institlüld ictterkcnny TeicneoUiochti institute lyit (.«Ulf C*»n«lon of Technology High performance liquid chromatography method development and chemometric analysis of ecstasy and cocaine Kim McFadden Supervisors Dr. B. F. Carney, Letterkenny Institute of Technology Dr. D. O'Driscoll, Forensic Science Laboratory, Dublin Submitted to Higher bducation and Training Awards Council (HETAC) in fulfilment for the requirements for the degree of Doctor of Philosophy HPLC method development & chemomelric analysis of ecstasy & cocaine Declaration I hereby declare that the work herein, submitted for Ph.D. in Analytical Science at Letterkenny Institute of Technology, is the result of my own investigation, except where reference is made to published literature. I also certify that the material submitted in this thesis has not been previously submitted for any other qualification. Kim McFadden 1 HPLC method development & chemometric analysis of ecstasy & cocaine Table of Contents Declaration 1 Abstract 5 List of Abbreviations 7 List of Figures 10 List of Tables 14 List of Presentations and Publications 16 Acknowledgments 18 Chapter 1: Literature review. 19 1.1. Introduction. 20 1.2. Drugs of abuse. 21 1.2.1. Narcotics. 22 1.2.2. Depressants. 24 1.2.3. Stimulants. 24 1.2.4. Hallucinogens. 25 1.2.5. Ecstasy. 26 1.2.6. Cocaine. 31 1.3. Current analytical techniques for forensic drug analysis. 34 1.3.1. Spectrometric methods. 34 1.3.2. Chromatographic methods. 36 1.4. Fundamentals of High Performance Liquid Chromatography. 40 1.4.1. Chromatographic interactions. 40 1.4.2. Mobile phase. 41 1.4.3. The column. 42 1.4.4. Applications of monolithic columns in the area of forensic drug analysis. 45 1.5. Chemometrics and statistical profiling. 50 1.5.1. Experimental design. 50 1.5.2. Drug classification. 52 1.5.3 Application of chemometrics to illicit drug profiling. 57 1.6. Legislation and control efforts in Ireland. 61 1.7. Aims and objectives. 63 2 HPLC method development & chemometric analysis of ecstasy & cocaine Chapter 2: Materials and methods. 66 2.1. General materials and instrumentation. 67 2.1.1. Chemicals and reagents. 67 2.1.2. Liquid chromatography system. 67 2.1.3. Atomic absorption spectrometry. 68 2.1.4. Miscellaneous instruments. 68 2.1.5. Computer software. 68 2.2. Univariate HPLC method development for the analysis of ecstasy. 69 2.2.1. Ecstasy standards and sample preparation. 69 2.2.2. Univariate method development for analysis of ecstasy. 71 2.2.3. Validation of univariate method-developed. 73 2.3. Multivariate HPLC method development for the analysis of cocaine. 74 2.3.1. Cocaine standards and sample preparation. 74 2.3.2. Multivariate method development for analysis of cocaine. 75 2.3.3. Validation of multivariate method developed. 79 2.4. Sugar analysis. 79 2.5. Metal analysis. 79 2.6. Data analysis and chemometric procedures. 81 2.6.1. Ecstasy data and pre-treatments. 81 2.6.2. Ecstasy data pattern recognition. 82 Chapter 3: HPLC method development. 85 3.1. Univariate HPLC method development for the analysis of ecstasy. 86 3.1.1. Chromatographic factors. 86 3.1.2. Selection of optimal HPLC conditions for ecstasy sample analysis. 92 3.1.3. Comparison of particulate and monolithic columns. 98 3.1.4. Validation of univariate method developed. 101 3.1.5. Conclusions. 105 3.2. Multivariate HPLC method development for the analysis of cocaine. 106 3.2.1. Multivariate method development. 106 3.2.2. Response surfaces. 113 3 HPLC method development & chemometric analysis of ecstasy & cocaine 3.2.3. Validation of multivariate method developed. 116 3.2.4. Conclusions. 118 Chapter 4: Physical and chemical analysis of illicit drug samples. 119 4.1. Ecstasy analysis. 120 4.1.1. Physical profile of ecstasy. 120 4.1.2. Chemical analysis of ecstasy. 124 4.1.3. General profiling of ecstasy. 128 4.1.4. Discussion. 132 4.2. Chemical analysis of cocaine. 136 4.2.1. Discussion. 139 4.3. Conclusions. 141 Chapter 5: Chemometrics and statistical profiling of ecstasy tablets. 143 5.1. Introduction. 144 5.2. Ecstasy data pre-treatment methods. 144 5.3. Ecstasy data pattern recognition methods. 149 5.3.1. Principal component analysis. 149 5.3.2. Hierarchal cluster analysis. 153 5.3.3. Pearson’s correlation coefficient. 159 5.4. Supervised pattern recognition methods. 165 5.5. Conclusions. 171 Chapter 6: Conclusions and future work. 173 6.1. Method development. 174 6.2. Analysis of illicit drug samples. 175 6.3. Chemometrics and statistical profiling of ecstasy samples. 176 6.4. Establishing a profiling protocol in a forensic laboratory. 177 6.5. Future studies on the current major drugs in Ireland. 