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Separation of Carnitine and Acylcarnitines by Ultra High Pressure and Monolithic Chromatographic Approaches and Their De...

Permanent Link: http://ufdc.ufl.edu/UFE0021473/00001

Material Information

Title: Separation of Carnitine and Acylcarnitines by Ultra High Pressure and Monolithic Chromatographic Approaches and Their Determination by Electrospray Ionization-Time-of-Flight Mass Spectrometry
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Cerutti, Estela S
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acylcarnitines, carnitine, monolithic, time, uhplc
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Carnitine is involved in the transport of fatty acids into the mitochondrial matrix, permitting long-chain fatty acid metabolism, via beta-oxidation, to produce energy. The hydroxyl group of carnitine is esterified for this pathway, and carnitine generates acylcarnitines with various chain lengths. A number of abnormal acylcarnitine profiles have been observed in patients with different metabolic disorders. Therefore, there is a need for improved sample analysis throughput for these metabolites. There are two emerging column technologies in liquid chromatography that have attracted much attention in recent years: small particle packed ( < 2 ?m) and monolithic columns. This study explored the use of these two approaches for the separation of carnitine-based compounds in combination with high resolution electrospray ionization (ESI)-orthogonal-acceleration (oa)-time-of-flight (TOF) analyses. It was feasible to generate satisfactory chromatographic resolution for the targeted analytes and both columns demonstrated comparable separation performance. The use of the developed approaches can be applied to the metabolic pattern analysis of carnitine and acylcarnitines in non-derivatized clinical samples, providing significant insights related to human health.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Estela S Cerutti.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Yost, Richard A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2017-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021473:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021473/00001

Material Information

Title: Separation of Carnitine and Acylcarnitines by Ultra High Pressure and Monolithic Chromatographic Approaches and Their Determination by Electrospray Ionization-Time-of-Flight Mass Spectrometry
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Cerutti, Estela S
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acylcarnitines, carnitine, monolithic, time, uhplc
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Carnitine is involved in the transport of fatty acids into the mitochondrial matrix, permitting long-chain fatty acid metabolism, via beta-oxidation, to produce energy. The hydroxyl group of carnitine is esterified for this pathway, and carnitine generates acylcarnitines with various chain lengths. A number of abnormal acylcarnitine profiles have been observed in patients with different metabolic disorders. Therefore, there is a need for improved sample analysis throughput for these metabolites. There are two emerging column technologies in liquid chromatography that have attracted much attention in recent years: small particle packed ( < 2 ?m) and monolithic columns. This study explored the use of these two approaches for the separation of carnitine-based compounds in combination with high resolution electrospray ionization (ESI)-orthogonal-acceleration (oa)-time-of-flight (TOF) analyses. It was feasible to generate satisfactory chromatographic resolution for the targeted analytes and both columns demonstrated comparable separation performance. The use of the developed approaches can be applied to the metabolic pattern analysis of carnitine and acylcarnitines in non-derivatized clinical samples, providing significant insights related to human health.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Estela S Cerutti.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Yost, Richard A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2017-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021473:00001


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SEPARATION OF CARNITINE AND ACYLCARNITINES BY ULTRA HIGH PRESSURE AND MONOLITHIC CHROMATOGRAPHIC APPROACHES AND THEIR DETERMINATION BY ELECTROSPRAY IONIZATION-TIME-OF-FLIGHT MASS SPECTROMETRY By ESTELA SOLEDAD CERUTTI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007 1

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2007 Estela Soledad Cerutti 2

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To my beloved people The important thing is not to stop questioning. Curiosity has its own reason for existing. One cannot help but be in awe when he contemplates the mysteries of eternity, of life, of the marvelous structure of reality. It is enough if one tries merely to comprehend a little of this mystery every day. Never lose a holy curiosity. --Albert Einstein 3

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ACKNOWLEDGMENTS This thesis would not have been possible without the guidance, encouragement, friendship, and trust I received from my academic advisors, Richard A. Yost and David H. Powell. I would like to sincerely express that I have learned from this experience is of great importance in both personal and professional aspects. I also have to thank the members of Powells (Julia, Joanna, Cris, and Jodie) and Yosts groups for their help in situations related to science, research, and life. I would like to extend my gratitude to Dr. Ben Smith for his advice, continuous assistance, kind suggestions, and for spending his valuable time serving on my committee. Many thanks also go to Dr. Peggy Borum for her priceless contribution to my research and for giving me the opportunity to interact with her amazing group of people. I am grateful to Fulbright/LASPAU, as academic program, and Sonia Wallenberg, as my program advisor, for their supportive and considerate presence through these two years. I also owe a debt of gratitude to my academic institution in Argentina; I would never have gotten here without the guidance I received from my people there. This journey in my life would not have been nearly as enjoyable without the presence of my great friends Erick and Lourdes, and their family, Jared, and Pablo. I thank my parents for providing me with the values to discover and achieve my goals in life. They deserve so much of the credit for my success. Gracias viejos! Last in sequence, but not least in importance, my thanks go to God for giving me the strength and the opportunity to modestly contribute to our community through this research. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES...........................................................................................................................7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT...................................................................................................................................13 CHAPTER 1 INTRODUCTION..................................................................................................................14 Mass Spectrometry-Based Metabolomics..............................................................................14 Time-of-Flight Mass Spectrometer.........................................................................................18 Background......................................................................................................................18 Orthogonal Acceleration-Time-of-Flight Analyzer (oa-TOF)........................................19 Electrospray Ionization...........................................................................................................23 Principles of Electrospray Ionization..............................................................................23 Nebulization....................................................................................................................23 Droplet Formation...........................................................................................................23 Droplet Charging.............................................................................................................24 Droplet Disintegration.....................................................................................................25 Further Mechanistic Details Related to Ion Formation in ESI........................................25 Advantages and Disadvantages of ESI............................................................................28 Liquid Chromatography..........................................................................................................29 Ultra High Pressure Liquid Chromatography..................................................................29 Monolithic Columns........................................................................................................33 Carnitine and Acylcarnitines..................................................................................................36 General Aspects...............................................................................................................37 Role of Carnitine.............................................................................................................38 Deficiencies in Carnitine.................................................................................................40 2 OPTIMIZATION OF MASS SPECTROMETER INSTRUMENTAL PARAMETERS......50 Historical Perspective and Basic Principles of Design of Experiments.................................50 Full Factorial Designs.............................................................................................................51 Fundamentals...................................................................................................................53 Sign of Interaction Effects...............................................................................................54 Experimental...........................................................................................................................54 Mass Spectrometer..........................................................................................................54 TOF Calibration...............................................................................................................55 HPLC...............................................................................................................................56 Standard Solutions...........................................................................................................56 Plasma Sample Preparation.............................................................................................57 Results and Discussion...........................................................................................................58 5

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Conclusions.............................................................................................................................59 3 COMPARISON OF PACKED AND MONOLITHIC COLUMNS......................................68 General Aspects of HPLC......................................................................................................68 Fast HPLC.......................................................................................................................69 Fast HPLC Analysis Using Small Particle Packedand Monolithic-Columns...............70 Comparison of Performance between Packed and Monolithic Columns........................73 Experimental Methods............................................................................................................74 Results and Discussion...........................................................................................................74 Optimization of the HPLC Experimental Conditions.....................................................75 Effect of Mobile Phase Composition.......................................................................75 Effect of Mobile Phase Buffer.................................................................................76 Effect of Flow Rate: van Deemter Plots...................................................................77 Chromatograms...............................................................................................................78 Figures of Merit...............................................................................................................78 Sample analysis...............................................................................................................79 Conclusions.............................................................................................................................80 4 CONCLUDING REMARKS AND FUTURE WORK..........................................................98 APPENDIX A SYNTHESIS OF L-ACETYLCARNITINE HYDROCHLORIDE.....................................101 B SYNTHESIS OF L-OCTANOYLCARNITINE HYDROCHLORIDE...............................103 C SYNTHESIS OF L-PALMITOYLCARNITINE HYDROCHLORIDE..............................105 D BLOOD ASSAY..................................................................................................................107 REFERENCES............................................................................................................................114 BIOGRAPHICAL SKETCH.......................................................................................................120 6

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LIST OF TABLES Table page 1-1. Historical developments in mass spectrometry..................................................................42 2-1. Chemical formula, structure, and pseudo-molecular ion of carnitine and acylcarnitines.....................................................................................................................61 2-2. Minimum and maximum levels for each factor................................................................61 2-3. Experimental design matrix..............................................................................................62 2-4. Analysis of variance corresponding to the analytical response for carnitine....................62 2-5. Analysis of variance corresponding to the analytical response for hexanoylcarnitine.....63 2-6. Analysis of variance corresponding to the analytical response for decanoylcarnitine.....63 2-7. ESI-TOF operational conditions.......................................................................................64 3-1. HPLC conditions and gradient program for the packed column with isopropanol as organic mobile phase.........................................................................................................81 3-2. HPLC conditions and gradient program for the packed column with acetonitrile as organic mobile phase.........................................................................................................81 3-3. HPLC conditions and gradient program for the monolithic column with isopropanol as organic mobile phase.....................................................................................................82 3-4. HPLC conditions and gradient program for the monolithic column with acetonitrile as organic mobile phase.....................................................................................................82 3-5. Figures of merit for the separation and determination of carnitine and acylcarnitines (packed column isopropanol as organic mobile phase) by UHPLC-ESI-TOF...............83 3-6. Figures of merit for the separation and determination of carnitine and acylcarnitines (monolithic column isopropanol as organic mobile phase) by HPLC-ESI-TOF............83 7

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LIST OF FIGURES Figure page 1-1. The omics cascade with its different omic levels.......................................................43 1-2. Main components and layout of typical oa-TOF systems with reflecting mass analyzer..............................................................................................................................44 1-3. Schematic representation of a generic electrospray ionization interface...........................45 1-4. Pathways for ion formation from a charged liquid droplet................................................46 1-5. Representation of the number of chromatographic monolith publications per year..........47 1-6. Chemical structure of L-carnitine......................................................................................47 1-7. Metabolism of L-carnitine..................................................................................................48 1-8. Representation of carnitine role.........................................................................................49 2-1. Agilent 6210 time-of-flight mass spectrometer.................................................................65 2-2. Pareto chart of standardized effects for carnitine..............................................................66 2-3. Pareto chart of standardized effects for hexanoylcarnitine................................................66 2-4. Pareto chart of standardized effects for decanoylcarnitine................................................67 3-1. van Deemter plots for the different chromatographic approaches.....................................84 3-2. Base peak chromatogram obtained with a packed column and isopropanol as organic mobile phase......................................................................................................................85 3-3. Base peak chromatogram obtained with a packed column and acetonitrile as organic mobile phase......................................................................................................................86 3-4. Base peak chromatogram obtained with a monolithic column and isopropanol as organic mobile phase.........................................................................................................87 3-5. Base peak chromatogram obtained with a monolithic column and acetonitrile as organic mobile phase.........................................................................................................88 3-6. Calibration curves for carnitine and acylcarnitines; packed column.................................89 3-7. Calibration curves for carnitine and acylcarnitines; monolithic column...........................90 3-8. Mass spectrum of carnitine with monolithic column.........................................................91 8

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3-9. Mass spectrum of lauroylcarnitine with monolithic column.............................................92 3-10. Mass spectrum of myristoylcarnitine with monolithic column.........................................93 3-11. Mass spectrum of palmitoylcarnitine with monolithic column.........................................94 3-12. Mass spectrum of stearoylcarnitine with monolithic column............................................95 3-13. Mass spectrum of myristoylcarnitine with packed column...............................................96 3-14. Mass spectrum of palmitoylcarnitine and stearoylcarnitine with packed column.............97 9

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LIST OF ABBREVIATIONS AC acylcarnitine Acyl-CoA acyl-coenzyme A ADC analog-to-digital converter Alpha () selectivity factor amu atomic mass unit ANOVA analysis of variance C 0 carnitine C 2 acetylcarnitine C 3 propionylcarnitine C 4 butyrylcarnitine C 6 hexanoylcarnitine C 8 octanoylcarnitine C 10 decanoylcarnitine C 12 lauroylcarnitine C 14 myristoylcarnitine C 16 palmitoylcarnitine C 18 stearoylcarnitine CAT carnitine acetyl transferase CE capillary electrophoresis CE-MS capillary electrophoresis-mass spectrometry CoA coenzyme A CoASH free coenzyme A COT carnitine octanoyl transferase CPT carnitine palmitoyl transferase 10

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CRM charged residue model CT carnitine-acylcarnitine translocase Da dalton DART direct analysis in real time DC direct current DESI desorption electrospray ionization DGFR drying gas flow rate DGT drying gas temperature DOE design of experiments ESI electrospray ionization FC free carnitine FT-IR Fourier transform-infrared spectroscopy GC gas chromatography H height equivalent to a theoretical plate HETP height equivalent to a theoretical plate HPLC high-performance liquid chromatography IEM ion evaporation model k retention factor L column length LC liquid chromatography LC-MS liquid chromatography-mass spectrometry LOD limit of detection LOQ limit of quantitation MAL metabolic assessment laboratory MS mass spectrometry 11

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MS/MS tandem mass spectrometry MSS mean sum of squares MW molecular weight m/z mass-to-charge ratio N number of theoretical plates NMR nuclear magnetic resonance NP nebulizer pressure oa orthogonal acceleration oa-TOF orthogonal acceleration-time-of-flight PEG polyethyleneglycol RP reversed phase R s resolution S/N signal-to-noise ratio SOP standard operating procedure TC total carnitine TOF time-of-flight TOF-MS time-of-flight mass spectrometry t r retention time UHPLC ultra high pressure liquid chromatography w peak width XIC extracted ion chromatogram 12

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SEPARATION OF CARNITINE AND ACYLCARNITINES BY ULTRA HIGH PRESSURE AND MONOLITHIC CHROMATOGRAPHIC APPROACHES AND THEIR DETERMINATION BY ELECTROSPRAY IONIZATION-TIME-OF-FLIGHT MASS SPECTROMETRY By Estela Soledad Cerutti August 2007 Chair: Richard A. Yost Major: Chemistry Carnitine is involved in the transport of fatty acids into the mitochondrial matrix, permitting long-chain fatty acid metabolism, via -oxidation, to produce energy. The hydroxyl group of carnitine is esterified for this pathway, and carnitine generates acylcarnitines with various chain lengths. A number of abnormal acylcarnitine profiles have been observed in patients with different metabolic disorders. Therefore, there is a need for improved sample analysis throughput for these metabolites. There are two emerging column technologies in liquid chromatography that have attracted much attention in recent years: small particle packed (<2 m) and monolithic columns. This study explored the use of these two types of approaches for the separation of carnitine-based compounds in combination with high resolution electrospray ionization (ESI)-orthogonal-acceleration (oa)-time-of-flight (TOF) analyses. It was feasible to generate satisfactory chromatographic resolution for the targeted analytes and both columns demonstrated comparable separation performance. The use of the developed approaches can be applied to the metabolic pattern analysis of carnitine and acylcarnitines in non-derivatized clinical samples, providing significant insights related to human health. 13

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CHAPTER 1 INTRODUCTION Mass Spectrometry-Based Metabolomics Dramatic technological advances in the biological sciences over the past few years have forged a new era of research including the emerging field of systems biology. Although the understanding of living organisms at the molecular system level is still in its infancy, it is evident that comprehensive investigations of the omic cascade (Figure 1-1) with genomics, transcriptomics, proteomics, and metabolomics are important building blocks and will play a central role in this new science [1]. The integrative analysis of an organisms response to a perturbation on the trascriptome, proteome, and metabolome levels will lead to a better understanding of the biochemical and biological mechanisms in complex systems. However, whereas genomics, transcriptomics, and proteomics have made significant strides in technology development, the tools for the comprehensive examination of the metabolome are still emerging [2]. The term metabolome was first used in 1998 [3] to describe the set of metabolites synthesized by an organism, in fashion analogous to that of the genome and proteome. This definition has been limited to the quantitative complement of all of the low molecular weight molecules present in cells in a particular physiological or developmental state [4]. Metabolomics was coined by Fiehn and defined as a comprehensive analysis in which all metabolites of a biological system were identified and quantified [5]. Historical approaches to metabolite analysis include metabolite profiling, metabolite fingerprinting, and target analysis. Metabolite fingerprinting aims to rapidly classify numerous samples using multivariate statistics, typically without differentiation of individual metabolites or their quantitation. It can be used as a diagnostic tool, for example, by evaluating a patients metabolic fingerprint in comparison to healthy and diseased subjects. Target analysis is 14

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constrained exclusively to the qualitative and quantitative analysis of a particular metabolite or metabolites. As a result, only a very small fraction of the metabolome is focused upon, signals from all other components being ignored. Metabolite profiling involves the identification and quantitation by a particular analytical procedure of a predefined set of metabolites of known or unknown identity and belonging to a selected metabolic pathway [5, 6]. By their nature, these approaches provide a restrictive non-comprehensive view of the metabolome. Nevertheless, metabolite profiling represents the oldest and most established approach and can be considered the precursor for metabolomics. The real power of metabolomics is realized when qualitative and quantitative analyses are performed. Numerous analytical platforms have been used for metabolomics applications, such as nuclear magnetic resonance (NMR) [7], Fourier transform-infrared spectroscopy (FT-IR) [8, 9] and mass spectrometry (MS) coupled to separation techniques, or using direct flow injection. The great advantages of NMR are the potential for high-throughput fingerprinting, minimal requirements for sample preparation, and the non-discriminating and non-destructive nature of the technique. However, only medium to high abundance metabolites will be detected with this approach and the identification of individual metabolites based on chemical shift signals that cause sample clustering in multivariate analysis is challenging in complex mixtures. Mass spectrometry-based metabolomics offers quantitative analyses with high selectivity and sensitivity and the potential to identify metabolites. Combination with a separation technique reduces the complexity of the mass spectra due to metabolite separation in a time dimension, provides isobar separation, and delivers additional information on the physicochemical properties of the metabolites. However, mass spectrometry-based techniques usually require a sample preparation step, which can cause metabolite losses, and based on the sample 15

