LC-MS/MS for Chromatographers

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1 LC-MS/MS for Chromatographers An introduction to the use of LC-MS/MS, with an emphasis on the analysis of drugs in biological matrices

2 LC-MS/MS for Chromatographers An introduction to the use of LC-MS/MS, with an emphasis on the analysis of drugs in biological matrices What does it cover? With the introduction of the mass spectrometer (MS) as a practical detector for a high-performance liquid chromatograph (LC or HPLC) in the early 1990s, LC-MS began to be used for routine applications. One form of LC-MS uses a tandem MS (LC-MS/MS), that has become the go-to instrument for quantitative analysis of drugs in biological matrices in the pharmaceutical laboratory. As they have become more widely used and understood, LC-MS and LC-MS/MS have found their way into environmental applications, synthetic chemistry, protein analysis, and many other areas. LC-MS/MS has moved from a technique available only to skilled experts in both HPLC and MS to a routine tool usable much as any other HPLC detector. You will find that this introductory course in LC-MS/MS will give you a good foundation of understanding the technique and how to use it in your daily work. We start out with an overview of different MS techniques, and ocus in the majority of the course on the quadrupole mass spectrometer. You will learn how MS works, how ions are produced and fragmented. Sections focus on qualitative, as well as quantitative analysis. Each part of the process, calibration, tuning, sample preparation, HPLC method development, and MS method development are covered. Many tricks and techniques of practical use are presented. Who should take this course? This course is designed to provide basic knowledge and practical applications of LC-MS for liquid chromatographers. Most of the examples emphasize the most popular use of LC-MS/MS today: the analysis of drugs in metabolites in biological extracts. Although no previous experience with HPLC is necessary, the course will be especially valuable to those with some chromatography experience. What do you get? You get full access to the 42 video modules and approximately 10.5 hours of instruction. You also get handouts containing copies of all of the approximately 515 PowerPoint slides used in the class. These are arranged for easy note-taking while you view each module and give you a valuable resource for future reference. Philosophy As we ve taught and revised this class over the past 15 years, we have found that a combination of descriptive, intuitive, and quantitative teaching approaches results in a class that appeals the beginner and more experienced laboratory worker alike. By starting at the basics and working to the specifics, you will learn how LC-MS/MS can be used as a practical tool in your laboratory. There are many learning styles, but with most of the information presented in graphic, textual, and audio forms, it is likely that every learning style will be served. We feel that learners need the ability to easily locate material for immediate or later review, either in its original video presentation form or through copies of the slides (with notes you took during the presentations). We feel that getting a true understanding of LC-MS/MS concepts is more important than being exposed to a large amount of detail when learning a new technique like this. If you need courses with more in-depth treatment of MS techniques, we will be happy to point you to more advanced classes for just this purpose. How is it organized? This is a unique web-based class. For convenient access and viewing, the course is split into 11 major sections plus a review, and each of these sections is broken down into 1-7 modules, ranging from 6-29 min, with an average running time of 15 min. The 42 modules contain approximately 10.5 hours of comprehensive instruction on LC-MS and LC-MS/MS. The first 6 sections cover the basic principles of LC-MS and LC-MS/MS, including operation principles, the difference between ion traps, quadrupoles, magnetic sector, and other detectors, the various parts of the instrumentation, ion formation, calibration and tuning, and qualitative analysis are covered in these sections. The remainder of the class uses this information to focus on how to set up an LC-MS method, including what is needed for the MS, the HPLC, sample preparation, quantitative analysis, and validation. We finish with a short review of the entire class.

3 Each of the 12 sections can be considered a stand-alone unit, but many proceed with the assumption that you ve already viewed the preceding sections. Each module in an individual section emphasizes a different topic, but they are divided more for convenience of viewing time than content. Except for review of material at a later time, we recommend against picking random modules to view. As a result, you will get the most out of this class by viewing all sections and modules in order. Then, if you need to review the material at some point, access to individual modules may be most appropriate. Here s what the course covers: MS operation, including the operation of the most popular LC-MS interfaces How a quadrupole mass filter works MS calibration and optimization Ion production, fragmentation, and detection Operation in MS, MS/MS, and MS/MS/MS modes Structure determination by product ion analysis Quantitative analysis Sample preparation Method development Validation Techniques to get the most from your LC-MS What will I get from this course? You will learn how a mass spectrometer works, the advantages (and limitations) of adding mass spectrometric detection to your liquid chromatographic analyses, and how to use MS to approach separation and detection problems intractable by other methods. By understanding the central roles of the LC-MS interface and the sample ion, you will have a solid grounding on which to further build your LC-MS expertise. You will receive a printed copy of the approximately 515 slides included in this course to supplement your note-taking as you view the class. Instructors The course was designed by John Dolan, Tom Jupille, and Lloyd Snyder. This class is taught by John Dolan, considered to be the one of the world s experts in HPLC. He has written more than 300 user-oriented articles on HPLC troubleshooting over the last 30 years in addition to more than 100 peer-reviewed technical articles on HPLC and related techniques. His three books (co-authored with Lloyd Snyder), Troubleshooting HPLC Systems, Introduction to Modern Liquid Chromatography (3rd edn), and High-Performance Gradient Elution, are standard references on thousands of desks around the world. He also was intimately involved in the preparation of Snyder, Glajch, and Kirkland s Practical HPLC Method Development (2nd edn) book. John is the author of Separation Science s popular HPLC Solutions articles. From a method development standpoint, he has worked in a clinical chemistry department, managed an HPLC instrument manufacturer s technical support laboratory, and written DryLab software. He has taught HPLC training classes around the world to more than 10,000 students. His casual style and ability to get information across in a practical and understandable manner make him in high demand as a training instructor and conference speaker. The late Michael Alexander, Ph.D., was one of the early practitioner s of LC-MS and LC-MS/MS, working with one multi-national and one startup pharmaceutical company as an LC-MS specialist. His combination of detailed understanding of all aspects of the MS, problem-solving capability, and easy communication of his vast knowledge are key factors in the continued success of this class. Course Detail Below are listed each section and sub-section of the class. A brief summary of each section is given first. After the subsection in parentheses is the approximate running time of that module. Section 1. Overview of Contemporary LC-MS We start with an overview of LC-MS. What has made LC-MS a practical tool in today s laboratory? Why is the interface a key development for reliable operation? How do quadrupoles, ion traps, magnetic sector, and other types of mass spectrometers compare? Why is the quadrupole the most popular instrument for quantitative analysis? Introduction to Quadrupoles (19 min) Overview of Other Types of Mass Spectrometers (15 min) The Challenges of LC-MS (9 min)

