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Applications in High
Resolution Mass
Spectrometry
This page intentionally left blank
Applications in High
Resolution Mass
Spectrometry
Food Safety and Pesticide
Residue Analysis

Edited by

Roberto Romero-González

Antonia Garrido Frenich

Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
Copyright © 2017 Elsevier Inc. All rights reserved.
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This book and the individual contributions contained in it are protected under copyright by
the Publisher (other than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and
experience broaden our understanding, changes in research methods, professional
practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in
evaluating and using any information, methods, compounds, or experiments described
herein. In using such information or methods they should be mindful of their own safety
and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or
editors, assume any liability for any injury and/or damage to persons or property as a
matter of products liability, negligence or otherwise, or from any use or operation of any
methods, products, instructions, or ideas contained in the material herein.

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Contents
List of Contributors.................................................................................. xi
Preface................................................................................................. xiii

CHAPTER 1 HRMS: Fundamentals and Basic Concepts................ 1

Franciso Javier Arrebola-Liébanas, Roberto Romero-González,
Antonia Garrido Frenich
1.1 Introduction (To High-Resolution Mass Spectrometry)............. 1
1.1.1 Basic Concepts (Units and Definitions) ......................... 1
1.1.2 Low-Resolution Mass Spectrometry Versus
High-Resolution Mass Spectrometry............................. 3
1.2 Resolution and Mass Resolving Power.................................. 6
1.3 Accurate Mass Measurement: Exact Mass and Mass Defect...... 7
1.4 Mass Calibration in High-Resolution Mass Spectrometry ......... 9
1.5 General Considerations......................................................12
Acknowledgments...................................................................13
References.............................................................................13
CHAPTER 2 HRMS: Hardware and Software ...............................15
Juan F. Garcı́a-Reyes, David Moreno-González, Rocı́o Nortes-
Méndez, Bienvenida Gilbert-López, Antonio Molina Dı́az
2.1 Introduction.....................................................................15
2.2 Principles of High-Resolution Mass Spectrometry
Analyzers........................................................................16
2.2.1 Time-of-Flight ........................................................ 17
2.2.2 Fourier Transform Ion Cyclotron Resonance ................ 19
2.2.3 Orbitrap................................................................. 21
2.3 Time-of-Flight Mass Spectrometry: Instrument
Configuration and Main Features ........................................22
2.3.1 Stand-alone Electrospray Ionization Time-of-Flight
and Hybrid Quadrupole Time-of-Flight
Instrumentation....................................................... 22
2.3.2 Improvements of Current (Quadrupole) Time-of-Flight
Instrumentation....................................................... 24
2.3.3 Ion Mobility Quadrupole Time-of-Flight ..................... 29
2.3.4 Hybrid Ion Trap Time-of-Flight ................................. 32
2.3.5 Gas ChromatographyeTime-of-Flight and Gas
ChromatographyeQuadrupole Time-of-Flight .............. 32

v
vi Contents

2.4 Orbitrap Analyzers: Instrument Configurations and Main

Features..........................................................................33
2.5 Acquisition Modes in High-Resolution Mass Spectrometry......39
2.5.1 Data-Dependent Acquisition...................................... 39
2.5.2 Data-Independent Acquisition.................................... 43
2.5.3 Postacquisition Approaches....................................... 44
2.6 Databases and the Internet Resources for High-Resolution
Mass Spectrometry ...........................................................44
Acknowledgments...................................................................50
References.............................................................................50
CHAPTER 3 Analytical Strategies Used in HRMS .......................59
Aan Agüera, Ana Belén Martı́nez-Piernas,
Marina Celia Campos-Mañas
3.1 Introduction.....................................................................59
3.2 Advantages of High-Resolution Mass Spectrometry
in Pesticide Analysis.........................................................60
3.2.1 Selectivity in High-Resolution Mass Spectrometry:
Accurate Mass and Resolution in Qualitative
Analysis ................................................................ 60
3.2.2 Improving Selectivity by Tandem Mass Spectrometry
Information ............................................................ 62
3.2.3 Quantitative Performance.......................................... 65
3.3 Data Analysis Workflows in High-Resolution Mass
Spectrometry ...................................................................68
3.3.1 Qualitative Screening Method Validation..................... 73
3.3.2 Nontarget Analysis .................................................. 75
3.4 Conclusions.....................................................................78
Acknowledgments...................................................................78
References.............................................................................78
Further Reading......................................................................81
CHAPTER 4 Current Legislation on Pesticides ...........................83
Helen Botitsi, Despina Tsipi, Anastasios Economou
4.1 Introduction.....................................................................83
4.2 Pesticides........................................................................83
4.2.1 Identity and Physicochemical Properties...................... 83
4.2.2 Pesticides Classification............................................ 84
4.2.3 Pesticide Metabolites and Transformation Products ....... 85
4.3 Legislation ......................................................................86
4.3.1 Pesticides Authorization ........................................... 86
 Contents vii

4.3.2 Maximum Residue Limits......................................... 88

4.3.3 Monitoring Programs ............................................... 90
4.4 Analytical Quality ControldMethod Validation ....................92
4.4.1 Guidelines for Pesticide Residue Analysis ................... 98
4.5 Mass Spectrometry in Pesticide Residue Analysis ................ 110
4.5.1 Mass Spectrometry Identification and Confirmation......110
4.5.2 Potential of High-Resolution Mass Spectrometry
in Pesticide Residue Analysis ...................................115
References........................................................................... 119
CHAPTER 5 Advanced Sample Preparation Techniques
for Pesticide Residues Determination by HRMS
Analysis .............................................................. 131
Renato Zanella, Osmar D. Prestes, Gabrieli Bernardi,
Martha B. Adaime
5.1 Introduction................................................................... 131
5.2 Matrix Effects and the Influence of Coextracted
Components .................................................................. 133
5.3 Sample Preparation Techniques for Pesticide Residue
Determination by Chromatographic Techniques Coupled
to High-Resolution Mass Spectrometry .............................. 135
5.3.1 Dilute-and-Shoot....................................................137
5.3.2 QuEChERS Method................................................138
5.3.3 Matrix Solid-Phase Extraction ..................................141
5.3.4 Solid-Phase Extraction ............................................142
5.3.5 Solid-Phase Microextraction and Stir Bar Sorptive
Extraction .............................................................147
5.3.6 Microwave-Assisted Extraction.................................149
5.3.7 Pressurized Liquid Extraction...................................150
5.4 Perspectives and Conclusions ........................................... 151
References........................................................................... 154
CHAPTER 6 Applications of Liquid Chromatography
Coupled With High-Resolution Mass
Spectrometry for Pesticide Residue Analysis
in Fruit and Vegetable Matrices ............................ 165
P. Sivaperumal
6.1 Introduction................................................................... 165
6.2 Applications of Pesticide Residue Analysis in Fruit
and Vegetable Samples by LC-HRMS................................ 168
viii Contents

6.3 Optimized Sample Preparation and Chromatographic

Conditions for Mass Analyzers ......................................... 186
6.4 Analytical Method Validation ........................................... 187
6.4.1 Matrix Effect.........................................................187
6.4.2 Evaluation of the Matrix Interferences by
UHPLC-HR/MS.....................................................189
6.4.3 Limit of Detection, Limit of Quantitation,
Accuracy, and Precision ..........................................192
6.5 Accurate Measurement of Pesticide Residues in Fruit
and Vegetable Samples.................................................... 192
6.5.1 Determination of Chlorine Isotope in Food
Samples................................................................192
6.5.2 Determination of Carbon, Chlorine, and
Bromine Isotope in Food Samples.............................195
6.6 Evaluation of Pesticide Residues in Fruit and Vegetable
Samples........................................................................ 195
6.7 Conclusion.................................................................... 197
Acknowledgments................................................................. 198
References........................................................................... 198
CHAPTER 7 Application of HRMS in Pesticide Residue
Analysis in Food From Animal Origin ..................... 203
Roberto Romero-González, Antonia Garrido Frenich
7.1 Introduction................................................................... 203
7.2 Instrumental Requirements............................................... 204
7.3 Analytical Procedures: Extraction and Chromatographic
Conditions..................................................................... 210
7.4 Quantitative and Qualitative Applications ........................... 215
7.5 Differences Between Low-Resolution Mass Spectrometry
and High-Resolution Mass Spectrometry Analytical
Methods........................................................................ 226
7.6 Overview and Future Perspectives..................................... 228
References........................................................................... 229
CHAPTER 8 Recent Advances in HRMS Analysis of Pesticide
Residues Using Atmospheric Pressure Gas
Chromatography and Ion Mobility ......................... 233
Lauren Mullin, Gareth Cleland, Jennifer A. Burgess
8.1 Introduction................................................................... 234
8.2 Atmospheric Pressure Gas Chromatography........................ 236
8.2.1 Introduction...........................................................236
8.2.2 Background...........................................................236
 Contents ix

8.2.3Current Design ......................................................237

8.2.4Ionization Mechanisms............................................238
8.2.5Ionization Trends for Pesticides ................................239
8.2.6Pesticide Screening Using Atmospheric Pressure Gas
Chromatography With High-Resolution Mass
Spectrometry .........................................................239
8.2.7 Improved Selectivity and Sensitivity With Atmospheric
Pressure Gas Chromatography ..................................245
8.2.8 Carrier Gas Flow Rate Increase ................................246
8.3 Time-of-Flight Mass Spectrometry .................................... 247
8.4 Ion Mobility Separation................................................... 249
8.4.1 Background and Theory ..........................................249
8.4.2 Application of Traveling Wave Ion Mobility
Spectrometry to Pesticide Screening ..........................251
8.4.3 Spectral Selectivity Enhancement..............................253
8.4.4 Protomer Visualization ............................................255
8.5 Summary and Conclusion ................................................ 258
Acknowledgments................................................................. 261
References........................................................................... 261
CHAPTER 9 Direct Analysis of Pesticides by Stand-Alone
Mass Spectrometry: Flow Injection and Ambient
Ionization ............................................................ 265
E. Moyano, M.T. Galceran
9.1 Introduction................................................................... 265
9.2 Flow Injection Analysis................................................... 268
9.2.1 FIA Method Development .......................................273
9.2.2 Sample Preparation for FIA-MS-Based
Methodologies .......................................................277
9.2.3 FIA-MS Methods Performance .................................279
9.2.4 FIA-MS Applications..............................................282
9.3 Ambient Mass Spectrometry ............................................ 283
9.3.1 Electrospray-Based Techniques.................................284
9.3.2 Plasma-Based Techniques ........................................290
9.3.3 Ambient Mass Spectrometry Method Development ......292
9.3.4 Sample Handling for Ambient Mass Spectrometry-
Based Methodologies..............................................298
9.3.5 Ambient Mass Spectrometry Method Performance.......302
9.3.6 Ambient Mass Spectrometry Applications ..................305
9.4 Final Remarks and Future Trends...................................... 307
Acknowledgments................................................................. 309
References........................................................................... 309
x Contents

CHAPTER 10 Identification of Pesticide Transformation Products

in Food Applying High-Resolution Mass
Spectrometry ..................................................... 315
Imma Ferrer, Jerry A. Zweigenbaum, E. Michael Thurman
10.1 Introduction................................................................. 315
10.2 Experimental ............................................................... 316
10.2.1 Chemicals and Reagents ...................................... 316
10.2.2 Greenhouse Study............................................... 316
10.2.3 Plant Extraction.................................................. 316
10.2.4 Liquid Chromatography/Quadrupole-Time-of-Flight
Mass Spectrometry Analysis................................. 317
10.2.5 Mass Profiler Software ........................................ 318
10.3 Imidacloprid Metabolites in Plants .................................. 318
10.3.1 Accurate Mass Databases..................................... 319
10.3.2 Mass Profiler Professional.................................... 326
10.3.3 Metabolite Distribution and Mass Balance
for Imidacloprid Metabolites ................................ 329
10.4 Imazalil Metabolites in Plants and Soil............................. 329
10.4.1 Chlorine Filter Approach ..................................... 330
10.4.2 Soil Metabolites ................................................. 332
10.5 Propiconazole Metabolites in Plants and Soil .................... 332
10.5.1 Chlorine Filter Approach ..................................... 332
10.6 Conclusions................................................................. 334
References........................................................................... 334

Index...................................................................................................337
List of Contributors

Martha B. Adaime
Federal University of Santa Maria, Santa Maria, Brazil
Ana Agüera
CIESOL, Joint Centre of the University of Almerı́a e CIEMAT, Almerı́a, Spain
Franciso Javier Arrebola-Liébanas
University of Almerı́a, Almerı́a, Spain
Gabrieli Bernardi
Federal University of Santa Maria, Santa Maria, Brazil
Helen Botitsi
General Chemical State Laboratory, Athens, Greece
Jennifer A. Burgess
Waters Corporation, Milford, MA, United States
Marina Celia Campos-Mañas
CIESOL, Joint Centre of the University of Almerı́a e CIEMAT, Almerı́a, Spain
Gareth Cleland
Waters Corporation, Milford, MA, United States
Anastasios Economou
National and Kapodistrian University of Athens, Athens, Greece
Imma Ferrer
University of Colorado, Boulder, CO, United States
Antonia Garrido Frenich
University of Almerı́a, Almerı́a, Spain
M.T. Galceran
University of Barcelona, Barcelona, Spain
Juan F. Garcı́a-Reyes
University of Jaén, Jaén, Spain
Bienvenida Gilbert-López
University of Jaén, Jaén, Spain
Ana Belén Martı́nez-Piernas
CIESOL, Joint Centre of the University of Almerı́a e CIEMAT, Almerı́a, Spain
Antonio Molina Dı́az
University of Jaén, Jaén, Spain

xi
xii List of Contributors

David Moreno-González
University of Jaén, Jaén, Spain
E. Moyano
University of Barcelona, Barcelona, Spain
Lauren Mullin
Waters Corporation, Milford, MA, United States
Rocı́o Nortes-Méndez
University of Jaén, Jaén, Spain
P. Sivaperumal
National Institute of Occupational Health, Ahmedabad, Gujarat, India
Osmar D. Prestes
Federal University of Santa Maria, Santa Maria, Brazil
Roberto Romero-González
University of Almerı́a, Almerı́a, Spain
E. Michael Thurman
University of Colorado, Boulder, CO, United States
Despina Tsipi
General Chemical State Laboratory, Athens, Greece
Renato Zanella
Federal University of Santa Maria, Santa Maria, Brazil
Jerry A. Zweigenbaum
Agilent Technologies Inc., Wilmington, DE, United States
Preface

Each problem that I solved became a rule, which served afterward to solve other
problems.
Rene Descartes

Currently, mass spectrometry is an essential tool in food safety and its output has
reached an unprecedented level, being the most adequate detection system for the
analysis of pesticide residues in food matrices. In the last few years, new analyzers,
ionization methods, and analytical strategies have been developed to increase the
scope of analytical methods as well as the reliability of the results.
Up to now, high-resolution mass spectrometry (HRMS) has been considered as a
complementary tool of conventional triple quadrupole (QqQ) analyzers. However,
time of flight and/or Orbitrap are replacing low-resolution mass spectrometry ana-
lyzers because they can increase the number of compounds simultaneously moni-
tored, retrospective analysis can be carried out, and unknown compounds can be
identified. All these tasks can be performed using one benchtop platform at suitable
sensitivity. In addition, if HRMS analyzers are coupled with ultrahigh-performance
liquid chromatography (UHPLC) the number of compounds analyzed in one single
run can increase considerably. Furthermore, when ambient ionization techniques are
used with HRMS analyzers, sample treatment could be minimized, increasing sam-
ple throughput.
The use of these HRMS analyzers opens a new scenario in pesticide residue anal-
ysis, and potential users should know the strategies that can be applied to get all the
information that these analyzers could provide, as well as the pros and cons in rela-
tion to QqQ.
Because of the fast application and implementation of these new procedures, we
consider that setting the main principles and strategies based on these approaches is
necessary to provide a comprehensive view of the cited tools, being a cornerstone in
the food safety field.
Thus, this book would provide a complete overview of the possibilities that
HRMS could offer in pesticide residue analysis in food, as well as the suitable work-
flow needed to achieve the goals proposed by scientists. Chapters 1e4 provide an
overview of HRMS, basic concepts, hardware, general approaches (target and
nontarget analysis), and current legislation related to this topic. Chapters 5e7 are
“applied chapters” where the main extraction procedures used, as well as the appli-
cation of HRMS in pesticide residue analysis in several types of matrices, are
described. Finally, Chapters 8e10 describe new advances on the use of HRMS
such as gas chromatography (GC)eHRMS, ambient ionization techniques, and
identification of transformation products. The content of each chapter is described
in more detail as follows.