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introduction system and the ionization technique used, specific metabolite classes may be discriminated. Therefore, parallel application of several techniques, for example, GC-MS and LC-MS is desired to study the metabolome comprehensively. Currently, mass spectrometry-based metabolomics is a rapidly emerging field with the number of annual MS publications exceeding published NMR-based investigations [1]. Liquid chromatography can be interfaced with the MS detector for metabolomic analysis. Liquid chromatography can reduce ion suppression caused by coeluting compounds, isobaric interferences in the case of low-resolving mass analyzers, and often can separate isomers. In addition, a good analytical separation will result in better detection limits and MS data quality due to reduced background noise. Reversed phase (RP) liquid chromatography using C 18 narrow bore columns with particle sizes of 3 m is widely used for metabolomic investigations. In addition, the application of a monolithic silica based C 18 capillary column for plant metabolomics has been reported [10]. However, conventional RP-HPLC separation is often insufficient for the separation of complex biological samples, resulting in poor metabolite resolution [1]. One approach to increase chromatographic resolution and peak capacity is to use small particle sizes, such as the 1.8-m particles used in ultra high pressure liquid chromatography (UHPLC). Sub-2 m particles provide narrow chromatographic peaks, which results not only in better resolution but also in lower detection limits. The downside of this approach is the high pressure (10,000,000 psi) needed to operate these columns, and thus special UHPLC systems are required. In summary, technological advances in NMR and mass spectrometry have opened a new chapter in biochemistry by introducing metabolomics as an approach to study metabolism and its regulation in relation to disease, genetic, and environmental factors. Since the metabolome is the 16

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most predictive of phenotype, metabolomics holds the promise to extensively contribute to the understanding of phenotypic changes as an organisms answer to disease, genetic changes, and nutritional, toxicological, environmental, and pharmacological influences. With regard to human health alone, multiple benefits of metabolomics investigations can be envisioned. It can deliver new tools to diagnose disease or monitor the success of nutritional and pharmacological interventions, for example, by using an unbiased metabolic fingerprinting approach. Based on research studies, metabolomics can provide new biomarkers to assess human health, and over time a powerful list of diagnostic markers will evolve, which can be measured using high-throughput assays. However, in order to proceed from the single biomarker concept to the global metabolome evaluation outside a research environment, the technology has yet to be developed to provide the clinician with the tools to assess entire wide classes of metabolites in biofluids and automatically process the data to evaluate the biochemical status of an individual. Many technical and methodological issues have to be addressed to create analytical platforms that readily answer biological questions efficiently. Besides the technological aspects, data standardization, export, and analysis are essential components of metabolomics [11]. While high-throughput and semi-quantitative proteomics, with automated identification of proteins by computer algorithms, is available now (e.g., SEQUEST algorithm search), data analysis and visualization tools, libraries, and databases for metabolomics have yet to be developed. However, as noted by Weckwerth [12], whether we model the reality or only a shadow of it, we form a better understanding of the intricate biochemical processes and their scattering in living systems. The main goal of this study is to contribute to the pattern recognition analysis of our target metabolites: carnitine and acylcarnitines, important biomarkers for the identification of specific 17

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metabolic diseases. Therefore, this work focuses on the development of chromatographic approaches and optimization of the mass spectrometer experimental conditions to be used for the separation and determination of the carnitine-based metabolites. The remainder of Chapter 1 presents an overview of mass spectrometry, focusing on the time-of-flight mass spectrometer, a description of the fundamentals of electrospray ionization, some general principles of liquid chromatography particularly for the application of small particle packed and monolithic columns-, a brief explanation of the metabolic role of carnitine and acylcarnitines; and, finally, the scope of the present work. Time-of-Flight Mass Spectrometer Background Mass spectrometry has a dynamic history dotted with Nobel laureates and a continually advancing technology that has made significant inroads into drug discovery, protein characterization, and even disease diagnosis. The history of the science clearly shows that MS had its roots in physics, branched into chemistry, and in the past two decades, has budded into biology [13]. Historical developments in mass spectrometry are summarized in Table 1-1. William E. Stephens of the University of Pennsylvania proposed the concept of time-of-flight mass spectrometer (TOF-MS) in 1946 [14]. Two years later, in 1948, Cameron and Eggers [15] announced the first time-of-flight mass spectra, demonstrating a mass resolution of around 5. In 1955, Wiley and McLaren [16] developed a technique to focus the spread of ionization position and initial ion energy, achieving a mass resolution of 300 or higher. This modification provided TOF MS with a practical resolving power, and Bendix began distribution of commercial models [17]. In the 1970s, a reflectron TOF-MS [18] was developed, featuring a resolution of a few thousand. In the late 1980s, Dawson and Guilhaus [19], and Dodonov [20] 18

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developed orthogonal acceleration (oa), which allowed an efficient combination of TOF-MS and continuous ionization sources. In a TOF analyzer, ions are separated on the basis of differences in their velocities as they move in a straight path toward a collector. TOF-MS is fast, capable of high resolving power and high accuracy, applicable to chromatographic detection and it is used for the mass determination of large biomolecules [21]. Orthogonal Acceleration-Time-of-Flight Analyzer (oa-TOF) The reflectron achieved a resolution of a few thousand in TOF-MS, approximately 1,000 times higher than Cameron's original design. However, the decline of ion efficiency in a system combining a continuous ionization source and TOF-MS was not corrected until Dawson and Guilhaus [19] and Dodonov [20] developed orthogonal acceleration. In the TOF-MS by Cameron and Eggers [15], most of the ions generated from the ion source were deflected by the chopper and blocked by the slit, allowing only a small portion of the ions generated to be introduced into TOF-MS. In the TOF-MS by Wiley and McLaren [16], while most of the ions generated flew inside TOF-MS and reached the detector, the duration of ion production was extremely short, and the sample molecules introduced to the ion source while the electron gate was closed were not ionized and were discarded. The utilization ratio of ions or sample molecules in these systems was probably 0.1% or less. The orthogonal acceleration method (oa) increased the ion utilization ratio to 25 to 50%. In the basic structure of an oa-TOF-MS the sample is continuously ionized by the ion source. Then, ions from the ion source pass into the flight tube, which is perpendicular to the incident beam direction. The perpendicular orientation of the source with respect to the flight tube reduces the dispersion in kinetic energy in the direction of the flight tube improving the mass resolution [22]. 19

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Underpinning all the developments in oa-TOF-MS over the last decade or so is the rapid advancement in the supporting technologies of nanoand pico-second timing electronics and the fast processing and storage of digitized time-transients afforded by advances in computer technology. It would be fair to say that the revolution in digital electronics has supported TOF-MS to a greater extent than most other forms of mass spectrometry that rely more heavily on analog signal processing. Challenges remain in many areas of oa-TOF-MS technology and it would be reasonable to expect further advances in instrument performance (notably speed, sensitivity and resolving power) as supporting technologies improve [22]. The oa-TOF-MS principles. The most striking departure of oa-TOF-MS from other attempts to combine continuous ion sources with TOF is the use of a separate direction (for the TOF analysis), orthogonal to the continuous ion-beam axis of the ion source. To initiate a shot an electrostatic field is created in nanoseconds (typically) and as a consequence, the ions in this field experience a force. The direction of the force is strictly orthogonal to the ion beam axis. The resulting orthogonal acceleration imparts a new component of velocity to the sampled ions and this component is vectorially independent of the axial velocity of the ion beam. Vectorial decoupling of the velocity of ions in the ion beam and TOF directions is an important feature of oa-TOF-MS. Ions sampled for TOF analysis will retain the velocity that they gained in the ion beam. For this reason, their spontaneous drift trajectories will be inclined from the TOF direction according to the ratio of their components of drift-velocity in the ion beam and TOF directions. The inclination of the drift trajectory should not be confused with the direction for TOF analysis. The latter is normal to the beam axis as is evident from the alignment of the detector plane which should always be parallel to the continuous ion beam as shown in Figure 1-2. If a reflecting 20

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geometry TOF analyzer is employed, the inclination of the drift trajectory conveniently allows placement of the detector so that it is alongside the orthogonal accelerator (Figure 1-2). Advantages and effects of orthogonal geometry. The independence of the TOF and source axes leads to a number of advantages inherent in: (i) the ability to reduce the average initial ion velocity component in the TOF direction to zero and, simultaneously, to reduce the spread of velocity components in the TOF direction; (ii) the ability to control independently the continuous ion beam energy and the ion drift energy so that approximately the same time is required for the ion beam to fill the orthogonal accelerator as is required for the ions to be accelerated and drift to the detector. The first of the above favorably affects resolving power as well as the repeatability and linearity of the TOF versus (mass-to-charge ratio (m/z)) 1/2 mass calibration. The second favors sensitivity. As mentioned earlier, an advantage of the oa-TOF-MS instrument geometry is the low spread in initial velocity in the TOF direction and the fact that the average initial velocity is zero in the direction that the ions are accelerated. The narrow spread of velocity increases the achievable resolving power, while the zero average velocity leads to a nearly ideal calibration relationship that requires only two well-separated mass-TOF points to be measured for a reliable calibration over the entire range of the instrument. At least two points are chosen, despite the fact that the calibration equation in TOF-MS is ideally a linear function, where time-of-flight is directly proportional to (m/z) 1/2 For a high resolution TOF-MS, experimental data give a relationship that is highly linear but not passing through the origin. A small offset is expected due to the finite rise-time of the push-out pulse and the time for the detector and the associated electronics to respond to the ion arrival event at the detector. There may also be a systematic timing error associated with sensing the start event by triggering on the pulse that is synchronous with sampling the ions in the orthogonal accelerator. The finite time for 21

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the push-out pulse to rise to its steady state amplitude will affect the linearity of the TOF versus (m/z) 1/2 for light ions that move a significant distance during the pulse rise time. This is a relatively minor effect that can only be observed on a high resolution oa-TOF instrument or at very low m/z. The resolving power in oa-TOF-MS has been shown to decrease towards low mass due to the constant contribution of the detector pulse width and jitter of the timing electronics [23]. Since the contribution is not correlated with the other dispersions that give rise to the limiting resolution of the ion optics, it adds to the peak width in quadrature and its effect thus diminishes rapidly as the ion arrival-time spread exceeds the pulse width and jitter. In practice, the mass resolving power as well as the simplicity and stability of the calibration are important factors that limit the mass accuracy. Coupling of electrospray with oa-TOF-MS. A. Electrospray applications account for most of the activity in oa-TOF-MS [22]. The coupling of electrospray with oa-TOF-MS has four main advantages: duty-cycle-related sensitivity, speed to facilitate MS and MS/MS with online liquid chromatography, high m/z capability, and excellent mass accuracy. It is an interesting observation that one of the earliest cited strengths of electrospray ionization was the predominance of ions in highly charged states. The ions observed typically occurred in m/z range accessible to modest performance quadrupole mass analyzers [24, 25]. Because quadrupole mass analyzers were often used for electrospray, ions formed with higher m/z than could be observed in the limited mass range simply could not have been observed. Now it is known that some biomolecules remain folded, when subjected to electrospray ionization, if the solvent conditions are nondenaturing. This gives rise to lower charge states [26] and simpler mass spectra containing m/z values of up to 10,000 or higher [27]. Such spectra can be far more informative about the biochemistry than those of the denatured species. Taking into account the speed, 22

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sensitivity, and mass range of oa-TOF, it is not surprising that these mass analyzers are becoming popular for electrospray with biomolecules. This is particularly true when speed and sensitivity are simultaneously required, such as in tandem mass spectrometry coupled to chromatography [28-30]. Electrospray Ionization Principles of Electrospray Ionization Electrospray ionization (ESI) has become one of the most important ionization techniques for the on-line coupling of liquid phase separation methods with mass spectrometry. It is a simple and elegant method that handles small and big molecules, operates at atmospheric pressure and at a moderate temperature, and is probably the most gentle ionization technique available for MS [31]. Nebulization In electrospray ionization, a dilute solution is sprayed from a fine needle as depicted in Figure 1-3, which carries a high potential (about 4 kV). If the needle carries a positive potential, the droplets will have an excess of positive charges, usually protons. Evaporation of the volatile solvent (e.g., H 2 O, CH 3 OH, CH 3 CN, or CH 2 Cl 2 ) results in increased columbic repulsion between the positive charges, which eventually results in fragmentation of the droplets and the generation of smaller droplets [32]. Droplet Formation Electrospray is the dispersion of a liquid into electrically charged droplets and, as such, combines two processes; droplet formation and droplet charging. The formation of small, micrometer-sized droplets does not present a problem if the liquids flow-rate, surface tension and electrolyte concentration are low. An increase in one or more of these variables makes it more difficult for the electric field to produce the desired charged aerosol for MS. The electric 23

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field strength at the sprayer tip can be increased to try and overcome the aforementioned three variables, but too high an electric field give rise to an electric discharge that accompanies the electrospray process. A discharge can be tolerated in some electrospray nebulization applications, but is detrimental in electrospray mass spectrometry. Electric discharge is particularly troublesome in the formation of negatively. In the negative-ion mode, the sprayer tip is at a high negative potential with respect to other parts of the source, and field emission of electrons from the sharp spray needle or from the sharp tip of the solvent front (the Taylor cone, [31]) is a facile process. Electrons are accelerated by the electric field between the sprayer and the surrounding source walls and ionize the mixture of gases and solvent vapors in the source. Droplet Charging From a macroscopic viewpoint, it is sufficient to assume excess positive charge to be present in the liquid front to produce the formation of a positively charged droplet. From a chemical viewpoint, it is necessary to define the mechanism of charged droplet formation and its relationship to the composition of the sample solution, which is, in turn, strongly dependent on the composition of the eluent used for the high performance liquid chromatographic separation. Does positive charge on a droplet imply that positive charge was indeed supplied to the liquid front? The supply of positive charge is possible if a metal spray capillary gradually dissolves, with concomitant formation of metal ions. Although this process can indeed take place, it is of minor importance in the practice of on-line LCMS or CEMS [31]. Under the majority of experimental LC-MS and CE-MS conditions, positive charge on droplets is generated by the removal of negative charge via electrochemical discharge of negative ions against the metal wall of the spray capillary. Under special conditions, electrons can be removed from sample molecules having very low ionization energy. When positively charged droplets hit the opposite 24

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plate, electrons are consumed to neutralize the positive ions in the droplet. As such, the ESI source is a special case of an electrolysis cell. When the ESI source is set up for the detection of negative ions, all power supplies are at reversed polarity and current flows in the opposite direction. Removal of positive ions inside the tip of the spray capillary provides droplets that are depleted of positive charge. The supply of negative charge to the solution may also take place; electrons released from the spray capillary can be captured by molecules having a high electron affinity. Droplet Disintegration When droplets separate from the liquid front at the tip of the spray capillary, electric repulsion has become larger than the cohesive force that keeps the liquid together. During its flight through gas at atmospheric pressure, the droplet undergoes size reduction by evaporation of solvent, so that charge density at the droplet surface increases. Furthermore, the droplets are subjected to shear forces by their flight through dense gas. As a result of both effects, the droplets undergo deformation, which leads to local high electric fields at protrusions on the surface. In cases where sufficient deformation and charge density electrostatic repulsion exceed the surface tension, the droplet becomes unstable and falls apart. The upper limit to charge on a droplet is called the Rayleigh stability limit [31]. Local deformation at the droplet surface may turn into a protrusion from which a small jet of microdroplets leaves the original parent droplet [31]. Further Mechanistic Details Related to Ion Formation in ESI There is still much debate on the mechanism(s) by which those gaseous ions are formed [33]. The two most favored of these mechanisms are embodied in the charged residue model (CRM) originally proposed by Malcolm Dole et al. in 1968 and 1970 [34, 35] and the ion evaporation model (IEM) suggested by Iribarne and Thomson in 1975 [36]. The CRM model 25

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suggested that singly ionized sample molecules remain after continuous solvent evaporation and droplet fragmentation. Alternatively, Iribane and Thomson proposed that ionized sample molecules are expelled from the droplets. A representation of the possible pathways for ion formation from a charged liquid droplet is shown in Figure 1-4. The CRM has its roots in a theoretical paper published by Lord Rayleigh [37] (John Williams Strutts) in 1882 [37]. In that paper, Rayleigh addressed the question of what would happen as solvent evaporates from a droplet of volatile liquid containing an excess of either anions or cations. He reasoned that the repulsive forces between those excess charges of like sign would cause their associated ions to be situated at equidistant intervals on the surface of the droplet. As the droplet size decreased by evaporation of solvent, those surface ions would get closer and closer together until the integral over the droplet surface of the coulomb repulsion forces between those surface ions would exceed the integral of surface tension of the droplet liquid over that same area. At that point (Rayleigh limit), the droplet would increase the available surface area by breaking up into a plurality of smaller offspring droplets. These offspring droplets also would undergo solvent evaporation until they, too, would reach the Rayleigh limit and subdivide into still smaller droplets. Dole et al.s [34, 35] idea was that a succession of such subdivisions of the original droplets would eventually lead to the formation of ultimate droplets so small that each of them would contain only one solute molecule. As the last solvent molecules evaporated from such an ultimate droplet, the residual solute molecule would retain some or all of the charges on that droplet to become a gas-phase solute ion. They also realized that this scenario might make possible the production of gas-phase ions of molecules too large to be ionized by the then-customary procedures based on gas-phase encounters between neutral molecules and sufficiently energetic electrons, photons, or other ions. The large oligomer 26

