Gdańsk University of Technology Faculty of Chemistry Department of Analytical Chemistry Ph.D thesis THE CHARACTERIZATION OF ENANTIOMERIC COMPOSITION AND IMPURITY PROFILE OF METHAMPHETAMINE AND ITS CHLORO-INTERMEDIATES SYNTHESIZED BY EMDE METHOD mgr inż. Justyna Małgorzata Płotka Supervisors: Prof. Marek Biziuk Ph.D Calum Morrison Gdańsk 2014 I would like to express my sincere gratitude to my advisors, Professor MAREK BIZIUK and Doctor CALUM MORRISON, for the continuous support of my Ph.D study and research, for their patience, motivation, enthusiasm, and immense knowledge. I would like to gratefully and sincerely thank Professor JACEK NAMIEŚNIK for his scientific advice and knowledge and many insightful discussions and suggestions. I am very grateful to Professor VASIL SIMEONOV for cooperation and advice during the project. Thanks to all of my FRIENDS from Department of Analytical Chemistry for spiritual support and creating a pleasant working atmosphere. I would like to thank my loved ones, MUM, DAD, SISTER, BROTHER, SISTER-IN-LAW, AND MY NEPHEWS who have supported me throughout entire process, both by keeping me harmonious and helping me putting pieces together. I will be grateful forever for your love. I would like to thank my husband ŁUKASZ for being with me in good and bad times, for his love and friendship, for support and patience. 2 TABLE OF CONTENTS ABSTRACT 6 LIST OF ABBREVIATIONS AND ACRONYMS 7 INTRODUCTION 12 I THEORETICAL PART 14 1. Amphetamine-type compounds as psychoactive stimulants 14 1.1. Methylamphetamine – abused drug of the early 21st century 16 1.1.1. History 16 1.1.2. Characterization 18 1.1.3. Pharmacology 20 1.1.4. Methylamphetamine synthesis route 21 1.1.5. Importance of stereochemistry of the Emde route of 25 methylamphetamine synthesis 1.1.6. Methylamphetamine- necessary to control 27 2. Gas and Liquid Chromatography used for the enantioseparation of 28 methylamphetamine 2.1. Gas chromatography as a technique of choice for MAMP 29 enantioseparation 2.1.1. Indirect separation of MAMP diastereomers using CSs and achiral 30 stationary phase 2.1.2. Direct separation of MAMP using CSPs 33 2.1.2.1 Separation of MAMP enantiomers using non-chiral derivatizing 35 reagents and CSPs 2.2. Liquid chromatography as a technique of choice for MAMP 35 enantioseparation 2.2.1. Indirect separation of MAMP diastereomers using CSs and achiral 35 stationary phase 2.2.2. Direct separation of MAMP using CSPs 39 2.2.3. Active mobile phase- chiral derivatizing reagent as an additive 44 3. Other techniques used for chiral separation of MAMP 45 4. Chemometric as a useful tool in analytical chemistry 46 5. Summary 48 II THE PURPOSE OF THE RESEARCH 49 III EXPERIMENTAL PART 50 1. Standards, chemical reagents and materials 50 2. Equipment used 50 3 3. Analytical procedures 52 3.1. Synthesis of chloro analogs of MAMP 52 3.2. Recrystalization procedure 53 3.3. IR analysis 53 3.4. NMR analysis 53 3.5. Achiral GC-MS analyses of chloro-intermediates of MAMP 53 3.6. Preparation of stock solutions 54 3.7. Optimization of the derivatization procedures 54 3.8. Effectiveness of derivatization 56 3.9. Chemometric analysis of derivatization procedure 56 3.10. Chiral GC-MS analyses 56 3.11. Method validation 57 IV RESULTS AND DISCUSION 57 1. Synthesis of chloro intermediates of MAMP and investigation of the 57 stereochemical course of part I of the Emde method 1.1. Synthesis of CE and CPSEP enantiomers 57 1.2. Identification of final products. Investigation of the stereochemical course 58 of the I part of Emde synthesis 1.2.1. NMR analysis 58 1.2.1.1. Analysis of 1H NMR spectra 59 1.2.1.2. Analysis of 13C NMR and 13C NMR with DEPT spectra 60 1.2.1.3. Analysis of COSY and HSQCED spectra 61 1.2.1.4. Analysis of 1D SELNOESY NMR spectra 67 1.2.2. Achiral GC-MS analyses of chloro-intermediates of MAMP 69 1.3. Assessment of the greenness of the synthesis 71 1.3.1. Green metrics 71 1.3.2. EcoScale 73 2. Enantioseparation of methylamphetamine, its chloro intermediates and 74 impurities 2.1. Selection of the CSP and initial chromatography conditions 74 2.2. Optimization of derivatization procedure 75 2.2.1. Chemometric interpretation of RRFs values calculated for TFAA 78 derivatization of analytes 2.2.2. Chemometric interpretation of RRFs values calculated for PFPA 80 derivatization of analytes 2.2.3. Chemometric interpretation of RRFs values calculated for CDFA 82 4 derivatization of analytes 2.2.4. General conclusions of chemometric data interpretation 85 2.3. Peak identification 86 2.4. Development of temperature-programmed chromatographic conditions 87 2.5. Validation of optimized chromatographic conditions 89 2.5.1. The uncertainty of measurement 91 2.6. Application of developed method to analyze MAMP samples 92 2.6.1. Identification