4 Section 2. Principles Quads and Traps Here we take a closer look at how the quadrupole and its most popular colleague, the ion trap, work. How is mass resolution, or resolving power, determined, and how does it differ from chromatographic resolution? Why are sensitivity and resolution inversely related? How the Quadrupole Works (15 min) How the Ion Trap Works (14 min) Mass Resolution (9 min) Compromises (19 min) Section 3. Basics of Quadrupole Instrumentation The operation of the two most popular interfaces, electrospray (ESI) and atmospheric pressure chemical ionization (APCI) are discussed. How do these create sample ions? What happens to things that don t evaporate? How does the vacuum work? How are data stored? We take a look at some of the popular instruments and see the important parts that are common to all MS detectors. The Interface (15 min) Practical Aspects of the Interface (19 min) Other Hardware Topics (17 min) Section 4. The Sample Ion What is the sample ion? How does it differ from a true molecular ion? When can we and can t we depend on differences in isotope mass? How does fragmentation take place in LC-MS/MS? Why does chemical ionization not always produce the same fragments and spectra? What it is the Sample Ion? (6 min) Isotopic Effects (11 min) Additional Confusion (12 min) Ion Fragmentation (8 min) Interpretation of Spectra (8 min) Section 5. Calibration and Tuning What is the difference between calibration and tuning? When do you perform each operation and how often? Calibration ((10 min) Tuning (14 min) Section 6. Qualitative Analysis Qualitative analysis (what s there) is one of the primary strengths of mass spectrometry in the analytical laboratory, so why is this the weak point when quadrupoles are used as the MS filter? It is easy to confirm that a particular peak is the same as a reference standard, and not too hard to tell if an unknown peak is structurally related to a known compound, but why is it so difficult to do structural analysis of completely unknown compounds? How can the limits of the tandem quadrupole be stretched by doing a MS/MS/MS experiment? Why is the ion trap so much better for MSn experiments? Confirming Identity (15 min) Establishing a Relationship (15 min) Proposing Structures (7 min) More on MSn (11 min) Section 7. Developing the MS Method Here we look at aspects of method development that focus on the MS portion of the instrumentation. How do we set up the instrument for a known method or a new method? How is calibration done with reference standards? Why is internal standardization the most popular way to standardize LC-MS? What makes a good internal standard? What is the difference between a chemical internal standard and an isotopic internal standard? Components of the MS Analysis (10 min) Calibration and Linearity (11 min) Sensitivity (15 min) Internal Standards (24 min)

5 Section 8. Quantitative Analysis Quantitative analysis is where the LC-MS/MS shows its real power. We take a look at integration and calibration of the method. Why is curve weighting popular with LC-MS, whereas it is rarely used with other LC detectors? How do you choose a weighting method? Integration (18 min) Calibration Curves and Curve Weighting (21 min) Section 9. Developing the LC Method A thorough treatment of LC method development is covered in the Advanced HPLC Method Development course in this series. Here we concentrate on LC method development as it focuses on the requirements of MS detection. We look at the starting conditions, including a module on how to quantitatively assess column selectivity differences. What LC variables are important in MS? Why is a scouting gradient so powerful, and how do we choose scouting conditions? How do we convert existing LC-UV methods to LC-MS? Where to Start (17 min) The Column (16 min) Other Variables (12 min) Scouting Gradients (29 min) Converting LC-UV to LC-MS (9min) Method Scaling (13 min) Odds & Ends (19 min) Section 10. Sample Preparation Sample preparation can be more of a challenge than either the LC or MS portions of the LC-MS method if the sample is in a challenging matrix. The key to sample preparation is to remove materials that will foul the interface, interfere with the chromatography, or cause ion suppression. We review 6 of the most popular sample preparation techniques. We look at how ion suppression occurs and how to test for it. Then we compare the effectiveness of several sample preparation techniques at reducing ion suppression. Dilution, Precipitation, & Liquid-Liquid Extraction (25 min) Solid-Phase Extraction (22 min) On-Line Extraction & QuEChERS (17 min) Ion Suppression (22 min) Section 11. Validation Validation in some form is likely to be needed for most LC-MS methods, even if they are used only for research purposes. We take a quick overview of the requirements for and process of validating LC-MS methods that will be used for quantification of drugs in biological matrices. Bioanalytical Method Validation (17 min) Precision, Accuracy, Recovery, & Selectivity (14 min) Recommendations (16 min) Section 12. Summary / Review (23 min) In this final section, we reprise the LC-MS class, reviewing key slides from each of the preceding sections. Frederick House, Princes Court Nantwich, CW5 6PQ, UK

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