niessen - liquid chromatography - mass spectrometry 3e [lcms] (crc, 2006)

Wilfried M.A. Niessen
hyphen MassSpec Consultancy
Leiden, The Netherlands
Liquid Chromatography–
Mass Spectrometry
Third Edition
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© 2006 by Taylor and Francis Group, LLC
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Library of Congress Cataloging-in-Publication Data
Niessen, W. M. A. (Wilfried M. A.), 1956-
Liquid chromatography mass spectrometry. 3rd ed. / Wilfried M.A. Niessen.
p. cm. (Chromatographic science series ; 97)
Includes bibliographical references and index.
ISBN-13: 978-0-8247-4082-5 (acid-free paper)
ISBN-10: 0-8247-4082-3 (acid-free paper)
1. Liquid chromatography. 2. Mass spectrometry. I. Title. II. Series: Chromato-
graphic science ; v. 97.
QD79.C454N54 2007
543’.84 dc22 2006013709
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CHROMATOGRAPHIC SCIENCE SERIES
A Series of Textbooks and Reference Books
Editor: JACK CAZES
1. Dynamics of Chromatography: Principles and Theory,
J. Calvin Giddings
2. Gas Chromatographic Analysis of Drugs and Pesticides,
Benjamin J. Gudzinowicz
3. Principles of Adsorption Chromatography: The Separation
of Nonionic Organic Compounds,

Lloyd R. Snyder
4. Multicomponent Chromatography: Theory of Interference,
Friedrich Helfferich and Gerhard Klein
5. Quantitative Analysis by Gas Chromatography, Josef Novák
6. High-Speed Liquid Chromatography,
Peter M. Rajcsanyi
and Elisabeth Rajcsanyi
7. Fundamentals of Integrated GC-MS (in three parts),
Benjamin J. Gudzinowicz, Michael J. Gudzinowicz,
and Horace F. Martin
8. Liquid Chromatography of Polymers and Related Materials,
Jack Cazes
9. GLC and HPLC Determination of Therapeutic Agents (in three parts),
Part 1
edited by Kiyoshi Tsuji and Walter Morozowich
, Parts 2 and 3
edited by Kiyoshi Tsuji
10. Biological/Biomedical Applications of Liquid Chromatography,
edited by Gerald L. Hawk
11. Chromatography in Petroleum Analysis,
edited by Klaus H. Altgelt
and T. H. Gouw
12. Biological/Biomedical Applications of Liquid Chromatography II,
edited by Gerald L. Hawk
13. Liquid Chromatography of Polymers and Related Materials II,
edited by Jack Cazes and Xavier Delamare
14. Introduction to Analytical Gas Chromatography: History, Principles,
and Practice,
John A. Perry
15. Applications of Glass Capillary Gas Chromatography,

edited by
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16. Steroid Analysis by HPLC: Recent Applications,
edited by
Marie P. Kautsky
17. Thin-Layer Chromatography: Techniques and Applications,
Bernard Fried and Joseph Sherma
18. Biological/Biomedical Applications of Liquid Chromatography III,
edited by Gerald L. Hawk
19. Liquid Chromatography of Polymers and Related Materials III,
edited by Jack Cazes
20. Biological/Biomedical Applications of Liquid Chromatography,
edited by Gerald L. Hawk
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21. Chromatographic Separation and Extraction with Foamed Plastics
and Rubbers,
G. J. Moody and J. D. R. Thomas
22. Analytical Pyrolysis: A Comprehensive Guide,
William J. Irwin
23. Liquid Chromatography Detectors,
edited by Thomas M. Vickrey
24. High-Performance Liquid Chromatography in Forensic Chemistry,
edited by Ira S. Lurie and John D. Wittwer, Jr.
25. Steric Exclusion Liquid Chromatography of Polymers,
edited by
Josef Janca
26. HPLC Analysis of Biological Compounds: A Laboratory Guide,
William S. Hancock and James T. Sparrow
27. Affinity Chromatography: Template Chromatography of Nucleic

Acids and Proteins,
Herbert Schott
28. HPLC in Nucleic Acid Research: Methods and Applications,
edited by Phyllis R. Brown
29. Pyrolysis and GC in Polymer Analysis,
edited by S. A. Liebman
and E. J. Levy
30. Modern Chromatographic Analysis of the Vitamins,
edited by
André P. De Leenheer, Willy E. Lambert, and Marcel G. M. De Ruyter
31. Ion-Pair Chromatography,
edited by Milton T. W. Hearn
32. Therapeutic Drug Monitoring and Toxicology by Liquid
Chromatography,
edited by Steven H. Y. Wong
33. Affinity Chromatography: Practical and Theoretical Aspects,
Peter Mohr and Klaus Pommerening
34. Reaction Detection in Liquid Chromatography,
edited by Ira S. Krull
35. Thin-Layer Chromatography: Techniques and Applications,
Second Edition, Revised and Expanded,
Bernard Fried
and Joseph Sherma
36. Quantitative Thin-Layer Chromatography and Its Industrial
Applications,
edited by Laszlo R. Treiber
37. Ion Chromatography,
edited by James G. Tarter
38. Chromatographic Theory and Basic Principles,
edited by

