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MSc ANALYTICAL TOXICOLOGY (2010 -11) The below gives a detailed description of each of the four taught modules and the project: Semester 1 (September to December) • • Module 1 - Principles of Analytical Toxicology Module 2 - Biochemical Toxicology Semester 2 (January to April) • Module 3 - Clinical and Forensic Toxicology • Module 4 - Advanced Analytical Toxicology The project (module 5) takes place following semester 2, from May to the beginning of September. Module 1 PRINCIPLES OF ANALYTICAL TOXICOLOGY Module Organisers: Dr Andrew Kicman Analytical Toxicology The aim of this module is to introduce the student to the major chromatographic and spectroscopic techniques used in analytical toxicology, the instrumentation used and the scientific principles underlying the instrumentation, and its use in sample analysis. At the end of the module the student should be able to: • • • • • Describe the chromatographic and spectroscopic instrumentation used and explain how it operates to enable analysis Explain the scientific principles underlying each chromatographic and spectroscopic technique Compare and contrast the various techniques giving advantages and disadvantages of each Decide which technique(s) would be most appropriate for the analysis of a given sample and explain the reasons for your decision Give examples of the uses for each technique in analytical science, with particular emphasis on analytical toxicology. Introduction to analytical toxicology • Introduction to course structure and content • Integration of analytical toxicology as covered by the four taught modules and the research project • Commonly applied techniques, past, present and future • Clinical and forensic perspectives Immunochemical techniques Immunoassay • Immunoglobulins • Competitive and non-competitive immunoassays Chromatography Introduction to separation science – • The role of analytical chemistry in analytical science • Analytical techniques used in analytical science • The elements of analysis • The pros and cons of the various techniques • Maximising information and evidential value What is separation science? –Basis of separation • Terminology • Definition of chromatography • Chromatographic theory The mechanisms of separation –Introducing the major separation techniques used in analytical chemistry • Exploring the basis of separation - intermolecular interactions (hydrogen bonding, van der Waals forces, ionic interactions) High Performance Liquid Chromatography (HPLC) –Introducing instrumentation • Normal phase HPLC • Reverse phase HPLC • Mechanisms of separation operating in HPLC • Stationary and mobile phases • Detecting the sample • Uses of HPLC Gas Chromatography (GC) – Instrumentation • Carrier gases • Injectors • Chromatographic columns and stationary phases • Use of temperature • Detectors • How to manipulate separation • Uses of GC (including headspace analysis) Chirality • Separation of enantiomers • Direct and indirect methods Sample Handling –Solvent and solid state extraction • Manipulation of pH to aid separation • Analysis of conjugates Spectroscopy and Spectrometry Introduction to spectroscopy – • The five properties of light • Spectral regions • Spectroscopic techniques • Energy levels Electronic Spectroscopy: Absorption – • UV/Visible absorption spectra • Chromophores • Absorbance • Beer’s Law and concentration • The measurement of spectra and application of UV/Visible spectroscopy • Comparison of instrument types: single beam, double beam, Diode array, Fourier-Transform Mass Spectrometry – • The technique • Instrumentation • Applications • Hyphenated Mass Spectrometric techniques and applications (LC-MS, GC-MS) • Application of techniques • Substance identification • Quantification Additional Spectrophotometry/spectrometry • Atomic Absorption and Emission Spectroscopy • Inductively Coupled Plasma Mass Spectrometry • Optical Emission Spectroscopy Mathematics for Analysts • • • Mathematical tools for analysts (chemometrics) Use of concentration units Chemical calculations for practical analytical work Reporting of results, use of concentration units and reference ranges Quality Assurance • • • • • • • • • Traceability Reference materials and drug standards. Quality Control procedures Quality Assurance Method validation Uncertainty budget – fit for purpose International Committee on Harmonisation (ICH), uncertainty and guidelines (e.g. ISO 17025) Laboratory accreditation (e.g. UKAS ) Specimen Preparation and Extraction • • Specimen collection, preservation and storage, chain of custody and documentation of specimens Specimen preparation and extraction techniques including how to develop