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molecules in which they were interested simply could not be vaporized by the usual methods without undergoing catastrophic thermal decomposition. In 1968 and 1970, Dole et al. [34, 35] published the results of their attempts to produce and mass analyze ions of polystyrene oligomers by means of this ESI technique. For several reasons, not realized until later, the molecular weight values they obtained were at odds with what was known about the probable values for those oligomers. Consequently, their results did not persuade other investigators to follow their lead until some years later. Sometime after Dole et al.s first two papers [34, 35] on ESI, Iribarne and Thomson [36] experimented briefly with the technique and in 1975 offered a somewhat different explanation for the possible production of gas-phase ions by evaporation of solvent from charged liquid droplets [36]. They argued that before a charged droplet became small enough to contain only one solute molecule, the charge density on its surface would become so high that the resulting field would be sufficiently intense to push one or more of those surface ions into the ambient gas, thereby forming gaseous ions of at least some of those solute molecules. Continued evaporation of solvent from successive generations of such charged droplets thus would ultimately result in driving many, if not most, of the surface cations (or anions) on the original droplets into the gas phase. The diagrams in Figure 1-4 attempt to illustrate schematically these two possible mechanisms by which nonvolatile solute species in a charged droplet could become free ions in the ambient gas. In the years after Dole et al.s papers [34, 35], there were several attempts to develop a satisfactory ionization technique based on these charged droplet scenarios but none of them were very successful. Then in the 1980s, building on the ideas of Dole et al. [34, 35] and Iribarne and Thomson [36], and drawing on Dr. Fenns group extensive studies on the free jet expansion of gases from relatively high pressure into vacuum, Fenns group, then at 27

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Yale, found the right combination of conditions and demonstrated the successful production and mass analysis of intact solute ions from molecules having a wide range of molecular weights [24, 38]. This success spawned much argument and discussion on the mechanisms by which such ions could be formed [33]. Over the past few years, the ion formation mechanism for a number of molecular species having a wide range of compositions and molecular weights has been investigated [39, 40]. After careful consideration of all of the results, it has been recently concluded by Prof. Fenns group [33] that for most, if not all, cases in which ESI is effective, gas-phase solute ions are formed from charged droplets according to the sequence of events described in the IEM of Iribarne and Thomson [36]. However, in the case of very large parent species, including polyethyleneglycol (PEGs) polymers with molecular masses as high as 5,000,000 Da, they believe that the CRM of Dole et al. [34, 35] may comprise the more likely ionization scenario [40]. Advantages and Disadvantages of ESI Advantages of ESI are as follows: a) analysis of compounds with a molecular weight up to about 310,000 amu is possible [41]; b) ESI is very sensitive; typical sample amounts range from low amol to pmol levels [42]; c) ESI is a very mild ionization technique; usually only sample molecules carrying multiple protons are generated. It is possible to observe native biological complexes bound by noncovalent interactions. Fragmentation can be induced by increasing the potential difference in the ion source; d) the mass accuracy is very good; errors of 0.05% or less are common without an internal standard; e) on-line coupling with liquid chromatography or capillary electrophoresis (CE) equipment is possible, because ESI is a solvent-based ionization technique. Disadvantages of ESI are as follows: a) analysis of mixtures is difficult, because each compound gives rise to several signals corresponding to sample molecules carrying a range of 28

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protons. Of course, LC or CE separation before sample ionization is the preferred method; b) the presence of salts, buffers, detergents, and other additives reduces the sensitivity dramatically. If buffers are needed, volatile buffers, such as ammonium acetate or ammonium formate are preferred. Liquid Chromatography Highly efficient separations are the cornerstone of many analytical methods that deal with extremely complex mixtures. Examples are the analysis of the metabolome of a cell, tissue or body fluid, which are currently the focus of many research activities in academic groups and pharmaceutical companies alike. Metabolomic studies continue to foster the development of novel technologies able to comprehensively analyze large sets of metabolites. These new analytical tools need to be fast and of sufficient resolving power to detect minute qualitative and quantitative changes in a metabolic profile. Monitoring such changes could lead to interesting new drug targets or diagnostic tools. Ultra High Pressure Liquid Chromatography High performance liquid chromatography has traditionally been performed in columns packed with 3 or 5 m particle diameters. The internal diameter of these columns is typically between 2 and 4.6 mm, although smaller column diameters are gaining popularity. High separation efficiency with a concomitant reduction in analysis time is achieved by reducing particle size [43]. Chromatographic packing materials with diameters in the range of 1-2 m are now commercially available. UHPLC uses the same separation methodology as conventional HPLC, but typically uses columns packed with particles smaller than 2m. These smaller particles dramatically increase column efficiency, which in turn increases mass sensitivity, analytical resolution, and speed. 29

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While instrumentation for UHPLC requires components similar to conventional HPLC, smaller column particle size results in higher system pressures and requires components, such as pumps, with high pressure ratings. In addition, the ideal pump should provide flow rates that are pulseless and stable. Band broadening model. The key to understanding the importance of using smaller column packing materials follows from the van Deemter equation [44], which is fundamental to chromatographic theory. According to the van Deemter equation: H = A + B/ + C (1-1) where: H is the columns plate height, and is the mobile phase linear flow rate. A, B, and C are constants. A smaller plate height H indicates higher separation power. The A term. The constant A is the eddy diffusion term. Eddy diffusion results from multiple flow paths in the column and is independent of mobile phase flow rate. Due to the shape of the packing particles, analyte molecules can follow multiple pathways of differing path lengths. These multiple pathways of differing length spread the analyte molecules apart and cause peak broadening. Voids in the stationary phase can further contribute to peak broadening due to channeling. In contrast, smaller particles offer smaller differences in path length, thus reducing peak broadening. The A term depends on the compactness of the stationary phase. The B term. The constant B is the longitudinal diffusion coefficient. It is related to the diffusion coefficient of the analyte molecules in the mobile phase. Shorter residence time of the analyte molecules in the column reduces peak broadening due to longitudinal diffusion. Faster mobile phase flow rates reduce residence time. This reduction contributes to better separation efficiencies, since the analyte molecules have less opportunity to diffuse, thus explaining the 1/ factor in this term. 30

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The C term. The constant C is the analyte mass transfer coefficient. It is related to the time needed for the analyte molecules to equilibrate between the mobile and stationary phases. If this equilibration is too slow, then some of the analyte molecules, which did not have enough time to bond to the stationary phase, will flow down the column with the mobile phase; whereas, the other molecules, which did not have enough time to detach from the stationary phase, are left behind. Higher mobile phase flow rates will contribute to the spreading of the analyte molecules, thus explaining the factor in this term. Smaller stationary phase particles are expected to reduce this equilibration time. It follows that smaller stationary phase particles should contribute to smaller A and C values. General advantages of smaller particles. The A term will contribute less to H and allow for higher resolution. As the C term becomes less significant to the value of H, an increase in mobile phase flow rates will not sacrifice separation performance as much. This would allow for faster separations with the same resolution. Even though smaller column particles may not directly affect the B value, the higher flow rates reduce this contribution to H. Smaller column packing material would allow for faster separations with higher resolution. Disadvantages. Smaller beads will pack with smaller interstitial spaces and offer higher resistance to solvent flow. In turn, this requires higher driving pressures. For optimal separation flow rates, column back pressure increases as an inverse cubic function of the stationary phase particle size. Typically, pressures of 20,000 psi and above are required, hence the name ultra high pressure liquid chromatography or UHPLC for short [45]. These smaller stationary phase particles can be packed in either capillary columns or in stainless steel columns. Capillary columns offer less solvent consumption and can assay tiny amounts of sample. Due to very low 31

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mobile phase consumption, capillary columns interface more naturally with mass spectrometer detectors. With the higher flow rates and pressure drops across the columns, Joule heating can occur in the column during an UHPLC analysis. The power of the generated heat is the product of the pressure drop and volumetric flow rate. Capillary columns are better for dissipating such heat due to their greater surface area to volume aspect ratio and lower volumetric flow rate (L min -1 ) for a given linear flow rate (mm sec -1 ). As a result of heating, stainless steel column types tend to be limited to microbore (< 2mm diameter bore) [45]. Stainless steel columns offer the advantage of assaying larger sample sizes due to higher carbon load, which in turn aids in detecting and quantifying minor components in a mixture. The choice of capillary or stainless steel columns gives UHPLC added flexibility. Despite the engineering challenges associated with using ultra-high pressures, micron-sized particles have been shown to be a viable route towards increasing speed and resolution in reversed-phase liquid chromatography. Additional modes of liquid chromatography, such as ion exchange, normal phase and size exclusion, should be investigated under ultra-high pressure conditions. Most critical to the development of this field, however, is the introduction of reliable commercial equipment capable of operation under ultra-high pressure conditions. While 7,000 bar (100,000 psi) is perhaps too ambitious a goal for commercial equipment in the immediate future, a commercial system capable of pressures of 2,000 bar (30,000 psi) should be a reasonable target for the near term. Such a system would permit the use of fairly long and efficient capillary columns packed with 1 m particles, and would provide a very significant and overdue improvement in the separation power of LC columns. 32

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Monolithic Columns Investigations and developments into new separation media continue to play a major role in separation sciences. Emerging technology is enabling very high separation efficiency and speed of analysis, surpassing the conventional particle-packed columns in high performance liquid chromatography. These included capillary electrochromatography [46], ultra high pressure liquid chromatography [47-49], and the use of monolithic columns [50, 51]. The word monolith was introduced by the Greeks a very long time ago and related mostly to large stony items (monolithos = single stone) [52]. Monolithic stationary phases are separation media in the format that can be compared to a single large particle that does not contain interparticular voids. As a result, all the mobile phase must flow through the stationary phase. In contrast to diffusion, which is the typical driving force for mass transfer within the pores of particulate stationary phases during chromatographic processes, convective flow through the pores enables a substantial increase in the speed of separation [53]. Monolithic columns have high column permeability and small-sized skeletons that decrease the diffusion path length of molecules in the stationary phase, resulting in a reduced contribution of both the Aand C-terms to band broadening [54]; thus, van Deemter curves for some monolithic columns are much flatter at high flow rates compared to conventional columns. These features allow the operation of monolithic columns at very high flow rates for fast separations with no significant loss in efficiency [54-57]. Interest in monolithic technology is reflected in the number of papers published per year, which has grown continuously since 1996 as shown in Figure 1-5. Based on the nature of their construction materials, monolithic columns can be classified as organic polymeror silica-based columns [50]. Silica and synthetic organic polymers have been utilized together with one of two different technologies: (i) packing with beads followed by 33

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their fixation to form a monolithic structure and (ii) the preparation of the monolith from low molecular weight compounds in situ. Interestingly, the organic polymer-based monoliths have always done a better job of separating larger molecules, such as proteins, nucleic acids, and synthetic polymers; silica-based monolithic columns enable fast separations of smaller molecules [50]. Thus, these two column technologies are complementary [53]. Polymer-based monolithic column. Originally, the highly crosslinked monolithic material was prepared from a polymerization mixture consisting of monovinyl and divinyl methacrylate monomers, a free-radical initiator, and a porogenic solvent in a flat or tubular mold that yielded a sheet or cylinder. The porous monolithic polymer was then removed from the mold, punched or sliced to obtain thin disks that were placed in a cartridge, and used for chromatographic separations. Continuing research led to the invention of monoliths in typical column formats; the monomers used were extended to styrene, divinylbenzene, and their derivatives [58]. In contrast to the disks mentioned earlier, these monoliths were polymerized in situ within a chromatographic tube or other tube, in which they remained after preparation was complete. Thanks to the slow thermally initiated polymerization in a vertical position, the initially formed cross-linked polymer nuclei could settle; thus, the liquid polymerization mixture was left on the top and around the solid phase. This mechanism readily compensated for the radial shrinkage that would otherwise create voids at the monolithcolumnwall interface and be deleterious to the chromatographic function of the monolithic column [59]. At the same time, Hjertn and his colleagues developed a process that led to the production of a highly crosslinked acrylamide-based matrix [60]. In their approach, a highly swollen cross-linked gel was prepared by the polymerization of aqueous solutions of N,N-methylene bisacrylamide and acrylic acid in the presence of a salt, typically ammonium sulfate; the gel was massively compressed to a 34

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fraction of its original volume. Such a compressed gel completely filled the cross section of the column and exhibited very good permeability to flow, despite the high degree of compression [61]. Silica-based monolithic column. Tanaka et al. made the next contribution to modern monolithic materials, finding their inspiration in a very popular inorganic support widely used in chromatography-silica [62]. In contrast to columns made of organic polymers, silica-based monolithic columns in typical analytical sizes cannot be prepared in situ because of the significant shrinkage when the material solidifies. Early studies with all these columns clearly demonstrated extremely fast chromatographic separations at high flow rates and at reasonably low back pressure. These advantageous features made monolithic columns particularly suitable for high-throughput applications [63]. Monolithic silica columns exhibit a tailor-made bimodal pore structure with both macropores or through pores and mesopores [64]. The large macropores are responsible for a low flow resistance and therefore allow the application of high eluent flow rate, while the small pores ensure sufficient surface area (300 m 2 /g approximately) for separation efficiency. Monolithic columns also have a significantly higher total porosity compared to conventional particulate columns, over 80% vs. ca. 65%, respectively. The most unique feature of these columns is their high permeability, which is nearly twice as high as that of packed columns [65]. Therefore, monolithic silica columns can be operated at high flow rates of up to 10 mL min -1 thus allowing fast separations of various mixtures [66]. Many applications, originally developed using packed columns, can be performed with a monolith while reducing the analysis time by a factor of 5. Monolithic silica columns are suitable for high throughput analysis as well as for two-dimensional HPLC methods [63]. 35

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Separation performance of silica-based monolithic columns and packed columns. Many authors have compared monolithic silica columns and conventional packed silica columns with respect to their physical and chromatographic properties [67-69]. All of them came to the conclusion that both types of the silica-based columns are comparable with respect to performance, selectivity, and reproducibility. Some authors even claim that monolithic silica columns are more stable than packed ones due to the rigid silica structure. Therefore, monolithic silica columns seem to display a great promise for the near future because further improvements may lead to enhanced efficiency which is needed in the challenging field of high throughput and bioanalytical analysis. Carnitine and Acylcarnitines Carnitine, an endogenous compound present in most mammalian tissues, is involved in the transport of activated fatty acids between cellular organelles and thus plays an important role in fatty acid metabolism and in cellular energy production. Carnitine binds fatty acids, generating various acylcarnitines with different chain lengths. Carnitine is also implicated in the maintenance of the cellular pool of free coenzyme A and in the elimination of potentially toxic acyl-CoA. In mammals, two-thirds of carnitine is provided by dietary intake and one-third by biosynthesis from the amino acids L-lysine and L-methionine. Since carnitine is present in most body tissues at much higher concentrations than in plasma, transport systems ensure its widespread distribution from sites of absorption and synthesis throughout the body. In many metabolic disorders, carnitine metabolism is greatly disturbed, leading to a redistribution of the carnitine and acylcarnitine pools. The determination of individual acylcarnitines in biological fluids is a powerful means to diagnose and monitor these disorders. 36

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General Aspects Carnitine (3-hydroxy-4-(N,N,N-trimethylammonio) butanoate) is a ubiquitous molecule within mammalian tissue, mainly located in skeletal muscle and heart [70-72], which was first discovered in skeletal muscle extracts in 1905 [73, 74]. Its chemical structure C 7 H 15 NO 3 was established in 1927 [75]. Carnitine is a small, water soluble, quaternary nitrogen-containing compound (Figure 1-6) that is present in both Land Dforms, L-carnitine being the biologically active form [76]. In humans, about 25% of L-carnitine is synthesized in liver, kidney, and brain from the amino acids lysine and methionine and ascorbic acid as a cofactor, but it can also be found in nutritional sources such as red meats and dairy products. The daily requirement for exogenous carnitine in humans is unknown [77]. Carnitines primary roles in the human body are in transporting long-chain fatty acids into the mitochondria for use as a fuel and buffering excess acyl-CoA accumulation within mitochondria [78]. Total carnitine (TC) is present as free carnitine (FC) and as esterified derivatives, or acylcarnitines (AC), with different chain lengths [79]. All these esters are equilibrated with L-carnitine by carnitine acyl transferase catalysis, with different specificities for each substrate [80]. Among these enzymes, most investigations have been carried out on carnitine acetyl transferase (CAT), carnitine octanoyl transferase (COT), and carnitine palmitoyl transferase (CPT), which are active on short-, medium-, and long-chain esters, respectively [81]. Through the action of acyltransferases, carnitine and acylcarnitines are rapidly interconvertible. Clinical carnitine deficiencies are predominantly due to enzyme deficiencies rather than dietary insufficiencies, except in cases of severe protein and energy malnutrition [77]. Figure 1-7 depicts the interconversion of L-carnitine and its ester. Free carnitine is the major carnitine pool representative [82].The proportion of acylcarnitines, with the acyl moiety ranging from short-chain to long-chain varies with 37