Jan Åke Jönsson
39. Field-Flow Fractionation: Analysis of Macromolecules and Particles,
Josef Janca
40. Chromatographic Chiral Separations,
edited by Morris Zief
and Laura J. Crane
41. Quantitative Analysis by Gas Chromatography, Second Edition,
Revised and Expanded,
Josef Novák
42. Flow Perturbation Gas Chromatography,
N. A. Katsanos
43. Ion-Exchange Chromatography of Proteins,
Shuichi Yamamoto,
Kazuhiro Naka-nishi, and Ryuichi Matsuno
44. Countercurrent Chromatography: Theory and Practice,
edited by N. Bhushan Man-dava and Yoichiro Ito
45. Microbore Column Chromatography: A Unified Approach
to Chromatography,
edited by Frank J. Yang
46. Preparative-Scale Chromatography,
edited by Eli Grushka
47. Packings and Stationary Phases in Chromatographic Techniques,
edited by Klaus K. Unger
48. Detection-Oriented Derivatization Techniques in Liquid
Chromatography,
edited by Henk Lingeman
and Willy J. M. Underberg
49. Chromatographic Analysis of Pharmaceuticals,
edited by
John A. Adamovics

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50. Multidimensional Chromatography: Techniques and Applications,
edited by Hernan Cortes
51. HPLC of Biological Macromolecules: Methods and Applications,
edited by Karen M. Gooding and Fred E. Regnier
52. Modern Thin-Layer Chromatography,
edited by Nelu Grinberg
53. Chromatographic Analysis of Alkaloids,
Milan Popl, Jan Fähnrich,
and Vlastimil Tatar
54. HPLC in Clinical Chemistry,
I. N. Papadoyannis
55. Handbook of Thin-Layer Chromatography,
edited by Joseph Sherma
and Bernard Fried
56. Gas–Liquid–Solid Chromatography,
V. G. Berezkin
57. Complexation Chromatography,
edited by D. Cagniant
58. Liquid Chromatography–Mass Spectrometry,
W. M. A. Niessen
and Jan van der Greef
59. Trace Analysis with Microcolumn Liquid Chromatography,
Milos KrejcI
60. Modern Chromatographic Analysis of Vitamins: Second Edition,
edited by André P. De Leenheer, Willy E. Lambert, and Hans J. Nelis
61. Preparative and Production Scale Chromatography,
edited by
G. Ganetsos and P. E. Barker

62. Diode Array Detection in HPLC,
edited by Ludwig Huber
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63. Handbook of Affinity Chromatography,
edited by Toni Kline
64. Capillary Electrophoresis Technology,
edited by Norberto A. Guzman
65. Lipid Chromatographic Analysis,
edited by Takayuki Shibamoto
66. Thin-Layer Chromatography: Techniques and Applications:
Third Edition, Revised and Expanded,
Bernard Fried
and Joseph Sherma
67. Liquid Chromatography for the Analyst,
Raymond P. W. Scott
68. Centrifugal Partition Chromatography,
edited by Alain P. Foucault
69. Handbook of Size Exclusion Chromatography,
edited by Chi-San Wu
70. Techniques and Practice of Chromatography,
Raymond P. W. Scott
71. Handbook of Thin-Layer Chromatography: Second Edition,
Revised and Expanded,
edited by Joseph Sherma and Bernard Fried
72. Liquid Chromatography of Oligomers,
Constantin V. Uglea
73. Chromatographic Detectors: Design, Function, and Operation,
Raymond P. W. Scott
74. Chromatographic Analysis of Pharmaceuticals: Second Edition,
Revised and Expanded,

edited by John A. Adamovics
75. Supercritical Fluid Chromatography with Packed Columns:
Techniques and Applications,
edited by Klaus Anton
and Claire Berger
76. Introduction to Analytical Gas Chromatography: Second Edition,
Revised and Expanded,
Raymond P. W. Scott
77. Chromatographic Analysis of Environmental and Food Toxicants,
edited by Takayuki Shibamoto
78. Handbook of HPLC,
edited by Elena Katz, Roy Eksteen,
Peter Schoenmakers, and Neil Miller
79. Liquid Chromatography–Mass Spectrometry: Second Edition,
Revised and Expanded,
Wilfried Niessen
80. Capillary Electrophoresis of Proteins,
Tim Wehr,
Roberto Rodríguez-Díaz, and Mingde Zhu
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81. Thin-Layer Chromatography: Fourth Edition, Revised and Expanded,
Bernard Fried and Joseph Sherma
82. Countercurrent Chromatography,
edited by Jean-Michel Menet
and Didier Thiébaut
83. Micellar Liquid Chromatography,
Alain Berthod
and Celia García-Alvarez-Coque
84. Modern Chromatographic Analysis of Vitamins: Third Edition,

Revised and Expanded, edited by
André P. De Leenheer,
Willy E. Lambert, and Jan F. Van Bocxlaer
85. Quantitative Chromatographic Analysis,
Thomas E. Beesley,
Benjamin Buglio, and Raymond P. W. Scott
86. Current Practice of Gas Chromatography–Mass Spectrometry,
edited by W. M. A. Niessen
87. HPLC of Biological Macromolecules: Second Edition,
Revised and Expanded,
edited by Karen M. Gooding
and Fred E. Regnier
88. Scale-Up and Optimization in Preparative Chromatography:
Principles and Bio-pharmaceutical Applications,
edited by
Anurag S. Rathore and Ajoy Velayudhan
89. Handbook of Thin-Layer Chromatography: Third Edition,
Revised and Expanded,
edited by Joseph Sherma and Bernard Fried
90. Chiral Separations by Liquid Chromatography and Related
Technologies,
Hassan Y. Aboul-Enein and Imran Ali
91. Handbook of Size Exclusion Chromatography and Related
Techniques: Second Edition,
edited by Chi-San Wu
92. Handbook of Affinity Chromatography: Second Edition,
edited by
David S. Hage
93. Chromatographic Analysis of the Environment: Third Edition,
edited by Leo M. L. Nollet

