This advanced application-oriented two-day course in LC-MS pays attention to all important aspects of quantitative bioanalysis by LC-MS, except for statistical aspects in data-processing. Starting from providing knowledge on important processes in analyte ionization by electrospray or APCI, the course provides a profound understanding of the important topics of quantitative bioanalysis using LC-MS. Quality control in instrument performance, method development, and systematic troubleshooting are key issues throughout the course. The theoretical parts are illustrated with a number of case studies. Experience with the LC-MS instrumentation is recommended prior to attending this course The aim is to learn the participants the general principles, background knowledge, and how to use this knowledge in practice.
The format of the course is interactive. This means that there will be lectures, which can be freely interrupted for questions and/or discussions, and exercises with interpretation examples, case studies, and other illustrations of the theory. These exercises are first done individually and subsequently discussed in the group.
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Typical Outline of a Two-day Course
Session 1: Introduction. Principles of Mass Spectrometry. System OverviewGeneral aspects of method development for quantitative analysis are introduced. General principles of mass spectrometry are discussed: analyte ionization techniques, quadrupole mass analysers, concepts of mass, and general aspects of data-acquisition and processing.
Session 2: Instrumentation and Chemistry of Electrospray Ionization for LC-MS.
Session 3: Information from LC-MS Mass Spectra.This session provides an overview of relevant instrumentation for electrospray ionization mass spectrometry, a profound discussion on the electrospray ionization mechanisms, and a start with discussion of matrix effects.
Session 4: Instrumentation and Chemistry of Atmospheric-pressure Chemical Ionization for LC-MS.In quantitative analysis, the analyte mass spectrum is a diagnostic tool. One should learn to appreciate the information that is present in the spectrum, e.g., adduct ions and adduct-bound dimers, but also topics related to average and monoisotopic mass, mass defects, and isotope peaks.
Session 5: Method Development. General Aspects. SRM Transition and Internal Standard.Instrumentation and ionization mechanisms for atmospheric-pressure chemical ionization (APCI) are discussed in this session. In addition, atmospheric-pressure photoionization is discussed. Attention is also paid to the choice between electrospray and APCI: are they really as different as they seem to be?
Session 6: Method Development: Mobile-Phase Composition.After acquiring the required background knowledge, the method development for quantitative bioanalysis can be discussed in depth, starting with topics related to selected reaction monitoring and the selected of an appropriate internal standard.
Session 7: Method Development: Matrix Effects. Importance of Sample Pretreatment.This session addresses mobile-phase compatibility issues and provides an overview of solvent systems that can be applied in LC-MS. The use of non-volatile mobile-phase additives is discussed, as well as the effect of various additives on analyte response. It provides directives for method development.
Session 8: Validation and Troubleshooting.Starting from an application example, ion suppression by matrix effects is discussed. Methods to evaluate sample pretreatment and separation methods with respect to matrix effects are discussed. Various approaches to (high-throughput) sample pretreatment are introduced.
Some aspects related to validation are discussed. However, the major issues in this session are recognition of problems, detection and solution to problems, approaches to systematic troubleshooting.