Applications Of Liquid Chromatography Tandem Mass Spectrometry For The Analysis Of Complex Mixtures

From inside our bodies to the environment we live in, there are a wide variety of unknown compounds that have been difficult to identify and characterize through current methods. We aim to study these compounds by liquid chromatography (LC) high-resolution tandem mass spectrometry (MS/MS). LC-MS/MS has become the gold standard for the analysis of complex mixtures due to its capabilities to decrease spectral overlap and high mass measurement accuracy. Proteins are of particular interest biologically because they give insight into various diseases and the health of our biological processes. Analysis of these proteins is hindered by the current methods implemented in laboratories due to the size and characteristics of these compounds and their natural behavior outside of biological systems. Just as import as what is inside the human body is what is in the environment, specifically the Earth's oceans. Just like proteins in the body, the Earth's oceans give us a picture into the health of the environment. We know that human activity has severally impacted our atmosphere and water sources. We aim to understand the connections with pollution and the effects it has on oceans, a major water source on Earth. First a broad overview of LC-MS/MS is presented in Chapter 1. The first section discusses the MS instrumentation used for the studies in the following chapters, the 14- and 21-Tesla Fourier Transform Ion Cyclotron Mass Spectrometer (14- and 21-T FT ICR MS). The custom-built system is designed to produce the highest resolution data for compound identification and structural information. Our facilities are also equipped with various liquid chromatography (LC) systems that are used to separate mixtures and decrease sample complexity. Lastly, a brief introduction to various complex mixtures analyzed by LC-MS. The chapters that follow cover the specific studies performed with the knowledge of LC-FT ICR-MS and compound identifications. Chapter 2 discusses a study into the benefits and limitations on the application of isotopic depletion for cultured proteins. Isotopic depletion is a method used to deplete compounds of various heavy isotopes of elements present, most commonly carbon, nitrogen, hydrogen, oxygen, and sulfur. Heavy isotopes effect MS experiments in multiple ways which are investigated in the study for proteins derived from multiple cell lines (E. coli and C. elegan). This work is novel in the application and analysis of isotopically depleted C. elegans proteins The methods used for the biological applications can be applied to the analysis of marine samples which is discussed in Chapters 3 and 4. Chapter 3 investigates the composition of sinking particles in the California Coastal Current, specifically metabolites. Sinking particles play a role in the regulation of biological processes in the water column but additionally have shown to regulate greenhouse emissions. We take an even deeper look with analysis of marine sediments to see how far pollutants travel down the water column and are stored at the bottom of the ocean, in Chapter 4. Marine sediment samples were collected from the Oregon continental shelf at established sample stations. The analysis of these environmental samples by LC-MS/MS is still in the developmental phase. Our work aims to develop novel LC-MS/MS methods that can give a fuller picture into environmental impacts of pollution. The overall goal is to expand the capabilities of LC-MS/MS experiments to complex mixtures. LC-MS/MS shows its true capabilities and potential in the experiments discussed here. From 300 Da to 40 kDa in size, LC-MS/MS can efficiently analyze a wide dynamic range of compounds which is critical to broad applicability. With this work, future experiments can analyze and identify a wide range of compounds by high-throughput LC-MS/MS analysis.

Due to its high sensitivity and selectivity, liquid chromatography–mass spectrometry (LC–MS) is a powerful technique. It is used for various applications, often involving the detection and identification of chemicals in a complex mixture. Ultra Performance Liquid Chromatography Mass Spectrometry: Evaluation and Applications in Food Analysis presents a unique collection of up-to-date UPLC-MS/MS methods for the separation and quantitative determination of components, contaminants, vitamins, and aroma and flavor compounds in a wide variety of foods and food products. The book begins with an overview of the history, principles, and advancement of chromatography. It discusses the use of UHPLC techniques in food metablomics, approaches for analysis of foodborne carcinogens, and details of UPLC-MS techniques used for the separation and determination of capsaicinoids. Chapters describe the analysis of contaminants in food, including pesticides, aflatoxin, perfluorochemicals, and acrylamide, as well as potentially carcinogenic heterocyclic amines in cooked foods. The book covers food analysis for beneficial compounds, such as the determination of folate, vitamin content analysis, applications for avocado metabolite studies, virgin olive oil component analysis, lactose determination in milk, and analysis of minor components of cocoa and phenolic compounds in fruits and vegetables. With contributions by experts in interdisciplinary fields, this reference offers practical information for readers in research and development, production, and routing analysis of foods and food products.