xiii
xiv Preface

To facilitate the introduction to the topics presented in this book, Chapter 1 de-
scribes the principles of HRMS, explaining several concepts such as monoisotopic
mass, resolution, mass accuracy, isotopic pattern, etc. Moreover, the differences be-
tween low- and high-resolution mass spectrometry are also discussed. In Chapter 2,
the several analyzers that could be used in HRMS are described, indicating the dif-
ferences between the HRMS systems from different vendors, as well as the software
that, nowadays, is available to process all the data provided by these analyzers.
Finally, online resources such as ChemSpider and MassBank are described. Target,
nontarget, and unknown analysis strategies are explained in Chapter 3, describing
how a database is prepared and the workflow commonly used for nontarget and un-
known analysis. Chapter 4 provides a complete overview of the general require-
ments indicated by international guidelines (i.e., SANTE) regarding the use of
HRMS in pesticide residue analysis, as well as the parameters that should be vali-
dated. A section describing pesticides legislation (indicating MRLs) is also included.
Bearing in mind that theoretically, unlimited number of compounds could be deter-
mined by HRMS, Chapter 5 describes the development of generic extraction
methods that allow the simultaneous extraction of a huge number of pesticides
that are needed. Chapters 6 and 7 cover the main applications describing the use
of HRMS coupled with UHPLC during the analysis of pesticide residue analysis
in fruits and vegetables (Chapter 6) and in food from animal origin (Chapter 7).
Although most of the current applications focused on pesticide residue applying
HRMS use liquid chromatography as the separation technique, in the last few years,
GC has also been utilized, because of the development of new ionization sources,
such as atmospheric pressure gas chromatography or new couplings such as GC-
Orbitrap. Therefore, Chapter 8 is devoted to this topic to highlight the potentiality
of GCeHRMS in pesticide residue analysis as well as new approaches such as
ion mobility. Because of the potentiality of HRMS, chromatographic step could
be removed from the conventional analytical method, and rapid detection of the
target compounds could be performed. This approach is interesting for pesticides
that cannot be commonly analyzed by multiresidue methods (i.e., very polar pesti-
cides), and the use of HRMS could simplify the analytical strategy. Chapter 9 is
focused on this approach as well as in the use of ambient mass spectrometry, which
allows for the direct analysis of samples without sample extraction and chromato-
graphic separation, especially when HRMS analyzers are used. Finally, Chapter
10 is dedicated to the advantages that HRMS provides for the identification of pesti-
cide transformation products.
The book is intended for researchers and professionals working with LCeMS,
such as food chemists, analytical chemists, toxicologists, food scientists, and
everyone who uses/needs this technique to evaluate food safety. Moreover, under-
graduate students would also be interested.
Finally, it has been a great pleasure to thank all the authors of this book for their
work. All of them are specialists and we really appreciate their effort and time. We
also give special thanks to the editorial and production teams of the publisher,
 Preface xv

Elsevier, especially Karen R. Miller, who started this adventure, and Jackie Trues-
dell, for her patience and help, allowing this work to come to fruition.

Roberto Romero-González
Antonia Garrido Frenich
Almerı´a, Spain
November, 2016.
This page intentionally left blank
 CHAPTER

HRMS: Fundamentals and

Basic Concepts

Franciso Javier Arrebola-Liébanas, Roberto Romero-González,

1
Antonia Garrido Frenich
University of Almerı´a, Almerı´a, Spain

1.1 INTRODUCTION (TO HIGH-RESOLUTION MASS

SPECTROMETRY)
1.1.1 BASIC CONCEPTS (UNITS AND DEFINITIONS)
Mass spectrometry (MS) is an analytical technique commonly used for qualitative
and quantitative chemical analysis. MS measures the massecharge ratio (m/z) of
any analyte, of both organic and inorganic nature, which has previously been
ionized. Only the ions are registered in MS, but the particles with zero net electric
charge (molecules or radicals) are not detected. Therefore, MS does not directly
measure mass, but it determines the m/z, being m the relative mass of an ion on
the unified atomic scale divided by the charge number, z, of the ion (regardless of
sign). The m/z value is a dimensionless number.
Because the mass of atoms and molecules is very small, the kilogram as standard
international (SI) base unit cannot be used for its measurement. For that, a non-SI
unit of mass, unified atomic mass unit (u) is used. At this point, in this introductory
section, it is worth clarifying some basic terms (units and definitions) in MS accord-
ing to the International Union of Pure and Applied Chemistry (IUPAC) recommen-
dations (IUPAC, 1997; Murray et al., 2013).
The u also called Dalton (Da), is defined as 1/12th of the mass of one atom of 12C
at rest in its ground state, being 1 u ¼ 1 Da ¼ 1.660538921 (73)  1027 kg (num-
ber in parentheses indicates the estimated uncertainty). In this way, the mass of other
atoms or molecules is expressed relative to the mass of the most abundant stable
isotope of carbon, 12C, and this value is dimensionless.
The z is defined as absolute value of charge of an ion divided by the value of the
elementary charge of the electron (e) rounded to the nearest integer, being
e ¼ 1.602177  1019 C. The m/z unit is the thomson (Th), although it is now a
deprecated term, being 1 Th ¼ 1 u/e ¼ 1.036426  108 kg/C. For that, use of the
dimensionless term m/z is accepted in the literature, and this criterion will be fol-
lowed throughout this book.
Other basic concepts that are commonly used in MS will be shortly described to
clarify the meaning of these throughout the following chapters.

Applications in High Resolution Mass Spectrometry. http://dx.doi.org/10.1016/B978-0-12-809464-8.00001-4 1

Copyright © 2017 Elsevier Inc. All rights reserved.
2 CHAPTER 1 HRMS: Fundamentals and Basic Concepts

• Atomic mass: The number that represents the element’s mass based on the
weighted average of the masses of its naturally occurring stable isotopes. For
example, the integer atomic mass of bromine is 80 Da. This is because there are
only two naturally occurring stable isotopes of bromine, 79Br and 81Br, which
exist in nature in about equal amounts. When the relative mass (Mr) of an ion,
molecule, or radical is reported, it is based on the atomic masses of its elements.
• Nominal mass: Mass of a molecular ion or molecule calculated using the isotope
mass of the most abundant constituent element isotope of each element
(Table 1.1) rounded to the nearest integer value and multiplied by the number
of atoms of each element. Example: nominal mass of H2O ¼ (2  1 þ 1  16)
u ¼ 18 u.
• Monoisotopic mass: Exact mass of an ion or molecule calculated using the mass
of the most abundant isotope of each element. Example: monoisotopic mass of
H2O ¼ (2  1.007825 þ 1  15.994915) u ¼ 18.010565 u. The exact mass of
the common elements and their isotopes are provided in Table 1.1.
• Exact mass: Calculated mass of an ion or molecule with specified isotopic
composition.
• Mass defect: Difference between the nominal mass and the monoisotopic mass of
an atom, molecule, or ion. It can be a positive or negative value.
• Relative isotopic mass defect (RDm): It is the mass defect between the mono-
isotopic mass of an element and the mass of its Aþ1 or its Aþ2 isotopic cluster
(Thurman & Ferrer, 2010). For instance, RDm for the pair 35Cl:37Cl is
0.0030 Da.
• Average mass: Mass of an ion or molecule weighted for its isotopic composition,
i.e., the average of the isotopic masses of each element, weighted for isotopic
abundance (Table 1.1). Example: average mass of
H2O ¼ (2  1.00794 þ 1  15.9994) u ¼ 18.01528 u.
• Accurate mass: Experimentally determined mass of an ion of known charge.
• Mass accuracy: Difference between the mass measured by the mass analyzer and
theoretical value.
• Resolution or mass resolving power: Measure of the ability of a mass analyzer to
distinguish two signals of slightly different m/z ratios.
• Mass calibration: Means of determining m/z values of ions from experimentally
detected signals using a theoretical or empirical relational equation. In general,
this is accomplished using a computer-based data system and a calibration file
obtained from a mass spectrum of a compound that produces ions of known m/z
values.
• Mass limit: Value of m/z above or below which ions cannot be detected in a mass
spectrometer.
• Mass number: The sum of the protons and neutrons in an atom, molecule, or ion.
If the mass is expressed in u, mass number is similar to nominal mass.
• Most abundant ion mass: The mass that corresponds to the most abundant peak
in the isotopic cluster of the ion of a given empirical formula.
 1.1 Introduction (To High-Resolution Mass Spectrometry) 3

Table 1.1 Nominal, Isotopic, and Average Masses of Some Common Stable
Isotopes
Nominal Isotopic Average
Element Isotope Abundance Mass Mass Mass
1
H H 99.9885 1 1.007825 1.00794
2
H 0.0115 2 2.014102
12
C C 98.93 12 12.000000 12.0110
13
C 1.08 13 13.003355
14
N N 99.632 14 14.003074 14.00674
15
N 0.368 15 15.000109
16
O O 99.757 16 15.994915 15.9994
17
O 0.038 17 16.999131
18
O 0.205 18 17.999160
19
F F 100 19 18.998403 18.9984
23
Na Na 100 23 22.989770 22.9898
28
Si Si 92.2297 28 27.976927 28.0855
29
Si 4.6832 29 28.976495
30
Si 3.0872 30 29.973770
31
P P 100 31 30.973762 30.9738
32
S S 94.93 32 31.972072 32.0660
33
S 0.76 33 32.971459
34
S 4.29 34 33.967868
35
Cl Cl 75.78 35 34.968853 35.4527
37
Cl 24.22 37 36.965903
79
Br Br 50.69 79 78.918336 79.9094
81
Br 49.32 81 80.916289
127
I I 100 127 126.904476 126.9045

1.1.2 LOW-RESOLUTION MASS SPECTROMETRY VERSUS HIGH-

RESOLUTION MASS SPECTROMETRY
It should be noted that mass measurements in MS can be carried out at either low
resolution (LRMS) or high resolution (HRMS). An LRMS measurement provides
information about the nominal mass of the analyte (Dass, 2007), i.e., the m/z for
each ion is measured to single-digit mass units (integer mass). However, exact
mass is measured by HRMS, i.e., the m/z for each ion is measured to four to six
decimal points (Ekman, Silberring, Westman-Brinkmalm, & Kraj, 2009). This is
very useful to structure elucidation of unknown compounds for analytes having
the same nominal mass, but with very small differences in their exact masses. As
a result, by LRMS measurements it is not possible to differentiate between imazalil,
C14H14Cl2N2O (14  12 þ 14  1 þ 2  35 þ 2  14 þ 1  16 ¼ 296 u), and flu-
nixin, C14H11F3N2O2 (14  12 þ 11  1 þ 3  19 þ 2  14 þ 2  16 ¼ 296 u),
4 CHAPTER 1 HRMS: Fundamentals and Basic Concepts

pesticides. However, this would be possible by using exact mass measurements,

imazalil C14H14Cl2N2O (14  12 þ 14  1.007825 þ 2  34.968852 þ 2 
14.003074 þ 1  15.994915 ¼ 296.048317 u) and flunixin C14H11F3N2O2 (14 
12 þ 11  1.007825 þ 3  18.998403 þ 2  14.003074 þ 2  15.994915 ¼
296.077262 u).
High-resolution mass spectrometers have evolved from the 1960s with the intro-
duction of double-focusing magnetic-sector mass instruments (Picó, 2015). Next,
Fourier transform ion cyclotron resonance (FT-ICR), time-of-flight (TOF), and Orbi-
trap mass analyzers were also introduced in the market. Also, hybrid HRMS instru-
ments, such as quadrupole TOF (Q-TOF), ion trap (IT)-TOF, linear trap quadrupole
(LTQ)-Orbitrap, or QeOrbitrap, have been developed. These last analyzers provide
tandem (MS/MS) or MSn spectra of high resolution, in addition to accurate mono-
isotopic mass measurements, of great applicability both for the confirmation of
target compounds and the identification of unknown compounds (Lin et al.,
2015). The TOF and Orbitrap analyzers, single or hybrid instruments, are the
most widely used in the analysis of organic contaminants, such as pesticide residues
(Lin et al., 2015; Picó, 2015).
Among the main characteristics that define the performance of a mass analyzer
are (Dass, 2007; de Hoffmann & Stroobant, 2007; Mcluckey & Wells, 2001) mass
range, speed, efficiency, linear dynamic range, sensitivity, resolution (or its mass
resolving power), and mass accuracy. The mass range is that over which a mass spec-
trometer can detect ions or is operated to record a mass spectrum. When a range of
m/z is indicated instead of a mass range, this should be specified explicitly. The
speed or scan speed is the rate at which the analyzer measures over a particular
mass range. Efficiency is defined as the product of the transmission of the analyzer
by its duty cycle, where the transmission is the ratio of the number of ions reaching
the detector and the number of ions entering the mass analyzer, and the duty cycle
can be described as the fraction of the ions of interest formed in the ionization step
that are subjected to mass analysis.
Linear dynamic range is considered as the range over which ion signal is linear
with analyte concentration. Sensitivity can be expressed as detection sensitivity or
abundance sensitivity; the first is the smallest amount of an analyte that can be
detected at a certain defined confidence level, while the second is the inverse of
the ratio obtained by dividing the signal level corresponding to a large peak by
the signal level of the background at one mass-to-charge unit lower or higher. A
summary of these characteristics of high-resolution mass analyzers is shown in
Table 1.2. As it can be observed, in terms of resolving power and accuracy, the
FT-ICR analyzer presents the best values, followed by the recently introduced tribrid
Orbitrap analyzer. TOF and Q-TOF analyzers have worse values, although the FT-
ICR analyzer comprises the worst sensitivity.
Last but not least, two key characteristics of high-resolution mass analyzers are
resolution (or its mass resolving power) and mass accuracy, which will be treated in
more detail in the following two sections.
 1.1 Introduction (To High-Resolution Mass Spectrometry)
Table 1.2 Comparison of the Characteristics of Some High-Resolution Mass Spectrometry Analyzers
Mass Linear Dynamic Resolving Power Accuracy
Analyzer Range Speed Range Sensitivity (FWHM) (ppm)
Magnetic sector 10,000 w1 s 109 106e109 100,000 <1
FT-ICR 10,000 w1 s 103e104 103e104 1,000,000 <1
TOF >300,000 Milliseconds 106 106 30,000 3e5
Q-TOF 10,000 wMilliseconds 103e104 106 30,000 3e5
IT-TOF w0.1 s 103e104 105 100,000 3e5
Exactive Orbitrap 4000 0.1 s >5000 106 100,000 <3
LTQ-Orbitrap 4000 0.1 s >5000 106 100,000/240,000 <3
Q-Orbitrap 8000 0.05 s 105 106 240,000 <2
Tribrid Orbitrap 6000 0.05 s 105 106e107 500,000 <1
FT-ICR, Fourier transform ion cyclotron resonance; FWHM, full width at half maximum; IT-TOF, ion trap TOF; LTQ, linear trap quadrupole; Q-TOF, quadrupole TOF;
TOF, time-of-flight. Adapted from Picó, Y. (2015). Advanced mass spectrometry. In Y. Picó (Ed.), Comprehensive analytical chemistry, Vol. 68. Amsterdam:
Elsevier.

