THERAPEUTIC DRUG MONITORING OF ANTIPSYCHOTICS HPLC ANALYTICAL METHOD DEVELOPMENT AND PHARMACEUTICAL ADVICES TO THE WARD CLINICIANS FOR SUCCESSFUL INDIVIDUALIZED THERAPY Therapeutisches Drug Monitoring von Antipsychotika – HPLC Analytische Methodenentwicklung und Pharmazeutische Beratung der Stationsärzte- für Erfolgreiche, Individualisierte Therapie Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftliche Fakultät IV - Chemie und Pharmazie- der Universität Regensburg Vorgelegt von Mary Remigius Agatha Onuoha aus Aba/ Nigeria im August 2016 Promotion gesucht eingereicht am: 01.08.2016 Die Arbeit wurde angeleitet von: Herrn Professor Dr. Dr. Ekkehard Haen Mündliche Prüfung am: 28.10.2016 Prüfungsausschuss: Herr Professor Dr. Sigurd Elz (Vorsitzender) Herr Professor Dr. Dr. Ekkehard Haen (1. Gutachter) Herr Professor Dr. Christoph Hiemke (2. Gutachter) Herr Professor Dr. Jens Schlossmann (3. Gutachter) For the less privileged of our society List of content List of content ................................................................................. I List of figures ................................................................................. V List of tables ................................................................................ VII List of abbreviation ....................................................................... X 1 Introduction .............................................................................. 1 1.1 General Description of Schizophrenia and Symptoms .......................... 1 1.2 Transmission of Neurotransmitters and Development of Schizophrenia ....................................................................................... 3 1.3 Pathways of Dopaminergic Neurotransmission .................................... 6 1.4 Phases of Schizophrenia and Therapeutic Goals ................................. 8 1.4.1 Therapy with Antipsychotics – Differentiation of schizophrenia from Psychosis ................................................... 10 1.4.2 The Use of Antipsychotics for the Treatment of Schizophrenia ............................................................................ 11 1.5 Indication for the Therapeutic Drug Monitoring of Antipsychotics ..................................................................................... 13 1.5.1 The AGNP Guidelines for the Application of TDM ..................... 14 1.5.2 Chemical Groups of Antipsychotic Drugs Analysed in this Thesis ................................................................................. 18 1.5.3 Determination of Serum Antipsychotic Concentrations .............. 20 1.5.4 Description of the Nine-Fold Table for the Interpretation of Antipsychotic TDM ........................................... 26 1.5.5 Serum Antipsychotic Concentration and Clinical Pharmacological Report ............................................................ 27 1.6 Different Analytical Methods ............................................................... 29 2 Aims and Objectives.............................................................. 32 Questions Raised ..................................................................... 34 3 Materials and Methods .......................................................... 35 3.1 Materials ............................................................................................. 35 3.1.1 Laboratory Instrumentation ........................................................ 35 3.1.2 Reagents and Chemicals .......................................................... 37 3.1.3 Human Serum for HPLC Analysis ............................................. 41 3.1.4 Study Antipsychotics ................................................................. 41 3.1.5 The Hospital and the Clinic Ward .............................................. 43 3.1.6 Materials for the Antipsychotics Data Analysis .......................... 44 3.2 Method ................................................................................................ 45 3.2.1 Current HPLC Method in TDM Laboratory Regensburg ............ 45 3.2.2 New Method Development by Automated Column Switching HPLC ......................................................................... 47 I 3.2.2.1 HPLC Separation Method for the Serum Determination of Antipsychotics ................................... 52 3.2.2.2 Validation of the Newly Developed HPLC Method ...................................................................... 54 3.2.2.3 Patients’ Test Samples and Measurment ..................... 59 3.2.3 TDM Routine Analysis with the Validated Method ..................... 59 3.2.3.1 Type of Study Patients ................................................. 59 3.2.3.2 TDM Request for the Study Substances ...................... 