94. Microfluidic Lab-on-a-Chip for Chemical and Biological Analysis
and Discovery,
Paul C.H. Li
95. Preparative Layer Chromatography,
edited by Teresa Kowalska
and Joseph Sherma
96. Instrumental Methods in Metal Ion Speciation,
Imran Ali
and Hassan Y. Aboul-Enein
97. Liquid Chromatography–Mass Spectrometry: Third Edition,
Wilfried M. A. Niessen
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© 2006 by Taylor and Francis Group, LLC
PREFACE TO THE THIRD EDITION
Before one starts to write the preface to the third edition of one’s book, one
obviously rereads the prefaces to the previous two editions. This third edition
significantly differs from the previous two editions. Most chapters are completely
new or have been extensively rewritten. With the new text and the update to current
developments, the orientation on technology and on the hyphenated character of
LC–MS, nowadays also including sample pretreatment and data processing, was
kept. In the first edition, the main focus was on (interface) technology. The second
edition still paid considerable attention to interface technology, but the application
section had grown to 200 pages. In this third edition, there are two application
sections, covering more than two-thirds of the text (420 out of the 600 pages). The
message that can be read from this is that the LC–MS technology has become
established and mature, whereas still rapid and exciting developments occur in its
many application areas.
This book provides a literature overview. The focus is on principles,
technologies, and especially applications and analytical strategies. Contrary to the
previous editions, I did not at all intend to achieve comprehensive literature

coverage in this third edition. Between 1998 and today, more than 15,000 papers
were published on the topics discussed in this book. It is impossible for me to read
all these papers, due to time limitations, and certainly to give proper attention to
their contents, due to space limitations. In each individual chapter, I have tried to tell
a story relevant to the topic of the chapter, providing a reasonable complete account
on LC–MS related developments in that field. The goal was to provide an
introduction and overview of the strategies and technologies important in each of the
selected application areas. Papers were more-or-less randomly selected to serve as
illustrations to the story and to help me in telling the story. In most cases, attention is
focussed on discussing the role of LC–MS in the selected application areas and to
highlight important analytical strategies, and not so much on the actual results
obtained. I have to apologize to the authors of so many excellent papers, that I could
not cite in the present text. There are far more applications than I could cover in this
edition of the book.
In the past years, LC–MS has definitively come out of the mass spectrometry
specialist’s laboratory to find its place in many chromatography laboratories. Small-
molecule application areas in environmental, food safety, and clinical analysis are
the clearest and most striking examples of this. Obviously, the huge impact of
LC–MS in pharmaceutical drug discovery and development continued. At the same
© 2006 by Taylor and Francis Group, LLC
time, the proteomics field developed, and LC–MS contributes significantly to these
developments.
This third edition is most likely also the last edition, at least in this form. The
exciting and spectacular growth of LC–MS in the past years is such that it is no
longer possible for one person to comprehensively cover and follow all relevant
developments in the wide variety of application areas.
Finally, I have to thank the many people who have inspired me over the years to
continue with my efforts in completing this book. This includes among others the
many people I meet during my courses and consulting work in LC–MS, my
colleagues and the Ph.D. students in my part-time job at the Free University in

Amsterdam, my international collaboration partners. I thank my wife and family,
who had to share me, because a large part of me was writing this book.
Wilfried Niessen
2006
© 2006 by Taylor and Francis Group, LLC
PREFACE TO THE SECOND EDITION
When the first edition of this book was published early 1992, LC–MS could
already be considered an important and mature analytical technique. However, at
that time, the great impact on LC–MS that electrospray and atmospheric-pressure
chemical ionization (APCI) would have could already be foreseen. Since then, the
versatility and application of LC–MS really exploded. Numerous LC–MS systems
have been sold in the past 6 years and have found their way into many different
laboratories, although the pharmaceutical applications of LC–MS appear to be most
important, at least in terms of instrument sales. LC–MS-MS in selective reaction
monitoring mode has now become the method of choice in quantitative bioanalysis.
This second updated, revised and expanded edition of this book on LC–MS was
written and finished in a period when interface innovations somewhat calmed down.
Electrospray and APCI have become the interfaces of choice. At present, no major
developments in interface technology can be foreseen that will lead to another
breakthrough in LC–MS. In terms of applications and versatility, innovations
continue to appear, e.g., in the use of LC–MS in characterization of combinatorial
libraries and in other phases of drug development, in the advent of electrospray time-
of-flight instrumentation for impurity profiling, in applications in the field of
biochemistry and biotechnology.
In view of these developments, older interfaces like thermospray, particle-beam
and continuous-flow fast-atom bombardment appear to be obsolete. Nevertheless, it
was decided to keep the second edition of this book as the comprehensive
introduction and review of all important aspects of LC–MS interfacing and as a
comprehensive guide through the complete field of LC–MS, covering all major
interfaces and paying attention to the history of the technique as well. However, all

