Ghent University Faculty of Pharmaceutical Sciences Quantitative analysis of new generation antidepressants using gas chromatography-mass spectrometry Applications in clinical and forensic toxicology Sarah Wille Pharmacist Thesis submitted to obtain the degree of Doctor in Pharmaceutical Sciences 2008 Dean: Promoter : Prof. Dr. Jean-Paul Remon Prof. Dr. Willy Lambert TABLE OF CONTENTS Table of contents Acknowledgements Copyright List of Abbreviations Structure Chapter I Introduction 1 Depression, use of antidepressants, and relevance of antidepressant monitoring I.1. Foreword 3 I.2. Onset of depression 3 I.3. Action mechanisms of antidepressants 4 I.3.1. Activation of transcription factors 5 I.3.2. Activation of neurotropic pathways 7 I.3.3. Increasing neurogenesis 8 I.4. Classification of antidepressants 8 I.5. Side-effects, drug-drug interactions and toxicity 10 I.6. Relevance of Therapeutic Drug Monitoring 13 I.7. Selection of antidepressants and relevant issues for TDM 14 I.7.1. Citalopram 15 I.7.1.1. Mechanism of action 16 I.7.1.2. Pharmacokinetics 16 I.7.1.3. Drug concentrations and clinical effects 17 I.7.1.4. Drug interactions, side-effects and toxicity 18 I.7.1.5. Analytical Methods 18 I.7.2. Fluoxetine 19 I.7.2.1. Mechanism of action 20 I.7.2.2. Pharmacokinetics 20 I.7.2.3. Drug concentrations and clinical effects 21 I.7.2.4. Drug interactions, side-effects and toxicity 22 I.7.2.5. Analytical Methods 22 I.7.3. Fluvoxamine 23 I.7.3.1. Mechanism of action 24 I.7.3.2. Pharmacokinetics 24 I.7.3.3. Drug concentrations and clinical effects 25 I.7.3.4. Drug interactions, side-effects and toxicity 25 I.7.3.5. Analytical Methods 26 I.7.4. Maprotiline 27 I.7.4.1. Mechanism of action 27 I.7.4.2. Pharmacokinetics 28 I.7.4.3. Drug concentrations and clinical effects 28 I.7.4.4. Drug interactions, side-effects and toxicity 28 I.7.4.5. Analytical Methods 29 I.7.5. Melitracen 30 I.7.6. Mianserin 30 I.7.6.1. Mechanism of action 31 I.7.6.2. Pharmacokinetics 31 I.7.6.3. Drug concentrations and clinical effects 31 I.7.6.4. Drug interactions, side-effects and toxicity 32 I.7.6.5. Analytical Methods 32 I.7.7. Mirtazapine 32 I.7.7.1. Mechanism of action 33 I.7.7.2. Pharmacokinetics 33 I.7.7.3. Drug concentrations and clinical effects 33 I.7.7.4. Drug interactions, side-effects and toxicity 34 I.7.7.5. Analytical Methods 35 I.7.8. Paroxetine 35 I.7.8.1. Mechanism of action 36 I.7.8.2. Pharmacokinetics 36 I.7.8.3. Drug concentrations and clinical effects 37 I.7.8.4. Drug interactions, side-effects and toxicity 38 I.7.8.5. Analytical Methods 38 I.7.9. Reboxetine 39 I.7.9.1. Mechanism of action 40 I.7.9.2. Pharmacokinetics 40 I.7.9.3. Drug concentrations and clinical effects 40 I.7.9.4. Drug interactions, side-effects and toxicity 41 I.7.9.5. Analytical Methods 41 I.7.10. Sertraline 42 I.7.10.1. Mechanism of action 42 I.7.10.2. Pharmacokinetics 42 I.7.10.3. Drug concentrations and clinical effects 43 I.7.10.4. Drug interactions, side-effects and toxicity 44 I.7.10.5. Analytical Methods 44 I.7.11. Trazodone 45 I.7.11.1. Mechanism of action 45 I.7.11.2. Pharmacokinetics 45 I.7.11.3. Drug concentrations and clinical effects 46 I.7.11.4. Drug interactions, side-effects and toxicity 46 I.7.11.5. Analytical Methods 47 I.7.12. Venlafaxine 48 I.7.12.1. Mechanism of action 48 I.7.12.2. Pharmacokinetics 49 I.7.12.3. Drug concentrations and clinical effects 49 I.7.12.4. Drug interactions, side-effects and toxicity 50 I.7.12.5. Analytical Methods 51 I.7.13. Viloxazine 51 I.7.13.1. Mechanism of action 52 I.7.13.2. Pharmacokinetics 52 I.7.13.3. Drug concentrations and clinical effects 52 I.7.13.4. Drug interactions, side-effects and toxicity 52 I.7.13.5. Analytical Methods 53 I.8. Relevance of antidepressant analysis in forensic toxicology 53 I.9. References 54 Chapter II Objectives 75 Chapter III Sample preparation 79 Development and optimization of a solid phase extraction procedure for several biological matrices III.1. Introduction 81 III.2. Experimental 82 III.2.1. Reagents 82 III.2.2. Stock solutions 83 III.2.3. Mixer, sonicator, vacuum manifold, evaporator 84 and centrifuge III.2.4. High Pressure Liquid Chromatography (HPLC) 85 III.2.5. Gas chromatography-Mass spectrometry (GC-MS) 85 III.3. Solid phase extraction development 87 III.3.1. Choice of SPE