60 3.2.3.3 Laboratory Measurments and Data Analysis ............... 60 3.2.4 Clinical Application of Laboratory Values through TDM ............. 63 3.2.4.1 Therapeutic Drug Monitorin at the Clinic Ward ............ 63 3.2.4.2 Routine Clinic Ward Visitation ...................................... 64 3.2.4.3 Preparation of Clinic Ward Questionnaires .................. 65 4 Results .................................................................................... 66 4.1 Method Development .......................................................................... 66 4.1.1 Choice of Mobile Phase and Analytical Column ........................ 66 4.1.2 Comparison of TEMED and Dihydrogenphosphate Buffer Solutions ......................................................................... 67 4.1.3 Column Selection and Measurements with Buffer Mobile Phase ............................................................................. 69 4.1.4 Column Selection with TEMED Mobile Phase ........................... 73 4.1.5 Influence of Column Particle Sizes on the Measurement ............................................................................. 77 4.1.6 Summary of Results of the Column Selection ........................... 80 4.1.7 Isocratic Separation of Butyrophenones .................................... 86 4.1.7.1 Wavelengths and Retention Times .............................. 86 4.1.7.2 Influence of the Temperature ....................................... 87 4.1.7.3 Influence of the Flow Rate ........................................... 88 4.1.7.4 Influence of pH ............................................................. 88 4.1.8 Application of the Gradient HPLC Separation Method ............... 90 4.1.9 Validation of the Newly Developed Isocratic HPLC- Method ..................................................................................... 96 4.1.9.1 Calibration and the Calibration Curve .......................... 97 4.1.9.2 Substance Recovery .................................................... 97 4.1.9.3 Intraday Precision ........................................................ 98 4.1.9.4 Interday Precision ...................................................... 100 4.1.9.5 The Accuracy of the Method ...................................... 101 4.1.9.6 Lower Detection (LOD) and Quantification Limit (LOQ) ................................................................ 102 4.1.9.7 Long-term Stability ..................................................... 102 4.1.9.8 Freeze/ Thaw Stability ................................................ 104 4.1.9.9 Test of Robustness .................................................... 105 4.1.9.10 Selectivity ............................................................... 106 4.1.9.11 Routine Application of the Validated Method .......... 106 4.2 Application of the Developed Method for TDM ................................. 114 II 4.2.1 Demographic Data of Study Patients ....................................... 114 4.2.2 Doses and Drug Concentrations .............................................. 115 4.2.2.1 Dose- concentration relationship of measured MLP sample and the DRR ......................................... 116 4.2.2.2 Dose- Concentration Relationship of Measured BPD Sample and the DRR ........................ 117 4.2.2.3 Dose- Concentration Relationship for HLP ................ 119 4.2.2.4 Dose- Concentration Relationship of Measured BRP Samples and the DRR ...................... 123 4.2.2.5 Dose- concentration relationship of measured FLT samples and the DRR ......................................... 124 4.2.2.6 Dose- concentration relationship of measured ZLT samples and the DRR ......................................... 133 4.2.3 Results of Antipsychotic TDM Data Evaluation........................ 135 4.2.3.1 The Konbest Data of Melperone ................................ 135 4.2.3.2 The Konbest Data of Benperidol ................................ 136 4.2.3.3 The Konbest Data of Haloperidol ............................... 136 4.2.3.4 The Konbest Data of FLT and ZLT............................. 148 4.2.3.5 ABDA Haloperidol Data Evaluation ............................ 152 4.2.3.6 AGATE Melperone-Benperidol Data Evaluation .................................................................. 153 4.2.4 Influence of Smoking on HLP and FLT serum Concentration .......................................................................... 154 4.2.4.1 Influence of Smoking on Haloperidol Serum Concentration ............................................................. 154 4.2.4.2 Influence of Smoking on Flupentixol Serum Concentration ............................................................. 158 4.3 Application of TDM at the Clinic Ward .............................................