated at 60 °C for 1 h before use. Samples were spotted in ethanol with Gilson pipettes. PROG metabolites were separated in the system cyclohexane; n-butyl acetate (1:2 v/v) whereas the sulphate esters of PREG and DUE A were separated from their corresponding free steroids in the system ethyl acetate: ethanol: ammonia (25:10:2 v/v). After chromatography, positions of the ^H-labelled compounds could be visualised by exposing the plates to a phosphoimager.

2.3 Extraction and fractionation of steroids from brain tissue

2.3.1 Tissue samples

Rats

Adult male Sprague-Dawley rats (250-450 g) came from the breeding colony at Biological Services, University College London. They were kept in a 12 h lighting regimen (lights on at 8.00 h) and were fed rat chow and water ad libitum. For experiments to test the extraction of endogenous ^H-PROG and its metabolites from brain tissue, rats were killed by CO 2 inhalation. All other rats were killed between 11.00

51 h and 15.00 h by cervical dislocation. The whole brain (including cerebellum but excluding olfactory bulbs) was rapidly removed and meninges carefully dissected out before storage at -70°C until use.

Humans

Approximately lOg slices of human frontal cerebral cortex were received from the Parkinson’s Disease Society Brain Research Centre at the Institute of Neurology, University College London. The samples of disease free subjects were taken approximately 2-8 h after death and stored at -70°C.

2.3.2 Tissue extraction

Ethyl acetate tissue extraction

Brain samples were homogenised with a high speed Polytron homogeniser (Kinematica,

CH) in 4 volumes ice-cold 0.1 M KH 2PO4 buffer, pH 7.4 at full speed for 10 seconds twice, letting the sample cool on ice for 1 minute after the first time. One volume of buffer was used to rinse the homogeniser probe and the fractions pooled. Tissue homogenates in phosphate buffer were then dripped into the same volume of ethyl acetate in a test tube in an ultra-sonication water bath. Extraction tubes were then shaken for 3 minutes by hand and vortexed for 1 minute, before centrifuging at 1500xg for 4 minutes. The supernatants were transferred to fresh tubes and the homogenate extracted twice more with ethyl acetate 1:1 (v/v). The extracts were washed by adding 0.5 volumes of H 2 O, shaking 3 minutes and centrifuging at 1500xg for 4 minutes. The pooled supernatants were dried down with a rotary evaporator for further processing.

Ethanol extraction

Frozen tissue samples were suspended in 4 volumes ice-cold ethanol (v/w) in polypropylene centrifugation tubes and homogenised with the Polytron homogeniser (10 seconds twice). The homogeniser probe was rinsed with one volume ethanol and this was pooled with the rest of the homogenate. The homogenate was left at -70°C for 30 minutes before centrifuging at 28000xg for 30 minutes at 4”C. The supernatant was then kept at -20°C until further processing. In some initial experiments, the pellet was resuspended in 5 volumes ice-cold 80% ethanol in potassium phosphate buffer (5 mM,

52 pH 7.4, v/v) by homogenising with the Polytron. After centrifugation, the supernatant was combined with the previous (10 volumes extracts).

Acetic acid/ethanol extraction

Pooled tissue samples were homogenised in 4.75 volumes potassium phosphate buffer (5 mM, pH 7) three times 10 seconds on ice with the Polytron and the probe rinsed with 0.25 volumes of the same buffer, which was then combined with the remainder. Small portions of the homogenates were taken off for determination of blood contamination of tissues as described above and stored at -70 °C. The remaining homogenate was dripped into 20 volumes acetic acid (3%, v/v) in 96% ethanol in polypropylene tubes in an ultra- sonicating water bath. After further sonication for 10 minutes the extracts were left at - 20°C overnight. They were then sonicated again with an MSE Soniprep probe 2 times 15 seconds, while being ice-cooled, left on ice for 30 minutes and then centrifuged at 28000xg for 30 minutes at 25 °C.

Estimation o f extraction recovery

To examine extraction recovery of ^H-PROG and its metabolites from brain tissue, rats were injected intraperitoneally with 0.7 MBq/kg ^H-PROG in phosphate buffered saline

(5 ml/kg). After 2.5-3 h they were killed by CO 2 inhalation, brains were removed quickly, dissected carefully free from meninges and divided along the midline before storage at - 70°C. In order to estimate total brain radioactivity, one half of each brain was homogenised with the Polytron homogeniser in 0.1 M potassium phosphate buffer pH 7.4, then solubilised in a minimum of 3 volumes of Soluene 350 (Packard, 0.5 M quarternary ammonium hydroxide in toluene). Samples were frequently mixed and incubated for at least 15 minutes at room temperature with Soluene 350. After adding hydrogen peroxide (final concentration of 1% w/v, BDH) to bleach the samples they were incubated for 2 h at 50°C. Sample aliquots of 0.3 ml were then counted in 3 ml of Ecoscint H to which had been added Triton X-100 6% v/v (BDH, scintillation grade), glacial acetic acid 0.6% v/v (AR grade) and butylated hydroxytoluene 2% w/v (Sigma) as antioxidant. The other brain halves were homogenised in phosphate buffer and extracted into acetic acid/ethanol or homogenised in ethanol as described above.

53 Estimation o f blood contamination o f brain tissue samples

Blood contamination of rat brain tissue was estimated by spectrophotometric determination of haemoglobin. A small portion (1 ml) of each rat brain homogenate in 5 volumes potassium phosphate buffer (5 mM, pH 7) was removed before extraction and stored at -20°C before measurement. After thawing on ice, samples were centrifuged (28000xg, 30 minutes, 4”C) and then 300 til of the supernatant placed in the chamber of a spectrophotometer. Haemoglobin was estimated by the change in absorbance between 560 and 578 nm upon reduction by the addition of sodium dithionite (to a final concentration of 10 mM). Blood content of the original brain samples could then be calculated assuming a haemoglobin concentration in rat blood of 157 g/1 [206].

2.3.3 Solvent partitioning

To test solvent partitioning between aqueous methanol and isooctane for the original extraction/purification procedure, standard steroids were dried down and redissolved in 0.3 volumes isooctane by sonication, followed by addition of 0.3 volumes 80 or 90% methanol in H 2 O (v/v) and again sonication. The solutions were transferred to clean tubes and the process repeated twice. The combined solutions were vigorously shaken three minutes, centrifuged 5 minutes at lOOOxg and the methanol phases transferred to fresh flasks. The isooctane phases were extracted twice more by shaking three minutes with an equal volume of 80 or 90% methanol. For all other extractions by the original procedure, the ethyl acetate extracts were dried down, and partitioned as above between 80% methanol against isooctane.

In the improved extraction procedure, ethanol extracts (5 volumes) were shaken together with 3.3 volumes of isooctane saturated with 80% ethanol in 5 mM potassium phosphate buffer for 3 minutes and vortexed for 1 minute. After centrifuging, the isooctane phases were removed and the partitioning repeated twice more. Solvent partitioning of extracts in acetic acid/ethanol is described in 2.3.7.

54 2.3.4 Solid phase extraction chromatography

Sep Pak CIS®

Sep Pak CIS® cartridges (1 cc, 30 mg, Waters Corp., Milford, USA) were activated and rinsed with 5 ml H 2 O, 5 ml methanol and 5 ml H 2 O at a flow rate of 10 ml/min in a vacuum manifold. Samples were loaded and eluted at 2 ml/minute flow rate as stated in relevant Sections. Care was taken that the cartridges did not dry out between washings.

Oasis HLB®

Oasis HLB® (3 cc, 60 mg or 5 cc, 200 mg. Waters Corp., Milford, USA) solid phase extraction cartridges were activated and rinsed with 15 ml (50 ml for 200 mg cartridges) methanol or 96% ethanol, 3 ml (10 ml) H 2 O and 5 ml (16.5 ml) of the solvent the samples were to be loaded in at a flow rate of 10 ml/minute in a vacuum manifold. Samples were loaded and eluted at 2 ml/minute flow rate as stated in the relevant Sections.

Oasis MAX®

Oasis MAX® cartridges (3cc, 60 mg. Waters Corp., Milford, USA) were activated and rinsed with 15 ml ethyl acetate, where this solvent was used for elution, and in all cases with 10 ml 0.25 M ammonium acetate buffer, pH 7.0, to exchange the stationary phase counter anion, 15 ml 96% ethanol and 3 ml H 2 O at a flow rate of 10 ml/minute. Samples were loaded and eluted at 2 ml/minute flow rate as stated in the relevant sections.

2.3.5 Celite chromatography

Celite powder was cleaned before use by soaking in 6 M HCl 1:1 (v/v) 12 h and then rinsed with H 2 O until the wash water was pH 7. It was then soaked in 99.9% ethanol 1:1 (v/v) 12 h, thereafter rinsed twice with the same volume of ethanol and twice with . After evaporating ether at room temperature the powder was heated at 200°C 12 h and subsequently stored in a desiccator until use. Before use, the powder was again activated by heating at 200°C for two hours. For chromatography, celite was impregnated with propylene glycol or ethylene glycol, which served as the stationary phase. This was done by thoroughly mixing the glycol and celite with a spatula, then leaving it at room temperature for 15 minutes. Columns were prepared as follows: Celite

55 impregnated with propylene and/or ethylene glycol was suspended in 10 ml isooctane, transferred to 5 ml disposable glass pipettes (plugged with glass wool or a small glass bead), allowed to settle under Nz-pressure and then packed tight with a glass rod. Thereafter the columns were kept in isooctane until use. Where indicated, water traps were packed below the glycol stationary phases. They consisted of 0.3 g or 0.6 g celite impregnated with H 2 O (3:1, w/v). Samples in 1.5 ml isooctane equilibrated with ethylene glycol were loaded on the columns, which were washed and eluted as indicated. In some cases, glycol eluted from the stationary phase was removed from the eluates by partitioning against H 2 O (7:1, v/v). The sample tubes were shaken for three minutes, centrifuged at HOOxg for 4 minutes and the supernatant taken off and dried down in a rotary evaporator.

2.3.6 Solvolysis

In the original extraction procedure the following solvolysis method for steroid sulphates was applied. After extraction of tissue homogenates in KH2PO4 buffer with ethyl acetate, the remaining aqueous phase was combined with the H 2 O wash, NaCl added to a concentration of 20% (w/v) and the pH lowered to 1 by addition of 2 M H2SO4. After checking the pH, steroid sulphate esters were extracted three times with ethyl acetate 1:1 (v/v) by shaking 3 minutes. After centrifugation at 1500xg, the ethyl acetate phase was transferred to a clean round bottom flask and incubated 16 h at 38”C. Following this incubation, the solution was neutralised by addition of approximately 1/32 volume pyridine. After checking the pH, the solution was then dried down in a rotary evaporator for further processing.

2.3.7 Final extraction and fractionation procedure for steroids from mammalian brain tissue

Samples were homogenised in phosphate buffer and extracted into acetic acid/ethanol as described above (p. 53). Supernatants were transferred to stoppered Pyrex tubes with Pasteur pipettes for solvent partitioning. The extracts were partitioned against 10 volumes of isooctane saturated with acetic acid/ethanol (3%, v/v) by shaking for 3 minutes and vortexing for 1 minute. After centrifuging at 1500xg, the isooctane phases

56 and precipitate layers formed at the phase interface were removed and the partitioning repeated twice more.

The partitioned extracts were dried down in round bottom flasks on a rotary evaporator and resuspended in 1.25 volumes 96% ethanol by swirling and sonication for 2 minutes.

H2 O (0.75 volumes) was added to give 60% ethanol (v/v) and the solution sonicated for further 2 minutes. The redissolving process was repeated once more and the combined fractions then centrifuged at lOOOxg for 12 minutes. The supernatant was loaded onto a primed 200 mg Oasis HLB® cartridge (corresponding to up to 8 g tissue). A further 4.4 volumes of 60% ethanol in KH 2 PO4 buffer (5 mM, pH 7, v/v) were passed through the cartridge and collected together with the loading eluate.

The eluates from the Oasis HLB® cartridges were dried down, the residues resuspended in 0.4 volumes 96% ethanol, sonicated 2 minutes, 1.475 volumes KH 2 PO4 buffer (5 mM, pH 7, v/v) added and sonicated again for 2 minutes. This process was repeated once more, all fractions were pooled and loaded onto a primed 60 mg Oasis MAX® cartridge (corresponding to up to 8 g tissue). A further 5 ml 20% ethanol in ammonium acetate buffer (20 mM, pH 7, v/v) were washed through, then free steroids eluted with 4 ml ethyl acetate. Any steroid glucuronides present were eluted with 20 ml 60% ethanol in formate/pyridine buffer (20 mM, pH 3, v/v), and after a wash with 2 ml ethyl acetate

(dried over Na 2 S0 4 ) to remove traces of water, steroid sulphates were eluted with 15 ml 50 mM BSA in acidified ethyl acetate.

Ethyl acetate eluates of Oasis MAX® cartridges were dried down under N 2 and derivatised by MO and TMSI or HFBA as described in 2.4.1. BSA/acidified ethyl acetate eluates (15 ml) of Oasis MAX® cartridges were dried over Na 2 S0 4 and solvolysed at 40°C for 16 h. After addition of 10 drops pyridine, the solution was taken to near dryness on a rotary evaporator. The residue was extracted three times with 2 ml ether by vortexing 30 seconds, sonicating 1 minute and centrifuging at 1500xg. The supernatants were combined and dried down for derivatisation by MO-TMSI as described in 2.4.1. For derivatisation by HFBA, the combined ether phases were washed three times with

H2 O. Each time, after addition of 1 ml H 2 O saturated with ether the samples were vortexed 30 seconds, centrifuged 2 minutes at 1200xg and the H 2 O phase removed and discarded. The ether phase was dried down and derivatised as described in 2.4.1 for HFBA.

57 2.4 Gas chromatography - mass specfom^etry

2.4.1 Sample derivatisation

Derivatisation with MO and TMSI

For quantitation and identification purposes, ijternaal standards were added to samples as

stated. Unless otherwise stated, samples were driedl down under a N 2 -stream and heating at 60°C, redissolved in 200 pi MO in pyridine (2%), w/v) and incubated at 60°C for 1 h. Then 100 pi TMSI were added and the mixture hejated a further 3 h at 100°C. Pyridine was evaporated under a N 2 -stream and heating; at 60°C and 1 ml cyclohexane : hexamethyldisilazane : pyridine 98:1:1 (v/v/v) ((CPH) added to the residue, before vortexing 30 seconds and sonication for 1 minute. Lipidex 5000® gel chromatography columns were prepared in Pasteur pipettes (0 5 cnn diameter) approximately 8 cm high. The solution was passed through a Lipidex coluimn under gravity. The residues of samples were twice more dissolved in 1 ml of the same solvent and passed through the column, unless otherwise stated. After drying down^ the pooled eluates were evaporated, dissolved in cyclohexane, transferred into autotsampler vials and stored at room temperature protected from light until ready for inje-ction onto the GC.

Microsolvolysis

In the method development sections of Chapters 5 and 4, steroid sulphate esters were sometimes treated by a microsolvolysis method. Steroid sulphate ester solutions were dried down, redissolved in 50 pi acidified ethyl acetate and incubated for 16 h at 40°C. MO in pyridine (2% w/v) (200 pi) was added, dried down to approximately 50 pi under

N2 and then incubated for 1 h at 60°C. After addition of 100 pi TMSI, incubation continued for 3 h at 100°C. The residue was purified with Lipidex chromatography as described above [169].

Derivatisation with HFBA or TFAA

After addition of the internal standard 16-DPREG, samples were dried under N 2 and heating at 60 °C and redissolved in 30 pi benzene followed by addition of 30 pi HFBA, and the reaction allowed to proceed for 30 niinut^s at 60 "C, unless otherwise stated.

The reaction solvents were evaporated under a N 2 -stream and heating at 60”C and I ml

58 cyclohexane : pyridine 98;2 (v/v) added to the residue, before vortexing 30 seconds and sonication for 1 minute, unless otherwise stated. Lipidex 5000® gel chromatography columns were prepared in Pasteur pipettes (0.5 cm diameter) approximately 8 cm high and washed with 6 ml cyclohexane ; pyridine 98:2 (v/v). The sample solution was passed through Lipidex columns under gravity. The residues of samples were twice more dissolved in 1 ml of the same solvent and passed through the column, unless otherwise stated. After addition of the internal standards tetracosane and octacosane, the pooled eluates were evaporated, dissolved in cyclohexane, transferred into autosampler vials and stored at room temperature protected from light until ready for injection onto the GC.

For derivatisation with TFAA, the samples were redissolved in 500 pi ethyl acetate and 50 |il TFAA was added for reaction at room temperature for 60 minutes. The solvents were evaporated off and the residues redissolved and stored in cyclohexane at room temperature until ready for injection, however no longer than two days.

Derivatisation with MTBSTFA

After addition of internal standards, samples were dried down and redissolved in 90 }xl

MTBSTFA containing 1 % TBDMSCl (w/v). 10 p.1 acetonitrile : pyridine : NH 4 I 5:15:1 (v/v/w) were added and the mixture left to react for 75 minutes at 80 °C. The reagents were freshly prepared each time before use. The samples were dried under N 2 and redissolved in cyclohexane for storage at room temperature until use.

Méthylation

Steroid glucuronides in ethanol were dried down, redissolved in 500 til BF 3 in methanol

(14%) and incubated at 100“C for 5 minutes. After addition of 500 |l i 1 H2 O saturated with NaCl the solution was extracted three times with ethyl acetate 1:1 (v/v). After drying down, derivatisation continued with the procedure for MO and TMSI.

2.4.2 Gas chromatography-mass spectrometry analysis

Some early analyses were carried out on an Hewlett Packard 5890 gas chromatograph (GC) coupled to an Hewlett Packard 5970 mass selective detector. All other analyses were carried out on a Shimadzu 17A GC coupled to a QP 5050A mass spectrometer (MS). Injections were done with a Shimadzu autosampler AOC-20s. The system was

59 controlled and data processed by the Shimadzu Class 5000 software. Samples were ionised by electron impact ionisation with an energy of 70 eV. The wall coated open tubular GC columns used were Chrompack CP SIL 5 CB 25 m in length, 0.25 mm inner diameter, 0.12 (j.m film thickness or Phenomenex Zebron ZBl 30 m in length, 0.25 mm inner diameter, 0.25 |um film thickness. Helium was used as the carrier gas.

The GC-conditions and MS parameters used in scan methods are shown in Table 2-2. This Table also contains GC-conditions used in SIM methods. The ion panels for all SIM methods are shown in Table 2-3.

Quality control

The continuous reproducible performance of the GC-MS system was monitored and ensured by the following several measures;

1. By tuning using perfluorotributylamine (PFTBA) as calibration gas before each set of samples. The absolute sensitivity, relative ion abundance (not only from PFTB A, but also due to leakage of H 2 O and N 2 from room air, see below), mass resolution and mass pattern adjustment of the MS were adjusted. Deteriorating performance could be noticed in the tuning results and cleaning allowed adjustment of optimal values. Leaks could also be detected here due to responses of H 2 O (18 m/z) and N 2 (28 m/z) ions.

2. GC-performance was monitored by injecting a mixture of standard alkanes C24, C26, C28, C30, C32, C34 (10 ng each in cyclohexane, Sigma), covering the retention time range of interest. Discrimination against the higher alkanes indicated deteriorating injection port or ion source performance usually due to build-up of sample residue. These alkanes were also regularly monitored for retention behaviour and if necessary SIM-protocols adjusted. Column problems could be seen from excessive baseline rise due to column bleed or changing peak shape of the above alkanes.

3. Overall performance of the system was then frequently verified using standard samples of steroid derivatives at low levels. This would also indicate specific problems (e.g. column active sites). Then signal-to-noise ratios of three steroids from across the retention time range were measured. These measures were used for routine performance

60 checks. An overall analytical method validation was carried out and is described in Chapter 3.

GC-MS maintenance

One or more solvent blanks were injected after each sample injection to purge residual sample from injector, column or ion source. All injection port parts and MS ion source were cleaned after approximately 50 sample injections or when deteriorating performance was detected by the above monitoring procedures. A clean injection port liner silanised with 5 mg glass wool was then inserted. The GC columns were conditioned when increased column bleed or excess blank activity due to sample residue was observed. This was done by heating the column 20°C above the end temperature of the GC-analysis for 3 hours. From time to time, a small section of the column on the injector side was removed. Also, the section of the column in the interface between GC and MS ion source sometimes caused problems due to generation of active sites and thus increased adsorption of analytes and carryover, probably due to the continuous exposure of this section of the column to high temperature. This problem was resolved by removing this section of column. Columns were replaced approximately every 12 months.

Analysis of results

Peak integrations were done manually using the Shimadzu Class 5000 MS software and used for identification and quantitation. Retention time and integration data were further processed in Microsoft Excel®. Graphs were prepared using Microcal Origin® and Microsoft Excel® software. For identification, qualifier to target ion ratios were calculated from their areas. Further identification was obtained from retention indices. For initial characterisation of derivatised steroids the Retention Index (RI) after Kovats was calculated as [94];

RI=100xN+100xnx [log t(A) - log t(N)] / [log t(N+n) - log t(N)l

where:

N ... number of carbon atoms in alkane eluting before compound of interest

61 n... increment in number of carbon atoms from alkane eluting before compound of interest to the one eluting after compound of interest t(A)... retention time (minutes) of compound of interest t(N)... retention time (minutes) of alkane eluting before compound of interest t(N+n)... retention time (minutes) of alkane eluting after compound of interest

In analysis of endogenous brain steroids, relative retention time (RRT) was calculated with respect to the closest of three internal standards which served as time reference peaks as the ratio of retention times in minutes of analyte and internal standard. For positive identification in selected ion monitoring, RRTs of analytes had to lie within ±0.5% of the RRTs of authentic standard compounds (see 5.2.3).

Further, for identification in selected ion monitoring compounds had to meet target values of qualifier to target ion ratios (Q/T) of standard compounds within 0.67 and 1.5 (±20% of relative abundance of target and qualifier ions). This is the recommended criterion of the working group of the UK National measurement system for mass spectral identification [208]. Confidence limits for RRT and Q/T were also calculated from a series of standard compounds to back up the above pragmatic criteria. The limits calculated were those within which a new observation from the same population is expected to lie [41]. The limits are given by

y„ ± t X V [ s^(y) X (l/n±l/m) ]

where

ÿn ... arithmetic mean of n standard measurements of y t ... appropriate t-statistic for the degrees of freedom and for the chosen level of probability s^(y)... variance of the mean standard measurements

62 n number of standard measurements m.... number of new observations

For quantitation, calibration curves were constructed of area ratios of standard steroid analytes and their respective internal standards against their amount ratios. Regression analysis was performed by the least square method. R-values were calculated as:

R = V[l-Z(yj-E(yÿ]/[(Zy')-(Zyi)'/n]

where

E (y i). .. estimated value of y\ y \ observed value n ... number of observations

For method evaluation, limits of detection were calculated from responses to standards of pure steroids. For calculation of limits of detection, signal to noise ratios (S/N) were determined using the tool provided in the MS software. The maximum noise was estimated within each sample run as three standard deviations from the equivalent signals in baseline areas of the chromatogram near the peak of the compound of interest (3 a noise criterion). Peak height was taken as signal. Prerequisite for positive identification was a lack of signal above noise at the RRT of interest in a solvent blank injection immediately prior to sample injection. Overall detection limits of the extraction and assay procedure were estimated from pure solution blanks run alongside the brain samples. The criterion for positive identification was that the area of the peak had to be three times of the one at the same RRT in the corresponding extraction blank. These three times blank values were converted into pg steroid using the calibration curves.

For comparison of sensitivities with different GC-MS parameters, sample responses were tested for significant difference by independent, two population Student’s t-test.

63 Table 2-1. Systematic and trivial names of standard steroids and their abbreviations used here, sources and relative molecular weights(M r ).

No Systematic name Trivial Name Abbreviation Source M r 1 5-Androsten-3 3-ol-17-one Dehydroepiandrosterone DHEA Sigma D-4000 288.4 2 5 P-Pregnan-3 P-ol-20-one 3 P,5 P-Tetrahydroprogesterone, 3p,5P-THPROG Sigma P-5385 318.5 3 5a-Pregnan-3a-ol-20-one 3 a, 5 a-T etrah\droprogesterone, 3a,5a-THPROG Sigma P-8887 318.5 4 5 P-Pregnan-3a-ol-20-one 3a,5 P-Tetrah\'droprogesterone, 3a,5P-THPROG Sigma P-8129 318.5 5 5 3-Pregnan-3,20-dione 5 P-Dihydroprogesterone 5P-DHPR0G Sigma P-6010 316.5 6 5-Pregnen-3 P-ol-20-one Pregnenolone PREG Sigma P-9129 316.5 7 5a-Pregnan-3,20-dione 5a-Dihydroprogesterone 5a-DHPR0G Sigma P-7754 316.5 8 4-Pregnen-3,20-dione Progesterone PROG Sigma P-0130 314.5 9 4-Pregnen-20P-ol-3-one 2 0 P -Dihydroprogesterone 20P-DHPROG Sigma P-6163 316.5 10 5P-Pregnan-3a, 21-diol-20-one 3a,5P-Tetrahydrodeox\'corticosterone, 3a,5p-THDOC Sigma P-9896 334.5 T etrahy drodeoxycorticosterone 11 5-Pregnen-3 P, 17a-diol-20-one 17a-Hydroxypregnenolone 17-OH-PREG Sigma H-5002 332.5 12 4-Pregnen-20a-ol-3 -one 2 Oa-Dihydroprogesterone 20a-DHPROG Sigma P-6288 316.5 13 5a-Pregnan-3a, 21-diol-20-one 3a,5a-Tetrahydrodeoxycorticosterone, 3a,5a-THDOC Sigma P-2016 334.5 Allotetrahydrodeoxycorticosterone 14 4-Pregnen-17a-ol-3,20-dione 17a-Hydroxyprogesterone 17-OH-PROG Aldrich 28622-2 330.5 15 5a-Pregnan-21 -ol-3,20-dione 5 a-Dihydrodeoxycorticosterone 5a-DHD0C Sigma P-4395 332.5 16 4-Pregnen-21 -ol-3,20-dione 11-Deoxycorticosterone DOC Sigma D-6875 330.5 17 4-Pregnen-11P ,21 -diol-3,20-dione Corticosterone CORT Sigma C-2505 346.5 18 4-Pregnen-16a-methyl-3,20-dione 16a-Methylprogesterone MEPROG Steraloids Q-2930 328.5 19 5-Pregnene-3 P,20P-diol 20P-Dihydropregnenolone 20P-DHPREG Sigma P-9004 318.5 20 4-Pregnen-11 P-ol-3,20-dione 11P -Hydroxyprogesterone IIP-OH-PROG Sigma H-5627 330.5 21 5-Pregnene-3 P,20a-diol 2 Oa-Dihydropregnenolone 20a-DHPREG Sigma P-8879 318.5 22 4-Pregnen-3a-ol-20-one 3a-Dihydroprogesterone 3a-DHPR0G Steraloids Q-3510 316.5 23 5a-Pregnan-20a-ol-3-one 5a,20a-Tetrahydroprogesterone 5a,20a-THPROG Sigma P-6413 318.5

64 Table 2-1 continued.

No Systematic name Trivial Name Abbreviation Source Mr 24 5a-Pregnane-3a,20a-diol 5 a-pregnane-3 a,20a- Sigma P-9015 320.5 diol 25 5a-Pregnan-3a, 17-diol-20-one 17(5a)-OH-pregnanolone 5a-pregnan-3a,17- SteraloidsP-2460 334.5 diol-20-one 26 5 P-Pregnan-3a, 17-diol-20-one 17(5 P)-OH-pregnanolone 5p-pregnan-3a,17- Sigma P-7379 334.5 diol-20-one 27 5 (3-Pregnane-3a,20a-diol 5 p-Pregnane-3a,20a-diol 5 p-pregnane-3 a,2 Oa- Sigma P-6379 320.5 diol 28 6a-Methyl-pregnen-17a-ol-3,20-dione 6a-Meth>’l-17-h\ drox\-progesterone ME-17-OH-PROG Sigma M-6013 344.5 29 5-Androsten-3P-ol-16-one 16-Dehydroepiandrosterone 16-DHEA Steraloids A8430 288.4 30 5,16-Pregnadien-3 P-ol-20-one 16-Dehydropregnenolone 16-DPREG Steraloids PI500 314.5 31 4-Pregnen-21 -ol-3,20-dione 21 -aldehyde,21 -hemiacetal DOC-aldehyde,hemiacetal Steraloids Q3466 360.5 32 4-Pregnen-20p-carbox-aldehyde-3-one Bisnorcholenaldehvde BNCA Sigma P-9890 328.5 33 11 P,17a,21 -trihydrox}'-4-pregnen-3,20-dione Hydrocortisone, cortisol cortisol Sigma H-4001 362.5 34 4-Androsten-17P-ol-3-one Testosterone TESTO Sigma T-1500 288.4 35 5a-Pregnan-3 p-ol-20-one 3 P,5a-T etrahydroprogesterone, 3p,5a-THPROG Sigma P-0666 318.5 Epiallopregnanolone 36 4-Pregnen-21 -ol-3,11,20-trione 11-Dehydrocorticosterone 11 -dehydro- Sigma D-9507 344.4 corticosterone 37 5 P-Pregnan-3 p,21 -diol-20-one 3 P,5 P-Tetrahydrodeoxycorticosterone, 3p,5p-THDOC Steraloids P-6950 334.5 EpitetrahydrodeoT^corticosterone 38 5a-Pregnan-3 P,21 -diol-20-one 3 p ,5 a-T etrahydrodeoxycorticosterone, 3p,5a-THDOC Sigma P-0648 334.5 Epiallotetrahydrodeoxycorticosterone 39 5a-Androstan-3 p-ol-17-one Epiandrosterone EpiA Sigma E-3375 290.4 40 4-Pregnen-17a,21 -diol-3,11,20-trione cortisone Sigma C-2755 360.45 41 4-Pregnen-17a,21 -diol-3,20-dione 11-Deoxycortisol 11-deoxycortisol Sigma R-0500 346.5 42 4-Pregnen-3 P-ol-20-one 3 P-Dihydroprogesterone 3P-DHPR0G Steraloids Q-3540 316.5

65 Table 2-1 continued.

No Systematic name Trivial Name Abbreviation Source Mr 43 5a-Pregnan-3a,l 1 (3-dioI-20-one 5a-Pregnan-3a,l ip-diol-20-one 5a-pregnan-3a,l 1P- Steraloids P-2340 334.5 dioI-20-one 44 5 (3-Pregnane-3a,20P-diol Pregnanediol 5P-pregnane-3a,20p- Sigma P-6879 320.5 diol 45 5a-Pregnan-3a-ol-11,20-dione Alphaxalone alphaxalone Sigma P-5052 332.5 46 4-Androsten-3,17-dione Androstenedione Adione Sigma A-9630 286.4 47 5 3-Pregnan-21 -ol-3,20-dione 5 P-Dihvdrodeox\ corticosterone 5P-DHD0C Steraloids P-8120 332.5 48 16, (5a)-Pregnen-3p-ol-20-one 16-Dehydroepipregnanolone 16-deh\dro- Steraloids Q-6650 316.5 epipregnanolone 49 1,4-Pregnadien-6a-methyl-11(3,17,21 -triol-3,20-dione 6a- MEPRED Steraloids P570 374.5 50 1,4-Pregnadien-11(3,17,21 -triol-3,20-dione , 1 -Dehydrocortisol PRED Steraloids P650 360.4 51 4-Pregnen-3,11,20-trione 11-Ketoprogesterone KETOPROG Steraloids Q4160 328.5 52 4-Pregnen-16a-methyl-l7-ol-3,20-dione acetate 16a-Methyl-17-acetoprogesterone 16a-methyl-17- Steraloids Q3021 386.5 acetoprogesterone 53 5-Androsten-3(3-ol-l 7-one sulphate, sodium salt Na-dehydroepiandrosterone sulphate DHEAS Sigma D-5297 390.5 54 1,3,5 f 101 -Oestratriene-3,17P-diol P-Oestradiol OESTR Sigma E-8875 272.4 55 5-Pregnen-3 P-ol-20-one sulphate, sodium salt Na-pregnenolone sulphate PREGS Sigma P-8508 418.6 56 5a-Androstan-l 7(3-ol-3-one 5a-Dihydrotestosterone 5-DHT Sigma A-83 80 290.4 57 5 - Androsten-3,11, 17-trione Androstenetrione Atrione MRC 300.4 58 4-Pregnen-6(3-ol-3,20-dione 6 P -Hydroxyprogesterone 6P-0H-PR0G MRC’ 330.5 59 5 (3-Pregnane-3a, 17,20a-triol 5P-pregnane- MRC^ 336.5 3a,17,20a-triol 60 5 (3-Pregnane-3 a, 1 la,20(3-triol 5P-Pregnane-3a,l la,20P-triol 5p-pregnane- MRC' 336.5 3a,lla,20p-triol 61 1,3,5 [ 101 -Oestr atrien-3 -ol-17-one Oestrone oestrone MRC' 270.4 62 5 (3-Androstan-3a-ol-17-one Etiocholan-3a-ol-17-one etiocholanolone MRC' 290.4 63 5a-Androstan-3a-ol-17-one Androsterone androsterone MRC' 290.4

66 Table 2-1 continued.

No Systematic name Trivial Name Abbreviation Source M r 64 5 P-Androstan-17a-ol-3-one 5 P-Androstan-17a-ol-3-one 5 P-androstan-17a-ol- MRC^ 290.4 3-one 65 4-Pregnen-16a-ol-3,20-dione 16a-Hydrox\progesterone 16a-0H-PR0G MRC’ 330.5 66 4-Pregnen-l 8-ol-3,20-dione 18-Hydroxyprogesterone 18-OH-PROG MRC’ 330.5 67 4-Pregnen-19-ol-3,20-dione 19-Hydrox\progesterone 19-OH-PROG MRC’ 330.5 68 4-Pregnen-2a-ol-3,20-dione 2a-Hydrox\progesterone 2a-0H-PR0G MRC’ 330.5 69 5a-Androstan-3a,l ip-diol-17-one 5 a -Androstan-3 a, 11 P-diol-17-one 5a-androstan-3a,l 1P- MRC’ 306.4 diol-17-one 70 4-Pregnen-3,6,20-trione 6-Ketoprogesterone 6-KETOPROG Steraloids Q4140 328.5 71 4-Androsten-3 P-ol-17-one 3 P-Dihydroandrostenedione 3P-dihydro- Steraloids A6850 288.4 androstenedione 72 5 -Pregnene-3 P, 17,20a-triol 5-Pregnene-3 P, 17,20a-triol 5-pregnene- Steraloids Q5890 334.5 3P,17,20a-triol 73 5-Cholesten-3p-ol Cholesterol CHOL Sigma C-8667 386.7 74 5-Cholesten-3 P-ol 3-butyrate Cholesteryl n-but>Tate CHOL BUT Sigma C-4758 456.8 MRC: Medical Research Council reference steroid collection, London, UK.

67 Table 2-2. GC and scan MS parameter settings in methods used for steroid analysis. Method Scan 1 SIMl Scan 2 Scan 3 Scan 4

Parameter

GC-column CPSil5CB CPSil5CB CPSil5CB ZBI ZBI Autosampler settings Viscosity compensation time (sec) - 0.8 0.8 5 5 Plunger injection speed - middle middle high high Syringe injeetion speed - high high high high GC settings Injection port dwell time (sec) - II 20 20 Injector temperature (°C) 250 250 250 280 250 Injection pressure (kPa) 47 100 400 400 400 Injection time (min) I I 2 4 1 Carrier gas pressure (kPa) 47 80 80 34.2 34.2 Time (minutes) --- 5 2 Rate I (kPa/min) --- 6.5 5.9

Final pressure 1 (kPa) --- 81 79 Final time 1 --- 0.33 0.33

Rate 2 (kPa/min) - - - 1.6 1.5

Final pressure 2 (kPa) - -- 111.7 98.0

Final time 2 - - - 4 5 Flow (ml/min) 1.0 1.5 1.5 0.7 0.7 Total flow (ml/min) 33 60 60 60 60 Linear Velocity (cm/s) 40.1 49.2 49.2 30.7 30.7 Initial oven temperature (°C) 70 70 70 70 70 Initial time (min) 3 3 3 5 2 Rate I (°C/min) 20 20 20 20 20 Final temperature I (”C) 220 220 220 220 220 Final time I (min) 0.33 0.33 0.33 0.33 0.33 Rate 2 (°C/min) 3 3 3 5 5 Final temperature 2 (°C) 280 285 285 315 285 Final time 2 (min) 3 3 3 4 5 Run time (min) 30.8 35.5 35.5 35.8 27.8 Interface temperature (°C) 280 280 285 315 285 MS parameters Deteetor voltage (kV) 2.2 1.9 1.55 1.55 Solvent cut (RI) 2000 2200 2200 2000

Scan parameters - Scan range (m/z) 99-800 50-800 99-800 50-800

Scan interval (sec) - 0.5 0.5 0.5 Threshold 500 1000 1000 1000

Scan speed (amu/sec) - 2000 2000 2000

68 Table 2-3. SIM MS settings of methods used in steroid analysis. For GC-parameters see method referred to in Table 2-2. Method SIM I SIM 2 SIM3 SIM 4 SIM 5 SIM 6 SIM 7 SIM 8 SIM 9 SIM 10 GC-parameters see SIM I see Scan 3 see Scan 3 see Scan 3 see Scan 3 see Scan 3 see Scan 3 see Scan 3 see Scan 3 see Scan 3 SIM parameters Detector voltage 1.6 1.7 1.7 1.7 1.7 1.7 1.7 1.7 1.7 1.6 Microscan width (amu) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Sampling rate (sec) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Ion set I Ions in ion set (m/z) 417.4, 417.40, 476.45, 358.35, 344.30, 389.35, 358.35, 389.35, 416.40, 358.35, 244.20, 244.25, 188.15, 268.25, 313.30, 358.35, 268.25, 358.35, 285.25, 268.25, 415.10, 388.35, 284.25, 360.35, 389.35, 343.30 260.25 268.25 326.30 260.25 400.40 298.30 269.25 270.25 268.25 Ion set end time (RI) 3200 2783 2790 2723 2760 2681 2716 2735 2720 2688 Ion set 2 Ions in ion set (m/z) 415.40, 415.40, 415.40, 415.40, 417.40, 415.40, 388.35, 476.45, 417.40, 384.35, 384.35, 384.35, 384.35, 244.20, 384.35, 298.25, 386.35, 244.20, 346.35, 284.25, 386.35, 343.30, 386.35, 294.25, 404.40 364.35 386.35 269.25 269.25 244.25 275.25 325.30 Ion set end time (RI) 2818 2836 2849 2860 2762 2813 2783 2784 2756 Ion set 3 Ions in ion set (m/z) 402.4, 462.45, 507.50, 372.35, 284.25, 343.30, 415.40, 415.40, 476.45, 386.35, 372.35, 358.35, 341.30, 269.25, 275.25, 384.35, 384.35, 386.35, 388.35, 343.35, 417.40, 303.30, 346.30, 288.25, 294.25, 294.25, 364.35 100.10 275.25 286.25 289.25 415.40, 325.30 325.30 384.35, 294.25, 325.30, 449.45 Ion set end time (RI) 2863 2895 2958 2945 2836 2840 2818 2825 2784 Ion set 4

69 Table 2-3 continued. Method SIM 1 SIM 2 SIM3 SIM 4 SIM 5 SIM 6 SIM 7 SIM 8 SIM 9 SIM 10 Ions in ion set (m/z) 402.40, 476.45, 443.45, 443.45, 462.45, 343.30, 402.40, 388.35, 415.40, 433.40, 188.15, 474.45, 474.45, 372.35, 275.25, 386.35, 298.25, 384.35, 462.45, 417.40, 476.45, 462.45, 332.30 288.25 312.30 404.40 294.25, 372.35 286.25 188.15 431.40 325.30, 284.25, 269.25, 346.35, 449.45 Ion set end time (RI) 2901 2960 3085 3147 2880 2890 2856 2864 2835 Ion set 5 Ions in ion set (m/z) 386.35, 443.45, 443.45, 531.50, 372.35, 303.30, 476.45, 462.45, 372.35, 296.30, 474.45, 474.45, 441.45, 341.30, 289.25, 507.50, 372.35, 462.45, 474.45, 462.45, 517.50, 513.50, 273.25 314.30 404.40, 332.30 332.30 362.35 431.40 427.45 603.60 386.35 Ion set end time (RI) 2944 3042 3238 3400 2914 2943 2950 2901 2890 Ion set 6 Ions in ion set (m/z) 429.45, 286.25, 513.50, 417.40, 429.40, 443.40, 296.25, 296.25, 339.35, 273.25, 603.60 301.30, 339.30, 474.45, 386.35, 386.35, 443.45, 370.35, 296.25 460.45, 353.35, 239.20, 239.20 474.45 339.30, 443.40, 384.35 474.45, 474.45, 362.35, 353.35, 384.35 384.35 Ion set end time (RI) 3142 3179 3400 2958 3051 3024 2929 2945 Ion set 7

70 Table 2-3 continued. Method SIM I SIM 2 SIM3 SIM 4 SIM 5 SIM 6 SIM 7 SIM 8 SIM 9 SIM 10 Ions in ion set (m/z) 513.50, 513.50, 443.40, 286.25, 476.45, 460.45, 443.40, 603.60, 603.60, 474.45, 398.35, 404.40, 445.40, 474.45, 515.50, 548.55, 353.35, 273.25 507.50 413.40 353.35, 605.60 517.50 384.35, 384.35 462.45, 431.40, 288.25 Ion set end time (RI) 3400 3400 3054 3189 3120 2960 3024 Ion set 8 Ions in ion set (m/z) 339.30, 513.50, 474.45, 443.40, 476.45, 370.35, 603.60, 443.40, 474.45, 404.40, 361.35 482.45, 300.30 353.35, 507.50 634.60 384.35 Ion set end time (RI) 3196 3400 3238 3031 3163 Ion set 9 Ions in ion set (m/z) 513.50, 513.50, 462.45, 513.50, 603.60, 603.60, 431.40, 603.60, 482.45, 482.45, 288.25 482.45, 634.60 634.60 634.60 Ion set end time (RI) 3400 3400 3159 3400 Ion set 10 Ions in ion set (m/z) 548.50, 517.50, 427.40 Ion set end time (RI) 3269 Ion set 11