methods, importance of pKa to liquid and solid phase extraction efficiency Practicals, Workshops and Tutorials Chemistry catch-up • Polarity, lipophilicity, pKa • • • Solubility Intermolecular interactions Functional groups and chemical nomenclature Calculations for analysts (Chemometrics) • Worked examples of chemical calculations • Common errors in calculations and their consequences Laboratory skills • Health and safety in the laboratory • Laboratory notebook keeping • Weighing – top pan and analytical balances • Correct use and care of pipettes. • Pipette skills exercise – simple quantification (mass and volume) • Volumetric work • Calibrating volumetric apparatus • Preparing standard solutions and determining the concentration of an unknown • Buffer preparation – weighing, use of pH meters, concentrations and dilutions Spot tests / Colorimetric • Analysis of a common drug • Determination of concentration using UV fixed wavelength Gas Chromatography (Direct injection of alcohol) • Analysis of a chemical mixture • Identification of components using retention time • Evaluation of data and preparation of reports Gas Chromatography-Mass Spectrometry: • Analysis of a chemical mixture • Identification of components using MS library searching • Evaluation of data and preparation of reports GC-MS data handling workshop - Matching MS spectra from samples against library spectra • Using MS spectra to select ions for selected ion monitoring (SIM) • Quantification using SIM data including QA High Performance Liquid Chromatography – model analytes, e.g. anticonvulsants • The elements of the instrument • Setting up methods • Sample injection • Determining plate number and resolution • Data analysis and interpretation High Performance Liquid Chromatography-MS o Analysis of a simple drug mixtures, e.g. drugs of abuse Spectroscopy – • UV-Vis: • • • • Instrument calibration – QA & QC Photometric calibration Resolution Determining concentration using UV-vis Specimen Preparation And Extraction • • Specimen collection, preservation and storage, chain of custody and documentation of specimens Specimen preparation and extraction techniques including how to develop methods, importance of pKa to liquid and solid phase extraction efficiency Introduction to the Principles of Qualitative and Quantitative Analysis o Substance identification o Quantification with and without use of internal standard Tutorials to support practicals MODULE 2 BIOCHEMICAL TOXICOLOGY Module Organisers: Dr Andrew Kicman Biochemical Toxicology The aims of this module are to provide an understanding of the basic principles of drug absorption, distribution, metabolism and excretion and elementary pharmacokinetics. The basic concepts, mechanisms and principles of toxicology and pharmacogenetics in relation to drug development, safety evaluation, requirements for toxicity testing and risk assessment, adverse drug reactions and toxicity. At the end of the module the student should be able to: • • • • • • • • • • • Understand the absorption, distribution and excretion of xenobiotics Identify the potential sites, reactions and factors which influence drug metabolism Appreciate the significance of drug metabolism on biological activity Understand the principles of pharmacokinetics and be able to define and calculate the commonly used pharmacokinetic parameters Have an understanding of the basic concepts and principles of toxicology Have an appreciation of the mechanisms underlying the toxicity of drugs and other xenobiotics Have a theoretical basis for understanding the role of drug metabolism in relation to toxicity Have an appreciation of risk assessment and the role of toxicological information Be able to interpret and integrate toxicological information Have gained problem solving abilities in relation to toxicology Be able to apply these skills in the assessment of actual and potential drug toxicity Chemistry for biologists / Biology for chemists Anatomy and physiology • Basic anatomy and physiology including major organ systems (eg brain, kidney, liver, lungs and GI tract) • Basic Introduction to clinical biochemistry Including blood, plasma, urine, electrolytes, fluid balance, proteins, enzymes, hormones • Medical terminology and acronyms Introduction to pharmacological classification and drug action • • • • Introduction to clinical pharmacology Drug classification and control Mechanisms of drug action Acute and chronic drug toxicity and potential for drug abuse Introduction to drug distribution • Basic definitions of absorption, distribution and elimination. • Drug transport in the blood, protein binding. • Transfer within body fluid compartments, drug tissue localization. • Blood-brain barrier, placental