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nutritional conditions, exercise, and diseases states [77]. Under normal metabolic conditions, acylcarnitines, which are mainly represented by acetylcarnitine, represent a mean value of 22% of the total carnitine pool in serum or plasma, 13% in muscle and liver and up to 60% in urine [83]. The relative amounts of acylcarnitine are often expressed as a ratio of acylcarnitine to free carnitine. Carnitine insufficiency can be diagnosed by measuring plasma and tissue carnitine concentrations [78]. Normal plasma concentration of total carnitine is 3090 M, free carnitine 26 M and acyl-carnitine esters 2 M [84-86] with TC concentrations lower than 20 M considered indicative of likely carnitine deficiency [87, 88]. A normal ratio of AC : FC in the fed state is less than 0.4, and ratios > 0.4 are believed to be indicative of a certain degree of limited FC availability [89]. Normal subjects show a large interand intra-individual variation in urinary excretion of carnitine (urinary FC 22 mol/day) [90, 91]. Role of Carnitine Carnitine is implicated in the transport of activated long-chain fatty acids from the cytosol, across the mitochondrial membranes, towards the mitochondrial matrix, where -oxidation takes place [92]. Fatty acid metabolism occurs in the mitochondrial matrix; however, the mitochondrial inner membrane is impermeable to fatty acids. Carnitine, by binding fatty acids, is essential in the transport of long-chain fatty acids into the mitochondrial matrix. Short, medium, and long-chain free fatty acids are transported through cell compartments as acylcarnitines esters (Figure 1-8). Transfer of the latter from peroxisomes to mitochondria is essential for cell energy production and membrane stabilization [92]. Cytosolic long-chain fatty acids are first activated by palmitoyl-CoA synthetase, located in the outer leaflet of the outer mitochondrial membrane, to acylcoenzyme A-derivatives (Ac-CoA). Ac-CoA can cross the outer mitochondrial membrane but needs to be converted to carnitine derivatives to be able to pass the inner mitochondrial membrane. For this purpose, the 38

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enzyme carnitine palmitoyltransferase I (CPT I), located in the inner side of the outer mitochondrial membrane, transesterifies the acyl-CoA to the respective acylcarnitines [93]. In this reaction, which is the rate-limiting step in the -oxidation of fatty acids and is inhibited by malonyl-CoA, the acyl moiety of the long-chain fatty acids is transferred from CoA to the hydroxyl group of carnitine. The long-chain acylcarnitines are then transported into the mitochondrial matrix by a specific transporter, carnitine-acylcarnitine translocase (CT), located in the inner mitochondrial membrane, which exchanges one long-chain acylcarnitine for one carnitine [94]. Once in the mitochondrial matrix, the enzyme carnitine palmitoyltransferase II (CPT II), located in the inner leaflet of the inner mitochondrial matrix, back converts the long-chain acylcarnitines into the respective long-chain acyl-CoAs. The released acyl-CoAs can undergo -oxidation and enter the respiratory chain within the matrix. The released carnitine can leave the mitochondrion via the above-mentioned translocase or be converted to acylcarnitines. Carnitine is the substrate for reactions catalyzed by carnitine acyltransferases that convert acyl-CoA to the corresponding acylcarnitine and free coenzyme A (CoASH), according to the following reversible reaction: Acyl-CoA + carnitine Acylcarnitine + CoASH (1-2) The L-carnitine pool reflects the CoA pool. This reversible exchange allows the cell to regulate its levels of free CoA using carnitine as a buffer, and, since carnitine is in most tissues in a much higher concentration than CoA, the extramitochondrial acetylcarnitine/carnitine ratio will prevent great fluctuations in the mitochondrial acetyl-CoA/CoA ratio by formation of acyl-CoA [95]. The carnitine buffering effect can be extended to the regulation of poorly metabolized and potentially toxic acyl-groups, resulting either from xenobiotics (e.g. pivalic acid and valproate) 39

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[96] or from blockage of a normal metabolic pathway (e.g. propionic acid in propionic academia [97]. These acyl-groups are converted to CoA-derivatives, thus depleting the cellular pool of free CoA. The subsequent transesterification of these acyl-CoAs to the corresponding acylcarnitines, followed by their excretion in the urine [98], replenish the free CoA pool, but lead to a carnitine deficiency that can be reversed through carnitine supplementation [99]. Deficiencies in Carnitine Based on the etiology, carnitine deficiencies are classified as primary and secondary [84, 88]. Primary carnitine deficiencies are genetic disorders of carnitine transport which include muscular and systemic carnitine deficiencies. The muscular form is characterized by progressive muscle weakness and lipid storage myopathy in type I fibers. The carnitine content in patients skeletal muscle is reduced but is normal in plasma and the liver [100]. The systemic form is characterized by low plasma and tissue (heart, skeletal, muscle, liver) carnitine concentrations. This deficiency is due to a defect in the specific high-affinity carnitine transporter that is expressed in fibroblasts, muscle, heart, and kidney. The secondary carnitine deficiency results from the high urinary excretion of carnitine esters. It is associated with (i) impaired oxidation of acyl-CoAs in mitochondria, (ii) defects in the mitochondrial respiratory chain, and (iii) excessive renal loss of carnitine. Deficiencies in carnitine lead to impaired transport of fatty acids into the mitochondria for oxidation. This can occur in (i) newborns and particularly in pre-term infants, (ii) in patients undergoing hemodialysis or exhibiting organic aciduria, and (iii) in pediatric cancer patients due to the neoplastic processes or the therapy, etc [101]. This thesis focuses on the development of novel chromatographic approaches and optimization of the mass spectrometer experimental conditions to be used for the separation and determination of carnitine and acylcarnitine. Commercially available monolithicand small particle size packed-columns for the separation of the analytes under study were used. In 40

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addition, an oa-TOF mass spectrometer was utilized for the accurate mass analyses of the mentioned above metabolites. Data were obtained in traditional positive electrospray ionization mode. The optimization of analytical procedures by multivariate techniques is faster, more economical and effective than the traditional one-at-a-time methodology. The multivariate optimization makes possible to understand circumstances that are not explained by the traditional approach, for example, the interactions between the factors that influence the analytical response. As part of our study, the optimization step of the MS parameters was performed using a 2-level full factorial design and this information is presented in Chapter 2. There are two emerging column technologies that have attracted much attention in recent years, one is the monolithic column, and the other is the use of small particle packed columns (sub-2-m particles). Herein, we explore these two chromatographic approaches. A comparison between the efficiency of a commercially available monolithic and a small particle packed columns for the carnitine and acylcarnitines separation was carried out. In addition, the application of the optimized chromatographic approaches as well as the MS parameters to the determination of the mentioned compounds in plasma samples is discussed in Chapter 3. Finally, Chapter 4 summarizes the work presented here, as well as some potential future research directions. 41

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Table 1-1. Historical developments in mass spectrometry (adapted from reference [13]) Investigator (s) Year Contribution J.J. Thomson 1899 First mass spectrometer A.J. Dempster 1918 Electron ionization and magnetic focusing F.W. Aston 1919 Atomic weights using MS W.E. Stephens 1946 Time-of-flight mass analysis H. Sommer, H.A. Thomas, J.A. Hipple 1949 Ion cyclotron resonance E.G. Johnson and A.O. Nier 1953 Double-focusing instruments W. Paul and H.S. Steinwedel 1953 Quadrupole analyzers J. Beynon 1956 High-resolution MS K. Biemann, C. Cone, B.R. Webster, and G.P. Arsenault 1966 Peptide sequencing M.S.B. Munson and F.H. Field 1966 Chemical ionization R.B. Dole 1968 Electrospray ionization H.D. Beckey 1969 Field desorption MS R.D. McFarlane and D.F. Torgesson 1974 Plasma desorption MS M.B. Comisarow and A.G. Marshall 1974 Fourier-transform ion cyclotron resonance mass spectrometry R.A. Yost and C.G. Enke 1978 Triple quadrupole MS M. Barber 1981 Fast atom bombardment K. Tanaka, M. Karas, and F. Hillenkamp 1983 Matrix-assisted laser desorption/ionization J. Fenn 1984 ESI on biomolecules S.K. Chowdhury, V. Katta and B.T. Chait 1990 Protein conformational changes (ESI-MS) M. Mann and M. Wilm 1991 MicroESI B. Ganem, Y.T. Li, and J.D. Henion B.T. Chait and V. Katta 1991 Noncovalent complexes with ESI-MS U. Pieles, W. Zurcher, M. Schar, and H.E. Moser 1993 Oligonucleotide ladder sequencing W.J. Henzel, T.M. Billeci, J.T. Stults, S.C. Wong, C. Grimley, and C. Watanabe 1993 Protein mass mapping G. Siuzdak, B. Bothner, S. Fuerstenau, and W.H. Benner 1996-2001 Intact viral analysis R. Caprioli 1999 Imaging mass spectrometry R.G. Cooks et al. 2004 Ambient ionization techniques (DESI) R.B. Cody et al. 2005 Ambient Ionization techniques (DART) 42

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43 Figure 1-1. The omics cascade with its different omic levels. It is shown in this hierarchical structure that the metabolome level is the most predictive of phenotype. Adapted from reference [1].

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44 Figure 1-2. Main components and layout of typical oa-TOF systems with reflecting mass analyzer. The ion beam enters from a source at the left. For positive ions, the beam is formed literally, or as though it originated from a source held at a positive potential of V beam and accelerated to enter the orthogonal accelerator (oa) at 0V. The beam optics make the beam more parallel before it enters the oa. The beam fills the first stage of the oa which is also at 0V until a bipolar push-out pulse pair is applied. A packet of ions of length l p is thus sampled and accelerated through grids to enter the drift region at a potential of V TOF Conventional reflecting TOF optics are used to bring the ions to a space-time focus on the detector. In this case the ion mirror shown has one stage though two stages could also be used. During the time that the ions are in the drift-region (and ion mirror) the oa is refilled with new beam. In the drift time for an ion of a particular m/z, isobaric ions in the beam travel a distance of l b The distances l b and l p together with the transmission of grids (G1G4) are primary factors that determine the mass analyzer efficiency of the instrument. Adapted from reference [22].

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45 Figure 1-3. Schematic representation of a generic electrospray ionization interface. Adapted from reference [32].

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46 Figure 1-4. A schematic representation of the possible pathways for ion formation from a charged liquid droplet. The diagram illustrate the ion formation mechanisms depicted in the charged residue model (CRM) of Dole et al. [34, 35] and the ion evaporation model (IEM) of Iribarne and Thomson [36], respectively. + represents a desolvated solute ion. The major difference between these two models is that the final ion in the latter model is produced by desorption, whereas the ion in the former model is produced by evaporation of solvent comprising the droplet. Adapted from reference [33].

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47 Figure 1-5. Representation of the number of chromatographic monolith publications per year from 1996 to 2007 (obtained by searching monolith columns for chromatography using SciFinder, Version 2006, American Chemical Society). OOHOHN+CH3CH3CH3 Figure 1-6. Chemical structure of L-carnitine.

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48 Short-chainACYLCARNITINES L-CARNITINE ACETYL-L-CARNITINEPROPIONYL-L-CARNITINEOCTANONYL-L-CARNITINEPALMITOYL-L-CARNITINE Long-chainACYLCARNITINES Medium-chainACYLCARNITINESCATCATCPTCOT Short-chainACYLCARNITINES L-CARNITINE ACETYL-L-CARNITINEPROPIONYL-L-CARNITINEOCTANONYL-L-CARNITINEPALMITOYL-L-CARNITINE Long-chainACYLCARNITINES Medium-chainACYLCARNITINESCATCATCPTCOT Figure 1-7. Metabolism of L-carnitine. Acetylcarnitines are products of reactions catalyzed by carnitine acyltransferases that utilize acyl-CoA. CAT: Carnitine Acetyltransferase; COT: Carnitine Octanoyltransferase; CPT: Carnitine Palmitoyltransferase. Adapted from reference [79].

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Figure 1-8. Carnitine plays a major role in the entry of activated long-chain fatty acids from the cytosol into mitochondria and is also involved in the transport of activated mediumand short-chain organic acids from peroxisomes to mitochondria; the ratio of acetyl-coenzyme A to coenzyme A is maintained by carnitine, which functions as a pool of activated acetyl units; the toxic effects of poorly metabolized acetyl groups are lowered through transesterification from CoA and excretion of acetylcarnitine esters by carnitine palmitoyltransferases (CPT I & CPT II) and carnitine acetyltransferase (CAT); carnitine-acylcarnitine translocase (CT) permits the entry and exit of short-chain acetylcarnitine esters in and out of mitochondria; OMM: outer mitochondrial membrane; IMM inner mitochondrial membrane. Adapted from reference [92]. 49

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CHAPTER 2 OPTIMIZATION OF MASS SPECTROMETER INSTRUMENTAL PARAMETERS Historical Perspective and Basic Principles of Design of Experiments Experimentation is a structured approach to the investigation process in which the relationship between inputs to the process that can be manipulated (excluding noise) and outputs that are observed needs to be determine. Experimental design and optimization are tools that are used to systematically examine different types of problems that arise within, e.g., research, development and production. It is obvious that if experiments are performed randomly the result obtained will also be random. Therefore, it is a necessity to plan the experiments in such a way that the interesting information will be obtained [102]. Sir Ronald A. Fisher introduced the topic of design of experiments (DOE) in the context of agricultural experiments in early 1920s [103]. While Fisher did not introduce the idea of full factorial experiments he clarified its methodology and paved the way for others like Yates and Eden to define it more precisely [104]. The idea with the use of designed experiments that are set up in advance knowing they will be statistically analyzed is that 1) no unnecessary runs should be required to obtain useful information, and 2) interactions can be identified between factors (factors are the experimental variables that can be changed independently of each other). In DOE, the variables are systematically varied simultaneously in predetermined combinations, thus making it possible to study interaction between variables. Analysis of the design of the experiments is built on the use of the analysis of variance (ANOVA), which was developed by Fisher in 1925 [105] to address the problem of comparing means. This statistical technique is central to the analysis of data from designed experiments to discover how changes in input variables (factors) affect the response (output) variables. The analysis comes in two forms, one telling the user which factor, factors, or 50

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interactions are more important than others by allocating a percentage of the overall variance to each factor or interaction. Secondly, an equation relating response to factors, namely a regression equation, can be obtained. ANOVA can be used to examine differences among the means of several different groups at once. The experimental data can be interpreted using graphs as a complement to ANOVA [106]; such graphical approaches include Pareto charts. The standardized Pareto chart contains a bar for each effect, sorted from most significant to least significant. The length of each bar is proportional to the standardized effect, which equals the magnitude of the t-statistic that would be used to test the statistical significance of that effect. A vertical line is drawn at the location of the 0.05 critical value for the corresponding statistical test. Any bars that extend to the right of that line indicate effects that are statistically significant at the 5% significance level. There are three basic principles of experimental design: randomization, replication, and blocking. Randomization means that the allocation of the treatments and experimental units should be done randomly so that the variation caused by potential factors is reduced. Replication means that each treatment is applied to a number of experimental units from the same population under study. It allows the experimental error to be estimated as it provides degrees of freedom for error. Blocking is a statistical technique that is used to minimize variation caused by nuisance factors and improves the precision of difference of comparisons among the treatments. The use of these principles helps to ensure the objectivity of the experimental data, and consequently the validity of the conclusions that are drawn from the experimental data. Full Factorial Designs It is reasonable to assume that the outcome of an experiment is dependent on the experimental conditions. This means that the result can be described as a function based on the experimental variables, 51

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)(x f y (2-1) The function f(x) is approximated by a polynomial function and represents a good description of the relationship between the experimental variables (x) and the responses (y) within a limited experimental domain. The simplest polynomial model contains only linear terms and describes only the linear relationship between the experimental variables (x 1 and x 2 ) and the responses. In a linear model, the two variables x 1 and x 2 are expressed as: residual x b x bby 22110 (2-2) where the residual term expresses the difference between the calculated and the experimental result. The next level of polynomial models contains additional terms that describe the interaction between different experimental variables. Thus, a second order interaction model contains the following terms: residua l x x b x b x bby 211222110 (2-3) The two models above are mainly used to investigate the experimental system, i.e., with screening studies, robustness tests or similar tests. To be able to determine an optimum maximum or minimum, quadratic terms have to be introduced in the model. By introducing these terms in the model, it is possible to determine non-linear relationships between the experimental variables and responses. The polynomial function below describes a quadratic model with two variables: residualxxbxbxbxbxbby 21122222211122110 (2-4) The polynomial functions described above contain a number of unknown parameters (b 0 b 1 b 2 etc.) that are to be determined. For the different models different types of experimental designs are needed. 52

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In any experimental procedure, several experimental variables or factors may influence the result. A screening experiment is performed in order to determine the experimental variables and interactions that have significant influence on the result, measured in one or several responses. At this point, when the variables to be investigated are selected, it is also decided which variables that should not be investigated. These variables have to be kept at a fixed level in all experiments included in the experimental design. When a list of variables to be investigated has been completed, an experimental design is chosen in order to estimate the influence of the different variables on the result. In screening studies, linear or second order interaction models are common, such as in full factorial or fractional factorial designs. The former design is limited to the determination of linear influence of the variables, while the latter allows for interaction terms between variables to be evaluated as well. Eventually, the variables with the largest influence on the procedure can be identified. Fundamentals In a full factorial design the influences of all experimental variables, factors, and interaction effects on the response or responses are investigated. If the combinations of k factors are investigated at two levels, a factorial design will consist of 2 k experiments. For larger experiments involving a great number of factors it may be desirable to reduce the number of tests to a subset of the full factorial, this is typically called fractional factorial. The run matrix considers only the factors and levels that need to be tested; the experimental matrix additionally considers the model being estimated as well as a unit vector for the intercept term in the regression equation. When a factor is numerical and tested at two levels the coding of the factors is given by (minus) for the lower value and +1 (plus) for the higher value. A zero-value is also included, a center, in which the factor is set at its mid-value. Three or four center experiments should always be included in factorial designs, for the following reasons: 53