71 Table 2-3 continued. Method SIM I SIM 2 SIM3 SIM 4 SIM 5 SIM 6 SIM 7 SIM 8 SIM 9 SIM 10 Ions in ion set (m/z) 513.50, 603.60, 482.45, 634.60 Ion set end time (RI) 3400

Method SIM 11 SIM 12 SIM 13 SIM 14 SIM 15 SIM 16 SIM 17 SIM 18 SIM 19 SIM 20 SIM 21 GC-parameters see Scan 3 see Scan 3 see Scan 3 see Scan 3 see Scan 3 see Scan 4 see Scan 4 see Scan 4 see Scan 4 see Scan 4 see Scan 4 SIM parameters Detector voltage 1.7 1.7 1.7 1.7 1.7 1.7 1.7 1.7 1.7 Microscan width (amu) 0.0 0.0 0.0 1.7 1.7 0.0 0.0 0.0 0.0 0.0 0.0 Sampling rate (sec) 0.2 0.2 0.2 0.0 0.0 0.2 0.2 0.2 0.2 0.2 0.2 Ion set 1 0.2 0.2 Ions in ion set (m/z) 360.35, 389.35, 476.45, 358.35, 417.40, 680.30, 298.25, 680.30, 298.25, 270.20, 298.25, 270.25, 358.35, 364.35, 268.25, 312.30, 451.15, 283.30, 451.15, 283.30, 255.20, 283.30, 376.35 268.25 507.50 260.25 326.30 127.25, 127.25, 127.25, 486.25, 127.25, 127.25, 141.25, 141.25 141.25, 442.25, 141.25 141.25 270.20, 270.20, 127.25, 255.20 255.20 141.25 Ion set end time (RI) 2699 2737 2752 2692 2756 2487 2515 2487 2517 2487 2517 Ion set 2 Ions in ion set (m/z) 388.35, 388.35, 388.35, 388.35, 476.45, 429.25, 496.35, 429.25, 496.35, 429.25, 496.35, 298.25, 298.25, 298.25, 298.30, 296.30, 712.45, 300.25, 712.45 514.40, 712.45 514.40, 404.4 404.40 404.40 404.40 364.35 496.35, 712.45, 300.25 300.25 514.35 697.45 Ion set end time (RI) 2766 2780 2783 2769 2784 2555 2586 2531 2563 2531 2563 Ion set 3

72 Table 2-3 continued. Method SIM 11 SIM 12 SIM 13 SIM 14 SIM 15 SIM 16 SIM 17 SIM 18 SIM 19 SIM 20 SIM 21 Ions in ion set (m/z) 284.25, 415.40, 415.40, 269.25, 415.40, 496.35, 296.25, 496.35, 469.25, 697.45, 712.45, 269.25, 384.35, 384.35, 449.45, 384.35, 469.25, 253.30, 712.45, 512.30, 712.45 697.45 346.30 294.25, 294.25, 284.25 284.25, 512.30, 496.35, 697.45, 712.45, 325.30 325.30 269.25, 514.35 481.20 514.35 697.45 346.35 Ion set end time (RI) 2791 2818 2825 2791 2804 2585 2625 2585 2586 2585 2586 Ion set 4 Ions in ion set (m/z) 415.40, 402.40, 388.35, 415.40, 386.35, 296.25, 442.05, 296.25, 296.25, 296.25, 296.25, 384.35, 386.35, 298.25, 384.35, 417.40, 253.30, 487.20, 253.30 253.30, 253.30 253.30, 294.25, 312.30 404.40 346.35, 312.30 708.25, 708.25, 496.35, 496.35, 325.30 269.20, 693.30 693.30, 481.20 481.20 284.25 496.35, 481.20 Ion set end time (RI) 2808 2871 2877 2818 2830 2622 2683 2622 2625 2622 2625 Ion set 5 Ions in ion set (m/z) 386.35, 303.30, 476.45, 296.30, 388.35, 510.35, 499.20, 298.25, 442.05, 298.25, 442.05, 244.20, 289.25, 507.50, 386.35, 298.25, 495.30, 257.25, 283.30 487.20, 283.30 487.20 417.40 314.30 404.40 312.30 404.40 298.30, 514.35 496.35, 283.25 481.20 Ion set end time (RI) 2839 2943 2918 2871 2877 2649 2734 2652 2689 2652 2689 Ion set 6 Ions in ion set (m/z) 507.50, 429.45, 507.50, 476.45, 303.30, 467.20, 528.40, 442.05, 499.20, 467.25, 499.20, 476.45, 339.30, 476.45, 507.50, 288.25, 514.40 467.25, 469.25, 514.35 514.35 514.35 386.35 460.45, 404.40 386.35, 314.30 722.30, 467.25, 443.40, 404.40 707.35, 514.35 474.45, 127.25, 353.30, 141.25 384.35 Ion set end time (RI) 2947 3051 2950 2950 2945 2675 2813 2675 2758 2675 2758

73 Table 2-3 continued. Method SIM II SIM 12 SIM 13 SIM 14 SIM 15 SIM 16 SIM 17 SIM 18 SIM 19 SIM 20 SIM 21 Ion set 7 Ions in ion set (m/z) 443.40, 286.25, 443.40, 443.40, 443.40, 499.20, 528.40, 499.20, 722.30, 499.20, 722.30, 474.45, 398.35, 474.45, 474.45 474.45 257.25 467.25, 257.25 707.35, 471.35 707.35, 353.35, 273.25 353.35, 526.40, 127.25, 127.25, 384.35 384.35 493.30 141.25 141.25 Ion set end time (RI) 3054 31II 3142 3024 3051 2725 2907 2729 2814 2729 2814 Ion set 8 Ions in ion set (m/z) 339.30, 517.50, 513.50, 476.45, 429.40, 526.40, 738.20, 526.40, 526.40, 526.40, 526.40, 370.35, 427.40, 603.60, 386.35, 460.45, 465.10 491.30 465.10 493.30 465.10 493.30 361.35 548.50 482.45, 507.50 286.25 634.60 Ion set end time (RI) 3192 3202 3400 3085 3189 2781 3100 2781 2880 2781 2880 Ion set 9

Ions in ion set (m/z) 513.50, 513.50, 517.50, 513.50, 499.20, 499.20, 499.20, 603.60, 603.60, 445.45, 603.60 257.25, 257.25, 471.35, 482.45, 482.45, 427.40 301.20, 127.25, 127.25, 634.60 634.60 255.15, 141.25 141.25 127.25, 141.25 Ion set end time (RI) 3400 3400 3235 3400 2852 2900 2900 Ion set 10 Ions in ion set (m/z) 513.50, 736.30, 603.60 595.35, 301.20, 255.15 Ion set end time (RI) 3400 3100

74 Table 2-3 continued. Method SIM 22 SIM 23 SIM 24 SIM 25 GC-parameters see Scan 3 see Scan 3 see Scan 3 see Scan 3 SIM parameters Detector voltage 1.7 1.7 1.7 1.7 Microscan width (amu) 0.0 0.0 0.0 0.0 Sampling rate (sec) 0.2 0.2 0.2 0.2 Ions in ion set (m/z) 638.60, 608.60, 593.60, 636.60, 607.60, 591.60, 610.60, 605.60, 590.55, 609.60, 595.60,581.55, 579.55, 567.55, 562.55, 578.55.566.55.561.55, 577.55.565.55, 560.55, 576.55, 564.55, 559.55, 552.55, 538.50, 531.50, 550.55, 536.50,548.55, 530.50, 534.50, 524.50,547.50, 533.50, 522.50, 100.15, 517.50,504.50, 520.50,516.50,503.50, 519.50,507.50,502.50, 518.50.505.50, 493.50, 491.50, 479.45,474.45, 490.45.478.45.473.45, 489.45, 477.45, 472.45, 488.45.476.45.475.45, 464.45,457.45,445.45, 462.45, 450.45,460.45, 444.45, 448.45, 443.45, 471.45.459.45.446.45, 436.40, 431.40, 426.40, 434.40, 430.40,419.40, 433.40, 429.40,417.40, 442.40, 432.40, 428.40, 415.40, 403.40, 397.40, 405.40, 402.40, 391.40, 414.40, 401.40,389.35, 416.40, 400.40, 388.35, 387.35.383.35.369.35, 386.35, 374.35, 360.35, 385.35, 372.35, 358.35, 384.35,371.35,357.35, 356.35.344.35, 338.30,355.35, 343.30, 329.30, 354.35.341.30, 327.30, 346.30, 340.30, 326.30, 315.30, 188.15 313.30,175.15 309.30, 129.15 295.30, 117.15 End time (RI) 3400 3400 3400 3400

75 Chapter 3 Development of a method for the analysis of steroids from mammalian brain tissue by gas chromatography - electron impact ionisation mass spectrometry (GC-EiMS)

3.1 Introduction

Steroids are frequently measured in biological materials using radioimmunoassays (RIA), with which high sensitivity in analysis can be achieved. Antibodies against neuroactive steroid metabolites have been raised [151], and assays have been useful in quantitation of usually individual or a few metabolites in brain tissue of mouse [47], rat [23,82,202] and human [22,98] from different physiological states. Unequivocal identification of steroids has, however only been achieved in conjunction with methods such as GC-MS [48,49]. RIA uses binding of radioactively labelled steroids to antibodies and competition of unlabelled steroids for the binding sites to estimate concentrations. Identification thus relies on the antibody specificity to the analyte. The high number of very similar steroid molecules frequently infers cross-reactivities of the antibodies (see for example [20,50,151]). Most frequently, steroids in a sample are separated by chromatographic methods such as celite partition or high performance liquid chromatography prior to assay by RIA to increase specificity (see Chapter 4, Introduction). Once the identities of compounds in a biological matrix have been established and possible interferences characterised, quantitations of those compounds can be carried out by RIA, which have the advantage of simplicity and speed in terms of analysis and sample preparation required compared with mass spectrometric methods. This Chapter describes a method which can be applied in the identification and assay of steroids in brain tissue (see Chapter 5).

Mass spectrometry (MS) is the technique of choice for unequivocal identification of steroids. There have been large numbers of reports of mass spectra of steroids which have led to characterisation of general steroid fragmentation pathways ([179] for review). These studies were mainly undertaken with electron impact ionisation (El). At the same time several different ionisation techniques including chemical, photo, field and fast atom bombardment ionisation have been developed, resulting in a widening of applications in

76 steroid analysis. Biological samples are usually too complex in composition for direct inlet MS and normally require prior fractionation by gas chromatography (GC) or high performance liquid chromatography (HPLC). These techniques will briefly be described here.

3.1.1 Gas chromatography

Steroids are usually derivatised to protect thermally labile compounds at the elevated temperatures in GC and may also avoid tailing of polar compounds or increase the response of a compound in the detection. Methods most commonly used for derivatisation of compounds for GC are silylation, alkylation, oxime formation, and acylation. The most versatile silylation reagent for steroids is trimethylsilylimidazole (TMSI). TMSI reacts with hydroxyl groups to form ethers and is usually used in combination with methoxyamine hydrochloride (MO), which reacts with keto-functions to give oximes [35]. TMSI on its own can also be used to react with keto-functions, forming enols. MO derivatisation has the disadvantage of formation of syn/anti -isomers that can separate in GC. Hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS) are milder silyl donors and are only used for few steroids. Higher alkyl- dimethylsilyl (e.g. ré?r/-butyldimethylsilyl (TBDMS)-donors) derivatising reagents have been investigated for use in steroid analysis. They react with hydroxyl groups to give TBDMS ethers, ketone functions react after énolisation [154,155], These TBDMS- derivatives are less susceptible to fragmentation in MS and give few highly abundant ions. This makes them useful for quantitation in high sensitivity analyses. However there is a loss in structural information and identification is less specific. Sensitivities achieved were reported in the range of 1 (testosterone) and 5 pg (progesterone) in analyses of TBDMS-derivatives with GC-EIMS [5].

Acylation with fluoro-organic acids has also been employed for high sensitivity steroid analysis (e.g. trifluoroacetic (TFAA), pentafluoropropionic (PFPA) and heptafluorobutyric acid (HFBA) (used in anhydride form)). These compounds are highly electronegative and are thus especially useful for electron capture detection and negative ion chemical ionisation (NICI) MS. In BIMS, they show little fragmentation and have prominent molecular ions. They also react with keto-groups to form enols. The solvent used with these reagents plays a reaction-modifying role. Using derivatisation by HFBA in ethyl acetate, PREG, PROG, 3a, 5a- and 3a,5p-THPROG could be detected in

77 extracts of cerebrospinal fluid by GC-NICIMS [195]. Several oestranes, androstanes and pregnanes could be derivatised with HFBA in acetonitrile at 80°C [32]. Cortisol [191], PREG, DHEA, PROG and 3a,5a-THPROG [106] were derivatised with HFBA by using acetone as solvent at room temperature. Several corticosteroids could successfully be derivatised using benzene as solvent and heating at 60°C [118]. Sensitivities achieved were 5 pg for the pregnanes in GC-NICIMS [195], 30 pg for cortisol by GC-EIMS [191] and between 0.5-2.5 pg for the corticosteroids by GC with electron capture detection [118].

Steroid conjugate derivatisation has been extensively investigated. Steroid sulphate conjugates were found to derivatise with TMSI and HFBA by exchange with the sulphate groups [171,192]. The resulting derivatives can be analysed as their respective free steroids. Steroids are also derivatised after cleavage of the sulphate groups prior to derivatisation with chemical hydrolysis (see Introduction, Chapter 4). A microsolvolysis method in a single step with MO/TMSI derivatisation has been described for sulphate esters of oestrogens [169]. MO in pyridine was added directly after incubation of the steroids in a small volume of acidified ethyl acetate. The yields were found as high as after conventional solvolysis and derivatisation. Steroid glucuronides can be derivatised after méthylation of the acid moiety with MO at ketone functions and at the hydroxylgroups with TMSI [169]. A direct derivatisation method using TBDMS donors was also devised [185].

GC-columns

Early problems in wall coated open tubular (WCOT) capillary columns of adsorption and stationary phase contraction have been overcome through surface deactivation and the introduction of fused silica as column support material, which has a more consistent composition than previously used materials. WCOT columns have low theoretical plate heights due to uniform mass transfer path lengths and high mass transfer rates. Furthermore, the small drop in pressure allows the use of long columns, which have higher numbers of theoretical plates. A large variety of stationary phases is available for GC. They are usually based on liquid polymers of methyl siloxane or ethylene glycol. To immobilise the phase in the column, increasing reproducibility of performance and life­ span, chemical bonding to the column material is now usually applied. Special low bleed columns, caused by cross-linking of the stationary phase to the support are now also

78 available for sensitive detectors such as MS. Such columns are used in the present study with CPSEL5 CB or equivalent stationary phases.

Samples in solvent can be applied to the GC-column in the split-, splitless- or on-column modes. In split injection, the injection volume (typically 1-2 pi) is reduced after evaporation in the heated injection chamber (typically 250-300°C) as the injector is constantly purged with a subsidiary carrier gas stream. A split-ratio of more than 100 is usually applied. The main disadvantage of this technique is the great loss of sample. In most cases this does not present a problem, except in trace analyses. A method allowing the great majority of the sample to be transferred onto capillary columns is the splitless technique devised by Grob [67]. A similar injector system as in split injection is used, but the split valve is initially kept closed. After this initial period, the injector is purged with the carrier gas stream to avoid tailing effects in the chromatogram and build up of sample residue in the injector. The technique uses a solute focusing effect that occurs as the solvent recondenses in the initial stretch of column that is held below the temperature of the sample solvent boiling point. Also for trace analysis, on-column injection can be performed, introducing the whole sample in liquid form into the column.

The most commonly used detector in capillary GC is not MS but the flame ionisation detector (FID) which has the widest range of applications. This GC method is usually used for quantitations in metabolic profiling of known urinary steroids [76]. Additional detectors for specific groups of compounds on GC are electron capture, phosphorus and flame photometry detectors.

3.1.2 High performance liquid chromatography

The use of high performance liquid chromatography (HPLC) for steroid separations is covered in Chapter 4. As opposed to capillary GC, the effluents from HPLC are not easily compatible with mass spectrometry for obvious reasons. Systems used for interfacing are, for instance, the moving belt, where the HPLC solvent is sprayed onto a heated belt that moves through zones of decreasing pressure. In the ion source, solutes are desorbed from the belt and subjected to various ionisation methods as described below [170]. Another interfacing method, thermospray ionisation is described in 3.1.3.

79 3.1.3 Mass spectrometry

Mass spectrometry can be principally described as a system in which molecules are first ionised, then the ions separated according to their mass-to-charge ratio (m/z) and finally their abundance measured.

In an electron impact (El) ion source, molecules cross a beam of electrons in an evacuated chamber. Electrons are emitted from a heated filament and consequently accelerated into a helical movement across the chamber by a voltage and a transverse magnetic field. The ionisation efficiency in El is about 1 in 1000 molecules at an electron energy of 70 eV, to which it is set by convention. At this energy ionisation efficiency has usually reached a plateau [128]. A high degree of fragmentation generally results from EL The resulting mass spectra are considered to be very specific. However, the degree of fragmentation depends also on the nature of the derivative of a compound (see e.g. [25]). Electrons interacting with neutral sample molecules transfer some of their kinetic energy to the molecules. Energy absorbed by the molecules leads to a transition to higher vibrational excitation states and/or higher energy ionised states. The excess energy can be higher than the energy difference needed for dissociation, thus this process can occur straight away. If the vibrational state coincides with the energy level of a different ion state, transition to this state can occur. The energy level in the different ion state might be above the dissociative energy. Some absorbed energy also is transferred to electrons in lower orbitals which are consequently excited into higher orbitals. The energy can be lost by emission of radiation or by transfer into vibrational energy upon relaxation of the electrons back into lower states. This interaction between electrons and molecules can be considered as the sole process of ionisation and fragmentation, molecule/molecule and molecule/ion interactions are very rare in El [164]. An example of the presumed EI- fragmentation of methoxyamine-trimethylsilyl-PREG is shown in Figure 3-7.

The fragmentation process can be generally schematically represented as follows;

ABC ABC^ (ionisation)

ABC^ -> AB^ + C (fragmentation)

AB + C (fragmentation)

AC + B^ (fragmentation and rearrangement)

80 AB" ^ A" + B

etc.

In calculating bond densities and orders, the weakest bonds can be found and in El these usually are cleaved first. For example, in oestrone the aromatic ring and the carbonyl function form the strongest bonds and thus are not prone to cleavage [105]. The fragmentation pathways have usually been empirically investigated by labelling specific positions in molecules with heavy isotopes (^H, ^^C). Generally the molecular ion is considered to be in the same configuration as the neutral molecule and daughter ion structures can be derived from simply splitting the parent ion in the appropriate position. However, there are several cases (mainly with aliphatic and aromatic ketones) where rearrangements of fragments occur (McLafferty-rearrangements) [179]. Also, in steroids derivatised with trimethylsilyl-groups migration of these groups has been observed [180]. Furthermore, hydrogen rearrangements occur non-specifically.

In chemical ionisation (Cl), molecules interact with ionised molecules of reactant gases that are produced by El. The kinetic energies transferred to the analyte molecules are much smaller than in El, resulting in less fragmentation. The softer ionisation leads to relatively more abundant molecular ions, which is of advantage when monitoring selected ions in analyses where increased sensitivity is required. Single ion monitoring with this method used in GC-MS has allowed analysis of 3a,5a-THPROG, e.g. in rat brain with high sensitivity [194,196].

Atmospheric pressure chemical ionisation (APCl) utilises carrier gas (GC) or liquid (HPLC) ionised by a corona discharge to ionise sample molecules [77]. This method lends itself to analysis of steroids in biological samples without extensive prior purification. Furthermore, it is possible when used in conjunction with HPLC to directly analyse conjugated steroids without prior cleavage of the conjugate group. For instance, PREG and PREG stearate were determined by this method in extracts from rat brain [174].

Another method with potential application in steroid MS resulting in high sensitivity is fast atom bombardment ionisation (FAB). This method also allows analysis of intact steroid conjugates, thus simplifying and reducing sample fractionation and purification. Corticosteroid mono- and diglucuronides [120] and several steroid glucuronides and

81 sulphates [171] could be analysed by FABMS using glycerol as the sample matrix. The latter study demonstrated resolution of mixtures of steroids in the same mass spectrum with this technique. However, this technique has the disadvantage that isomers would interfere in each other’s analysis. Complex biological samples are probably not suitable for this method.

In electrospray ionisation (ESI), an effluent of sample solution in a polar solvent (e.g. HzO/methanol) is nebulised through a jet assisted by a coaxial gas stream [62]. A strong electrical field causes the liquid droplets to become highly charged on the surface, as they get smaller and smaller. Ionisation occurs probably as the highly charged solvent evaporates from droplets containing few or even single sample molecules. This method has the advantage that it is applicable to highly polar and non-volatile compounds (i.e. steroid sulphates/glucuronides) [175]. Due to the possibility of introduction of polar liquids into the ion source this method is particularly suited for combination with HPLC.

Decreasing the carrier solution flow (-20 nl/min) and the capillary diameter results in decreased fragmentation and higher responses of individual ions in nanoelectrospray ionisation (nanoESI). For analysis of steroid sulphates, extremely high sensitivities have been reported in the femtomolar [66] and even attomolar range [37]. The method was used in tandem MS mode. However, free steroids need to be sulphated as only negatively charged molecules can be determined. Free steroids derivatised to their oximes were also analysed with nanoESI-MS with slightly higher detection limits [107].

In thermospray ionisation liquid sample is introduced into the ion source through a nozzle. The sample solvent and contained buffer salts are used to create chemical ionisation upon evaporation. Low fragmentation results from the process. The method also allows liquid chromatography to be combined to MS [24].

There are several further ionisation methods including matrix-assisted laser desorption ionisation (MALDI), field desorption, desorption chemical ionisation, plasma desorption, they have found less application in steroid analysis though [135].

Mass analysis

Ions formed in the ion source are extracted through electrical potential differences, then focused and finally accelerated by another potential difference into the mass analyser. In a quadrupole mass separator, superimposed electrical fields determine the path of the

82 ions (see Figure 3-1). The 4 rod electrodes are arranged diagonally opposed. DC and radio frequency voltages are applied between adjacent rods, with opposite rods electrically connected. In the resulting field, ions entering the quadrupole are oscillating in X - and y-direction, with only one m/z ratio following a stable path at certain DC and RF-voltages and reaching the electron multiplier for detection. Ions of other m/z ratios follow unstable trajectories and leave the quadrupole before the ends. Mass scanning is achieved by varying the DC and RF voltages while keeping their ratio constant. Advantages of quadrupole mass separators are that high speeds of mass scanning are possible and ease of rapid switching between selected discrete m/z ratios, which is useful in selected ion monitoring (SIM see below). However, they can only be used in low resolution MS [135].

Magnetic sector instruments use the principle of charged particles of a certain kinetic energy being deflected in a magnetic field according to their mass to charge (m/z) ratio. By variation of the magnetic field strength, different m/z over time are analysed in the detector fixed in one position. Magnetic sector instruments in conjunction with electrostatic deflectors (double focusing instruments) are used in high resolution mass spectrometry (HRMS) [170]. Ion trap mass separators are similar to quadrupole MS, except that here all ions are held between a set of hyperbolic electrodes and only ions whose trajectories are made unstable reach the electron multiplier. Masses are scanned by changing a RF voltage applied to a ring electrode around the ion trap, making sequential m/z ratio trajectories unstable. The advantage of this technique compared to quadrupole MS is that potentially all ions produced are detectable and thus a highly increased sensitivity, especially in the total ion monitoring mode is achieved [117]. Further mass analysing methods are time-of-flight (TOF), which is mostly used in conjunction with MALDI, and Fourier transform MS.

Ions produced by the ion source and separated in the mass analyser are most widely detected in an electron multiplier detector. In the latter, impact of ions in the first dynode results in the emission of electrons. The electrons are then accelerated by an applied voltage through a chain of dynodes, resulting in a great amplification of the initial electron current. The dynodes are usually made from beryllium-copper. The electron multiplier voltage applied in El-quadrupole MS is usually in the range of 1-3 kV [36].

83 Tandem mass spectrometry (MS-MS) basically consists of two MS in series. Instead of leading ions into the detector they are guided into another ionisation process from a first mass sorting, and resulting secondary ions are separated and finally analysed in a detector. Thus information is gained about both precursor and successor molecules of a fragment. In principle any combination of MS can be used, if ionisation in the second step occurs in the gas phase. In practice, collision induced dissociation is frequently used to yield product ions, which are separated and detected by various methods. The process of linking MS in series can be continued further (MS"). The methods give increased specificity and can be applied for samples with complex matrices, direct analysis of compounds in a mixture or analysis of fragmentation pathways [135]. GC-MS-MS has been applied to determine PREG and DHEA in rat brain [177].

Selected ion monitoring (SIM)

SIM can be used instead of scanning, if higher sensitivity is required. A prerequisite is the knowledge of the mass spectral properties of a compound in a particular ionisation mode. SIM requires a mass analysing system that can be set quickly to selected discrete m/z values. In quadrupoles this can be achieved and magnetic sector instruments are also suitable for this purpose, but often in a restricted mass range. For selection of ions, such m/z values should be chosen at which interferences from electrical noise and from sample background are minimal. Where possible, sample blanks should be investigated prior to setting up the SIM method. If interferences at certain m/z values cannot be avoided, higher sample purification can be considered. Generally, fragments of high m/z (>300) are less likely to show interferences from chemical background due to the nature of the fragmentation process. Derivatisation can be utilised to achieve sufficient numbers of ions in this mass area. There are several derivatisation reagents causing high mass increments, examples of which were mentioned above (see 3.1.1). In any case, ions carrying a major portion of the total ion current (TIC) are preferable for SIM, as they give higher sensitivity in the assay. In positive or negative ion Cl modes fewer fragmentations occur than in El and thus ions with higher proportions of the ion current can be obtained. Thus these ionisation methods are especially well suited for SIM. They are, however, less specific than methods where more diagnostic ions are obtained.

84 3.1.4 Mass spectrometric identification

There have been extensive studies on mass spectral fragmentations of organic molecules since the introduction of the technique which can be used as a tool in identifying structural elements of unknowns in the scanning mode (for review see [179]). Molecular ions are often not abundant in EI-MS, and thus for this identification help other ionisation methods are useful in addition (e.g. Cl), as the high degree of fragmentation in El is very informative. Mass spectral identification in the scanning mode is now often achieved by comparison with spectra of standard compounds. There are large databases of mass spectra (-200 000 compounds) which can be consulted for comparison.

In order to decrease the limit of detection in many quantitative MS analyses, SIM has to be employed. Identification in SIM is based on monitoring of diagnostic ions. There is a wide variety of approaches on how identification is finally performed, including single, two, three and four or more ion monitoring, with and without the control of relative ion abundances (e.g. [49,106,119,194,196] all using GC-MS). The approach also depends on the context of the analytical method. A systematic approach to evaluate the specificity of SIM was used by Sphon [184]. A rising number of ions was included for a compound and it was investigated how many compounds out of a large mass spectral database were identified when those ions were monitored. The relative abundance ratios of those ions were also looked at with decreasing windows of variability. It was shown that by monitoring three ions and including relative ion abundance variabilities of ±10%, only the target compound fell within those criteria. This study was later applied to updated much larger mass spectral databases (-60 000 spectra) by the same author and by others with the same criteria except applying relative ion abundance variabilities of ±20% on several compounds and gave the same result [9,207,208]. However these criteria only consider the mass spectrometric endpoint measurement. In practice often GC or LC are used in conjunction with MS. This infers additional specificity due to the high resolving power, especially in the case of GC. Furthermore, the preliminary sample preparation steps can provide varying degrees of selectivity, with solid phase extraction one of the most specific methods [9]. A further accepted method for definitive identification is to use two ion monitoring of derivatives of two different derivatisation methods.

These methods are also the basis of identification (together with retention time criteria, see below) in the detection of illegal hormone residues in cattle using low-resolution GC-

85 MS as outlined in the regulations of the European Union [1], one of the few regulatory frameworks to exist on the issue of chemical identification of substances in biological materials.

In GC-MS the retention volume or time of compounds on gas chromatographic columns can be used for further identification. However, the retention time (RT) is not only dependent on the structure of a compound and column stationary phase, but also on column temperature and carrier gas pressure. Small variations in these parameters can cause RT variations even between consecutive analyses on the same day with the same settings of the instrumental parameters. For qualitative purposes, it is thus better to use relative RTs, such as the retention index (RI) [94], sometimes expressed in methylene units, which are the RI divided by a factor of 100. This provides a linear relation between the logarithm of the retention time and the number of carbons in molecules of the alkane series (see Section 2.4.2). Relative retention time (RRT) provides a linear relation between the retention times of two compounds. They are somewhat more pressure and temperature dependent than the RI. Criteria for positive identification using RRT are again not unique across the field. The criterion specified by the EU on detection of illegal hormone residues in cattle using low-resolution GC-MS [1], is for the RRT of the unknown to lie within ± 0.5% of the RRT of a reference compound analysed under the same conditions.

3.1.5 Quantitative mass spectrometry

Calibration

To reliably quantify compounds by GC-MS, the internal standard method has to be used with appropriate compounds. This is necessary to correct for variabilities in the injection volumes and transfer of gas phases from the injection port onto the GC-column [128]. There are two main types of internal standard (IS). Compounds of the first type are isotopically labelled analogues to the compound of interest. The second type of internal standards are isomers or homologues of the compound of interest not endogenously occurring in the sample matrix. In the first category, stable isotope labelled compounds are mostly used. They are considered to have identical response factors to the compounds of interest in mass spectrometry when used in the same order of magnitude of concentration (within of approximately 10). A further advantage of these compounds

86 is their near identical behaviour in the sample preparation and GC-separation steps and can thus be used to correct for recovery losses [128]. There have also been reports of carrier effects of the labelled compounds, resulting in increased sensitivity [179]. The main disadvantages of these types of IS are that they are expensive and only few isotopically labelled steroids are commercially available. Especially for complex steroid mixtures as under investigation here, the use of this type of IS is thus not practical. Within the second class of IS it has to be aimed to find closely related but not naturally occurring compounds to the analytes of interest. However the behaviour of those compounds is difficult to predict and it has thus to be carefully investigated what isomers or homologues of compounds allow quantitation with sufficient reproducibility.

Limits of detection

The level of detectability of a compound in GC-MS is determined by electrical noise, sample background, column and interface adsorption, background introduced by the column stationary phase and by reduction of sensitivity in the ion source through the carrier gas [36,128]. In the mass spectrometric practice, there is wide variability in the determination of analytical detection limits. Frequently, responses of peaks of compounds of interest are compared to variability of the baseline in nearby regions of the chromatograms (signal to noise ratios, S/N) and the concentrations at defined S/Ns used as detection limits. S/Ns of five [191,194], three [40,87], 2.4 [211], two [5], etc. have been reported. Sometimes the variabilities of blank matrices are determined for calculation of the detection limit. Three standard deviations of the blank matrix [91] or a fixed factor of 2.5 x blank signal have been employed [32]. The EU states in its regulations for growth promoting residue analysis from cattle that S/N of 3 has to be used or if it is not possible to determine, to use the mean plus 3 standard deviations of the signals from at least 20 blank determinations as detection limit [1].

Ion statistics, chemical, electrical background etc. translate probabilistically into a detection limit as follows. The baseline noise signals are usually considered as normally distributed. The variability of the noise can be expressed as a (or standard deviation) and the detection limit determined by multiplying with a factor depending on the level of confidence achieved. Thus 95% confidence is achieved with a factor of 1.65, 99% with 2.32 and 99.86% with a factor 3.0 x a. In the latter case, the probability that a signal caused by the blank is considered as a signal caused by a sample component is 0.14 %. A

87 factor of 3.0 X a has been proposed by Kaiser for general use in calculations of detection limits [85]. It has been confirmed later by various investigators and extended to limits of quantitation. To reduce the error of quantitation it was proposed to use a limit of quantitation of > 10 x c, with the area 3-10 x a being used for detection only [108].

3.2 Results and discussion

3.2.1 Development of a method for derivatisation of steroids to methyloxime trimethylsilyl ethers for gas chromatography-mass spectrometry in the electron impact mode

As a first step towards the development of a method for determination of brain steroids by GC-EIMS, a wide range of free steroids which could be encountered in mammalian tissue were derivatised by formation of the methyloxime trimethylsilyl ethers. The steroids are listed in Chapter 2, Table 2-1, compounds 1-50. This range of steroids was chosen from products of known enzymes in the mammalian CNS and peripheral glands (see Figure 1-2), their isomers (e.g. 3(3,5P-THDOC) as well as possible products of enzymes from other pathways even though they have not yet been demonstrated in brain (e.g. 5a-pregnan-3a,l ip-diol-20-one, 5P-pregnane-3a,20a-diol). Also shown in the Table are non-biological steroids used as internal standards for the determination of detection limits and quantitation of endogenous compounds (see below). The other compounds in the Table were used as standards for comparison in the screening of unknown compounds by multiple ion screening later in this study (Chapter 5).

After formation of the methyloxime derivatives of keto-groups (except C-11), hydroxyl­ groups react with TMSI to form the silyl ether. In a pilot experiment, 20a-DHPREG (1 jUg) was reacted with MO (200 p,l pyridine solution) for 1 h at 60°C and TMSI (100 jil) for 3h at 100°C. TMSI is not volatile. Thus this and other excess reagents were removed by Lipidex 5000® chromatography [8]. The steroid was loaded and eluted with 2 ml cyclohexane: pyridine hexamethyldisilazane 98:1:1 (v/v/v, CPH). An aliquot corresponding to 50 ng steroid was injected onto the GC-MS (Figure 3-2). A single peak was observed. Other steroids (e.g. PROG) gave low responses when derivatised in this way. Lipidex 5000® conditions were then evaluated for all compounds under investigation in this study (Chapter 2, Table 2-1, compounds 1-50). Each steroid (1.25 pg) was derivatised by MO and TMSI as above for 20a-DHPREG. The residue was dissolved in CPH and loaded onto Lipidex 5000® columns. The columns were eluted with 5 ml of the same solvent with 1 ml fractions collected sequentially for separate GC- analysis. After addition of 250 ng tetracosane, the fractions were dried down, reconstituted in cyclohexane and aliquots injected onto the GC-MS. Area ratios to tetracosane were compared to their total from all elution fractions for each compound to give the elution profiles shown in Figure 3-3. It was found that 3 ml CPH is sufficient to elute all steroids of interest with at least 97% of the total eluted from the Lipidex columns. After 2 ml elution volume, different proportions of different compounds had eluted. For some compounds, only 30% could be recovered after 2 ml CPH, whereas the yield was above 70% for others. Among the later eluting compounds were the less polar PROG and 5-reduced PROG. More polar compounds such as the THDOC isomers were retained less strongly. For representative compounds, it was investigated whether the elution in the above Lipidex purification procedure matched the absolute recovery of MO-TMSI derivatised steroids. Radiolabelled tracers (41000 dpm ^H- CORT and 200000 dpm ^H-PROG) were thus derivatised with MO-TMSI and purified by Lipidex. Aliquots of radioactivity were counted before derivatisation and after Lipidex chromatography. After elution of Lipidex 5000® columns with 3 ml CPH, 108 ± 2.3 % ^H-CORT and 78.5 ± 10.8 % ^H-PROG (mean ± SD, n=3 and n=4, respectively) were recovered. From the experiments with unlabelled and labelled reference compounds it was concluded, that the method with these conditions can be used for derivatisation with high yield of a wide range of steroids. They span a wide range of polarity and it is assumed most of the other compounds of interest show similar behaviour. No residue was visible after drying down of the eluate. Thus it was assumed that involatile excess derivatisation reagents are successfully removed by the applied procedure.

3.2.2 Optimisation of gas chromatographic and mass spectrometric conditions for analysis of steroid MO- and TMS- derivatives

GC-MS conditions

In order to be able to quantify simultaneously as wide a range of steroids as shown in Table 2-1 from brain tissue, an analysis method of the highest sensitivity was required.

89 Thus an initial check on the sensitivity of GC-MS analysis of steroid MO-TMS- derivatives was carried out. The influence of several parameters of both GC and MS in the selected ion monitoring (SIM) mode on analysis sensitivity was investigated. 3a- DHPROG (ion 417 m/z) was used to carry out this optimisation.

Temperature gradient and injection pressure

The signal to noise ratio (S/N) given by ion 417 m/z of 3a-DHPR0G is shown in Figure 3-4. In analysis A the parameter-settings of GC and MS were used as described in 2.4.2 (SIM method 1). This Figure also shows S/Ns when the settings were varied one by one. The various parameters did not seem to influence the sensitivity of the steroid analysis except for gas chromatographic temperature gradient and injection pressure. Lowering of the temperature gradient by l“C/min decreased the S/N slightly. An increase in injection pressure to 400 kPa produced a doubling of the S/N compared to the S/N observed with 100 kPa injection pressure. The decrease in S/N with a decreased temperature gradient is to be expected as the vapour pressure increase during the chromatographic run is lower compared to a separation with as tee per temperature gradient. This, in turn leads to longer retention times and thus an increase in peak widths and decrease of peak maxima. However, the temperature gradient is crucial to separation of analytes. Generally lower T-gradients give better peak top resolution. In view of the increased peak widths at these gradients the overall resolution might not get better, or even be worse. Thus it is not easily predictable how compound separations are affected by this parameter and so they will be investigated in a later section. A possible explanation for the increase in sensitivity with increased injection pressure is that due to increased pressure, less solvent and sample molecules evaporate at the column starting temperature during the initial sampling time. The object was to increase the solvent focusing effect and consequently obtain sharper peaks in the chromatographic elution.

Gas velocity and mass spectrometric parameters

Peak resolution in a given column is determined among other factors by gas velocity and is lowest at the optimum gas velocity. As resolution is obviously important for analysis of complex mixtures, the effect of gas velocity on sensitivity was investigated (via column flow). As the viscosity of Helium increases with temperature, column flow will decrease with time in temperature gradient analyses. Changes in pressure gradients (50-100 kPa and 80-150 kPa) did not affect sensitivity of the analysis.

90 Detector and quadrupole pre-rod repeller voltages did not affect sensitivity. They presumably change the overall current reaching the detector, not the proportion between analyte and background noise currents.

Injection temperature

Initially separation of the MO-TMS derivatives of the compounds listed in Table 2-1 by gas chromatography was investigated on a 25m column with 0.25mm inner diameter, 0.12|im film thickness and a CPSil 5CB stationary phase. With the conditions used initially (injection temperature 230°C, injection pressure 400 kPa, temperature gradient after 65°C for 3 min, 2 0 “C/min to 220°C, 3°C/min to 285°C, 285 °C for 3 minutes; column flow rate 1.5 ml/minute; pressure constant at 80 kPa), a significant fraction of the chromatogram was reflected in subsequent GC-analyses, indicating high carryover amounts. The resulting chromatogram is shown in Figure 3-5 a). The carryover cleared only after several blank injections, which was unacceptable. Not only did it cause blank problems, but also indicated that significant amounts of the sample were lost, thus decreasing sensitivity. The carryover problem was relieved after examination of various parameter setting changes (see Table 3-1). Those settings were applied in several combinations. In Figure 3-5 b) a large increase in the late/early eluting compounds response ratios can be seen compared to Figure 3-5 a). This also much decreased the carryover into following GC-runs. Conditions used in this analysis different from those in the analysis shown in Figure 3-5 a) were an injection temperature of 280°C and column start temperature 70”C. The peaks of the later eluters were sharper (this was confirmed by the decreased area/height ratio determined for cortisol, peak 80), thus increased sensitivity of those compounds could be obtained. An increased sensitivity ratio late/early eluting compounds is more favourable as sensitivity generally decreases with retention time in GC (as can be seen in Figure 3-5 from the negative slope of responses versus retention time) and thus this is a limiting factor for the analytical performance.

Injection speed, column temperature gradient

A further large improvement could be achieved with changes to injection temperature (280°C), injection speed (high) and column temperature gradient 5°C/min instead of 3°C/min (Figure 3-5 c). In Figure 3-5 b) sharper peaks for late eluting compounds had been observed compared to initial GC-runs. However, the area to height ratio of early compounds (measured for EpiA, peak 15) actually had increased. In Figure 3-5 c), they

91 were found to be lower for both late and early eluting compounds compared to Figure 3- 5 a) and b). Many other parameter setting changes had no effect on the chromatogram.

The improvement of discrimination against later eluting compounds observed between GC runs of Figure 3-5 a) and b) shows the discrimination was at least in part due to incomplete transfer from injection port to column. Later eluting compounds possess higher boiling points. If the injection temperature is below those boiling points the decreased rate of evaporation causes incomplete sample transfer to the column and peak broadening. Thus the higher temperature in the latter analysis improved the situation. However, in the analysis of Figure 3-5 b), sensitivity for lower boiling compounds actually decreased (as judged from area to height ratios of peaks). A possible explanation for this is that at the injection temperature much higher than the boiling point of some compounds the evaporation rate is very high and causes sample vapour to progress through the column before the temperature gradient is started. This was resolved by the conditions employed in Figure 3-5 c). Injection speed and column temperature gradient (from 3 to 5“C/min) during the main part of elution were changed compared to the run of Figure 3-5 b). The peak broadening of low boiling compounds was presumably counteracted by the steeper temperature gradient. Thus high injection temperatures and speeds largely resolved the carryover problem. The latter prevent sample solvent starting to boil in the needle, leaving higher boiling sample molecules behind and sticking to the needle. With the higher injection speed employed, it is assumed that the sample is pushed out of the syringe before it can start to boil and allow it to spread through the injection chamber. As a further preventative measure the syringe dwell time was set to 2 0 seconds. This would allow the syringe needle to be purged of any residual components at high temperature, thus decreasing general background noise.

GC-column

Having improved carryover and sensitivity, resolution of the steroids derivatised with MO and TMSI was further investigated using a 30m column with 0.25mm inner diameter and 0.25|im film thickness with a ZBl stationary phase as compared to a 25m length,

0.25mm inner diameter, 0 . 1 2 p.m film thickness column with a CPSil 5CB stationary phase. This column was predicted to have a higher capacity for samples of high concentration due to the higher film thickness and better resolving power due to the increased length. The chemistry of the stationary phase is the same for both columns.