of impurities of MAMP classified as synthesized by 96 Emde method V CONCLUSIONS AND SUMMARY 100 VI FUTURE STUDY 103 VII LITERATURE 104 VIII STRESZCZENIE 112 IX SCIENTIFIC ACHIEVEMENTS 114 5 ABSTRACT Because the number of people who abuse methylamphetamine (MAMP) is increasing every year, it has become a global threat. Background knowledge about MAMP is significant for many reasons with the most important issues being associated with human health in terms of prediction and protection applied to reduction in drug addiction. The project aims to develop and optimize the new analytical methodology in order to create a comprehensive characterization of MAMP in terms of impurity and chirality profile, and also enantiomeric characterization of chloro-intermediates of MAMP that are not commercially available, and necessary as reference materials for the further analysis. The configuration of chloro-intermediates were determined by 1D and 2D NMR analysis in combination with achiral GC-MS analysis. It was shown that chlorination of the ephedrine (EP)/pseudoephedrine (PSEP) compounds occurs via inversion and retention of configuration around the α carbon and mixture of diastereoisomers (chloroephedrine and chloropseudoephedrine) were formed, with the ratio of the resulting compounds dependent on the precursors used. A GC-MS method using a γ-cyclodextrin chiral stationary phase was developed and optimized for the simultaneous enantiomeric separations of MAMP and its common precursors, EP and PSEP, as well as its chloro-intermediates formed during MAMP synthesis by Emde method, after derivatization with trifluoroacetic anhydride. Cluster analysis and principal components analysis were used as multivariate statistical methods in the interpretation of data obtained from the derivatization process. The new methodology in comparison with other existing procedures is innovative, because it is more economical and ecological (the chiral separation of analytes of interest is performed in a single analysis run in short time, what has not been presented in any literature data). Moreover, the new methodology is easy to apply and rapid. Methylamphetamine samples were successfully analyzed using the proposed method. Several new impurities were detected in this study in MAMP classified as manufactured by Emde. The developed analytical procedure and characterization of CEP analogs synthesized by the Emde method leads to an indication of the method of synthesis as well as which precursors were used for MAMP manufacture indicating that this research would provide valuable information to law enforcement agencies regarding the provenance of MAMP seizures. 6 LIST OF ABBREVIATIONS AND ACRONYMS 1 2 3 Abbreviations/ English term Polish term acronyms 1D One dimensional Jednowymiarowa 2D Two dimensional Dwuwymiarowa α Separation factors Współczynnik rozdzielenia Ac.B Acetate buffer Bufor octanu ADHD Attention Deficit Hyperactivity Zespół nadpobudliwości Disorder psychoruchowej z deficytem uwagi AE Atom Economy Ekonomia atomów AE,exp Experiment Atom Economy Ekonomia atomów, wartość doświadczalna AE’ Atom Efficiency Efektywność atomowa AITC 2,3,4-tri-O-acetyl-α-D- Izotiocyjanian 2,3,4-tri-O-acetylo-α- arabinopyranosyl isothiocyanate D-arabinopiranozylo AM Amphetamine Amfetamina ATS Amphetamine – type stimulant Fenetylaminy psychoaktywne B Blood Krew CA Cluster analysis Analiza skupień CAN Acetonitrile Acetonitryl CB Carbethoxyhexafluorobutyryl Chlorek chloride karboetoksyheksafluorobutyrylu CD Cyclodextrin Cyklodekstryny CDFA Chlorodifluoroacetic anhydride Bezwodnik kwasu chlorodifluorooctowego CE Capillary electrophoresis Elektroforeza kapilarna CE- β -CD Carboxyethyl- β -cyclodextrin Karboksyetylo-β-cyklodekstryna CEP Chloroephedrine Chloro efedryna Chiral-CBH Chiral protein phase Chiralna faza proteinowa CM- β -CD Carboxymethyl- β -cyclodextrin Karboksymetylo-β-cyklodekstryna CNS Central Nervous System Centralny układ nerwowy COSY Correlation spectroscopy Spektroskopia korelacyjna CPSEP chloropseudoephedrine Chloropseudoefedryna 7 LIST OF ABBREVIATIONS AND ACRONYMS – CONT. 1 2 3 CS Chiral selector Selektor chiralny CSP Chiral stationary phase Chiralna faza stacjonarna CV Coefficient of variation Współczynnik zmienności DA Discriminant analysis Analiza dyskryminacyjna DAD Diode-array detector Detektor z matrycą diodową DEPT Distortionless enhancement by Bezzakłóceniowe wzmocnienie polarization transfer sygnału (jąder niskoczułych) poprzez transfer polaryzacji d-MTPAA D-α-methoxy-α- Kwas d-α-metoksy-α- (trifluoromethyl)phenyl acetic acid (trifluorometylo) fenylooctowy DOM 2,5- dimethoxy-4- 2,5 - dimetoksy-4- methylamphetamine metyloamfetamina DR Derivatization reagent Odczynnik derywatyzujący E -factor Environmental factor Czynnik Środowiskowy EA