5
6 CHAPTER 1 HRMS: Fundamentals and Basic Concepts

1.2 RESOLUTION AND MASS RESOLVING POWER

Resolution or resolving power is the capacity of a mass analyzer to yield distinct sig-
nals for two ions with a small m/z difference (de Hoffmann & Stroobant, 2007). Un-
fortunately, there is confusion about these two concepts and also between mass
resolving power and resolving power in MS, because the definitions provided by
different documents are not exactly the same.
Dass (2007) defines the mass resolution of a mass spectrometer as its ability to
distinguish between two neighboring ions that differ only slightly in their mass
(Dm). According to this definition, it is the inverse value of the resolving power,
RP ¼ m/Dm, where m is the average of the accurate masses, (m1 þ m2)/2, of the
two neighboring ions. Xian, Hendrickson, and Marshall (2012) define the resolution
as the smallest mass difference, m2  m1 or Dm, between two mass spectral peaks
such that the valley between their sum is a specified fraction (e.g., 50%) of the
height of the smaller individual peak. A similar definition is given by Marshall,
Hendrickson, and Shi (2002), as the minimum mass difference between two equal
magnitude peaks such that the valley between them is a specified fraction of the
peak height.
The IUPAC recommendations (Murray et al., 2013) define resolution as m/Dm,
where m is the m/z of the ion of interest. Although depending on the method of mea-
surement of D(m/z), it is possible to differentiate between the two concepts (Murray
et al., 2013; Price, 1991). On one hand, resolution, as 10% valley, is the (m/z)/D(m/z)
value measured for two peaks of equal height in a mass spectrum at m/z and m/
z þ D(m/z) that are separated by a valley for which the lowest point is 10% of the
height of either peak, i.e., the peaks are resolved when the valley between the two
m/z values is 10% of the height of either one (Fig. 1.1). For peaks of similar height
separated by a valley, let the height of the valley at its lowest point be 10% of the
lower peak, and the resolution should be given for a number of values of m/z.
This 10% valley definition for the resolution is used with magnetic-sector analyzers
(Ekman et al., 2009).
On the other hand, resolution, as peak width, expresses the (m/z)/D(m/z) value for
a single peak, where D(m/z) is the width of the peak at a height, which is a specified
fraction (50, 5, or 0.5%) of its maximum peak height (Fig. 1.1). The used fraction is
often 50%, and D(m/z) is named as full width at half maximum (FWHM). FT-ICR,
TOF, and Orbitrap analyzers use this 50% valley definition for set resolution (Ekman
et al., 2009).
In addition, there is controversy in the definition of mass resolving power and
resolving power in MS (IUPAC, 1997). The definition of the first term is similar
to the definition of resolution indicated earlier (Murray et al., 2013), i.e., as a dimen-
sionless ratio between m/Dm. Resolving power in MS is the ability of an instrument
or measurement procedure to distinguish between two peaks differing in the quotient
m/z by a small increment and expressed as the peak width in mass units. However,
both terms have been unified in the current IUPAC definition as a measure of the
ability of a mass spectrometer to provide a specified value of mass resolution.
 1.3 Accurate Mass Measurement: Exact Mass and Mass Defect 7

FIGURE 1.1
Methods of calculating mass resolving power.
Reprinted from Picó, Y. (2015). Advanced mass spectrometry. In Y. Picó (Ed.), Comprehensive analytical
chemistry, Vol. 68. Amsterdam: Elsevier, with permission from Elsevier.

1.3 ACCURATE MASS MEASUREMENT: EXACT MASS AND

MASS DEFECT
It is important to differentiate between accurate mass and exact mass. The first is the
experimentally determined mass of an ion of known charge (Bristow and Webb,
2003; Sparkman, 2006) and it refers to a measured mass, while the second is the
calculated mass of an ion or molecule with specified isotopic composition (Kim,
Rodgers, & Marshall, 2006), and it refers to a calculated mass. Therefore, although
an LR mass spectrometer can measure integer relative mass with high accuracy, the
information obtained is not so complete as the measurement of accurate relative
mass offered by HR mass spectrometers (Herbert & Johstone, 2003). The difference
between the nominal mass and the monoisotopic mass of an atom, molecule, or ion,
positive or negative value, is the mass defect.
8 CHAPTER 1 HRMS: Fundamentals and Basic Concepts

The value of accurate mass measurement is illustrated in the following examples:

(1) to distinguish compounds with the same integer nominal (molecular) mass in the
same sample; (2) to determine the molecular formula or elemental composition for
an unknown compound, which is helpful for its identification; and (3) to find out the
fragmentation routes. As an example, Fig. 1.2 shows the fragmentation pattern for
the flonicamid pesticide, which can be elucidated by HRMS.
In HRMS, the mass accuracy is the difference between the m/z value measured
by the mass spectrometer and the theoretical m/z value. It can be reported as an
absolute value; for instance, in millimass units (mmu) or millidalton (mDa):

Mass accuracyðmmuÞ ¼ ðm=zmeasured  m=ztheoretical Þ  103

Also, it can be expressed as a relative value in parts per million (ppm):
 
m=zmeasured  m=ztheoretical
Mass accuracyðppmÞ ¼ 106
ðm=ztheoretical Þ

FIGURE 1.2
Fragmentation pattern for the flonicamid pesticide (TFNA: 4-trifluoromethylnicotinic acid;
TFNA-AM: 4-trifluoromethilnicotinamide).
 1.4 Mass Calibration in High-Resolution Mass Spectrometry 9

In general, an acceptable value of the measured mass should be within 5 ppm of

the accurate mass (Gross, 1994). A key point to minimize error in accurate mass
measurement is ensuring that the target ion is completely free of interfering ions,
because these ions shift the mass of the target peak.
In general, high mass resolution and high mass accuracy depend on each other,
because the latter tends to improve as the former is improved. HR allows to separate
neighboring ions, and accurate mass can deliver molecular formulas (Gross, 2011).
Therefore, it is important to note that HR alone does not equally imply measuring
the accurate mass.

1.4 MASS CALIBRATION IN HIGH-RESOLUTION MASS

SPECTROMETRY
Mass calibration is a relevant process in every mass spectrometer for a proper rep-
resentation of ions in the m/z axis. It also results in a very important fact in HRMS
where not only high mass resolution but also high mass accuracy is critical (Gross,
2011). For that, typically, mass reference compounds with a compilation of well-
known m/z values are needed (Busch, 2004, 2005).
Calibration is frequently performed in an automatic or semiautomatic way by the
mass spectrometer software when the list of ions of those mass calibration com-
pounds are correlated with experimentally obtained m/z values. It is called external
mass calibration if the mass calibration is stored in a calibration file for further mea-
surements and the mass calibration standard is not used during acquisition of exper-
imental mass spectra. Frequency of recalibration has influence on mass accuracy of
the mass analyzer. The selection of the mass calibration compound depends on the
ionization method and, of course, the mass analyzer used. However, (1) they should
yield sufficient regularly spaced abundant ions across the entire scan range; (2) the
reference ions should have negative mass defects to prevent overlap with typical
compounds containing C, H, N, and O; and (3) they should be readily available,
chemically inert, and sufficiently volatile. Some of the most common calibration
standards and their masses and relative abundances can be found in literature
(Dass, 2007). For example, perfluorokerosene (PFK) is often established as a
mass calibration standard in electron ionization (EI). PFK provides numerous frag-
ment ions that may be used up to m/z 700e1100 depending on the type of mixture
used (commercially available from low to high boiling grades). Also, perflourotribu-
tylamine (FC-43) is also proposed as mass calibration standard thanks to its charac-
teristics ions up to 614 in an EI spectrum (Sack, Lapp, Gross, & Kimble, 1984).
When a high-mass calibration (i.e., up to 3000 u) is required, triazines and a mixture
of fluorinated phosphazenes called Ultramark can be used as reference calibrants.
For electrospray instruments, the most typical calibration standards are CsI, poly(-
ethylene glycol) (PEG), poly(ethylene glycol) bis(carboxymethyl ether), poly(eth-
ylene glycol monomethyl ether), and poly(propylene glycol). MALDI users also
have several reference compounds available, such as a-CHCA matrix (dimer þ Hþ),
10 CHAPTER 1 HRMS: Fundamentals and Basic Concepts

4-hydroxy-3-methoxycinnamic acid (trimer þ Naþ), angiotensin I and II, bradyki-

nin, substance P, desArg1-bradykinin, gramicidin, and autodigestion products of
trypsin.
As an alternative, internal mass calibration can be performed. For that, the mass
calibration standard is introduced using a second inlet system into the ion source, for
instance, as a volatile standard. As an alternative, it can be mixed with the analyte
before analysis. This last option presents more limitations than the use of alternative
inlet systems.
Typical mass accuracy obtained by internal mass calibration used to be better
than those obtained by external calibration. Some examples are 0.1e0.5 ppm
with FT-ICR, 0.5e1 ppm with Orbitrap, 0.5e5 ppm with magnetic sector, or
1e10 ppm with TOF analyzers.
Fast atom bombardment instruments are sometimes internally calibrated with
good mass accuracy by using the matrix peaks for a mass calibration but it is
preferred a mixture of the standard with the analyte (matrix) if unwanted reactions
are not observed and proper solubility of the analyte and standard. One typical mass
calibration standard used is PEG with an average molecular weight of 600 u (PEG
600). In this sense, the reproducibility of mass calibration after several scan cycles is
improved because of affection of magnets by hysteresis.
MALDI mass calibration can be compromised if thick sample layers are used
with on-axis TOF instruments. However, orthogonal acceleration TOF analyzers
present better results. In some cases, for example, in the analysis of synthetic poly-
mers, the formation of evenly spaced oligomer ions can be used as internal mass
calibration (Dienes et al., 1996). In the case of TOF analyzers, the conversion
from a measured flight time to mass requires a mass calibration. The computer
makes the calculations using proper algorithms once the values of flight time for
a few calibrant masses are known. Sometimes, a second calibration step is requested
to achieve enough accuracy (Ferrer & Thurman, 2009). It must be carefully
controlled changes in flight distances or accelerating potentials to obtain mass accu-
racies of 1 ppm, but again, the internal mass calibration can correct such instrument
factors with an automatic data processing carried out at the same time as that of the
analysis of the sample. Generally, a mass calibration per day or week is enough to
obtain a proper accuracy of m/z for many TOF instruments, but it is adequate to use
internal mass calibration, especially when long analyses are performed (Chernush-
evich, Loboda, & Thomson, 2001).
FT-ICR mass analyzers with superconducting magnets are frequently very stable
for many days of use in normal applications. In this case, a mass accuracy better than
1 ppm can be achieved in a wide mass range (Rodgers, Blumer, Hendrickson, &
Marshall, 2000).
Some HR mass spectrometers such as double focusing systems (DFS) can be
operated by multiple ion detection (MID) mode where the intensities of some
ions typical of a target analyte can be continuously monitored to increase sensitivity,
precision, and selectivity of the method. A monitoring window is selected if the
 1.4 Mass Calibration in High-Resolution Mass Spectrometry 11

instrument is coupled to a chromatographic inlet device (gas or liquid chromato-

graph). For data acquisition, the magnet gets blocked in one mass and electric scans
are carried out modifying the acceleration voltage. Each scan suffers a rectification
of the mass calibration at the same time that experimental data are obtained. This
technique is called lock-mass technique and improves mass accuracy increasing reli-
ability of mass spectrometric data obtained. Resolution of the instrument can also be
recalculated on each scan. It can also be improved doubling the calibration masses in
a technique called Lock-plus-cali mass technique. This scan-to-scan mass calibra-
tion processed in the background improves confidence of the analytical data. For
this internal mass calibration, the calibration standard is leaked continuously from
the reference inlet system into the ion source. Two ion masses are selected from
the reference substance: one mass that is below the analyte target masses and another
one above the analyte target masses. The lowest mass is called “lock mass” and the
highest is named “calibration mass.” The magnet is locking the magnet at the start of
each MID process and performing a mass calibration based on the lock mass. All
analyzer jumps to the calibration and target masses by fast electrical jumps of the
acceleration voltage. It provides a fine calibration in only a very few milliseconds
(Thermo Fisher Scientific Inc., 2007). Fig. 1.3 shows the typical sequence of steps
during a MID process in DFS instrument.

FIGURE 1.3
Internal mass calibration and target mass detection during a multiple ion detection (MID)
process in a double focusing system (DFS) instrument: A, magnet locking and lock mass
sweep. Mass calibration and resolution determination; B, electrical jump to calibration
mass; C, calibration mass sweep and mass calibration; D, electrical jumps to target
masses; E, electrical jump to calibration mass for mass calibration.
12 CHAPTER 1 HRMS: Fundamentals and Basic Concepts

1.5 GENERAL CONSIDERATIONS

Empirical determination of a molecular formula for a substance can be very useful in
organic MS. HRMS can be an alternative to traditional chemical methods based on
the tedious, slow, and often inaccurate process of breaking down of a known weight
of a molecule into its constituent elements and weight of them (Herbert & Johstone,
2003). It should be mentioned that the mass of an electron is very small compared
with masses of any element, and therefore, frequently, the mass of Mþ is considered
the same as that of M. Therefore, an HR mass spectrometer can be used to measure
relative atomic, molecular, or fragment ion masses with high accuracy. An accept-
able value of the measured mass should be within 5 ppm of the accurate mass (Gross,
1994).
It is essential that the ion of interest is properly resolved from all other neigh-
boring ions because any interfering ion would introduce an error in mass measure-
ment. Therefore, a high resolving power is very important for accurate mass
measurements. For small molecules, a resolving power higher than, i.e., 10,000 is
not critical. For example, a resolving power of 770 would be enough for mass-
resolving C2 H4 þ (28.031300) and COþ (27.994915). Nevertheless, a resolving po-
 

wer of at least 5500 is needed to separate C13 H16 eC2 H4 þ and C13 H16 eCOþ ions,
 

even if the difference between both masses is 0.036385 u. Another difficulty is that
the possible elemental composition becomes quite larger as the mass increases.
Full scan can be one of the simplest acquisition modes to obtain an accurate mass
value when an internal mass calibration is carried out. Accurate masses of all ions
can be determined in one single chromatographic run, and elemental composition
can be achieved with an adequate accuracy of less than 5 ppm. Peak-matching
mode is a more accurate mass measurement technique with a typical accuracy lower
than 0.3 ppm. For that, only one ion is determined at a time, for example, using the
MID technique described earlier. It is recommended that the mass of the mass cali-
bration ion must be within 2% of the unknown mass to give the highest accuracy. A
combination of slow scanning of the accelerating voltage and computer programs
that improve signal averaging, smoothing, and peak centroiding improves mass ac-
curacy (Hammar, Pettersson, & Carpenter, 1974).
A nominal mass may result from several combinations of elements, but only one
composition can match an accurate mass. Nevertheless, more possible combinations
can occur with an increased number of atoms in a molecule. A list of potential mo-
lecular formulas for various masses has been proposed (Beynon & Williams, 1963).
Diverse algorithms and computer programs are available for elemental composition;
many of them are available online.
Finally, it should be mentioned that RDm is also a valuable tool for confirmation
purposes. The sum of RDm of all isotopes of the molecule is defined as the isotopic
mass average (IMA), and Padilla-Sánchez et al. (2012) used this approach for the
reliable identification of small pesticides such as ethephon and N-acetyl-glufosinate,
obtaining an experimental IMA(Aþ2) value of 0.00286 Da for ethephon, which was
within the theoretical interval of 0.0029  0.0001 Da.
 References 13

ACKNOWLEDGMENTS
The authors gratefully acknowledge Andalusian Regional Government (Regional Ministry of
Innovation, Science, and Enterprise) and FEDER for financial support (Project Ref. P-12-
FQM 1838).