92 Initially many compounds did not resolve on this column (see Figure 3-6 a). The improved injection conditions from the previous experiments were used again. Column temperature was set to 70°C for 3 minutes, then 20°C/minute to 260°C followed by 5°C/minute to 310°C. Column pressure was constantly at 80 kPa. Variations of temperature gradient combinations did not significantly improve the resolution, with only some groups of compounds separated. Finally in the injection shown in Figure 3-6 b) good resolution of most compounds previously unresolved was observed. Several groups of steroids were resolved better in Figure 3-6 b) than Figure 3-5 a), thus the higher peak number reflects better MO-TMS steroid resolution. In this method, column temperature gradients differed to the previous with 20°C/minute to 220°C and 5°C/minute to 315°C. Additionally it was attempted to keep the same linear velocity (30.7 cm/s) throughout the run. This was achieved by applying a pressure gradient from 34.2 kPa (kept for 5 minutes) to 81 kPa at 6.5 kPa/min and 1.6 kPa/min to 111.7 kPa. The resulting column flow was 0.7 ml/minute. Peaks unresolved in the chromatogram shown in Figure 3-5 c) that could be resolved here were: 3(3-DHPROG and 5p-pregnane-3a,20a-diol (peaks 89 and 91 in Figure 3-6 b) and peak 56 in Figure 3-5 c), 5p-pregnan-3a,17-diol-20-one, 3a- DHPROG and 3p,5P-THPROG (82-84 vs. 50), 3a,5P-THDOC and 5a-pregnan-

3a,lip-diol-20-one (98, 99 vs. 6 6 ), DOC and lip-OH-PROG (111, 112, 113 vs. 84, 85). Some compounds could still not be resolved however e.g. alphaxalone and 20a- DHPREG (96), 16-DPREG and 5a-pregnane-3a,20a-diol (89), 20p-DHPREG and 5a- DHPROG (94). Their spectra were sufficiently different though that diagnostic ions could be found for development of selected ion monitoring methods for their unequivocal identification, as described in later sections. All isomers (peaks 82,86; 83,91; 84,85,86,93; 87,89,90; 91,94; 94,96; 98,99,99a, 108; 99a, 100; 103,109) which have identical spectra could be satisfactorily resolved with this GC-method (scan method 3, see 2.4.2). All above analyses had been carried out at least three times.

As mentioned in the beginning of this section, gas velocity is a crucial factor in determining peak resolution during gas chromatographic analysis. Thus by keeping a constant linear velocity the separation could be largely improved in Figure 3-6 b) compared to Figure 3-6 a). The further analyses in this project were carried out using the ZBl column under the improved final conditions described above (with exception of some early experiments carried out on the CPSil 5CB column).

93 3.2.3 Characterisation of MO-TMS- derivatives of steroids by GC-EIMS

The MO-TMS-derivatives of the above chosen potential metabolites as well as non- endogenous steroids as potential internal standards were initially characterised by their El-mass spectra and GC-retention behaviour. The spectra were obtained by scanning of fragments arriving from the ion source over 99-800 m/z (scan method 3). It has been found all steroid derivatives investigated could be stably derivatised as methoximes and/or trimethylsilyl ethers. The analysis of the mass spectra showed that all ketogroups (positions 3-, 16-, 17-, 20- in the group of steroids under investigation) gave derivatives with MO, with the only exception being the ketofunction at position 11, as described previously [179]. The keto-function at position 11 is sterically hindered and thus does not react with MO. TMSI reacted with OH-groups in all positions investigated, i.e. C-3, C-11, C-17, C-20, C-21 in the MO-TMSI reaction.

Examples of mass spectra and the proposed fragmentation patterns are shown in Figure 3-7 for the MO-TMSI derivatives of PREG and 5a-pregnan-3a,17-diol-20-one. It can be seen, that ionisation by electron impact results generally in a number of fragments, giving good diagnostic information. The molecular ions were generally weak (see e.g. m/z 417 for MO-TMS-PREG in Figure 3-7 a). For MO-TMS-PREG the base peak, ion 386 is derived by loss of a methoxy- group (-31). This, together with loss of a methyl group (-15, ion 402 in this case) was one of the most commonly observed fragmentations of MO-TMSI- steroids Also very common is the loss of trimethylsilanol (-90, ion 327). The ions 312 and 296 in Figure 3-7 a) originated most likely from combined losses of individual fragments (-90 and -15, -90 and -31). Mainly for 3P-ol-5-ene steroids, a fragment 129 is seen from a break in ring A, but can also occur at 11- and 17- positions substituted with TMS. In some of the fragmentations, transfer of an H-ion occurred, e.g. fragment 100. Arising from cleavage of the D-ring of the steroid structure, this fragmentation was found to be characteristic for steroids with a carbonyl function at C- 20 derivatised with MO. Fragments with 156 and 188 m/z were found in spectra of MO- TMSI derivatised C-21 steroids with C-21- or C-17- hydroxyl-groups and a carbonyl - function at C - 2 0 (see for instance Figure 3-7 b) for 5a-pregnan-3a,17-diol-20-one). The latter fragment (188 m/z) is especially good as a diagnostic for these structures with usually high relative abundance.

The El-mass spectral fragmentations of all steroid MO-TMS derivatives analysed are

94 summarised in Table A-1 in Appendix 1. Spectra of isomers contain the same ions, but their relative ion abundances were found to differ (not shown). Also listed in the Table are the Kovats retention indices (RI) obtained after analysis according to the GC-method optimised in 3.2.2. All isomers could be separated from each other.

Several steroids gave double peaks in the gas chromatogram. In most cases, those arose from syn- and anti-conformations of derivatives with an MO at position C3. Usually the peak areas were similar, in the range from 1 - 2 : 1 . However, the THDOC-isomers gave double peaks with a relatively small second peak (peak areas - 1 0 :1).

The above information was used to set up selected ion monitoring (SIM), as described below (3.2.5).

3.2.4 Derivatisation of conjugated steroids by MO and TMSI

Having set up methods for gas chromatographic separation and mass spectrometric detection of free steroid MO-TMS derivatives, experiments were performed to determine whether the sulphuric, glucuronic and fatty acid conjugated steroids could be directly derivatised by MO and TMSI for GC-EIMS analysis. It was an aim in this study to develop a method for analysis of steroids both in their free and sulphate conjugated form and thus the sulphate esters that are too polar for gas chromatography need to be converted to non polar derivatives. Derivatisation of fatty acid esters and glucuronides of steroids was also important in order to estimate interference of those compounds from brain extracts with the analysis of free steroids and sulphate esters by GC-MS, especially if the bound groups were removed by the derivatisation reaction. Finally, derivatisation of fatty esters and glucuronides would allow their monitoring by GC-MS during development of brain extraction and fractionation procedures.

95 Derivatisation o f sulphate esters

DHEAS and PREGS together with the same amount of tetracosane were derivatised as described in Chapter 2 . Analysis by GC-MS revealed peaks eluting at the same time as the respective free steroids with the same mass spectra. However, the yield of MO-TMS- PREG or -DHEA was low from their respective sulphates (see Table 3-2) and so further procedures were investigated for cleavage of the sulphate esters before derivatisation.

A microsolvolysis method in a single step with MO-TMSI derivatisation has been described for sulphate esters of oestrogens [ 169] and this was used to determine whether cleavage of the sulphate esters could be achieved. DHEAS, PREGS and tetracosane (10 |ig each) were microsolvolysed and derivatised as described in 2.4.1. The response ratios of the MO-TMS-derivatives to tetracosane in GC-MS were compared to those observed after direct derivatisation by MO-TMSI and other derivatisation methods which are described below and the results are shown in Table 3-2. A considerable increase in yield compared to the direct derivatisation methods was observed with microsolvolysis followed by derivatisation with MO-TMSI. By contrast, microsolvolysis followed by TBDMS- derivatisation was not successful. These relative yields between direct derivatisation of PREGS and DHEAS by MO-TMSI with and without prior microsolvolysis are comparable to those reported for oestrogens [169].

It is assumed that the microsolvolysis works similar to traditional solvolysis methods. Due to the small volume of solvent, neutralisation of the sample can be done directly with the derivatisation reagent solution. No further purification prior to derivatisation is necessary. The partial evaporation step before reaction completion is assumed to be necessary to remove ethyl acetate to a great part, which would otherwise inhibit reaction completion. TMSI has been shown to replace the S04^'-group of the 3-0H-A5-steroids PREGS and DHEAS under the above used reaction conditions. However, this transestérification is only partial as the results of the microsolvolysis experiments demonstrated. Solvolysis including the findings here is further investigated in Chapter 4 (4.2.2.4) in the context of previous sample treatments (solid phase extraction, etc.).

Derivatisation of steroid glucuronides

Replacement of steroid glucuronide functions by TMSI has been described [25]. This could cause potential interferences in analyses of steroid tissue extracts with other

96 fractions. The MO-TMSI method used here was thus applied to standard PREG glucuronide. Analysis of the reaction products by GC-MS could detect no steroid derivative from injection of 1 p.g equivalent steroid. It was estimated that with this method approximately 0.75 ng of the equivalent free steroid would have been detected (scan mode).

It was then investigated whether derivatisation with the glucuronide ether intact would be more successful. The acid moiety can be guarded by méthylation [25]. This method was adapted here (see 2.4.1). After méthylation of the acid moiety with boron trifluoride- methanol, MO-TMS derivatisation yielded a compound with a double peak in the chromatogram (RIs -3190 and 3245, scan method 2 , column CPSil 5 CB). The gas chromatogram, however, showed several other peaks of which the identity could not be clarified and the signal of the putative derivative was comparatively low. These facts indicate a low yield from the reaction and formation of reaction artefacts.

Derivatisation of fatty acid ester steroid conjugates

MO-PREG acetate yielded a single peak (RI 2854 compared to 2834 of the free steroid, scan method 2, column CPSil 5 CB) with high response (detectability 1.8 ng in scan mode), thus indicating high reaction efficiency. Its mass spectrum could be clearly assigned to the proposed fragmentation pattern. Structural fragments, e.g. 1 0 0 m/z indicated derivatisation by MO at C-20, however not trimethylsilylation at C-3. The acetylated carbonyl function according to this fragmentation did not react with MO. On the contrary, PREG stearate could not be detected as MO- or TMS-derivative by GC- MS (detection limit 0.75 ng of free steroid equivalent in scan mode). The GC separation was extended 1 0 minutes beyond the normal time program isothermally.

If the derivatisation reaction had worked in a similar manner to PREG acetate, a possible explanation for lack of detection of this steroid is its retention by Lipidex 5000® in the routine cleanup procedure after MO-TMS derivatisation. The intact steroid ester is both a significantly larger molecule and more hydrophobic one than the corresponding free steroid. Size exclusion and polarity are the underlying mechanisms of chromatography by Lipidex. AJternatively it could be that the applied GC conditions (scan method 2 extended by 10 minutes) were not suitable for detection of this derivative. Larger molecules of similar chemical structure are expected to have longer elution times/higher elution temperatures. Even after the above GC run extension the steroid derivative might

97 not have progressed to the detector. However, it can be concluded that steroid long chain fatty acid esters do not interfere with analysis of free steroids or steroid sulphates from brain by GC-MS.

3.2.5 Development of two ion selected ion monitoring methods for analysis of steroid MO-TMS- derivatives by GC-EIMS

Selected ion monitoring (SIM) is well known to increase the sensitivity of the GC-MS assay compared with scanning acquisition due to the longer dwell times on acquiring signals from each ion. This approach was thus used here. The aim was to establish two diagnostic ions for each MO-TMS-steroid for identification purposes, selected, if possible, from the higher mass region (> 300 m/z) of the spectrum. The response of the major ion is used in quantitation. Ions in this region usually show higher selectivity (see e.g. the mass spectra in Figure 3-7). Even though the gas chromatographic elution of the above steroids had been optimised, several compounds were still slightly overlapping (Figure 3-6 b). Great care had thus to be taken in the choice of ions. Table A-1 (Appendix I) shows all the major ions from the mass spectrum of each compound. It can be seen that there are many possibilities for interferences between closely eluting compounds. Only ions not appearing in spectra of close-by or co-eluting compounds (difference in RI < 5) in the chromatogram could therefore be used for SIM, including their isotopic ion fragments (up to ±2 m/z). After that particular diagnostic ions were chosen according to the above description of diagnostic fragment formation (3.2.3). In most cases the molecular ions (M) were not high enough in relative abundance for use in SIM. In some cases the molecular ion was used, as there was no other choice (e.g. 20a/p-DHPROG, 3[3,5)3- and 3a,5a-THDOC). In most cases fragments M-15, M-31, or M-3I-90 were used (DHEA, EpiA, 17-OH-PROG, cortisol, 11-deoxycortisol, cortisone, etc.). For 3a,5a-, 3a,5p-, 3p,5p- and 3 p ,5 a - THPROG 388 (M-31) and 100 would be the clear choice, but ion 1 0 0 was subject to high interferences in the chromatogram at times when the former three compounds eluted and thus ion 298 (M-31-89-transfer hydrogen) was used in those cases.

In the case of C-20 reduced compounds the choice of ions presented a special problem due to the high relative abundance of fragment 117. Generally, this ion could not be used for SIM. Higher molecular fragments had more or less very weak abundance. For 20ct/p

98 -DHPROG only the fragment where presumably the side chain together with C-17 (117 +14) were cleaved could be found as specific ions apart from the molecular ions. In the case of 5p-pregnane-3a,20a-diol and 5p-pregnane-3a,20p-diol only ions 284 and 269 (presumably M-89-89 then -2 transfer hydrogens and M-89-89-13) gave significant signals apart from 117. However background interferences could not be avoided due to the relatively low mass of those ions. In the case of the THDOC-isomers ions 476 (M- 31) and 188 are clearly the pair of choice. For the 3a,5a- and 3P,5p-isomers there were interferences from neighbouring compounds in the chromatogram, so ions 507 (M) and 358 (M-149) (presumably loss of the methyloxime at C-20 and C-21 with the trimethylsilyloxygroup) were used here. For lip-OH-PROG and DOC 429 and 460 are selective ions in the high mass range with high relative abundance. But due to their similar retention times interferences cannot be avoided. Ions 370 (M-89 - 1 transfer hydrogen), 339 (M-89-1 transfer hydrogen -31) and 273 (presumably M-188 + 1 transfer hydrogen), 286 (M-173), respectively, were used instead. Further interferences from neighbouring ions precluded the use of the following: 386 (3a-DHPR0G and 5p- pregnan-3a,17-diol-20-one), 476 (5a-pregan-3a, 11 p-diol-20-one, interference from isotopic ion of 17-OH-PREG) All target and qualifier ions distinguished in this way are listed in Table A-1.

For maintaining high analytical sensitivity, the number of ions monitored was limited to four at a time. However, these could be changed to advantage at set times (in terms of RI) through a SIM method. To achieve this, four separate runs had to be applied to monitor all the ions of all compounds. In exceptional cases six ions had to be detected in one ion set (see Table A-2). For compounds giving double peaks, both peaks were monitored in case they were both major. For the THDOC-isomers, only the major peaks were monitored (see 3.2.3). Steroid identification in these SIM methods is achieved by means of GC-retention behaviour and the relative responses of the two steroid ions monitored. However, although these ratios have good reproducibility in analysis of pure standards, there were variabilities when analysing tissue extracts due to remaining background interferences that could not be excluded in this method. These two ion SIM methods (SIM methods 2-5) were thus used for initial screening of brain extracts and the identity of analytes confirmed with more definitive methods (see below).

99 Specificity of two ion selected ion monitoring of steroid MO-TMS-derivatives

Through the above described choice of specific ions for two ion SIM for a wide range of steroids, possible interferences from other steroids could be excluded. Furthermore, ions with high background interferences from the sample matrix were identified and excluded from the SIM protocols as far as possible. Interferences from reagents were minimised by choice of highest purity reagents and optimisation of the sample purification procedure (see Chapter 4). Minimal interference was seen from column or septum bleed, mass spectral noise and sample solvent with the ions chosen. The interferences of the reagent blanks from the brain extraction and purification procedure described in Chapter 4 determine the detection limit of the assay and are examined in Chapter 5.

3.2.6 Multiple ion screening methods for identification of novel compounds in mammalian brain

The above two ion SIM procedure was designed to detect a wide range of known steroids. In order to screen for other compounds which might have been missed in this selection, SIM methods were set up looking simultaneously for four groups of 32 ions. Molecular ions of possible steroids were predicted from the results shown in Table A-1

as follows. The relative molecular weights (M r ) of the pregnane, androstane and

oestrane nuclei are 288, 260, 246 respectively. The resulting M rs of all permutations of substitutions with functional groups (hydroxyl and ketone) were calculated, followed by introduction of double bonds. TMS-derivatised hydroxyl functions give a mass increment

of 8 8 , MO-derivatised keto-functions of 43. Hydroxyl and ketone functions were

considered at all chemically possible positions (hydroxyl: C-1, 2, 3, 4, 5, 6 , 7, 1 1 , 1 2 , 15,

16, 17, 18, 19, 20, 21 for pregnanes, at C-1, 2, 3, 4, 5, 6 , 7, 11, 12, 15, 16, 17, 18, 19

for androstanes and at C- 1, 2 , 3, 4, 6 , 7, 1 1 , 1 2 , 15, 16, 17, 18 for oestranes; ketones:

C- 1 , 2 , 3, 4, 6 , 7, 1 1 , 1 2 , 15, 16, 18 aldehyde, 19 aldehyde, 20 for pregnanes, at C-1, 2 ,

3, 4, 6 , 7, 1 1 , 12, 15, 16, 17, 18 aldehyde, 19 aldehyde for androstanes and at C- 6 , 7, 11, 12, 15, 16, 17, 18 aldehyde for oestranes. The maximum number of substitutions, however, was considered to be five hydroxyl groups or three keto-groups or mixed

substitutions of the two with up to five in total, and 2 functional groups the minimum (C- 3, C-20 in pregnanes, C-3, C-17 in androstanes and oestranes). Double bonds were considered possible at 4-5, 5-6 for pregnanes and androstanes. Oestrogens with the aromatic A-ring were assumed to have no other double bonds. Ketones at C-11 and 12

100 were thus assumed to stay underivatised (giving a mass increment of 14). One of the most common losses for MO-derivatised keto-groups is the methoxy-group (31 m/z) and was considered to occur for all compounds containing derivatised keto-groups. For steroids with only hydroxyl-groups derivatised, the most common loss of trimethylsilanol (-90) was predicted. The expected molecular ions and possible fragments used for multiple ion screening are shown in Table 3-3.

3.2.7 Methods for confirmation of brain steroids

For all the compounds tentatively identified with the above two and multiple ion SIM methods, formal identification was attempted. Two approaches were undertaken to achieve this: analysis of MO-TMS derivatised steroids by three ion SIM and/or analysis of steroids derivatised by a second method by two ion SIM. Two methods were chosen for alternative derivatisation, pertluoroacylation and /t?r/-butyl-dimethylsilylation.

3.2.7.1 Derivatisation of steroids for GC-EIMS using pertluoroacylation

Two derivatisation methods were chosen because of the resulting high sensitivity in GC- NICIMS, acylation by the perfluoroacyl compounds trifluoroacetic (TFAA) and heptafluorobutyric acid anhydride (HFBA) [194]. These reagents react to the acyl esters with steroid hydroxylgroups. Ketogroups were transformed into enols by the reagents and subsequently form the acyl esters. Due to the high density of electronegative functions, they are especially well suited for NICIMS. The potential of these derivatives for EIMS was examined. Initially derivatives were formed by incubation with 500 pi ethyl acetate and 50 pi HFBA or TFAA at room temperature for 60 minutes. Derivatives of both compounds gave single peaks. The El mass spectrum of HFB-PROG and proposed fragmentation pathways are shown in Figure 3-8.

The HFB-reaction of PROG leads to énolisation of the keto-group and consequently an isomérisation of the A4- to a A3, A5- configuration. As can be seen from the spectrum, the derivatives have higher masses than MO-TMS derivatives and thus generally have high signal to noise ratios when used in SIM. Commonly fewer fragments are formed than in El of the same steroids as MO-TMS compounds. Only few prominent ions are observed in the higher mass region (>300). The heptafluorobutyryl group fragments

101 easily. This is presumably due to a weakening of the C-3-0 bond due to the multiple electronegative centres in the derivative group.

The HFB-derivatives eluted later from the GC than the TFA-derivatives and gave higher responses compared to tetracosane. Thus HFBA was used in further experiments for derivatisation.

Further examination of derivatisation led to the finding that although the above used compounds gave excellent properties on El-MS, other compounds did not yield derivatives with the above used HFBA-derivatisation method (e.g. CORT). As an alternative derivatisation method to pertluoroacylation, tert (t) -butyldimethylsilylation of steroids was investigated, as described in the next Section (3.2.7.2).

3.2.7.2 Derivatisation of steroids for GC-EIMS using ferf-butyldimethylsilylation

N-methyl-N-(rm-butyl-dimethylsilyl)- trifluoroacetamide (MTBSTFA) was previously described as a TBDMS- donor in a reaction catalysed by r-butyldimethylsilylchloride

(TBDMSCl) in pyridine and acetonitrile [ 1 0 1 ]. NH4I was added as reducing agent. Using this method, TBDMS-PROG was prepared. The total ion chromatogram, El mass spectra (50-800 m/z) and proposed fragmentations are shown in Figure 3-9. The resulting compounds give five peaks in the GC. From the spectra it was concluded that there are presumably 2 derivatives formed under the above conditions, 3-TBDMS-PROG and 3-,20-di-TBDMS-PRGG, and isomers. Three of the four isomers of 3-,20-di- TBDMS-PROG separate on the GC under the employed conditions. Only one peak was detected for 3-TBDMS-PROG. Isomérisation took place between A3-4, A5-6 and A2-3, A4-5 in the A-ring and for the double-derivatives also between A17-20 and A20-21 in the D-ring.

Retention indices of the main peaks, major and diagnostic ions of several steroids derivatised with MTBSTFA are shown in Table A-4 in Appendix 1. Analysis on GC of several compounds from this Table revealed that several compounds could not be resolved under these conditions (25 m, 0.25 mm ID, 0.12 pm film thickness CP SIL 5 CB column. GC-conditions: He carrier gas pressure 80 kPa, column flow rate 1.5 ml/min splitless, injector purge on after 2 minutes; GC temperature gradient 70°C for 3 minutes, 20°C/minute to 220°C, 3°C/minute to 285°C, hold for 13 minutes), among them steroids

102 with the same mass spectral ions (e.g. TBDMS-5a-DHPR0G and TBDMS-PREG). A preliminary estimation of the sensitivity of the method in selected ion monitoring (using high mass selective diagnostic ions) using the 3 a noise criterion gave values of approximately 3 and 5 pg for 3a,5a-THPROG and 3a,5a-THDOC respectively.

TBDMS-derivatives of steroids gave simple mass spectra compared to MO-TMS derivatives and ions of relatively high molecular mass. They possess prominent molecular ions under the GC-EIMS conditions employed. Several steroids showed loss of a methyl- group, resulting in highly abundant M-15 ions. Another very characteristic ion found in several spectra was M-57 which accounts for loss of a tert-huiy\ group. Those ions were usually in the >500 m/z range and thus good diagnostic ions for identification and for quantitation in SIM. Ion 199 was observed in spectra of all C-21 steroids with a 20- keto-group. A likely origin would thus be a fragment arising from cleavage of the D-ring. The base peak in all spectra investigated here was ion 73. This ion is presumably a dimethylsiloxy-fragment (minus a transfer hydrogen). Hydroxyl groups at C-17 and C-11 in C-21 -steroids were found not to be derivatised by the reagent mixture used. This finding is contrary to the situation after TMSI derivatisation, where these hydroxyls react. However, these groups are sterically hindered (compare e.g. reaction of C-11 ketone with MO). Thus the larger size of the TBDMS-group compared to TMS could possibly prevent reaction.

Keto-functions at C-3 or C-20 led to formation of two double-bond isomers, which separated on the GC in many cases (as discussed above for PROG, see Figure 3-9). All C-21 steroids formed two derivatives, once with all hydroxyl- and ketones, if present, derivatised and once partially derivatised at either only C-3 or C-20. Those derivatives separated on the GC (see for example Figure 3-9). The formation of isomers and partial derivatives is unfavourable for sensitivity, as the peak height decreases relative to the amount of the compound. The method is still more sensitive than analysis of MO-TMS derivatives when the machine detection limits for pure compounds only are considered. However several analytes from the range of interest could not be separated by virtue of GC retention time or monitoring of ions unique to the compounds. Furthermore reliable quantitation was virtually impossible for more than two or three compounds in an analysis, as separation of isomers of the same derivative sometimes into four peaks plus one or two from a single derivative of the same compound made it impossible to design SIM programs detecting at least all the major peaks.

103 After the initial investigations of steroid derivatisation methods, it was found that HFBA derivatisation was advantageous over the TBDMS method for the given analytical problem. The HFBA derivatisation method was examined further so as to optimise detection of most steroids of the range described in Table 2-1 (see 3.2.7.3).

3.2.7.3 Optimisation of HFBA derivatisation of steroids

Several methods [32,106,118,191,195] (see introduction to this Chapter) were evaluated to achieve the conditions for analysis of the wide range of compounds under investigation. The response ratios of the resulting derivatives relative to tetracosane as standard were monitored in GC-EIMS and are shown in Table 3-4. With the initial conditions (3.2.7.1 and method 2 in Table 3-4), several of the investigated compounds gave poor or no responses in GC-MS analysis. Those compounds were then used for further development of the method. As can be seen in Table 3-4, method 5 (30pi HFBA + 30 pi benzene, 60°C for 30 minutes) gave the highest responses for the range of compounds, except for 5a-DHPR0G. This compound did not give a significant derivative yield with any method. However with the former method all derivatives of the compounds in Table 2-1 could be formed except the already mentioned 5a-DHPR0G and further SP-DHPROG, 11-deoxycortisol and cortisone (see Appendix 1, Table A-5).

The reproducibility of the derivatisation and assay method was estimated by analysing several solutions of PREG (10 ng) derivatised by HFBA with method 5 in Table 3-4. The inter-assay coefficient of variation (CV) found was 4.6% (n=5). This is an acceptable CV-value and compares well with the one found for the MO-TMS-derivatisation and assay method (see Table 3-6).

Gas chromatographic conditions for separation o f HFB-derivatives of steroids

GC-conditions for separation of steroid HFB- derivatives were optimised using a 30 m, 0.25 mm inner diameter, 0.25 pm film thickness ZBl column (the result being scanning method 4, Chapter 2, Table 2-2). With this method, separation of most compounds could be achieved. 3a-DHPR0G and 3p-DHPR0G could not be separated by this method or by the other methods investigated. 5P-pregnan-3a,17-diol-20-one could not be separated from the second peak of 5a-pregnan-3a,17-diol-20-one. For the other compounds not resolved by GC (5a-pregnan-3a, 11 p-diol-20-one and 5p-pregnane-3a,20a-diol; 16-

104 DPREG, 20P-DHPROG and 20P-DHPREG; PROG and PREG; 5a-pregnan-3a,17-diol- 20-one, 20a-DHPROG and 20a-DHPREG; 17-OH-PROG and 17-OH-PREG; 11- dehydrocorticosterone and 5a-DHD0C) interference can be avoided by monitoring different ions. This is consistent with previous reports where six HFB-derivatised steroids were analysed and HFB-PREG and HFB-PROG could not be resolved by GC [106].

The sensitivities observed with this GC-MS method were higher than with all other methods investigated for most compounds. It was found that a decrease in injector (280°C->250°C) and interface (315°C->285°C) temperatures compared to the method employed for GC-MS of MO-TMS derivatives (see 3.2.2) led to increased sensitivity. However, no carryover into subsequent GC-runs was observed after analysis of samples of reference compound mixtures derivatised by HFBA even at high concentrations. These facts together led to the conclusion that HFB-derivatives are more temperature- sensitive and volatile than MO-TMS- and TBDMS-derivatives. The resulting sensitivities in SIM analysis are shown in Table A-5, Appendix 1.

Characterisation of HFB- derivatives of steroids by GC-EIMS and development of selected ion monitoring methods

The ion ft-agmentation patterns in GC-EIMS (50-800 m/z) of the HFBA derivatives of all the compounds listed in Chapter 2, Table 2-1 (compounds 1-50) were analysed individually and are shown together with their RI in Table A-5 in Appendix 1. SIM methods were developed after the initial two ion screening of brain steroids (5.2.1) and are described in Appendix 1 (Table A- 6 ).

3.2.7.4 Alternative derivatisation methods for conjugated steroids

To confirm the steroid sulphate esters found by two ion SIM, again two approaches were chosen: analysis of the steroid esters by two ion SIM of different derivatives and three ion SIM of MO-TMS derivatives. Thus it was investigated whether the above described derivatisation methods for free steroids, fluoroacylation and tert- butyldimethylsilylation could be used for sulphate conjugated steroids. Steroid sulphate groups have been reported to be replaced by HFBA [25] by transestérification. Here it was first investigated whether the steroid sulphate esters could be derivatised with the

105 methods used above for free steroids. DHEAS and PREGS were incubated with 50 ( 0,1 TFAA or HFBA in 500 (ol ethyl acetate for 30 minutes at room temperature. Their retention times and mass spectra were equal to the ones of the respective free steroid derivatives. Replacement of the steroid sulphate group was also attempted using MTBSTFA. Since the TMS-group of TMSI was found to replace the steroid sulphate group, it was reasoned that the same could occur with the TBDMS-group of MTBSTFA. Using the same reaction conditions as for the free steroids (see Chapter 2), DHEAS and PREGS were incubated. No TBDMS-derivatives detectable by GC-EIMS were formed by the above compounds. Similarly, derivative formation was attempted with MTBSTFA after microsolvolysis (see 3.2.4). The response ratios relative to tetracosane in GC-MS compared to those obtained after other derivatisation methods are shown in Table 3-2.

It can be seen from Table 3-2 that analysis after microsolvolysis and MO-TMS derivatisation gave the highest responses. Low or no recovery was found for TBDMS- derivatisation after microsolvolysis. Out of the direct derivatisation attempts, the highest results were achieved with TFAA and HFBA. Thus it seems cleavage procedures such as solvolysis prior to derivatisation can increase the yield considerably. However the microsolvolysis method is presumably not compatible with pertluoroacylation. The high acid content in the solvolysis solvent most likely prevents ester formation. Neutralisation as performed after microsolvolysis for MO-TMS derivatisation would not be possible as the resulting salt content would also interfere with TFA/HFB- reaction. However this could be resolved by removal of acid residues and is described together with investigation of other solvolysis methods after sample extraction procedures in 4.2.2.4.

To test for possible interference with free steroid analysis, reaction of fatty acid ester steroid conjugates with HFBA was also investigated. PREG acetate gave a single peak (RI 2765, scan method 2, column CPSil5 CB). Its mass spectrum contained some ions that also occurred in the spectrum of the derivative of the free steroid. Structural fragments indicated derivatisation by HFBA at C-20, but not any other position in the molecule. Steroid short chain fatty acid esters are presumably not present in the mammalian nervous system as they have not been found to date in analyses of the fatty acids contained in steroid esters [200]. Still, in the event that they exist and that short chain fatty acid esters are not completely removed by the tissue fractionation procedures, there is no interference in analyses of steroids derivatised by the employed HFBA

106 method. The RI is >100 different from the free steroid derivative. On the contrary, PREG stearate could not be detected as HFB-derivative by GC-MS (detection limit approximately 65 pg for free steroid equivalent).

3.2.7.5 Three ion selected ion monitoring for MO-TMS- derivatives of steroids

The second approach for confirmation of identity of brain steroids used was SIM of MO- TMS-derivatised compounds from brain extracts using three rather than two diagnostic ions. Three ion SIM of MO-TMS steroids was developed using mass spectral data of reference compounds identified after the two and multiple ion screening approaches (5.2.1, 5.2.2) and is described in Appendix 1.

3.2.8 Method for quantitation of brain steroids

Compounds formally identified with the above three ion monitoring of MO-TMS- derivatives and/or two ion monitoring of HFB-derivatives were generally quantified by two ion monitoring of MO-TMS-derivatives. In calibrating the SIM- analysis method, the responses of the target ions of the compounds to be quantitated relative to the IS ME-17-OH-PROG target ions were plotted against their relative amounts. Curves were fitted by linear regression (except for 17-OH-PREG, fitted by polynomial regression) using the least square method and correlation coefficients calculated. Figure 3-10 shows the calibration curve for DHEA as an example. The equations of the curves and R-values are given in Table 3-5. Linearity was tested in the range 0.5-200 ng. All calibration curves except the one for the above mentioned compound were linear in this range. For higher accuracy, calibration curves were fitted for the range 0.5-10 ng for quantitation of steroids from brain extracts, however.

Accuracy of the two ion SIM method was determined by assaying several mixtures of standard steroids containing varying amounts and comparison of the measured to the calculated amounts in % (see Table 3-5).

The intra-assay reproducibility at 0.5 ng and the inter-assay reproducibility across 0.5-10 ng were determined by calculating the coefficients of variation (CV) of the assayed amounts of mixtures of standard steroids in pure solution and are shown in Table 3-6.

107 Factors influencing the level of detectability of a compound in GC-MS were introduced in Section 3.1. The limits of detection here were determined of the two ion SIM methods in GC-MS for analysis of steroids derivatised by MO and TMSI by comparing the signal of compounds at 0.5 ng to three standard deviations of the base line noise in a neighbouring region of the peak. Samples were analysed with the apparatus at its most sensitive settings (SIM methods 2-5) and the resulting detection limits are shown in Table 3-6. The estimates of the peak-to-peak assay sensitivities shown in the Table were all done in pure solutions of standard compounds and describe the absolute lower limit of detection of the GC-MS system. The limits of detection of the overall analytical procedure including extraction from tissue and fractionation were determined for analyses of brain extracts and are shown in Chapter 5.

The quantitations were accurate for the majority of compounds, with the values within acceptable ranges. The determinations were less accurate for cortisol and 5a-pregnan- 3a,llp-diol-20-one at the lowest level (0.5 ng), 11-dehydrocorticosterone (0.5 and 1.6 ng) and DOC (0.5 and 4 ng). Intra- and inter-assay reproducibilities of the method are satisfactory for most compounds examined, with exceptions 1 1 -dehydrocorticosterone and 5a-DHD0C at 0.5 ng. In those cases the poor accuracy and reproducibility is mostly due to the poor peak shape that is achieved near to the detection limits for the compounds concerned. With exception of 5a-DHD0C all of the mentioned compounds have relatively high detection limits. Peak broadening and lowering of the peak maximum occurs with increasing retention times. The above compounds all have long retention times. This is generally observed for corticosteroids and 11-hydroxylated compounds. Poor accuracy and reproducibility, however, is not observed for all compounds of this class. Some of the detection limits are low (e.g. THDOC-isomers). The detection limits are determined not only by factors discussed in 3.1.5 general to the instrumentation but also by steroid specific factors, such as the proportion of the ion current carried by an ion. Compounds with a spectral distribution of intensities limited to a low number of ions give generally higher responses for those ions. The sensitivity of the method described here is generally very good, except for the above discussed corticosteroids and Ilp-OH- PROG and 5a-pregnan-3a, 11 P-diol-20-one. The machine detection limits are generally higher than for the HFB-derivatives, as would be predicted. The HFB-compounds have less complex fragmentation patterns than MO-TMS-derivatives. In addition the HFBA derivatisation reagent and the reaction solvent give less chemical noise during GC-MS

108 analysis than MO and TMSI. However for certain corticosteroids and 11-hydroxylated compounds the detection limits are similarly high with both derivatisation methods.

3.3 Conclusion

Although a wide range of closely related compounds was analysed simultaneously, high specificity was achieved in three ion monitoring. This is due to the good mass spectral properties given by the MO-TMS-derivatives on electron impact and successful gas chromatographic separation of isomers. At the same time the procedures described here gave good sensitivity ( 1 0 0 - 1 0 0 0 femtomoles) and are thus suitable for the determination of brain steroids, which are expected to be present in the low ng/g range (low picomoles/g).

Femtomole (10-100 with a few exceptions) sensitivity for HFB -derivatives was observed. The sensitivity achieved here is slightly lower compared to NICI-MS using single ion monitoring (low femtomole) [194], however this technique has been reported to lack robustness [106].

A disadvantage of GC is that sulphate conjugated steroids cannot be directly analysed. LC-MS can potentially simplify the sample preparation procedures. For the separation of the wide range of both free and sulphate conjugated compounds in the present study however it was found to be necessary to use GC due to its superior resolving power compared to LC and convert the sulphated compounds into their unconjugated analogues prior to analysis.

The methods for screening, identification and quantitation are suitable for steroids extracted and fractionated from mammalian brain, as described in the following Chapters.

109 Table 3-1. GC-conditions employed in optimisation of separation of MO-TMS-steroids. Other parameter values equal in all methods: injection pressure 400 kPa, column start temperature time 3 minutes (5 minutes when sampling time 4 minutes), initial temperature program after column start time 20"C/min to 220“C, final column temperature 285"C, final column temperature time 3 minutes, column pressure after sampling time 80 kPa, column flow 1.5 ml/minute, column CPSil 5 CB, 25 m, 0.25 mm inner diameter, 0.12 pm film thickness.

Injection Start Temperature Sampling Injection Syringe temperature temperature gradient time (min) speed dwell time

CC) CC) CC/min) (s) 230 55 3 2 low 0 250 65 5 4 high 20 280 70

Table 3-2. Responses of steroid sulphate esters (n=2) after different derivatisation methods or after microsolvolysis and derivatisation relative to tetracosaneJV.D.: not detectable.

Response ratio (in % of microsolvolysis) TFAA HFAA MO-TMS Microsolvolysis, MO- Microsolvolysis, TMS TBDMS

PREGS 61.2 63.5 33.7 1 0 0 N.D.

DHEAS 45.2 36.3 23.5 1 0 0 5.16

110 Table 3-3. Predicted EIMS -ions (m/z) of MO-TMS-derivatives of steroids for multiple ion screening of brain extracts for new compounds. Molecular ions of nuclear structures (M) of pregnanes, androstanes or oestranes with substitutions and/or unsaturations (0-ketone, OH-hydroxyl, DB-double bond) followed by their -31 or -90 fragments in the rows underneath. Not shown are ions of structures where keto-groups are at positions C-11 or C-12. The ions are monitored in four different injections (SIM methods 22-25). Substitutions

Structure M 2 0 20+DB 30 30+DB 30H+20 30H+20 20H+20 20H+20 30H+0 30H+0+ 30H 30H+ +DB +DB DB DB Pregnane 288 374 372 417 415 638 636 550 548 595 593 552 550 Androstane 260 346 344 389 387 610 608 522 520 567 565 524 522 Oestrane 240 326 369 590 502 547 504

Mass fragments Pregnane 343 341 386 384 607 605 519 517 564 562 462 460 Androstane 315 313 358 356 579 577 491 489 536 534 434 432 Oestrane 295 338 559 471 516 414

Ill Table 3-3 continued.

Substitutions Structure M 20H+0 20H+0+ 20H+30 20H+30 20H 20H+D lOH+30 lOH+30 lOH+20 lOH+20 lOH+10 lOH+10 DB +DB B +DB +DB +DB Pregnane 288 507 505 593 591 464 462 505 503 462 460 419 417

Androstane 260 479 477 436 434 477 475 434 432 391 389 Oestrane 240 459 416 457 414 371

Mass fragments

Pregnane 476 474 562 560 374 372 474 472 431 429 388 386 Androstane 448 446 346 344 446 444 403 401 360 358 Oestrane 428 326 426 383 340

112 Table 3-4. Relative response ratios of free steroids (10 ng) derivatised with HFBA in different reaction conditions as shown and tetracosane (10 ng) in GC-EIMS. ETAC: ethyl acetate, Benz; benzene, CH3CN: acetonitrile. ND: not determined.