chapters have been extensively revised and expanded. The discussions on interface
technology and ionization methods have been integrated. Experimental parameters
and optimization are covered in much more detail in the various interface-related
chapters. Another major change concerns the attention paid to applications: instead
of one 50-page chapter, like in the first edition, the major fields of application of
LC–MS, i.e., in environmental, pharmaceutical, biochemical and biotechnological
analysis and in the analysis of natural products and endogenous compounds, are
reviewed in five chapters, covering almost 200 pages, in this second edition.
© 2006 by Taylor and Francis Group, LLC
The author would like to thank the people who reviewed some of the new
chapters and whose valuable comments were used to enhance the quality of the text:
Dr. Jaroslav Slobodník (Environmental Institute, Koš, Slovak Republic), Dr. Arjen
Tinke (Yamanouchi Europe, Leiderdorp, the Netherlands), and Dr. Maarten Honing
(AKZO-Nobel Organon, Oss, the Netherlands).
Wilfried Niessen
1998
© 2006 by Taylor and Francis Group, LLC
PREFACE TO THE FIRST EDITION
In the early 1970s several groups started research projects aiming at the
development of the on-line coupling of liquid chromatography and mass
spectrometry (LC–MS). These research efforts were mainly inspired by the great
success of combined capillary gas chromatography mass spectrometry (GC–MS) in
solving analytical problems. However, the development of on-line LC–MS turned
out to be a demanding and challenging task. In the past 20 years many approaches to
LC–MS have been described. Some of these are successful and commercially
available. LC–MS is no longer a highly sophisticated technique being used in
laboratories of specialists only. LC–MS has grown to become a mature and routinely
used technique in many areas of applications. LC–MS still is a rapidly developing
technique, expanding its analytical power and attracting more and more users.
In a period of rapid developments, this book on LC–MS is written. The core of

this book is therefore focussed more on principles and strategies than on reviewing
applications. All aspects of LC–MS are covered in this comprehensive review,
giving a survey of the field from various angles and both for newcomers and
experienced users. For the newcomers, the text affords a comprehensive introduction
and review of all important aspects in LC–MS interfacing. Experienced users will
find an extensive review of the various aspects, and perhaps some new viewpoints
and inspiration for new experiments to develop and optimize LC–MS. Since the
field of LC–MS is moving extremely fast, some of the chapters will unfortunately
need updating on appearance of this volume. This is certainly true for the Ch. 9 and
10. In principle, all literature available to us by the end of 1990 is incorporated in
this text. In some chapters, some later appeared papers have been included, either by
brief mention in the text or in the applications tables and review.
This text is written from the 'true hybrid' philosophy on LC–MS. For that reason,
concise introductions in liquid chromatography (Ch. 1) as well as mass spectrometry
(Ch. 2) precede a general discussion on interfacing chromatography and mass
spectrometry (Ch. 3). Subsequently, the various interfaces for LC–MS are discussed
from a technological point of view. After a historical overview, in which all
approaches to on-line LC–MS are discussed (Ch. 4), the commercially available and
therefore most widely applied LC–MS interfaces are discussed, i.e., the moving-belt
interface (Ch. 5), direct liquid introduction (Ch. 6), thermospray (Ch. 7), continuous-
flow fast atom bombardment (Ch. 8), particle-beam interfaces (Ch. 9) and
electrospray and related methods (Ch. 10). Developments in combining supercritical
© 2006 by Taylor and Francis Group, LLC
fluid chromatography and capillary electrophoresis to mass spectrometry are
reviewed as well (Ch. 11 and 12) to fit LC–MS in the whole analytical framework of
separation methods coupled with mass spectrometry. Next, the field of LC–MS is
approached from the ionization point of view. Attention is paid to specific aspects of
ionization under LC–MS conditions. In this respect, attention is paid to electron
impact ionization (Ch. 13), chemical ionization (Ch. 14), ion evaporation (Ch. 15),
and fast atom bombardment (Ch. 16), while a chapter on various ways to induce

fragmentation (Ch. 17) closes the section on ionization. The third angle on LC–MS
is from the application point of view. Applications from the fields of environmental,
pharmaceutical and biochemical analysis as well as the analysis of natural products
are discussed (Ch. 18), not to provide in-depth information in that particular field of
application, but from a general analytical point of view, allowing the comparison of
the different interfaces and the assessment of applicability ranges of the various
LC–MS interfaces. Finally, LC–MS is considered as a hybrid technique. First, some
aspects related to mobile phase compatibility problems are reviewed (Ch. 19). Then,
LC–MS is considered from a general point of view. The various experimental
parameters related to the separation, the interface, the ionization, and the mass
analysis as well as aspects related to data handling are considered from the hybrid
point of view. Developments in the various fields, that are combined in LC–MS as a
hybrid technique, are reviewed. Important areas of future research are indicated (Ch.
20). Each chapter is written as a separate unit, that can be read apart from the other
chapters, while extensive cross-referencing is provided.
Finally, this text could not have been completed without the inspiration, research
activities, help and advice from many of the people in our laboratories at the Leiden
University (Center for Bio-Pharmaceutical Sciences) and the department of structure
elucidation and instrumental analysis at TNO. We would like to thank especially
U.R. Tjaden, C.E.M. Heeremans, E.R. Verheij, R.A.M. van der Hoeven, P.S.
Kokkonen, A.C. Tas, G.F. La Vos, L.G. Gramberg, M.C. ten Noever de Brauw, A.P.
Tinke, D.C. van Setten, J.J. Pot and his people at the photography and drawing
department of the Gorlaeus Laboratories, Ms. P. Jousma-de Graaf and M. van der
Ham-Meijer.
Wilfried Niessen
Jan van der Greef
August 1991
© 2006 by Taylor and Francis Group, LLC
CONTENTS
ABBREVIATIONS

