FULLTEXT01

DETERMINATION OF BIOMARKERS FOR
LIPID PEROXIDATION AND OXIDATIVE STRESS
− Development of analytical techniques and methods −
Kristina Claeson Bohnstedt
Doctoral Thesis
Department of Analytical Chemistry
Stockholm University
Stockholm, 2005
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Doctoral Dissertation, 2005
Department of Analytical Chemistry
Stockholm University
106 91 Stockholm
Akademisk avhandling som för framläggande av filosofie doktorsexamen vid

Stockholms universitet offentligen försvaras i Magnélisalen, Kemiska
övningslaboratoriet, Svante Arrhenius väg 12, torsdagen den 27 januari 2005.
ISBN 91-7265-988-2
© Kristina Claeson Bohnstedt, 2005
Intellecta DocuSys AB, Sollentuna
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Table of contents
Abstract
Preface - List of papers
Part One
1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3 What is a biomarker? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4 Biological samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.1 Brain tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.2 Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.3 Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.4 Cerebrospinal fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5 Lipid peroxidation biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.1 Aldehydes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.1.1 MDA - formation, properties and analysis . . . . . . . . . . . . . . . 20
5.1.2 Hydroxynonenal - formation, properties and analysis . . . . . . 22
5.2 Isoprostanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
5.2.1 Isoprostanes - formation, properties and analysis . . . . . . . . . . 23
Part Two
6 Preparation of biological samples for CE or LC . . . . . . . . . . . . . . . . . . 29
6.1 Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
6.2 Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
6.3 Liquid-liquid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
6.4 Solid phase extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
6.5 Column switching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
6.6 Derivatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
6.7 Other techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
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7 Capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
7.1 Introduction to CE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
7.2 Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
7.3 In capillary sample concentration . . . . . . . . . . . . . . . . . . . . . . . . . 39
7.3.1 Field amplified stacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
7.3.2 Isotachophoretic stacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
7.4 Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
7.5 Detection in CE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7.6 Qualitative and quantitative aspects in CE . . . . . . . . . . . . . . . . . . 44
8 LC-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
8.1 The effect of column diameter in LC . . . . . . . . . . . . . . . . . . . . . . 46
8.2 Porous graphitic carbon as a packing material in LC . . . . . . . . . 48
8.2.1 Manufacture and structure of PGC . . . . . . . . . . . . . . . . . . . . . 48
8.2.2 Performance of PGC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.3 ESI as an ionization technique for MS detection . . . . . . . . . . . . . 51
8.4 Ion supression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
8.5 PGC and ESI-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
8.6 Qualitative and quantitative aspects in ESI-MS . . . . . . . . . . . . . 56
Part Three
9 Summary of the papers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
9.1 Paper I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
9.2 Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
9.3 Paper III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
9.4 Paper IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
9.5 Paper V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
9.6 Unpublished results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
10 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
11 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
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Abstract
Oxidative stress can be defined as a state of disturbance in the pro-oxidant/
antioxidant balance in favour of the former, leading to potential damage. Pro-
cesses associated with oxidative stress involve reactive oxygen species and
radicals and can result in elevated levels of oxidatively modified or toxic mol-
ecules that can cause cellular malfunction, and even cell death. Destruction of
membrane lipids, lipid peroxidation, caused by reactive oxygen species and
radicals has been coupled to many diseases and also normal ageing.
The measurement of low molecular weight biomarkers of lipid peroxidation

present in complex matrices such as brain tissue, plasma, urine or cerebrospi-
nal fluid is a delicate and difficult task and there is a need for improved analyti-
cal tools in this field of research.
The major foci of this thesis and the work underlying it are the development of
analytical techniques and methods for determining biomarkers for oxidative
stress and lipid peroxidation. Aspects of particular concern include the effects
of sample treatments prior to analysis, evaluation of the developed methods
with respect to possible artefacts, and the scope for results to be misinterpreted.
The specific research goals and issues addressed are detailed in five papers,
which this thesis is based upon.
Paper I focuses on malondialdehyde, describing and evaluating two new sim-