Method 1 2 3 4 5 6 7 8 9 1 0 11 1 2

HFBA (|nl) 50 50 1 0 0 1 0 0 30 30 30 30 30 50 30 50

Solvent ETACETACETAC ETAC Benz CH3CN Acetone Acetone CH3CN CH3CN CH3CN -

Solvent (m-I) 500 500 500 500 30 30 30 30 30 2 0 0 30 0 TCC) 25 25 25 25 60 25 60 25 80 80 25 25 Time(min) 30 60 30 60 30 60 60 60 60 60 60 60 Steroid DHEA 0.554 0.560 0.615 0.562 0.992 0.961 ND ND ND ND ND ND 3a,5a-THPROG 0.414 0.457 0.509 0.472 0.946 0.860 ND ND ND ND ND ND

PREG 0.910 1.401 1 .0 1 1 1 . 0 2 0 1.961 2.204 ND NDND ND ND ND PROG 0.131 0.145 0.105 0.069 0.485 0.606 ND ND NDNDNDND

5a-DHPR0G 0 . 0 0 0 0.014 0 . 0 1 0 0 . 0 1 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 2 0 . 0 0 1 0.005 0.006 0 . 0 0 1 0.004

3a,5a-THDOC 0.045 0.123 0.079 0.042 0.932 0.684 0.696 0.575 0.820 0.761 0.889 ND

CORT 0 . 0 0 0 0.033 0.019 0 . 0 1 1 0.166 0.062 ND ND ND ND ND ND

113 Table 3-5. Calibration curve equations, R values and accuracy (as % of added amount) of two ion SIM analysis of steroids in GC-EIMS. Numbers in left hand

Calibration curve equation R Accuracy (%) Amount (ng) 0.5 1.6 4.0 10.0 No Steroid Mean SEM n Mean SEM n Mean SEM n Mean SEM n 22 3a-DHPR0G y = 0.2298X 0.996895 107.7 6.2 5 90.2 1.8 5 95.1 4.4 5 101.0 1.8 5

2 3 3 ,5 3 -THPROG y = 0.9773x 0.996444 111.9 2.7 5 100.1 3.0 5 99.2 6.7 5 100.1 1.4 5 3 3a,5a-THPROG y = 0.8373x + 0.0013 0.997898 97.6 3.2 5 94.9 4.6 5 103.3 5.1 5 99.6 0.6 5 4 3a,53-THPROG y = 1.0571x 0.998048 118.5 2.8 5 99.6 3.7 5 101.4 4.7 5 99.7 1.0 5 24 5a-pregnane-3 a, 20a-diol y = 0.1428x 0.993076 125.5 5.0 5 98.9 2.3 4 117.0 6.2 4 97.8 1.2 5 6 PREG y = 0.2594x + 0.0007 0.993479 95.0 1.5 5 92.6 1.5 5 105.0 7.2 5 94.6 5.2 5 35 33,5a-THPROG y= 1.1964X-0.0018 0.998599 121.7 3.2 5 98.1 1.8 5 96.9 3.6 5 100.5 0.9 5 45 AJphaxalone y = 0.8403x 0.996845 117.0 1.8 5 102.1 2.0 5 105.5 4.0 5 99.0 2.0 5 21 20a-DHPREG y = 0.2982x 0.994233 119.4 2.7 5 99.0 3.8 5 109.4 5.1 5 98.5 2.4 5 43 5a-pregnan-3a, 113-diol-20-one y = 0.7764x + 0.0015 0.983616 181.1 4.9 5 116.9 4.0 5 95.0 4.1 5 90.5 2.4 5 11 17-OH-PREG y = -0.4562x^ + 0.2919x - 0.988433 118.6 2.8 5 88.8 5.7 5 102.8 10.0 5 100.4 5.0 5 0.0005 14 17-OH-PROG y = 0.8573x - 0.0016 0.965556 112.45 2.7 5 93.9 8.2 5 100.7 7.7 5 N.D. N.D. 33 Cortisol y = 0.0985x 0.966902 247.1 9.4 4 135.7 12.3 5 115.4 1.3 4 96.7 5.3 5 26 53-pregnan-3a, 17-diol-20-one y = 1.6444x - 0.0018 0.988838 109.3 7.4 5 95.6 3.8 5 100.5 4.3 5 79.6 6.2 5 25 5a-pregnan-3a, 17-diol-20-one y = 1.644x - 0.0011 0.984378 103.0 7.0 5 96.3 3.3 5 100.4 5.2 5 82.5 6.8 5 44 53-pregnane-3a,203-diol y = 0.3898x 0.980153 127.6 4.3 5 118.4 6.6 5 122.6 7.4 5 104.2 1.0 5 27 5 3 -pregnane-3 a, 20a-diol y= 0.3332X - 0.0002 0.960677 110.2 2.1 5 95.8 5.5 5 100.6 8.8 5 81.5 1.2 5

19 2 0 3 -DHPREG y = 0.4312x 0.996895 120.2 3.3 5 100.3 2.9 5 108.9 3.4 5 98.5 1.4 5 7 5a-DHPR0G y = 0.4975x 0.994786 110.2 3.3 5 88.1 3.6 5 109.6 2.0 5 98.7 2.8 5 10 3a,53-THDOC y = 0.5229X 0.980408 118.2 8.0 5 104.8 3.7 5 110.4 6.1 5 97.7 6.5 5

47 5 3 -DHDOC y = 0.3148x 0.995188 122.5 6.7 5 97.1 8.3 5 100.5 7.5 5 99.9 1.3 5

114 Tab e 3-5 continued. Calibration curve equation R Accuracy (%) Amount (ng) 0.5 1.6 4.0 10.0 No Steroid Mean SEM n Mean SEM n Mean SEM n Mean SEM n 16 DOC y = 0.3164x 0.97355 208.5 8.5 4 101.6 10.0 4 145.8 9.5 5 89.7 7.3 5 20 113-OH-PROG y = 0.0879X + 0.0002 0.990707 130.3 9.0 5 91.5 1.8 4 97.3 10.8 4 100.5 3.1 5 17 CORT y = 0.0619X + 7E-05 0.997346 110.7 2.7 5 101.5 1.5 4 106.2 11.2 5 100.3 1.0 5 1 DHEA y = 0.3828x 0.99559 124.4 3.8 5 102.4 1.6 5 108.4 3.4 5 98.5 2.4 5 39 EpiA y = 1.179x 0.998699 109.6 2.5 5 98.9 1.2 5 104.3 3.6 5 99.3 0.6 5 42 33-DHPROG y= 0.313x 0.994636 118.8 6.4 5 102.5 4.3 5 109.4 7.1 5 98.4 0.9 5 37 33,53-THDOC y = 0.3105x + 2E-05 0.993076 110.1 2.6 5 99.1 2.9 4 101.8 2.6 5 99.9 4.0 5 13 3a,5a-THDOC y = 0.2542X 0.989394 112.7 5.4 5 99.8 0.7 4 106.1 6.0 5 98.8 4.2 5 12 20a-DHPROG y = 0.4007x 0.991262 117.5 3.8 5 104.4 3.8 5 95.7 6.3 5 100.5 4.0 5 38 33,5a-THDOC y = 0.5492x 0.986103 115.4 6.0 5 102.1 6.9 5 102.9 7.1 5 99.4 5.2 5 41 11 -deoxycortisol y = 0.6382X 0.997747 119.6 2.6 5 106.3 5.4 5 102.5 2.7 5 99.4 1.8 5 36 11 -dehydrocorticosterone y = 0.0338X 0.980816 297.3 14.2 4 154.5 15.2 5 176.9 21.7 5 106.8 6.0 4 46 Adione y = 0.467x 0.99368 108.5 3.6 5 100.8 1.6 3 110.9 5.5 5 98.2 2.3 5 34 TESTO y = 0.8078x 0.998399 114.7 3.3 5 102.4 1.5 3 104.6 2.6 5 99.2 1.3 5 5 53-DHPROG y = 0.4375x 0.997146 126.1 5.0 5 104.6 4.3 3 100.1 3.5 5 99.8 2.1 5 8 PROG y = 0.3575x - 3E-05 0.998499 115.6 1.9 5 97.0 0.5 3 97.8 3.6 5 100.3 1.2 5 23 5a,20a-THPROG y = 0.257x + 0.0004 0.997647 107.8 2.5 5 99.9 3.2 3 99.0 5.4 5 100.2 1.1 5 15 5a-DHD0C y = 0.0458x 0.962705 103.5 24.3 4 95.1 2.1 3 116.3 10.9 4 95.4 3.4 5 40 Cortisone y = 0.3041x 0.998198 116.2 3.1 5 104.1 3.3 3 99.1 0.8 5 100.0 2.0 5

115 Table 3-6. Intra- and inter-assay reproducibility as well as detection limits of two ion SIM analysis of steroids in GC-EIMS. Intra-assay Inter-assay Detection reproducibility reproducibility limit (pg) Added amount (ng) 0.50 0.50 1.6 4.0 10.0 Steroid Mean SEM CV(%) n Mean SEM CV(%) n Mean SEM CV(%) n Mean SEM CV(%) n Mean SEM CV(%) n 3a-DHPR0G 0.41 0.03 16.4 4 0.54 0.03 12.9 5 1.4 0.0 4.5 5 3.8 0.2 10.4 5 10.1 0.2 4.1 5 24.2

3 3 ,5 3 -THPROG 0.57 0.03 12.2 5 0.56 0.01 5.5 5 1.6 0.0 6.6 5 4.0 0.3 15.2 5 10.0 0.1 3.2 5 7.4 3a,5a-THPROG 0.55 0.02 6.9 5 0.49 0.02 7.3 5 1.5 0.1 10.9 5 4.1 0.2 11.0 5 10.0 0.1 1.4 5 8.3 3a,53-THPROG 0.52 0.03 12.0 5 0.59 0.01 5.2 5 1.6 0.1 8.3 5 4.1 0.2 10.4 5 10.0 0.1 2.2 5 8.7 5a-pregnane-3a- 1.05 0.04 6.8 4 0.63 0.03 9.0 5 1.6 0.0 5.1 4 4.7 0.3 11.9 4 9.8 0.1 2.8 5 73.6 20a-diol PREG 0.48 0.03 14.8 5 0.47 0.01 3.6 5 1.5 0.0 3.7 5 4.2 0.3 15.3 5 9.5 0.5 12.3 5 21.5 33,5a-THPROG 0.50 0.02 9.7 5 0.61 0.02 5.9 5 1.6 0.0 4.1 5 3.9 0.1 8.3 5 10.1 0.1 2.0 5 8.0 Alphaxalone 0.58 0.03 10.1 5 0.58 0.01 3.5 5 1.6 0.0 4.3 5 4.2 0.2 8.6 5 9.9 0.2 4.5 5 11.3 20a-DHPREG 0.50 0.03 12.2 5 0.60 0.01 5.0 5 1.6 0.1 8.5 5 4.4 0.2 10.5 5 9.8 0.2 5.5 5 64.8 5a-pregnan- 0.88 0.06 14.5 5 0.91 0.02 6.1 5 1.9 0.1 7.7 5 3.8 0.2 9.6 5 9.1 0.2 5.9 5 119.0 3a, 113-diol-20-one 17-OH-PREG 0.65 0.05 14.6 4 0.59 0.01 5.2 5 1.4 0.1 14.3 5 4.1 0.4 21.7 5 10.0 0.5 11.0 5 52.9 17-OH-PROG 0.68 0.03 11.4 5 0.56 0.01 5.4 5 1.5 0.1 19.6 5 4.0 0.3 17.1 5 N.D. N D N.D. - 43.5 Cortisol 1.16 0.09 16.9 5 1.31 0.08 14.3 5 2.2 0.2 20.2 5 4.6 0.1 2.6 4 9.7 0.5 12.3 5 64.4 53-pregnan-3a,17- 0.60 0.02 8.1 5 0.55 0.04 15.1 5 1.5 0.1 9.0 5 4.0 0.2 9.5 5 8.0 0.6 17.4 5 1.7 diol-20-one 5a-pregnan-3a,17- 0.59 0.02 7.0 5 0.52 0.03 15.1 5 1.5 0.1 7.7 5 4.0 0.2 11.5 5 8.3 0.7 18.3 5 1.4 diol-20-one

116 Table 3-6 continued. Intra-assay Inter-assay Detection reproducibility reproducibility limit (pg) Added amount (ng) 0.50 0.50 1.6 4.0 10.0 Steroid Mean SEM CV(%) n Mean SEM CV(%) n Mean SEM CV(%) n Mean SEM CV(%) n Mean SEM CV(%) n 5p-pregnane- 0.00 4 0.64 0.02 7.5 5 1.9 0.1 12.5 5 4.9 0.3 13.4 5 10.4 0.1 2.1 5 50.5 3a,20p-diol 5p-pregnane- 0.40 0.02 11.4 4 0.55 0.01 4.3 5 1.5 0.1 12.9 5 4.0 0.4 19.6 5 8.2 0.1 3.3 5 48.8 3a,20a-diol 20P-DHPREG 0.36 0.01 2.5 4 0.60 0.02 6.2 5 1.6 0.0 6.4 5 4.4 0.1 6.9 5 9.9 0.1 3.2 5 28.5 5a-DHPR0G 0.59 0.03 9.4 4 0.55 0.02 6.6 5 1.4 0.1 9.2 5 4.4 0.1 4.1 5 9.9 0.3 6.4 5 68.7 3a,5P-THDOC 0.66 0.03 10.1 4 0.59 0.04 15.1 5 1.7 0.1 7.8 5 4.4 0.2 12.3 5 9.8 0.7 14.9 4 7.0 5P-DHD0C 0.45 0.05 20.2 4 0.61 0.03 12.3 5 1.6 0.1 19.1 5 4.0 0.3 16.8 5 10.0 0.1 2.8 5 40.3 DOC 0.91 0.05 10.2 4 1.04 0.05 9.1 4 1.6 0.2 22.0 4 6.0 0.4 13.9 4 9.3 0.7 17.8 5 217.9 1 Ip-OH-PROG 1.01 0.11 10.4 4 0.65 0.04 15.4 5 1.5 0.0 4.4 4 3.9 0.5 24.7 4 10.0 0.3 6.9 5 262.4 CORT 0.56 5.6 3 0.55 0.01 5.4 5 1.6 0.0 3.2 4 4.2 0.4 23.6 5 10.0 0.1 2.3 5 79.4 DHEA 0.70 0.04 12.7 5 0.62 0.02 6.9 5 1.6 0.0 3.5 5 4.3 0.1 6.9 5 9.9 0.2 5.4 5 23.6 EpiA 0.66 0.04 13.2 5 0.55 0.01 5.1 5 1.6 0.0 2.7 5 4.2 0.1 7.8 5 9.9 0.1 1.4 5 7.6 3P-DHPR0G 0.59 0.03 9.7 5 0.59 0.03 12.1 5 1.6 0.1 9.5 5 4.4 0.3 14.4 5 9.8 0.1 2.1 5 26.7 3P,5P-THDOC 0.52 0.04 14.2 4 0.55 0.01 5.4 5 1.6 0.0 6.6 5 4.1 0.1 5.8 5 10.0 0.4 9.0 5 6.5 3a,5a-THDOC 0.50 0.03 11.5 4 0.56 0.03 10.8 5 1.6 0.0 1.6 5 4.2 0.2 12.6 5 9.9 0.4 9.5 5 7.1 20a-DHPROG 0.57 0.04 13.7 4 0.59 0.02 7.3 5 1.7 0.1 8.1 5 3.8 0.3 14.7 5 10.1 0.4 8.9 5 24.4 3P,5a-THDOC 0.39 0.01 6.8 4 0.58 0.03 11.6 5 1.6 0.1 15.1 5 4.1 0.3 15.4 5 9.9 0.5 11.8 5 5.5 11-deoxycortisol 0.67 0.06 17.6 4 0.60 0.01 4.8 5 1.7 0.1 11.4 5 4.1 0.1 6.0 5 9.9 0.2 4.0 5 16.2 11 -dehydro­ 0.88 43.3 3 1.49 0.08 10.7 4 2.5 0.3 22.0 4 6.5 0.8 25.1 4 10.7 0.7 12.6 4 213.6 corticosterone Adione 0.65 0.03 10.0 4 0.54 0.02 7.3 5 1.6 3.6 3 4.4 0.2 11.0 5 9.8 0.2 5.1 5 33.2

117 Table 3-6 continued.

Intra-assay Inter-assay Detection reproducibility reproducibility limit (pg) Added amount (ng) 0.50 0.50 1.6 4.0 10.0 Steroid Mean SEM CV(%) n Mean SEM CV(%) n Mean SEM CV(%) n Mean SEM CV(%) n Mean SEM CV(%) n TESTO 0.69 0.03 8.2 4 0.57 0.02 6.4 5 1.6 3.2 3 4.2 0.1 5.6 5 9.9 0.1 3.0 5 10.9 5P-DHPR0G 0.66 0.04 12.1 4 0.63 0.03 8.9 5 1.7 9.3 3 4.0 0.1 7.9 5 10.0 0.2 4.6 5 27.4 PROG 0.65 0.06 18.8 4 0.58 0.01 3.7 5 1.6 1.1 3 3.9 0.1 8.2 5 10.0 0.1 2.6 5 43.8 5a,20a-THPROG 0.60 0.07 22.4 4 0.54 0.01 5.2 5 1.6 7.1 3 4.0 0.2 12.2 5 10.0 0.1 2.4 5 28.2 5a-DHD0C 1.21 0.15 25.5 4 0.52 0.12 47.0 5 1.5 4.9 3 4.7 0.5 20.9 4 9.5 0.3 8.0 5 1.7 Cortisone 0.60 0.02 6.7 4 0.58 0.02 5.9 5 1.7 7.1 3 4.0 0.0 1.8 5 10.0 0.2 4.5 5 32.3

118 Figure 3-1. Quadrupole mass separator. From: [170].

o ■ 3

C) 3 Ü.

Ion Lenses Quadrupole electrodes Electron source multiplier

Figure 3-2. Total ion chromatogram of TMS-20a-DHPREG as analysed by GC-MS. Column: CPSilSCB, temperature gradient 3C/min (scan method 1).

Abundance

2000000 -

1500000

1000000

500000

1 8.00 20.00 22.00 26.0024.00 28.00 30.00

119 Figure 3-3. Elution of MO and TMSI derivatised reference steroids from Lipidex 5000® chromatography with cyclohexane : pyridine : HMDS (98:1:1, v/v/v) as analysed by 2 ion SIM. Percentage cumulative recovery of total amounts eluted against elution volume. Data points represent mean&t SD, n=3. See Table 2-1 for steroid abbreviations.

100 -

80- ■- -DHEA • EpiA A- Adone 80- T- TESTO // ■I «■

20 /■

2 3 4 SLijQnVfelLme(ml)

100-

80- - ■--5p-pregTan-3a,17- diol-20-one GO- • 3a-DHPROG - A - 3p,5p-THPROG - T- 3a,5a-THPROG I 40- - ♦ -3a,5p-THPROG

20 -

0 -

1 2 3 54 GUionVjLme(ml)

120 Figure 3-3 continued.

100 -

80-

—■— 5a-pregnan-3a,17-diol-20-one 60- - - 5p-pregnane-3a,20p-diol A 16-DPREG - T- 5a-pregiane-3a,20a-diol I 43 - - 5p-pregTane-3a,20bt-ciol

2 0 -

1 2 3 4 5 BiiîQnUiLme(nnl)

100-

80- — 3P-DHPR0G - «--Sp-DHPROG 60- A-PREG - T- 3p,5a-THPROG - 20P-DHPREG I 43 -

20 -

0 -

1 2 3 4 5 Biüon'udLme(nnl)

121 Figure 3-3 continued.

100 -

80-

—■— 5a-DHPROG - «--alphaxalone 60- A 20tx-DHPREG -• ▼— PROG - 5a,20bi-THPROG 40-

20 -

0 -

1 2 3 4 5 Bubon Uiiime (ml)

100 -

80-

— 3a.5p-THDOC 60- - •--3p,5p-THDCC A 17-OH-PREG - ▼- 5a-pregnan-3a,1lp- diol-20-one 40- - 3a,5a-THDOC

2 0 -

0 -

1 2 43 5 Bubon Uiune (ml)

122 Figure 3-3 continued.

100-

æ- —■— 20p-DHPROG - •--20tx-DHPROG A 17-OH-PROG 0 0 - - ▼- 5P-DHDOC - ME-17-0H-PR0G I

2D-

0 -

1 4 523 BiücnVfclLfTie(ml)

100 -

80-

— 3p,5a-THIDOC

00 - - # -5a-DHDOC A DOC - T - 11P-OH-PROG - 11-deoxycoMisol 40-

20 -

0 -

1 4 523 BiJicn\AdLme(ml)

123 Figure 3-3 continued.

100 -

80-

— 11-dehydro corticcsterone 0 0 - - •--cortisone A CORT - ▼- PRED 40- - cortisol

2 0 -

1 2 3 4 5 BubcnU]|LiTe(ml)

Figure 3-4. Signal to noise ratios for 3a -DHPROG ion 417 m/z (50 pg) analysed by GC-MS, n=5, * significantly different to response using condition A with p=0.05. Conditions in determination A (SIM method 1): GC-injector temperature 250 C, injector pressure (Inj. p) 100 kPa, column initially at 70 “C for 3 minutes, then gradient (dT) 20 “C/minute until 220 "C, then 3“C/minute until 285 ”C, 5 minutes. Elution pressure (p) of 80 kPa (column flow 1.5 ml/minute, linear velocity 49.2 cm/second) constant; MS prerod voltage (Rep.V) -3.5 V, detector voltage (Det.V) 1.6 kV; sani|)ling rate 0.2 seconds and microscan width (M.scan) 0.2 amu. Conditions in other determinations same as in A with exceptions as indicated in Figure.

2 0 -

15-

1 0 -

o

124 Figure 3-5. GC-separation of MO-TMS-derivatives of reference steroids on a 25 m, 0.25 mm ID, 0.12 |im film thickness CP SIL 5 CB column. a) GC-conditions: injection temperature 230®C, injection speed low, He carrier gas pressure 80 kPa, column flow rate 1.5 ml/min splitless, injector purge on after 3 min; GC temperature gradient 65"C for 3 min, 20“C/min to 220“C, 3®C/min to 285"C, hold 3 min; MS acquisition mode: scan 50-800 m/z, detector voltage 1.9 kV. Numbers above peaks denote the following steroids: 25-DHEA, 26-EpiA, 30-Adione, 31-Adione, 34-TESTO, 35- TESTO, 40-5p-pregnan-3a,17-diol-20-one, 3a-DHPR0G, 3p,5p THPROG, 41-3a,5a- THPROG, 42-3a,5p-THPROG, 43-5a-pregnan-3a,17-diol-20-one, 44-5p-pregnane-3a,20P- diol, 45-16DPREG, 5a-pregnane-3a,20a-diol, 46-5p-DHPROG, 47-5P-pregnane-3a,20a- diol, 3P-DHPR0G, 48-PREG, 50 3p,5a THPROG, 51, 5a-DHPR0G, 52 5a DHPROG, 52 20P-DHPREG, 54-alphaxalone, 20-DHPREG, 55-PROG, 56 -5a,20a-THPROG, 57- 5a,20a-THPROG, 59-3a,5p-THDOC, 5a-pregnan-3a,llp-diol-20-one, 17-OH-PREG, 20p- DHPROG, 60- 3p,5p-THDOC, 61-3a,5a-THDOC, 20a-DHPROG, 63- 17-OH-PROG, 64- 17 OH-PROG, 65-ME-17-OH-PROG, 66-5P-DHDOC, 68-ME-17-OH-PROG, 69-3p,5a- THDOC, 70-5a-DHDOC, 71-5a-DHDOC, 73-DOC, lip-OH-PROG, 74-1 Ip-OH PROG, 75-11-deoxycortisol, 76-11-deoxycortisol, 78-11-dehydrocorticosterone, 80-cortisone, 81- cortisone, 82-CORT, 83-CORT, 84-PRED, 85-cortisol. b) GC-MS conditions different to a): injection temperature 280”C, column start temperature 70"C. Numbers above peaks denote the following steroids: 14-DHEA, 15-EpiA, 18-Adione, 20-TESTO, 21-TESTO, 24-5p-pregnan-3a,17-diol-20-one, 3a-DHPR0G, 3p,5p-THPROG, 25-3a,5a-THPROG, 26-3a,5P-THPROG, 27-5a-pregnan-3a,17-diol-20-one, 28-5P- pregnane-3a,20p-diol, 29-5a-pregnane-3a,20a-diol, 16-DPREG, 30-5p-pregnane-3a,20a- diol, 3P-DHPR0G, 5P-DHPR0G, 31-PREG, 32-3p,5a-THPROG, 33 5a-DHPR0G, 34-5a- DHPROG, 34-20P-DHPREG, 35-20a-DHPREG, alphaxalone, 36-PROG, 37-5a,20a- THPROG, 38-5a,20a-THPROG, 39-20p-DHPROG, 40-3a,5P-THDOC, 20a-DHPROG, 5a-pregnan-3a,lip-diol-20-one, 17-OH-PREG, 41-3p,5P-THDOC, 3a,5a THDOC, 43-17- OH-PROG, 44 17 OH-PROG, 45-ME-17 OH PROG, 46 5P-DHDOC, 48-ME 17-OH PROG, 51-3p,5a-THDOC, 52-5a-DHDOC, 54-5a DHDOC, 57-D0C,l 1 p OH-PROG, 58- 1 Ip OH-PROG, 61-11-deoxycortisol, 62-11-deoxycortisol, 68-11-dehydrocorticosterone, 73-cortisone, 74-cortisone, 75-CORT, 76-CORT, 79-PRED, 80-cortisol. c) GC-MS conditions different to a): injection temperature 280®C, injection speed high, column start temperature 70“C, temperature gradient between 220“C and 280®C 5”C/min.. Numbers above peaks denote the following steroids: 38-DHEA, 39-EpiA, 43-Adione, 44- Adione, 46-TESTO, 47-TESTO, 50-5p-pregnan-3a,17-diol-20-one, 3a-DHPROG, 3p,5p- THPROG, 51-3a,5a-THPROG, 52-3a,5p-THPROG, 53-5a-pregnan-3a,17-diol-20-one, 54-5p-pregnane-3a,20p-diol, 55-5a-pregnane-3a,20a-diol,16-DPREG, 56-5p-pregnane- 3a,20a-diol, 3p-DHPR0G, 5p-DHPR0G, 58-PREG, 59-3p,5a-THPROG, 60-5a- DHPROG, 61-5a-DHPROG, 20p-DHPREG, 62-20a-DHPREG, alphaxalone, 63-PROG, 64-5a,20a-THPROG, 65-5a,20a-THPROG, 66-3a,5P-THDOC, 5a-pregnan-3a,lip-diol- 20-one, 17-OH-PREG, 20P-DHPROG, 67-3p,5p THDOC, 68-3a,5a-THDOC, 20a- DHPROG, 70-17-OH-PROG, 71-17-OH-PROG, ME-17-OH-PROG, 72 5p-DHD0C, 74- ME-17-OH PROG, 80-3p,5a-THDOC, 81 5a-DHD0C, 82 5a-DHD0C, 84-DOC, lip OH- PROG, 85-11 p-OH-PROG, 87-11-deoxycortisol, 88-11-deoxycortisol, 93-11- dehydrocorticosterone, 96- cortisone, 97-cortisone, 98-CORT, 99-CORT, 102-PRED, 103- cortisol

125 Figure 3-5 continued. a) Rel. abundance

8Q8%3

17.5 20 22,525 27.5 Retention time (min) 394.718.804 Rel. abundance

4950 >9 BO rJ1 72, 7778

17.5 20 22.5 27.525 Retention time (min) c)

Rel. abundance

50 114

109 113 125

112 123 tmi |Ja 120 122

17.5 22.5 Retention time (min)

126 Figure 3-6. GC-separation of MO-TMS-derivatives of reference steroids on a 30 m, 0.25 mm inner diameter, 0.25 p,m film thickness ZBl column. a) GC-conditions: injection temperature 280"C, injection speed high, He carrier gas pressure 80 kPa, column flow rate 1.3 ml/minute splitless, injector purge on after 5 min, GC temperature gradient 70“C for 5 minutes, 20"C/minute to 260”C, 5®C/minute to 310‘*C, hold 5 minutes; MS acquisition mode: scan 50-800 m/z, detector voltage 1.9 kV. Numbers above peaks denote the following steroids: 55-DHEA, 56-EpiA, 59-Adione, 60-TESTO, 62-5P- pregnan-3a,17-diol-20-one, 63-3a-DHPROG, 64-5a-pregnan-3a,17-diol-20-one, 3^,5^- THPROG, 3a,5a-THPROG, 3a,5P-THPROG, 65-5Ppregnane-3a,20p-diol, 67-5a- pregnane-3a,20a-diol, 5P-pregnane-3a,20a-diol, 16-DPREG, 68-3P DHPROG, 5p- DHPROG, 69-PREG, 70-3p,5a-THPROG, 71-5a DHPROG, 20p DHPREG, 72-5a- DHPROG, 20a-DHPREG, alphaxalone, 73-PROG, 3a,5P-THDOC, 5a,20a-THPROG, 74- 3p,5p THDOC, 5a-pregnan-3a,llp-diol-20-one, 17-OH-PREG, 20p-DHPROG, 75-3a,5a- THDOC, 5a,20a-THPROG, 77-20a-DHPROG, 78-17-OH-PROG, ME-17 OH-PROG, 80- ME 17 OH PROG, 84-5P-DHDOC, 86-5a-DHDOC, DOC, 87 5a-DHD0C, 11 p-OH- PROG, 88-1 ipOH-PROG, 89-11-deoxycortisol, 94-11-dehydrocorticosterone. b) GC-MS conditions different to a): He carrier gas pressure 34.2 kPa for 5 minutes, 6.5 kPa/minute to 81 kPa, 1.6 kPa/minute to 111.7 kPa, hold 5 minutes, column flow rate 0.7 ml/minute splitless, GC temperature gradient 20"C/minute to 220"C, 5"C/minute to 315“C, hold 5 minutes (scan method 3). Numbers above peaks denote the following steroids: 75 - DHEA, 76-EpiA, 80-Adione, 81-TESTO, 82-5p-pregnan-3a,17-diol-20-one, 83-3a- DHPROG, 84-3p,5p-THPROG, 85-3a,5a-THPROG, 86-3a,5P-THPROG, 5a-pregnan- 3a,17-diol-20-one, 87-5P-pregnane-3a,20p-diol, 89-5a-pregnane-3a,20a-diol, 16-DPREG, 90-5p-pregnane-3a,20a-diol, 91-3p-DHPROG, 5p-DHPR0G, 92-PREG, 93-3p,5a- THPROG, 94-5a-DHPROG, 20p-DHPREG, 95-5a-DHPROG, 96-20a-DHPREG, alphaxalone, 97-PROG, 98-3a,5p-THDOC, 5a,20a-THPROG, 99-3p,5p-THDOC, 5a,20a- THPROG, 5a-pregnan-3a,lip-diol-20-one, 17-OH-PREG, 99a-3a,5a-THDOC, 20p- DHPROG, 100-20a-DHPROG, 101-17 OH PROG, 102-17-OH-PROG, ME-17 OH PROG, 103 5P-DHD0C, 105-ME-l 7-OH-PROG, 108 3p,5a THDOC, 109-5a-DHDOC, 110-5a- DHDOC, 111-DOC, 112-1 ip OH-PROG, 113-lip OH-PROG, 114-11-deoxycortisol, 115- 11-deoxycortisol, 121-11-dehydrocorticosterone, 123-cortisone, 124-cortisone, 125-CORT, 127-PRED, 128-cortisol.

127 Figure 3-6 continued.

a) 186,996,366 Rel. abundance

50

78 79

17 M 8182, 0 21 22 23 24 25 26 27 Retention time (min)

b) 211,884,946 Rel. abundance

50 99a 110 122 133

100 144 127 158 108 117 142 135 121

0 225 25 27.530 32.5 35

Retention time (min)

128 Figure 3-7 a). El- mass spectrum (99-800 m/z) of PREG derivatised with MO and TMSI and proposed fragmentation pathways.

O-CH 3 CH3 v^ N --0 -C H 3

Abundance 386

500000 :■ 100 4-

400000

239 312 129 402 300000

296, 200000

197 417 100000 279 327

497 705 797605 m /z~>0 100 150 200 250 300 350 400 450 500 550 600 650 700 750

129 Figure 3-7 b). El- mass spectrum (99-800 m/z) of 5a-pregnan-3a,17-diol-20-one derivatised with MO and TMSI and proposed fragmentation pathways. indicates loss of fragment.

T .N-}-0 CH; CH. CM: O—Si— CH I O — Si— CH; CH, CH. CM; □

Rel. abundance

50 ■ 4 7

364 107 172 298 14^ 255 386 507 200 492 579 661 100 200 300 400 500 600 700

130 Figure 3-8. El-mass spectrum (SO-SOOm/z) of HFB -PROG and possible fragmentations, indicates loss of fragment.

Rel. 183124 abundance

281505 739 793 I I I'r rr | I i i i i i i i'r y T t i I i i i i i | 100 200 300 400 500 GOO 700 800 m/z

131 Figure 3-9. Total ion chromatogram and El- mass spectra (3-TBDMS-PROG, 3-,20-di-TBDMS-PROG, 50-800 m/:^ for PROG derivatised withMTBSTFA/ TBDMSCl/ NHjI/ pyridine/ acetonitrile together with their proposed fragmentation pathways.

Rel. abundance 428 1,949,724

504 504

'.1.131 791 '2 [ 46 492 543 620 683 \ Î59 626 Rel. i*i|rM 111 II ifi II 111 II i|i II III II i|i II III II abundance 100 200 300 400 500 600 700 800 100 200 300 400 500600 700 800 m/z m/z 35,220,400 30 H V

20 H

loH

20 3 0 Retention time (min)

132 Figure 3-10. Calibration curve for MO-TMS-DHEA, ion 358 m/z to IS MO-TMS-ME 17-OH PROG ion 443 m/z from 0.5-10 ng, IS amount 50 ng. y=0.3828*x, R=0.99559, n=20.

0.09 -r

0.08 --

0.07 --

0.06 --

5 0.05

■S 0.04 --

0.03 --

0.02 -■

0.01 --

0 0.02 0.04 0.06 0.08 0 1 0.12 0.14 0.16 0.18 0.2

133 Chapter 4 An improved procedure for the extraction and fractionation of brain steroids

4.1 Introduction

As mentioned in Chapter 1, steroid analysis from nervous tissue poses several problems. Not only are sensitive and specific assay methods necessary, but steroids need to be extracted, purified and fractionated prior to analysis.

4.1.1 Extraction

Steroids are difficult to extract from tissues. The physical and chemical interactions caused by the complexity of the matrix are vast and ideally an extraction medium is required that perturbs all these. If total steroid content is analysed, the situation is further complicated in that the steroid molecules cover a wide range of polarity not only between conjugated and unconjugated, but also between steroids of the same class. For example, with free steroids polar corticosteroids exist at one end and the more hydrophobic progestins at the other end of the polarity scale. Extractions from nervous tissue have mainly been done using intermediate polarity solvents such as ethyl acetate [46,49,119,159]. Tissue samples are homogenised in hypotonic buffers or water prior to extraction with solvents. The above solvent is presumed to extract the free steroids and leave steroid sulphate esters behind in the aqueous tissue homogenate. The latter are then extracted by the salting out-method again with ethyl acetate. Usually they are then solvolysed before further treatment.

After separation from hydrophobic conjugates, the free steroid fractions require further purification/separation before analysis by GC-MS or RIA. One problem with extraction with solvents such as ethyl acetate is the solubility of proteolipid, causing difficulties in the physical separation of the phases.

Other methods used arc extraction of aqueous tissue homogenates with two solvent systems such as : methanol and hexane ; isopropanol [72,174], since they show good penetration of both aqueous and lipidic phases of tissues before they are

134 separated by centrifugation. More recently, combinations of liquid-solid extraction of steroids from tissues have been used. For instance, Andersson and Sjovall [4] reported homogenisation of testis in hexane ; isopropanol before drying down the homogenate together with Lipidex 1000 ® gel for subsequent fractionation by differential elution from this matrix. Rat brains were homogenised [48] with acetone : ethanol (1:1), followed by re-extraction of the pellet with chloroform : methanol (1:1). After removal of protein by centrifugation, the extracts were adsorbed onto Sephadex LH-20 columns. The steroids were then fractionated by differential elution. Others have used adsorption onto SePak or Isolute CIS cartridges ® (see below) after extraction into ethanol

[23,130], methanol : H 2 O [196] or methanol : acetic acid [106] after removal of protein by centrifugation. All the above two solvent or liquid-solid procedures extract both free and steroid conjugate fractions. One of their advantages is that some purification is achieved already in the initial extraction step. Another application with potential interest is the use of super critical fluid extraction (CO 2 ) for steroid extraction from tissues [187]. By adjusting the conditions, almost any analyte-matrix combination specificity can be reached and this method significantly reduces sample preparation time and the use of large volumes of organic solvents. However, it poses extensive apparatus requirements.

The extraction of endogenous steroid from brain tissue is difficult to measure as there are no valid indirect methods. Few studies have reported efficiency estimations [23,106]. Addition of labelled compounds to tissue homogenates cannot be expected to reach equilibration with endogenous compounds and so such estimates should be treated with caution. Only after injection of animals with labelled compounds and comparison of the extracted radioactivity with that found in digests of the tissue can valid estimations be made and even then such measurements have to assume isotopic equilibrium (see [179]). After intraperitoneal injection of rats with ^H-PROG 100% extraction of the labelled steroid from brain was subsequently achieved when the tissue was incubated in ethanol for 7 days at -20°C [23]. Similarly, approximately 100% extractions of ^H-PREG from brains of rats injected with this label was reported after homogenisation of samples in methanol/1% acetic acid [106].

135 4.1.2 Purification procedures

Solvent partitioning

Steroid purification usually employs liquid chromatographic techniques and solvent extractions in various combinations. Ethyl acetate and similar solvents co-extract lipidic material including cholesterol (CHOL), its fatty acid esters, fatty acids etc. along with the steroids of interest. These usually interfere with the final assay method to varying degrees. Carried over hydrophilic interferents are usually removed from the hydrophobic extraction solvent with a water wash. Solvent partitioning between an alcohol phase and a more hydrophobic solvent is often used to clean up the free steroid fraction prior to additional purification procedures [50,213]. This removes hydrophobic steroid conjugates though and if they are to be analysed is less favourable. Extractions of steroids into more hydrophilic solvents such as ethanol require subsequent fractionation of steroids.

Solid phase extraction

Solid phase extraction (SPE) is a method with high capacity for fractionation. SPE stationary phases have been developed in parallel with high performance liquid chromatography (HPLC). Commonly used for steroid extraction are phases based on CIS alkyl chains on a silica support matrix (SePak®, Isolute®, see Figure 4-1 a). This allows chromatography in the reversed phase mode. Typical elution solvents used are based on methanol:H20 mixtures [50,106,119,175,196]. After direct extraction of steroids onto the solid phase cartridge, hydrophilic contaminants can be removed with an aqueous wash step. This is necessary when the tissue has been homogenised in a relatively hydrophilic solvent (e.g. ethanol). Free steroids are usually eluted with 70-85% methanol.

Fatty acid esters and other hydrophobic conjugates are retained on the cartridges and can be eluted with 100% methanol [119]. A disadvantage of the C l8 columns is that the matrix is prone to collapse, especially if it dries and conditions have thus to be kept meticulously. Recently, a polymer based SPE stationary phase has become available. The poly(divinylbenzene-co-N-vinylpyrrolidone) (Oasis®, see Figure 4-1 b) stationary phase has both hydrophilic and hydrophobic properties and is stable even long time after drying out, thus avoiding the problems mentioned above (see present study).

136 High performance liquid chromatography (HPLC)

Several studies used HPLC to further purify steroids prior to analysis by GC-MS [106,119], Usually HPLC is used in the reversed phase mode for steroid analysis and a huge variety of stationary phases is now available, which should allow highly specific separations. Reported stationary phases used are ones like the above mentioned CIS long alkyl chains bonded to silica supports [173] or hydroxylsubstituted diol [46,106], Elution is performed using mixtures of solvents such as hexane-isopropanol either continuous [119] or in a gradient [106], methanol-H20 [173], MeCN-HiO [176], Whereas there is great potential for HPLC methods in steroid purification/analysis, they are usually limited to only few steroids at a time due to the need for optimisation of conditions for each compound [46,106,119], A potential advantage of the technique is direct analysis of conjugated steroids (see 4,1,3),

Partition chromatography

Purification based on polarity can be achieved using partition chromatography on Celite, Celite impregnated with propylene or ethylene glycol is typically used as stationary phase, with varying proportions of polar/non-polar solvents (e g, isooctane/ethyl acetate) used for elution. The method is more commonly used for separation of individual steroids prior to analysis by e g, RIA, but can also be used for group separation, Celite with propylene glycol (1:1, w/v) columns, e g, fractionated 5a-DHPR0G, PROG, PREG and 3a,5a-THPROG by elution with isooctane and isooctane : toluene (40:60) extracted from rat brains in ethanol for analysis by RIA [202],

Gel chromatographic systems have been frequently employed in steroid purification, Sephadex® gel was used with various substituents. One of the most common substituents is hydroxypropyl Sephadex® (LH-20), This was for instance used to purify acetone : ethanol and chloroform : methanol rat brain extracts for DHEA and subsequent fractionation from its conjugates prior to RIA [48], A more hydrophobic stationary phase is obtained by hydroxyalkyl (C12+C14) substitution of Sephadex® (Lipidex® 1000 and 5000, substituted with 10 and 50% w/w, respectively). By drying down a hexane : isopropanol homogenate of testicular tissue with Lipidex® 1000, all steroids and their conjugates could be extracted and partially purified by subsequent elution with water followed by methanol:H20 (85:15) of the gel connected to a Cl 8 SPE column in

137 series. Cholesterol and fatty acids could then be removed by passage through a Lipidex® 5000 column with 70% methanol containing 0.5% acetic acid. Reversed phase systems have also been used to purify steroids. Rat brain extracts in ethyl acetate were dried down and applied onto a Lipidex® 5000 column in hexane : chloroform (8:2) for purification prior to GC-MS [49].

4.1.3 Fractionation of free and conjugated steroids

As mentioned above, hydrophobic solvents are presumed to leave hydrophilic steroid conjugates behind and extract free steroids, their fatty acid and other possibly present nonpolar conjugates from aqueous tissue homogenates [49,119], The steroidal sulphate esters are too polar to be analysed by GC-MS. Thus the general procedure is to cleave the ester and perform analysis of the liberated free steroid. Sulphatase enzymes are available from bacteria {E. coli) and molluscs {Helix pomatid). They all result in partial hydrolysis of the steroids. They possess glucuronidase activity at the same time and can thus be used for treatment of both fractions if present. Chemical hydrolysis of ketosteroid sulphates was optimised by Burstein and Lieberman [28]. Their methods are based on solvent extraction and result in quantitative yields. The aqueous solution of the steroid sulphates is acidified to approximately pH 1 with H 2 SO4 and extracted with organic solvents such as ether or ethyl acetate. The experiments showed that the catalysis was not due to product removal by the solvents. Instead it was found to occur upon solvolysis in the solvent. After the incubation period, the organic phase can be worked up as described for free steroids. The solvolysis procedures have been shown capable of cleaving A-ring saturated and unsaturated Cl 9-steroids and should be applicable for most other steroids. Problems of solvolysis to be avoided are inhibition by traces of moisture and low extraction efficiency.

Other separation techniques of conjugate classes and free steroids have also been used. This is necessary when relatively hydrophilic solvents are used for initial extraction from tissues.

Hydrophobic gel ion exchange chromatography

Sephadex LH20 substituted with diethylaminoethyl (DEAE) groups are frequently employed. With these columns, separation of steroid monoglucuronides, mono- and

138 disulphates and free steroids is possible [59]. With the stronger anion exchanger triethylaminohydroxypropyl Sephadex (TEAP LH-20) it was possible to separate phenolic and neutral steroids in addition to the above conjugates [7].

Solid phase extraction (SPE)

Hydrophobic steroid conjugates that are co-extracted in most solvents are usually separated from other fractions by reversed phase SPE [106,119]. Lieberman and co­ workers [119] eluted free steroids from CIS SPE cartridges with 80% methanol and then the lipid- and possible sulpholipid conjugates with 100% methanol. Lipid esters are cleaved by saponification with methanolic base and the obtained free steroids processed fiirther for analysis. Backstrom and co-workers [202] have attempted to use the above described SPE method on CIS stationary phase to separate sulphate conjugates, free steroids and steroid fatty acid esters by differential elution with mixtures of methanol :

H2 O. The steroids from an ethanol brain extract are applied to the SPE cartridge in 5% methanol. After a wash with this solvent, steroid sulphates were displaced with 40%, free steroids with S5% and steroid fatty acid esters with 100% methanol.