sorbent 87 III.3.2. Choice of loading, washing and eluting conditions 90 III.3.3. Final SPE method of ADs spiked in water samples 93 III.4. Optimization of the SPE procedure for extraction of ADs 95 from biological matrices III.4.1. SPE optimization for plasma samples 96 III.4.2. SPE optimization for blood samples 98 III.4.3. SPE optimization for brain samples 99 III.4.4. SPE optimization for hair samples 100 III.4.5. Recovery of ADs using SPE from plasma, blood 103 brain tissue III.5. Conclusion 104 III.6. References 106 Chapter IV Derivatization 109 Development and optimization of a solid phase extraction procedure for several biological matrices IV.1. Introduction 111 IV.2. Experimental 114 IV.2.1. Reagents 114 IV.2.2. Preparation of standard solutions 114 IV.2.3. Instrumentation 115 IV.2.4. Gas chromatographic parameters 115 IV.2.5. Mass spectrometric parameters 116 IV.3. Acetylation 116 IV.3.1. Optimization of acetylation reaction 116 IV.3.2. Acetylation reaction with antidepressants 116 IV.3.2.1. ADs containing an alcohol function 117 IV.3.2.2. ADs containing a primary amine function 119 IV.3.2.3. ADs containing secondary amine functions 121 IV.3.2.4. Tertiary amines 122 IV.3.3. Conclusion 123 IV.4. Heptafluorobutyrylation 124 IV.4.1. Optimization of HFBI reaction 124 IV.4.1.1. Experimental 124 IV.4.1.2. Results 124 IV.4.2. Optimization of HFBA reaction 126 IV.4.2.1. Experimental 126 IV.4.2.2. Results 126 IV.4.3. Heptafluorobutyrylation of antidepressants 127 IV.4.3.1. ADs containing an alcohol function 128 IV.4.3.2. ADs containing a primary amine function 130 IV.4.3.3. ADs containing secondary amine functions 130 IV.4.3.4. Tertiary amines 131 IV.4.4. Conclusion 131 IV.5. Choice of acylation procedure 133 IV.5.1. Acetylation versus heptafluorobutyrylation 133 IV.5.2. Heptafluorobutyrylimidazole versus heptafluoro- 134 butyric anhydride IV.5.2.1. Experimental 134 IV.5.2.2. Results 134 IV.5.3. Conclusion 136 IV.6. Final derivatization procedure 137 IV.7. Validation of final derivatization procedure 137 IV.7.1. Precision 137 IV.7.1.1. Experimental 137 IV.7.1.2. Results 137 IV.7.2. Linearity 138 IV.7.2.1. Experimental 138 IV.7.2.2. Results 138 IV.7.3. Stability of the derivatives 139 IV.7.3.1. Experimental 139 IV.7.3.2. Results 139 IV.8. Conclusion 141 IV.9. References 142 Chapter V Gas chromatographic-mass spectrometric 145 method development V.1. Introduction 147 V.2. Experimental 148 V.2.1. Reagents 148 V.2.2. Stock solutions 149 V.2.3. Equipment 149 V.3. Gas chromatographic parameters 150 V.3.1. Sample introduction 150 V.3.1.1. Cold on-column versus split/splitless injection 150 V.3.1.2. Splitless injection optimization 152 V.3.2. Chromatographic separation 157 V.3.2.1. Column choice 158 V.3.2.2. Choice of carrier gas and flow rate 159 V.3.2.3. Optimization of temperature program 159 V.3.3. Internal standard choice 162 V.3.4. Conclusion: gas chromatographic method 163 V.4. Mass spectrometric parameters 164 V.4.1. Optimization of mass selective detector parameters 167 V.4.2. Spectra of the derivatized ADs after electron 168 ionization V.4.2.1. Venlafaxine and O-desmethylvenlafaxine 168 V.4.2.2. Viloxazine 170 V.4.2.3. Fluvoxamine 171 V.4.2.4. Fluoxetine, fluoxetine-d6 and desmethylfluoxetine 172 V.4.2.5. Mianserin, mianserin-d3 and desmethylmianserin 175 V.4.2.6. Mirtazapine and desmethylmirtazapine 177 V.4.2.7. Melitracen 179 V.4.2.8. Reboxetine 179 V.4.2.9. Citalopram, desmethylcitalopram and dides- 180 methylcitalopram V.4.2.10. Maprotiline and desmethylmaprotiline 182 V.4.2.11. Sertraline and desmethylsertraline 184 V.4.2.12. Paroxetine and paroxetine-d6 185 V.4.2.13. Trazodone and m-chlorophenylpiperazine 186 V.4.3. Spectra of the derivatized ADs after positive ion 188 chemical ionization V.4.3.1. Venlafaxine and O-desmethylvenlafaxine 190 V.4.3.2. Viloxazine 192 V.4.3.3. Fluvoxamine 192 V.4.3.4. Fluoxetine, fluoxetine-d6 and desmethylfluoxetine 193 V.4.3.5. Mianserin, mianserin-d3 and desmethylmianserin 196 V.4.3.6. Mirtazapine and desmethylmirtazapine 198 V.4.3.7. Melitracen 199 V.4.3.8. Reboxetine 200 V.4.3.9. Citalopram, desmethylcitalopram and dides- 201 methylcitalopram V.4.3.10. Maprotiline and desmethylmaprotiline 204 V.4.3.11. Sertraline and desmethylsertraline 206 V.4.3.12. Paroxetine and paroxetine-d6 207 V.4.3.13.