INTRODUCTION
Ch. 1 Liquid chromatography and sample pretreatment 3
Ch. 2 Mass spectrometry 23
TECHNOLOGY
Ch. 3 Strategies in LC–MS interfacing 53
Ch. 4 History of LC–MS interfaces 73
Ch. 5 Interfaces for atmospheric-pressure ionization 105
Ch. 6 Atmospheric-pressure ionization 141
APPLICATIONS: SMALL MOLECULES
Ch. 7 LC–MS analysis of pesticides 179
Ch. 8 Environmental applications of LC–MS 215
Ch. 9 LC–MS in drug discovery and development 233
Ch. 10 LC–MS in drug metabolism studies 257
Ch. 11 Quantitative bioanalysis using LC–MS 289
Ch. 12 Clinical applications of LC–MS 331
Ch. 13 LC–MS analysis of steroids 359
Ch. 14 LC–MS in food safety analysis 381
Ch. 15 LC–MS analysis of plant phenols 413
APPLICATIONS: BIOMOLECULES
Ch. 16 LC–MS analysis of proteins 441
Ch. 17 LC–MS analysis of peptides / Enabling technologies 463
Ch. 18 LC–MS in proteomics 493
Ch. 19 LC–MS for identification of post-translational modifications 523
Ch. 20 LC–MS analysis of oligosaccharides 545
Ch. 21 LC–MS analysis of lipids and phospholipids 565
Ch. 22 LC–MS analysis of nucleic acids 583
© 2006 by Taylor and Francis Group, LLC
ABBREVIATIONS
2D two-dimensional
ADME adsorption, distribution, metabolism and excretion

AES atomic emission spectrometry
AfC affinity chromatography
ALS acid-labile surfactant
AmOAc ammonium acetate
ANIS analogue internal standard
APCI atmospheric-pressure chemical ionization
API atmospheric-pressure ionization
APPI atmospheric-pressure photoionization
BIRD black-body infrared radiative dissociation
BLAST Basic Local Alignment Search Tool
BSA bovine serum albumin
CE capillary electrophoresis
Cf-FAB continuous-flow fast-atom bombardment
CI chemical ionization
CID collision induced dissociation
CIEF capillary isoelectric focussing
CYP cytochrome P450 complex
DAD photodiode array detection
DCI direct chemical ionization
DDA data-dependent acquisition
DLI direct liquid introduction
ECD electron-capture dissociation
ECNI electron-capture negative ionization
EDC endocrine disrupting compound
EHI electrohydrodynamic ionization
EI electron ionization
ELSD evaporative light scattering detection
ESA electrostatic analyser
ESI electrospray ionization
FAB fast-atom bombardment

FAC frontal affinity chromatography
FAIMS high-field asymmetric-waveform ion-mobility spectroscopy
© 2006 by Taylor and Francis Group, LLC
FD field desorption ionization
FT-ICR-MS Fourier-transform ion-cyclotron resonance mass spectrometry
FWHM full-width at half maximum
GC gas chromatography
GE gel electrophoresis
H/D hydrogen/deuterium exchange
HFBA heptafluorobutyric acid
HILIC hydrophilic interaction chromatography
HPAEC high-performance anion-exchange chromatography
IAC immunoaffinity chromatography
ICAT isotope-coded affinity tag
ICP inductively coupled plasma
ID internal diameter
IEC ion-exchange chromatography
IEF isoelectric focussing
IEV ion evaporation ionization
ILIS isotope-labelled internal standard
IMAC immobilized metal-ion affinity chromatography
IMER immobilized enzyme reactor
IRMPD infrared multiphoton dissociation
IS internal standard
LC liquid chromatography
LCxLC comprehensive liquid chromatography
LINAC linear acceleration collision cell
LIT linear ion trap
LLE liquid-liquid extraction
LOQ lower limit of quantification

MAGIC monodisperse aerosol generation interface for chromatography
MALDI matrix-assisted laser desorption ionization
MBI moving-belt interface
MRL maximum residue level
MS mass spectrometry
MS-MS tandem mass spectrometry
MSPD matrix solid-phase dispersion
MTBE methyl-t-butyl ether
MudPIT multidimensional protein identification technology
MUX multiplexed electrospray interface
NMR nuclear magnetic resonance spectroscopy
PAGE polyacrylamide gel electrophoresis
PBI particle-beam interface
PBMC peripheral blood mononuclear cells
PD plasma desorption ionization
PEG poly(ethylene) glycol
PFK perfluorokerosene
PFTBA perfluorotributylamine
PMF peptide mass fingerprinting
© 2006 by Taylor and Francis Group, LLC
PPG poly(propylene) glycol
PSA peptide sequence analysis
PS-DVB poly(styrene–divinylbenzene)
PTM post-translational modification
Q-LIT quadrupole-linear-ion-trap hybrid
Q-TOF quadrupole-time-of-flight hybrid
RAM restricted-access material
RF radiofrequency
RPLC reversed-phase liquid chromatography
S/N signal-to-noise ratio