High performance liquid chromatography (HPLC)

PREG, PREGS and PREG stearate were assayed in different applications [173,175,176]. PREG was measured in methanolic extracts of rat brain after prior sequential purification with CS SPE, Sephadex PHP-LH-20 and CIS SPE chromatography steps. PREGS was measured intact after a similar procedure in extracts with ethanol, but needed two further preparative HPLC separation steps before the final analysis. Similarly, PREG stearate, after extraction of brain with ethyl acetate from aqueous homogenate, required several preparative HPLC steps, normal and reversed phase, before final analysis. Steroids are usually not very responsive to HPLC UV detectors. However, by derivatisation with fluorescent derivatives a large increase in sensitivity can be obtained. In the above mentioned studies of PREG [176] and PREG stearate [173], anthroyl cyanide and dansyl derivatives, respectively, were prepared. PREGS in brain extracts was determined directly with a mass spectrometric detector [175]. This technique is further described in the introduction to Chapter 3.

139 Ion exchange chromatography

Recently, silica based SPE columns with ion exchanger groups have become available. A method for fractionation of steroid sulphates, glucuronides and unconjugated steroids by strong anion exchange (SAX) SPE columns has been described [197]. This method is faster and uses less solvent than the above described Sephadex anion exchange methods. After removal of unconjugated steroids with methanol, glucuronide conjugates are eluted with formic acid in 50% methanol ; H 2 O. The stronger acidic steroid sulphate esters are finally desorbed by 0.25 M triethylammonium sulphate. A novel polymer based anion exchange cartridge has also recently been introduced (Oasis mixed mode anion exchange (MAX)®, see Figure 4-1). This cartridge gives hydrophilic-lipophilic balance based separation possibilities in addition to the anion exchanger function. It should also again avoid problems with stationary phase stability compared to the silica based columns (see present study).

In the initial stages of the present project, the aim was to set up a procedure for extraction, fractionation and purification of steroids and their sulphate esters from brain tissue, for subsequent assay by GC-MS, using conventional ethyl acetate extraction, solvent partitioning, solid phase extraction and partition chromatography with propylene and/or ethylene glycol on celite as stationary phase on celite (original procedure). For this, the individual steps were first evaluated for their suitability in achieving the above tasks, and an overall procedure set up according to the results from this was tested on brain extracts. During the course of this work, it was found that several problems persisted with this conventional procedure, particularly in the extraction of interfering lipidic material from brain and in the low recoveries of particular steroids. Further, it was found the conventional SPE on CIS could be improved with new polymer based adsorbents and that such SPE products opened the possibility of co-extraction and fractionation of free steroids together with their sulphate esters. The structure of the SP adsorbents used in this project are shown in Figure 4-1. The polymer based Oasis® products avoid danger of collapse of the alkyl chains on drying of the cartridge. Oasis HLB® and MAX® polymers can be used for clean-up and fractionation of steroids, as will be shown in this Chapter.

140 4.2 Results and discussion

4.2.1 Original procedure for extraction, fractionation and purification of steroids from mammalian brain tissue

4.2.1.1 Steroid extraction and solvent partitioning of extracts

Ethyl acetate extraction of steroids from aqueous media/brain homogenates is well established (see 4.1.1). Here, extraction of ^H-labelled PROG into ethyl acetate from 0.1 M phosphate buffer, pH 7.4 gave a recovery of 97.9 ± 4.3 % (mean ± S.E.M., n=5) in three extraction steps 1:1 (v/v). In further experiments free steroids were extracted from brain homogenates in 0.1 M phosphate buffer, pH 7.4 after this protocol.

Solvent partitioning was required to purify the samples of lipidic material before the following chromatographic steps. The standard steroids 3a,5a-THPROG, 5 a- DHPROG, 3a,5a-THDOC, PREG, PROG, 20a-DHPROG, 5a-DHD0C, DOC and

17-OH-PROG (500 ng each) in methanol (80% or 90% in H 2 O, v/v) were partitioned against isooctane 1:1 (v/v). After removal of the methanol phase, the isooctane phase was extracted twice more with an equal volume of the aqueous methanol. After addition of internal standards, the combined methanol phases were dried down, derivatised and analysed by GC-MS. Recoveries calculated by comparison of the response ratios of the above steroids to ones obtained by analysis of pure standard solutions are shown in Table 4-1. This revealed that isooctane did not remove large amounts of free steroids from solutions of 80% or 90% methanol after this protocol. Similar recoveries of the steroids investigated in this Section were observed in 80% and 90% methanol.

4.2.1.2 Solid phase extraction

Sep Pak CIS® solid phase extraction

As a major clean-up step for free steroids from hydrophobic matrix interferences in brain extracts, solid phase extraction was used. Recoveries of steroids from Sep Pak C l8® chromatography were monitored using steroid standard solutions. The standard steroids 3a,5a-THPROG, 5a-DHPR0G, 3a,5a-THD0C, PREG, PROG, 20a-DHPROG (lOpg) were loaded onto Sep Pak C l8® cartridges (30 mg) in 10 ml 80% methanol in

141 H2 O (v/v). The eluate solution was collected. Steroids were further eluted with 5 ml 80% methanol. Steroid content in the combined eluate was analysed by GC-MS. The recoveries are shown in Table 4-2 and revealed low and variable recoveries for the steroids investigated.

Purification of brain extracts with and without a Sep Pak C l8® solid phase extraction was also investigated. Figure 4-2 shows the comparison of rat brain extracts purified by isooctane partitioning and celite chromatography with or without a Sep Pak C l8® solid phase extraction step. The solid phase extraction can be seen to have reduced the CHOL content of the extract.

The removal of CHOL from the extracts of rat brain prepared here is necessary as the GC-column is overloaded by the amounts contained. The elution of several compounds of interest would be obscured by the presence of the large amounts of CHOL. However, removal of CHOL in brain extracts by C l8 solid phase extraction leads to low and variable recovery of several steroids. It is known that the stationary phase of C18 SPE cartridges is prone to disintegration, especially when not completely solvated at all times or the pH of the mobile phase is slightly acidic. Even though great care is being applied in the handling of the elution process from the SePak Cl 8® cartridges, instability of the stationary phase might occur and lead to the variability of the recoveries. Another solid phase extraction cartridge based on a polymer (poly(divinylbenzene-co-N- vinylpyrrolidone)) Oasis Hydrophilic Lipophilic Balance (HLB)® was therefore tested for separation of lipids and lipid esters.

Oasis HLB® solid phase extraction

^H-PROG or ^^C-CHOL with 100 ng unlabelled PROG and 1 pg CHOL butyrate were redissolved in 80% methanol in phosphate buffer (5 mM, pH 7.4, v/v), loaded onto a cartridge and further eluted with 80% methanol followed by 100% methanol (see Figure 4-3). ^H-PROG-recovery in 15 ml 80% methanol was 99.1 % (mean, n=3), ^^C-CHOL was not eluted by this solvent at 80%, but came off readily in 100% methanol.

It has been shown that a good separation of a free steroid (^H-PROG) from ^"^C-CHOL can be achieved with Oasis HLB® solid phase extraction and this method was further employed as the SPE purification step in brain extraction procedures.

142 4.2.1.3 Partition chromatography on celite

Partition chromatography with propylene and/or ethylene glycol on celite as stationary phase was employed to fractionate free steroids and remaining cholesterol and its esters as well as other hydrophobic compounds in brain extracts. Elution of free steroids from celite columns was investigated using the radioactive tracers ^H-PROG, ^H-5a- DHPROG and ^H-CORT. Celite columns (1 g) were prepared with an ethylene glycol stationary phase (1:1, w/v). 10000 dpm each of the above steroids were redissolved in 1.5 ml isooctane saturated with ethylene glycol and loaded onto the celite columns. After a wash with 2 ml isooctane, the steroids were eluted with 40 ml 30 % ethyl acetate in isooctane (v/v). Fractions of 1 ml were collected, dried down and redissolved for counting of radioactivity. Elution of the above steroids after this procedure is shown in Figure 4-4.

PROG and 5a-DHPR0G are the most apolar C21- steroids and elute earliest of the steroids investigated in the chromatographic system employed. Thus, this system appears to be suitable for the removal of interfering lipidic substances retained by the stationary phase in a wash step with a hydrophobic elution solvent such as isooctane prior to elution of the above mentioned steroids.

The above elution solvent mixture of 30% ethyl acetate in isooctane eluted some of the glycol stationary phase from the celite columns. Ethylene or propylene glycol need to be removed because of interference in the following derivatisation reactions and GC-MS assay. To prevent this leakage of the stationary phase, columns were set up with a small water impregnated celite section following the actual stationary phase. Celite columns, 1 g with ethylene glycol stationary phase (1:1, w/v) were prepared with either 0.3 g or 0.6 g celite impregnated with H 2 O (3:1, w/v) packed below. Several reference steroids (5 |ig) were loaded and eluted from the columns as above. After addition of internal standards, the eluate was dried down, derivatised by MO and TMSI and analysed by GC- MS for calculation of recoveries (see Table 4-3). Low and variable recoveries were found. Another attempt to remove ethylene or propylene glycol from the eluted celite fractions was to wash the solvent with water. The label ^H-CORT (87000 dpm) was dissolved in 300 fil ethylene glycol, to which 20 ml 30% ethyl acetate in isooctane (v/v) and 3 ml H 2 O were added. After extracting, 99.7 % (mean, n=2) of the radioactivity was found in the organic phase, without any residue of ethylene glycol. The fact that

143 CORT was not removed by the water wash indicates that most steroids of interest here (see Table 2-1, Chapter 2) would be successfully recovered, as CORT is one of the most polar of this range.

Elution of ^H-CORT (14800 dpm) and ^H-5a-DHPR0G (76500 dpm) from celite columns with ethylene and propylene glycol stationary phases was then investigated. Table 4-4 shows the recoveries obtained. This showed that ^H-CORT and ^H-5a- DHPROG could be recovered well in all the solvent - stationary phase systems investigated. The elution profile of ^H-5a-DHPR0G with isooctane was monitored on ethylene (2:1, w/v) and ethylene/propylene glycol (4:1:1, w/v/v) columns and is shown in Figure 4-5. Elution of 5a-DHPR0G from the ethylene (2:1, w/v) glycol columns started after 2 ml isooctane, from the ethylene/propylene glycol (4:1:1, w/v/v) columns after 3 ml isooctane, volumes which can be used as wash to remove hydrophobic interferents. The ethylene/propylene glycol (4:1:1, w/v/v) column was used for further experiments, as it allowed a higher wash volume.

Elution of several reference steroids at 600 ng including CHOL and cholesterol butyrate (CHOLBUT) (20 pg) from celite (1 g) columns with an ethylene/propylene glycol stationary phase (4:1:1, w/v/v) was then examined. Samples were loaded in 1.5 ml isooctane and the columns washed with 1 ml isooctane before elution with 5 ml isooctane, 6 ml 20% ethyl acetate in isooctane (v/v) and 12 ml 50% ethyl acetate in isooctane (v/v). Steroid content was analysed by GC-MS after partitioning against H 2 O and derivatisation for determination of recovery (see Table 4-5). This experiment showed that a good recovery for a wide range of free steroids could be achieved with the Celite system employed. A complete separation of free steroids from CHOL and CHOLBUT was however not possible.

4.2.1.4 Overall performance of original procedure for extraction and fractionation of steroids from brain tissue

Figure 4-6 gives a flow diagram of the original procedure developed at the start of this project using ethyl acetate extraction, isooctane partitioning. Oasis HLB® solid phase extraction and celite chromatography for isolation and fractionation of steroids and their conjugates from brain tissue. The procedure was evaluated by measuring recoveries of free steroids added to human brain tissue homogenate and determination of endogenous

144 steroids in unspiked portions of these samples (see Table 4-6). The selected ion traces of the derivative of the endogenous steroid DHEA is shown in Figure 4-7.

This evaluation showed that high recoveries for several compounds could be obtained through the extraction/purification procedure developed. However, low recoveries for non-polar compounds such as PROG and 5a-DHPR0G were observed. Furthermore interfering lipids were not sufficiently removed and caused problems in analyses of some compounds. This is presumably due to the lack of separation of hydrophobic substances from steroids on Celite chromatography, as has been shown for CHOL and CHOLBUT previously (see Table 4-5).

In an investigation in the laboratory (carried out by R. Ibrahim, O. Clowry and M. Little) it was found that after addition of ^H-PREGS to rat brain homogenate in phosphate buffer followed by ethyl acetate extraction, 14.7±0.5% of the radioactivity was contained in the organic phase and 69.6±3.7% in the aqueous phase. After extraction of homogenates to which ^H-PREG was added the content of radioactivity in the organic phase was 101.4+2.3%, and 84.8+2.1% after a water wash. Thus the separation of free and sulphate-conjugated steroids by partitioning with aqueous buffers and ethyl acetate is only partial. The content of label in the ethyl acetate phase after extraction of ^H- PREGS could be reduced by water washes. The potential risk of interference is quite significant, however.

4.2.2 Improved and simplified procedure for the extraction, purification and fractionation of steroids and their sulphate esters from mammalian brain tissue

The problems highlighted in the evaluation of the original extraction and fractionation procedure (incomplete separation of free and sulphated steroids, low recoveries of non­ polar steroids, incomplete separation from CHOL and its esters) led to a need to develop an improved and simplified procedure. The following sections describe an extraction based on ethanol followed by solid phase extraction for purification and separation of free and sulphate conjugated steroid fractions for analysis by GC-MS.

145 4.2.2.1 Extraction of steroids from brain tissue in ethanol or ethanol/acetic acid

Combined extraction of free and conjugated steroids from brain into ethanol [23,130], or methanol/acetic acid [106] has been reported. Extraction of ^H-label from brain tissue after injection of animals with ^H-PROG using either ethanol or acetic acid in ethanol was investigated in the present project. It was found that extraction into 20 volumes 3% acetic acid in ethanol gave 88.2% yield compared to 42.4% and 104.2% with 14 and 30 volumes 3% acetic acid in ethanol, respectively. Extraction by homogenisation into 5 volumes ethanol gave only 1.9% recovery (all values mean, n > 2). Extraction experiments using 3% acetic acid in ethanol were from here on all carried out with 20 and not 30 volumes for practical reasons (to reduce large amounts of solvents being used).

In the following method development sections sometimes ethanol brain extracts were used as a matrix to evaluate elution behaviour, etc. The results were used for preliminary method development steps. In the final evaluation of the overall extraction and fractionation procedure after extraction of tissue into ethanol/acetic acid, the validity of the individual steps was then confirmed.

4.2.2.2 Oasis HLB® solid phase extraction

Oasis HLB® solid phase extraction (SPE) cartridges had already been investigated for use as sample purification step to remove hydrophobic interferences using ^H-PROG and ^^C-CHOL as tracers (see 4.2.1.2). The elution of those compounds with 80% and 100% methanol has already been shown in Figure 4-3. In order to evaluate the use of HLB cartridges in the new procedure based on ethanolic extracts, elution of ^H-PROG was examined with ethanol in potassium phosphate buffer (5 mM, pH 7.4). Rat brain extracts (5 volumes + 5 volumes from re-extraction of pellet after centrifugation, w/v) were dried and redissolved in 60 or 80% ethanol in potassium phosphate buffer (5 mM, pH 7.4, v/v), loaded onto cartridges (corresponding to 7 mg tissue/mg stationary phase) and eluted further with the same solvents. Cumulative recovery of the radioactivity is shown in Figure 4-8.

With 80% ethanol, ^H-PROG was completely eluted within 15 volumes, with 60% within 35 volumes, thus giving an indication what volumes to apply in a purification protocol. It

146 can further be seen that ^H-PROG is interacting with the Oasis stationary phase in 60% ethanol, consistent with the chromatographic process. This is in contrast to the situation with 80% ethanol, where virtually no retention is observed.

Purification of the brain extracts by 60 and 80% ethanol elution through Oasis HLB® from hydrophobic interferences was evaluated by GC-MS. A total of 20 ml 80% ethanol rat brain extracts (5 volumes + 5 volumes from re-extraction of pellet after centrifugation) was passed through 3 cartridges (corresponding to 35 mg tissue/mg stationary phase) successively and further eluted with two volumes 80% ethanol in potassium phosphate buffer each time. Four volumes were taken off after each elution, dried down and analysed by GC-MS after derivatisation with MO and TMSI. ^H-PROG spiked samples were processed in parallel to monitor recovery. Recoveries were 100% after each step of this procedure. High amounts of CHOL and its esters were observed after each elution, however, with no significant decrease after subsequent SPE steps (data not shown). Thus there seems to be insufficient retention of hydrophobic substances on the Oasis HLB® cartridges due to preferential partitioning into 80% ethanol and /or the cartridges are overloaded with the contents of these brain extracts so that no interaction with the stationary phase takes place.

Next, ethanol extracts (5 volumes + 5 volumes from re-extraction of pellet after centrifugation) corresponding to 7, 14, 21 and 35 mg tissue/mg stationary phase were dried down, redissolved in 133 pl/mg stationary phase 60% ethanol in potassium phosphate buffer and passed through cartridges, followed by further elution with 200 p.l/mg stationary phase of the same solvent. Portions corresponding to 1% of the original sample were analysed by GC-MS after derivatisation. The samples containing 21 and 35 mg tissue/mg stationary phase overloaded the GC-column with the derivative of CHOL. However, CHOL was increasingly retained with higher ratios of solid phase adsorbent to brain tissue.

It was estimated in order to detect steroids of interest > 1 g tissue is needed as starting material. When extracts corresponding to 20, 12, and 8 mg tissue were injected, the corresponding signal of interfering CHOL from 1 g tissue extract in each case would obscure the chromatogram and damage the ionisation system. At 7 mg tissue/mg stationary phase CHOL was retained at an acceptable level.

147 4.2.2.3 Solvent partitioning

Excess lipidic material could also be removed from ethanol/acetic acid (3 % v/v) brain extracts by solvent partitioning prior to Oasis HLB© SPE. It was shown above (4.2.1.1) that isooctane partitioning 1 ; 1 (v/v) did not remove excessive amounts of polar or non­ polar free steroids from 80% or 90% methanol solutions. This was taken as basis to use the technique for extracts in 80% ethanol.

Purity of rat brain extracts after isooctane partitioning, redissolving in 60% ethanol in potassium phosphate buffer and passing through Oasis HLB® cartridges (samples corresponding to 35 mg tissue/mg stationary phase) was judged by GC-MS after separation of free steroid and steroid sulphate fractions by Oasis MAX® and is described in Section 4.2.2.4.

4.2.2.4 Separation of free and sulphate conjugated steroids by hydrophilic/hydrophobic balance and/or ion exchange chromatography

Separation of free steroids and their sulphate esters was previously described in using SePak C l8® cartridges and differential elution [202]. For separation of free from sulphate conjugated steroids after brain extraction with ethanol or ethanol/acetic acid, Oasis HLB® or Oasis MAX® anion exchange solid phase extraction cartridges were investigated here (after prior purification of extracts with isooctane partitioning and a solid phase extraction step with Oasis HLB®).

Oasis HLB® solid phase extraction

The separation of steroid sulphates from free steroids by differential elution from Oasis HLB® cartridges was attempted. Steroids were eluted from the cartridges with solvents at alkaline pH to keep steroid sulphates in the ionised form, ensuring maximal polarity differences. First, elution of ^H-PROG from the cartridges was further examined. ^H- PROG (1.1x10^ dpm) were added to 30 ml 40% ethanol in potassium phosphate buffer (5 mM, pH 7.4, v/v) containing 2% (v/v) ammonia. This solution was passed through an

Oasis HLB® cartridge (60 mg) and after washing with 3 ml H 2 O, elution followed with 9 ml 100% ethanol. In the 40% ethanol loading fraction, 6.8 ± 0.5% of the radioactivity were recovered, 0.1 ± 0.02 % in the water wash and 94.6 ± 0.5% in the 100% ethanol

148 elution fraction (all values mean ± S.E.M., n=4). This indicated that free steroids and steroid sulphates could be separated by differential elution from Oasis HLB® cartridges.

Separation was tested by spiking ethanolic rat brain extracts with PREG, DHEA and their sulphate esters. After purification by isooctane partitioning andan Oasis HLB® solid phase extraction step, eluates from the first Oasis HLB® (10 volumes, corresponding to 35 mg tissue/mg stationary phase) in 60% ethanol in potassium phosphate buffer (5 mM, pH 7.4, v/v) were diluted to 30% and loaded onto a second HLB cartridge. After a wash with 2.5 volumes of 30% ethanol, steroid sulphates were eluted with 4 volumes 40% ethanol in ammonium acetate buffer (10 mM, pH 9, v/v), free steroids with 5 volumes 60% ethanol in ammonium acetate buffer (10 mM, pH 9, v/v). The recovery of reference steroids that had been added to brain homogenates in the sulphate and free steroid fractions as monitored by GC-MS after derivatisation of both sulphates and free steroids to the MO-TMS-derivatives was as follows: DHEA 77.3%, PREG 79.1%, DHEAS 38.7%, PREGS 38.0%. There were no major interferences of hydrophobic or hydrophilic compounds in samples processed through this procedure without prior addition of reference compounds.

Separation of free steroids and steroid sulphates on Oasis HLB® was finally checked using different compounds in the free steroid and sulphate fraction. The reference steroids PREGS, DHEAS, EpiA, TESTO, 3a,5a-THPROG, PROG, 3a,5a-THDOC and CORT were fractionated in a similar procedure to above. The resulting elution profiles are shown in Figure 4-9. This experiment showed an inability of this elution system to separate free steroids and steroid sulphate esters. Of the sulphated steroids, PREGS, which is more lipophilic than DHEAS, was incompletely eluted with 40% ethanol and carried through into the 60% ethanol fraction. The most polar of the free steroids investigated, CORT and TESTO are partially carrying over into the 40% ethanol fraction. It is doubtful that a group separation of free and sulphate conjugated steroids on the Oasis HLB® cartridges is possible with another solvent system. In order to prevent elution of CORT and other polar free steroids, the polarity of the first elution solvent needs to be increased. As PREGS did not elute completely with the applied volume of 40% ethanol, it is unlikely that it and other apolar steroid sulphates could be completely eluted with a more polar solvent. Even if apolar steroid sulphates could be eluted with a large volume of a more polar solvent, this would very likely start to elute polar free steroids.

149 The lack of separation of PREG and PREGS is contrary to the finding of the previous experiment. It has to be concluded that the carryover was not noticed due to the fact that free and sulphate conjugated steroids are analysed in the same way by the employed GC- MS method.

Oasis Mixed Mode Anion Exchange ®

In view of the problem described above in obtaining a complete separation of free steroids and steroid sulphates, investigations were carried out on the Oasis Mixed Mode Anion Exchange (MAX®) polymer. This is the HLB® polymer modified to incorporate an anion exchange function (see Figure 4-1).

Oasis MAX® loading conditions

Free steroid and steroid sulphate elution were initially examined using the steroids PREG and ^H-DHEAS (diluted to a specific activity of 1.1x10* dpm/nmol with unlabelled compound). They were loaded onto the MAX columns in 30% ethanol in potassium phosphate buffer (5 mM, pH 7.4, v/v, 660 pl/mg stationary phase) and washed with the same solvent (83 pl/mg stationary phase). Recovery of ^H-PREG was 1.3% in the combined load and wash fractions, whilst recovery of ^H-DHEAS was 0.4% (means, n=2). Thus, free steroids and sulphate esters are retained on the MAX cartridges after loading and washing in pure solution of 30% ethanol in potassium phosphate buffer. An experiment was performed to see whether steroids are retained on the MAX stationary phase under these or other conditions in the presence of brain extract as matrix. The brain extracts (5 volumes in ethanol) were partitioned against isooctane and loaded onto and eluted from Oasis HLB in 60% ethanol before addition of ^H-DHEA. The HLB eluates were diluted to different concentrations of ethanol and loaded onto MAX cartridges. Figure 4-10 shows elution of ^H-DHEA from the cartridges after loading in these solvents.

As can be seen from Figure 4-10, considerable amounts of the polar ^H-DHEA were lost into the load and wash fractions upon loading in 25% and 30% ethanol. The lowest

percentage of ethanol ( 2 0 %) can be used as loading solvent mixture without significant losses of free steroid. Having determined the most suitable solvent mixture to load brain extracts onto MAX cartridges, elution of free steroids was further examined.

150 Oasis MAX® free steroid elution

^H-PREG and ^H-DHEAS were loaded onto the MAX columns and after a wash, elution followed with 60% ethanol in ammonium acetate buffer (20 mM, pH 9, v/v, 0.33 ml/mg stationary phase). Recoveries of ^H-PREG and ^H-DHEAS were 92.2% and 0.9%, respectively. Other solvents for free steroid elution were investigated. ^H-PROG (diluted to a specific activity of 4.4x10^ dpm/nmol with unlabelled compound) was loaded onto an Oasis MAX cartridge, and after wash elution followed with either 40% ethanol in ammonium acetate buffer (20 mM, pH 7), 100% ethanol or ethyl acetate. Within 0.166 ml/mg stationary phase of 40% ethanol, 84.5% of the radioactivity were recovered, within 0.33 ml/mg stationary phase 92.4% and within 0.5 ml/mg stationary phase 94.9%. The recoveries in 0.083 ml/mg stationary phase and 0.166 ml/mg stationary phase 100% ethanol were 96.1 and 99.8%, respectively and in 0.066 ml/mg stationary phase ethyl acetate, 110.9 % (all values mean, n=2). Representative free steroids (^H-PREG and ^H- PROG) could be eluted with high recovery in one or several solvents. Elution of the steroid sulphate ^H-DHEAS is negligible in a low ionic strength solvent of medium polarity.

Steroid conjugate elution from Oasis MAX® and solvolysis of steroid sulphate esters

Steroid sulphate elution from the MAX stationary phase was examined next after loading ^H-PREGS or ^H-DHEAS. The binding of the anions can be displaced by either high salt concentration or low pH (> 2 below pK of analyte) eluents. ^H-DHEAS elution with 60% ethanol in ammonium acetate buffer (0.25 M, pH 7, v/v) gave 52.2% recovery (mean, n=2), elution of ^H-PREGS with 60% ethanol in low pH buffers (pH 2, 3, 4 or 5, 20 mM, v/v) recoveries < 1% with 0.5ml/mg stationary phase. Elution with 60% ethanol in pyridine/acetate buffer (1 M, pH 5, v/v) or 60% ethanol in ammonium carbonate (0.25 M, v/v, 0.5ml/mg stationary phase) gave 31.8% and 71.9% recovery (mean, n=2), respectively.

In the above solvents, the steroid sulphates should be recovered intact after elution from Oasis MAX® cartridges. Determination of steroid sulphates by GC-EIMS was previously examined (see 3.2.4) and it was found that these conjugates are best analysed as derivatives of their free equivalents after cleavage of the sulphate group. Derivatisation efficiency was analysed after elution from Oasis MAX® in 60% ethanol in

151 ammonium carbonate and microsolvolysis. PREGS and DHEAS (1.25|Lig) in 30 ml 60% ethanol in ammonium carbonate were dried down after addition of internal standards, transferred to derivatisation tubes by 3x1 ml ethanol containing 0.5% NH 3 (v/v) and dried down again. They were then microsolvolysed and derivatised by MO-TMSI as described in Chapter 2 . After analysis by GC-MS, peak area ratios to internal standards were calculated and compared to those of equimolar amounts of corresponding free steroids to give derivatisation efficiency. The efficiency found was low compared to microsolvolysis and derivatisation of steroids dried from stock solution ( 1 0 0 pg/ml ethanol/NHs 0.5%) and even direct derivatisation of the steroid sulphates with MO and TMSI (see Table 4-7).

A preliminary investigation into solvent systems suitable for solvolysis was then carried out. Subsequently experiments were performed to check whether these solvents can elute steroid sulphates from MAX® cartridges and results are summarised in Table 4-7. It is established that solvolysis can occur in ethyl acetate acidified with sulphuric acid [28]. However, elution experiments with this solvent mixture of ^H-DHEAS from MAX (0.33 ml/mg stationary phase) gave low recovery ( 6 .8 %, mean, n= 2 ).

Solvolysis in ethyl acetate can also be done after extraction of steroid sulphates from an aqueous medium. PREGS and DHEAS (1.25 pg each) were added to 8 ml 60% ethanol/0.5 M NaCl/ 0 . 1 M H2 SO4 , a possible eluent for steroid sulphates from Oasis MAX®. The solution was extracted into ethyl acetate (three times with 0.6 volumes) after dilution to 30% ethanol/ 0.1 M H 2 SO4 and addition of NaCl to 20% w/v. The extract was dried over Na 2 S0 4 , acidified ethyl acetate ( 2 ml) was added and incubation allowed to proceed at 40®C for 16 h. The incubation mix was neutralised with pyridine, dried down and transferred to another tube with ethyl acetate. This solution was dried down either directly after addition of internal standards or after a water wash (0.5 volumes) before derivatisation with MO and TMSI. The reaction yielded virtually no derivatives (see Table 4-7, solvolysis with or without wash).

Solvolysis in acidified ethyl acetate containing benzene sulphonic acid (BSA) (50 mM), which was predicted to be a high affinity ligand of the MAX stationary phase cation was then investigated. PREGS and DHEAS (1.25 pg each) were incubated in 15 ml of this solution at 40“C for 16 h. After neutralisation with pyridine, the solution was taken to near dryness and the residue extracted three times with ether. After drying down and

152 addition of internal standards, the mixture was derivatised with 200 |il MO/pyridine for 1 h at 60°C and 100 pi TMSI for 3 h at 100”C. Derivatisation efficiency for this procedure is also shown in Table 4-7 (Solvolysis in BSA/ acidified ethyl acetate). The yield from this procedure could be further increased by incubation of the solution in BSA/acidified ethyl acetate as above but in the presence of Na 2 S0 4 (Table 4-7, Solvolysis in BSA/ acidified ethyl acetate/ Na 2 S0 4 ). This last method gave the highest yield found for all the methods that were compatible with elution from Oasis MAX® and could be accepted for further investigation.

After this preliminary examination, the recovery obtained in the above (Solvolysis in

BSA/ acidified ethyl acetate/ Na 2 S0 4 ) method was confirmed in several further experiments. The recoveries found were 81.2 ± 3.0 % and 63.0 ± 4.2 (means ± S.E.M, n=4) for DHEAS and PREGS, respectively.

Finally, this method was examined for use with HFBA-derivatisation. After incubation in

BSA/ acidified ethyl acetate in the presence of Na 2 S0 4 , drying down and ether extraction as described above, the ether phase was partitioned with ether saturated H 2 O three times to remove pyridine and other salts, dried down and incubated with 30 pi benzene and 30 pi HFBA a 60°C for 30 minutes. The relative response ratios of DHEAS and PREGS after this procedure are again shown in Table 4-7 (Solvolysis in BSA/ acidified ethyl acetate/ Na2 S0 4 followed by H 2 O wash and derivatisation by HFBA).

The suitability of BSA in acidified ethyl acetate for steroid sulphate ester elution from MAX® cartridges and separation from free steroids was then examined. Other known steroid conjugates are fatty acid esters and glucuronic acid ethers. Steroid fatty acid esters are more hydrophobic than their free equivalents and are most likely removed by the purification procedures (isooctane partitioning. Oasis HLB® chromatography) prior to the Oasis MAX® step. However, these compounds have been shown to not interfere with the GC-MS analysis of MO-TMS- or HFB-derivatives of free and sulphate conjugated steroids employed here (see Chapter 3). Steroid glucuronides are not known to be present in mammalian nervous tissue at present. Nevertheless, an enzyme for glucuronidation of steroids is expressed in mammalian brain (see Chapter 1) and to avoid any possible contamination, their elution from Oasis MAX® cartridges was investigated here.

153 As no radioactively labelled steroid glucuronides were available, elution of those compounds as well as free and sulphated steroids from Oasis MAX® was monitored by analysing urine samples of five human subjects where these compounds are expected. Urine extracts were loaded in 20% ethanol and the cartridge washed with the same solvent. Free steroids were eluted with ethyl acetate, steroid glucuronides with 60% ethanol in formate/pyridine buffer, pH 3 and after a wash with ethyl acetate, steroid sulphates with benzene sulphonic acid in acidified ethyl acetate. This is described in Appendix 2, where the successful separation of the three steroid classes is confirmed. Comparison with traditional methods of urinary steroid separation indicated high recovery in all fractions.

Finally, to check elution of interferences by the above solvents for free steroids, rat brain extracts previously isooctane partitioned and passed through Oasis HLB® (corresponding to 35 mg tissue/mg stationary phase) were loaded onto MAX® cartridges in 20% ethanol in ammonium acetate buffer (20 mM, pH 7), and eluted with either 20 volumes 40% ethanol in ammonium acetate buffer (20 mM, pH 7) or 4 volumes ethyl acetate. This was followed in either case by 20 volumes 60% ethanol in pyridine/formate buffer (20 mM, pH 3, v/v; glucuronide fraction), 2 volumes ethyl acetate (wash) and 15 volumes benzene sulphonic acid in acidified ethyl acetate (50 mM; sulphate fraction). The free steroid and sulphate eluates were dried down, derivatised and analysed by GC-MS. Similar patterns of interferences were observed in the free and sulphate steroid fractions after elution with 40% ethanol or ethyl acetate, acceptable for brain sample analysis.

Thus ethyl acetate was used for further experiments in elution of free steroids from Oasis MAX® for practical reasons (smaller solvent volumes, solvent easier to evaporate).

4.2.2.5 Purification of HFBA-derivatives of steroids extracted and fractionated from mammalian brain tissue

Preliminary analyses of rat brain extracts in ethanol/acetic acid processed by isooctane partitioning. Oasis HLB® chromatography and MAX® fractionation according to the above optimised protocols showed high interferences when derivatised by HFBA. For removal of remaining interferences Lipidex 5000® chromatography was employed. After derivatisation by the method developed in 3.2.7.3, mixtures of all compounds in Table A-

154 1 were eluted from Lipidex 5000® columns by 10% ethyl acetate in cyclohexane (v/v) or 2% pyridine in cyclohexane (v/v). The elution profiles for the HFB-derivatives are shown in Figure 4-11 and good recovery for all compounds within 3 ml from the employed Lipidex columns by the procedure using 10% ethyl acetate in cyclohexane as eluent can be seen. In the procedure using 2% pyridine in cyclohexane as eluent, for some compounds only low recoveries could be achieved.

Next the ability of these Lipidex procedures to remove interfering compounds from brain extracts derivatised with HFBA was investigated after elution with the above two solvent mixtures. Aliquots of free steroid fractions of brain extracts were compared before and after purification by Lipidex 5000® chromatography with the above mobile phases.

Only 2% pyridine in cyclohexane gave a satisfactory clean-up of the sample and was used in the extractions of rat brains described in Chapter 5.

4.2.2.6 Overall recovery of improved procedure for extraction and fractionation

of steroids from brain tissue

The final procedure for extraction and separation of free steroids and their sulphate esters, as devised after consideration of the preceding experiments, is summarised in Figure 4-12. Extraction efficiency for endogenous steroids by acetic acid/ethanol was shown in 4.2.2.1 for rats injected i.p. with ^H-PROG. To evaluate the overall fractionation procedure for a wide range of steroids, a cocktail of standard compounds at known concentrations was added to rat brain homogenates. The recovery was then estimated by GC-MS and two ion SIM and is shown in Chapter 5, Table 5-7 beside quantitations of endogenous brain steroids. As a final evaluation of this procedure, its application to the survey of steroids in the mammalian brain is described in Chapter 5.

4.3 Conclusion

An improved and simplified procedure for the extraction, purification and fractionation of free steroids and their sulphate esters from mammalian nervous tissue for analysis by GC-MS could be developed. A combined extraction of free and conjugated steroids into acetic acid/ethanol is followed by purification through solvent partitioning and a single column chromatographic step using the polymer based hydrophilic lipophilic balance

155 Oasis® cartridges. Fractionation of free and conjugated steroids is then done using the novel Oasis MAX® hydrophilic lipophilic balance anion exchange chromatography cartridges.

This method presents advantages over traditional procedures based on liquid-liquid extraction methods using organic solvents such as ethyl acetate. Free and sulphate linked steroids can be processed through purification procedures, which usually are required for both fractions, in the same sample, reducing greatly the time and materials needed for sample preparation. The initial extraction step reduces the amount of interfering lipidic material present compared to extraction with ethyl acetate and similar solvents, thus further decreasing the degree of extract purification required.

Fractionation of steroids by Oasis MAX® hydrophilic lipophilic balance anion exchange chromatography has been used for the first time on brain extracts. This technique is more reliable for separating free steroids and their sulphate esters than liquid-liquid extraction between aqueous phases and ethyl acetate. Furthermore it allows separation of these steroid classes from steroid glucuronide conjugates. The Oasis MAX® fractionation should also be superior to silica based strong anion exchange (SAX) fractionation, the application of which to fractionation of steroids from biological fluids has been previously reported [197], but not from nervous tissue, in that the stationary phase is more stable and does not collapse on drying out.

The novel procedure has been shown to be suitable for a wide range of steroids of varying polarities and thus can be applied as described in Chapter 5.

156 Table 4-1. Recovery (%) of steroids in 90% or 80% methanol in H2 O after solvent partitioning against isooctane. Steroid content was determined by GC-MS in the scan mode after derivatisation by MO and TMSI. Values represent means (n=3 for 90% methanol, n=2 for 80% methanol).

Steroid 90% methanol 80% methanol 20a-DHPROG 97.0 97.4 3a,5a-THDOC 95.7 90.9 17-OH-PROG 97.7 99 2 PROG 96.7 94.8 3a,5a-THPROG 93 3 83 9 PREG 95 3 89.7 5a-DHPR0G 90.7 79.5

Table 4-2. Recovery of steroids from Sep Pak CIS® chromatography. Steroids were loaded in 10 ml methanol (80% in H%0, v/v) and eluted with 5 ml 80% methanol. Steroid content was determined by GC-MS. Values represent mean ± SEM, n=5.

Steroid Recovery (%) 3a,5a-THPROG 50.7+3.8 5a-DHPR0G 58.3+3.0 3a,5a-THDOC 81.2+4.9 PREG 56.8±3.0

PROG 58.2±4.2 20a-DHPROG 93.9±3.9

157 Table 4-3. Recovery of selected steroids from celite chromatography with ethylene glycol stationary phase (1:1, w/v). Water traps of 0.3 g or 0.6 g celite (celite : H2 O 3:1, w/v) were installed below the stationary phases. Steroids (5 pg) were loaded in 1.5 ml isooctane saturated with ethylene glycol. After washing with 2 ml isooctane, elution followed with 40 ml 30% ethyl acetate in isooctane (v/v). Steroid recoveries were calculated from peak area ratios to internal standards in the eluate compared to standard samples as analysed by GC-MS in the SIM mode after derivatisation with MO and TMSI. Values are means (ranges), n=3 (n=2 for 0.3 g 2HO trap).

Steroid 0.3 g H2 O trap 0.6 g H2 O trap lip-OH-PROG 10.8(3.6) 24.4(13.4) 5 P-pregnan-3a, 17-diol-20-one 32(0) 19.7 (7.2)

20P-DHPROG 17.4 (2) 34.7 (7.2)

3a,5a-THPROG 30.2(14.4) 15.6(2) 3a-DHPR0G 13.6(5.2) 27.3 (2.4) DHEA 10.2(2.8) 15.0(1.4) EpiA 19.2(4.4) 26.4 (2.4) PREG 13,2(1.6) 26.1 (4.8)

Table 4-4. Recoveries (%) of H-CORT and H 5a DHPROG from ethylene and propylene glycol stationary phases on celite (1 g). Ethylene and propylene glycol (4:1:1, w/v/v), elution with 5 ml isooctane, 6 ml 20 % ethyl acetate in isooctane (v/v) and 12 ml 50 % ethyl acetate in isooctane (v/v); ethylene glycol (2 : 1, w/v), elution with 5 ml isooctane, 8 ml 20 % ethyl acetate in isooctane (v/v) and 8 ml 50 % ethyl acetate in isooctane (v/v); ethylene glycol (1:1, w/v), elution with 6 ml isooctane, 14 ml 20 % ethyl acetate in isooctane (v/v) and 15 ml 50 % ethyl acetate in isooctane (v/v). Samples were loaded in 1.5 ml isooctane saturated with ethylene glycol and the columns washed with 2 ml isooctane before elution (not for ^H-5a-DHPR0G). The eluate was partitioned against H2 O before drying down and redissolving for counting of radioactivity. Data represent mean ± SD from 3 experiments or single observations (*). - not determined.

Stationary phase ^H-CORT ^H-5a-DHPR0G (celite ; ethylene glycol : propylene glycol) 4:1:1 109.2 ±0.1 109.4* 2:1:0 112.9 + 0.1 96.4* 1:1:0 115.9 + 0.0 -

158 Table 4-5. Recoveries (%) of reference steroids from celite (1 g) columns with ethylene and propylene glycol (4:1:1, w/v/v) stationary phases. Samples were loaded in 1.5 ml isooctane saturated with ethylene glycol and the columns washed with 1 ml isooctane before elution with 5 ml isooctane, 6 ml 20% ethyl acetate in isooctane and 12 ml 50 % ethyl acetate in isooctane.