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Sack, T. M., Lapp, R. L., Gross, M. L., & Kimble, B. J. (1984). A method for the statistical
evaluation of accurate mass measurement quality. International Journal of Mass Spec-
trometry and Ion Processes, 61(2), 191e213.
Sparkman, O. D. (2006). Mass spec desk reference (2nd ed.). Pittsburgh: Global View
Publishing.
Thermo Fisher Scientific Inc.. (2007). [Online], Available from http://tools.thermofisher.com/
content/sfs/brochures/high-resolution-mid-data-acquisition-for-target-compound-
analysis-with-the-dfs-gcms-system.pdf.
Thurman, E. M., & Ferrer, I. (2010). The isotopic mass defect: A tool for limiting molecular
formulas by accurate mass. Analytical and Bioanalytical Chemistry, 397, 2807e2816.
Xian, F., Hendrickson, C. L., & Marshall, A. G. (2012). High resolution mass spectrometry.
Analytical Chemistry, 84, 708e719.
 CHAPTER

HRMS: Hardware and

Software

Juan F. Garcı́a-Reyes, David Moreno-González, Rocı́o Nortes-Méndez,

2
Bienvenida Gilbert-López, Antonio Molina Dı́az
University of Jae´n, Jae´n, Spain

2.1 INTRODUCTION
Undoubtedly, the establishment of electrospray ionization (ESI) in the late 1980s (Fenn,
Mann, Meng, Wong, & Whitehouse, 1989, p. 64; Yamashita & Fenn, 1984, p. 4451)
and its enormous potential for biomedical applications prompted the development and
growth of liquid chromatographyemass spectrometry (LCeMS) instrumentation.
Several manufacturers launched LCeMS instrumentation in the mid-1990s with
either ion trap or triple quadrupole (QqQ) analyzers, which established itself as the
gold standard for quantitative trace analysis. Meanwhile, the use of high-resolution
LCeMS instrumentation with time-of-flight (TOF) analyzers was commercially
available from 1996 with the development of first hybrid quadrupole time-of-flight
(Q-TOF) instruments. Micromass commercialized the first Q-TOF mass spectrometer
in 1996 (Q-TOF-1), which was furnished with ESI source, quadrupole mass filter,
hexapole collision cell, orthogonal acceleration, and microchannel plate detector. It
featured a resolving power of 5000. Unfortunately, these instruments were originally
applicable only to qualitative purposes such as the determination of molecular mass
and elemental composition through accurate mass analysis. There were several issues
(linear dynamic range due to detector saturation and scarce robustness) that limited
the use of such instrumentation for quantitative purposes.
The last two decades have witnessed an unprecedented growth of mass
spectrometric instruments of increasing high-mass resolving power. Such ability
enabling accurate mass measurements permits the determination of elemental
compositions for small molecules up to 500e1000 Da in mixtures containing
thousands of chemical components (Marshall & Hendrickson, 2008, p. 579). There
has also been an outstanding growth in the use of high-resolution instrumentation for
quantitative application. This fact has been supported by relevant improvements of
instrument performance in terms of resolution, sensitivity, and speed. Among them,
in 2004, several vendors (Agilent, Bruker, Waters) offered updated and upgraded
versions of TOF instruments, with improvements on electronics to prevent detector
saturation, temperature control of flight tube, and continuous accurate mass
calibration systems (dual sprayer, MassLock, etc.), which broadened the range of
applications and enable more versatile and user-friendly operation. In 2005, the

Applications in High Resolution Mass Spectrometry. http://dx.doi.org/10.1016/B978-0-12-809464-8.00002-6 15

Copyright © 2017 Elsevier Inc. All rights reserved.
16 CHAPTER 2 HRMS: Hardware and Software

orbital ion trapping analyzer (Orbitrap), an alternative concept of high-resolution

instrumentationdprobably one of the major breakthrough in mass spectrometry in
the last decadesdwas launched by Thermo Fisher Scientific. Its advantaging features,
particularly related to resolving power, have fostered lately the development of high-
performance TOF analyzers with enhanced performance and solutions to challenge
the current Orbitrap lead.
Besides TOF and Orbitrap, high-resolution mass spectrometry (HRMS) can be also
carried out using Fourier transform ion cyclotron resonance (FT-ICR) first commercial-
ized in 1983 (Nier, Yergey, & Gale, 2015) and magnetic sector instruments. Provided
the current literature and the number of instruments sold and manufacturers offering
these technologies, it seems that the interest for these technologies is diminishing. In
gas chromatographyehigh-resolution mass spectrometry (GCeHRMS), for years GC
has been coupled to HR magnetic sector instruments, mostly for dioxin analysis. Yet,
GCeHRMS is the reference instrumentation for analysis of many persistent organic
pollutants (Eljarrat & Barceló, 2002, p. 1105; Hernández et al., 2012, p. 1251;
Van Babel & Abad, 2008, p. 3956) particularly dioxins, including polychlorinated
dibenzo-p-dioxins and polychlorinated dibenzofurans. However, recently there has
been a growing interest in GCeHRMS with TOF and Orbitrap mass analyzers
(Mol, Tienstra, & Zomer, 2016, p. 161; Peterson, Balloon, Westphall, & Coon,
2014, p. 10,044; Peterson, Mcalister, Quarmby, Griep-Raming, & Coon, 2010, p.
8618; Van Mourik, Leonards, Gaus, & De Boer, 2015, p. 259) partly associated
with the introduction of atmospheric pressure chemical ionization (APCI) sources
(Van Babel et al., 2015, p. 9047), which ultimately may replace magnetic sector
instruments within the next decade.
In this chapter, an overview of the more commonly used HRMS instrumentation
viz. those capable of featuring resolving power above 10,000 (full width at half
maximum, FWHM) is provided, including orthogonal acceleration/reflectron TOF
mass spectrometers and Fourier transform high-resolution mass spectrometers
(Orbitrap and FT-ICR instruments) coupled with either LC or GC. Along with
the ability to provide accurate mass measurements, these instruments offer several
hardware combinations yielding hybrid instruments [coupled with ion traps and/or
quadrupoles and even ion mobility (IM) spectrometry modules] providing
interesting possibilities in terms of acquisition mode to face different qualitative
and quantitative applications. On the other hand, the combination of accurate
mass measurements with available databases (often free on the Web) permits
unprecedented capability to scrutinize samples and their content in an automated
fashion.

2.2 PRINCIPLES OF HIGH-RESOLUTION MASS

SPECTROMETRY ANALYZERS
In this section, the principles of the main HRMS analyzers used nowadays are briefly
outlined. A thorough explanation of the physical principles and the involved
 2.2 Principles of High-Resolution Mass Spectrometry Analyzers 17

technical aspects related to TOF, Orbitrap, and FT-ICRMS instruments is provided

elsewhere (Fjeldsted, 2016, p. 19; Hu, Noll, Li, Makarov, & Hardman, 2005, p. 430;
Perry, Cooks, & Noll, 2008, p. 661).

2.2.1 TIME-OF-FLIGHT
TOF is a relatively mature technology (Fjeldsted, 2016, p. 19). Actually, the concept
of TOF mass analysis was first proposed by Stephens in 1946 (Stephens, 1946, p.
691), and subsequently developed by Cameron and Eggers and referred to as a
“velocitron” in 1948 (Cameron & Eggers, 1948, p. 605). The basic principle of
TOF instrument relies on the velocity-dependent separation and subsequent detection
of ions as they travel through a flight tube. Charged ions produced are accelerated into
TOF tube and are allowed to drift along the path. Provided that velocity is dependent
on mass-to-charge (m/z) ratio, the lighter ions will fly faster and reach the detector
earlier. There are two main types of TOF analyzers: linear and of orthogonal
acceleration. Whereas the use of linear time-TOFs was associated with pulsed
ionization methods such as laser beams with matrix-assisted laser desorption/
ionization (MALDI) experiments, the latter is the most commonly used configuration
nowadays. Fig. 2.1 shows the scheme of an orthogonal acceleration TOF. It consists of

FIGURE 2.1
Scheme of an orthogonal acceleration time-of-flight instrument.
18 CHAPTER 2 HRMS: Hardware and Software

an ionization interface, which is followed by an ion transfer section (ion optics) and
focusing region. The continuous ion beam emerging from the ion source is sharpened
within the ion optic path and sent toward the pulser. A high-voltage pulse is applied to
the pusher plate to orthogonally accelerate pockets of ions into the flight tube.
Wiley and McLaren (1955, p. 1150) realized the importance of space focusing.
They found that small differences on the position of the ions created in the ion source
and the distances to the detector affected resolving power dramatically. Ions created
at the back of the ion source volume had a longer distance to travel to the detector
than those that were created closer to the front of the ion source volume. They
demonstrated that with the appropriate increase in voltage at the back pusher plate
within the ion source it was possible to significantly reduce the loss of resolution
because of the origin of the ions within the source. This advance was connected
to the early development of the first TOF commercial instrument offered by Bendix
in the mid-1950s (Fjeldsted, 2016, p. 19; Wiley, 1956, p. 817).
Therefore, during TOF analysis, it is important that all ions receive an equal
acceleration to, as much as possible, reduce spreading in time. The focusing of
the ion beam before reaching the pusher and the following orthogonal acceleration
become critical to ensure equal starting conditions for all ions regardless of their
individual masses and thermal energy. Thus, the initial spatial and ion energy spread
among the ions are minimized. Mamyrin (1966) proposed the use of an ion mirror
(referred to as a reflectron) in the flight path to compensate for the spread in the
initial energy of the ions. Mamyrin showed that when a proper reflecting field
was established, ions with greater energy proportionally travel further into the
reflectron before being reflected with the effect that the arrival time of the ions at
the detector shows a greater tolerance to the effects of their initial energy spread.
Therefore, a careful design of the reflectron permits a compensation of speed
differences, as caused by the remaining energy difference among the accelerated
ions. In addition, the implementation of the reflectron doubled the flight path length
to achieve longer flight times and therefore increased resolution.
TOF instruments are of pulsed nature. The use of ion packets is central to TOF
analyzers. Initially, the creation of ion packets was generated by employing a pulsed
ionization source such as an electron ionization filament or a beam deflector. In the
latter approach, a pulsed ion packet was created through deflection by gating, or
momentarily steering, the ion beam into the TOF analyzer. In this sense, the concept
of orthogonal acceleration was proposed by Dawson and Guilhaus (1989, p. 155). A
continuous beam of ions was momentarily pushed in a direction perpendicular to the
direction of the continuous ion beam. The packet of orthogonally directed ions
enters the TOF tube that is therefore oriented perpendicular to the continuous ion
beam rather than in-line. With this configuration, the energy spread is substantially
minimized. The introduction of the orthogonal TOF geometry was of paramount
importance for adapting TOF to atmospheric pressure ionization sources, such as
ESI, and the subsequent growth in TOF applications in LC/MS.
The detectordlocated at the end of the flight pathdrecords the arrival time and
the number of incoming ions. The square of the flight time is proportional to the m/z
 2.2 Principles of High-Resolution Mass Spectrometry Analyzers 19

of the detected ion. Hence, masses (m/z) can simply be calculated after an instrument
calibration process. Because of the fast ion flight times, a single push cannot be used
to record a spectrum. Hundreds of consecutive pushes are summed to produce a
single, averaged spectrum instead (corresponding to one chromatographic data
point). A single pulser push accelerates only the ions passing above the surface of
the pusher plate. The next push should not be started before the heaviest ion
accelerated by the preceding push has reached the detector. This results in duty
cycles clearly below 100%. The very short ion flight times within the flight tube
put extreme importance on the frequency of the detection device and the following
signal processing. In the past, fast time-to-digital conversion (TDC) detectors were
only able to measure the flight time, but could not count the number of the
simultaneously incoming ions at a given time. Such devices have been replaced
by analog-to-digital conversion (ADC) detectors, which unlike TDC devices can ac-
count for the number of ions simultaneously hitting the detector plate, thus enabling
the expansion of the dynamic range to up to four decades. Yet this point remains as
one of the main weaknesses of TOF detectors; they undergo saturation from a
threshold intensity, which not only affects the measured signal amplitude but also
can result in a relevant mass shift of the ions being affected by saturation.

2.2.2 FOURIER TRANSFORM ION CYCLOTRON RESONANCE

FT-ICR was first described by Marshall and Comisarow in 1974 (Comisarow &
Marshall, 1974, p. 282; Comisarow & Marshall, 1996, p. 581). Yet, it is the
instrument providing the highest performance in terms of resolving power. In fact,
the resolving power of either a magnetic sector or a TOF depends on the path length
during the experiment, 7 m being the path length of the highest resolution magnetic
sector and between 1 and 2.5 m in current TOF analyzers (leaving aside multireflec-
tron TOF instruments). In contrast, an ion with m/z 1000 at a standard 9.4 T magnet
exhibits cyclotron frequencies of 144,346 Hz and thus, has an effective path length
higher than 9 km in 1 s (2p
0.01 m
144,346 Hz ¼ 9070 m).
FT-ICR enables the accurate determination of m/z of ions based on the cyclotron
frequency of the ions in a fixed magnetic field (Comisarow & Marshall, 1996, p. 581;
Marshall & Hendrickson, 2008, p. 579). The ions are trapped in a Penning trap
(a magnetic field with electric trapping plates) where they are excited (at their
resonant cyclotron frequencies) to a larger cyclotron radius by an oscillating electric
field orthogonal to the magnetic field. After the excitation field is removed, the ions
are rotating at their cyclotron frequency in phase (as a “packet” of ions). These ions
induce a charge (detected as an image current) on a pair of electrodes as the packets
of ions pass close to them. The resulting signal is called a free induction decay,
transient, or interferogram that consists of a superposition of sine waves. The signal
is digitalized and subjected to discrete fast Fourier transformation to yield a
spectrum of ion cyclotron frequencies, which may then be converted to a spectrum
of m/z (Marshall, Hendrickson, & Jackson, 1998, p. 1). The expression that describes
20 CHAPTER 2 HRMS: Hardware and Software

the rotation of the ions at a cyclotron frequency [nc (Hz)] in the spatially uniform
magnetic field (B) is:
nc ¼ ezB=2pm
in which B is the magnetic field, m is the mass, and e is the elementary charge. At
room temperature, typical ion cyclotron orbital radii are at the submillimeter level.
Considering that ions with a certain m/z rotate with random phase, it is necessary to
resonantly excite the ions with an oscillating or rotating electric field to yield a
spatially coherent packet of ions of each given m/z. The motion of the different
ion packets gives rise to a time-domain signal consisting of the difference in current
induced on a pair of opposed electrodes (Fig. 2.2).
Nowadays, only one vendor commercializes a portfolio of FT-ICR instruments
(Bruker Daltonics, Bremen, Germany). Up to three different models are offered
including a hybrid quadrupole FT-ICR 7-Tesla (T) (solariX XR) and 12-T or 15-T
high-field solariX XR with resolving power up to 600,000 at m/z 400 with 1 s
transient (and up to 10,000,000 resolving power depending on magnet and
acquisition time). To enable a flexible range of experiments, these instruments
permit a wide range of fragmentation techniques including in-source collision-
induced dissociation (CID), CID in collision cell, electron-capture dissociation
(ECD), electron-transfer dissociation (ETD), and sustained off-resonance irradiation
(SORI) CID in cell. In the past, Thermo also offered a hybrid linear ion trap (LTQ)-
FT-ICRMS, but it was withdrawn in 2014, with the emergence of high-field
Orbitrap, which nearly matched the performance of FT-ICR systems. The
applications foreseen for this type of instrumentation are petroleomics (Cho,
Ahmed, Islam, & Kim, 2015, p. 248; Nikolaev, 2015, p. 421; Schwemer, Rüger,
Sklorz, & Zimmermann, 2015, p. 11,957) and state-of-the art proteomics research
(Marshall & Chen, 2015, p. 410; Nicolardi, Bogdnov, Deelder, Palmblad, & Van

(A) (B) (C) (D)

Image
Current

FT
0

Time (ms) Frequency (kHz) m/z

FIGURE 2.2
Summary of Fourier transform mass spectrometry based detection of ions in Fourier
transform ion cyclotron resonance mass spectrometry: (A) excited ion cyclotron rotation,
(B) differential image-current signal from opposed detection electrodes, (C) frequency-
domain signal, and (D) mass spectrum obtained by calibrated frequency-to-m/z
conversion. A typical acquisition of 1 s compatible with liquid separation permits resolving
power above 400,000 (depending on the magnet used). Higher acquisition time permits
increased values of resolving power above 1,000,000. For details, see Comisarow and
Marshall (1996, p. 581) and Marshall and Hendrickson (2008, p. 579).
 2.2 Principles of High-Resolution Mass Spectrometry Analyzers 21

Der Burgt, 2015, p. 27,133), being the application of FT-ICR in pesticide testing
scarcely addressed as the requirements and maintenance cost of such sophisticated
instrumentation are not met by standard routine laboratories (Gilbert-López et al.,
2013, p. 419).