drug transfer • Renal and biliary excretion. Biological fate of xenobiotics • Introduction to basic concepts in xenobiotic biotransformation Functionalisation reactions • Oxidations • Reductions • Hydrolysis as Phase I or functionalisation reactions Conjugation reactions • Glucuronidation • Sulphation • N-acetylation • Glutathione conjugation • Methylation • Amino acid conjugation Factors affecting drug metabolism • Physiological and environmental factors affecting biotransformation Pharmacological significance of xenobiotic biotransformation • Effect of disease and genetic polymorphism on human xenobiotic biotransformation Introduction to Pharmacokinetics • Simplifying assumptions • The concept of compartments • Intravenous drug administration Definitions of pharmacokinetic parameters • Elimination rate constant half-life • Volume of distribution • Clearance Intravenous drug administration • Compartmental models Oral drug administration • Determination of absorption rate • Bioavailability • Use of AUC to determine bioavailability • Urinary drug excretion Multiple dosing • Drug accumulation • • Steady-state Dose regimens Basic principles of toxicology • Introduction to the science of toxicology and its importance • Dose response relationship and what may be derived from it Types of toxic response • The adverse responses to chemical and drug exposure which may be manifested by mammalian organisms such as humans Reactive intermediates • The types of chemically reactive intermediates that may be produced by metabolism of a xenobiotic and the consequences of this Genotoxicity • Mechanisms of DNA modification by xenobiotics • Chemical carcinogenesis N-Oxidation and drug toxicity • Mechanisms of arylamine mediated hepatic and bladder carcinogenesis • Induction of ferrihaemoglobinemia by arylhydroxylamines Sulphur toxins • Toxic sulphur conjugates derived from glutathione: MDMA and dopamine Target organ toxicity • Liver • Kidney • Lung • CNS - The reasons that specific organs are targeted are discussed Toxicokinetics • Discussion of the relationship between pharmacokinetics and the toxicity of drugs and chemicals Specific examples of drug toxicity • Particular drugs will be studied in detail in relation to their toxicities and mechanisms underlying them MODULE 3 CLINICAL AND FORENSIC TOXICOLOGY Module Organisers: Professor Robin Braithwaite The aim of this module is to provide an in depth understanding of common drugs and chemical poisons relevant to both clinical and forensic toxicology. A strong emphasis will be placed on the application of modern analytical methods to the measurement of drugs, chemical poisons and their metabolites in biological fluids in both a clinical and medicolegal context. Although this is a standalone module it is designed to be integrated with Module 4 (Advanced Analytical Toxicology), which has a high laboratory practical element. The emphasis of this module is to integrate aspects of drug pharmacology and metabolism with extensive use of both clinical and forensic case reports. The module will include assessed coursework, data evaluation, report preparation and presentation, tutorials and workshops. At the end of the module the student should be able to: • • • • • • • • Understand the clinical and forensic issues associated with the laboratory investigation of acute and chronic poisoning, including drug and substance misuse Understand the main clinical and biochemical features associated with acute and chronic poisoning due to the ingestion of important drug and chemical poisons Understand the pharmacokinetics and metabolism of important drug and chemical poisons and relationship to acute and chronic toxicity, including sudden or unexpected deaths Critically review and evaluate the significance of analytical data from human poisoning cases Present the significance of analytical findings accurately and concisely, including court reporting Demonstrate an understanding of the various analytical approaches to the detection and quantitation of drugs and metabolites in biological fluids and tissues Understand the role of both laboratory testing and point of care testing in the management and detection of drug misuse Understand the principles and practice of therapeutic drug monitoring Drug misuse, poisoning and its epidemiology • The use and abuse of substances used as both medicines and poisons • International epidemiology of poisoning and its medical definitions and current trends • Available information sources, for example Toxbase, poisons information services • Current issues including chemical poisons and counterfeit pharmaceuticals Clinical management of poisoning • Signs and symptoms of poisoning • Antidotes, enhanced