SALSA scoring algorithm for spectral analysis
SBSE stir-bar sorptive extraction
SCX strong cation-exchange chromatography
SDS sodium dodecylsulfate
SEC size exclusion chromatography
SFC supercritical fluid chromatography
SILAC stable isotope labelling with amino acids in cell cultures
SIM selected-ion monitoring
SIMS secondary-ion mass spectrometry
SNP single nucleotide polymorphisms
SORI sustained off-resonance irradiation
SPE solid-phase extraction
SPME solid-phase microextraction
SRM selected-reaction monitoring
SS-LLE solid-supported liquid-liquid extraction
STP sewage treatment plant
TAG triacylglycerides
TCA trichloroacetic acid
TDM therapeutic drug monitoring
TFA trifluoroacetic acid
TFC turbulent flow chromatography
TMT tandem mass tags
TOF time-of-flight mass analyser
TSP thermospray ionization
UV ultraviolet detection
© 2006 by Taylor and Francis Group, LLC
INTRODUCTION
© 2006 by Taylor and Francis Group, LLC
3
1

LIQUID CHROMATOGRAPHY
AND SAMPLE PRETREATMENT
1. Introduction 3
2. Instrumentation for liquid chromatography 4
3. Separation mechanisms 9
4. Other modes of liquid chromatography 12
5. Sample pretreatment strategies 15
6. References 21
1. Introduction
Chromatography is a physical separation method in which the components to be
separated are selectively distributed between two immiscible phases: a mobile phase
is flowing through a stationary phase bed. The technique is named after the mobile
phase: gas chromatography (GC), liquid chromatography (LC), or supercritical fluid
chromatography (SFC). The chromatographic process occurs as a result of repeated
sorption/desorption steps during the movement of the analytes along the stationary
phase. The separation is due to the differences in distribution coefficients of the
individual analytes in the sample. Theoretical and practical aspects of LC have been
covered in detail elsewhere [1-5].
This chapter is not meant to be a short course in LC. Some aspects of LC,
important in relation to combined liquid chromatography–mass spectrometry
(LC–MS), are discussed, e.g., column types and miniaturization, phase systems and
separation mechanisms, and detection characteristics. In addition, important sample
pretreatment techniques are discussed. Special attention is paid to new developments
in LC and sample pretreatment.
© 2006 by Taylor and Francis Group, LLC
Ch. 14
2. Instrumentation for liquid chromatography
In LC, the sample is injected by means of an injection port into the mobile-phase
stream delivered by the high-pressure pump and transported through the column
where the separation takes place. The separation is monitored with a flow-through

detector. In designing an LC system, one has to consider a variety of issues:
C The separation efficiency is related to the particle size of the stationary phase
material. A higher pressure is required when the particle size is reduced. With a
typical linear velocity in the range of 2–10 mm/s, a pressure drop over the
column can exceed 10 MPa, obviously depending on the column length as well.
C In order to maintain the resolution achieved in the column, external peak
broadening must be reduced and limited as much as possible. In general, a 5%-
loss in resolution due to external peak broadening is acceptable. In practice, this
means that with a 3–4.6-mm-ID column, a 20-µl injection volume and a 6–12 µl
detector cell volume can be used in combination with short, small internal-
diameter connecting tubes. Avoiding external peak broadening is especially
important when the column internal diameter is reduced [6].
C The quality of the solvents used in the mobile phase is important in LC–MS.
Phthalates and other solvent contaminants can cause problems [7]. Appropriate
filtering of the solvents over a 0.2–0.4-µm filter is required. Degassing of the
mobile phase is required to prevent air bubble formation in the pump heads, but
also in interface capillaries.
Table 1.1:
Characteristics of LC columns with various internal diameters
Type ID
(mm)
F (µl/min) V
inj
(µl) C
max
at
detector
1
Relative
loading

capacity
2
Conventional 4.6 1000 100 1 8333
Narrowbore 2.0 200 19 5.3 1583
Microbore 1.0 47 4.7 21.2 392
Microcapillary 0.32 4.9 0.49 207 41
Nano-LC 0.05 0.120 0.012 8464 1
1
Based on column ID;
2
Based on given injection volume.
© 2006 by Taylor and Francis Group, LLC
Liquid chromatography and sample pretreatment 5
C High-throughput LC–MS analysis demand for high-pressure pumps capable of
delivering an accurate, pulse-free, and reproducible and constant flow-rate. A
small hold-up volume is needed for fast gradient analysis. High-pressure mixing
devices are to be preferred. Modern LC pumps feature advanced electronic
feedback systems to ensure proper functioning and to enable steep solvent
gradients.
C Injection valves with an appropriate sample loop volume, mainly determined by
the external peak broadening permitted, are used. Reduction of sample memory
and carry-over is an important aspect, especially in quantitative analysis. Modern
autosamplers allow a more versatile control over the injection volume by the
application of partially filled loops and enable reduction of carry-over by needle
wash steps.
2.1 The column
The column is the heart of the LC system. It requires appropriate care.
Conventionally, LC columns are 100–300-mm long and have an internal diameter of
3–4.6 mm with an outer diameter of
1