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a) the risk of missing non-linear relationships in the middle of the intervals is minimized, and b) repetition allows for determination of confidence intervals. What + and should correspond to for each factor is defined from what is assumed to be a reasonable variation to investigate. In this way the size of the experimental domain has been settled. For two and three factors the experimental domain and design can be illustrated in a simple way. For two variables the experiments will describe the corners in a quadrate while in a design with three variables they are the corners in a cube. Sign of Interaction Effects The sign for the interaction effect between variable 1 and variable 2 is defined as the sign for the product of variable 1 and variable 2. The signs are obtained according to normal multiplication rules. By using these rules it is possible to construct sign columns for all the interactions in factorial designs. Experimental Mass Spectrometer The instrument used for all experiments was an Agilent 6210 Time-of-flight mass spectrometer (Agilent Technologies Inc., Santa Clara, CA) configured with an ESI source; a schematic diagram of the time-of-flight mass spectrometer is shown in Figure 2-1. Ions were simultaneously generated from the sample eluting from an HPLC by the first capillary nebulizer and from reference compounds, introduced via the second capillary nebulizer. Sprayers were positioned 7.5 mm in front of the spray nozzle shield and 15 mm apart at an angle of 60 between the respective sprayer needle tips. The nebulizers were held at ground potential. Ions were then drawn into a metal-plated, glass dielectric capillary inlet (0.6mm aperture) by a combination of voltage (capillary voltage: 4.0 kV) and pressure differentials. Heated nitrogen drying gas (300 C) flowing at 10 L min -1 around the capillary inlet was used to desolvate the droplets and aid the 54

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formation of gas-phase ions. The fragmentor voltage (the voltage differential between the capillary exit and the skimmer) was set to 120 V to focus ions into the skimmer. An octopole ion guide confined ions in the second vacuum stage and transported them to a second ion guide traversing the third and fourth vacuum stages. The octopole radiofrequency (RF) was 250Vpp, ensuring transmission of a broad mass-to-charge range (60 amu). The ion beam was shaped in the fourth vacuum stage using a DC quadrupole lens. The flattened ion beam then passed through two slits before entering the ion pulser in the TOF analyzer vacuum stage (operating at a vacuum of 3.3 10 -5 Pa using a separate 255 L s -1 turbomolecular pump). Positive ions were injected into the two-stage ion pulser and orthogonally accelerated into the flight tube. The other end of the flight tube was attached to a two-stage reflectron, or ion mirror, that focused ions onto the detector. One-dimensional wire grids were used in both the ion pulser and mirror to optimize transmission and maintain beam focus. The active area of the microchannel plate ion detector amplified secondary electrons generated from ion impact and accelerated them to a scintillator. The resulting light was focused onto a high-speed photomultiplier tube photocathode to allow for its isolation from the flight tube voltage. The potential placed across the photomultiplier tube determined the final gain adjustment of the detector. Signal was acquired using a fast analog-to-digital converter (ADC); the ADC did not require any dead time corrections and was configured to record up to 10,000 transients per second. TOF Calibration An Agilent Technologies ES Tuning Mix (P/N G2421A) containing betaine (CAS #: 107-43-7, MW: 117.1), hexamethoxyphosphazene (CAS #: 957-13-1, MW: 321.0), hexakis(2,2-difluoroethoxy)phosphazene (CAS #: 186817-57-2, MW: 621.0), hexakis(1H, 1H, 3H55

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tetrafluoropropoxy)phosphazene (CAS #: 58943-98-9, MW: 921.9), hexakis(1H, 1H, 5H-octafluoropentoxy)phosphazene (CAS #: 16059-16-8, MW: 1521.0), hexakis(1H, 1H, 7H-dodecafluoroheptoxy)phosphazene (CAS #: 3830-74-8, MW: 2121.4), and hexakis(1H, 1H, 9H-perfluorononyloxy)phosphazene (CAS #: 186043-67-4, MW: 2720.1), provided mass reference ions for a baseline calibration of the mass axis. HPLC An Agilent Technologies 1200 Series Rapid Resolution LC system (Agilent Inc., Santa Clara, CA) equipped with a binary pump provided solvent delivery and separations. Two columns, a Zorbax Eclipse XDB-C 18 column (Agilent Technologies, Santa Clara, CA), 50 mm 4.6 mm, with 1.8 m particle diameter and a C 18 -Phenomenex Onyx monolithic column (Phenomenex Inc., Torrance, CA), 100 mm 4.6 mm, were employed for separation of the analytes under study. All chemicals were obtained from Burdick and Jackson (Muskegon, MI). Standard Solutions Laboratory-made standards for carnitine and acylcarnitines were used for all the experiments (the synthesis procedure is detailed in Appendix-a). Stock standard solutions for each analyte were prepared by dissolving an accurately weighed mass of the standard in methanol. The prepared stock solutions were stored at 4C. A list containing the chemical information for the analytes under study as well as the mass of its pseudo-molecular ions in positive ESI mode is detailed in Table 2-1. A mixed standard solution was prepared by mixing exactly known volumes of the different stock standard solutions to create a final solution which was 1.0 mg L -1 in each analyte. This mixed solution was prepared freshly on the day of analysis. 56

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Whenever necessary, the working solutions used in the calibration, containing a mixture of all analytes under study, were prepared by dilution of the corresponding stock solutions. Solutions for calibration ranged from 5 to 1000 g L -1 in each analyte. Plasma Sample Preparation Piglet plasma samples were treated according to the Standard Operating Procedure (SOP) established to provide standards that are used in the Metabolic Assessment Laboratory (MAL) -under the direction of Dr. Peggy Borum in Food Science and Human Nutrition at the University of Floridaas required by Section 58.81 of the Good Laboratory Practice for Non-clinical Laboratory [107] (the sample preparation procedure is described in Appendix-b-d.) In order to favor the dissolution of the polar and less polar compounds, for MS analysis the plasma samples were solubilized with a mixture of 20 L of water, 140 L of methanol, and 140 L of acetonitrile. The samples were split into two 150 L aliquots. One aliquot was spiked with a known volume (concentration) of the mixture of standards; the standard solution containing the mixture of analytes was placed in the appropriate vial for MS analysis, evaporated under a gentle stream of nitrogen until dryness, and then the plasma aliquot was added to the vial. Blank solutions were prepared in the same way. It is important to notice that no derivatization procedure was used before or after column separation for the analytes under study. This fact made an essential difference between our procedure and those approaches reported in the literature for the separation and/or determination of carnitine and carnitine-based compounds in clinical samples [108]. 57

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Results and Discussion Considering the MS parameters, a two-level full factorial design with 8 runs (2 3 ) and 3 replicates of the central point was developed in order to determine the influence of the factors and their interactions on the system. Three factors were studied: nebulizer pressure (NP), drying gas flow rate (DGFR), and drying gas temperature (DGT). The factorial design was evaluated using the mass spectrometer signal (peak area counts) as analytical response. In this sense, the analytical response related to the peak area of three different compounds was taking into consideration. Based on their chemical properties, an analyte from each of the three main groups (-short, medium-, and long-chain) of carnitine and acylcarnitine compounds was selected; thus, carnitine, hexanoylcarnitine, and decanoylcarnitine were used as the representative compound of each group. In addition, based on the results obtained from previous experiments, the value for the fragmentation voltage (voltage differential between the capillary exit and the skimmer) was fixed at 120 V to achieve the best transmission and the minimum fragmentation for all the analytes, regardless of their chemical structure. Minimum and maximum levels of each factor as shown in Table 2-2 were chosen according to preliminary experimental data. The experimental sequence of the experiments, matrix design, and the analytical signal (the relative response (%) was calculated taking the maximum value obtained as 100%; and the remaining values showed in the table were calculated from it) are shown in Table 2-3, The experiments were carried out in a random order. Significances of the effects were checked and evaluated by ANOVA (Table 2-4, 2-5, and 2-6) and the standardized Pareto charts (Figures 2-2, 2-3, and 2-4). In these charts, the effects are plotted sorted (in absolute value) in descending order. The probability value (p) is used to determine the significance of the individual factors and their interactions. In hypothesis testing, 58

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the probability value (p-value) is the probability of obtaining a statistic different from the parameter () specified in the null hypothesis ( 1 2 = 0) as the statistic obtained in the experiment. The probability value is computed assuming the null hypothesis is true. If the probability value is below the significance level traditionally, experimenters use the 0.05 level (5% level) then the null hypothesis is rejected. In summary, if the probability is less than or equal to the significance level, then the null hypothesis is rejected; if the probability is greater than the significance level then the null hypothesis is not rejected. When the null hypothesis is rejected, the outcome is said to be "statistically significant"; when the null hypothesis is not rejected then the outcome is said be "not statistically significant." If the outcome is statistically significant, then the null hypothesis is rejected in favor of the alternative hypothesis ( 1 2 ). The smaller the p-value, the more significant is the factor. The results of this study demonstrated that, in the studied levels, all these variables and their interactions are not statistically significant (p > 0.05). This way, the nebulizer pressure recommended is 50 psi, the drying gas flow rate chosen is 10 L min -1 and the drying gas temperature selected is 300 C. A list containing all the ESI-TOF parameters used for the determination of carnitine and carnitine-based compounds is depicted in Table 2-7. Conclusions Factorial design approach is a useful tool to establish and improve analytical procedures. Although it seems more complex than the univariate procedure from the operative point of view, it is advantageous since it makes use of fewer experiments and provides important information on interactions among the studied variables. 59

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The application of a 2-level full factorial design allowed the optimization of parameters that influence the performance of the mass spectrometer. Employing the ANOVA and Pareto charts, it was possible to evaluate the influence of each variable and the combination of variables in the analytical response obtained. The conditions selected in the study allowed the maximization of the analytical response of the mass spectrometer for the representative group of compounds. 60

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Table 2-1. Chemical formula, structure, and pseudomolecular ion of carnitine and acylcarnitines # R ACYLCARNITINES Structure Theoretical [M+H] + C 0 OH Free Carnitine 162.1125 C 2 OCOCH 3 Acetylcarnitine 204.1230 C 3 OCOCH 2 CH 3 Propionylcarnitine 218.1387 C 4 OCO(CH 2 ) 2 CH 3 Butyrylcarnitine 232.1543 C 6 OCO(CH 2 ) 4 CH 3 Hexanoylcarnitine 260.1856 C 8 OCO(CH 2 ) 6 CH 3 Octanoylcarnitine 288.2169 C 10 OCO(CH 2 ) 8 CH 3 Decanoylcarnitine 316.2482 C 12 OCO(CH 2 ) 10 CH 3 Lauroylcarnitine 344.2795 C 14 OCO(CH 2 ) 12 CH 3 Myristoylcarnitine 372.3108 C 16 OCO(CH 2 ) 14 CH 3 Palmitoylcarnitine 400.3421 C 18 OCO(CH 2 ) 16 CH 3 Stearoylcarnitine 428.3734 OOHOHN+CH3CH3CH3 OROHN+CH3CH3CH3 Table 2-2. Minimum and maximum levels for each factor Variable Minimum (-) Central (0) Maximum (+) Nebulizer Pressure (psi) 50 55 60 Drying Gas Flow Rate (mL min -1 ) 10 11 12 Drying Gas Temperature (C) 300 325 350 61

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Table 2-3. Experimental design matrix Experiment (NP) a (DGFR) b (DGT) c (RR (%)) 1 (RR (%)) 2 (RR (%)) 3 1 + + + 93.1 100 99.7 2 + + 98.6 91.4 97.3 3 + + 97.1 97.2 94.6 4 + 91.9 92.3 97.7 5 + + 88.6 90.6 94.8 6 + 94.8 95.4 92.6 7 + 77.4 96.9 92.3 8 100 99.9 100 9 0 0 0 97.5 95.7 95.2 10 0 0 0 86.1 99.2 93.4 11 0 0 0 84.7 98.5 99.7 (NP) a : Nebulizer Pressure; (DGFR) b : Drying Gas Flow Rate (mL min -1 ); (DGT) c : Drying Gas Temperature (C); (RR (%)) 1 : Relative Response (%) based on area (counts) [M+H] + = 162.1125 Carnitine; (RR (%)) 2 : Relative Response (%) based on area (counts) [M+H] + = 260.1862 Hexanoylcarnitine; (RR (%)) 3 : Relative Response (%) based on area (counts) [M+H] + = 316.2488 Decanoylcarnitine Table 2-4. Analysis of variance corresponding to the analytical response for carnitine Factor SS a df b MS c F-ratio p d NP 56.4379 1 56.4379 1.152805 0.395315 DGFR 12.9561 1 12.9561 0.264642 0.658155 DGT 116.7805 1 116.7805 2.385369 0.262478 NP x DGFR 0.4895 1 0.4895 0.009998 0.929474 NP x DGT 91.4371 1 91.4371 1.867703 0.305092 DGFR x DGT 2.4016 1 2.4016 0.049054 0.845274 Lack of Fit 127.5944 2 63.7972 1.303128 0.434192 Pure Error 97.9140 2 48.9570 Total SS 506.0110 10 SS a : sum of squares; df b : degrees of freedom; MS c : mean of squares; p d : probability value 62

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Table 2-5. Analysis of variance corresponding to the analytical response for hexanoylcarnitine Factor SS a df b MS c F-ratio p d NP 0.5327 1 0.53272 0.15743 0.729870 DGFR 10.4914 1 10.49141 3.10040 0.220337 DGT 4.2696 1 4.26963 1.26175 0.378041 NP x DGFR 20.6834 1 20.68338 6.11231 0.131979 NP x DGT 57.0376 1 57.03761 16.85564 0.054521 DGFR x DGT 0.4538 1 0.45382 0.13411 0.749317 Lack of Fit 15.0474 2 7.52372 2.22340 0.310232 Pure Error 6.7678 2 3.38389 Total SS 115.2838 10 SS a : sum of squares; df b : degrees of freedom; MS c : mean of squares; p d : probability value Table 2-6. Analysis of variance corresponding to the analytical response for decanoylcarnitine Factor SS a df b MS c F-ratio p d NP 11.98835 1 11.98835 1.140449 0.397382 DGFR 0.00010 1 0.00010 0.000009 0.997826 DGT 4.19312 1 4.19312 0.398891 0.592224 NP x DGFR 12.47651 1 12.47651 1.186889 0.389731 NP x DGT 3.20627 1 3.20627 0.305012 0.636234 DGFR x DGT 30.96688 1 30.96688 2.945874 0.228234 Lack of Fit 2.03910 2 1.01955 0.096990 0.911586 Pure Error 21.02390 2 10.51195 Total SS 85.89422 10 SS a : sum of squares; df b : degrees of freedom; MS c : mean of squares; p d : probability value 63

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Table 2-7. The ESI-TOF operational conditions Ionization mode ESI (positive mode) Fragmentor (V) 120 OCT RF V (V) 250 Gas temperature (DGT, C) 300 Drying gas flow rate (DGFR, L min -1 ) 10 Nebulizer pressure (NP, psi) 50 Capillary (V) 4000 64

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Figure 2-1. Schematic diagram of an Agilent 6210 time-of-flight mass spectrometer. 65

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Effect Estimate (Absolute Value) -.099988 .2214823 .5144339 1.073688 1.366639 -1.54446 p=.05 NP x DGFRDGFR x DGTDGFRNPNP x DGTDGT -0.50.00.51.01.52.02.53.03.54.04.55.0 Figure 2-2. Pareto chart of standardized effects for variables in the optimization of MS parameters using carnitine as source of the analytical response (Table 2-3; RR (%) 1 ). Effect Estimate (Absolute Value) .3662125 -.396772 1.123277 -1.7608 2.472309 4.105562 p=.05 DGFR x DGTNPDGTDGFRNP x DGFRNP x DGT -0.50.00.51.01.52.02.53.03.54.04.55.0 Figure 2-3. Pareto chart of standardized effects for variables in the optimization of MS parameters using hexanoylcarnitine as source of the analytical response (Table 2-3; RR (%) 2 ). 66

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Effect Estimate (Absolute Value) .0030751 .552279 -.631578 1.067918 1.089444 1.716355 p=.05 DGFRNP x DGTDGTNPNP x DGFRDGFR x DGT -0.50.00.51.01.52.02.53.03.54.04.55.0 Figure 2-4. Pareto chart of standardized effects for variables in the optimization of MS parameters using decanoylcarnitine as source of the analytical response (Table 2-3; RR (%) 3 ). 67

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CHAPTER 3 COMPARISON OF PACKED AND MONOLITHIC COLUMNS General Aspects of HPLC Liquid chromatography (LC) is a separation technique where analytes are separated by virtue of differing solubilities between a liquid mobile phase and a liquid or solid stationary phase [109]. High-performance liquid chromatography (HPLC) is one mode of chromatography. HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components. The amount of resolution is important, and is dependent upon the extent of interaction between the solute components and the stationary phase. The stationary phase is defined as the immobile packing material in the column. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures [110, 111]. HPLC, as compared with the classical technique (LC), is characterized by: a) small diameter (2 5 mm), reusable stainless steel columns, b) column packings with very small (3, 5 and 10 m) particles and the continual development of new substances to be used as stationary phases, c) relatively high inlet pressures and controlled flow of the mobile phase, d) precise sample introduction without the need for large samples, e) special continuous flow detectors capable of handling small flow rates and detecting very small amounts, f) automated standardized instruments, e) rapid analysis, and f) high resolution [109-111]. Initially, pressure was selected as the principal criterion of modern liquid chromatography and thus the name was "high pressure liquid chromatography" or HPLC. This was, however, an 68

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unfortunate term because it seems to indicate that the improved performance is primarily due to the high pressure. This is, however, not true. In fact high performance is the result of many factors: very small particles of narrow distribution range and uniform pore size and distribution, high pressure column slurry packing techniques, accurate low volume sample injectors, sensitive low volume detectors and, of course, good pumping systems. Naturally, pressure is needed to permit a given flow rate of the mobile phase; otherwise, pressure is a negative factor not contributing to the improvement in separation. Recognizing this, most experienced chromatographers today refer to the technique as high performance liquid chromatography still permitting the use of the acronym HPLC [109-111]. Although many chromatographers feel high performance liquid chromatography is a mature technique, chromatography research continues to make progress. Fast HPLC There is no clear definition for fast analysis. The term fast HPLC is a relative one. Fast analysis refers to decreasing the analysis time of a method. Analysis time by itself is sometimes a poor measurement of chromatographic parameters; rather the important parameter is the number of compounds separated per unit time. Nevertheless, it should be noted that the terms fast LC, high speed HPLC, fast HPLC and ultra-fast HPLC are commonplace in the literature without a formal definition. Nowadays, there is a competition between two means for fast LC analysis, namely, HPLC with monolithic phases and small particle phases (< 2 m) used in ultra high pressure liquid chromatography (UHPLC). With both phase types a substantially faster analysis, faster method development, reduced solvent consumption, and increased mass sensitivity can be achieved. 69