elimination techniques • Clinical care of poisoned patients Alcohol (ethanol) • The clinical and forensic toxicology of alcohol, its abuse and consequences • Measurement of alcohol in body fluids and it relationship to effects & toxicity • Pharmacokinetics and metabolism of alcohol • Acute and chronic alcohol toxicity and suspected alcohol related deaths Methanol and Glycols • The clinical and forensic toxicology of methyl and other alcohols including ethylene, propylene and diethylene glycol • Laboratory techniques for the measurement of alcohols and glycols in body fluids • Pharmacokinetics and metabolism of methanol and ethylene glycol • Acute and chronic toxicity and fatalities Amphetamines • The clinical and forensic toxicology of amphetamine and closely related drugs including methamphetamine, and MDMA (ecstasy) • Laboratory techniques used in the investigation of suspected poisoning or abuse • The pharmacokinetics and metabolism of amphetamines • Acute and chronic toxicity and fatalities Cannabis and Cannabinoids • The clinical and forensic toxicology of cannabis • • • Cocaine • • • • Laboratory techniques used in the investigation of suspected cannabis abuse and intoxication The pharmacokinetics and metabolism of cannabinoids Acute and chronic toxicity The clinical and forensic toxicology of cocaine Laboratory techniques used in the investigation of suspected cocaine abuse and intoxication The pharmacokinetics and metabolism of cocaine Acute and chronic toxicity and fatalities Opiates • The clinical and forensic toxicology of opiates including morphine , heroin , codeine and dihydrocodeine • Laboratory techniques used in the investigation of suspected opiate abuse and overdosage • The pharmacokinetics and metabolism of opiates • Acute and chronic toxicity and fatalities Opioids • The clinical and forensic toxicology of important opioid drugs including methadone, dextropropoxyphene and buprenorphine • Laboratory techniques used in the investigation of suspected opioid abuse and overdosage • The pharmacokinetics and metabolism of opioids • Acute and chronic toxicity and fatalities Newer drugs: GHB, Ketamine and Piperazines • The clinical and forensic toxicology of GHB, ketamine and piperazines • Laboratory techniques used in the investigation of suspected abuse and overdosage • The pharmacokinetics and metabolism of GHB, ketamine and piperazines • Acute and chronic toxicity and fatalities Benzodiazepines • The clinical and forensic Toxicology of benzodiazepines and related hypnotic drugs such as zopiclone, zolpidem and related drugs. • Laboratory techniques for the detection and measurement of these drugs in biological fluids in the investigation of suspected abuse and overdose. • The pharmacokinetics and metabolism of this group of drugs and relationship to acute and chronic toxicity and suspected fatalities will be reviewed Psychotropic drugs • The clinical and forensic toxicology of major groups of psychotropic drugs such as the antidepressants and antipsychotics • Laboratory techniques for the detection and measurement of these drugs in biological fluids in the investigation of suspected poisoning and overdosage • The pharmacokinetics and metabolism of important drugs and relationship to acute and chronic toxicity and suspected fatalities will be reviewed Pesticides: OP’s , Paraquat , Phosphides , Strychnine • The clinical and forensic toxicology of important pesticides • Laboratory techniques for detection and measurement of these agents in biological fluids in the investigation of suspected poisoning • The pharmacokinetics and metabolism of these agents and relationship to acute and chronic toxicity and suspected fatalities will be reviewed Non opioid analgesics • The clinical and forensic toxicology of important non-opioid analgesics such as paracetamol and aspirin • Laboratory techniques for the detection and measurement of these drugs and their metabolites in biological fluids in the investigation of suspected poisoning • The pharmacokinetics and metabolism of these drugs and relationship to acute and chronic toxicity and suspected fatalities will be reviewed Cardiovascular agents • The clinical and forensic toxicology of important cardiovascular agents, including beta blockers, antiarrhythmic agents, calcium antagonists and digoxin • Laboratory techniques for the detection and measurement of various agents in biological fluids • The pharmacokinetics and metabolism of cardiovascular agents and relationship to acute and chronic toxicity including fatalities will be reviewed Metallic poisons • The clinical and forensic toxicology of important metals, including arsenic, lead, mercury and thallium • Laboratory techniques for