/
4
inch. In LC–MS, and especially in
quantitative bioanalysis, shorter column are used, e.g., 30–50 mm, and packed with
3–5 µm ID packing materials. A variety of other column types, differing in column
inner diameter, are applied. Some characteristics of these columns are compared in
Table 1.1.
The microcapillary packed and nano-LC columns are made of 0.05–0.5-mm-ID
fused-silica tubes. The packing geometry of these columns differs from that of a
larger bore column, resulting in relatively higher column efficiencies. These type of
columns are frequently used in LC–MS applications with sample limitations, e.g., in
the characterization of proteins isolated from biological systems.
With respect to packing geometry and column efficiency, microbore columns are
equivalent to conventional columns, except with respect to the internal diameter.
Since most electrospray (ESI) interfaces are optimized for operation with flow-rates
between 50 and 200 µl/min, the use of 1–3-mm-ID microbore columns is
advantageous, because no post-column solvent splitting is required.
Asymmetric peaks can have a number of causes: overloading, insufficient
resolution between analyte peaks, unwanted interactions between the analytes and
the stationary phase, e.g., residual silanol groups, voids in the column packing, and
external peak broadening.
In most cases, the use of a guard column is advised. It is placed between the
injector and the analytical column to protect the latter from damage due to the
injection of crude samples, strong adsorbing compounds, or proteins in biological
samples that might clog the column after denaturation. In this way, the performance
and lifetime of the expensive analytical column can be prolonged. Guard columns
inevitably result in a loss of efficiency.
© 2006 by Taylor and Francis Group, LLC
Ch. 16
2.2 General detector characteristics

The detector measures a physical parameter of the column effluent or of
components in the column effluent and transforms it to an electrical signal. A
universal detector measures a bulk property of the effluent, e.g., the refractive index,
while in a specific detector only particular compounds contribute to the detector
signal.
A detector can be either a concentration sensitive device, which gives a signal
that is a function of the concentration of an analyte in the effluent, or a mass-flow
sensitive device, where the signal is proportional to the mass flow of analyte, i.e., the
concentration times the flow-rate.
The analyte concentration at the top of the chromatographic peak c
max
is an
important parameter, related to the dilution in the chromatographic column. It can be
related to various chromatographic parameters:
where M is the injected amount, N is the plate number of the column with an internal
diameter d
c
, a length L, and a column porosity
g
, and k’ is the capacity ratio of the
analyte. Guided by this equation, a particular detection problem can be approached
by optimizing the separation parameters, e.g., amount injected, column diameter,
plate number, and capacity ratio. It also is an important equation in appreciating the
use of miniaturized LC column.
Other important characteristics of a detector for LC are:
C The noise, which is the statistical fluctuation of the amplitude of the baseline
envelope. It includes all random variations of the detector signal. Noise generally
refers to electronic noise, and not to the so-called 'chemical noise', although the
latter generally is far more important in solving real-life analytical problems.
C The detection and determination limits, which are generally defined in terms of

signal-to-noise ratios (S/N), e.g., an S/N of 3 for the detection limit and of 5–10
for the determination limit of lower limit of quantification.
C The linearity and linear dynamic range. A detector is linear over a limited range
only. In ESI-MS, the linearity is limited inherent to the ionization process. A
linear dynamic range of at least 2–3 order of magnitude is desirable.
C The detector time constant. The detector must respond sufficiently fast to the
changes in concentration or mass flow in the effluent, otherwise the peaks are
distorted.
© 2006 by Taylor and Francis Group, LLC
Liquid chromatography and sample pretreatment 7
2.3 Detectors for LC
Next to the mass spectrometer, which obviously is considered being the most
important LC detector in this text, a number of other detectors [8] are used in
various applications:
C The UV-absorbance detector is the most widely used detector in LC, which is a
specific detector with a rather broad applicability range. The detection is based
on the absorption of photons by a chromophore, e.g., double bonds, aromatic
rings, and some hetero-atoms. According to the equation of Lambert-Beer, the
UV detector is a concentration-sensitive device.
C The fluorescence detector is a specific and concentration-sensitive detector. It is
based on the emission of photons by electronically excited molecules.
Fluorescence is especially observed for analytes with large conjugated ring
systems, e.g., polynuclear aromatic hydrocarbons and their derivatives. In order
to extend its applicability range, pre-column or post-column derivatization
strategies have been developed [9].
C Evaporative light-scattering detection (ELSD) is a universal detector based on
the ability of particles to cause photon scattering when they traverse the path of a
polychromatic beam of light. The liquid effluent from an LC is nebulized. The
resulting aerosol is directed through a light beam. The ELSD is a mass-flow
sensitive device, which provides a response directly proportional to the mass of