The eluate was partitioned against H2 O before drying down and redissolving for derivatisation and analysis by GC-MS. Data represent mean ± SD, n=3. Steroid % Recovery DHEA 81.4 ±9.9

3a,5a-THPROG 92.1 ±6.3 3a,5a-THDOC 107.9 ±8.2

5a-DHPR0G 85.2 ±8.1 CORT 89.8 ±5.9 CHOL 57.8 ±8.1 CHOLBUT 63.0 ±8.3

Table 4-6. Recoveries of free steroids added to and concentrations of endogenous free steroids in human cerebral cortex (approx. 5 g each) extracted by original procedure. In this extraction. Oasis HLB cartridges were eluted with 80% ethanol and celite/propylene glycol columns (1:1, w/v) were eluted with 48 ml 10% ethyl acetate in isooctane after a 2 ml isooctane wash. Steroids were analysed as their MO-TMS-derivatives by GC-MS in two ion SEM. * This value could not be obtained due to an error in the analysis. INT.* tissue matrix interference prevented analysis. Recovery (%) Concentration (ng/g) DHEA 90.4 0.46 3a,5a-THPROG 38.0 TNT 3a,5p-THPROG 43.4 INT 5a-DHPR0G 12.8 INT 17-OH-PREG 27.4 INT 3a,5a-THDOC 65 9 INT

3a,5p-THDOC 52.4 INT PREG * 4.48 PROG 31.5 INT 20P-DHPROG 49.1 INT

20a-DHPROG 49.3 INT 17-OH-PROG 28.9 INT DOC 62.4 INT

159 Table 4-7. Responses of DHEA or DHEAS and PREG or PREGS after derivatisation with MO and TMSI to the internal standard ME-17-0H-PR0G after different pre-treatments on GC- MS. Values are relative, with responses of the free steroid derivatives set as 100. For comparison, responses of DHEAS and PREGS after HFBA-derivatisation and solvolysis were also calculated. In this case, the ratios were calculated to the internal standard tetracosane and compared to response ratios of the free steroid MO-TMS-derivatives to the same amount of tetracosane set as 100. For further explanation of sample pre-treatments see text.

Sample pre-treatment DHEA/DHEAS PREG/PREGS Free steroids: None 100.0 100.0 Steroid sulphates: None 20.3 10.2 Microsolvolysis 75.2 35.5 Microsolvolysis after drying down 11.9 6.4 from 60% ethanol/NHiCOs solution Solvolysis without wash 0.3 0.3 Solvolysis with wash 0.5 1.2 Solvolysis in BSA/ acidified ethyl 47.2 21.7 acetate Solvolysis in BSA/ acidified ethyl 73.9 49.7

acetate/ Na2 SÛ4 Solvolysis in BSA/ acidified ethyl 172.7 114.8

acetate/ Na2 S0 4 followed by H 2 O wash and derivatisation by HFBA

160 Figure 4-1. Stationary phases of solid phase extraction cartridges, a) Sepak CIS®, b) Oasis HLB® (poly(divinylbenzene-co-N-vinylpyrrolidone)), c) Oasis MAX®.

a)

\ Si-Q—Si— (CH2)i7CH3 0 Si-OH / b)

c)

161 Figure 4-2. Removal of cholesterol from rat hrain extracts by Sep Pak C18© solid phase extraction. Ethyl acetate extracts of approx. 4 g tissue were isooctane partitioned and either (a) first passed through C18 cartridges in 80% methanol (corresponding to 100 mg tissue/mg stationary phase) or (b) directly loaded onto celite columns after drying down and reconstitution in 1 ml 10% ethyl acetate in isooctane. Elution from celite columns followed in 48 ml 10% ethyl acetate in isooctane. The eluates were dried down after addition of internal standard, derivatised by MO-TMSI and analysed by GC-MS in two ion SIM. Cholesterol eluted at 26-30 minutes.

a)

7000000

soooooo

5000000

SOOOOOO

2000000

1000000

15.00 18.00 20.00 2 2 .0 0 24.00 28.00 30.00 32.00 34.00 b)

A b un dan ce

4 50000

4 00000

350000

300000

2 50000

200000

150000

1OOOOO-

5 0000 -

Tim e—> 16.00 18.00 20.00 22.00 24.00 26.00 28 OO 30 OO 32 OO 34 OO 36 OO

162 Figure 4-3. Recovery (%) of ^H-PROG and ‘‘‘C-CHOL from Oasis HLB® cartridges. 67000 dpm ^H-PROG (light grey columns) or 10000 dpm CHOL (dark grey columns) and 100 ng PROG and 1 |ig CHOL butyrate were loaded in 10 ml 80% methanol in phosphate buffer (5 mM, pH 7.4, v/v) and further eluted with 5 ml of the same solvent and 10 ml of 100% methanol. H PROG and ' C CHOL were loaded on different columns. The eluate was collected in fractions of 10 ml and then 5 ml and aliquots were taken and counted for radioactivity. Data points represent means ± SD, n=3.

80% methanol 100 % methanol 100

80

^ 60

I0) a: 40

20

Elution fraction number

163 Figure 4-4. Elution profiles (% recovery) of ^H-5a-DHPR0G (5P), ^H-PROG (P) and ^H- CORT (C) from celite with ethylene glycol stationary phase (1:1, w/v). Samples were loaded in 1.5 ml isooctane saturated with ethylene glycol, elution in 30% ethyl acetate in isooctane as indicated. P and C were loaded in the same sample.

Isooctane 30 % ethyl acetate in Isooctane 80-1 5P P

80-

20 -

0 10 20 30 43 30 Biüon\^Lrne(ml)

Figure 4-5. Elution of ^H-5a-DHPR0G from celite (1 g): ethylene glycol : propylene glycol (circles: 2:1:0, squares: 4:1:1) stationary phases. Samples were loaded in 1.5 ml isooctane saturated with ethylene glycol and then eluted in isooctane.

80-

43-

2 4 6 8 10 12 14 BilicnUiLme(mi)

164 Figure 4-6. Original procedure for extraction and separation of free steroids and their sulphate esters from nervous tissue.

Homogenise tissue in 5 volumes phosphate buffer (0.1 M, pH 7.4) I Extract with ethyl acetate 1:1 three times

Aqueous phase * 4- Organic phase I I Wash with 0.5 volumes H 2O Add NaCl to 20% (w/v), H^SO^ to I PHl I Dry down, redissolve in 0.9 volumes isooctane and 0.9 Extract with ethyl acetate 1:1 three times volumes 80% methanol (3 min shaking) I Shake 3 minutes, vortex 1 4 ---- Incubate at 38°C, 16 h minute repeat T twice I Transfer methanol phase to clean tube, add 0.9 volumes — Neutralise with pyridine, dry down 80% methanol to isooctane phase I Redissolve in 2 volumes 80% methanol, load onto Oasis HLB Load pooled methanol phase onto Oasis (corresponding to 35mg tissue/mg HLB (corresponding to 35mg tissue/mg stationary phase), collect eluate stationary phase), collect eluate I I Elute Oasis with 0.5 volumes 80% methanol I I Dry down pooled eluate I T Redissolve in 1.5 ml isooctane saturated with ethylene glycol, load celite/ethylene glycol/propylene glycol column (4:1:1, w/v/v) I I Wash celite column with 1 ml isooctane 1 I Elute celite column with 5 ml isooctane, 6 ml 20% ethyl acetate in isooctane (v/v) and 12 ml 50% ethyl acetate in isooctane (v/v).

1 \ Wash eluate witli HjO 1:6 (v/v)

1 ^ Steroids from sulphate Free steroids conjugates

165 Figure 4-7. Selected ion chromatograms of MO-TMS-DHEA from human cerebral cortex extracted by the original procedure and analysed by GC-MS in two ion SIM.

A bundance Ion 358.35 (iii/z) 1000

600

2001 17.30 17.35 17.40 17.45 17.50 T im e- Abundance Ion 268.20 (nVz) 1000

600

2001—— Time- 17.25 17.30 17.40 17.45 17.5017.35

Figure 4-8. Recoveries of H-PROG in rat hrain extracts from Oasis HLB® cartridges with a) 60 % (circles) and h) 80% (squares) ethanol in potassium phosphate buffer (5mM, pH 7.4, v/v). H PROG ( a) 223000 or b) 1.2*10* dpm) were added to ethanol extracts (10 xolumes, w/v), after which they were dried down and redissolved in a) 10 volumes or b) 5 volumes elution solvent. The solutions were loaded (corresponding to 7 mg tissue/mg stationary phase) and the cartridges further eluted with a) 40 volumes b) 20volumes of the same solvent and in the case of b) then with 10 volumes of 100% ethanol. Fractions of 2 ml were collected, dried down and counted for radioactivity which was used to calculate cumulative recoveries. Each point is the mean of 2 observations.

120 n

100 -

80 H

0 0 -

^ 4 0 -

2 0 -

0 -

I 10 15 20

But)on vdLire (ml)

166 Figure 4-9. Elution of a) steroid sulphate esters and b) free steroids from Oasis HLB® cartridges. Compounds are indicated in the Figure. Samples were loaded in 3 ml 20% ethanol in potassium phosphate buffer (5 mM, pH 7, v/v) and washed with 5 ml of the loading solvent; steroid sulphates were eluted with 8 ml 40% ethanol in potassium phosphate buffer (5 mM, pH 7, v/v), free steroids with 8 ml 60% ethanol in potassium phosphate buffer (5 mM, pH 7, v/v). The eluates were collected in 4 ml fractions, dried down and analysed as their MO-TMS- derivatives by GC-MS. Relative elution (%) was calculated by comparison of area ratios to standards in each fraction to the overall eluate. Data points represent means ± SD, n=3 or single observations. a) 40 % EtOH: 60 % EtOH: 5 mM KH^PO, 5 mM KH2PO, 100-, buffer pH 7 buffer pH 7

80- — DHEAS - • - PREGS

c 60 - o

40-

2 0 -

0 1 2 3 4 Elution fraction number b)

40 % EtOH 60 % EtOH: 5 mM KH2PO4 5 mM KH2PO4 buffer pH 7 buffer pH 7

—■— EpiA - • - TESTO ■ A--3a,5a-THPROG - T - PROG - 3a,5a-THDOC CORT

2 3 Elution fraction number

167 Figure 4-10. Elution of ^H-DHEA from Oasis MAX© in different load solvents. ^H-DHEA (8*10^ dpm) was added to rat brain extracts previously isooctane partitioned and passed through Oasis HLB®. Samples were loaded in 60 ml 30% ethanol (•), 40 ml 30% ethanol (+), 48 ml 25% ethanol (■) and 60 ml 20% ethanol ( ^ , all in potassium phosphate buffer 5 mM, pH 7.4, v/v; ) and washed with 5 ml of the loading solvent; elution continued with 16 ml 60% ethanol in ammonium acetate buffer (20 mM, pH 7, v/v), 12 ml 60% ethanol in formate/pyridine buffer (20 mM, pH 3, v/v) and 8 ml 60% ethanol in 0.1 M H 2SO4/O.5 M NaCl (v/v). Elution fractions were dried down and counted for radioactivity to calculate % elution.

100-I Load Wash i Eluate; Eluate: Eluate 90- 80- 70- Î 0 0 - TD 50- 40- 1 30- 2 0 -

10 -

0 0.1 0.2 0.30.4 0.5 0.6 0.7 0.8 0.9 1 Relative el Lticn vd un e

Figure 4-11. Elution of HFB-derivatised reference steroids from Lipidex 5000® chromatography columns. Elution with 10% ethyl acetate in cyclohexane (v/v, panels on the left) or 2% pyridine in cyclohexane (v/v, panels on the right) as analysed by 2 ion SIM. Percentage cumulative recovery against elution volume of single observations.

168 Figure 4-11 continued.

100-, lOOn

80 80 -■ — TESTO # - DHEA ■ - TESTO 60- A 5p-pregnane- k 60 • - DHEA 3a.20p-diol I A 5p-pregnane- i 40- ▼- 3a,5a-THPROG Ë 40- 3a,20p-diol 3p,5p-THPROG ▼ 3a.5a-THPROG I + 20P-DHPROG % - 20p-DHPROG 20 2 0 -

OH 0 - #" -T" 3 4 Elution Volume (ml) Elution Volume (ml) 80-1 60- PROG PROG 60- — — _ — — — — PREG PREG - A- 3a,5a-THDOC 40- 3a,5a-THDOC T- 3a,5p-THDOC o ▼ - 3a,5p-THDOC | 40- - ♦ - 17-OH-PROG 17-OH-PROG 2 - + - 3p,5a-THDOC - 4--- 3p,5a-THDOC / •| 20- 5p-DHD0C CO X Sp-DHDOC 2 0 - a # 0 - “I---- ' I ' I ' I ' r 1 2 3 4 5 6 1 2 3 4 5 Elution Volume (ml) Elution Volume (ml)

169 Figure 4-11 continued.

80n

5a-DHD0C 60 * —■— 5a-DHD0C 60- • ' 5a-pregnane- » - • - 5a-pregnane- 3a,20a-diol 3a,20a-diol ^ 5p-pregnane- 40 -A 5p-pregnane- 40- 3a,20a-diol 3a,20a-diol ▼- 20P-DHPREG - 20p-DHPREG ♦- 5a-pregnan- ♦- 5a-pregnan- % 204 3a,17-diol-20-one 3a,17-diol-20-one 4- 20a-DHPROG + 20a-DHPROG X 20a-DHPREG X 20a-DHPREG ^ OH

~i----'----- 1---'----- 1--- “ I r 2 3 4 5 Elution Volume (ml) Elution Volume (ml)

50- 100- 3p,5p-THDOC -A— 3p,5p-THDOC 5a ,20a-THPROG 40- T" 5a,20a-THPROG g 80- 17-OH-PREG ♦ 17-OH-PREG DOC 30- 4 - DOC î 60- llp-OH-PROG X 11p-0H-PR0G .A- CORT 2 0 - * CORT % 40- 16 mt '-y I 1 0 - 20- 4 d 0 - 0- T---- 2 3 4 5 Elution Volume (ml) Elution Volume (ml)

170 Figure 4-12. Final procedure for extraction and separation of free steroids and their sulphate esters from nervous tissue.

Homogenise tissue in 5 volumes phosphate buffer I Extract into 20 volumes 3% acetic acid/ethanol, centrifuge i Supernatant partition against 10 volumes isooctane three times I Dry down, dissolve in 4 volumes 60% ethanol, centrifuge

Load supernatant onto Oasis HLB (corresponding to 35mg tissue/mg stationary phase), collect eluate I Elute Oasis with 4.4 volumes 60% ethanol I Dry down pooled eluate i Redissolve in 20% ethanol, load onto Oasis MAX cartridge (corresponding to 10.5 mg tissue/mg stationary phase) I Wash cartridge with 5 ml 20% ethanol i Elute cartridge with 4 ml ethyl acetate —► Free steroids I Elute cartridge with 20 ml 60%ethanol __ ^ Steroid glucuronides in formate/pyridine (20 mM, pH 3) I Wash cartridge with 2 ml ethyl acetate dried over Na 2 S0 4

Elute cartridge with 15 ml BSA/acidified Incubate 16 h at 40°C, Na 2 S0 4 ethyl acetate (50 mM) dried over Na 2 S0 4 + Neutralise with pyridine, dry down I Wash with H 2 O three times - Extract with ether three times I I Steroids from sulphate Steroids from sulphate conjugates for derivatisation conjugates for derivatisation with HFBA with MO/TMSI

171 Chapter 5 A survey of steroids in the mammalian brain

5.1 Introduction

Dehydroepiandrosterone (DHEA) was the first steroid to be characterised in mammalian brain (Chapter 1). The sulphate ester of DHEA was also reported. Since then, pregnenolone (PREG) has been the steroid found at the highest level in most species, in accordance with its role as precursor of most other steroids (see Table 5-1, values irrespective of steroid source). Other steroids in brain of male rats demonstrated by unequivocal methods include PROG, 5a-DHPR0G, 3a,5a-THPROG, 3P,5a-THPROG and EpiA. Considerable amounts of sulphate and lipid conjugates of the first two mentioned steroids were also determined. Similar levels of steroids were found in male mouse brain, except for PROG which seems to be somewhat lower than in the rat [212]. In humans, DHEA, DHEAS, PREG, PREGS have been determined in several regions of the brain and were found to be in the same ranges as in rat brain. Values were found to be considerably higher than in plasma [98], suggesting an ability for independent synthesis sirnilar to rat. PROG, 5a-DHPR0G and 3a,5a-THPROG were found to show significant variations between different regions of the female human brain and in different endocrine states [22]. Significant correlations between brain and plasma PROG, but not between brain 3a,5a-THPROG and plasma PROG overall were found. Several steroids have also been demonstrated in guinea pig, dog, pig and monkey brain [12,119].

The aims of this Chapter were to establish the free and sulphate conjugated steroid metabolites in male rat brain. This was attempted by applying the improved procedures for extraction, fractionation and analysis of steroids from nervous tissue developed in the previous Chapters. After preliminary screening for individual steroids by two ion selected ion monitoring (SIM) (in 5-7 panels each in 4 injections) and for generic steroids by 32 ion SIM (in 4 injections), preliminarily identified compounds were formally identified by applying three ion SIM and again two ion SIM after derivatisation by a different method. Also shown.are quantitations of positively identified compounds.

172 5.2 Results and discussion

5.2.1 Two ion selected ion monitoring of steroids in rat brain

As an initial screen, free steroid and steroid sulphate fractions of rat brain extracts were monitored for steroids by GC-MS with two ion SIM (SIM methods 2-5). These extracts of adult male rat whole brain were prepared by the improved extraction and fractionation procedure developed as described in Chapter 4 and summarised in Figure 4-12. The steroids were then derivatised with MO and TMSI. The development, specificity and sensitivity of the two ion SIM methods for a wide range of steroids was shown in 3.2.5 and 3.2.8.

Examples for the coelution of the diagnostic ions of possible endogenous steroid metabolites are shown in Figure 5-1 for the free steroids and Figure 5-2 for steroid sulphate esters. In some cases, interferences were observed for one of the two ions. The compounds concerned were still considered in the formal identification procedures presented below. The tentative identities of the compounds as analysed here are shown in Table 5-2.

5.2.2 Multiple ion screening of steroids in rat brain

Although as wide a range of endogenous metabolites as possible was screened in the above two ion SIM procedures, other compounds might have been missed. A different approach to look for unknown steroids in the unconjugated fraction was to use multiple ion screening of brain extracts. In this method, four groups of 32 ions are monitored simultaneously (SIM methods 22-25). The ions were predicted from the fragmentations of MO-TMS-derivatised steroids (Table A-1) taking into account different steroid nuclear structures and all possible permutations of substitutions. Examples of co-elutions of potential diagnostic ions from those extracts are shown in Figure 5-3. As expected, these analyses showed peaks for ions of some of the free steroids already detected in the two ion SIM analyses (e.g. PROG, Figure 5-3 AP-dione). In addition, other diagnostic ions were found to co-elute and are shown in Table 5-3 alongside their probable generic structures. Attempts were made to match those newly revealed analytes with standard compounds, and these are also shown in Table 5-3 for comparison of their relative retention times (RRT). Two of these putative analyte steroids had diagnostic ions which

173 elute within ±1% of the RRT of ions from a standard steroid: 4-androsten-3 p-ol-17-one and 4-pregnene-6P-ol-3,20-dione. Here a slightly wider variability of RRT of ±1% compared to ±0.5% used in the other SIM methods (see 5.2.3) was applied for identification to allow for the fact that RRTs across the whole range were calculated to a single internal standard (ME-17-OH-PROG). Although only one peak of co-eluting ions indicative of the latter compound was detected, it was considered for further investigation with three ion SIM, as it is possible that the second peak was below detection limit. The 32 ion SIM methods have to be considered less sensitive than respective two ion SIM methods.

It was beyond the scope of the project to analyse all the possible standard compounds matching the structures deduced from the co-elution of ions in those multiple ion screens. Furthermore the possibility that some of the ion peaks detected stem fi'om isotopes of fragments with a different nominal mass cannot be excluded. Co-eluting ions for which standards with the possible structures could not be analysed remain subject to further confirmation of identity.

5.2.3 Confirmation and quantitation of brain steroids

Definitive identification of the compounds revealed in brain extracts in the above two ion SIM and multiple ion screens was attempted by monitoring co-elution of three ions of these MO-TMS-derivatives (SIM methods 6-9 for free steroids, SIM methods 10-13 for steroid sulphates and 14-15 after adjustments, see below, p. 176). Additional confirmation was sought by using a second derivatisation method on these brain extracts using HFBA and two ion SEM (SIM methods 16-17 for free steroids, SIM methods 18- 19 for steroid sulphates and 20-21 after adjustments). The additional ions for SEM of the MO-TMS derivatives were chosen after the same criteria as for the two ion SIM methods (see 3.2.5). Similarly, specific ions for two ion SEM of EEFBA-derivatives were chosen (see Appendix 1).

The criteria for identification were both RRT and qualifier to target ion ratios (Q/T). The first criterion used was for the RRT of a potential endogenous compound to be within ± 0.5% of the RRT of a standard compound run alongside the tissue sample. The second criterion was for the endogenous compound to be within ± 20% relative abundance of

174 both T and Q ions. The lower and upper limits of the respective Q/T ratios are therefore 0.67 and 1.5 x the standard value.

Examples of RRTs and Q/Ts for unconjugated and sulphate conjugated endogenous rat brain steroids for which all ions monitored were above the detection limit are shown in Table 5-4 and Table 5-5. Table 5-4 shows the RRT results for derivatives from several extracts, alongside the values given by standard compounds. These identification limits were developed as pragmatic limits in this study by analysis of numerous samples of reference compounds. In order to test and finther strengthen the basis of those pragmatic limits, confidence limits (at the 99.9% level) for RRTs using parametric statistics were also calculated from a number of standard samples. These are shown alongside the above pragmatic limits. As the examples show, the former are in good agreement with those confidence limits. The identification results were in agreement when either the pragmatic 0.5% or the 99.9% parametric confidence limits were applied in 82 out of 87 cases. For the number of analyses carried out, it can be expected for practical reasons that some standard compounds will show a narrower distribution of relative retention times than given by the above pragmatic limits. Generally, this was taken as a good backup for the pragmatic 0.5% limits and these can be justifiedly applied.

Table 5-5 shows the Q/T values of several derivatives from brain extracts alongside values from standard compounds. Again, pragmatic limits are used and compared with parametric 99.9% confidence limits calculated from various standard runs. The identification results were the same with both the pragmatic 20% and the 99.9% parametric confidence limits except in 4 cases out of 102, thus providing further confidence for the use of these identification criteria.

Overall definitive identification was then assumed, if either co-elution of three ions of the MO-TMS-derivative within the RRT- and Q/T- limits or co-elution of two ions within the RRT- and Q/T-limits from both of two different derivatisation methods (MO-TMSI and HFBA) was observed. Tentative identification of compounds was suggested where RRT- and Q/T-criteria were fulfilled in two ion SIM of HFB-derivatives. The identification procedures of both RRT and Q/T analyses of brain steroids and the final decision on identification are summarised in Table 5-6. Examples of the co-eluting ion peaks of both MO-TMS- and HFB- derivatives of endogenous free and sulphate conjugated steroids are shown in Figure 5-4 and Figure 5-5 respectively.

175 Among the free steroids, 5a-DHPR0G showed co-elution with the Q/T-ratios somewhat outside the limits. However, this compound did not derivatise with HFBA, thus could not be confirmed in this way and was presented as tentatively identified. The free steroids unequivocally confirmed are DHEA, 3a,5a-THPROG, PREG, PROG, 3a,5a-THDOC, CORT, 20P-DHPREG, 20a-DHPREG, TESTO, 5a-pregnan-3a,17-diol-20-one. Tentative identification is suggested for 5a-DHPR0G, 20a-DHPROG, 5a,20a- THPROG, 5a-pregnane-3a,20a-diol, 3p,5a-THPROG, 3p,5a-THDOC, 5a-pregnan- 3a, 11 p-diol-20-one.

In the sulphate fraction only DHEA was unequivocally identified. Analysis of steroid sulphate conjugates gave interferences for several compounds in some ion responses. In subsequent analyses different ions were chosen in those cases as described below. For PREGS after MO-TMSI derivatisation, ions 402, 386, 312 were detected, but large interferences seen for ion 402. Thereafter ion 296 was monitored for the compound instead. However, Q/T ratios did not fulfil the diagnostic criteria. Furthermore, signals for the compound in brain extracts after HFB-derivatisation were below detection limit. The MO-TMS derivative of 3a-DHPR0GS ion 417 was detected in extract 5, but interferences were observed for ions 244 and 386. In analyses of subsequent extracts, the ions were thus changed to 417, 312, 326. However, no signal above detection limit was detected in those extracts for those ions HFB-derivatives of the compound were not analysed as they could not be resolved from the 3P-DHPR0G derivative. Interferences were also observed for ions 386 and 364 of MO-TMS- 5a-pregnan-3a,17-diol-20-one- S. Other ions monitored were 476, 188 and 296 in subsequent analyses, but in those were below detection limit. The HFB derivative of this steroid gave a signal in analysis of a brain extract for only the target ion (442). The qualifier ion (487) was below detection limit. For the MO-TMS derivatives of 5a-pregnane-3a,20a-diol-S and 5p-pregnane- 3a,20a-diol-S two ions (269, 284) were detected, but a third ion was below detection limit (449). However, this ion has an expected relative abundance of approximately 10- 15% and thus might be easily missed. There are no other unique ions with higher relative abundance for those compounds and with 269, 284 and 346 as diagnostic ions the compound could not be further confirmed. No detectable signals were observed for the HFB-derivative of 5a-pregnane-3a,20a-diol-S. For the HFB-derivative of 5p-pregnane- 3a,20a-diol-S only ion 712 was above detection limit. Ion 469 was observed for 5a- pregnan-3a,l lp-diol-20-one-S as HFB-derivative, but the compound could not be

176 confirmed as MO-TMS-derivative. Various ions (386, 244, 417, 296, 312) of MO-TMS- 3p-DHPR0GS were monitored in different brain extracts. However, there were either interferences or no detectable signals in the three ion monitoring of the compound. This compound was not determined in two ion monitoring after HFB-derivatisation, because it could not be resolved from 3a-DHPR0G as mentioned earlier. For compounds where all ion peaks were above detection limit and peaks were not distorted by interference, the RRTs and Q/Ts are shown in Table 5-4 and Table 5-5.

Both confirmed and tentatively identified compounds were quantified in either two or three ion SIM. Quantitation was done using area ratios of the target ions of MO-TMS- derivatives to the internal standards and calibration curve functions shown in Table 3-5. For quantitation in two ion SIM of HFB derivatives and certain compounds in three ion SIM of MO-TMS-derivatives single point calibrations were used. All extracts had been prepared as described in Methods (2.3.7), except extract 8 which was twice subjected to Lipidex chromatography after HFBA derivatisation. The concentrations of endogenous steroids in male adult rat whole brain from several extracts are shown in Table 5-7. Recoveries of standard steroids added to brain hoinogenates and carried through the extraction, fractionation and assay procedure are also shown in this Table.

Detection limits for the two ion SIM methods of all the compounds listed in Table A-1 and carried in pure solution reagent blanks alongside extracts of rat brain were determined for both the free steroid and the steroid sulphate fractions. Compounds were not considered to be detected, unless their ion areas were at least three times the equivalent signals at the same relative retention time in reagent blanks analysed alongside the tissue samples (for consideration of detection limits see 3.1). The concentrations at those levels were determined from calibrations performed using linear regression (with the exception of 17-OH-PREG which gave a quadratic curve, calibration functions see Table 3-5) and the minimal values from four free steroid and three steroid sulphate extractions carried out are also shown in Table 5-7.

Blood contamination of brain samples was estimated by the spectrophotometric measurement of haemoglobin and found to be less than 0.6% (v/v). Other estimates of blood contamination of rat brain samples [202] from assays of peroxidase activity have yielded similar values (0.3-0.7 %). The blood contamination is for most steroids thus unlikely to contribute significantly to the brain concentrations measured (it was estimated

177 for PREG, DHEA, TESTO and found to be maximally about 1%, using plasma values reported in [12,103,190,213]). An exception is CORT. The CORT concentration reaches values of up to approximately 115 ng/ml at the peak of the daily cycle at 20.00 h in regimens of lights on from 7.00 - 19.00 h [103]. At this level the blood content of the brain samples of this study would amount to 12% of the mean concentration determined. The samples in this study were collected around about the nadir of the plasma CORT diurnal cycle (11.00 h), where its concentration is typically about 13 ng/ml [103], however it cannot be definitely excluded that higher plasma concentrations were present in the animals used.

As can be seen from Table 5-7, the most abundant steroid found in the present study of male rat brain was CORT. The second most abundant compound was PREG, followed by TESTO, PROG, 5a-DHPR0G and 3(3,5a-THDOC. Somewhat lower were then 20a- DHPREG, 20P-DHPREG, 3a,5a-THPR0G, 3p,5a-THPROG, 5a,20a-THPROG and 3a,5a-THDOC. The lowest levels were found for DHEA, 20a-DHPROG, 5a-pregnane- 3a,20a-diol, 5a-pregnan-3a,17-diol-20-one and 5a-pregnan-3a, 11 p-diol-20-one. DHEAS was somewhat higher than DHEA. The concentrations found were varying notably between analyses of extracts (see Table 5-7), even though animals were kept strictly under the same controlled conditions. Variabilities of steroid concentrations in brain extracts from animals kept under the same conditions were previously reported, however. Shimada et al. [176] found PREG levels to vary between animals under the same treatment by a factor of ~10. Variations can also be seen in Table 5-1 summarising previous reported identifications of steroids. Consistent with previously reported findings the levels of PREG and DHEAS were much higher compared to DHEA [48,106]. However, the previously reported high concentrations of PREGS (see 5.1, for example [50,119]) could not be confirmed here and no further sulphate esters could be formally described, with exception of DHEAS. The recovery of PREGS in the extraction procedure here was relatively low, but even after correction for procedural losses levels of 0.25 ng/g would have been detected, which is much lower than in some previous reports. It is possible that the lack of detection of this steroid here is due to natural variation. Another possible reason for the discrepancy is the difference in the methodologies used. The major difference in the sample preparation between the present and other studies on steroids in brain is the separation of free steroids and their conjugates. Here Oasis MAX® ion exchange chromatography was used for the complete

178 separation of free steroids from sulphate esters as opposed to the partial separation of organic solvent extraction from aqueous phases or polarity based solid phase extraction used for this task in previous reports (see Chapter 4). Complete separation of free steroids from the sulphate esters on the MAX cartridges used in the present study has also been confirmed by analysis of urine samples (see Appendix 2).

Criteria for unequivocal identification in SIM are a subject of wide debate (see for instance [19]) and there is a wide variability of criteria used throughout the literature. For mass spectrometric identification of steroids in brain, several methods have been employed. Whereas PREG, DHEA, their sulphate and fatty acid esters have been subject to many studies and have been well characterised including by 5 ion SIM, other compounds have only been measured with single ion monitoring. The more ions are used in SIM the higher the specificity achieved. However, there is a limit to the practicability of this approach, as there is a decline in sensitivity as more ions are monitored. Furthermore, it is in most cases not possible to find several specific ions that are without interference from neighbouring compounds when a large number of close-by eluting compounds are monitored simultaneously. In the above mentioned analyses using 5 ion SIM a single compound was characterised using pooled tissue from several animals.

An investigation into specificity of low-resolution MS has shown that three ion monitoring with ion response ratios within ± 20% of those of standards allows unique identification (see 3.1.4). Additional specificity is introduced if MS three ion monitoring is used in conjunction with LC or GC and selective sample preparation steps. Three (or more) ion monitoring has not been applied so far for identification of brain steroids, except in the case of PREG, DHEA, 3a,5a-THPROG, 3p,5a-THPROG and EpiA. In those cases relative ion variabilities were not shown, however. In the past few years several methods for measurement of steroids in brain tissue based on GC-MS or LC-MS have been developed (see for example [38,66,106,130,175,177,194,196]). These methods, however, are all limited to the quantitation of the already identified compounds PREG, DHEA, PROG, 3a,5a-THPROG or PREGS and DHEAS. In this study it was for the first time attempted to identify a wider range of compounds with unequivocal identification methods.

179 Previously identified compounds in rat brain could be confirmed and several further compounds were identified. Further evidence was presented for a wide range of analytes for further confirmation.

The source of the identified steroids in the present study could not be assigned to entry via the BBB after synthesis by peripheral steroidogenic glands or to synthesis within the brain. However, knowledge of steroid metabolising enzymes found in brain (see Section 1.3.1.5) allows some speculation as to the possible pathways generating the compounds reported here (see Figure 5-6).

Based on the present results, the main metabolites of PREG would be PROG, 20a- DHPREG and 20p-DHPREG. 20P-HOR activity has not yet been discovered in rat brain, but the possibility for the presence of the enzyme remains, especially as expression of the enzyme was described in mouse and neonatal pig brain (see Chapter 1). PROG is possibly metabolised further to 20a-DHPROG and 5a-DHPR0G. Furthermore, two reduced metabolites of 5a-DHPR0G were identified, 3a,5a-THPROG and 3P,5a- THPROG (with the latter to be confirmed). Another PROG metabolite, DOC, as well its 5a-reduced form, 5a-DHD0C could not be identified. This is consistent with lack of brain P450c21 expression. The downstream metabolite 3a,5a-THDOC was confirmed, however and potentially also 3p,5a-THDOC. The latter two compounds would thus seem likely to enter the brain from the circulation after peripheral synthesis rather than in situ synthesis.

17-OH-PROG and another compound arising by the action of P450cl7, 17-OH-PREG could not be detected. This is consistent with the lack of detection of P450cl7 in the adult rat CNS so far despite many attempts. However, the downstream product of 17- OH-PREG, DHEA was found in the brain. DHEA was the first documented neurosteroid [48], that remains in brain long after removal of peripheral glands. Thus the findings here are consistent with the hypothesis that DHEA arises via a different pathway than CNS P450cl7 (see 1.3.1.5.1). Nevertheless, a different 17-hydroxylated compound has been found, 5a-pregnan-3a,17-diol-20-one. This finding could be explained by the compound entering the brain via the BBB or by the existence of another mechanism of 17- hydroxylation than via P450cl7. Formation of the compound in peripheral glands and secretion has not been reported. Furthermore, a precursor of the compound, 3a,5a- THPROG is present in brain, thus it is possible that the compound is formed in brain.

180 The significance of 3a,5a-THPROG and 3a,5a-THDOC is well established with their potent GABAa-R modulatory properties, that are the basis for their anxiolytic, sedative and hypnotic actions (see 1.3.2.1, 1.3.4). The findings here point to the possibility of the origins of the two steroids, at least in part to be different, with 3a,5a-THPROG being partially or completely produced within the brain and 3a,5a-THDOC most likely not, as described above. This leads to the questions of the functions of those compounds. One possible reason could be that 3a,5a-THDOC is produced by the adrenal as part of the stress response and 3a,5a-THPROG at least partially within the brain as a localised signalling function. The 3 p-isomers of the above compounds are inactive at the GABAa- R. They can, however possibly act as inhibitors of the action of the respective 3a- isomers [148]. Furthermore, 3p,5a-THPROG was shown to be a substrate for 3p,5a- diol-hydroxylase, the most abundant P450 steroid hydroxylase in the brain [204]. Hydroxylation by this enzyme at positions C-6 or C-7 is a major catabolic pathway in prostate and possible could serve this function in brain, acting to reduce the levels of 3a,5a-THPROG [IBS]. This function could be fulfilled in the following way: by up- regulation of 3p-H0R activity, 3p,5a-THPROG (and possibly 3p,5a-THD0C) is produced and acts as antagonist of the 3a,5a-reduced isomer to reduce GABAa-R potentiation. To remove the 3a,5a-isomers, the levels of their precursors 5a-DHPR0G and/or 5a-DHD0C are reduced by increased production of 3P,5a-metabolites, which in turn are removed by action of 3p,5a-diol-hydroxylase. The 3a-H0R reaction is then tilted in the oxidative direction due to reduced levels of the products of the reaction in this direction, thus reducing levels of 3a,5a-isomers.

The significance of 5a-pregnane-3a,20a-diol (the 20-reduced metabolite of 3a,5a- THPROG) found here (to be confirmed), is not known. Pregnanes reduced at C-20 have a reduced efficacy compared to the 20-ketones in enhancing the current induced by

GAB A at recombinant GABAa-R expressed in Xenopus oocytes [15]. The maximum potentiation of GABA-stimulated ^^Cl'-uptake and currents is also significantly lower than for 3a,5a-THPROG, suggesting it to be a partial agonist [14,15]. As expected from their effects on GABAa-R modulation, 3a,5a/p,20a/p-reduced pregnane-diols were found to decrease anxiety in the Vogel test upon intracerebroventricular administration in rat. The 3a,5a,20a-reduced compound had the highest potency of the four isomers. They were lower in potency than the corresponding 20-ketones, consistent with the findings for GABAa-R modulation [30]. It was suggested that the strong GABAa-R

181 modulatory effects of 3a,5a-THPROG provides little margin for production of levels that leads to anxiolytic effects but not sedation [15]. Thus the existence of less efficacious GABA a -R agonists could be a mechanism for a fine regulation of the neuroregulatory activity of these endogenous steroidal compounds.

These findings together indicate the possibility that there are several metabolites with different grades of potency at GABA a -R modulation. The evidence for the presence of 3a,5a-THDOC, 3a,5a-THPROG and 5a-pregnane-3a,20a-diol in the brain of rats suggests that this is a physiologically relevant action of those compounds. 5a-pregnane- 3a,20a-diol is likely to be synthesised from 5a,20a-THPROG, which was also found (to be confirmed). But it is also possible that locally the activity of 3a,5a-THPROG is regulated by the 20-reduction pathway. This could be a fast means of acute modulation of the GABA a -R potentiation by steroids. The finding of 5a,20a-THPROG is consistent with the higher reactivity of 20a-DHPROG compared to PROG with respect to 5a- reduction [39].

The 20-reduction pathway could also be of significance in the regulation of the level of

PROG and/or the GABA a-R active PROG metabolites (see above). Similarly 20a- DHPREG and 20p-DHPREG could be formed in order to regulate the concentration of

PREG and/or the GABA a -R active PROG metabolites, as this is a direct precursor of 20a-DHPROG. Further 20p-reduced pregnanes have not been detected.

The significance of the 11 p-hydroxylase pathway is possibly increased by the present obtained evidence for 5a-pregnan-3a,l ip-diol-20-one (to be confirmed). It is possible that this is formed locally as significant levels of the precursor 3a,5a-THPROG have been detected. Hydroxylation at C-11 leads to a loss of the potentiating effect on

GABA a -R mediated Cl'-uptake (see 1.3.2.1). 3a,5a-THPROG hydroxylated at position

C-17, 5a-pregnan-3a,17-diol-20-one also is inactive in potentiating GABA a -R agonist mediated Cl'-flux. Thus the 11-hydroxylation and 17-hydroxylation are likely to be inactivation pathways for the neuromodulator 3a,5a-THPROG. Alternatively these compounds could have actions on neurotransmitter-receptors/ion channels other than

GABA a -R or other signalling pathways thus far undetected.

The possibility thus exists for a variety of pathways that might regulate the activity of the

GABA a -R active metabolites 3a,5a-THPROG and 3a,5a-THDOC. A requirement for

182 multiple and tight regulation of their activity is consistent with their extremely high potency as modulators of the GABA a -R function. Those pathways (20-reduction, 17- hydroxylation, 11-hydroxylation, 3 (3-reduction for antagonists and to decrease levels of 3a-isomers) result in a modulation of the efficacy of their GABA-modulatory activity or to inactivation. It can be imagined that the pathways of inactivation differ in the time- scale of their regulation.

One of the highest concentrations of a compound found was of TESTO. This is a major steroid in the circulation and thus most likely the brain levels arise through entry from the blood, especially since its immediate precursor was not detected in brain. Many of its effects on the male rat brain are thoughlto be mediated via aromatisation to OESTR (see Chapter 1). However, no OESTR could be detected in the present study, although its presence in the adult rat is likely as TESTO was found and aromatase activity is present in adult male rat brain. Thus OESTR exists possibly at very low levels. Furthermore, production could be highly localised and the levels could be tightly regulated by catabolism. Thus overall brain concentration could be too low to be detected. This is supported by the fact that aromatase expression is highly localised (see Chapter 1). As discussed in Chapter 1, oestrogens could be acting locally in a paracrine manner to modulate neurotransmission. Furthermore, small concentrations of OESTR are likely because the changes of behaviour after castration of the rat can be reversed by application of extremely small doses of OESTR [83]. Androgen receptors also exist in the brain. TESTO feedback regulates luteinising hormone secretion probably via AR.

CORT was detected, not cortisol, which is consistent with the fact that rats are able to produce the former in the body, but not the latter. CORT is a major peripheral steroid and probably the main source of the brain content is peripheral adrenal production. It could be made in situ as 11 p-hydroxylase activity is present in the nervous system (see Chapter 1). Its precursor, DOC was not detected though. Although some 21-hydroxylase activity has been shown in brain, P450c21 expression is not responsible for this activity and the production of DOC is still disputed (1.3.1.5.1). Maybe it is there highly localised, too low to detect at the whole brain level. The in situ synthesis of CORT might have significance locally, especially since the enzyme for its formation seems to be located exclusively in neurones and thus there might add to autocrine/paracrine signalling. As described in Chapter 1, glucocorticoids have profound effects on the nervous system, influencing behaviour, cognition and emotion.

183 Glucocorticoid inactivation by oxidation at C-11 is thought to be an important mechanism in brain to prevent activation of MR by these steroids. It is possible that this reaction also protects brain areas from deleterious effects of excess glucocorticoids [75]. The oxidation product of CORT, 11-dehydrocorticosterone however could not be detected in the brain extracts prepared in this study. However, it has recently been shown that lip-HO R has a specific expression pattern, with certain cerebral cortical and hippocampal areas only showing high expression [131]. Furthermore, the enzyme was demonstrated to function reversibly in hippocampus in oxidative and reductive directions at low levels of CORT [81]. It is expected that the enzyme becomes active mainly in the oxidative direction at high levels such as those reached in stress. However, highly localised production of 11-dehydrocorticosterone is still likely in certain brain regions, but the amounts would not add up to a significant whole brain level.

As summarised in Figure 5-6, the present results indicate several pathways of steroid metabolism in brain consistent with known enzyme activities. Further studies are now necessary on the brains of gonadectomised and adrenalectomised animals to evaluate the interaction of central pathways with peripheral sources of steroids.