the detection and measurement of heavy metals in biological fluids and tissues • The pharmacokinetics and metabolism of important heavy metals and relationship to acute and chronic toxicity, including fatalities will be reviewed Therapeutic Drug Monitoring • Anticonvulsants • Lithium • Cardiovascular agents • Cancer therapy Fire deaths: Carbon monoxide and Cyanide • The clinical and forensic toxicology of carbon monoxide and cyanide • Laboratory techniques for the detection and measurement of carbon monoxide and cyanide in biological fluids in the investigation of suspected poisoning • The pharmacokinetics and metabolism of these agents and relationship to acute and chronic toxicity and suspected fatalities will be reviewed Volatile substance abuse • The clinical and forensic toxicology of volatile substance abuse. Laboratory techniques for the identification and measurement of common volatile substances in biological fluids and tissues • The pharmacokinetics and metabolism of volatile substances and relationship to acute and chronic toxicity, including fatalities will be reviewed Drugs in Sport • The toxicology of anabolic steroids, protein hormones (human growth hormone, insulin and erythropoietin) and other drugs used by bodybuilders and athletes. • Laboratory techniques employed for the detection of doping, as developed by laboratories accredited by the World Anti-doping Agency Poisonous plants and herbal medicines • The clinical and forensic toxicology of major poisonous plants and common herbal medicines • Laboratory and other techniques for the identification and analysis of plant material including the analysis of biological fluids Role of laboratory investigations • • • The role of the laboratory investigations in the diagnosis and management of suspected poisoning The role of the laboratory in the investigation of sudden or unexpected deaths Best practice for the provision of laboratory investigations including the techniques used and their application to patient care Legislation and medico-legal aspects of drug abuse and poisoning • International conventions on drugs and poisons • Role of the coroner and medical examiner • Role of the toxicologist in court • Preparation of legal reports and oral presentation Laboratory visit To be arranged. Workshops Investigation of drug misuse - analytical, clinical, occupational and forensic aspects . • Introduction to trends in drug misuse • Specimen collection , adulteration and chain of custody • Analytical techniques for screening and confirmation Post - mortem Toxicology • The legal system and introduction to the role of the coroner /medical examiner, coroner’s officers and police. • The post-mortem examination , post mortem changes and physiology and collection of specimens . The role of the pathologist • Laboratory investigation of unexpected or sudden death – What to look for , Selection of analytical techniques, Choice of specimens and Interpretation of results .The role of the toxicologist . • Problems and pitfalls in the interpretation of post mortem findings for drugs and alcohol e.g. putrefaction , post – mortem redistribution and exhumation - some illustrative cases . • Investigation of heroin related deaths, mechanisms of death and current clinical and forensic issues • Use of hair analysis in understanding historical drug use in the deceased . Forensic Evidence • Writing court reports and presentation of evidence in court. • Exercise based on a real case . MODULE 4 ADVANCED ANALYTICAL TOXICOLOGY Module Organisers: Dr Andrew Kicman & Dr Mark Parkin The aim of Module 4 is to provide the student with an understanding of sample handling, immunoassay, on-site testing for drugs and alcohol and mass spectrometry. At the end of the module the student should be able to: • • • • • • • • • Describe the principles and practice of sample handling Describe the principles of immunoassay, the importance of specificity and know how to interpret the data Compare the advantages to the disadvantages of on-site drug testing Describe the mass spectrometric instrumentation used and explain how it operates to enable analysis Compare and contrast the various ionisation techniques giving advantages and disadvantages of each technique Compare and contrast the various mass analysers giving advantages and disadvantages of each technique Explain the scientific principles underlying the technique Decide which technique(s) would be most appropriate for the analysis of a given chemical/biological sample and explain the reasons for your decision Give examples of the uses for each technique in analytical chemistry, with particular emphasis on analytical science. Advanced sample handling • Consideration of matrices (hair, oral fluid, blood, urine, vitreous humor) • Solid Phase Extraction (reverse phase, normal phase, ion exchange, mixed-mode) • Solid Phase Microextraction • Critical Evaluation of Published Methods • Dilute and inject – “dilute and shoot” (for mass spectrometry) Immunoassay • Heterogenous and homogenous assays • Non-isotopic versus isotopic assays • Immunoglobulin Affinity, including Scatchard Plot • Antibody Titre • Specificity • Data Analysis On-site testing for drugs • Near patient testing devices; roadside testing • Technology • Specificity • Matrices (urine versus saliva); breath alcohol • Advantages and disadvantages • Future role ADVANCED MASS SPECTROMETRY Ionisation techniques in practice • Electron ionisation • Chemical ionisation • Atmospheric pressure ionisation • Atmospheric pressure chemical ionisation (APCI) • Electrospray ionisation (ESI) • Desorption electrospray ionisation (DESI) and Direct Analysis in Real Time (DART) • Inductively coupled plasma resonance MS • Collision cell • Ionisation and hyphenated techniques Mass analysers • Linear quadrupoles • 3D ion traps • 2D ion traps • Magnetic sector instruments (isotope ratio mass spectrometry) • Time Of Flight (TOF) including reflectron and delayed extraction MS in Analytical Toxicology • Chromatography and mass spectrometry refresher • Gas Chromatography-Mass Spectrometry (GC-MS) • Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) • Liquid Chromatography-Time Of Flight Mass Spectrometry (LC-TOFMS) • Inductively Coupled Plasma Mass Spectrometry (ICP-MS) • Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) • Direct Analysis in Real Time (DART) n MS techniques in practice • Selected reaction monitoring • Product ion scanning • Precursor ion scanning • Neutral loss scanning • Effect of time domain & energies • Collision Activated Dissociation (CAD) • Software Interpretation of Mass Spectra • Isotope Abundance • Accurate Mass And Elemental Composition` • Fragmentation Pathways Method Development and Validation • Infusion • Chromatography • Instrument Tuning • Matrix Effects • Identification Criteria for Confirmatory Purposes • Quantification Large Molecule Mass Spectrometry • Protein identification • Analysis of intact and digested proteins • Peptide sequencing • Peptide mass mapping • Post-translational modifications • Examples, e.g. Insulin Practicals and Workshops In these practical classes and workshops, you will have the opportunity to gain hands on in order to learn the key elements of sample extraction, immunoassay, on-site testing and mass spectrometry. These will include: • • • • • • • • • • • Sample Extraction using Reverse Phase, Normal Phase, Ion-Exchange and Mixed Mode Cartridges Competitive Immunoassay for Recreational Drugs Handling of On-site Testing Devices, including alcometers Mass Spectrometry including infusion and hyphenated Separation Techniques Tuning the Mass Spectrometer Resolution, sensitivity and mass accuracy Mass Range, Scan and Selected Ion Monitoring Sensitivity and speed MS/MS – selected reaction monitoring Identification of a Substance in a Matrix Quantification of target analytes MODULE 5 Project All students have an analytical toxicology placement, either of a clinical or forensic nature between May and August. The total length of the placement is 16 weeks, which includes preparation of the project report. This academic year, placements will begin in May. Students generally undertake the placement in a laboratory or organisation exterior to King's, although staff at King's also offer placements that may be of interest to students and some organisations offer placements where some of the practical work takes place in the organisation and some takes place at King’s. You must recognise that it is not always possible to assign you your first or second (or sometimes even your third) placement choice, although we will do our best. If you refuse to select an alternative placement from those on offer, one will be assigned to you and you will be expected to take it, irrespective of where in the UK it is located. Security clearance is mandatory for placements in some laboratories. The host laboratory will arrange for the security clearance. It should be noted that we reserve the right to refuse an external placement. In these circumstances, the individual will carry out their placement within King's. Assessment Assessment will be via a written review of the literature pertaining to the research topic; submission of a journal article; presentation of a poster with oral questioning by assessors. • • • • Literature Review Research Article, in the style of a journal publication Poster presentation, including oral examination Supervisors evaluation