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Faster analysis. Typical isocratic assays can be performed in 1 min and gradient runs in 3 min. Fast HPLC offers a way to increase resolution without switching to a longer column by increasing analyte retention. Rapid method development and validation. Because resolution is controlled primarily by the mobile phase, HPLC method development is mostly a process of finding the right mobile phase and adjusting its solvent strength. The big advantages for fast LC here are quick feedback and rapid column equilibration. The analyst can change the mobile-phase conditions, wait just a couple of minutes for the column to equilibrate, re-inject the sample, and examine the resulting chromatogram for rapid feedback. In method validation, analytical performance data such as precision, accuracy, robustness, linearity, and sensitivity are gathered, and hundreds of assays are required. Here, fast HPLC methods can be validated in days rather than weeks. Reduced solvent consumption and enhanced mass sensitivity. The shorter analysis times in fast HPLC translate to a 50% reduction in solvent use, which lowers the cost of purchase and waste disposal. Mass sensitivity. The smaller column volume means less dilution of the analyte peaks, resulting in a higher mass sensitivity (taller peak heights). Fast HPLC Analysis Using Small Particle Packedand Monolithic-Columns According to the van Deemter equation (Chapter 1, equation 1-1) [44], as the particle size decreases to less than 2.5 m, not only is there a significant gain in efficiency, but the efficiency does not diminish at increased flow rates or linear velocities. By using smaller particles, speed and peak capacity (number of peaks resolved per unit of time in gradient separations) can be extended to new limits, termed ultra high pressure liquid chromatography [112]. 70

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The technology takes full advantage of chromatographic principles to run separations using columns packed with smaller particles and/or higher flow rates for increased speed, with superior resolution and sensitivity. Efficiency (or theoretical plates, N) is the primary separation parameter behind UHPLC since it relies on the same selectivity () and retentivity (k) as HPLC. In the fundamental resolution (R s ) equation: tentivityySelectivitEfficiencySystemskkNRRe114 (3-1) Resolution is proportional to the square root of N. But since N is inversely proportional to particle size (d p ): pdN1 (3-2) as the particle size is lowered, N and R s are increased. N is also inversely proportional to the square of the peak width (w): 21wN (3-3) This illustrates that the narrower the peaks are, the easier they are to separate from each other. Also, peak height (H) is inversely proportional to the peak width: wH1 (3-4) So as the particle size decreases to increase N and subsequently R s an increase in sensitivity is obtained, since narrower peaks are taller peaks. Narrower peaks also mean more peak capacity per unit time in gradient separations. Still another equation comes into play when migrating toward smaller particles: 71

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poptdF1 (3-5) This relationship reveals that as particle size decreases, the optimum flow (F opt ) to reach maximum N increases. But since back pressure is proportional to flow rate, smaller particle sizes require much higher operating pressures, and a system properly designed to capitalize on the efficiency gains. There is an upper limit to the pressures that can be applied at the column inlet; analysts must choose a compromise between the analysis time and the column efficiency [113]. They can trade one for the other but cannot have at once fast and highly efficient analyses beyond the limits that were clearly defined by Giddings [114] and, later, by Knox and Saleem [115, 116]. Now, imagine that we make a porous bed that is the converse of a packed column bed. All the parts of a packed column bed that were solid become available to the mobile phase while all the parts of that bed that were liquid become a porous solid similar in internal structure to the packed particles. Admittedly, we must further imagine that the contact points between the particles are replaced by openings in the solid network that have a significant size and let through the flow of the mobile phase, otherwise the constriction would hinder the flow too severely or even stop it. The external porosity of the column becomes approximately 0.60. The internal porosity decreases because the solid lumps of adsorbent, the elements of the silica skeleton occupy a smaller volume fraction of the column (approximately 40 instead of 60%). The throughpores are less tortuous. They are still constricted but probably less. For all these reasons, the permeability of the new column bed is markedly improved. This new bed is continuous, that is monolithic [113]. The efficiency of this column is controlled by the average sizes of the porons or mesopores, the globs of porous material, and the throughpores, the channels for bulk flow. The major advantage of this new approach to bed design is that we might be able to choose these 72

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two sizes independently of each other, provided that we have enough control on the preparation process of monolithic beds [46, 50-53, 58-64]. This change in the type of chromatographic bed used offers an entirely new realm of possibilities, but it is still the beginning of a long period of research endeavors during which the immense number of possible approaches to design and prepare these monolithic columns will be optimized. This empirical optimization process will eventually provide a variety of columns suitable for the many different separations that have to be performed by HPLC. Comparison of Performance between Packed and Monolithic Columns The performance of chromatographic columns are characterized by their efficiency, which indicates their separation power, by their permeability, which informs on the maximum column length and/or flow rate that can be used with a given column type and instrument, and by their phase ratio, which is related to the pattern of solute retention factors, the possibility of resolving certain pairs of compounds, and the column saturation capacity. These characteristics are well known for packed columns. Most of the information available for these columns can be extended to monolithic columns with suitable adjustments [116]. On a purely technical basis, the monolithic columns have two major advantages over the packed columns, a high permeability and a low resistance to mass transfer, but they still suffer from one important drawback, a high eddy diffusion contribution to their HETP. The external porosity of monolithic columns is about 50% higher than that of packed columns; hence they exhibit a lower specific surface area. That might be a drawback in some rare cases since retention decreases with decreasing density of centers of interactions between analytes and the stationary phase. In almost all cases, however, some minor adjustments of the mobile phase composition allow the achievement of suitable retention factors. The high porosity of monolithic columns is the direct cause of their high permeability. As a result, unless they are extremely long 73

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(i.d., a few meters or longer), monolithic columns can be operated at high mobile phase velocity, if needed. On a direct, practical basis, monolithic columns seem to have over packed columns the serious advantage of being more resistant to fouling by complex, real samples and of requiring a lesser degree of sample clean-up. This is particularly useful for samples of biological or clinical origin e.g., plasma samples. Combined with the fast mass transfer kinetics across monolithic columns, this high permeability allows the easy achievement of fast separations, hence of a high analysis throughput. Alternately, long monolithic columns could be used to achieve high column efficiency hence separations exhibiting high resolution. Because the mass transfer kinetics in monolithic columns is fast, these columns exhibit a high efficiency and, moreover, their efficiency decreases with increasing mobile phase velocity more slowly than that of packed columns. This difference is quite important. The Hs (columns plate height) of the available monolithic silica columns are generally lower than those of conventional columns packed with 4 or 5 m particles while they have the permeability of columns packed with 10 or 12 m particles. The current drawback of monolithic columns is that the eddy diffusion contribution is larger than for packed columns [117, 118]. As a result, the maximum efficiency of monolithic column is markedly lower than it should be if their beds were more homogeneous. Experimental Methods The experimental section as detailed in Chapter 2 is also applicable to Chapter 3. Results and Discussion Experiments were designed to compare two commercially available columns, a monolithic (C 18 -Phenomenex Onyx monolithic column, 100 mm 4.6 mm. Phenomenex Inc., Torrance, CA) and a particulate column (Zorbax Eclipse XDB-C 18 column, 50 mm 4.6 mm, with 1.8 m. Agilent Technologies, Santa Clara, CA). 74

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There were four main objectives to these studies: (1) optimize the experimental conditions to achieve an efficient separation of carnitine and carnitine derivatives; (2) use van Deemter plots to illustrate both columns behavior at high flow rates; (3) acquire the figures of merits for the different chromatographic approaches; (4) demonstrate the applicability of the optimized methodologies to the analysis of carnitine and acylcarnitines in plasma samples. Optimization of the HPLC Experimental Conditions The effectiveness of HPLC separation was tested using a standard solution mixture containing 1 mg L -1 of each analyte (carnitine and acylcarnitines); 15 L was fixed as the injection volume. The gradient elution profile was optimized to obtain the highest resolution of compounds and the shortest time of analysis. Effect of Mobile Phase Composition The choice of the organic component of the mobile phase is important in any method development. A linear gradient elution method was used in this study, the entire HPLC effluent stream, when not diverted to waste, was directed into the MS interface. The polarity of carnitine and acylcarnitines is quite different. In comparison with the rest of the compounds, carnitine and short-chain acylcarnitines could be considered compounds of polar nature. The non-polar character, and consequent strong retention on the C 18 functionalized stationary phase, is more noticeable for the mediumand long-chain acylcarnitines due to their extended hydrocarbonated chain. To achieve the separation of this mixture of polar and less polar compounds, the selection of an adequate organic solvent was be evaluated. Several organic solvents were considered for the preliminary experiments. Based on the best results obtained, isopropanol (IPA) and acetonitrile were selected as the non-polar component (B) in the eluent system for both columns, 75

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and their composition was varied between 5 to 100% (v/v) with water (A) to optimize the separation of carnitine and acylcarnitines. The use of a gradient from 5% to 100% of organic phase provided a fast separation with sufficient resolution. Additionally, it was noticeable the effect of acetonitrile on sharpening the peak shape, this effect may be due to the fact that acetonitrile decreases non-specific binding to silanol groups. However, the benefic effect of isopropanol on separating some interferent peaks, even better than acetonitrile, was also observed. Effect of Mobile Phase Buffer As a rule of thumb, mobile phase pH should be selected so that it is at least 1.5 pH units from the analytes pKa. This assures that the analytes are either 100% ionized or 100% non-ionized and should help control run-run reproducibility. Due to the zwitterionic nature of acylcarnitines (pKa ~ 3.8), acidic conditions were used to promote protonation of the carboxylic group, which resulted in the production of positively charged quaternary amines. Thus, the positive ESI mass spectra of acylcarnitines were dominated by their [M+H] + ions with minimal fragmentation. Different organic acids at several concentrations were tested: formic acid, ammonium acetate, and acetic acid. Buffer additives were incorporated in both A and B solvents in the gradient to maintain a constant concentration throughout the gradient. Acetic acid gave the best resolution and improvement in peak shape for the separation of carnitines and acylcarnitines when compared with the mentioned above compounds. As a result, 1% (0.17 mol L -1 ; pH ~ 2.8) acetic acid was added to the mobile phases. In summary, our previous results indicated that chromatographic gradient system composed of water and isopropanol or acetonitrile as organic mobile phase, when adding 1% acetic acid, could sharpen peak shapes and improve analytical sensitivity and resolution for the 76

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HPLC analysis of carnitine and acylcarnitine compounds for both type of columns. The optimum gradient programs for the separation of the compounds under study, taking into account each column, as well as each mixture of solvents are summarized in Tables 3-1 3-4. Effect of Flow Rate: van Deemter Plots The effect of the mobile phase flow rate on the separation of the carnitine and acylcarnitines was evaluated. Fifteen microliters of the standard sample mixture was injected onto the HPLC system (packed or monolithic column) at varying flow rates, from 0.8 to 1.6 mL min -1 at a gradient mobile phase composition (see conditions for each column and organic mobile phase in tables 3-1 3.4). The height equivalent to a theoretical plate (H or HETP) was determined using the following equations for N and H: N LH (3-6) 22/154.5 wtNr (3-7) where L is the column length (m), t r is the retention time for the corresponding analyte (min), and w 1/2 is the peak width at height (min). The behavior of the chromatographic system at varying flow rates is depicted in Figure 3.1. As it is possible to observe in this graph, a flow rate equal to 1.3 mL min -1 gave the smaller HETP (m) value for all cases. In addition, the performance of the monolithic column using isopropanol as organic phase was slightly better than that of the same column using acetonitrile or that of the packed column using isopropanol or acetonitrile. However, in a more general sense, the chromatographic performance of these columns could be comparable at 1.3 mL min -1 77

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Chromatograms Under the optimum conditions detailed above, satisfactory chromatographic resolution for the targeted compounds was obtained. An example of the base peak chromatograms obtained is illustrated in Figures 3-2 3.5. Figures of Merit The calibration curves were tested over the range 5 1000 g L -1 Each point of the calibration graph corresponded to the mean value from three independent peak measurements (Figures 3-6 and 3-7). The linearity relationship between peak areas and concentrations was good, and the regression coefficients (R), were greater than 0.9991 for all the curves. The detection limits (at a signal-to-noise ratio of three), limits of quantitation (at a signal-to-noise ratio of ten), and linear ranges for all the compounds under study and considering the different chromatographic approaches are shown in Tables 3-5 and 3-6. As mentioned above satisfactory results were obtained with the different types of chromatographic approaches; however, it was very noticeable that the limit of detections for propionylcarnitine (C 3 ) and butyrylcarnitine (C 4 ) were almost ten times higher than those for the rest of the compounds. Some tentative explanations could be proposed for this issue: (i) propionylcarnitine and butyrylcarnitine exhibit a mid-polar nature when compared with the rest of the acylcarnitines and, maybe, this condition could have a detrimental effect on the electrospray process, (ii) propionylcarnitine coeluted with carnitine (C 0 ) and acetylcarnitine (C 2 ); consequently, an effect of ion suppression resulting in a low ionization efficiency and signal attenuation of C 3 might be possible, and (iii) a combination of (i) and (ii) effects for propionylcarnitine. 78

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Although the reasons for the observed lack of sensitivity for propionylcarnitine and butyrylcarnitine have not yet been clarified, the limits of quantitation achieved for these metabolites are suitable for their determination in plasma samples [82-119]. On the other hand, decanoylcarnitine (C 10 ) showed to be the most intense peak in all of the experiments. It could be ascribed to the effect of the optimized ESI-MS parameters on the analytical response of the system for this analyte, as well as to the non-polar nature of C 10 and its more favorable interaction with stationary and mobile phases than the rest of the compounds. The intra-day precision of the proposed method was tested with five repeated injections of standard solution mixture at the concentration level of 1.0 mg L -1 The inter-day precision was studied by analyzing 1 mg L -1 standard solution mixture, with seven injections randomly executed in a 21-day period. The intra-day relative standard deviations were below 3% for and the inter-day relative standard deviations were below 5% for both columns. Accuracy was also determined by the same procedure, and the accuracy data were calculated as: 100min(%)ionconcentratalonionconcentra t analyzedmeanAccuracy (3-8) For all the analytes, considering intra-day and inter-day experiments, the average accuracy was better than 95%. The obtained results demonstrate that the proposed analytical method provide good validation. Sample analysis Piglet plasma samples were prepared as described in Chapter 2. The above-mentioned optimized parameters for the separation and determination of carnitine and acylcarnitines by LC-ESI-TOF were applied. Mass spectra of the analytes are shown in Figures 3-8 3.14. These experiments were part of a study carried out in collaboration with Dr. Borums group in the Food 79

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Science and Human Nutrition at University of Florida; the same was focused on the improvement of the sample preparation procedure. Conclusions A simple, relatively rapid and accurate HPLC method was successfully developed and validated. Packed and monolithic columns were used for the separation of carnitine and acylcarnitines and their subsequent determination by positive ESI-oa-TOF. Under optimum conditions both columns demonstrated comparable separation performance. To the best of our knowledge, this is the first time that gradient HPLC methods, using small particle packedand monolithic-columns, have been applied to the simultaneous separation of carnitine and acylcarnitines. Based on the results obtained, it is feasible to assure that the metabolic pattern analysis of carnitine and carnitine derivatives in plasma and other clinical samples is currently under way. 80

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Table 3-1. The HPLC conditions and gradient program for the packed column with isopropanol as organic mobile phase HPLC conditions Column Zorbax Eclipse XDB-C 18 RRHT 600 bar, 4.6 100 mm, 1.8 m Solvent A 1% (v/v) acetic acid in water Solvent B 1% (v/v) acetic acid in isopropanol Injection volume (L) 15 Flow rate (mL min -1 ) 1.3 Standard mixture (mg L -1 ) 1.0 Gradient program Time (min) %A %B 0:00 95 5 5:00 95 5 10:00 50 50 25:00 0 100 30:00 95 5 40:00 95 5 Table 3-2. The HPLC conditions and gradient program for the packed column with acetonitrile as organic mobile phase HPLC conditions Column Zorbax Eclipse XDB-C 18 RRHT 600 bar, 4.6 100 mm, 1.8 m Solvent A 1% (v/v) acetic acid in water Solvent B 1% (v/v) acetic acid in acetonitrile Injection volume (L) 15 Flow rate (mL min -1 ) 1.3 Standard mixture (mg L -1 ) 1.0 Gradient program Time (min) %A %B 0:00 95 5 5:00 95 5 10:00 50 50 19:00 0 100 30:00 95 5 35:00 95 5 81

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Table 3-3. The HPLC conditions and gradient program for the monolithic column with isopropanol as organic mobile phase HPLC conditions Column Phenomenex ONIX monolithic column-C 18 4.6 100 mm Solvent A 1% (v/v) acetic acid in water Solvent B 1% (v/v) acetic acid in isopropanol Injection volume (L) 15 Flow rate (mL min -1 ) 1.3 Standard mixture (mg L -1 ) 1.0 Gradient program Time (min) %A %B 0:00 95 5 6:50 95 5 25:50 0 100 30:50 95 5 35:00 95 5 Table 3-4. The HPLC conditions and gradient program for the monolithic column with acetonitrile as organic mobile phase HPLC Conditions Column Phenomenex ONIX monolithic column-C 18 4.6 100 mm Solvent A 1% (v/v) acetic acid in water Solvent B 1% (v/v) acetic acid in acetonitrile Injection volume (L) 15 Flow rate (mL min -1 ) 1.3 Standard mixture (mg L -1 ) 1.0 Gradient program Time (min) %A %B 0:00 95 5 6:50 95 5 25:50 0 100 30:50 95 5 35:00 95 5 82