This book is intended both to be an introduction to techniques and applications of liquid chromatography/mass spectrometry and to serve as a reference for future workers. When we undertook its writing, we chose not to cover the field, particularly applications, exhaustively. Rather we wished to produce a book that would be of use to people just beginning to use the technique as well as to more advanced practitioners. In this regard, we have sought to highlight techniques and applications that are of current importance, while not neglecting descriptions of approaches that may be of significance in the future. We hope that we have succeeded in this. At the same time we hope that the bibliography, with indexes classified by author and title, will make this book of value to those who may disagree with our emphasis. ACKNOWLEDGMENTS. One of us (C. G. E. ) wishes to acknowledge the en couragment of Professor J. A. McCloskey in undertaking this project. All four of us are grateful for the continuous and expert assistance of V. A. Edmonds in the preparation of the Bibliography. Alfred L. Y ergey Bethesda, Maryland Charles G. Edmonds Richland, Washington lvor A. S. Lewis London, England Marvin L. Vestal Houston, Texas v Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . 2. Direct Liquid Introduction Interfaces . . . . . . . . . . 5 . . . 2. 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2. 2. Operating Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2. 3. Specific DLI Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2. 3. 1. Capillary Inlets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2. 3. 2. Diaphragm Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2. 3. 3. Nebulizing Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Liquid Chromatography: Applications, Second Edition,is a single source of authoritative information on all aspects of the practice of modern liquid chromatography. It gives those working in both academia and industry the opportunity to learn, refresh, and deepen their knowledge of the wide variety of applications in the field. In the years since the first edition was published, thousands of papers have been released on new achievements in liquid chromatography, including the development of new stationary phases, improvement of instrumentation, development of theory, and new applications in biomedicine, metabolomics, proteomics, foodomics, pharmaceuticals, and more. This second edition addresses these new developments with updated chapters from the most expert researchers in the field. Emphasizes the integration of chromatographic methods and sample preparation Explains how liquid chromatography is used in different industrial sectors Covers the most interesting and valuable applications in different fields, e.g., proteomic, metabolomics, foodomics, pollutants and contaminants, and drug analysis (forensic, toxicological, pharmaceutical, biomedical) Includes references and tables with commonly used data to facilitate research, practical work, comparison of results, and decision-making

This fully updated volume describes methods and protocols for a number of drugs and toxins in a stepwise manner. Exploring the versatility and flexibility of mass spectrometry, the book covers the advantages of this technology, which typically include elimination of the need for special reagents such as antibodies, increased sensitivity and specificity, and multi-component analysis enabling the screening of tens to hundreds of compounds in a single assay run. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Clinical Applications of Mass Spectrometry in Drug Analysis: Methods and Protocols, Second Edition serves as a valuable resource for laboratory professionals who are already utilizing mass spectrometry or considering bringing this technology to their labs.

First explaining the basic principles of liquid chromatography and mass spectrometry and then discussing the current applications and practical benefits of LC-MS, along with descriptions of the basic instrumentation, this title will prove to be the indispensable reference source for everyone wishing to use this increasingly important tandem technique. * First book to concentrate on principles of LC-MS * Explains principles of mass spectrometry and chromatography before moving on to LC-MS * Describes instrumental aspects of LC-MS * Discusses current applications of LC-MS and shows benefits of using this technique in practice

This dissertation focuses on coupling separation techniques such as ion mobility (IM) and liquid chromatography (LC) to mass spectrometry and their application to characterization of complex mixtures. Non-covalent complexes between poly(ethylene imine) (PEI) and single stranded oligodeoxynucleotides (ODNs), as well as components from black raspberries, were characterized utilizing ion mobility mass spectrometry (IM-MS) and liquid chromatography mass spectrometry (LC-MS), respectively. Interfacing these separation methods to mass spectrometry allows for detection and identification of isobaric species and species present in low concentration. Non-covalent complexes between low molecular weight poly(ethylene imine) (PEI 400 and 800) and single-stranded oligodeoxynucleotides (ODNs) were investigated for five ODNs, including d(TTTTT), d(CCCCC), d(AAAAA), d(GGGGG) and d(GCGAT). In chapter 4 the compositions, as well as solution and gas-phase stabilities of the complexes (termed polyplexes) were examined by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS2). Independent of the mixing ratio of the reactants, the polyplex with 1:1 polymer-to-nucleotide stoichiometry, PN, is the dominant product. The gas-phase stabilities, assessed by MS2 and collisionally activated dissociation, follow the same order, providing evidence that the polyplex structures in aqueous solution and the more hydrophobic environment of the gas phase are very similar. Non-covalent complexes with different composition but the same molecular mass were corroborated by ion mobility mass spectrometry (IM-MS). In chapter 5 of this dissertation an investigation of the expanded non-covalent system, ternary complexes, of poly(ethylene imine), single-stranded oligodeoxynucleotides and glutamic acid entities, is discussed. The solution stabilities and gas-phase stabilities of the ternary complexes (termed terplexes) were examined by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS2). In addition, higher order ternary structures with multiple units of polymer and/or nucleotide present within the terplex were identified utilizing IM-MS. Finally, chapter 6 provides information on characterization of non-anthocyanin components from black raspberry fractions by interfacing liquid chromatography (LC) to mass spectrometry. The black raspberry extracts were provided from the lab of Dr. Joseph C. Scheerens at Ohio Agricultural Research and Development Center, Wooster, Ohio. Combination of LC-MS, ESI-MS and MS2 provided structural information for the corresponding components.