2.2.3 ORBITRAP
The Orbitrap mass analyzer is the first high-performance mass analyzer that employs
trapping of ions in electrostatic fields, together with a sophisticated ion injection
process, which enables high resolution, mass accuracy, and excellent sensitivity
for addressing numerous analytical applications in both research and routine
analysis. The principle of Orbitrap is based on the Kingdon ion trap described in
1923 (Kingdon, 1923, p. 408). It was a trapping device consisting of a charged
wire stretched along the axis of an enclosed metal can. The wire establishes an
electrostatic field within the can, and ions that possess sufficiently high tangential
velocity orbit the wire, rather than directly colliding with it (Eliuk & Makarov,
2015, p. 61). It was first commercially introduced in 2005 (Eliuk & Makarov,
2015, p. 61). It provides a very convenient balance between the advantages of a
benchtop instrument featuring high resolution approaching FT-ICR performance,
whereas the instrument size, laboratory space requirements, and regular operational
maintenance are more comparable to Q-TOF instruments.
A thorough explanation of the mass analysis process is detailed elsewhere (Eliuk
& Makarov, 2015, p. 61). This mass analyzer is based on the confinement of ions in
an electrostatic potential well (Makarov, 1999, US 5886346; Makarov, 2000, p.
1156; Makarov & Hardman, 2006, US 6998609 B2) created between two carefully
shaped electrodes: an inner coaxial spindled-shaped electrode and an outer (barrel-
like) electrode, which is actually composed of two symmetrical halves electrically
isolated from each other, set out for two purposes: establishment of the ion trapping
fields and as receiver plates for image-current detection. The electrodes are precisely
machined so that the electrostatic attractions of the ions to inner electrode are finely
balanced by centrifugal forces, which cause the ions to orbit around the spindle.
Previously, ions are first accumulated on an external injecting device (C-trap) that
traps the ions in gas-filled quadrupole being then injected tangentially into the
mass analyzer in short pulses (Fig. 2.3). Besides, an axial field causes the ions to
oscillate harmonically along the spindled-shaped electrode. The outer electrodes
allow differential image-current detection. These image currents produced by the
oscillating ions are detected, followed by a fast Fourier transform (FT) to convert
the time-domain signal to frequency domain and then to m/z spectrum. Here the
resolution is directly proportional to number of harmonic oscillations detected.
The resolving power can be enhanced by increasing the gap between inner and outer
electrodes providing higher field strength for a given voltage. As the maximum
acquisition time is limited in Orbitrap, the resolution power is not as high as
FT-ICR. However, commercial Orbitrap analyzer provides a nominal resolution
power as high as 500,000 at FWHM (Table 2.1).
22 CHAPTER 2 HRMS: Hardware and Software

FIGURE 2.3
Cross section of the C-trap ion accumulation device and the Orbitrap mass analyzer with
an example of an ion trajectory. During the voltage ramp, the ion packets enter the
Orbitrap mass analyzer forming rings that induce current which is detected by the
amplifier.
Reprinted with permission from Thermo Fisher Scientific, Copyright © 2016.

2.3 TIME-OF-FLIGHT MASS SPECTROMETRY: INSTRUMENT

CONFIGURATION AND MAIN FEATURES
2.3.1 STAND-ALONE ELECTROSPRAY IONIZATION TIME-OF-FLIGHT
AND HYBRID QUADRUPOLE TIME-OF-FLIGHT INSTRUMENTATION
The Micromass Q-TOF-1 (1996) was the first commercially available Q-TOF
instrument launched in 1996, featuring a resolving power of 5000 (Morris et al.,
1996, p. 889). Later, other small manufactures including Perceptive, Analytica or
Bradford, Sensar, and JEOL introduced ESI-TOFs. In 1999, Micromassdpart of
Watersdupdated the Q-TOF system (Q-TOF-2) with an increase in the resolving
power up to 10,000 and presented a stand-alone TOF instrument (Waters LCT). A
similar instrument was also commercialized by Applied Biosystems (now Sciex)
around 2000 (Applied Biosystems Mariner ESI-TOF). In 2001, Waters upgraded
the performance of Q-TOF (Q-TOF Ultima) using W-type flight path providing a
resolving power of 17,500. After that, in 2002 Applied Biosystems (now Sciex)
launched the Q-STAR XL, its first Q-TOF, which was updated in 2008 (Q-STAR
Elite) with improvements in detector design and increased resolving power.
Table 2.1 Overview of the Technical Specifications and Characteristics of Time-of-Flight (TOF) and Orbitrap
Mass Accuracy
Mass Resolving Power
Analyzer Instrument (FWHM Defined Internal External m/z Acquisition
Type Manufacturer Name at m/z) Calibration Calibration Range Speed (Hz)
Q-TOF Bruker MicroOTOF-Q II 20,000 (m/z 922) <2 <5 50 20
Daltonics e20,000
MaXis impact 40,000 (m/z 386) <1 <3 50 50
e20,000
MaXis 4G 60,000 (m/z 1222) <0.6 <2 50 30 (MS), 10
e20,000 (MS/MS)
Waters XEVO G2 Q-TOF 22,500 (m/z 956) <1 e 20 30
e16,000
Synapt G2-S 50,000 (m/z 956) <1 e 20 30
HDMS e100,000

2.3 Time-of-Flight Mass Spectrometry

Agilent 6500 Q-TOF 42,000 (m/z 922) <1 e 50 50
series e10,000
Sciex TripleTOF 4600 30,000 (full-range) <0.5 <1 5e40,000 100
TripleTOF 5600 35,000 (full-range) <0.5 <2 5e40,000 100
TripleTOF 6600 40,000 (full-range) <0.5 <2 5e40,000 100
IT-TOF Shimadzu LCeMS-IT-TOF 10,000 (m/z 1000) 3 5 50e5000 10
Orbitrap Thermo Exactive <1 <5 50e4000 12 at RP
scientific 17,500
Q-Orbitrap Q-Exactive 140,000 (m/z 200) <1 <5 50e4000 12 at RP
17,500
LTQ- Orbitrap Elite 240,000 (m/z 400) <1 <3 50e4000 8 at RP
Orbitrap 17,500
Tribrid- Orbitrap Fusion 500,000 (m/z 200) <1 <3 50e6000 18 at RP
Orbitrap Lumos tribrid 17,500
FWHM, full width at half maximum; HDMS, high-definition mass spectrometry; IT-TOF, ion trap time-of-flight; LTQ-Orbitrap, linear ion trap Orbitrap.
Reproduced from Royal Society of Chemistry with permission, Copyright 2015 [Lin, L., Lin, H., Zhang, M., Dong, X., Yin, X., Qu, C. et al. (2015). Types, principle,
and characteristics of tandem high-resolution mass spectrometry and its applications. RSC Advances, 5(130), 107623e107636].

23
24 CHAPTER 2 HRMS: Hardware and Software

The second generation of TOF instruments emerged by 2004, enabling a

significant enhancement of both linearity and robustness. Three major vendors
(Waters, Agilent Technologies, and Bruker) offer either completely new or
completely transformed instruments. Waters first came up with both LCT TOF
Premier and Q-TOF Premier using 4 GHz TDC detection and programmable
enhanced dynamic range, which decreases the ion beam intensity to reduce detector
saturation, extending the quantitation capabilities of the instrument. These TOF
instruments featured continuous accurate mass calibration (MassLock). Both Bruker
and Agilent introduced ESI-TOF instruments in 2004 with 10,000 resolving power,
being the equivalent Q-TOF instruments launched in 2005 (micrOTOF-Q) and 2006
(Agilent 6510 Q-TOF), respectively, with resolving power of c. 15,000 in both cases.
Follow-up products (microOTOF-Q II operating at 2 GHz and 6520 Q-TOF
operating at 4 GHz) enabled resolutions of about 20,000. These two instruments
used ADC instead of TDC, featuringdaccording to manufacturersda dynamic
range of up to five decades. A comprehensive explanation on the details of each
technology is given elsewhere (Fjeldsted, 2003, 2016, p. 19). An example of the
typical hardware configuration of a current Q-TOF instrument is illustrated in
Fig. 2.4, where the schematics of Q-TOF Agilent 6550 is shown including a dual
ion funnel ion sampling and a two-stage ion mirror (Fjeldsted, 2016, p. 19).

2.3.2 IMPROVEMENTS OF CURRENT (QUADRUPOLE) TIME-OF-

FLIGHT INSTRUMENTATION
The improvement of TOF instrumentation can be attributed to different aspects, which
are interrelated so that it is difficult to isolate the contribution of each individual factor
to the overall performance.
Ionization step. First of all, ion production and atmospheric sampling is a key
aspect on the overall sensitivity. Significant progress has been made from early
electrospray experiments in the 1980s. The use of a nebulizing gas was proposed
to increase the efficiency of ion production. To accelerate droplet size reduction,
heated gases were then used (Turbo V source, Sciex). Then, different manufacturers
came up with similar heated ESI based sources, such as the Jet Stream from Agilent,
first implemented in 2008 Q-TOF 6530. Jet Stream (Agilent) consists of an enhanced
ESI technology employing concentrically applied heated gas providing an average
fivefold sensitivity increase compared with nonthermally enhanced ESI design
(although this may vary depending on each individual compound).
Ion sampling. Major efforts have also been carried out for the improvement of ion
sampling in the mass spectrometer. For this purpose, a balance between sampling
orifice/inlet capillary diameter and vacuum demands must be achieved. Additionally,
the ion transfer step has also been improved with more efficient ion guide technologies
such as the ion funnels (Agilent) and other ion guide devices [QJet ion guide
(Sciex) or Stepwave (Waters)], permitting an increase in the ions entering the mass
spectrometer with the subsequent improvement of sensitivity.
 2.3 Time-of-Flight Mass Spectrometry 25

FIGURE 2.4
Key elements of modern Quadrupole time-of-flight (Q-TOF) mass spectrometer.
Reprinted with permission from Elsevier, Copyright © 2016 [Fjeldsted, J.C. (2016). Advances in time-of-flight
mass spectrometry. In S. Pérez, P. Eichhorn, & D. Barceló (Eds.), Comprehensive analytical chemistry. Appli-
cations of time-of-flight and Orbitrap mass spectrometry in environmental, food, doping and forensic analysis
(p. 19). Amsterdam: Elsevier].

The use of radio frequency (RF) fields exerts a force on ions over a wide range of
pressure and mass. Based on this principle, the QJet ion guide designed by Sciex
improved efficiency in separating ions from the noncharged species (neutrals),
improving the focusing of captured ions into the mass spectrometer. Another
approach (StepWave) was presented by Waters and first implemented in 2008 in
the Synapt G2 hybrid instrument based on the use of a set of ion guides to shift
the ion beam axis offset to the atmospheric pressure sampling inlet. RF and direct
current (DC) potential are applied onto a set of ion concentric rings with overlapping
apertures, so that the ions are shifted off the main axis into smaller off-axis
concentric apertures, which focus the ion beam and direct it to the subsequent
mass analysis stages while neutral gas passes along the principle axis being diverted
to the exhaust.
Based also on the use of stacked ring RF ion guides, R.D. Smith et al. showed
that ion confinement could be achieved at pressures in the range from 0.1 to
30 torr (Kelly, Tolmachev, Page, Tang, & Smith, 2010, p. 294), when constructing
26 CHAPTER 2 HRMS: Hardware and Software

a set of ring electrodes having a linear decrease in the inner diameters in the last sec-
tion of the stack (the so-called ion electrodynamic funnel), which could be used indi-
vidually or as a pair in succession (dual ion funnel) with the second having a lower
operating pressure than the preceding funnel. These RF ion funnel technologies
enable highly efficient ion capture and may provide up to a 10-fold increase in
the atmospheric sampling efficiencies and overall sensitivity (Fjeldsted, 2016, p.
19). The use of multiplexed (hexabore) capillaries has also been proposed to in-
crease ion sampling efficiency (Kelly et al., 2010, p. 294).
Resolving power and flight path length. Unlike Orbitrap, the resolving power
in TOF analyzer increases with m/z values. Several solutions have improved the
overall performance of commercial TOF instrumentation particularly in terms of
resolving power, attaining values up to 50,000 or even higher. Most of the improve-
ments should be attributed to both ion optics (focusing of ions before TOF analysis)
and detection electronics including the increase in the detector operation frequency
of ADC TOF detectors (from 1 to 5 GHz) (Fjeldsted, 2016, p. 19). Besides, there is
another obvious strategy that increases resolution (increasing the flight path length),
although at the expense of sensitivity. This can be accomplished by using either
longer flight tubes (up to 1.5 m) or multiple reflection by placing a second mirror
between the ion pusher and detector, reflecting the beam twice, thus doubling the
effective flight path length [such as W optics mode in Waters, N-optic design (Sciex)
or folded path technology enabling up to 64 reflections (LECO)]. Ion mirror
(reflectron) has also been improved through the use of two-stage mirrors, which
compensate ion energy spread from the ion pusher. Nevertheless, reflectrons not
only double the flight path length, but also are responsible for a relevant loss of
recorded ion abundances. Hence, users of such multireflectron instruments have
to come across a compromise between sensitivity and selectivity. Last, but not least,
flight tube temperature control in the system also reduces the internal energy spread
of the ions with the same mass, so that an increase in resolution is also achieved.
Ion detection. Ion detection and digitization have also undergone significant
improvements in the last decade. It seems that most of the manufacturers have found
that multichannel plateebased detectors with ADC are the best choice to avoid
detector saturation and lack of linearity (Fjeldsted, 2016, p. 19). However, the use
of very fast TDC conversion and a multianode detector is also used in instruments
offering excellent performance such as Sciex TripleTOF 5600 Q-TOF (Fig. 2.5)
(Andrews, Simons, Young, Hawkridge, & Muddiman, 2011, p. 5442). A detailed
discussion on this matter is described by Fjeldsted (2003, 2016, p. 19).
Accurate mass measurements and mass calibration. Mass accuracy is central
to HRMS. This fact is particularly important in TOF instruments, as they are
generally less robust than FT-MS instruments in terms of the stability of accurate
mass measurements and the subsequent accuracy (ppm mass errors). This is a key
parameter, as small changes in the experimental conditions (such as laboratory
temperature) usually affect the actual time measurements of ions TOF, provided
the change in drift tube path length due to temperature changes (small thermal
expansion or contraction of the flight tube length). A high-performance temperature
 2.3 Time-of-Flight Mass Spectrometry 27

FIGURE 2.5
(A) Schematic of a Quadrupole time-of-flight instrument from Sciex (TripleTOF 5500)
using multianode time-to-digital conversion (TDC) detection. (B) An image of the
machined TripleTOF MS instrument platform.
Reproduced with permission from American Chemical Society, Copyright © 2011.

control assembly of the drift tube is mandatory to assess mass accuracy performance
of TOF instruments.
For this purpose, mass axis calibration is an important process for obtaining
reliable results. Mass calibration consists of comparing the observed experimental
mass measurement of one or two known ions with their (theoretical) exact mass.
Calibration may be internal (i.e., the reference masses are for ions of known
elemental composition in the same mass spectrum of the analyte) or external (i.e.,
reference masses from a mass spectrum of another analyte acquired under similar
conditions). Internal calibration is typically at least twofold more accurate than
external (Marshall et al., 1998, p. 1). Accurate mass correction is usually performed
in full-scan (FS) data, as the ions selected are available in all acquired spectra
through the run. However, when recording accurate mass spectra in MS/MS mode
with precursor ion selection, the lack of such calibrating ions hampers the accuracy
of product ion spectra.
In the first generation of TOF instruments, external mass calibration was applied
before the chromatographic run. This approach did not deliver results better than
5e10 ppm relative mass error because of the significant drifting of instrumental
status (e.g., flight tube temperature). To overcome this situation, various strategies
have been proposed to provide continuous accurate mass calibration in commercial
28 CHAPTER 2 HRMS: Hardware and Software

TOF instruments, allowing them to feature routine relative mass errors in the range
of 1e3 ppm, when using mass calibration devices such as MassLock (Waters), the
dual sprayer solution (Agilent Technologies), or similar technologies presented by
other manufacturers such as the TwinSprayer (Sciex), which is an independent
calibrant delivery path proposed in the recently commercialized compact Sciex Q-
TOF (Sciex X500R Q-TOF) dedicated for small molecule applications (Fig. 2.6).
These assemblies provide a compensating mechanism to keep flight times constant
for a given fixed mass (reference solution). First, Waters proposed the discontinuous
periodical infusion of a lock mass solution by a switching device that alternatively
permitted the flow from two independent ESI sprayers (the reference solution and
the analytical sample) (Eckers et al., 2000, p. 3683; Wolff et al., 2001, p. 2605).
This strategy is not flawless because it introduces mass signalsdunrelated to the
sampledinto both the spectrum and the data file, which are used to calibrate the
entire data set at the end of the run. Likewise, Agilent proposed the use of a dual
sprayer assembly, where a calibrant solution was continuously sprayeddorthogonal
to the analytical sprayerdand introduced in the mass spectrometer (Fig. 2.6). This
solution provided excellent results even enabling the measurement of the mass of an
ion using twin fragment ions (Ferrer & Thurman, 2005, p. 3394). However, although
they are orthogonal, there might be some interaction between the analytical and
reference sprayers, which may yield signal suppression if the concentration/
abundance of the reference solution analytes is not controlled. As mentioned
previously, the newest Sciex Q-TOF (Sciex X500R Q-TOF) makes use of an

FIGURE 2.6
Examples of continuous calibration systems for mass calibration in time-of-flight
instrumentation: (A) Agilent MSD TOF (2004); (B) Sciex X500R (2016).
Reprinted with permission from Sciex and Agilent Technologies, Copyright © 2016.
Another Random Scribd Document
with Unrelated Content
volunteers, in order to destroy the Mississinaway towns. They
proceeded and performed the duty, and took some prisoners. And
here is the evidence of the manner in which they treated them.