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Table 3-5. Figures of merit for the separation and determination of carnitine and acylcarnitines (packed column isopropanol as organic mobile phase) by UHPLC-ESI-TOF Analyte LOD a (S/N b =3) (mol L-1)/ (pg)c LOQ d (S/N=10) (mol L-1) Linear range (mol L -1 ) Carnitine 0.062 / 250 0.12 0.06 6.21 Acetylcarnitine 0.099 / 503 0.20 0.10 4.92 Propionylcarnitine 0.230 / 1249 1.38 0.23 4.61 Butyrylcarnitine 0.214 / 1250 0.93 0.09 4.33 Hexanoylcarnitine 0.058 / 376 0.13 0.06 3.86 Octanoylcarnitine 0.017 / 122 0.06 0.02 3.48 Decanoylcarnitine 0.016 / 126 0.16 0.02 3.17 Lauroylcarnitine 0.015 / 129 0.26 0.02 2.91 Myristoylcarnitine 0.014 / 130 0.04 0.01 2.69 Palmitoylcarnitine 0.013 / 130 0.04 0.01 2.50 Stearoylcarnitine 0.012 / 128 0.05 0.01 2.34 a LOD: Limit of detection; b S/N: Signal-to-noise ratio; c mass (volume = 25L); d LOQ: Limit of quantitation Table 3-6. Figures of merit for the separation and determination of carnitine and acylcarnitines (monolithic column isopropanol as organic mobile phase) by HPLC-ESI-TOF Analyte LOD a (S/N b =3) (mol L-1) / (pg)c LOQ d (S/N=10) (mol L-1) Linear range (mol L -1 ) Carnitine 0.031 / 75 0.07 0.03 6.21 Acetylcarnitine 0.148 / 451 0.22 0.15 4.92 Propionylcarnitine 1.382 / 4501 2.77 1.38 4.61 Butyrylcarnitine 0.461 / 1605 1.51 0.02 4.33 Hexanoylcarnitine 0.039 / 152 0.06 0.04 3.86 Octanoylcarnitine 0.017 / 73 0.05 0.02 3.48 Decanoylcarnitine 0.024 / 114 0.07 0.02 3.17 Lauroylcarnitine 0.029 / 149 0.27 0.03 2.91 Myristoylcarnitine 0.014 / 78 0.04 0.01 2.69 Palmitoylcarnitine 0.013 / 78 0.04 0.01 2.50 Stearoylcarnitine 0.012 / 77 0.04 0.01 2.34 a LOD: Limit of detection; b S/N: Signal-to-noise ratio; c mass (volume = 15L)); d LOQ: Limit of quantitation 83

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0.80.91.01.11.21.31.41.51.602468101214161820 PC.ACN MC.ACN MC.IPA PC.IPAHETP (m)Flow Rate (mL min-1) Figure 3-1. van Deemter plots for the different chromatographic approaches. HETP: height equivalent to a theoretical plate; PC.ACN: packed column and acetonitrile as organic mobile phase; MC.ACN: monolithic column and acetonitrile as organic mobile phase; MC.IPA: monolithic column and isopropanol as organic mobile phase; PC.IPA: packed column and isopropanol as organic mobile phase. 84

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Figure 3-2. Base peak chromatogram obtained with a packed column and isopropanol as organic mobile phase; concentration standard mixture: 1 mg L-1; injected volume: 15 L; mobile phase flow rate: 1.3 mL min-1; C0: carnitine; C2: acetylcarnitine; C3: propionylcarnitine; C4: butyrylcarnitine; C6: hexanoylcarnitine; C8: octanoylcarnitine; C10: decanoylcarnitine; C12: lauroylcarnitine; C14: myristoylcarnitine; C16: palmitoylcarnitine; C18: stearoylcarnitine. 85 Figure 3-2. Base peak chromatogram obtained with a packed column and isopropanol as organic mobile phase; concentration standard mixture: 1 mg L -1 ; injected volume: 15 L; mobile phase flow rate: 1.3 mL min -1 ; C 0 : carnitine; C 2 : acetylcarnitine; C 3 : propionylcarnitine; C 4 : butyrylcarnitine; C 6 : hexanoylcarnitine; C 8 : octanoylcarnitine; C 10 : decanoylcarnitine; C 12 : lauroylcarnitine; C 14 : myristoylcarnitine; C 16 : palmitoylcarnitine; C 18 : stearoylcarnitine. C 18 C 14 C 16 C 12 C 10 C 8 C 6 C 4 C 0 C 2 C 3

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C 3 C 18 C 16 C 14 C 12 C 10 C 8 C 6 C 4 C 0 C 2 86 Figure 3-3. Base peak chromatogram obtained with a packed column and acetonitrile as organic mobile phase; concentration standard mixture: 1 mg L -1 ; injected volume: 15 L; mobile phase flow rate: 1.3 mL min -1 ; C 0 : carnitine; C 2 : acetylcarnitine; C 3 : propionylcarnitine; C 4 : butyrylcarnitine; C 6 : hexanoylcarnitine; C 8 : octanoylcarnitine; C 10 : decanoylcarnitine; C 12 : lauroylcarnitine; C 14 : myristoylcarnitine; C 16 : palmitoylcarnitine; C 18 : stearoylcarnitine.

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87 Figure 3-4. Base peak chromatogram obtained with a monolithic column and isopropanol as organic mobile phase; concentration standard mixture: 1 mg L -1 ; injected volume: 15 L; mobile phase flow rate: 1.3 mL min -1 ; C 0 : carnitine; C 2 : acetylcarnitine; C 3 : propionylcarnitine; C 4 : butyrylcarnitine; C 6 : hexanoylcarnitine; C 8 : octanoylcarnitine; C 10 : decanoylcarnitine; C 12 : lauroylcarnitine; C 14 : myristoylcarnitine; C 16 : palmitoylcarnitine; C 18 : stearoylcarnitine. C 4 C 18 C 16 C 14 C 12 C 10 C 8 C 6 C 2 C 0

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C 18 C 16 C 14 C 12 C 10 C 8 C 6 C 4 C 2 C 0 88 Figure 3-5. Base peak chromatogram obtained with a monolithic column and acetonitrile as organic mobile phase; concentration standard mixture: 1 mg L -1 ; injected volume: 15 L; mobile phase flow rate: 1.3 mL min -1 ; C 0 : carnitine; C 2 : acetylcarnitine; C 3 : propionylcarnitine; C 4 : butyrylcarnitine; C 6 : hexanoylcarnitine; C 8 : octanoylcarnitine; C 10 : decanoylcarnitine; C 12 : lauroylcarnitine; C 14 : myristoylcarnitine; C 16 : palmitoylcarnitine; C 18 : stearoylcarnitine.

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010020030040050060070080090010000100000020000003000000400000050000006000000700000080000009000000 10000000 C0 C2 C3 C4 C6 C8 C10 C12 C14 C16 C18Peak Area (counts)Concentration (g L-1) Figure 3-6. Copanol as organic mobile phase); concentration standard mixture ranged between 5 and 10L-1; injected volume: 25 L; mobile phase flow rate: 1.3 mL min-1; C0: carnitine; Cacetylcarnitine; C3: propionylcarnitine; C4: butyrylcarnitine; C6: hexanoylcarni alibration curves for carnitine and acylcarnitines; packed column (isopr00 g 2: ; t ineC8: octanoylcarnitine; C10: decanoylcarnitine; C12: lauroylcarn i tine; C14: myristoylcarnitine; C16: palmitoylcarnitine; C18: stearoylcarnitine. 89

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0100200300400500600700800900100001500003000004500006000007500009000001050000120000013500001500000 C0 C2 C3 C4 C6 C8 C10 C12 C14 C16 C18Peak Area (counts)Concentration (g L-1) Figure 3-7. Calibration curves for carnitine and acylcarnitines; monolithic column (isopropanol as organic mobile phase); concentration standard mixture ranged between 5 and 1000 g L -1 ; injected volume: 15 L; mobile phase flow rate: 1.3 mL min -1 ; C 0 : carnitine; C 2 : acetylcarnitine; C 3 : propionylcarnitine; C 4 : butyrylcarnitine; C 6 : hexanoylcarnitine; C 8 : octanoylcarnitine; C 10 : decanoylcarnitine; C 12 : lauroylcarnitine; C 14 : myristoylcarnitine; C 16 : palmitoylcarnitine; C 18 : stearoylcarnitine. 90

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Figure 3-8. Mass spectrum of carnitine (theoretical [M+H] + : 162.1125) in piglets plasma samples; column: monolithic column (acetonitrile as organic mobile phase); injected volume: 15 L. 91

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Figure 3-9. Mass spectrum of lauroylcarnitine (theoretical [M+H] + : 344.2795) in piglets plasma samples; column: monolithic column (acetonitrile as organic mobile phase); injected volume: 15 L. 92

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Figure 3-10. Mass spectrum of myristoylcarnitine (theoretical [M+H] + : 372.3108) in piglets plasma samples; column: monolithic column (acetonitrile as organic mobile phase); injected volume: 15 L. 93

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Figure 3-11. Mass spectrum of palmitoylcarnitine (theoretical [M+H]+: 400.3421) in piglets plasma samples; column: monolithic column (acetonitrile as organic mobile phase); injected volume: 15 L. 94

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Figure 3-12. Mass spectrum of stearoylcarnitine (theoretical [M+H]+: 428.3734) in piglets plasma samples; column: monolithic column (acetonitrile as organic mobile phase); injected volume: 15 L. 95

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Figure 3-13. Mass spectrum of myristoylcarnitine (theoretical [M+H] + : 372.3108) in piglets plasma samples; column: packed column (isopropanol as organic mobile phase); injected volume: 15 L. 96

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Figure 3-14. Mass spectrum of palmitoylcarnitine and stearoylcarnitine (theoretical [M+H] + : 400.3421 and 428.3734, respectively) in piglets plasma samples; column: packed column (isopropanol as organic mobile phase); injected volume: 15 L. Figure 3-14. Mass spectrum of palmitoylcarnitine and stearoylcarnitine (theoretical [M+H] + : 400.3421 and 428.3734, respectively) in piglets plasma samples; column: packed column (isopropanol as organic mobile phase); injected volume: 15 L. 97 97

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CHAPTER 4 CONCLUDING REMARKS AND FUTURE WORK LC-MS methods were developed for improving the efficiency of separation of carnitine and acylcarnitines in a metabolic context. High precision and accuracy of the proposed chromatographic approaches, together with the optimization of the mass spectrometer operational parameters were demonstrated. The results clearly suggest that both UHPLC (small particle-packed column approach) and HPLC (monolithic column approach) methods can serve as an advantageous complement to conventional methods for separation and determination of the carnitine-based compounds. The chromatographic approaches described in the present manuscript dealt with samples without any kind of derivatization (neither prior, nor after column separation), thus achieving a less complex, less expensive, and less time-consuming way to carry out the analysis of the mentioned above metabolites when compared with conventional reported methods [108]. Using the features provides for the proposed chromatographic techniques plus the mass-accuracy and sensitivity characteristics of the oa-TOF mass spectrometer, carnitine and acylcarnitines were efficiently separated and accurately detected. The advantages of using small particle-packed and monolithic columns in the analysis of carnitine and carnitine-based compounds were also demonstrated and confirmed. Based on the separation performance shown by the two mentioned columns, it was possible to establish efficient methodologies applicable to LC systems capable or incapable of withstanding high backpressure values. Additionally, the benefits related with the use of different organic mobile phase compositions were described. As a next step, the separation and reliable quantitative determination of the mentioned compounds for clinical applications will be performed. In addition, the extension of the 98

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separation and determination methodologies to other metabolites is currently under way. This type of study will constitute what is known as metabolic pattern analysis. The aim of this thesis is to achieve a complete understanding of the metabolic role of known and unknown carnitine and carnitine derivatives, and other metabolites, present in clinical samples by using all the available analytical tools to accomplish such goal. In this sense, it is well known that tandem mass spectrometry provides the ability to positively identify the analytes with the correct compound assignments using collision-induced dissociation of the more stable ion. The combination of results obtained from a mass accurate instrument (oa-TOF), together with the unambiguous analytes identification from a triple quadrupole mass spectrometer will effectively promote the achievement of the mentioned goals. Research concerned with all aspects of the separation technology (e.g. stationary phases, column physical characteristics, etc.), plus a more detailed study of intra-day and inter-day precision will also be performed. It is known that biological samples are complex matrices and precautions should be taken into consideration before starting its preparation. In this particular project, the handling of these samples will be focused on the fact that carnitine, its related compounds, and other metabolites, must be separated, identified, and quantified with great reliability. The development of sample preparation assays in agreement with regulatory and MS requirements will be an important aspect of the present research work. In summary, implementation of appropriate sample preparation procedures, efficient separation techniques, suitable ionization modes, and mass-accurate analysis complemented with 99

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tandem mass spectrometry investigation will help to achieve a better understanding of the metabolic pathway and biological role of the compounds in a metabolic context. 100

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APPENDIX A SYNTHESIS OF L-ACETYLCARNITINE HYDROCHLORIDE Calculations. L-carnitine. MW = 162.0 g mol -1 4 mg used (4.13 x 10 -3 g) / 162.0 g mol -1 = 2.55x10 -5 mol. Acetyl chloride. MW = 78.50 g mol -1 d = 1.104 g ml -1 [1.104 g ml -1 ] / 78.50g mol -1 = [(1.406 x10 -2 mol ml -1 ) (x ml)] / 2.55x10 -5 mol = 7.6 /1 X = 14 L to be used. There is a 7.6:1 ratio of acetyl chloride: L-carnitine. Microscale Procedure modified from reference [120]. Materials needed. L-carnitine [4.13 mg] Lonza CAS: [541-15-1], Batch 508714; trifluoroacetic acid [7 L] Acros CAS: [76-05-1] Lot # 81054/1; acetyl chloride [14 L] Acros CAS: [75-36-5] Lot# A011219401; acetone; ethyl ether; methanol. Procedure. Weigh out approx. 4.13 mg of carnitine and dissolve in 7 L of trifluoroacetic acid and mix with 14 L of the acetyl chloride in a 2 mL-capacity vial. Leave in a water-bath set at 40-50 C overnight. [Label vial as C2:0, weight with cap on = 3.3836g]. Keep moisture out by blowing N 2 gas into vial and cap vial overnight. Cool the reaction vessel to RT and evaporate the contents with N 2 gas at RT (1 hour). Add 167 L of cold acetone and leave the capped vial on ice for 10-15 min. Transfer contents of vial into a clean vial labeled C2:0 [weight = ______g ] in order to remove any undissolved material. Add cold ethyl ether to the supernatant at RT to promote crystallization (333 L). After crystallization had started, keep adding ethyl ether until vial is almost completely filled (approximately 167 L). Precipitation occurred. Leave to sit overnight to drive complete crystallization. Separate solvent from crystals by collecting the supernatant in another vial (use Hamilton syringe) [Label as C2:0_R] and leave on ice in cold room overnight to allow for crystallization. Keep the crystals in the same vial [C2:0] they were in a dessicator overnight. Collect crystals from C2:0_R into C2:0. Dissolve crystals in 33 L of methanol and add an appropriate amount of acetone (133-167 L maximum) (acetone was used 101

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to avoid formation of oils upon the addition of ether). 167 L mL of ethyl ether was then added again to promote recrystallization. After recrystallization has started, another 333 L of ethyl ether was added and the vial was kept on ice overnight. Separate supernatant from crystals using a Hamilton Syringe. Collect supernatant in another vial and save. [Label vials as C2:0_Sup]. Air-dry the crystals and take small amount of crystals for NMR analysis. Dissolve small amounts of the crystals in d 6 -DMSO and obtain spectrum. Use a portion of the crystals for mass spectrometry analysis. Obtain spectrum. 102

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APPENDIX B SYNTHESIS OF L-OCTANOYLCARNITINE HYDROCHLORIDE Calculations. L-carnitine: MW = 162.0 g mol -1 4 mg used (5 x 10 -3 g) / 162.0 g mol -1 = 3.09 x 10 -5 mol Octanoyl chloride: MW = 162.66 g mol -1 d = 0.953 g mL -1 [0.953g ml -1 ] / 162.66g mol -1 = [(5.86 x10-3 mol mL -1 ) (x mL)]/ 3.09 x 10 -5 mol = 7.6 / 1 X = 40 L to be used. There is a 7.6:1 ratio of octanoyl chloride: L-carnitine. Microscale Procedure modified from reference [120]. Materials needed. L-carnitine [5 mg] Lonza CAS: [541-15-1], Batch 508714; trifluoroacetic acid [20 L] Aldrich Catalog # 30,203-1, CAS: [76-05-1] Lot # EI00641BI; octanoyl chloride [40 L] Aldrich Catalog # 0-473-3, CAS: [111-64-8], Lot# 04005BU; acetone; ethyl ether; methanol. Procedure. Weigh out approx. 5 mg of carnitine and dissolve in 20 L of trifluoroacetic acid and mix with 40 L of the octanoyl chloride in a 2 mL-capacity vial. Leave in a water-bath set at 40-50 C overnight. [Label vial as C8:0, weight with cap = 3.32492g]. Keep moisture out by blowing N 2 gas into vial and cap vial overnight. Cool the reaction vessel to RT and evaporate the contents with N 2 gas at RT (1 hour). Add 167 L of cold acetone and leave the capped vial on ice for 10-15 min. Transfer contents of vial into a clean vial labeled C8:0 [weight = ______g ] in order to remove any undissolved material. Add cold ethyl ether to the supernatant at RT to promote crystallization (333 L). After crystallization had started, keep adding ethyl ether until vial is almost completely filled (approximately 167 L). Precipitation occurred. Leave to sit overnight to drive complete crystallization. Separate solvent from crystals by collecting the supernatant in another vial (use Hamilton syringe) [Label as C8:0_R] and leave on ice in cold room overnight to allow for crystallization. Keep the crystals in the same vial [C8:0] they were 103