184 Table 5-1. Typical concentrations of steroids in adult male rat brain as characterised by GC-MS in reports by other authors. Values represent mean in ng/g wet tissue. ND- not detectable. Steroid sulphate esters- after solvolysis; steroid fatty acid esters- after saponification. PREG DHEAPROG 5a-DHPR0G 3a,5a-THPROG 3p,5a-THPROG EpiA Reference Free steroids 65.0 12.0 3.0 2.5 2.5 [119] 4.1 0.45 1.95 [106] 2.5 1.9 [50] Steroid sulphate ester 21.0 4.0 ND 2.7 [119] 8.26 2.47 [106] 0.53 [130] Steroid fatty acid ester 3.0 7.0 ND 0.5 [119]

185 Table 5-2. Results of screening in male rat whole brain extracts in GC-MS two ion selected ion monitoring after MO-TMS-derivatisation. Extracts were prepared and fractionated as described in Section 2.3.7. Screening was performed in four different brain extractions for the free steroid (FS) and three for the steroid sulphate (SS) fraction. For extracts brains from four animals were pooled, average weights 7.08 g (FS) and 7.35 g (SS). YES or NO denotes compounds tentatively identified or excluded by this screening procedure. TBC- denotes compounds for which the screening did not give a clear indication of their presence (to be confirmed). Steroid numbers correspond to steroids in Table 2-1. N/A -not applicable. No. Steroid FS SS No. Steroid FS SS

22 3a-DHPR0G YES TBC 26 5P-pregnan-3a, 17-diol- NO TBC 20-one 2 3p,5P-THPROG TBC TBC 25 5a-pregnane-3a,17- YES YES diol-20-one 3 3a,5a-THPROG YES TBC 44 5p-pregnane-3a,20P- YES YES diol 4 3a,5p-THPROG TBC TBC 27 5p-pregnane-3a,20a- NO YES diol 6 PREG YES TBC 19 20P-DHPREG YES TBC

35 3p,5a-THPROG YES TBC 7 5a-DHPR0G YES N/A

45 Alphaxalone NO NO 10 3a,5P-THDOC TBCNO

21 20a-DHPREG YES NO 9 20P-DHPROG TBC NO

43 5a-pregnan-3a,l ip-diol- YES TBC 47 5P-DHD0C TBC NO 20-one 11 17-OH-PREG YES NO 16 DOCTBC TBC

14 17-OH-PROG YES TBC 20 llp-OH-PROG TBC NO

33 Cortisol NO NO 17 CORT YES NO

1 DHEA YES YES 46 Adione NO N/A

39 EpiA NO TBC 34 TESTO YES TBC

42 3P-DHPR0G NO YES 5 5P-DHPR0G TBC N/A

37 3p,5P-THDOC TBC TBC 8 PROG YES N/A

13 3a,5a-THDOC YES YES 23 5a,20a-THPROG TBC TBC

12 20a-DHPROG TBC YES 15 5a-DHD0C TBCNO

38 3p,5a-THDOC YES TBC 40 Cortisone NO NO

41 11-deoxycortisol NO TBC 24 5a-pregnane-3a,20a- YES YES diol 36 11-dehydro­ TBC NO corticosterone

186 Table 5-3. Comparison of relative retention times (to ME-17-0H-PR0G) given by the diagnostic ions of putative free steroids from rat whole brain extracts in comparison to known standard steroids. Analysed by multi ion SIM-methods (see Section 5.2.2). P-pregnane, A-androstane, E-oestrane. A indicates double bond. Diagnostic Rat whole brain free steroid extract Probable structure Known standards for comparison ions RRT RRT Steroid name m/z m/z RRT peak 2"** peak 372 341 0.8846 0.9541 0.9660 A? -dione

415 384 0.8906 0.9004 0.9154 0.9203 0.9742 1.1824 1.2701 A? -trione 1.0717 4-pregnen-3,6,20-trione 593 562 1.1840 AP-triol-one/P-diol-trione 552 462 1.0417 1.0590 P-triol 0.9368 0.9950 5 (3-pregnane-3 a, 17,20a-triol 0.9898 5p-pregnane-3a,l la,20p-triol

550 460 1.0419 1.0594 1.1519 AP-triol 1.0100 5-pregnene-3 p, 17,20a-triol

505 474 0.9854 AP-diol-one

591 560 1.2260 1.2567 1.2636 AP-diol-trione 464 374 1.1104 1.3553 P-diol 462 372 0.8250 0.9439 0.9536 1.0439 1.2410 AP-diol 503 472 0.9150 AP-ol-trione 462 431 0.8062 1.0553 P-ol-dione 460 429 0.8064 0.9779 1.0774 1.0844 1.1314 1.1517 AP-ol-dione 0.9825 0.9892 4-pregnene-6p~ol-3,20-dione

187 Table 5-3 continued. Diagnostic Rat whole brain free steroid extract Probable structure Known standards for comparison ions RRT RRT Steroid name m/z m/z RRT r ‘ peak 2^^ peak 1.0200 1.0265 4-pregnene-19-ol-3,20-dione 1.0000 4-pregnene-2a-ol-3,20-dione

1.0325 1.0348 4-pregnene-16a-ol-3,20-dione

419 388 0.9453 0.9551 1.2370 AP-ol-one 417 386 0.9185 0.9742 1.0752 AP-ol-one

389 358 0.8227 0.8760 0.8850 0.9056 0.9180 0.9650 1.0596 A-trione/ AA-ol-one 0.8316 4-androsten-3 p-ol- 17-one

387 356 0.7644 0.8227 1.1931 AA-trione 0.9088 0.9160 5-androsten-3,11,17-trione

520 489 1.2578 1.2651 AA-diol-dione 524 434 0.9458 A-tiiol 522 432 0.9462 0.9642 AA-triol 479 448 1.0583 A-diol-one 0.8882 5a-androstan-3a, 11 p-diol-17-one

432 401 0.9184 AA-ol-dione 391 360 0.8260 0.9650 A-ol-one 0.8568 0.8629 5a-androstan-17 P-ol-3-one

188 Table 5-3 continued. Diagnostic Rat whole brain free steroid extract Probable structure Known standards for comparison ions RRT RRT Steroid name m/z m/z RRT r ‘ peak 2”*^ peak 0.8052 5a-androstan-3a-ol-17-one

0.7904 0.7930 5 p-androstan-17a-ol-3-one

326 295 0.8124 0.8790 0.8860 0.9050 1.1040 E-dione 590 559 1.2377 1.2619 1.3060 E-triol-dione 502 471 1.1767 E-diol-dione 504 414 1.2944 1.3206 E-triol 459 428 1.2345 E-diol-one 416 326 0.8261 0.8441 0.886 0.9054 0.9189 1.1317 1.1813 E-diol 0.8656 1,3,5 [ 10] -oestratriene-3,17p-diol

457 426 0.8099 0.8864 0.9953 1.2234 1.3356 E-ol-trione 414 383 0.8800 1.1817 1.2691 1.3474 1.3531 E-ol-dione 371 340 0.9152 1.0261 1.1780 1.2772 E-ol-one 0.8572 1,3,5 [ 10] -oestratrien-3 -ol-17-one

189 Table 5-4. Examples of identification of free and sulphate conjugated (as indicated in Table) steroids in adult male rat brain. Relative retention indices (RRT) for endogenous compounds derivatised with MO and TMSI or HFBA of typical rat brain extracts and standard steroids are shown. For compounds giving double peaks the RRTs of both peaks are shown. RRTs are given for standard compounds (Std) run alongside sample with upper and lower limits (±0.5%) and parametric confidence limits (see text) determined from several standard samples. For comparison, differences of upper and lower parametric confidence limit from Std mean (99.9% conf. limit) and brain compound RRT from Std mean (Tissue - mean Std) are given. Extract 1 was analysed by 2 ion SIM of MO-TMS-derivatives, extracts 5, 6 by 3 ion SIM of MO-TMS-derivatives, extracts 7, 8 by 2 ion SIM of HFB-derivatives. Extracts 7 and 8 were each prepared from the same pool of tissue homogenate. Steroid numbers correspond to steroids in Table 2-1.

Free steroids 0.5% limit Parametric 99.9% confidence limit Extract 6 Tissue Std Lower limit Upper limit Std mean S.E.M. n 99.9% conf. Tissue - No. Steroid RRT RRT RRT limit mean Std 1 DHEA 0.909 0.911 0.907 0.916 0.911 0.0001 9 0.002 -0.002 6 PREG 1.015 1.016 1.011 1.021 1.016 0.0001 9 0.001 -0.001 8 PROG 0.964 0.965 0.960 0.970 0.964 0.0002 9 0.003 0.000 23 5a,20a-THPROG 0.972 0.967 0.962 0.972 0.966 0.0002 9 0.004 0.006 0.986 0.973 0.969 0.978 0.973 0.0001 9 0.003 0.013

13 3a,5a-THDOC 0.978 0.979 0.974 0.983 0.977 0.0002 9 0.003 0.001

21 20a-DHPREG 0.954 0.955 0.950 0.960 0.954 0.0003 9 0.005 0.001

7 5a-DHPR0G 1.028 1.028 1.023 1.034 1.029 0.0002 9 0.002 -0.001 1.034 1.035 1.029 1.040 1.036 0.0002 9 0.003 -0.001 16 DOC 1.056 1.057 1.051 1.062 1.057 0.0001 9 0.002 -0.001 17 CORT 0.986 0.987 0.982 0.992 0.986 0.0001 9 0.002 0.000

190 Table 5-4 continued. Free steroids 0.5% limit Parametric 99.9% confidence limit Extract 6 Tissue Std Lower limit Upper limit Std mean S.E.M. n 99.9% conf. Tissue - No. Steroid RRT RRT RRT limit mean Std CORT (2) 0.988 0.989 0.984 0.994 0.988 0.0002 9 0.004 0.000 Extract 8 1 DHEA 1.050 1.050 1.045 1.056 1.050 0.0000 7 0.001 0.000 3 3a,5a-THPROG 0.960 0.958 0.954 0.963 0.958 0.0001 7 0.002 0.002

2 3P,5p-THPROG 0.961 0.975 0.970 0.980 0.975 0.0002 4 0.005 -0.014 9 20P-DHPROG 0.989 1.001 0.996 1.006 1.001 0.0001 4 0.004 -0.012

47 Sp-DHDOC 1.009 1.017 1.012 1.022 1.017 0.0001 4 0.002 -0.009

24 5a-pregnane-3a,20a-diol 0.968 0.969 0.964 0.974 0.968 0.0001 7 0.002 -0.001

27 5 p-pregnane-3 a, 20a-diol 0.968 0.978 0.973 0.982 0.978 0.0001 7 0.001 -0.010

25 5 a-pregnan-3 a- 17-diol- 1.032 1.032 1.027 1.037 1.032 0.0003 7 0.005 0.001 20-one

12 20a-DHPROG 1.029 1.030 1.024 1.035 1.030 0.0001 4 0.002 0.000

23 5a,20a-THPROG 0.955 0.952 0.947 0.957 0.952 0.0002 7 0.003 0.003

191 Table 5-4 continued.

Steroid sulphates 0.5% limit Parametric 99.9% confidence limit Tissue Std Lower limit Upper limit Std mean S.E.M. n 99.9% conf. Tissue - No. Steroid RRTRRT RRT limit mean Std Extract 1 1 DHEA 0.914 0.915 0.910 0.919 0.911 0.0005 9 0.008 0.003 Extract 5 6 PREG 1.016 1.016 1.010 1.021 1.016 0.0001 9 0.001 0.000 Extract 6

38 3j3,5a-THDOC 1.039 1.033 1.028 1.038 1.032 0.0001 9 0.002 0.007 Extract 7

3 3a,5a-THPROG 0.956 0.959 0.954 0.963 0.958 0.0001 8 0.002 -0.002 Extract 8

1 DHEA 1.051 1.050 1.045 1.056 1.050 0.0000 7 0.001 0.001 38 3p,5a-THDOC 1.321 1.323 1.317 1.330 1.324 0.0002 8 0.003 -0.003

192 Table 5-5. Examples of identification of free and sulphate conjugated (as indicated in Table) steroids in adult male rat brain. Qualifier (Q) to target ion (T) ratios (Q/T) for standard steroids and endogenous compounds of typical rat brain extracts are shown. In the case of 3 ion SIM, the 2 Q ions are listed one below the other. Q/Ts are given for standard compounds (Std) run alongside samples with pragmatic upper and lower limits (±20% limits; 1.5 and 0.67x Std Q/T, see text for explanation) and parametric confidence limits (see text) determined from several standard samples. For comparison, differences of upper and lower parametric confidence limit from Std mean (99.9% conf. limit) and brain compound Q/T from Std mean (Tissue - mean Std) are given. Extracts 1,3 were analysed by 2 ion SIM of MO-TMS-derivatives, extracts 5, 6 by 3 ion SIM of MO-TMS-derivatives, extracts 7, 8 by 2 ion SIM of HFB-derivatives. Extracts 7 and 8 were prepared from the same pool of tissue homogenate. Steroid numbers correspond to steroids in Table 2-1. BD-below detection limit. Free steroids 20% limits Parametric 99.9% confidence limits Extract 6 m/z Tissue Std Lower limit Upper limit Std mean S.E.M. n 99.9% conf. Tissue -

No. Steroid T Q Q/T Q/T Q/T limit mean Std 1DHEA 358 268 101.0 175.3 117.4 262.9 195.2 6.5 6 118.5 -94.2 260 70.0 136.3 91.3 204.5 146.3 4.5 6 82.2 -76.3 6 PREG 402 386 165.4 184.2 123.4 276.3 193.9 4.3 6 78.5 -28.5 312 118.2 107.1 71.8 160.7 105.8 4.3 6 77.2 12.4 8 PROG 372 341 97.3 92.4 61.9 138.5 61.6 7.4 6 134.2 35.6 273 65.6 54.9 36.8 82.4 54.9 3.1 6 56.2 10.7 23 5a,20-THPROG 303 289 110.5 32.9 22.0 49.3 42.2 2.0 6 35.9 68.4 314 145.8 20.0 13.4 30.0 26.1 1.7 6 31.3 119.7 13 3a,5a-THDOC 476 507 27.8 31.8 21.3 47.7 27.1 1.6 6 29.3 0.7 404 18.8 20.9 14.0 31.3 19.4 0.4 6 7.7 -0.6

193 Table 5-5 continued. Free steroids 20% limits Parametric 99.9% confidence limits Extract 6 m/z Tissue Std Lower limit Upper limit Std mean S.E.M. n 99.9% conf. Tissue -

No. Steroid T Q Q/T Q/T Q/T limit mean Std 21 20a-DHPREG 462 372 316.7 236.0 158.1 354.0 230.6 14.9 6 269.7 86.1 332 94.4 86.6 58.0 129.8 94.1 3.8 6 68.8 0.3 7 5a-DHPR0G 343 275 122.2 59.7 40.0 89.6 67.6 3.5 6 64.1 54.6 288 153.8 79.5 53.2 119.2 85.4 4.4 6 79.4 68.4

16 DOC 286 398 108.3 20.4 13.7 30.6 20.4 - 1

273 141.5 71.8 48.1 107.7 71.8 - 1 17 CORT 548 517 131.7 107.0 71.7 160.5 123.6 8.7 6 158.1 8.1 427 140.7 128.4 86.0 192.5 155.4 11.0 6 199.6 -14.7 Extract 8 1 DHEA 270 255 19.5 18.9 12.6 28.3 19.4 0.2 7 3.0 0.1

3 3a,5a-THPROG 496 514 42.0 44.3 29.7 66.5 44.2 0.6 7 10.0 -2.2

1 '3(3,5P-THPROG 496 514 43.1 34.8 23.3 52.3 36.5 0.7 4 19.3 6.6

9: 20P-DHPROG 708 693 16.4 12.2 8.2 18.3 13.1 0.5 4 14.8 3.3

475P-DHD0C 301 255 50.5 96.6 64.7 144.9 139.4 14.7 4 426.6 -88.9

194 Table 5-5 continued. Free steroids 20% limits Parametric 99.9% confidence limits Extract 8 m/z Tissue Std Lower limit Upper limit Std mean S.E.M. n 99.9% conf. Tissue -

No. Steroid T Q Q/T Q/T Q/T limit mean Std 24 5a-pregnane- 712 697 59.3 68.7 46.0 103.1 66.0 1.0 7 16.2 -6.7 3a,20a-diol 27 SP-pregnane- 712 697 59.2 42.9 28.7 64.3 42.8 0.4 7 5.9 16.4 3a,20a-diol 25 5a-pregnan- 442 487 220.7 217.6 145.8 326.4 275.4 44.6 7 752.1 -54.8 3a,17-diol-20-one

12 20a-DHPROG 708 693 16.6 20.4 13.6 30.5 19.3 0.6 4 17.1 -2.7

23 5a,20-THPROG 499 514 168.2 158.3 106.0 237.4 141.1 4.3 7 72.8 27.1

Steroid sulphates 20% limits Parametric 99.9% confidence limits Extract 1 m/z Tissue Std Lower limit Upper limit Std mean S.E.M. n 99.9% conf. Tissue -

No. Steroid T Q Q/T Q/T Q/T limit mean Std 1 DHEA 358 268 203.6 176.0 117.9 264.0 181.8 4.3 9 68.8 21.8

195 Table 5-5 continued. Steroid sulphates 20% limits Parametric 99.9% confidence limits m/z Tissue Std Lower limit Upper limit Std mean S.E.M. n 99.9% conf. Tissue -

No. Steroid I Q Q/T Q/T Q/T limit mean Std Extract 3 38 3p,5a-THDOC 476 188 3946.9 117.0 78.4 175.5 144.4 10.3 9 164.4 3802.5 Extract 5 6 PREG 402 386 17.1 220.5 147.7 330.8 195.0 5.7 6 103.7 -178.0 312 19.0 126.1 84.5 189.2 121.1 2.2 6 40.4 -102.1 Extract 6 38 3p,5a-THDOC 476 507 35.0 24.1 16.1 36.1 25.1 1.0 6 18.6 -10.9 386 BD Extract 7 3 3a,5a-THPROG 496 514 162.6 47.7 31.9 71.5 44.5 0.8 7 12.9 118.0 Extract 8 1 DHEA 270 255 126.6 19.7 13.2 29.5 19.0 0.4 7 6.8 107.6

38 3p,5a-THDOC 499 471 91.3 15.8 10.6 23.7 17.1 0.8 3 50.1 74.2

196 Table 5-6. Summary of identification of free and sulphate conjugated steroids in adult male rat whole brain. Indications are given as to whether a compound met the ±0.5%-criteria of RRT and ±20% of Q/T as presented in Tables 5-4 and 5-5 for selected examples. The overall decision takes into account all the individual results as described in the text. Extracts 1, 3, 9,10 were analysed by 2 ion SIM of MO-TMS-derivatives, extracts 4 (only steroid sulphate fraction), 5, 6 by 3 ion SIM of MO-TMS-derivatives, extracts 7, 8 by 2 ion SIM of HFB-derivatives. Extracts 7, 8 and 9, 10, respectively were prepared from the same pool of tissue homogenate. IN denotes those within, OUT those outside RRT or Q/T limits. In extracts 5 and 6, data for both Q ions are shown (separated by /). IDENT-identified (see text for details). * only 2 ions monitored. Steroid numbers correspond to steroids in Table 2-1. Free steroids RRT Q/T No. Steroid Extract Extract Decision 5 6 7 8 9 10 5 6 7 8 9 10 1DHEA IN IN IN OUT/OUT OUT IN IDENT 2 3P,5P-THPR0G IN OUT OUT IN Not present

3 3a,5a-THPROG IN IN IN OUT/IN IN IN IDENT 4 3a,5p-THPROG OUT OUT IN OUT Not present 6PREG ININ ININ IN/IN IN/IN IN IN IDENT

7 5a-DHPR0G IN OUT/OUT Tentative 8 PROG IN ININ IN/IN OUT IN IDENT 920P-DHPROG IN OUT OUT IN Not present

12 20a-DHPROG INININ IN/OUT OUT IN Tentative

13 3a,5a-THDOC IN IN IN IN/IN OUT OUT IDENT 16DOC IN OUT/OUT Not present

197 Table 5-6 continued.

Free steroids RRT Q/T No. Steroid Extract Extract Decision

5 6 7 8 9 10 5 6 7 8 9 10 17 CORT IN IN/IN IDENT

1920P-DHPREG IN IN IN IN IN/IN IN/IN ININIDENT

20 IIP-OH-PROG IN OUT OUT OUT Not present 21 20a-DHPREG IN IN IN IN IN/OUT IN/IN IN IN IDENT

22 3a-DHPR0G OUT OUT/OUT Not present 23 5a,20a-THPROG IN* OUT OUT IN OUT* OUT/OUT OUT IN Tentative

24 5a-pregnane-3a,20a- E4 IN IN IN IN IN OUT OUT Tentative diol

25 5a-pregnane-3a, 17- IN IN OUT IN OUT/OUT OUT/IN IN IN IDENT diol-20-one

27 5 P-pregnane-3 a, 20a- OUT OUT IN IN Not present diol 34 TESTOIN IN IN IN IN/IN IN/IN OUT OUT IDENT 35 3p,5a-THPROG IN IN Tentative

198 Table 5-6 continued.

Free steroids RRT Q/T No. Steroid Extract Extract Decision

5 6 7 8 9 10 5 6 7 8 9 10

38 3p,5a-THDOC OUT IN OUT/IN OUT/OUT Tentative

43 5a-pregnan-3a, 11P- OUT IN OUT IN Tentative diol-20-one

47 Sp-DHDOC OUT OUT IN OUT Not present

58 6P-0H-PR0G IN OUT/IN Not present

Steroid sulphates RRT Q/T No. Steroid Extract Extract Decision 1 3 4 5 6 7 8 1 3 4 5 6 7 8 IDHEA IN IN IN IN OUT OUT IDENT

3 3a,5a-THPROG IN OUT Not present 6 PREG IN IN OUT/OUT OUT/OUT Not present 38 3p,5a-THDOC OUTIN OUT IN OUT OUT IN* OUT Not present

199 Table 5-7. Quantitation of steroids in adult male rat whole brain. Concentrations (Cone.) of MO-TMS- or HFB-derivatives of steroids listed in the left hand column in eight adult male rat brain extracts analysed by 2 SIM in GC-MS are given in ng/g tissue using calibration curves constructed as described in Table 3-5. For some compounds, quantitation was done in 3 ion SIM after MO-TMS derivatisation and/or 2 ion SIM after HFBA derivatisation. Extracts1,2, 3, 4 were quantified by 2 ion SIM of MO-TMS-derivatives, extracts 5,6 by 3 ion SIM of MO-TMS-derivatives, extracts 7,8 by 2 ion SIM of HFB-derivatives. In the steroid sulphate fraction only extract 1 analysed by 2 ion SIM showed a positive response. Extracts 7 and 8 were prepared from the same pool of tissue homogenate, but underwent a slightly different purification procedure (see text for details). Also shown are minimal detection limits (Det.l., ng/g tissue) of MO-TMS-derivatives of steroids determined from reagent blanks run alongside tissue samples in 2 ion SIM and recoveries (Recov., %, mean ±S.E.M, n=4) of the entire extraction and analysis procedure. ND-not detectable. Blank fields: not determined. N/A -not applicable. ^ determined after HFB-derivatisation in two ion SIM using single point calibration. ^ determined after MO-TMS-derivatisation in 3 ion SIM using single point calibration. Free steroids Steroid sulphates Cone, (ng/g) Det.l. Recov. Cone. Det.l. Recov. (ng/g) (%) (ng/g) (ng/g) (%) Extract No. Steroid 1 2 3 4 5 6 7 8 Mean S.E.M.

1 DHEA ND ND ND ND ND 0.03' 0.03 0.004' 59.9+0.7 0.16 0.14 51.3+2.3

34 TESTO ND0.91 0.54 0.29 ND 0.58 0.18 0.007 53.0+1.2 ND 0.17

6 PREG 1.65 0.42 0.67 1.04 0.95 0.27 0.05 39.8+1.0 ND 0.05' 28.6+2.2

21 20a-DHPREG 0.46 ND 0.31 ND 0.10 0.20 0.27 0.08 0.05 38.1+1.1 ND 0.04

19 20P-DHPREG 0.34 0.10 0.14 0.10 0.17 0.06 0.03 36.8+0.7 ND 0.17

8 PROG 0.84 ND 0.10 0.49 0.48 0.21 0.09 63.1+3.4 NDN/A

7 5a-DHPR0G ND ND ND ND 0.52^ 0.52 0.12 31.5+3.9 ND N/A

200 Table 5-7 continued. Free steroids Steroid sulphates Cone, (ng/g) Det.l. Recov. Cone. Det.l. Recov. (ng/g) (%) (ng/g) (ng/g) (%) Extract No. Steroid 1 2 3 4 5 6 7 8 Mean S.E.M.

3 3a,5a-THPROG ND 0.30 ND ND 0.25'0.18' 0.24 0.03 0.06 39.311.1 ND 0.21

35 3p,5a-THPROG ND NDNDND 0.16' ND 0.16 0.05' 36.4+0.8 ND 0.16

12 20a-DHPROG ND ND 0.10 ND 0.004' o.io' 0.07 0.03 0.002' 46.5+0.7 ND 0.19

23 5a,20a-THPROG ND ND ND ND ND 0.43' 0.43 O.Ol' 40.9+2.6 ND 0.09

24 5a-pregnane-3a,20a-diol NDND ND 0.09 0.02' 0.37' 0.16 0.11 0.07 55.9+3.2 ND 0.11

25 5a-pregnan-3a, 17-diol-20-one ND ND ND ND 0.03^ 0.06^ ND 0.08' 0.06 0.01 0.02' 49.9+1.8 ND 0.18

43 5a-pregnan-3a, 11 P-diol-20-one NDND NDND ND O.Ol' 0.01 0.005' 43.3+2.5 ND 0.08

13 3a,5a-THDOC 0.26 0.08 0.24 0.36 0.24 0.06 0.006 41.4+2.1 ND 0.02

38 3p,5a-THDOC 0.56 ND 0.80 0.80 0.72 0.08 0.03 37.0+2.4 ND 0.03

17 CORT ND 4.62 ND 3.89 ND 8.74 5.75 1.51 1.02 43.7+5.8 ND 0.32

39 EpiA NDNDND ND 0.006 57.8+0.5 ND 0.02

201 Table 5-7 continued. Free steroids Steroid sulphates Cone, (ng/g) Det.l. Recov. Cone. Det.l. Recov. (ng/g) (%) (ng/g) (ng/g) (%) Extract No. Steroid 1 2 3 4 5 6 7 8 Mean S.E.M.

46 Adione ND ND ND ND 0.03 71.2+1.3 ND N/A

22 3a-DHPR0G ND ND ND ND 0.09 21.0+2.3 ND 0.16

42 3P-DHPR0G NDND ND ND 0.06 27.3+3.1 ND 0.06

5 5P-DHPR0G ND ND ND ND 0.27 35.2+2.0 NDN/A

20 lip-OH-PROG ND ND ND ND 0.33 63.8+4.7 ND 0.55

11 17-OH-PREG ND ND ND ND 0.07 47.6+1.4 ND 0.06

14 17-OH-PROG NDND ND ND 0.12 58.5+1.4 ND 0.15

9 20P-DHPROG ND ND NDND 0.01 ND 0.01

2 3P,5P-THPROG ND ND NDND 0.07 42.2+1.1 ND 0.21

4 3a,5P-THPROG ND ND ND ND 0.04 39.4+1.1 ND 0.02

45 Alphaxalone ND ND NDND 0.009 48.5+1.9 ND 0.02

26 5 P-pregnan-3a, 17-diol-20-one ND ND ND ND 0.04 52.0+4.9 ND 0.03

202 Table 5-7 continued. Free steroids Steroid sulphates Cone, (ng/g) Det.l. Recov. Cone. Det.l. Recov. (ng/g) (%) (ng/g) (ng/g) (%) Extract No. Steroid 1 2 3 4 5 6 7 8 Mean S.E.M.

44 5p-pregnane-3a,20(3-diol NDND NDND 0.14 36.3±3.8 ND 0.06

27 5 P -pregnane-3 a, 20a-diol NDND ND ND 1.02 29.5+0.8 ND 1.11

16 DOCND NDND ND 0.35 109.9+33 ND 0.81

15 5a-DHD0C ND NDNDND 0.44 45.0+1.1 ND 0.25

47 SP-DHDOC ND NDND ND 0.03 35.9+0.8 ND 0.01

10 3a,5p-THDOC NDND NDND 0.005 35.4+2.7 ND 0.005

37 3p,5p-THDOC ND ND ND ND 0.03 44.3+4.0 ND 0.004

33 Cortisol ND ND NDND 0.04 12.8+2.3 ND 0.04

41 11-deoxycortisol ND ND ND ND 0.06 49.0+4.4 ND 0.05

36 11-dehydrocorticosterone ND ND ND ND 3.06 34.0+9.3 ND 1.50

40 Cortisone ND ND ND ND 0.10 21.9+3.4 ND 0.05

203 Figure 5-1. Selected ion chromatograms of male rat whole brain extract free steroid fractions derivatised by MO/TMSI and analysed in two ion SIM. Tissue (~8 g) was extracted and fractionated as described in Section 2.3.7. Ion (m/z as shown) abundance is plotted against RRT to one of three internal standards indicated in Table A-2. * or number indicates expected RRT. Compounds are indicated by their abbreviated name and reference number as given in Table 2-1. The results of the screening are also indicated: tent, denotes compounds tentatively identified by this procedure, TBC denotes compounds for which the screening did not give a clear indication of their presence (to be confirmed). For clarity of presentation, in some cases traces are shown base-shifted. In those cases, no y-axis is drawn.

PREG (6), tent. 3(5,5a-THPROG (35), tent.

50-

1.015 1.025 1.015 1.025

20a-DHPREG (21), tent. 17-OH-PREG (11), tent.

462.45- 1.0 372.35- 1.0

100-

50-

0.953 0.962 0.972 0.981

5p-pregnane-3a,20a-diol (27), not present. 20(3 -DHPREG(19), tent. 462.45- 1.0 ^ 372.35- 1.0 *

100 25-

1.006 1.016 1.026 1.036

204 Figure 5-1 continued.

3a,5P-THDOC(10), TBC. 20P-DHPROG (9), TBC. *

0.963 0.972 0.972 0.982 0.991

3P-DHPR0G (42), not present. 3p,5p-THDOC (37), TBC, 3a,5a-THDOC (13), tent. 37 13 386.35

244.25

^ 507.50- V 358.35-

1------1 I------1.005 1.009 1.013 0.964 0.973 0.982

TESTO (34), tent. 11 -deoxycortisol (41 ), not present.

517.50- 1.0 427.45- 1 0 50-

25-

1 1 1---- 0.946 0.956 0.966 1.057 1.066 1.076

PROG (8), tent. 5a-pregnane-3a,20a-diol (24), tent. 372.35- 1.0 341.30- 1.0

100-

100-

W

0.955 0.964 1.003 1.011

205 Figure 5-2. Selected ion chromatograms of male rat whole brain extract steroid sulphate fractions derivatised by MO/TMSI and analysed in two ion SIM. Tissue (~8 g) was extracted and fractionated as described in Section 2.3.7. Ion (m/z as shown) abundance is plotted against RRT to one of three internal standards indicated in Table A-2. * or number indicates expected RRT. Compounds are indicated by their abbreviated name and reference number as given in Table 2-1. The results of the screening are also indicated: tent, denotes compounds tentatively identified by this procedure, TBC denotes compounds for which the screening did not give a clear indication of their presence (to be confirmed). For clarity of presentation, in some cases traces are shown base-shifted. In those cases, no y-axis is drawn.

PREG (6), TBC. 20a-DHPREG (21), not present.

402.40" 1.0 386.35" 1.0 * 100- A

1.015 1.025 0.953 0.962

5p-pregnan-3a,17-dioi-20-one (26), TBC, 5(3-pregnane-3a,20a-dioi (27), tent. 5a-pregnan-3a,]7-diol-20-one (25), tent.

188.15" 1.C 26 25

0.963 0.973 0.984 0.996 1.000 1.004 1.008

5p-DHD0C (47), not present. 1P-OH-PROG (20), not present.

370.35" 1.0 339.30" 1.0

50- 50-

1.010 1.028 1.057 1.066

206 Figure 5-2 continued.

DHEA(l), tent. 3P-DHPROG (42), tent.

16.35" 1.0 4.25" 1.0

20 -

358.35" 10- 268.25"

0.903 0.914 0.991 1.002 1.012

Figure 5-3. Typical selected ion chromatograms of the free steroid fraction of rat brain extracts analysed in GC-MS by multiple ion screening. Pooled tissue of rats (~8 g) was extracted and fractionated as described in Section 2.3.7. Ion (m/z as shown) abundance is plotted against RRT to an internal standard (ME-17-OH-PROG). Co-elution of diagnostic ions are indicative of generic structures denoted in Figure. Identification was attempted by comparison to GC-MS properties of authentic reference standards (see Table 5-3). For clarity of presentation, traces are shown base-shifted.

207 Figure 5-3 continued.

AP -dione AP-triol

37235" 550.50’ 341.30" 460.45’ 0.961 0.970 1.0301.039 1.049

AP-ol-dione A-trio ne/ AA-cl-cne

429.40’ 460.45’ 389.35’

0.970 0.989 0.816 0.819 0.823 3.827

AP-ol-trione A-diol-one

479.45’ 503.50’ 448.45’ 472.45= T 0.911 0.915 0.919 1.048 1.067 1.086

E-triol E-ol-one

414.40’ i\ 504 50’ 340.30’ T 1.286 1.296 1.305 0.906 0.915 0925

208 Figure 5-4. Typical selected ion chromatograms of rat brain extracts analysed in GC-MS by two or three ion monitoring of MO-TMS-derivatives for identification. Traces of endogenous compounds are shown below those of reference compounds. Ion (m/z as shown) abundance is plotted against RRT to one of three internal standards as indicated in Table A-3.* indicates expected RRT. Compounds are indicated by their abbreviated name and reference number as given in Table 2-1. The overall decisions on identification based on the diagnostic RRT and Q/T criteria are also shown. For clarity of presentation, in most cases traces are shown base- shifted (no y-axis drawn).

209 Figure 5-4 continued.

DHEA ( 1 ), identified. 3a,5a-THPROG (3), identified.

358.35-

388.35’ V k', y

^ 260.25 404.40-

0.906 0.917 0.927 0.968 0.972 0.976

358.35 388.35- 268.25

404.40-

260. 25- 0.906 0.910 0.967 0.977

210 Figure 5-4 continued.

PREG (6), identified. TESTO (34), identified.

402.40’ 389.35’

386.35’ 358 35’ / \ / ''312.30' 268.25’

T I 1 ' —..- 1---- 1.014 1.019 1.023 0.938 0.948 0.959

402.40 389.35’' 386.35’ 358.35’'

312.30’ 268,25='

TT 1.008 1.019 0.948 0.952 0.956

211 Figure 5-4 continued.

20P-DHPREG (19), identified. 5a-DHPR0G (7), tentative.

462.45 343.30’

275.25’ ,332.30"

T 1.023 1.034 1.023 1.034 1.044

372.35’

\ ' 462.45’

332,30’

- 288.25’

1.020 1.031 1.028 1.038

212 Figure 5-4 continued.

PROG (8), identified. 20a-DHPROG(12), tentative.

273.25" 1.C

372.35= 341.30" 417.40=

'296.25=

0.958 0.962 0.965 0.969 0.984 0.994

83,855

10

372.35= 341.30’

273.25=

0.957 0.962 0.967 0.980 0.989

213 Figure 5-4 continued.

3a,5a-THDOC (13), identified. 3p,5a-THDOC (38), tentative.

476.45^ - 476.45’ 507.50^ 404.40’ 404 40^ 386 35: 507.50’ TT 0.975 0.977 0.979 1.026 1.036 1.045

476.45’ 476.45’

507.50= - 404.40’ \ -404.40’ 386.35’' 507.50' 0.976 0.980 0.983 1.027 1.046

214 Figure 5-4 continued.

CORT (17), identified. 20a-DHPREG (21), identified.

548.50"

517,50" ^ 427 40- 462.45“

372.35’

.332 30“

0.955 0.972 0.990 0.950 0.954 0.958

372.35“

548.50“ 462.45“

332.30“ 517.50“ 427.40“

0.973 0.982 0.990 0.952 0.956 0.960

215 Figure 5-4 continued.

5a-pregnan-3a, 17-diol-20-one (25), 5p-DHPROG (5), not present. identified.

343.30’ 275.25’

476.45’ 288.25’

364 35" 0.976 0.981 0.986 1.010 1.015 1.019

343.30’ r-"i ‘-'C.i 275.25=

288.25’

TT 0.971 0.982 1.006 1.010 1.014 1.018

216 Figure 5-4 continued.

5a-DHDOC (15), not present. 6P-OH-PROG (58), not present.

* *

462.45 460.45=

431.40* K- 445.40=

'■■'V- 413.40=

1.036 1.046 1.056 0.977 0.981 0.992

Æ 288.25= 460.45= 'i 445.40=

1.036 1.046 0.979 0.983 0.986 0.990

217 Figure 5-4 continued.

3a-DHPROG (22), not present. 3p,5a-THDOCS (38 S), not present.

476.45= 507,50=

100-

417.40= 244.20=

■V 386.35=

0.968 0.970 0.982 1.026 1.034

476.45" 1.0 29,667 507.50" 1.0

50-

.r 386.35"

0.970 0.974 0.979 1.036 1.040 1.044

218 Figure 5-4 continued.

DHEAS ( 1 S), identified. PREGS (6 S), not present.

386.35’' 1.0 203.824- 31230" 1.0 296.30’' 1.0 1004

358.35’

ir" ■ 268.25’

0.912 0.923 1.018 1.022

296.30’' 1.0 754 386.35" 1.0 312.30" 1.0

504

358.35’ 254

268.25’

0.903 0.914 1.014 1.025

219 Figure 5-5. Typical selected ion chromatograms of rat brain extracts analysed in GC-MS by two ion monitoring of HFB-derivatives for identification. Traces of endogenous compounds are shown below those of reference compounds. Ion (m/z as shown) abundance is plotted against RRT to one of three internal standards as indicated in Table A-6. * indicates expected RRT. Compounds are indicated by their abbreviated name and reference number as given in Table 2-1. The overall decisions on identification based on the diagnostic RRT and Q/T criteria are also shown. For clarity of presentation, in most cases traces are shown base-shifted (no y-axis drawn).

220 Figure 5-5 continued.

5a-pregnan-3a,l ip-diol-20-one (43), 20a-DHPREG (21), identified. tentative.

512.30"

481.45" 0.971 0.976 1.028 1.041

496.45"

512.30'

1 I 0.966 0.971 0.977 0.983 1.022 1.036

221 Figure 5-5 continued.

5a-pregnane-3a,20a-diol (24), 20P-DHPREG (19), identified. tentative.

712 45' ^ 496.35’

.-.-697 45' 481.35’' 0.954 0.982 1.000 1.014

496.35’ 712.45'

481.35* 697.45= 0.951 0.979 0.995 1.009

222 Figure 5-5 continued.

5a-pregnan-3a, 17-diol-20-one (25), 20a-DHPROG ( 12), tentative. identified.

708,25= 487.20’“

693.30“

1.024 1.038 1.052 1.024 1.038 1.052

442.05“

693.30“ 1.021 1.035 1.021 1.035

223 Figure 5-5 continued.

5a,20a-THPROG (23), tentative. PREG (6), identified.

499.20= 298.30"

283.25" 514.35=

0.942 0.947 0.952 0.957 1.016 1.021 1.027

[499.20" '514.35"

298.25=

283.20" 0.938 0.950 1.014 1.020 1.025

224 Figure 5-5 continued.

3p,5a-THPROG (35), tentative. 3a,5a-THPROG (3), identified.

11,556

100- 1496.35"

514.35"

1.035 1.041 1.046 0.926 0.954

467.45" 1.0 12,345 514.50" 10

100-

496.35’

514.35’

1.030 1.035 0.927 0.954

225 Figure 5-5 continued.

PROG (8), identified. 5p-pregnane-3a,20p-diol (44), not present.

510.35= 429.25=

495.30= 712.45=

1.016 1.021 1.027 0.926 0.940

429.30= 1.0 712.45= 1.0

510.35= 100-

495.30=

1.010 1.016 1.022 0.927 0.954

226 Figure 5-5 continued.

3(3,5p-THDOC (37), not present. 1 ip-OH-PROG (20), not present.

499,20* 493.45*

0.937 0.950 1.014 1.038

526.50*

493.45*

1 1--- 0.938 0.950 1.010 1.022 1.034

227 Figure 5-5 continued.

DOC (16), not present. CORT (17), not present.

738.70” 1.0 * 491.45” 1.0

100-

75-

722.30’

707.35”

0.962 0.987 1.086 1.111 1.135 1.159

722.30* 738.20’ . Æ 491.30’

I 1------1------0.963 0.987 1.086 1.111 1.135

228 Figure 5-5 continued.

3p,5p-THPROG (2), not present 20P-DHPROG (9), not present.

708.25=

y v 693.30= 514.35= 1 1------r T 0.970 0.975 0.981 0.986 0.996 1.010

496.35= 708.40=

514.35= 693.40=

0.965 0.971 0.976 0.982 0.988 0.995 1.002 1.009

229 Figure 5-5 continued.

DHEA (1), identified. DHEAS (1 S), identified.

270.20’ 270.20’

255,20’ 255.20’

r — 1.035 1.052 1.035 1.052

^ ^ 1270 . 2 5 ’' 1.0 178.229 255.25" 1.0

50-

270.20’ 25-

1.032 1.048 1.025 1.041 1.056

230 Figure 5-5 continued.

3a,5a-THPROGS (3 S), not present.