178 References 181 Appendices 209 4 HPLC method development & chemometric analysis of ecstasy & cocaine Abstract Consumption of illegal drugs of abuse remains a major social issue aligned with a global law-enforcement priority. Forensic analysts are faced with the challenge of continually developing sophisticated methods of analysis to combat the increasing variability that occurs in illicit drug samples. Research work for this thesis has focused on the development of High Performance Liquid Chromatography (HPLC) methods for the analysis of major drug constituents associated with ecstasy and cocaine illicit drug samples. Emphasis has been placed on method development with strategies of univariate or multivariate experimental approaches used in the selection and optimisation of procedures. Considerations with regard to the choice of chromatographic factors, solutes under investigation and the provision of quality assurance data throughout the research work have been the main criteria in methods developed. Two HPLC methods were developed to qualitatively and quantitatively assay for the major drug components and analogue derivatives found in ecstasy and cocaine. Methods developed have undergone validation studies including intra- and inter- reproducibility, accuracy, and linearity of calibration, limit of detection (LOD) and limit of quantification (LOQ) and the use of internal standards. Applications of methods to ecstasy and cocaine samples seized in Ireland ensured their suitability for routine analysis of illicit drug samples. As part of this study, chemical profiling of 183 ecstasy tablets seized in Ireland during 2002-2004 were recorded as discrete data sets. Chemical data sets include both the quantification and occurrence in individual tablets of the major amphetamine components (i.e. MDA, MDMA, MDEA, MBDB methamphetamine and amphetamine), adulterant components (i.e. caffeine, phenacetin, acetaminophen and acetylsalicylic acid), excipients components (i.e. sucrose, glucose, lactose, fructose, mannitol, sorbitol and inositol) and inorganic components (i.e. Al, Zn, Fe, Mg, Ca, Cr, Pb, Na and K ). Chemometrics, including unsupervised methods of principal component analysis (PCA), hierarchical cluster analysis (HCA) and Pearson’s correlation coefficient, as well as supervised methods of linear discriminant analysis (LDA) and artificial neural networks (ANN) was applied to the chemical data sets to demonstrate the ability of the statistical approach to linking sample seizures. HCA and ANN were the numerical methods that most efficiently distinguished between 5 HPLC method development & chemometric analysis of ecstasy & cocaine linked and unlinked seizures. Eleven groups were identified from the chemical data sets with group classification dependant on the main amphetamine, adulterant and excipient components present. The benefits from this study can provide strategic intelligence and an understanding of the operational level on the Irish ecstasy market and help evaluate the changing profile or dynamics associated with this illegal market supply. 6 HPLC method development & chemometric analysis of ecstasy & cocaine List of Abbreviations 2C-B 4-bromo-2, 5-dimethoxyphenethylamine A Amphetamine AA Atomic absorption ACN Acetonitrile ADRU Alcohol and drug research unit ANN Artificial neural networks ASA Acetylsalicylic acid ATS Amphetamine - type - stimulants BZP 1 - Benzylpiperazine CE Capillary electrophoresis CV Coefficient of variation DAD Diode array detection DAP Drug awareness programmme DEA Drug enforcement agency DIMS Drugs information monitoring system DOB 4-bromo-2, 5-dimethoxyamphetamine DOM 4-Methyl-2, 5-dimethoxyamphetamine ECD Electron capture detector ELSD Evaporative light scattering detection EMCDDA European monitoring centre for drugs and drug addiction ENFS1 European network of forensic science institutes FID Flame ionization detector GBL Gamma - butyrolactone GC Gas chromatography GC-MS Gas chromatography-mass spectrometry GHB Gamma hyrdoxybutyric acid HCA Hierarchal cluster analysis HPLC High performance liquid chromatography HETP Height equivalent to theoretical plate HRB Health research board ICH International conference on harmonisation IEC Ion-exchange chromatography 7 HPLC method development & chemometric analysis of ecstasy & cocaine INCB International narcotics control board INEF Irish needle exchange forum IR Infra red k’ Retention factor k’ave? Average retention factor KNN K-nearest neighbour LC Liquid chromatography LC-MS Liquid chromatography-mass spectrometry LDA Linear discriminant analysis LLE Liquid-liquid extraction LOD Limit of detection LOQ Limit of quantification LSD Lysergic acid diethylamide MAOC-N Maritime analysis and operations centre - narcotics MDA 3, 4-Methylenedioxyamphetamine MDEA 3, 4-Methylenedioxy-N-ethylamphetamine MDMA 3,4-Methylenedioxymethamphetamine MBDB N-methyl-1 -1 -(1, 3-benzdioxoI-5-yl)-2-butanamine