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in a dessicator overnight. Collect crystals from C8:0_R into C8:0. Dissolve crystals in 33 L of methanol and add an appropriate amount of acetone (133-167 L maximum) (acetone was used to avoid formation of oils upon the addition of ether). 167 L mL of ethyl ether was then added again to promote recrystallization. After recrystallization has started, another 333 L of ethyl ether was added and the vial was kept on ice overnight. Separate supernatant from crystals using a Hamilton Syringe. Collect supernatant in another vial and save. [Label vials as C8:0_Sup. Air-dry the crystals and take small amount of crystals for NMR analysis. Dissolve small amounts of the crystals in d 6 -DMSO and obtain spectrum. Use a portion of the crystals for mass spectrometry analysis. Obtain spectrum. 104

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APPENDIX C SYNTHESIS OF L-PALMITOYLCARNITINE HYDROCHLORIDE Calculations. L-carnitine. MW = 162.0 g mol -1 4 mg used (5.15 x 10 -3 g)/162.0 g mol -1 = 3.18 x10 -5 mol. Palmitoyl chloride. MW = 274.88 g mol -1 d = 0.906 g mL -1 [0.906g mL -1 ] / 274.88 g mol -1 = [(3.30 x10 -3 mol mL -1 ) (x mL)]/ 3.18 x10 -5 mol = 7.6 / 1 X = 72 L to be used. There is a 7.6:1 ratio of palmitoyl chloride: L-carnitine. Microscale Procedure modified from reference [120]. Materials needed. L-carnitine [5.15 mg] Lonza CAS: [541-15-1], Batch 508714; trifluoroacetic acid [36 L] Aldrich Catalog # 30,203-1, CAS: [76-05-1] Lot # EI00641BI; palmitoyl chloride [72 L] Aldrich Catalog # P7-8, CAS: [112-67-4], Lot# 02912PU; acetone; ethyl ether; methanol. Procedure. Weigh out approx. 5.15 mg of carnitine and dissolve in 36 L of trifluoroacetic acid and mix with 72 L of the palmitoyl chloride in a 2 mL-capacity vial. Leave in a water-bath set at 40-50 C overnight. [Label vial as C16:0, weight with cap = 3.37805g]. Keep moisture out by blowing N 2 gas into vial and cap vial overnight. Cool the reaction vessel to RT and evaporate the contents with N 2 gas at RT (1 hour). Add 167 L of cold acetone and leave the capped vial on ice for 10-15 min. Transfer contents of vial into a clean vial labeled C16:0 [weight = ______g ] in order to remove any undissolved material. Add cold ethyl ether to the supernatant at RT to promote crystallization (333 L). After crystallization had started, keep adding ethyl ether until vial is almost completely filled (approximately 167 L). Precipitation occurred. Leave to sit overnight to drive complete crystallization. Separate solvent from crystals by collecting the supernatant in another vial (use Hamilton syringe) [Label as C16:0_R] and leave on ice in cold room overnight to allow for crystallization. Keep the crystals in the same vial [C16:0] they were in a dessicator overnight. Collect crystals from C16:0_R into C16:0. Dissolve 105

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crystals in 33 L of methanol and add an appropriate amount of acetone (133-167 L maximum) (acetone was used to avoid formation of oils upon the addition of ether). 167 L of ethyl ether was then added again to promote recrystallization. After recrystallization has started, another 333 L of ethyl ether was added and the vial was kept on ice overnight. Separate supernatant from crystals using a Hamilton Syringe. Collect supernatant in another vial and save. [Label vials as C16:0_Sup]. Air-dry the crystals and take small amount of crystals for NMR analysis. Dissolve small amounts of the crystals in d 6 -DMSO and obtain spectrum. Use a portion of the crystals for mass spectrometry analysis. Obtain spectrum. 106

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APPENDIX D BLOOD ASSAY Purpose. The purpose of the Standard Operating Procedure (SOP) is to provide standard procedures that are used in the Metabolic Assessment Laboratory (MAL) as required by Section 58.81 of the Good Laboratory Practice for Non-clinical Laboratory. Purpose. The purpose of the Standard Operating Procedure (SOP) is to provide standard procedures that are used in the Metabolic Assessment Laboratory (MAL) as required by Section 58.81 of the Good Laboratory Practice for Non-clinical Laboratory. Scope. This procedure provides instructions for the blood assay procedure Responsibilities. It is the responsibility of Metabolic Assessment Laboratory personnel to follow this procedure. It is the responsibility of supervisory personnel to ensure compliance with this procedure and to train employees and students responsible for performing this procedure. References. Department of Health and Human Services, Food and Drug Administration. Good laboratory practice regulations. Final Rule. Federal Register 52(172): 33768-33779, September 4, 1987. Reagents and materials. 1. Methanol. 2. Acetonitrile. 3. 5 inch Pasteur pipets. 4. Glass beakers. 5. P1000 Pipette. 6. P200 Pipette. 7. 1000 L Disposable pipette tips. 8. 200 L disposable pipette tips. 9. Plastic test tubes. 10. Plastic test tube top. 11. Deionized water. 12. Acetic acid. 13. Microcentrifuge tubes. 14. Transfer pipettes. 15. Glass green cap vial. 16. Glass black cap vial. 17. Aluminum foil. 18. 25 G needle. 19. Elution columns: 40m Bond Elut Si bulk density (g mL -1 ): 0.36-0.44, surface pH: 6.5-7.5, average pore diameter: 54-66, washable residue (mg g -1 ): 1.5. Equipment. 1) Microcentrifuge. 2) Vortexer. 3) Evaporating manifold. 4) Nitrogen tank. 5) Balance beam. 6) Face mask. 7) Gloves. 107

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Safety precautions. Members of the MAL have been trained extensively in the procedures described in this SOP. Definitions. A. Standard Operating Procedure (SOP) Standard Operating Procedure is a document that provides instructions for completing a specific task in the lab. B. Metabolic Assessment Laboratory (MAL) The Metabolic Assessment Laboratory is the laboratory that will use this SOP. Procedure. A. Setup. A.1. Prepare labels according to the label template for hemolysate samples in Appendix 1. A.1.a. Be sure to label the hemolysate plastic tubes with the date prepared, piglet number, and hemolysate #. A.1.b. Remember to use gloves and a facemask when handling tubes and vials containing sample material. B. Blood separation. B.1. Prepare plasma and RBC from blood sample according to SOP located at G:\GLP_G\G2\WPtoWORD\L007A-00.G2.doc. C. Day 1 Preparation of RBC Hemolysate. C.1. Fill black ice buckets with ice from pilot plant. C.1.a. Obtain a plastic beaker from Cabinet 19 and fill with water and ice for thawing. C.2. Obtain appropriate RBC tubes from freezer in room 425. C.1.a. Be sure to always differentiate between Portal and Central blood. C.3. Add approximately an equal amount of deionized water to the tubes of RBCs. C.2.a. Remember to use gloves and a facemask when handling tubes and vials containing sample material. C.4. Thaw the RBCs slowly and completely until the mixture is homogeneous. C.3.a. Rub the tube between hands quickly, and swirl in the beaker of ice water. However, do not allow the tube to become too warm. C.5. Vortex, and freeze overnight. C.4.a. Tubes can be combined with a transfer pipette to make more appropriate volumes about 2/3 full. However, do not fill the tubes too much as the liquid will expand in the freezer. Never mix portal and central blood samples, or combine blood from different sources (e.g. different piglet surgeries). C.4.a. Remember to use gloves and a facemask when handling 108

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tubes and vials containing sample material. D. Day 2 Preparation of RBC hemolysate/plasma carnitine extracts and the Hemoglobin hemolysate standard. D.1. Thaw hemolysate/plasma samples according to procedures described in B.1., B.2., and B.4. D.2. Prepare a plastic tube of Hgb corresponding to each hemolysate tube used. D.1.a. Label a HgB plastic test tube for each hemolysate tube. Include the piglet number, HgB, and date the HgB was made. D.1.b. Pipet 450 L deionized water into the HgB tube. D.1.D. Pipet 50 L of a hemolysate sample into the HgB tube. D.1.d. Vortex the HgB tube for 10 seconds and freeze overnight. D.3. Prepare a 100 L sample of RBC hemolysate and/or plasma for carnitine analysis. D.3.a. Prepare and label 1.6 mL microcentrifuge tubes for each sample. D.3.a.i. See labeling document for Day 2 labels for addition of A/M in Appendix 1. D.3.a.ii. Label P + Sample # + Assayer ID + Batch Number. 9.D.3.a.iii. Remember to use gloves and a facemask when handling tubes and vials containing sample material. D.3.b. Prepare the Acetonitrile/Methanol solution. D.3.b.i. See A/M Protocol located in Appendix 2. D.3.D. Pipet 1 mL of 3:1 A/M solution to each microcentrifuge tube. D.3.d. Vortex the hemolysate/plasma tube, and pipet 100 L of hemolysate/plasma into the microcentrifuge tube once thawing is complete. D.3.d.i. In order to make things go smoothly, one person should pipet the sample, while the other vortexes and opens the test tubes. D.3.d.ii. Change pipet tips in between each volume of blood pipette. D.3.d.iii. Remember to use gloves and a facemask when handling tubes and vials containing sample material. D.3.e. Vortex thoroughly to mix, and freeze overnight. E. Day 3 Protein separation, column elution, and evaporation. E.1. Remove the samples from the freezer. E.2. Prepare the samples for the first centrifugation. E.2.a. Place samples on centrifuging rack and balance on the beam balance. E.2.a.i. Use empty microcentrifuge tubes filled with water in each rack to balance properly. E.2.b. Place the racks into the centrifuge making sure that the samples are arranged properly in 109

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the rack so that they balance in the centrifuge. E.2.b.i. The tubes should be positioned so that when they are placed in the centrifuge, the racks are 180 degrees apart and the tubes are on the same row in the rack but in the position exactly alternate the one in the other rack. Tube 1 should be exactly opposite the empty spot in row one and tube 2 should be exactly opposite the empty spot in row 1. E.2.c. Set the centrifuge to speed (12,000rpm), at 15 minutes. Turn on the centrifuge, push the time button, and flip the start button. E.2.E. While the centrifuge is running, prepare the microcentrifuge tubes for the second centrifugation. E.2.E.i. Label according to Appendix 1. E.2.E.ii. Prepare Pasteur pipets with bulbs for each sample. E.2.E.iii. Remember to use gloves and a facemask when handling tubes and vials containing sample material. E.2.e. When the first centrifugation is finished, remove the samples carefully from the centrifuge without jostling the pellets. E.2.f. Remove the supernatant from the sample and transfer to the microcentrifuge tubes for the second centrifugation. E.2.f.i. Uncap the empty tube and while holding both tubes in one hand, transfer the supernatant from the first tube into the second tube. E.2.f.ii. Make sure to obtain the entire liquid sample while avoiding the pellet. E.2.f.iii. Slant the tube with the supernatant to one side so that the liquid can be drawn out of the tube without disturbing the supernatant. Also make sure the pipette is touching the side of the tube without residue to avoid pulling in the pellet. E.2.f.iv. Be sure to record the appearance of the pellet and supernatant in the research notebook page. E.2.f.v. Remember to use gloves and a facemask when handling tubes and vials containing sample material. E.2.g. Repeat for all samples and replace them in the centrifuge rack to balance on the balance beam. E.2.h. Centrifuge a second time for 15 minutes at 12,000rpm. E.2.i. Prepare microcentrifuge tubes labeled with initials and sample number for the third centrifugation. E.2.i.i. Remember to use gloves and a facemask when handling tubes and vials containing sample material. E.2.j. Remove samples from the 110

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centrifuge and repeat steps 9.E.2.e-9.E.2.g. E.2.j.ii. Record whether or not there was pellet in the sample tube. E.2.k. Centrifuge a third time for 15 minutes at 12,000rpm. E.2.l. Prepare the 10:9:1 W/M/AA mixture and columns. E.2.l.i. When the samples have centrifuged for 8 minutes, prepare the W/M/AA mixture according to the protocol in Appendix 3. E.2.l.ii. Place columns in the waste collected tubes and pipet 0.5 mL of methanol into each column, rotating the column to wash the sides. Cover with aluminum foil. E.2.l.iii. Place the labeled sample collected vials in the sample collected test tubes and cover with aluminum foil. E.2.l.iv. See the labeling document for proper labels. E.2.m. Prepare Pasteur pipets with bulbs for each sample for transferring. E.2.n. Remove the samples from the centrifuge and transfer them to the columns. 9.E.2.n.i. Record the times for each sample. E.2.n.ii. Remember to use gloves and a facemask when handling tubes and vials containing sample material. E.2.o. As each sample finishes eluting, add the second 0.5 mL methanol making sure to rotate the column as you wash the sides with solvent. Record times for each sample. E.2.p. Add the W/M/AA mixture to the column. E.2.p.i. As each sample finishes the second wash, transfer the columns to the sample collected glass tubes, attaching a 25 G needle to the end of the column. E.2.p.ii. Pipet 1 mL of the W/M/AA mixture to each column, recording the times. E.2.p.iii. It is faster if one person does the pipetting and the other transfers the column and attaches the needle. E.2.p.iv. Remember to use gloves and a facemask when handling tubes and vials containing sample material. E.2.q. Finish capping the samples and prepare for evaporation. E.2.q.i. Push the column down into the sample vial and pull up both to remove the vial from the test tube. E.2.q.ii. Cap the vial and discard the needle and column in the red sharps box. E.2.q.iii. Remember to use gloves and a facemask when handling tubes and vials containing sample material. F. Evaporation of samples. F.1. Setup. F.1.a. Prepare nitrogen tank for use. F.1.a.i. Make sure nitrogen tank is full or more than 100 lb/psi before use; 111

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any less may not be enough for the evaporation. F.1.a.ii. Record the start time and starting pressure on the nitrogen tank tracking sheet. F.1.b. Tips for using nitrogen tank F.1.b.i. Any tank with air should be bolted down to the ground using an appropriate harness. F.1.b.ii. The main valve is used to turn the tank on/off. F.1.b.iii. The regulator increases/decreases the nitrogen pressure as needed. F.1.b.iv. The gauge shows the pressure of the air being released. F.1.b.v. At 1000 lbs/psi, reorder nitrogen. F.1.c. Prepare the block manifold for use. F.1.c.i. Attach nitrogen tank to the manifold using tubing. The tubing is attached to a spigot on the back of the block manifold. F.1.c.ii. Determine number of samples that will be evaporated and arrange needles on the nitrogen air releasing apparatus. Space them evenly and plug the remaining unused air releasers. F.2. Sample evaporation. F.2.a. Place the sample vials in the block holder. F.2.b. Lower the air releasing apparatus into the sample vials so that the bottom of the needle is not lower than the neck of the vial. F.2.b.i. The needle should not come in contact with the liquid. F.2.b.ii. Use a technique of taping the appropriate height for the needle to be placed into each vial. F.2.c. Turn on the nitrogen tank. F.2.c.i. Turn the main valve in the open direction. F.2.c.ii. Adjust the pressure to between 2,000 and 2,500 lb/psi using the regulator. F.2.c.iii. Adjust the knob on the manifold so that the nitrogen is being released through the needles and into the sample. The sample should not be bubbling under the nitrogen air. A puddle should be formed where nitrogen is hitting the sample uniformly. F.2.d. Allow the sample to evaporate with the fume hood completely down, checking the samples every 15 minutes until the sample is dry. F.2.d.i. Lower the needles so that the samples are constantly under efficient evaporation but not to the point of bubbling. F.2.F. Turn off nitrogen tank. F.2.F.i. Decrease the pressure by turning the regulator. F.2.F.ii. Turn off the main valve by rotating the valve to the close position. F.2.F.iii. Increase the pressure by turning the regulator to aspirate any nitrogen in the tank 112

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through the opening of the manifold by bleeding the line. F.2.F.iv. Record the stop pressure and time on the nitrogen tank tracking sheet. Sample Assay Labels. Day 1 Sample Label: PXXX; Sample PXXXX; Central/Portal RBC Hemolysate; XX/XX/XX. Day 2 and Day 3 Sample Label: PXXXXL(B). 113

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BIOGRAPHICAL SKETCH Estela Soledad Cerutti was born in San Luis, Argentina, on December 17, 1976. She was the first child of Lilia and Luis Cerutti. At age of ten, after playing with her cousins chemistry set and ruining a tables surface, Soledad first discovered her love for the subject. Soledad attended Universidad Nacional de San Luis, graduating with a Bachelors degree in chemistry in March, 2002. After graduation, she immediately started working toward a Ph.D. and completed her studies with a doctoral degree in chemistry on December 15, 2006. Her Argentinean advisor, Dr. Luis Dante Martinez, was the driving force that made Soledad realizes the necessity of travel to United States to explore the field of mass spectrometry. Keeping those ideas in mind, she applied for a Fulbright scholarship and, after an arduous selection process, was awarded with a grant to study in the United States. Soledad moved to Gainesville and started with her Masters program at University of Florida in August, 2005. She worked under the supervision of Dr. Richard Yost and Dr. David Powell developing chromatographic approaches for the separation of metabolites and their detection by time-of-flight mass spectrometry. Though initially intending to only attain a masters degree, her positive personal and scientific experiences have convinced Soledad to continue with her research. She is currently working toward her Ph.D. degree in mass spectrometry at University of Florida. 120