Mass spectrometry is an analytical technique that can be used for the structural characterization and quantification of a wide range of molecules. The technique is extensively used by chemists for the analysis of small and volatile organic compounds. Mass spectrometry has long been an important technique for the identification of materials ranging from pure compounds to complex mixtures. Mass spectrometry can be used to determine molecular weight of compounds or using different ionization conditions, can provide more structural details through the analysis of fragmentation patterns. This level of detail can be attained for pure compounds and some mixtures. Mass spectrometry can also be combined with separation techniques such as gas chromatography or liquid chromatography to allow more complex mixtures to be examined. These hyphenated techniques provide a range of options for the characterization of complex materials.

Mass Spectrometry for the Clinical Laboratory is an accessible guide to mass spectrometry and the development, validation, and implementation of the most common assays seen in clinical labs. It provides readers with practical examples for assay development, and experimental design for validation to meet CLIA requirements, appropriate interference testing, measuring, validation of ion suppression/matrix effects, and quality control. These tools offer guidance on what type of instrumentation is optimal for each assay, what options are available, and the pros and cons of each. Readers will find a full set of tools that are either directly related to the assay they want to adopt or for an analogous assay they could use as an example. Written by expert users of the most common assays found in a clinical laboratory (clinical chemists, toxicologists, and clinical pathologists practicing mass spectrometry), the book lays out how experts in the field have chosen their mass spectrometers, purchased, installed, validated, and brought them on line for routine testing. The early chapters of the book covers what the practitioners have learned from years of experience, the challenges they have faced, and their recommendations on how to build and validate assays to avoid problems. These chapters also include recommendations for maintaining continuity of quality in testing. The later parts of the book focuses on specific types of assays (therapeutic drugs, Vitamin D, hormones, etc.). Each chapter in this section has been written by an expert practitioner of an assay that is currently running in his or her clinical lab. Provides readers with the keys to choosing, installing, and validating a mass spectrometry platform Offers tools to evaluate, validate, and troubleshoot the most common assays seen in clinical pathology labs Explains validation, ion suppression, interference testing, and quality control design to the detail that is required for implementation in the lab

Mass spectrometry is fast becoming an indispensable field for medical professionals. The mass spectrometric analysis of metabolites and proteins promises to revolutionize medical research and clinical diagnostics. As this technology rapidly enters the medical field, practicing professionals and students need to prepare to take full advantage of its capabilities. Medical Applications of Mass Spectrometry addresses the key issues in the medical applications of mass spectrometry at the level appropriate for the intended readership. It will go a long way to help the utilization of mass spectrometry in medicine.The book comprises five parts. A general overview is followed by a description of the basic sampling and separation methods in analytical chemistry. In the second part a solid foundation in mass spectrometry and modern techniques of data analysis is presented. The third part explains how mass spectrometry is used in exploring various classes of biomolecules, including proteins and lipids. In the fourth section mass spectrometry is introduced as a diagnostic tool in clinical treatment, infectious pathogen research, neonatal diagnostics, cancer, brain and allergy research, as well as in various fields of medicine: cardiology, pulmonology, neurology, psychiatric diseases, hemato-oncology, urologic diseases, gastrointestinal diseases, gynecology and pediatrics. The fifth part covers emerging applications in biomarker discovery and in mass spectrometric imaging. * Provides a broad look at how the medical field is benefiting from advances in mass spectrometry.* Guides the reader from basic principles and methods to cutting edge applications.* There is NO comparable book on the market to fill this fast growing field.