496.35'

yS 514.35=

0.926 0.954

496.35- 1.0 514.35- 1.0

1004

0.950 0.955 0.961

231 Figure 5-6. Possible pathways of steroid synthesis and metabolism in adult male rat brain. Compounds identified in the present study are shown in red and those tentatively identified are shown in green. Enzymes previously identified at activity, protein or mRNA levels by others (see Chapter 1.3.1.5) are also shown in red. Dehydroepi- Fatty acid ester of Cholesterol Pregnenolone sulphate androsterone Dehydroepiandrosterone Fatty acid ester sulphate of Pregnenolone P450SCC

CH3\..0H CH3N. .OH CH3 OH Acyl- 20P-HOR CH3 .OH transferase CHg^O

HO" Dehydroepi­ 3», 5(1.20(1- 5(1.20(1- 20p-Dihydro- 20a-Dihydro- Pregnenolone 17a-OH-pregnenolone androsterone pregnanediol THPROG pregnenolone pregnenolone 17a-0Hlase I sp.HSD 17.20-lyase 20a-HOR 30-HSD I 3P-HSD CH3s,0 CHsv^O CHi^O CHo^O 0 CH3 .OH 20a-HOR r -O H

HO' Androstenedione Progesterone 17tt-OH-progesterone 5a-pregnan-3(x,17- 20(i-Dihydro- 5(i-pregnan-3(i,11h- diol-20-one progesterone 21-OHIase------diol-20-one 17P-HOR 17a-0Hlase

CH3 n^O CH3s_0 HOCHo.^0 HOCHz^O OH

5(i-Reductase HO' o j d ? T estosterone 5(i-Dihydro- 11-Deoxycorticosterone 11-Deoxycortisol 3|i,5(i-Tetrahydro- 3a,5a-Tetrahydro- progesterone progesterone 1ip-OHIase ------progesterone 3a-HOR Arom atase 3P-HOR I-CCH2 ^ o HOCHsv^O

H0CH,_O OH

HO'" 3p,5(i-Tetrahydrodeoxy- 3a,5a-Tetrahydrodeoxy- 5a-Dihydrodeoxy- corticosterone corticosterone corticosterone Corticosterone Cortisol Oestradiol

232 Chapter 6 Conclusion

In this work, methods for the extraction, fractionation and GC-MS analysis of steroids were developed and evaluated for the identification and assay of these compounds in mammalian brain. It was demonstrated that it is possible to analyse simultaneously and accurately in the same sample over forty different steroids. Efficient separation of this range of analytes on GC could be achieved using pressure and temperature programming. The quantitation method was linear over a wide range for virtually all compounds and detection limits of the GC-MS method were in the low pg range for most compounds. The high sensitivity was achieved due to using high pressure injection. The detection limits for analysis of tissue extracts were somewhat higher due to interferences from reagents. However, low interferences were present in analyses of HFB-derivatised extracts due to a slightly different purification procedure and the lower chemical background of these derivatisation reagents and overall detection limits of the latter method were in the 1-lOOpg/g tissue range for most compounds. Quantitation of steroids can probably be optimised. Recently, the steroids PROG, 5a-DHPR0G, 3a,5a- THPROG, 3a,5P-THPROG and 3a,5a-THDOC have become available as stable isotope labelled analogues. These compounds could be used as internal standards in an improved quantitation procedure, allowing higher precision analyses.

It was shown that ethanol/acetic acid is an effective solvent mixture for combined extraction of unconjugated and conjugated steroids. Highly reliable separation of free and sulphate conjugated steroids was achieved using mixed mode anion exchange solid phases (MAX®).

The methods were applied to brain samples of male rat. This showed that there is probably a more complex pattern of steroids present in the mammalian brain than previously thought. This study might thus help to elucidate the roles of steroids in the nervous system under physiological and clinical conditions. Among the brain steroids are the potent neuromodulatory 3a,5a-reduced PROG metabolites 3a,5a-THPROG, 3a,5a-THDOC as well as their possible competitive antagonists 3p,5a-THPROG and 3p,5a-THDOC. It was also found that the former compounds are metabolised in a variety of ways (11-, 17-hydroxylation, 20-reduction). Several further 20-reduced

233 compounds were found as well as CORT and TESTO. On the other hand, previously demonstrated high levels of sulphated steroids could not be confirmed. It remains open as to whether PREGS was not detected due to differences in the methodology used compared to previous studies or to natural variation in the brain content.

The study aimed to establish the brain content of steroids, but the determination of their origin was beyond the scope of this study. The experiments have been performed on brains from intact animals and thus compounds could be derived from peripheral sources as well as de novo synthesis from within the brain. Further the study was restricted to whole brains of male rat; individual brain regions, spinal cord or peripheral nervous system were not analysed.

A further application of the method could be investigation of the diurnal regulation of the major brain steroids. As already mentioned, previous studies in the laboratory [47] have revealed diurnal fluctuations in the content of PROG and its 3,5-reduced metabolites in mouse brain. However, it is not known if all brain steroids show such fluctuations or how they might relate to diurnal rhythms in plasma content. Brain and plasma samples obtained from rats killed in intervals during 24 hours can be analysed after extraction as described above by SIM of HFB-derivatives. Such a study requires analysis of a high number of samples of small size. The HFB A derivatisation method was found to result in higher sensitivity in the GC-MS analysis and is thus more suitable for the analysis. It is not anticipated that major modifications of the analytical method will be required for these experiments. However, slight adaptations of the Lipidex 5000® purification step after HFB -derivatisation are necessary to improve the recovery of e.g. 3a,5a-THDOC, 3(3,5a-THD0C and CORT to allow their sensitive analysis by this method. The sensitivity for the remaining metabolites should allow quantitation of the metabolites identified in this study in single brains. For the analysis of plasma samples the Oasis HLB ® SPE method is expected to be used for extraction/purification by modifying the existing method. Thereafter the samples can be processed in the same way as tissue extracts, using Oasis MAX® and solvolysis procedures prior to HFB-derivatisation and GC-MS SIM.

The next step is the evaluation of central versus peripheral sources of brain steroids. Rats will be adrenalectomised and gonadectomised, then left for several days for peripheral steroids to decline. They will then be killed at times determined by the previous diurnal

234 study and samples of plasma and brain (together with those from sham-operated animals) taken for analysis by the procedures described above.

In conclusion, procedures are now developed to elucidate the regulation of steroids in mammalian brain.

235 References

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249 Appendices

Appendix 1

Gas chromatographic-mass spectrai data of steroids derivatised by MO/TMSi, TBDMS- and HFBA and seiected ion monitoring methods

The major ions of all steroid MO-TMS derivatives analysed are summarised in Table A- 1. Also listed in the Table are the Ko vats retention indices (RI) obtained after analysis according to the GC-method optimised in 3.2.2. In Table A-2, two ion monitoring methods developed for the steroids in Table A-1 are described.

Confirmation of compounds identified after two and multiple ion screening of brain steroids (5.2.1, 5.2.2) was done using three ion SIM of MO-TMS-derivatives and two ion SIM of HFB-derivatives (see below). Using the mass spectral data in Table A-1 and of compounds identified by multiple ion SIM a target and two qualifier ions were selected for each steroid in free and sulphate ester fractions (see Table A-3, SIM methods 6-13). The ions were chosen so as no interference would occur from ions of overlapping compounds in the GC-elution. Examples of qualifier to target ion area ratios in addition to relative retention times to internal standards used for identification of these compounds are shown in Chapter 5, Table 5-4 and Table 5-5.

The sensitivity of the three ion SIM procedure for MO-TMS -derivatives was determined for the compounds of interest in pure solution and is also shown in Table A-3. The limit of detection of the overall sample purification and analysis procedure of endogenous steroids in brain extracts using two ion SIM is shown in Chapter 5, Table 5-7.

The major ions of selected steroids as their TBDMS-derivatives are shown together with their RI and diagnostic qualifier and target ions in Table A-4.

Table A-5 shows the major ions in GC-EIMS (50-800 m/z) of the HFBA derivatives of all the compounds listed in Chapter 2, Table 2-1 (compounds 1-50) together with their RI, diagnostic target and qualifier ions and detection limits for two ion SIM.

250 Using the mass spectral data in the Table, target and qualifier ions for two ion SIM methods for analysis of HFB-derivatised steroids were identified after similar criteria as used above for MO-TMS-derivatives (3.2.5). Compounds identified in brain after initial two ion screens of MO-TMS-derivatives (5.2.1) were then subjected to two ion SIM after HFBA derivatisation. In order to maintain high analytical sensitivity, again it was aimed to limit the number of ions monitored at a time to four. Two separate SIM runs were used to monitor all the ions of all compounds in order to maintain the low number of ions monitored at a time (Table A-6) (SIM methods 16-19). In exceptional cases six ions had to be detected in one ion set. The resulting detection limits achieved in these SIM methods determined by using the three a noise definition are also shown in Table A-5.

251 Table A-1. Retention index after Kovats (RI), major ions, target and qualifier diagnostic ions (m/z) for two-ion SIM of MO-TMS-derivatives of steroids in GC- EIMS. Fifteen most abundant ions are listed in order of descending relative abundance. RI on 30 m ZBl column, 0.25 mm inner diameter, 0.25 |im film thickness. MS scan range 99-800 m/z (scan method 3). Steroid numbers correspond to steroids in Table 2-1. No. Steroid RI r ' R12'^^ Diagnostie Major ions peak peak ions (m/z) (m/z) 1 DHEA 2631 358 268 129 268 358 260 105 130 269 131 284 374 119 359 117 143 211 39 EpiA 2645 360 270 360 270 361 271 105 107 213 100 119 133 109 145 362 108 131 46 Adione 2690 2693 344 313 344 125 137 313 153 151 105 138 345 314 120 134 119 126 139 34 TESTO 2713 2717 389 268 389 153 125 137 129 151 268 105 390 358 147 119 131 138 133 26 5 P-pregnan-3a, 17-diol- 2740 476 188 476 156 188 477 158 364 105 296 172 386 507 107 170 147 478 20-one 22 3a-DHPR0G 2746 417 244 100 143 142 127 417 244 105 386 144 145 107 296 195 119 169 2 3(3,5(3-THPROG 2752 388 298 100 388 298 389 107 105 241 113 101 133 243 119 147 121 173 3 3a,5a-THPROG 2757 388 298 100 388 389 107 105 241 101 113 173 133 147 298 215 117 121 4 3a,5P-THPROG 2764 388 298 100 388 298 389 105 107 241 113 119 101 133 147 173 243 131 25 5 a-pregnan-3 a, 17-diol- 2766 476 188 476 477 156 188 158 364 296 107 105 172 170 386 507 478 255 20-one 44 5 P-pregnane-3 a,20P-diol 2779 284 269 117 118 119 116 103 284 105 107 269 346 347 121 133 147 109 30 16-DPREG 2802 415 400 129 415 286 294 384 105 310 119 131 400 134 133 107 117 145 24 5 a-pregnane-3 a,2 Oa-diol 2803 346 269 117 118 119 116 269 346 107 105 374 147 103 109 121 135 347 27 5 P-pregnane-3 a,2 Oa-diol 2811 284 269 117 118 119 116 284 269 105 107 346 347 133 109 121 147 103 42 3P-DHPR0G 2818 386 244 100 143 142 127 386 105 417 244 144 145 119 169 107 131 181 5 5P-DHPR0G 2820 343 275 100 343 288 275 105 244 107 344 289 119 145 108 131 122 187 6 PREG 2834 402 386 100 129 386 105 312 402 296 288 131 119 107 239 145 143 387 35 3p,5a-THPROG 2847 388 100 100 388 389 107 105 101 113 119 173 147 243 121 133 241 114 7 5a-DHPR0G 2859 2872 343 275 100 343 288 275 344 105 289 113 126 119 276 133 108 107 106 19 20P-DHPREG 2860 372 462 117 118 129 119 372 462 105 143 107 282 267 243 373 103 131 45 Alphaxalone 2880 402 433 100 402 105 147 403 138 107 129 101 121 136 109 119 122 131 21 20a-DHPREG 2882 372 462 117 129 118 119 372 105 462 143 282 267 107 131 145 120 130

252 Table A-1 continued. No. Steroid R ir ' RI2"‘*Diagnostic Major ions peak peak ions (m/z) (m/z) 8 PROG 2900 372 341 372 341 153 100 125 151 120 286 137 273 105 131 117 122 342 10 3a,53-THDOC 2907 2977 476 188 476 188 175 158 105 507 107 358 477 103 144 119 133 159 121 23 5a,20a-THPROG 2907 2920 303 289 117 303 118 119 289 304 105 107 126 121 314 147 271 101 106 43 5 a-pregnan-3 a, 11 P-diol- 2921 386 296 100 386 115 476 105 296 143 387 107 131 145 147 223 119 213 20-one 11 17-OH-PREG 2921 362 474 474 475 156 158 188 129 105 362 294 143 157 505 384 159 476 37 3P,5P-THDOC 2924 2984 507 358 476 175 188 477 158 507 107 105 103 283 119 144 133 108 358 9 20P-DHPROG 2927 417 286 117 153 417 125 118 137 119 296 301 151 105 138 286 418 126 13 3a,5a-THDOC 2929 2993 507 358 476 188 175 477 158 507 105 107 103 133 144 283 404 119 358 12 20a-DHPROG 2947 417 286 117 153 125 118 417 137 105 119 286 301 126 418 151 296 131 14 17-OH-PROG 2967 2975 429 339 429 430 273 339 158 460 170 156 317 105 172 188 200 117 144 28 ME-17-0H-PR0G 2970 3004 443 474 443 287 474 158 444 353 170 188 331 105 156 172 200 445 151 47 5P-DHD0C 2987 431 462 431 188 288 462 103 119 144 432 145 175 359 105 275 107 158 38 3p,5a-THDOC 3045 3098 476 188 476 188 175 158 105 507 107 358 477 103 144 119 133 159 121 15 5a-DHD0C 3058 3071 462 431 431 288 462 188 158 105 175 432 275 107 359 117 126 113 156 16 DOC 3098 273 286 429 286 460 103 125 105 153 188 273 145 137 430 151 107 120 20 IIP-OH-PROG 3105 3111 370 339 100 339 460 152 370 143 131 120 429 105 153 192 151 119 159 41 11-deoxycortisol 3124 3136 517 427 517 103 114 518 427 168 105 281 143 151 182 243 125 159 131 36 11 -dehydrocorticosterone 3196 443 474 474 152 295 207 188 103 209 137 165 228 191 217 443 132 147 40 Cortisone 3223 3242 531 441 114 531 208 168 441 104 243 459 120 103 143 533 152 119 112 17 CORT 3247 3258 548 517 103 427 105 188 143 517 158 152 120 131 148 117 104 153 548 50 PRED 3280 513 603 129 193 105 603 121 149 147 175 168 115 131 220 260 355 513 33 Cortisol 3290 605 515 605 281 515 246 240 198 145 191 146 103 197 245 170 105 165

253 Table A-2. Two ion SIM methods for the analysis of MO-TMS-derivatives of steroids. Ion set time windows, steroid RIs and target and qualifier ions as well as indications of IS used to calculate RRT (1-16-DPREG, 2-ME-17-OH-PROG, 3-Pl fŒD). Steroic numbers correspond to steroids in Table 2-1 SIM method Ion set No. Steroid End time (RI) RID'peak RI 2"'^ peak Target ion (m/z) Qualifier Ion (m/z) IS 1 I 22 3a-DHPR0G 2783 2746 417 244 1 2 3(3,5|3-THFRQG 2752 388 298 1 3 3a,5a-THPROG 2757 388 298 1 4 3a,53-THPROG 2764 388 298 1 2 30 16-DPREG 2818 2802 415 384 1 24 5a-pregnane-3a,20a-diol 2803 346 269 1 3 6 PREG 2863 2834 402 386 1 35 33,5a-THPROG 2847 388 100 1 4 45 Alphaxalone 2901 2880 402 433 1 21 20a-DHPREG 2882 462 372 2 5 43 5 a-pregnan-3 a, 113-diol-20-one 2944 2921 296 386 2 II 17-OH-PREG 2921 474 362 2 6 28 ME-17-OH-PROG 3142 2970 3004 443 474 2 14 17-OH-PROG 2967 2975 429 339 2 7 50 PRED 3400 3280 513 603 3 33 Cortisol 3290 605 515 3 2 I 26 5 P-pregnan-3a, 17-diol-20-one 2790 2740 476 188 1 25 5a-pregnan-3a,17-diol-20-one 2766 476 188 1 44 5 3-pregnane-3a,20P-diol 2779 284 269 1 2 30 16-DPREG 2836 2802 415 384 1 27 5 3-pregnane-3a,20a-diol 2811 284 269 1 3 19 203-DHPREG 2895 2860 462 372 1 7 5a-DHPR0G 2859 2872 343 275 1 4 10 3a,53-THDOC 2960 2907 476 188 2 9 203-DHPROG 2927 417 286 2 5 28 ME-17-0H-PR0G 3042 2970 3004 443 474 2 47 53-DHDOC 2987 462 431 2 6 16 DOC 3179 3098 286 273 2

254 Table A-2 continued. SIM method Ion set No Steroid End time (RI) R ir'peak R12"^peak Target ion (m/z) Qualifier Ion (m/z) IS 20 IIP-OH-PROG 3105 3111 370 339 2 7 17 CORT 3400 3247 3258 548 517 3 50 PRED 3280 513 603 3 3 I I DHEA 2723 2631 358 268 1 39 EpiA 2645 360 270 1 2 30 16-DPREG 2849 2802 415 384 1 42 3P-DHPR0G 2818 386 244 1 3 37 3P,53-THDOC 2958 2924 507 358 2 13 3a,5a-THDOC 2929 507 358 2 12 20a-DHPROG 2947 417 286 2 4 28 ME-17-OH-PROG 3085 2970 3004 443 474 2 38 3p,5a-THDOC 3045 476 188 2 5 41 11 -deoxvcortisol 3238 3124 3136 517 427 2 36 11 -deh\'drocorticosterone 3196 474 443 3 6 50 PRED 3400 3280 513 603 3 4 I 46 Adione 2760 2690 2693 344 313 1 34 TESTO 2713 2717 389 268 1 2 30 16-DPREG 2860 2802 415 384 1 5 53-DHPROG 2820 343 275 1 3 8 PROG 2945 2900 372 341 2 23 5a,20a-THPROG 2907 2920 303 289 2 4 28 ME-I7-0H-PR0G 3147 2970 3004 443 474 2 15 5a-DHD0C 3058 3071 462 431 2 5 40 Cortisone 3400 3223 3242 531 441 3 50 PRED 3280 513 603 3

255 Table A-3. Target (T) and two qualifier ions (Ql, 2), ion set end times, retention indices (RI) and detection limits for three ion SIM methods used to confirm identities of a) unconjugated and b) sulphate conjugated brain steroids as well as indications of IS used to calculate RRT (1-16-DPREG, 2-ME-17-OH-PROG, 3- PRED). For IS 4 ions were monitored. Numbers correspond to those listed in Table 2-1.

SIM method Ion set No Steroid End-time (RI) RI H^peak R12"‘^peak T 01 02 03 Detection limit (pg) IS 1 1 71 3 P-dihvdroandrostenedione 2681 2616 389 358 343 65.8 1 2 22 3a-DHPR0G 2762 2746 417 244 386 46.7 1 3 44 5P-pregnane-3a,20p~diol 2779 284 269 346 49.5 1 3 30 16-DPREG 2802 415 384 294 325 1 3 24 5a-pregnane-3a,20P-diol 2803 269 346 449 57.5 1 3 27 5 P-pregnane-3a,20a-diol 2836 2811 284 269 449 48.5 1 4 19 20P-DHPREG 2880 2860 462 372 332 37.6 1 5 8 PROG 2914 2900 372 341 273 71.4 2 6 9 20P-DHPROG 2927 417 301 296 94.3 2 6 12 20a-DHPROG 2958 2947 417 301 296 100.0 2 7 28 ME-17-OH-PROG 2970 3004 443 474 353 384 2 7 47 5P-DHD0C 3054 2987 462 431 288 43.9 2 8 20 llp-OH-PROG 3196 3105 3111 339 370 361 142.9 2 9 50 PRED 3400 3280 513 603 482 634 3 2 I I DHEA 2716 2631 358 268 260 9.9 1 2 30 16-DPREG 2813 2802 415 384 294 325 1 3 5 5P-DHPR0G 2840 2820 343 275 288 24.9 1 4 7 5a-DHPR0G 2890 2859 2872 343 275 288 25.4 1 5 23 5a,20a-THPROG 2943 2907 2920 303 289 314 58.8 2 6 14 17-OH-PROG 2967 2975 429 339 460 73.5 2 6 28 ME-17-0H-PR0G 3051 2970 3004 443 474 353 384 2 7 16 DOC 3189 3098 286 398 273 113.6 2 8 50 PRED 3400 3280 513 603 482 634 3 3 I 34 TESTO 2735 2713 2717 389 268 358 38.8 1 2 2 3p,5P-THPROG 2752 388 298 404 18.7 1 2 3 3a,5a-THPROG 2757 388 298 404 57.5 1

256 Table A-3 a) continued SIM method Ion set No Steroid End-time (RI) RI H^peak RI 2^^^ peak T Ql Q2 Q3 Detection limit (pg) IS 2 4 3a,5p-THPROG 2783 2764 388 298 404 17.4 I 3 30 16-DPREG 2818 2802 415 384 294 325 I 4 6 PREG 2856 2834 402 386 312 23.8 I 5 10 3a,5(3-THDOC 2907 476 507 404 41.7 2 5 37 33.53-THDOC 2924 507 476 386 11.2 2 5 13 3a.5a-THDOC 2950 2929 507 476 404 38.5 2 6 28 ME-17-OH-PROG 3024 2970 3004 443 474 353 384 2 7 38 3fl5a-THDOC 3120 3045 476 404 507 42.4 2 8 36 11 -deh\’drocorticosterone 3238 3196 474 443 300 152.8 3 9 50 PRED 3400 3280 513 603 482 634 3 4 1 54 OESTR 2720 2690 416 285 326 78.5 1 2 25 5 a-pregnan-3 a, 17-diol - 2 0 -one 2784 2766 476 386 364 II.3 I 3 30 16-DPREG 2825 2802 415 384 294 325 1 4 35 33,5a-THPROG 2864 2847 388 298 404 64.1 I 5 21 20a-DHPREG 2901 2882 462 372 332 32.1 2 6 43 5a-pregnan-3 a, 113-dioI-20-one 2921 296 386 239 39.1 2 6 II 17-OH-PREG 2929 2921 474 362 384 34.0 2 7 58 63-OH-PROG 2960 2936 2950 460 445 413 108.7 2 8 28 ME-17-OH-PROG 3031 2970 3004 443 474 353 384 2 9 15 5a-DHD0C 3159 3058 3071 462 431 288 82.0 2 10 17 CORT 3269 3247 3258 548 517 427 94.3 3 II 50 PRED 3400 3280 513 603 482 634 3

Table A-3 b) SIM method Ion set No Steroid End time (RI) RI H‘peak RI2'’‘*peak T Ql Q2 Q3 Detection limit (pg) IS I I I DHEA 2688 2631 358 268 260 9.9 I 2 22 3a-DHPR0G 2756 2746 417 244 386 46.7 I 3 25 5a-pregnan-3a,17-dioI-20-one 2784 2766 476 386 364 11.3 I 4 30 16-DPREG 2802 415 384 294 325 I

257 Table A-3 b) continued SIM method Ion set No Steroid End time (RI) RI peak RI 2"^ peak T Ql Q2 Q3 Detection limit (pg) IS 4 24 5 a-pregnane-3 a,2 Oa-diol 2803 269 346 449 57.5 1 4 27 5 P-pregnane-3a,20a-diol 2836 2811 284 269 449 48.5 1 5 19 20(3-DHPREG 2890 2860 462 372 332 37.6 1 6 43 5a-pregnan-3a,11 P-diol-20-one 2945 2921 296 386 239 39.1 2 7 28 ME-17-OH-PROG 3024 2970 3004 443 474 353 384 2 8 38 33,5a-THDOC 3163 3045 476 404 507 42.4 2 9 50 PRED 3400 3280 513 603 482 634 3 2 1 39 EpiA 2699 2645 360 270 376 28.4 1 2 2 33,5P-THPROG 2766 2752 388 298 404 18.7 1 3 44 5P-pregnane-3a,20P-diol 2791 2779 284 269 346 49.5 1 4 30 16-DPREG 2808 2802 415 384 294 325 1 5 42 3P-DHPR0G 2839 2818 386 244 417 44.2 1 6 37 3P,5P-THDOC 2947 2924 507 476 386 11.2 2 7 28 ME-17-OH-PROG 3054 2970 3004 443 474 353 384 2 8 20 IIP-OH-PROG 3192 3105 3111 339 370 361 142.9 2 9 50 PRED 3400 3280 513 603 482 634 3 3 I 34 TESTO 2737 2713 2717 389 268 358 38.8 1 2 3 3a,5a-THPROG 2780 2757 388 298 404 57.5 1 3 30 16-DPREG 2818 2802 415 384 294 325 1 4 6 PREG 2871 2834 402 386 312 23.8 1 5 23 5a,20a-THPROG 2943 2907 2920 303 289 314 58.8 2 6 14 17-OH-PROG 2967 2975 429 339 460 73.5 2 28 ME-17-OH-PROG 3051 2970 3004 443 474 353 384 2 7 16 DOC 3111 3098 286 398 273 113.6 2 8 41 11-deoxycortisol 3202 3124 3136 517 427 548 73.5 3 9 50 PRED 3400 3280 513 603 482 634 3 4 I 26 5 P-pregnan-3a, 17-diol-20-one 2752 2740 476 364 507 6.0 1 2 4 3a,5p-THPROG 2783 2764 388 298 404 17.4 1 3 30 16-DPREG 2825 2802 415 384 294 325 1

258 Table A-3 b) continued.

SIM method Ion set No Steroid End time (RI) RI r ' peak RI 2"^ peak T Ql Q2 Q3 Detection limit (pg) IS 4 35 3|3,5a-THPROG 2877 2847 388 298 404 64.1 I 5 10 3a,53-THDOC 2918 2907 476 507 404 41.7 2 6 13 3a,5a-THDOC 2950 2929 507 476 404 38.5 2 7 28 ME-I7-0H-PR0G 3142 2970 3004 443 474 353 384 2 8 50 PRED 3400 3280 513 603 482 634 3

259 Table A-4. Retention index after Kovats (RI) of main peak, major ions, diagnostic (target, T and qualifier, Q) ions (m/z) for SIM of TBDMS-derivatives of steroids in GC-EIMS. Major ions listed in order of descending relative abundance. Numbers in left hand column correspond to those listed in Table 2-1. RI on CP SIL 5

No. Steroid RI T Q Major ions 1 DHEA 3140 459.4 516.5 73 459 460 59 253 131 105 211 147 81 251 157 159 145 516 3 3a,5a-THPROG 3255 531.6 489.45 73 531 532 199 399 59 74 131 489 76 107 413 93 67 103 30 16-DPREG 3376 486.35 527.5 73 486 527 471 485 59 141 105 91 119 140 147 159 487 57 7 5a-DHPR0G 3401 529.55 544.6 73 529 530 59 199 531 185 57 127 105 544 85 81 107 115 6 PREG 3422 529.55 544.6 73 529 199 530 59 531 127 185 159 115 105 487 91 81 544 8 PROG 3453 527.6 542.6 73 199 542 528 59 543 529 527 200 544 115 91 119 127 143 12 20a-DHPROG 3463 544.65 487.4 73 544 545 159 59 103 115 57 55 105 76 542 487 93 199 11 17-OH-PREG 3543 503.45 411.45 143 73 503 279 119 157 144 504 185 57 133 145 59 115 411 21 20a-DHPREG 3589 503.45 411.45 73 143 117 57 503 59 81 55 119 95 89 147 105 411 85 14 17-OH-PROG 3589 501.45 409.4 73 143 501 502 59 119 57 117 157 503 409 55 115 144 105 28 ME-17-0HPR0G 3611 572.55 515.55 73 572 143 573 515 59 57 357 157 185 574 119 145 159 93 13 3a,5a-THDOC 3683 619.6 676.75 73 147 619 74 620 115 57 133 676 81 59 621 149 55 677 15 5a-DHD0C 3889 617.5 674.75 73 147 617 57 207 115 618 59 133 55 149 674 148 619 93 16 DOC 3927 615.5 672.75 73 147 615 616 115 672 59 499 673 500 617 133 143 148 149 17 CORT 4026 631.55 689.45 73 147 74 631 689 59 690 632 115 143 57 119 133 149 207

260 Table A-5. Retention index after Kovats (RI) and major ions of HFB-derivatives of steroids in GC-EIMS. 15 major ions listed in order of descending relative abundance. Also listed are target (T), qualifier (Q) ions (m/z) and detection limits of steroids that are monitored with confirmatory SIM methods. Numbers in left

No. Steroid RI RI2*'‘^ T Q Det. limit Major ions peak peak (pg) (m/z) 33 Cortisol 2320 132 91 341 77 191 117 135 109 189 84 159 113 299 325 73 164 44 5 (3-pregnane-3a,20(3-diol 2358 2504 429 712 2.2 429 93 215 55 107 81 67 69 121 79 95 91 105 68 122 712 34 TESTO 2453 680 451 1.2 105 91 69 680 133 119 55 320 107 79 168 146 131 665 467 451 46 Adione 2465 87 100 70 510 91 526 105 79 55 67 81 69 74 93 57 119 1 DHEA 2470 270 255 5.1 270 121 91 55 105 79 93 107 67 271 143 69 255 199 77 81 22 3a-DHPR0G 2476 298 283 91 159 298 105 93 107 147 77 283 299 67 145 115 165 128 132 42 3P-DHPRGG 2476 298 283 298 105 79 91 121 145 85 81 55 147 119 107 135 213 92 283 39 EpiA 2492 67 55 93 79 91 107 486 81 108 97 105 69 147 442 109 95 43 5 a-pregnan-3 a, 11 P-diol-20- 2506 2568 469 512 5.0 105 131 79 91 81 95 93 213 55 143 58 117 67 132 469 512 one 3 3a,5a-THPROG 2543 496 514 5.0 84 81 67 93 79 95 55 71 107 91 215 105 69 496 119 514 4 3a,53-THPROG 2555 496 300 6.5 84 71 67 55 79 81 93 215 95 107 91 105 496 121 69 300 24 5 a-pregnane-3 a,2 Oa-diol 2558 712 697 1.0 429 107 215 93 121 81 55 67 95 79 69 91 147 430 712 697 2 3P,5P-THPRGG 2567 496 514 5.7 84 71 67 79 81 93 55 95 107 91 105 215 496 121 69 514 27 5 P-pregnane-3a,20a-diol 2570 712 697 2.3 429 93 107 81 215 55 67 79 121 69 95 91 68 149 712 697 30 16-DPREG 2603 296 253 7.5 296 91 105 133 93 159 145 117 119 297 107 131 79 121 81 253 9 20P-DHPRGG 2605 708 693 3.7 161 708 91 69 105 121 79 119 81 169 93 175 107 159 147 693 19 20P-DHPREG 2606 496 481 5.6 496 121 105 91 93 107 79 55 69 161 497 67 81 145 133 481 45 Alphaxalone 2628 67 91 81 95 79 528 55 105 93 71 122 147 109 69 107 119 8 PRGG 2638 510 495 15.5 69 510 91 147 105 55 85 79 67 93 131 133 107 81 95 495 6 PREG 2640 298 283 11.8 298 91 105 93 55 79 67 85 107 145 81 121 95 299 131 283 12 20a-DHPRGG 2641 2647 708 693 1.2 69 161 105 91 119 55 169 708 121 93 81 147 67 107 79 693 25 5 a-pregnan-3 a, 17-diol-20- 2644 2652 442 487 17.6 469 67 93 79 55 91 135 100 95 81 107 105 161 215 442 487 one 26 5 P-pregnan-3a, 17-diol-20- 2652 2658 469 530 67 81 93 71 55 79 231 107 442 69 95 91 230 135 469 530 one

261 Tab e A-5 continued. No. Steroid RI R12"'^ T Q Det. limit Major ions peak peak (pg) (m/z) 21 20a-DHPREG 2653 496 481 18.3 496 121 105 91 107 55 93 69 145 79 161 81 119 67 481 497 35 33,5a-THPROG 2659 467 514 43.5 84 71 55 67 79 93 81 107 95 91 69 85 105 134 467 514 13 3a,5a-THDOC 2691 2751 499 257 8.1 257 55 95 93 81 79 149 499 91 67 107 258 69 121 105 109 10 3a,53-THDOC 2705 2762 499 257 10.5 257 81 93 67 55 95 499 107 163 149 79 91 109 147 258 57 37 33,53-THDOC 2714 2779 499 257 6.2 257 81 107 67 95 93 109 55 69 121 79 91 58 119 149 499 23 5a,20a-THPROG 2725 499 514 9.1 55 231 57 105 93 208 137 81 109 232 163 124 79 123 514 499 14 17-OH-PROG 2745 526 465 11.4 119 69 91 169 157 129 526 117 67 81 104 55 79 115 95 465 11 17-OH-PREG 2746 2751, 528 467 153.8 253 213 105 467 91 271 133 145 143 79 119 69 157 197 107 528 2833 16 DOC 2792 722 707 58.8 69 147 55 722 91 119 105 169 157 107 79 57 93 143 723 707 38 33,5a-THDOC 2817 2879 499 257 2.0 257 69 81 95 55 67 79 93 107 133 163 91 147 109 121 499 47 53-DHDOC 2828 301 255 2.6 55 255 81 67 69 95 301 91 79 93 107 121 83 119 53 77 20 113-OH-PROG 2835 526 493 133.3 526 69 95 91 81 93 79 107 55 117 71 145 77 119 155 493 36 11-dehvdrocorticosterone 2877 736 595 400.0 208 75 265 69 169 107 117 157 103 89 115 141 275 133 595 736 15 5a-DHD0C 2881 2946 301 255 3.1 55 67 163 95 69 301 81 255 79 123 119 93 56 105 107 273 17 CORT 2979 738 491 150.9 69 55 738 91 169 105 81 95 119 79 107 129 67 121 93 491

262 Table A-6. Two ion SIM methods for the analysis of HFB-derivatives of free (a) and sulphate conjugated (b) steroids. Ion set time windows, steroid RIs and diagnostic target (T) and qualifier (Q) ions as well as indications of IS used to calculate RRT (1-tetracosane, 2-16- DPREG, 3-octacosane).

SIM Ion set Steroid/I S End time (RI) RI T Q IS method I I Tetracosane 2487 2400 127 141 I TESTO 2453 680 451 1 DHEA 2470 270 255 1 2 5 P-pregnane-3a,20P-diol 2555 2504 429 712 2 3a,5a-THPROG 2543 496 514 2 3 3p,5p-THPROG 2585 2567 496 514 2 5 a-pregnan-3 a, 11 P-diol-20-one 2568 469 512 2 4 20P-DHPROG 2622 2605 708 693 2 16-DPREG 2603 296 253 2 5 PROG 2649 2638 510 495 2 PREG 2640 298 283 2 6 3p,5a-THPROG 2675 2659 467 514 2 7 3a,5a-THDOC 2725 2691 499 257 2 3a,5p-THDOC 2705 499 257 3 8 17-OH-PROG 2781 2745 526 465 3 9 3p,5a-THDOC 2852 2817 499 257 3 5P-DHD0C 2828 301 255 3 Octacosane 2800 127 141 3 10 11 -dehydrocorticosterone 2950 2877 736 595 3 5a-DHD0C 2881 301 255 3 2 I Tetracosane 2400 127 141 1 3a-DHPR0G 2515 2476 298 283 I 3P-DHPR0G 2476 298 283 I 2 3a,5p-THPROG 2586 2555 496 300 2 5 a-pregnane-3 a,20a-diol 2558 712 697 2 5 P -pregnane-3 a,20a-diol 2570 712 697 2 3 16-DPREG 2625 2603 296 253 2 20P-DHPREG 2606 496 481 2 4 5 a-pregnan-3 a, 17-diol-20-one 2683 2644 442 487 2 20a-DHPROG 2647 708 693 2 20a-DHPREG 2653 496 481 2 5 3p,5p-THDOC 2734 2714 499 257 3 5a,20a-THPROG 2725 499 514 3 6 17-OH-PREG 2813 2746 528 467 3 DOC 2792 722 707 3 Octacosane 2800 127 141 3 7 17-OH-PREG 2907 2833 528 467 3 IIp-OH-PROG 2835 526 493 3 8 CORT 2979 738 491 3

263 Table A-6 b) continued.

SIM method Ion set Steroid/IS End time (RI) RI T Q IS 1 I Tetracosane 2487 2400 127 141 I TESTO 2453 680 451 I DHEA 2470 270 255 I 2 5 P-pregnane-3a,20|3-diol 2531 2504 429 712 2 3 5 a-pregnane-3 a,20a-diol 2585 2558 712 697 2 3p,5P-THPRQG 2567 496 514 2 4 16-DPREG 2622 2603 296 253 2 5 PREG 2652 2640 298 283 2 6 5 P-pregnan-3a, 17-diol-20-one 2675 2658 442 469 2 3p,5a-THPROG 2659 467 514 2 7 3a,5a-THDOC 2729 2691 499 257 2 3a,5P-THDOC 2705 499 257 3 3p,5P-THDOC 2714 499 257 3 8 17-OH-PROG 2781 2745 526 465 3 9 Octacosane 2850 2800 127 141 3 3p,5a-THDOC 2817 499 257 3 2 I Tetracosane 2517 2400 127 141 I 3a-DHPR0G 2476 298 283 I 3P-DHPR0G 2476 298 283 I EpiA 2492 486 442 I 2 3a,5a-THPROG 2563 2543 496 514 2 3a,5p-THPROG 2555 496 300 2 3 5a-pregnan-3a,I ip-diol-20-one 2586 2568 469 512 2 5p-pregnane-3a,20a-diol 2570 712 697 2 4 16-DPREG 2625 2603 296 253 2 20P-DHPREG 2606 496 481 2 5 5a-pregnan-3a,I7-diol-20-one 2689 2644 442 487 2 20a-DHPREG 2653 496 481 2 6 5a,20a-THPROG 2758 2725 499 514 3 7 DOC 2814 2792 722 707 3 Octacosane 2800 127 141 3 8 IIP-OH-PROG 2880 2835 526 493 3

264 Appendix 2

Application of method for separation of free steroids and their sulphate and giucuronide conjugates to human urine samples

As no radioactively labelled steroid glucuronides could be obtained, elution of those compounds as well as free and sulphated steroids from Oasis MAX® (see 4.2.2.4) was monitored by analysing urine samples of five human subjects. Urine samples (from 24 h collections) were passed through SePak C l8® cartridges, which were then washed with

H2 O and eluted with 100% ethanol. After dilution of the eluates to 20% ethanol, they were loaded onto Oasis MAX® cartridges. The cartridges were washed with 20%

ethanol in 20 mM KH 2PO4 buffer pH 7.4, and free steroid, giucuronide and sulphate fractions eluted with ethyl acetate, 60% ethanol in 20 mM formic acid/pyridine pH 3 and after a wash with ethyl acetate, with 50mM BSA/acidified ethyl acetate, respectively. The free steroid fractions were analysed by GC-MS after drying down and derivatising by MO and TMSI. The steroid giucuronide fractions were, after drying down, redissolved in 0.5 M sodium acetate buffer, pH 6 and incubated at 37°C for 24 h with glucuronidase enzyme. The solution was then passed through SePak CIS® cartridges, which were after a water wash eluted with 100% ethanol. After drying down, the sample was derivatised with MO and TMSI. The steroid sulphate fraction was incubated 16h at

40°C in the presence of Na 2 S0 4 , then neutralised with pyridine and dried down. After extraction with ether three times, the sample was dried down and derivatised by MO and TMSI.

Total steroid profiles were also acquired. These were not processed through Oasis MAX®, but treated with glucuronidase/sulphatase enzyme after the initial elution from SePak CIS®. The gas chromatograms of the free steroid, giucuronide and sulphate fractions obtained by Oasis MAX® chromatography are shown in Figure A-1.

Samples 1 and 2 were women about 30 weeks into pregnancy. The samples showed elevated levels of PROG and certain cortisol metabolites appearing mainly in the giucuronide fraction, as would have been expected (see Figure A-1 a and b). The former metabolites appear in the chromatograms from about 17-20 minutes, the latter from 20- 24 minutes. Sample 3 was a normal adult male, with several androgen (14-17 minutes).

265 PROG (17-20 minutes) and cortisol metabolites (20-24 minutes) mainly as glucuronides and very few in sulphate and free steroid fractions (Figure A-1 c). Sample 4 was of 2 weeks old female with hyponatraemia and hyperkalaemia. However, normal levels of adrenal androgen and cortisol metabolites were observed. These appear mainly as sulphates, as expected for a neonate (see Figure A-1 d). Sample 5 was a 2 weeks old male with suspected congenital adrenal hyperplasia (CAR). Elevated 17- hydroxyprogesterone and 21-deoxycortisol metabolites could be observed and no cortisol and ALDO metabolites, consistent with 21-hydroxylase deficiency (which would cause CAR). These metabolites appear in the giucuronide fraction (Figure A-1 e).

The metabolites monitored were thus detected in the fractions (free, sulphate or giucuronide) they were expected to be in. This gives confirmation for the procedure using Oasis MAX® anion exchange solid phase extraction cartridges to be suitable for fractionation of free, sulphate and giucuronide conjugated steroids (see 4.2.2.4).

266 Figure A-1. Total ion chromatograms of urine steroids. Urines had been passed through Sep Pak CIS® cartridges, eluted with ethanol, diluted to 20% with potassium phosphate buffer (5 mM, pH 7.4, v/v) and loaded onto Oasis MAX® and eluted as described in the text. Free steroids are in the left, steroid sulphates in middle and steroid glucuronides in right panels. Urine samples were from subjects as follows and are further detailed in text. Numbers above peaks denote the following compounds: a) Female 30 weeks pregnancy; l-Sp-pregnane-3a,20a-diol, 2-oestriol b) Female 30 weeks pregnancy; l-Sp-pregnane-3a,20a-diol, 2-oestriol c) Normal adult male; 1-androsterone, 2-etiocholanolone, 3-11 p OH-androsterone, 4-1 ip- OH-etiocholanolone, 5-5p-pregnane-3a,17,20a-triol, 6-3a,17a,21-trihydroxy-5p-pregnan- 11,20-dione (), 7-3a,lip,17a,21-tetrahydroxy-SP-pregnan-20-one (), 8-3a,17a,20a,21-tetrahydroxy-5p-pregnan-ll-one (cortolone) d) 2 weeks old female; 1-16-OH-DHEA, 2-5-androstene-3(3,16a,17P-triol, 3-16,18-di-OH- DHEA, 4-tetrahydrocortisone, 5- 6a-OH-tetrahydrocortisone e) 2 weeks old male with suspected CAR; l-5P-pregnan-3a,17-diol-20-one (17-OH- pregnanolone), 2-15p,17a-di-OH-pregnanolone, 3-5p-pregnane-3a,17,20a-triol, 4-11-oxo- pregnanetriol

267 Figure A-1 a).

.Ll. JlL l . uwww Él^ 21 X_a 34 16 ]■ Jn 14 l« I l J* I» 20 12 24 16 IB

268 Figure A-1 b).

JÈjLLlii

269 Figure A-1 e).

272