‘But the character of this gallant detachment, exhibiting, as it did,

perseverance, fortitude, and bravery, would, however, be incomplete, if, in the
midst of victory, they had forgotten the feelings of humanity. It is with the
sincerest pleasure that the general has heard, that the most punctual obedience
was paid to his orders, in not only saving all the women and children, but in
sparing all the warriors who ceased to resist: and that even when vigorously
attacked by the enemy, the claims of mercy prevailed over every sense of their
own danger, and this heroic band respected the lives of their prisoners. Let an
account of murdered innocence be opened in the records of heaven, against our
enemies alone. The American soldier will follow the example of his government,
and the sword of the one will not be raised against the fallen and the helpless, nor
the gold of the other be paid for scalps of a massacred enemy.’

I hope, sir, the honorable gentleman will now be able better to

appreciate the character and conduct of my gallant countrymen,
than he appears hitherto to have done.

But, sir, I have said that you have no right to practice, under
color of retaliation, enormities on the Indians. I will advance in
support of this position, as applicable to the origin of all law, the
principle, that whatever has been the custom, from the
commencement of a subject, whatever has been the uniform usage,
coeval and coexistent with the subject to which it relates, becomes
its fixed law. Such is the foundation of all common law; and such,
I believe, is the principal foundation of all public or international law.
If, then, it can be shown that from the first settlement of the
colonies, on this part of the American continent, to the present time,
we have constantly abstained from retaliating upon the Indians the
excesses practiced by them towards us, we are morally bound by
this invariable usage, and cannot lawfully change it without the most
cogent reasons. So far as my knowledge extends, from the first
settlement at Plymouth or at Jamestown, it has not been our
practice to destroy Indian captives, combatants or non-combatants.
I know of but one deviation from the code which regulates the
warfare between civilized communities, and that was the destruction
of Indian towns, which was supposed to be authorized upon the
ground that we could not bring the war to a termination but by
destroying the means which nourished it. With this single exception,
the other principles of the laws of civilized nations are extended to
them, and are thus made law in regard to them. When did this
humane custom, by which, in consideration of their ignorance, and
our enlightened condition, the rigors of war were mitigated, begin?
At a time when we were weak, and they comparatively strong; when
they were the lords of the soil, and we were seeking, from the vices,
from the corruptions, from the religious intolerance, and from the
oppressions of Europe, to gain an asylum among them. And when is
it proposed to change this custom, to substitute for it the bloody
maxims of barbarous ages, and to interpolate the Indian public law
with revolting cruelties? At a time when the situation of the two
parties is totally changed—when we are powerful and they are weak
—at a time when, to use a figure drawn from their own sublime
eloquence, the poor children of the forest have been driven by the
great wave which has flowed in from the Atlantic ocean almost to
the base of the Rocky mountains, and, overwhelming them in its
terrible progress, has left no other remains of hundreds of tribes,
now extinct, than those which indicate the remote existence of their
former companion, the mammoth of the new world! Yes, sir, it is at
this auspicious period of our country, when we hold a proud and
lofty station among the first nations of the world, that we are called
upon to sanction a departure from the established laws and usages
which have regulated our Indian hostilities. And does the honorable
gentleman from Massachusetts expect, in this august body, this
enlightened assembly of christians and Americans, by glowing
appeals to our passions, to make us forget our principles, our
religion, our clemency, and our humanity? Why is it that we have not
practiced towards the Indian tribes the right of retaliation, now for
the first time asserted in regard to them? It is because it is a
principle proclaimed by reason, and enforced by every respectable
writer on the law of nations, that retaliation is only justifiable as
calculated to produce effect in the war. Vengeance is a new motive
for resorting to it. If retaliation will produce no effect on the enemy,
we are bound to abstain from it by every consideration of humanity
and of justice. Will it then produce effect on the Indian tribes? No;
they care not about the execution of those of their warriors who are
taken captive. They are considered as disgraced by the very
circumstance of their captivity, and it is often mercy to the unhappy
captive to deprive him of his existence. The poet evinced a profound
knowledge of the Indian character, when he put into the mouth of a
son of a distinguished chief, about to be led to the stake and
tortured by his victorious enemy, the words:

‘Begin, ye tormentors! your threats are in vain:

The son of Alknomook will never complain.’

Retaliation of Indian excesses, not producing then any effect in

preventing their repetition, is condemned by both reason and the
principles upon which alone, in any case, it can be justified. On this
branch of the subject much more might be said, but as I shall
possibly again allude to it, I will pass from it, for the present, to
another topic.

It is not necessary, for the purpose of my argument in regard to

the trial and execution of Arbuthnot and Ambrister, to insist on the
innocency of either of them. I will yield for the sake of that
argument, without inquiry, that both of them were guilty; that both
had instigated the war; and that one of them had led the enemy to
battle. It is possible, indeed, that a critical examination of the
evidence would show, particularly in the case of Arbuthnot, that the
whole amount of his crime consisted in his trading, without the limits
of the United States, with the Seminole Indians, in the accustomed
commodities which form the subject of Indian trade, and that he
sought to ingratiate himself with his customers by espousing their
interests, in regard to the provision of the treaty of Ghent, which he
may have honestly believed entitled them to the restoration of their
lands. And if, indeed, the treaty of Fort Jackson, for the reasons
already assigned, were not binding upon the Creeks, there would be
but too much cause to lament his unhappy if not unjust fate. The
first impression made, on the examination of the proceedings in the
trial and execution of those two men, is, that on the part of
Ambrister there was the most guilt, but, at the same time, the most
irregularity. Conceding the point of guilt of both, with the
qualification which I have stated, I will proceed to inquire, first, if
their execution can be justified upon the principles assumed by
general Jackson himself. If they do not afford a justification, I will
next inquire, if there be any other principles authorizing their
execution; and I will in the third place make some other
observations upon the mode of proceeding.

The principle assumed by general Jackson, which may be found

in his general orders commanding the execution of these men, is,
‘that it is an established principle of the law of nations, that any
individual of a nation making war against the citizens of any other
nation, they being at peace, forfeits his allegiance, and becomes an
outlaw and a pirate.’ Whatever may be the character of individuals
waging private war, the principle assumed is totally erroneous when
applied to such individuals associated with a power, whether Indian
or civilized, capable of maintaining the relations of peace and war.
Suppose, however, the principle were true, as asserted, what
disposition should he have made of these men? What jurisdiction,
and how acquired, has the military over pirates, robbers, and
outlaws? If they were in the character imputed, they were alone
amenable, and should have been turned over, to the civil authority.
But the principle, I repeat, is totally incorrect, when applied to men
in their situation. A foreigner connecting himself with a belligerent,
becomes an enemy of the party to whom that belligerent is opposed,
subject to whatever he may be subject, entitled to whatever he is
entitled. Arbuthnot and Ambrister, by associating themselves,
became identified with the Indians; they became our enemies, and
we had a right to treat them as we could lawfully treat the Indians.
These positions are so obviously correct, that I shall consider it an
abuse of the patience of the committee to consume time in their
proof. They are supported by the practice of all nations, and of our
own. Every page of history, in all times, and the recollection of every
member, furnish evidence of their truth. Let us look for a moment
into some of the consequences of this principle, if it were to go to
Europe, sanctioned by the approbation, express or implied, of this
house. We have now in our armies probably the subjects of almost
every European power. Some of the nations of Europe maintain the
doctrine of perpetual allegiance. Suppose Britain and America in
peace, and America and France at war. The former subjects of
England, naturalized and unnaturalized, are captured by the navy or
army of France. What is their condition? According to the principle of
general Jackson, they would be outlaws and pirates, and liable to
immediate execution. Are gentlemen prepared to return to their
respective districts with this doctrine in their mouths, and to say to
their Irish, English, Scotch, and other foreign constituents, that they
are liable, on the contingency supposed, to be treated as outlaws
and pirates?

Is there any other principle which justifies the proceedings? On

this subject, if I admire the wonderful ingenuity with which
gentlemen seek a colorable pretext for those executions, I am at the
same time shocked at some of the principles advanced. What said
the honorable gentlemen from Massachusetts (Mr. Holmes), in a cold
address to the committee? Why, that these executions were only the
wrong mode of doing a right thing. A wrong mode of doing a right
thing! In what code of public law; in what system of ethics; nay, in
what respectable novel; where, if the gentleman were to take the
range of the whole literature of the world; will he find any sanction
for a principle so monstrous? I will illustrate its enormity by a single
case. Suppose a man, being guilty of robbery, is tried, condemned,
and executed, for murder, upon an indictment for that robbery
merely. The judge is arraigned for having executed, contrary to law,
a human being, innocent at heart of the crime for which he was
sentenced. The judge has nothing to do, to insure his own acquittal,
but to urge the gentleman’s plea, that he had done a right thing in a
wrong way!

The principles which attached to the cases of Arbuthnot and

Ambrister, constituting them merely participes in the war, supposing
them to have been combatants, which the former was not, he
having been taken in a Spanish fortress, without arms in his hands,
all that we could possibly have a right to do, was to apply to them
the rules which we had a right to enforce against the Indians. Their
English character was only merged in their Indian character. Now, if
the law regulating Indian hostilities be established by long and
immemorial usage, that we have no moral right to retaliate upon
them, we consequently had no right to retaliate upon Arbuthnot and
Ambrister. Even if it were admitted that, in regard to future wars,
and to other foreigners, their execution may have a good effect, it
would not thence follow that you had a right to execute them. It is
not always just to do what may be advantageous. And retaliation,
during a war, must have relation to the events of that war, and must,
to be just, have an operation on that war, and upon the individuals
only who compose the belligerent party. It becomes gentlemen,
then, on the other side, to show, by some known, certain, and
recognised rule of public or municipal law, that the execution of
these men was justified. Where is it? I should be glad to see it. We
are told in a paper emanating from the department of state, recently
laid before this house, distinguished for the fervor of its eloquence,
and of which the honorable gentleman from Massachusetts has
supplied us in part with a second edition, in one respect agreeing
with the prototype—that they both ought to be inscribed to the
American public—we are justly told in that paper, that this is the first
instance of the execution of persons for the crime of instigating
Indians to war. Sir, there are two topics which, in Europe, are
constantly employed by the friends and minions of legitimacy against
our country. The one is an inordinate spirit of aggrandizement—of
coveting other people’s goods; the other is the treatment which we
extend to the Indians. Against both these charges, the public
servants who conducted at Ghent the negotiations with the British
commissioners, endeavored to vindicate our country, and I hope
with some degree of success. What will be the condition of future
American negotiators, when pressed upon this head, I know not,
after the unhappy executions on our southern border. The
gentleman from Massachusetts seemed yesterday to read, with a
sort of triumph, the names of the commissioners employed in the
negotiation at Ghent. Will he excuse me for saying, that I thought he
pronounced, even with more complacency, and with a more gracious
smile, the first name in the commission, than he emphasized that of
the humble individual who addresses you?

[Mr. Holmes desired to explain.]

There is no occasion for explanation; I am perfectly satisfied.

[Mr. Holmes, however, proceeded to say that his intention was, in pronouncing
the gentleman’s name, to add to the respect due to the negotiator that which was
due to the speaker of this house.]
 To return to the case of Arbuthnot and Ambrister. Will the
principle of these men having been the instigators of the war, justify
their execution? It is a new one; there are no landmarks to guide us
in its adoption, or to prescribe limits in its application. If William Pitt
had been taken by the French army, during the late European war,
could France have justifiably executed him on the ground of his
having notoriously instigated the continental powers to war against
France? Would France, if she had stained her character by executing
him, have obtained the sanction of the world to the act, by appeals
to the passions and prejudices, by pointing to the cities sacked, the
countries laid waste, the human lives sacrificed in the wars which he
had kindled, and by exclaiming to the unfortunate captive, you,
miscreant, monster, have occasioned all these scenes of devastation
and blood? What has been the conduct even of England towards the
greatest instigator of all the wars of the present age? The
condemnation of that illustrious man to the rock of St. Helena, is a
great blot on the English name. And I repeat what I have before
said, that if Chatham, or Fox, or even William Pitt himself, had been
prime minister in England, Bonaparte had never been so
condemned. On that transaction history will one day pass its severe
but just censure. Yes, although Napoleon had desolated half Europe;
although there was scarcely a power, however humble, that escaped
the mighty grasp of his ambition; although in the course of his
splendid career, he is charged with having committed the greatest
atrocities, disgraceful to himself and to human nature, yet even his
life has been spared. The allies would not, England would not,
execute him upon the ground of his being an instigator of wars.