the non-volatile analyte. Because it can detect compounds that are transparent to
other detection techniques, the ELSD is frequently used in conjunction with
LC–MS to obtain a complete analysis of the sample [10].
C Nuclear magnetic resonance spectroscopy (NMR) coupling to LC has seen
significant progress in the past five years [11]. Continuous-flow NMR probes
have been designed with a typical detection volume of 40–120 µl or smaller. The
NMR spectrum is often recorded in stop-flow mode, although continuous-flow
applications have been reported as well.
C An inductively-coupled plasma (ICP) is an effective spectroscopic excitation
source, which in combination with atomic emission spectrometry (AES) is
important in inorganic elemental analysis. ICP was also considered as an ion
source for MS. An ICP-MS system is a special type of atmospheric-pressure ion
source, where the liquid is nebulized into an atmospheric-pressure spray
chamber. The larger droplets are separated from the smaller droplets and drained
to waste. The aerosol of small droplets is transported by means of argon to the
torch, where the ICP is generated and sustained. The analytes are atomized, and
ionization of the elements takes place. Ions are sampled through an orifice into
an atmospheric-pressure–vacuum interface, similar to an atmospheric-pressure
ionization system for LC–MS. LC–ICP-MS is extensively reviewed, e.g., [12].
© 2006 by Taylor and Francis Group, LLC
Ch. 18
Table 1.2:
Separation mechanisms in LC
adsorption selective adsorption/desorption on a solid phase
partition selective partition between two immiscible liquids
ion-exchange differences in ion-exchange properties
ion-pair formation of ion-pair and selective partition or sorption of
these ion-pairs
gel permeation /
size exclusion

differences in molecular size, or more explicitly the ability
to diffuse into and out of the pore system
Table 1.3:
Phase systems in various LC modes
Mechanism Mobile phase Stationary phase
adsorption
(normal-phase)
apolar organic solvent with
organic modifier
silica gel, alumina
bonded-phase material
adsorption
(reversed-phase)
aqueous buffer with organic
modifier, e.g., CH
3
OH or
CH
3
CN
bonded-phase material,
e.g., octadecyl-modified
silica gel
ion-pair aqueous buffer with organic
modifier and ion-pairing
agent
reversed-phase bonded-
phase material
partition liquid, mostly nonpolar liquid, physically coated
on porous solid support

ion exchange aqueous buffers cationic or anionic
exchange resin or
bonded-phase material
size exclusion non-polar solvent silica gel or polymeric
material
© 2006 by Taylor and Francis Group, LLC
Liquid chromatography and sample pretreatment 9
3. Separation mechanisms
A useful classification of the various LC techniques is based on the type of
distribution mechanism applied in the separation (see Table 1.2). In practice, most
LC separations are the result of mixed mechanisms, e.g., in partition
chromatography in most cases contributions due to adsorption/desorption effects are
observed. Most LC applications are done with reversed-phase LC, i.e., a nonpolar
stationary phase and a polar mobile phase. Reversed-phase LC is ideally suited for
the analysis of polar and ionic analytes, which are not amenable to GC analysis.
Important characteristics of LC phase systems are summarized in Table 1.3.
3.1 Intra- and intermolecular interactions
Various intra- and intermolecular interactions between analyte molecules and
mobile and stationary phase are important in chromatography [5] (Figure 1.1):
C The covalent bond is the strongest molecular interaction (200–800 kJ/mol). It
should not occur during chromatography, because irreversible adsorption and/or
damage to the column packing material takes place.
C Ionic interactions between two oppositely charged ions is also quite strong
(40–400 kJ/mol). Such interactions occur in ion-exchange chromatography,
which explains the sometimes rigorous conditions required for eluting analytes
from an ion-exchange column.
C Ions in solution will attract solvent molecules for solvation due to ion-dipole
interactions (4–40 kJ/mol).
Figure 1.1: Intra- and intermolecular interactions important in chromatography.
Based on [5].

© 2006 by Taylor and Francis Group, LLC
Ch. 110
Table 1.4:
Interactions between analytes and stationary phase packing materials.
(
Ø Primary Interaction; Ï Secondary Interaction; e Silanol Activity)
Packing
Non-
polar
Polar Anion Cation
Exchange
Octadecyl (C
18
), octyl (C
8
), and
phenyl (–C
6
H
5
)
ØÏ e
Ethyl (C
2
), cyano (–C=N), and
diol (2× – OH)
ØØ e
Silica (–Si–OH) Øe
Amino (–NH
2

), and
diethylaminopropyl (DEA)
ÏØ Ø e
Quaternary Amine (SAX) ÏÏ Ø e
Carboxylic Acid (CBA) ÏÏ Ø
Benzenesulfonic (SCX) ØÏ Ø
C
The hydrogen atom can interact between two electronegative atoms, either within
one or between two molecules. Hydrogen bonding can be considered as an
important interaction (4–40 kJ/mol) between analyte molecules and both the
mobile and the stationary phase in LC. In reversed-phase LC, both water and
methanol can act both as acceptor and donor in hydrogen bonding, while
acetonitrile can only accept, not donate.
C The third type of medium-strong interaction (4–40 kJ/mol) is the Van der Waals
interaction, which are short-range interactions between permanent dipoles, a
permanent dipole and the dipoles induced by it in another molecule, and
dispersive forces between neutral molecules.
C Weaker interactions (0.4–4 kJ/mol) are longer range dipole–dipole and
dipole–induced dipole interactions.
Alternatively, intermolecular interactions can be classified as:
C polar interactions, where hydrophilic groups like hydroxy, primary amine,
carboxylic acid, amide, sulfate or quaternary ammonium groups are involved.
C nonpolar interactions, where hydrophobic groups like alkyl, alkylene, and
aromates are involved.
C nonpolar interactions were carbonyl, ether, or cyano groups are involved.
C ionic interactions, i.e., between cations and anions.
Along these lines, the interactions in various column packing materials can be
classified (Table 1.4). The most important LC modes are briefly described below.
© 2006 by Taylor and Francis Group, LLC