Introduction

The agenda includes a series of interactive tutorials interspersed between more formal lectures, designed to give delegates a deeper understanding of bioanalytical tools employed within the DMPK discipline. The tutorials aim to reinforce lecture material, but in a more practical form, including a tutorial on “method development”, which will describe real examples from lecturers to illustrate some of the more (or less) common problems encountered in the bioanalytical process. Tutorials will be conducted in groups of 8-10 and the tutorial group will remain together throughout the course.

Outline of course material: the course will cover the following topics

  1. Physico-chemical Properties of Drugs: How the structure of a molecule affects its physico-chemical properties and how these properties can be used to best effect in ~ developing good extraction, separation and detection strategies. Principles of chirality and the impact on bioanalysis and/or metabolism are also covered.

  2. Fundamental Aspects of Mass Spectrometry (MS): Realigned to concentrate on atmospheric pressure ionisation (API) mass spectrometry, in particular electrospray and atmospheric pressure chemical ionization (APCI) techniques. Covering ionisation modes, different types of mass analyser, scan modes and an explanation of the jargon that surrounds this critical technology. An introduction to the increasingly important matrix-assisted laser desorption ionisation (MALDI) mass spectrometry is also provided.

  3. Sample Preparation & Retention Mechanisms: An overview of sample preparation techniques commonly used in bioanalysis, including protein precipitation, liquid/liquid extraction and solid phase extraction (SPE), together with methods for dealing with different types of samples e.g. extraction of tissue and solid samples. Why sample preparation is required and why different techniques may be used for different analytes, together with how to exploit retention mechanisms in SPE and HPLC, are all described in detail. Lectures also provide an overview of automated methods used in bioanalysis and in vitro screening, including robotic systems, column switching and parallel strategies.

  4. HPLC & UHPLC Theory and Practice: An introduction to HPLC, its apparatus and theory; from plumbing to peak shape. Covering basic principles of the chromatographic processes with some practical tips and help with common problems. Including how to get the best from your HPLC system - focusing on resolution, efficiency and performance in quantitative bioanalysis. Lectures cover reverse-phase, normal phase, ion-pair, ion-exchange, chiral HPLC and hillic chromatography. Dedicated attention is also given to UHPLC, using small particle packed columns.

  5. Quantitative Mass Spectrometry: Or more broadly “how to use your API LC/MS to support drug discovery and development”. The pros and cons of LC/MS v LC/MS/MS, APCI v electrospray are looked at, and the challenges of ion suppression are covered in depth. Optimisation of LC methods for LC/MS/MS, the effect of mobile phase on ionisation, and automated MS method development are also examined.

  6. Biopharmaceutical Analysis: Several lectures are now devoted to the analysis of larger biopharmaceutical molecules, as a prelude to the DMDG “Large Molecule” training course. Introduction to this topic includes peptides, oligonucleotides and large intact proteins, comparing both LC/MS and ligand binding approaches.

  7. Qualitative Mass Spectrometry (Metabolite Identification): Covers practical aspects of metabolite identification by mass spectrometry (focusing on API methods) and includes detailed discussions on the use of tandem MS (concepts, instrumentation and experimentation). Also includes the use of accurate mass measurements, on-line radiochemical detection, integration with nuclear magnetic resonance (NMR) spectroscopy and stable isotope labels.

  8. Strategies for Bioanalytical Method Development: Summing it all up, we plot a course through the development of a good bioanalytical method by LC/MS. Initial considerations and experiments, chromatographic options, optimisation, validation and application of the final method will be covered.

  9. Strategies for Metabolite Isolation: Focuses on the isolation of metabolites from biological samples and integration of these methods into a generalized strategy for metabolite identification using LC/MS (and NMR). Also highlights sample selection criteria and experiment types (in vivo, in vitro, in situ) for successful metabolite identification.