The mode of the trial and sentencing these men was equally
objectionable with the principles on which it has been attempted to
prove a forfeiture of their lives. I know the laudable spirit which
prompted the ingenuity displayed in finding out a justification for
these proceedings. I wish most sincerely that I could reconcile them
to my conscience. It has been attempted to vindicate the general
upon grounds which I am persuaded he would himself disown. It
has been asserted, that he was guilty of a mistake in calling upon
the court to try them, and that he might have at once ordered their
execution, without that formality. I deny that there was any such
absolute right in the commander of any portion of our army. The
right of retaliation is an attribute of sovereignty. It is comprehended
in the war-making power that congress possesses. It belongs to this
body not only to declare war, but to raise armies, and to make rules
and regulations for their government. It is in vain for gentlemen to
look to the law of nations for instances in which retaliation is lawful.
The laws of nations merely lay down the principle or rule; it belongs
to the government to constitute the tribunal for applying that
principle or rule. There is, for example, no instance in which the
death of a captive is more certainly declared by the law of nations to
be justifiable, than in the case of spies. Congress has accordingly
provided, in the rules and articles of war, a tribunal for the trial of
spies, and consequently for the application of the principle of the
national law. The legislature has not left the power over spies
undefined, to the mere discretion of the commander-in-chief, or of
any subaltern officer in the army. For, if the doctrines now contended
for were true, they would apply to the commander of any corps,
however small, acting as a detachment. Suppose congress had not
legislated in the case of spies, what would have been their
condition? It would have been a casus omissus, and although the
public law pronounced their doom, it could not be executed, because
congress had assigned no tribunal for enforcing that public law. No
man can be executed in this free country without two things being
shown—first, that the law condemns him to death; and, secondly,
that his death is pronounced by that tribunal which is authorized by
the law to try him. These principles will reach every man’s case,
native or foreign, citizen or alien. The instant quarters are granted to
a prisoner, the majesty of the law surrounds and sustains him, and
he cannot be lawfully punished with death without the concurrence
of the two circumstances just insisted upon. I deny that any
commander-in-chief, in this country, has this absolute power of life
and death, at his sole discretion. It is contrary to the genius of all
our laws and institutions. To concentrate in the person of one
individual the powers to make the rule, to judge and to execute the
rule, or to judge and execute the rule only, is utterly irreconcilable
with every principle of free government, and is the very definition of
tyranny itself; and I trust that this house will never give even a tacit
assent to such a principle. Suppose the commander had made even
reprisals on property, would that property have belonged to the
nation, or could he have disposed of it as he pleased? Had he more
power, will gentlemen tell me, over the lives of human beings than
over property? The assertion of such a power to the commander-in-
chief is contrary to the practice of the government. By an act of
congress which passed in 1799, vesting the power of retaliation in
certain cases in the president of the United States—an act which
passed during the quasi war with France—the president is authorized
to retaliate upon any of the citizens of the French republic, the
enormities which may be practiced, in certain cases, upon our
citizens. Under what administration was this act passed? It was
under that which has been justly charged with stretching the
constitution to enlarge the executive powers. Even during the mad
career of Mr. Adams, when every means was resorted to for the
purpose of infusing vigor into the executive arm, no one thought of
claiming for him the inherent right of retaliation. I will not trouble
the house with reading another law, which passed thirteen or
fourteen years after, during the late war with Great Britain, under
the administration of that great constitutional president, the father
of the instrument itself, by which Mr. Madison was empowered to
retaliate on the British in certain instances. It is not only contrary to
the genius of our institutions, and to the uniform practice of the
government, but it is contrary to the obvious principles on which the
general himself proceeded; for, in forming the court, he evidently
intended to proceed under the rules and articles of war. The extreme
number which they provide for is thirteen, precisely that which is
detailed in the present instance. The court proceeded not by a bare
plurality, but by a majority of two thirds. In the general orders issued
from the adjutant general’s office, at head quarters, it is described
as a court-martial. The prisoners are said, in those orders, to have
been tried, ‘on the following charges and specifications.’ The court
understood itself to be acting as a court-martial. It was so
organized, it so proceeded, having a judge advocate, hearing
witnesses, and the written defence of the miserable trembling
prisoners, who seemed to have a presentiment of their doom. And
the court was finally dissolved. The whole proceeding manifestly
shows, that all parties considered it as a court-martial, convened and
acting under the rules and articles of war. In his letter to the
secretary of war, noticing the transaction, the general says, ‘these
individuals were tried under my orders, legally convicted as exciters
of this savage and negro war, legally condemned, and most justly
punished for their iniquities.’ The Lord deliver us from such legal
conviction, and such legal condemnation! The general himself
considered the laws of his country to have justified his proceedings.
It is in vain then to talk of a power in him beyond the law, and
above the law, when he himself does not assert it. Let it be
conceded that he was clothed with absolute authority over the lives
of those individuals, and that, upon his own fiat, without trial,
without defence, he might have commanded their execution. Now, if
an absolute sovereign, in any particular respect, promulgates a rule,
which he pledges himself to observe, if he subsequently deviates
from that rule, he subjects himself to the imputation of odious
tyranny. If general Jackson had the power, without a court, to
condemn these men, he had also the power to appoint a tribunal.
He did appoint a tribunal, and became, therefore, morally bound to
observe and execute the sentence of that tribunal. In regard to
Ambrister, it is with grief and pain I am compelled to say, that he
was executed in defiance of all law; in defiance of the law to which
general Jackson had voluntarily, if you please, submitted himself,
and given, by his appeal to the court, his implied pledge to observe.
I know but little of military law, and what has happened, has
certainly not created in me a taste for acquiring a knowledge of
more; but I believe there is no example on record, where the
sentence of the court has been erased, and a sentence not
pronounced by it carried into execution. It has been suggested that
the court had pronounced two sentences, and that the general had a
right to select either. Two sentences! Two verdicts! It was not so.
The first being revoked, was as though it had never been
pronounced. And there remained only one sentence, which was put
aside upon the sole authority of the commander, and the execution
of the prisoner ordered. He either had or had not a right to decide
upon the fate of that man, with the intervention of a court. If he had
the right, he waived it, and having violated the sentence of the
court, there was brought upon the judicial administration of the
army a reproach, which must occasion the most lasting regret.

However guilty these men were, they should not have been
condemned or executed without the authority of the law. I will not
dwell, at this time, on the effect of these precedents in foreign
countries; but I shall not pass unnoticed their dangerous influence in
our own country. Bad examples are generally set in the cases of bad
men, and often remote from the central government. It was in the
provinces that were laid the abuses and the seeds of the ambitious
projects which overturned the liberties of Rome. I beseech the
committee not to be so captivated with the charms of eloquence,
and the appeals made to our passions and our sympathies, as to
forget the fundamental principles of our government. The influence
of a bad example will often be felt, when its authors and all the
circumstances connected with it are no longer remembered. I know
of but one analogous instance of the execution of a prisoner, and
that has brought more odium than almost any other incident on the
unhappy emperor of France. I allude to the instance of the execution
of the unfortunate member of the Bourbon house. He sought an
asylum in the territories of Baden. Bonaparte despatched a corps of
gen-d’armes to the place of his retreat, seized him, and brought him
to the dungeons of Vincennes. He was there tried by a court-martial,
condemned, and shot. There, as here, was a violation of neutral
territory; there, the neutral ground was not stained with the blood of
him whom it should have protected. And there is another most
unfortunate difference for the American people. The duke d’Enghein
was executed according to his sentence. It is said by the defenders
of Napoleon, that the duke had been machinating not merely to
overturn the French government, but against the life of its chief. If
that were true, he might, if taken in France, have been legally
executed. Such was the odium brought upon the instruments of this
transaction, that those persons who have been even suspected of
participation in it, have sought to vindicate themselves from what
they appear to have considered as an aspersion, before foreign
courts. In conclusion of this part of my subject, I most cheerfully
and entirely acquit general Jackson of any intention to violate the
laws of the country, or the obligations of humanity. I am persuaded,
from all that I have heard, that he considered himself as equally
respecting and observing both. With respect to the purity of his
intentions, therefore, I am disposed to allow it in the most extensive
degree. Of his acts, it is my duty to speak, with the freedom which
belongs to my station. And I shall now proceed to consider some of
them, of the most momentous character, as it regards the
distribution of the powers of government.

Of all the powers conferred by the constitution of the United

States, not one is more expressly and exclusively granted, than that
which gives to congress the power to declare war. The immortal
convention who formed that instrument, had abundant reason,
drawn from every page of history, for confiding this tremendous
power to the deliberate judgment of the representatives of the
people. It was there seen, that nations are often precipitated into
ruinous war, from folly, from pride, from ambition, and from the
desire of military fame. It was believed, no doubt, in committing this
great subject to the legislature of the union, we should be safe from
the mad wars that have afflicted, and desolated, and ruined other
countries. It was supposed, that before any war was declared, the
nature of the injury complained of, would be carefully examined, and
the power and resources of the enemy estimated, and the power
and resources of our own country, as well as the probable issue and
consequences of the war. It was to guard our country against
precisely that species of rashness which has been manifested in
Florida, that the constitution was so framed. If, then, this power,
thus cautiously and clearly bestowed upon congress, has been
assumed and exercised by any other functionary of the government,
it is cause of serious alarm, and it becomes this body to vindicate
and maintain its authority by all the means in its power; and yet
there are some gentlemen, who would have us not merely to yield a
tame and silent acquiescence in the encroachment, but even to pass
a vote of thanks to the author.

On the twenty-fifth of March, 1818, the president of the United

States communicated a message to congress in relation to the
Seminole war, in which he declared, that although, in the
prosecution of it, orders had been given to pass into the Spanish
territory, they were so guarded as that the local authorities of Spain
should be respected. How respected? The president, by the
documents accompanying the message, the orders themselves
which issued from the department of war to the commanding
general, had assured the legislature that, even if the enemy should
take shelter under a Spanish fortress, the fortress was not to be
attacked, but the fact to be reported to that department for further
orders. Congress saw, therefore, that there was no danger of
violating the existing peace. And yet, on the same twenty-fifth day
of March, (a most singular concurrence of dates,) when the
representatives of the people received this solemn message,
announced in the presence of the nation and in the face of the
world, and in the midst of a friendly negotiation with Spain, does
general Jackson write from his head-quarters, that he shall take
St. Marks as a necessary depot for his military operations! The
general states, in his letter, what he had heard about the threat on
the part of the Indians and negroes, to occupy the fort, and declares
his purpose to possess himself of it, in either of the two
contingences, of its being in their hands, or in the hands of the
Spaniards. He assumed a right to judge what Spain was bound to do
by her treaty, and judged very correctly; but then he also assumed
the power, belonging to congress alone, of determining what should
be the effect and consequence of her breach of engagement.
General Jackson generally performs what he intimates his intention
to do. Accordingly, finding St. Marks yet in the hands of the
Spaniards, he seized and occupied it. Was ever, I ask, the just
confidence of the legislative body, in the assurances of the chief
magistrate, more abused? The Spanish commander intimated his
willingness that the American army should take post near him, until
he could have instructions from his superior officer, and promised to
maintain, in the mean time, the most friendly relations. No!
St. Marks was a convenient post for the American army, and delay
was inadmissible. I have always understood that the Indians but
rarely take or defend fortresses, because they are unskilled in the
modes of attack and defence. The threat, therefore, on their part, to
seize on St. Marks, must have been empty, and would probably have
been impossible. At all events, when general Jackson arrived there,
no danger any longer threatened the Spaniards, from the miserable
fugitive Indians, who fled on all sides, upon his approach. And, sir,
upon what plea is this violation of orders, and this act of war upon a
foreign power, attempted to be justified? Upon the grounds of the
conveniency of the depôt and the Indian threat. The first I will not
seriously examine and expose. If the Spanish character of the fort
had been totally merged in the Indian character, it might have been
justifiable to seize it. But that was not the fact; and the bare
possibility of its being forcibly taken by the Indians, could not justify
our anticipating their blow. Of all the odious transactions which
occurred during the late war between France and England, none was
more condemned in Europe and in this country, than her seizure of
the fleet of Denmark, at Copenhagen. And I lament to be obliged to
notice the analogy which exists in the defences made of the two
cases. If my recollection does not deceive me, Bonaparte had
passed the Rhine and the Alps, had conquered Italy, the
Netherlands, Holland, Hanover, Lubec, and Hamburg, and extended
his empire as far as Altona, on the side of Denmark. A few days’
march would have carried him through Holstein, over the two Belts,
through Funen, and into the island of Zealand. What then was the
conduct of England? It was my lot to fall into conversation with an
intelligent Englishman on this subject. ‘We knew (said he) that we
were fighting for our existence. It was absolutely necessary that we
should preserve the command of the seas. If the fleet of Denmark
fell into the enemy’s hands, combined with his other fleets, that
command might be rendered doubtful. Denmark had only a nominal
independence. She was, in truth, subject to his sway. We said to her,
give us your fleet; it will otherwise be taken possession of by your
secret and our open enemy. We will preserve it, and restore it to you
whenever the danger shall be over. Denmark refused. Copenhagen
was bombarded, gallantly defended, but the fleet was seized.
Everywhere the conduct of England was censured; and the name
even of the negotiator who was employed by her, who was
subsequently the minister near this government, was scarcely ever
pronounced here without coupling with it an epithet, indicating his
participation in the disgraceful transaction. And yet we are going to
sanction acts of violence, committed by ourselves, which but too
much resemble it! What an important difference, too, between the
relative condition of England and of this country! She, perhaps, was
struggling for her existence. She was combating, single-handed, the
most enormous military power that the world has ever known. With
whom were we contending? With a few half-starved, half-clothed,
wretched Indians, and fugitive slaves. And, whilst carrying on this
inglorious war, inglorious as it regards the laurels or renown won in
it, we violate neutral rights, which the government had solemnly
pledged itself to respect, upon the principle of convenience, or upon
the light presumption that, by possibility, a post might be taken by
this miserable combination of Indians and slaves.

On the eighth of April, the general writes from St. Marks, that he
shall march for the Suwaney river; the destroying of the
establishments on which will, in his opinion, bring the war to a close.
Accordingly, having effected that object, he writes, on the twentieth
of April, that he believes he may say that the war is at an end for
the present. He repeats the same opinion in his letter to the
secretary of war, written six days after. The war being thus ended, it
might have been hoped that no further hostilities would be
committed. But on the twenty-third of May, on his way home, he
receives a letter from the commandant of Pensacola, intimating his
surprise at the invasion of the Spanish territory, and the acts of
hostility performed by the American army, and his determination, if
persisted in, to employ force to repel them. Let us pause and
examine the proceeding of the governor, so very hostile and
affrontive in the view of general Jackson. Recollect that he was
governor of Florida; that he had received no orders from his
superiors, to allow a passage to the American army; that he had
heard of the reduction of St. Marks; and that general Jackson, at the
head of his army, was approaching in the direction of Pensacola. He
had seen the president’s message of the twenty-fifth of March, and
reminded general Jackson of it, to satisfy him that the American
government could not have authorized all those measures. I cannot
read the allusion made by the governor to that message, without
feeling that the charge of insincerity, which it implied, had at least
but too much the appearance of truth in it. Could the governor have
done less than write some such letter? We have only to reverse
situations, and to suppose him to have been an American governor.
General Jackson says, that when he received that letter, he no longer
hesitated. No, sir, he did no longer hesitate. He received it on the
twenty-third, he was in Pensacola on the twenty-fourth, and
immediately after set himself before the fortress of San Carlos de
Barancas, which he shortly reduced. Veni, vidi, vici. Wonderful
energy! Admirable promptitude! Alas, that it had not been an energy
and a promptitude within the pale of the constitution, and according
to the orders of the chief magistrate. It is impossible to give any
definition of war, that would not comprehend these acts. It was
open, undisguised, and unauthorized hostility.

The honorable gentleman from Massachusetts has endeavored to

derive some authority to general Jackson from the message of the
president, and the letter of the secretary of war to governor Bibb.
The message declares, that the Spanish authorities are to be
respected wherever maintained. What the president means by their
being maintained, is explained in the orders themselves, by the
extreme case being put of the enemy seeking shelter under a
Spanish fort. If even in that case he was not to attack, certainly he
was not to attack in any case of less strength. The letter to governor
Bibb admits of a similar explanation. When the secretary says, in
that letter, that general Jackson is fully empowered to bring the
Seminole war to a conclusion, he means that he is so empowered by
his orders, which, being now before us, must speak for themselves.
It does not appear that general Jackson ever saw that letter, which
was dated at this place after the capture of St. Marks. I will take a
momentary glance at the orders. On the second of December, 1817,
general Gaines was forbidden to cross the Florida line. Seven days
after, the secretary of war having arrived here, and infused a little
more energy into our councils, he was authorized to use a sound
discretion in crossing or not. On the sixteenth, he was instructed
again to consider himself at liberty to cross the line, and pursue the
enemy; but, if he took refuge under a Spanish fortress, the fact was
to be reported to the department of war. These orders were
transmitted to general Jackson, and constituted, or ought to have
constituted, his guide. There was then no justification for the
occupation of Pensacola, and the attack on the Barancas, in the
message of the president, the letter to governor Bibb, or in the
orders themselves. The gentleman from Massachusetts will pardon
me for saying, that he has undertaken what even his talents are not
competent to—the maintenance of directly contradictory
propositions, that it was right in general Jackson to take Pensacola,
and wrong in the president to keep it. The gentleman has made a
greater mistake than he supposes general Jackson to have done in
attacking Pensacola for an Indian town, by attempting the defence
both of the president and general Jackson. If it were right in him to
seize the place, it is impossible that it should have been right in the
president immediately to surrender it. We, sir, are the supporters of
the president. We regret that we cannot support general Jackson
also. The gentleman’s liberality is more comprehensive than ours.
I approve with all my heart of the restoration of Pensacola. I think
St. Marks ought, perhaps, to have been also restored; but I say this
with doubt and diffidence. That the president thought the seizure of
the Spanish posts was an act of war, is manifest from his opening
message, in which he says that, to have retained them, would have
changed our relations with Spain, to do which the power of the
executive was incompetent, congress alone possessing it. The
president has, in this instance, deserved well of his country. He has
taken the only course which he could have pursued, consistent with
the constitution of the land. And I defy the gentleman to make good
both his positions, that the general was right in taking, and the
president right in giving up, the posts.

[Mr. Holmes explained. We took these posts, he said, to keep them from the
hands of the enemy, and, in restoring them, made it a condition that Spain should
not let our enemy have them. We said to her, here is your dagger; we found it in
the hands of our enemy, and, having wrested it from him, we restore it to you in
the hope that you will take better care of it for the future.]

The gentleman from Massachusetts is truly unfortunate; fact or

principle is always against him. The Spanish posts were not in the
possession of the enemy. One old Indian only was found in the
Barancas, none in Pensacola, none in St. Marks. There was not even
the color of a threat of Indian occupation as it regards Pensacola
and the Barancas. Pensacola was to be restored unconditionally, and
might, therefore, immediately have come into the possession of the
Indians, if they had the power and the will to take it. The gentleman
is in a dilemma from which there is no escape. He gave up general
Jackson when he supported the president, and gave up the
president when he supported general Jackson. I rejoice to have seen
the president manifesting, by the restoration of Pensacola, his
devotedness to the constitution. When the whole country was
ringing with plaudits for its capture, I said, and I said alone, in the
limited circle in which I moved, that the president must surrender it;
that he could not hold it. It is not my intention to inquire, whether
the army was or was not constitutionally marched into Florida. It is
not a clear question, and I am inclined to think that the express
authority of congress ought to have been asked. The gentleman
from Massachusetts will allow me to refer to a part of the
correspondence at Ghent different from that which he has quoted.
He will find the condition of the Indians there accurately defined.
And it is widely variant from the gentleman’s ideas on this subject.
The Indians, inhabiting the United States, according to the
statement of the American commissioners at Ghent, have a qualified
sovereignty only, the supreme sovereignty residing in the
government of the United States. They live under their own laws and
customs, may inhabit and hunt their lands; but acknowledge the
protection of the United States, and have no right to sell their lands
but to the government of the United States. Foreign powers or
foreign subjects have no right to maintain any intercourse with
them, without our permission. They are not, therefore, independent
nations, as the gentleman supposes. Maintaining the relation
described with them, we must allow a similar relation to exist
between Spain and the Indians residing within her dominions. She
must be, therefore, regarded as the sovereign of Florida, and we
are, accordingly, treating with her for the purchase of it. In
strictness, then, we ought first to have demanded of her to restrain
the Indians, and, that failing, we should have demanded a right of
passage for our army. But, if the president had the power to march
an army into Florida, without consulting Spain, and without the
authority of congress, he had no power to authorize any act of
hostility against her. If the gentleman had even succeeded in
showing that an authority was conveyed by the executive to general
Jackson to take the Spanish posts, he would only have established
that unconstitutional orders had been given, and thereby transferred
the disapprobation from the military officer to the executive. But no
such orders were, in truth, given. The president acted in conformity
to the constitution, when he forbade the attack of a Spanish fort,
and when, in the same spirit, he surrendered the posts themselves.

I will not trespass much longer upon the time of the committee;
but I trust I shall be indulged with some few reflections upon the
danger of permitting the conduct on which it has been my painful
duty to animadvert, to pass without a solemn expression of the
disapprobation of this house. Recall to your recollection the free
nations which have gone before us. Where are they now?
 ‘Gone glimmering through the dream of things that were,

A school-boy’s tale, the wonder of an hour.’

And how have they lost their liberties? If we could transport

ourselves back to the ages when Greece and Rome flourished in
their greatest prosperity, and, mingling in the throng, should ask a
Grecian if he did not fear that some daring military chieftain, covered
with glory, some Philip or Alexander, would one day overthrow the
liberties of his country, the confident and indignant Grecian would
exclaim, no! no! we have nothing to fear from our heroes; our
liberties will be eternal. If a Roman citizen had been asked, if he did
not fear that the conqueror of Gaul might establish a throne upon
the ruins of public liberty, he would have instantly repelled the
unjust insinuation. Yet Greece fell; Cæsar passed the Rubicon, and
the patriotic arm even of Brutus could not preserve the liberties of
his devoted country! The celebrated Madame de Stael, in her last
and perhaps her best work, has said, that in the very year, almost
the very month, when the president of the directory declared that
monarchy would never more show its frightful head in France,
Bonaparte, with his grenadiers, entered the palace of St. Cloud, and
dispersing, with the bayonet, the deputies of the people, deliberating
on the affairs of the state, laid the foundation of that vast fabric of
despotism which overshadowed all Europe. I hope not to be
misunderstood; I am far from intimating that general Jackson
cherishes any designs inimical to the liberties of the country.
I believe his intentions to be pure and patriotic. I thank God that he
would not, but I thank him still more that he could not if he would,
overturn the liberties of the republic. But precedents, if bad, are
fraught with the most dangerous consequences. Man has been
described, by some of those who have treated of his nature, as a
bundle of habits. The definition is much truer when applied to
governments. Precedents are their habits. There is one important
difference between the formation of habits by an individual and by
governments. He contracts it only after frequent repetition. A single
instance fixes the habit and determines the direction of
governments. Against the alarming doctrine of unlimited discretion in
our military commanders when applied even to prisoners of war,
I must enter my protest. It begins upon them; it will end on us.
I hope our happy form of government is to be perpetual. But, if it is
to be preserved, it must be by the practice of virtue, by justice, by
moderation, by magnanimity, by greatness of soul, by keeping a
watchful and steady eye on the executive; and, above all, by holding
to a strict accountability the military branch of the public force.

We are fighting a great moral battle, for the benefit not only of
our country, but of all mankind. The eyes of the whole world are in
fixed attention upon us. One, and the largest portion of it, is gazing
with contempt, with jealousy, and with envy; the other portion, with
hope, with confidence, and with affection. Everywhere the black
cloud of legitimacy is suspended over the world, save only one
bright spot, which breaks out from the political hemisphere of the
west, to enlighten, and animate, and gladden the human heart.
Obscure that, by the downfall of liberty here, and all mankind are
enshrouded in a pall of universal darkness. To you, Mr. Chairman,
belongs the high privilege of transmitting, unimpaired, to posterity,
the fair character and liberty of our country. Do you expect to
execute this high trust, by trampling, or suffering to be trampled
down, law, justice, the constitution, and the rights of the people? by
exhibiting examples of inhumanity, and cruelty, and ambition? When
the minions of despotism heard, in Europe, of the seizure of
Pensacola, how did they chuckle, and chide the admirers of our
institutions, tauntingly pointing to the demonstration of a spirit of
injustice and aggrandizement made by our country, in the midst of
an amicable negotiation. Behold, said they, the conduct of those who
are constantly reproaching kings. You saw how those admirers were
astounded and hung their heads. You saw, too, when that illustrious
man, who presides over us, adopted his pacific, moderate, and just
course, how they once more lifted up their heads with exultation and
delight beaming in their countenances. And you saw how those
minions themselves were finally compelled to unite in the general
praises bestowed upon our government. Beware how you forfeit this
exalted character. Beware how you give a fatal sanction, in this
infant period of our republic, scarcely yet two score years old, to
military insubordination. Remember that Greece had her Alexander,
Rome her Cæsar, England her Cromwell, France her Bonaparte, and
that if we would escape the rock on which they split, we must avoid
their errors.

How different has been the treatment of general Jackson, and

that modest, but heroic young man, a native of one of the smallest
states in the union, who achieved for his country, on lake Erie, one
of the most glorious victories of the late war. In a moment of
passion, he forgot himself, and offered an act of violence which was
repented of as soon as perpetrated. He was tried, and suffered the
judgment to be pronounced by his peers. Public justice was thought
not even then to be satisfied. The press and congress took up the
subject. My honorable friend from Virginia (Mr. Johnson), the faithful
and consistent sentinel of the law and of the constitution,
disapproved in that instance, as he does in this, and moved an
inquiry. The public mind remained agitated and unappeased, until
the recent atonement so honorably made by the gallant commodore.
And is there to be a distinction between the officers of the two
branches of the public service? Are former services, however
eminent, to preclude even inquiry into recent misconduct? Is there
to be no limit, no prudential bounds to the national gratitude? I am
not disposed to censure the president for not ordering a court of
inquiry, or a general court-martial. Perhaps, impelled by a sense of
gratitude, he determined, by anticipation, to extend to the general
that pardon which he had the undoubted right to grant after
sentence. Let us not shrink from our duty. Let us assert our
constitutional powers, and vindicate the instrument from military
violation.

I hope gentlemen will deliberately survey the awful isthmus on

which we stand. They may bear down all opposition; they may even
vote the general the public thanks; they may carry him triumphantly
through this house. But, if they do, in my humble judgment, it will
be a triumph of the principle of insubordination, a triumph of the
military over the civil authority, a triumph over the powers of this
house, a triumph over the constitution of the land. And I pray most
devoutly to Heaven, that it may not prove, in its ultimate effects and
consequences, a triumph over the liberties of the people.
 ON SOUTH AMERICAN
AFFAIRS.
IN THE HOUSE OF REPRESENTATIVES,
FEBRUARY 9, 1819.
[THE house being in committee of the whole, on the bill to increase the
salaries of certain officers of government, Mr. Clay rose and said:]

IT had been his settled intention to renew, pending this bill, the
proposition which he had the honor of submitting at the last session,
having for its object the recognition of the independence of the
United Provinces of South America. He was restrained from
executing that intention, by two considerations; one was his
personal indisposition, but another and more important one, was,
the small portion of the session yet remaining, to transact the public
business. Whilst he was up, he would say, that so far from his
opinions, expressed on the former occasion, having undergone any
change, they had been strengthened and confirmed, by all the
occurrences which had subsequently taken place. He had been
anxious, if time had permitted, to examine what appeared to him
very exceptionable reasons assigned for declining to recognise our
sister republic, in a paper entitled to the most profound respect, the
message of the president at the opening of congress. He was
desirous, also, of noticing the still more exceptionable grounds taken
in a paper recently transmitted to the house, from the department of
state (it ought to be laid on our table; why it was not, he did not
know; he hoped our worthy clerk would, in his future contract for
the public printing, guard against the delay to which we have so
often been subjected). From that paper it appeared, that even a
consul could not be received from the southern republic, because
the grant of an exequator implied recognition! We receive her flag,
we admit her commerce, and yet refuse the consular protection
which that flag and commerce necessarily drew with them! But to
submit his proposition, would be to occasion, perhaps, a protracted
debate. And considering the few days yet left us, the pressing and
urgent, though not more important business yet to be done, he
should not hold himself excusable to the house and to the country,
after having himself so materially contributed to the consumption of
time in debate, if he were even the unintentional instrument of
preventing the passage of what might be thought essential laws. He
would like exceedingly to contrast the objections urged against the
reception of the Venezuelean minister, with the more forcible and
stronger personal ones that lay to the present Spanish minister. But
deep as the interest which he heretofore had felt and still felt, in the
success of that great struggle to the south, he must, for the reasons
assigned, forbear to press any proposition upon the house, at
present. Should it be necessary at another session, and should he
have the honor of a seat on this floor then, he pledged himself to
bring up the subject, unless adverse causes should render it highly
inexpedient.
 ON THE SPANISH TREATY.
IN THE HOUSE OF REPRESENTATIVES, APRIL 3, 1820.
[A PERUSAL of this speech will be always gratifying and instructive, to all who
would wish to be well informed in the political history of the United States. While it
shows, in a striking manner, the foresight and sagacity of Mr. Clay, as an American
statesman, it contains facts of much importance with regard to the settlement of
the southern boundaries of the United States and the acquisition of Florida. It will
be seen, that Mr. Clay disapproved of the treaty between this country and Spain,
made in 1819, by the administration of Mr. Monroe, for reasons stated in this
speech, which was made before the treaty was ratified by Spain. His principal
objection to the treaty appears to have been, that it relinquished our claim to
Texas, which territory Mr. Clay considered of much greater value to us than
Florida. The settlement of these questions, by the subsequent ratification of the
treaty, in October, 1820, by which we relinquished Texas and acquired Florida,
does not diminish the value of this record of Mr. Clay’s views on a subject, which
has increased in importance since the independence of both Mexico and Texas has
been established.]

THE house having resolved itself into a committee of the whole,

on the state of the union, and the following resolutions, submitted
some days ago by Mr. Clay (the speaker) being under consideration:

First, resolved, that the constitution of the United States vests in

congress the power to dispose of the territory belonging to them;
and that no treaty, purporting to alienate any portion thereof, is valid
without the concurrence of congress:

Second, resolved, that the equivalent proposed to be given by

Spain to the United States in the treaty concluded between them, on
the twenty-second of February, 1819, for that part of Louisiana lying
west of the Sabine, was inadequate; and that it would be
inexpedient to make a transfer thereof to any foreign power, or to
renew the aforesaid treaty:

Mr. Clay said, that, whilst he felt very grateful to the house for
the prompt and respectful manner in which they had allowed him to
enter upon the discussion of the resolutions which he had the honor
of submitting to their notice, he must at the same time frankly say,
that he thought their character and consideration, in the councils of
this country, were concerned in not letting the present session pass
off without deliberating upon our affairs with Spain. In coming to the
present session of congress, it had been his anxious wish to be able
to concur with the executive branch of the government in the
measures which it might conceive itself called upon to recommend
on that subject, for two reasons, of which the first, relating
personally to himself, he would not trouble the committee with
further noticing. The other was, that it appeared to him to be always
desirable, in respect to the foreign action of this government, that
there should be a perfect coincidence in opinion between its several
coördinate branches. In time of peace, however, it might be
allowable, to those who are charged with the public interests, to
entertain and express their respective views, although there might
be some discordance between them. In a season of war there
should be no division in the public councils; but a united and
vigorous exertion to bring the war to an honorable conclusion. For
his part, whenever that calamity may befall his country, he would
entertain but one wish, and that is, that success might crown our
struggle, and the war be honorably and gloriously terminated. He
would never refuse to share in the joys incident to the victory of our
arms, nor to participate in the griefs of defeat and discomfiture. He
conceded entirely in the sentiment once expressed by that illustrious
hero, whose recent melancholy fall we all so sincerely deplore, that
fortune may attend our country in whatever war it may be involved.
 There are two systems of policy, he said, of which our
government had had the choice. The first was, by appealing to the
justice and affections of Spain, to employ all those persuasives which
could arise out of our abstinence from any direct countenance to the
cause of South America, and the observance of a strict neutrality.
The other was, by appealing to her justice also, and to her fears, to
prevail upon her to redress the injuries of which we complain—her
fears by a recognition of the independent governments of South
America, and leaving her in a state of uncertainty as to the further
step we might take in respect to those governments. The unratified
treaty was the result of the first system. It could not be positively
affirmed, what effect the other system would have produced; but he
verily believed that, whilst it rendered justice to those governments,
and would have better comported with that magnanimous policy
which ought to have characterized our own, it would have more
successfully tended to an amicable and satisfactory arrangement of
our differences with Spain.

The first system has so far failed. At the commencement of the

session, the president recommended an enforcement of the
provisions of the treaty. After three months’ deliberation, the
committee of foreign affairs, not being able to concur with him, has
made us a report, recommending the seizure of Florida in the nature
of a reprisal. Now the president recommends our postponement of
the subject until the next session. It had been his intention,
whenever the committee of foreign affairs should engage the house
to act upon their bill, to offer, as a substitute for it, the system which
he thought it became this country to adopt, of which the occupation
of Texas, as our own, would have been a part, and the recognition
of the independent governments of South America another. If he did
not now bring forward this system, it was because the committee
proposed to withdraw their bill, and because he knew too much of
the temper of the house and of the executive, to think that it was
advisable to bring it forward. He hoped that some suitable
opportunity might occur during the session, for considering the
propriety of recognising the independent governments of South
America.

Whatever he might think of the discretion which was evinced in

recommending the postponement of the bill of the committee of
foreign relations, he could not think that the reasons, assigned by
the president for that recommendation, were entitled to the weight
which he had given them. He thought the house was called upon, by
a high sense of duty, seriously to animadvert upon some of those
reasons. He believed it was the first example, in the annals of the
country, in which a course of policy, respecting one foreign power,
which we must suppose had been deliberately considered, has been
recommended to be abandoned, in a domestic communication from
one to another coördinate branch of the government, upon the
avowed ground of the interposition of foreign powers. And what is
the nature of this interposition? It is evinced by a cargo of scraps,
gathered up from this chargé d’affaires, and that; of loose
conversations held with this foreign minister, and that—perhaps
mere levee conversations, without a commitment in writing, in a
solitary instance, of any of the foreign parties concerned, except
only in the case of his imperial majesty; and what was the character
of his commitment we shall presently see. But, he must enter his
solemn protest against this and every other species of foreign
interference in our matters with Spain. What have they to do with
them? Would they not repel as officious and insulting intrusion, any
interference on our part in their concerns with foreign states? Would
his imperial majesty have listened with complacency, to our
remonstrances against the vast acquisitions which he has recently
made? He has lately crammed his enormous maw with Finland, and
with the spoils of Poland, and, whilst the difficult process of
digestion is going on, he throws himself upon a couch, and cries out,
don’t, don’t disturb my repose.

He charges his minister here to plead the cause of peace and

concord! The American ‘government is too enlightened’ (ah! sir, how
sweet this unction is, which is poured down our backs,) to take hasty
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