plified sample pre-treatment regimes for the determination of malondialdehyde
in rat brain tissue by capillary electrophoresis with UV detection. The effects
of sample storing and handling are also considered.
Paper II describes the synthesis, characterization and implementation of a new

internal standard for the determination of malondialdehyde in biological samples
using electrophoretic or chromatographic separation techniques. The useful-
ness of the internal standard is demonstrated in analyses of rat brain tissue
samples.
5
Paper III presents a method for the determination of 4-hydroxynon-2-enal in
brain tissue from rats employing micellar electrokinetic chromatography sepa-
ration and laser-induced fluorescence detection.
Paper IV is focused on the development of a new methodology for determin-

ing the stereoisomeric F2-isoprostanes in human urine samples employing chro-
matographic separation on porous graphitic carbon and detection by electrospray
ionization-tandem mass spectrometry. The results from this study conflict with
the hypothesis that peripheral isoprostanes are elevated in patients with
Alzheimer’s disease.
Paper V describes porous graphitic carbon chromatography-tandem mass spec-

trometry for the determination of isoprostanes in human cerebrospinal fluid. A
new simplified sample pre-treatment regime, involving a column switching
technique, is presented that allows direct injection of a relatively large volume
of CSF into the chromatographic system.
6
Preface
This thesis is divided into three parts. The first presents an introduction to
oxidative stress, and then discusses types of biological samples and compounds
that have been used to monitor such stress and to elucidate associated pro-
cesses. The second is dedicated to the analytical tools used in the studies un-
derlying the thesis, and summarises the results obtained. Finally, the third part
discusses insights into oxidative stress provided by these and previous investi-
gations and issues that remain to be clarified.
This thesis is based upon the following publications
I Kristina Claeson, Fredrik Åberg, Bo Karlberg.

Free malondialdehyde determination in rat brain tissue by capillary
zone electrophoresis: evaluation of two protein removal procedures.
Journal of Chromatography B, 740 (2000) 87-92.
The author was responsible for the idea, the laboratory work, data evalu-
ation and writing of this paper.
II Kristina Claeson, Gunnar Thorsén, Bo Karlberg.

Methyl malondialdehyde as an internal standard for the determination of
malondialdehyde.
The author was responsible for the idea, the laboratory work (in collabo-
ration with Gunnar Thorsén), data evaluation (except for the NMR and
MS data) and writing of this paper.
III Kristina Claeson, Gunnar Thorsén, Bo Karlberg.

Micellar electrokinetic chromatography separation and laser induced fluo-
rescence detection of the lipid peroxidation product 4-hydroxynonenal.
The author and Gunnar Thorsén contributed equally to the laboratory work,
data evaluation and writing of this paper.
7
IV Kristina Claeson Bohnstedt, Bo Karlberg, Lars-Olof Wahlund,
Maria Eriksdotter Jönhagen, Hans Basun, Staffan Schmidt.
Determination of isoprostanes in urine samples from Alzheimer patients
using porous graphitic carbon liquid chromatography-tandem mass spec-
trometry.
The author and Staffan Schmidt were jointly responsible for the idea, and
the author was responsible for the laboratory work, data evaluation and
writing of this paper.
V Kristina Claeson Bohnstedt, Bo Karlberg, Hans Basun,

Staffan Schmidt.
Porous graphitic carbon chromatography-tandem mass spectrometry for
the detection of isoprostanes in human cerebrospinal fluid
Journal of Chromatography B, submitted.
The author and Staffan Schmidt were jointly responsible for the idea, and
the author was responsible for the laboratory work, data evaluation and
writing of this paper.
Paper not included in this thesis
Joanna Olsson, Kristina Claeson, Bo Karlberg, Ann-Caroline

Nordström.
Determination of indole-3-acetic acid and indole-3-acetylaspartic acid in
pea plant with capillary electrophoresis and fluorescence detection.
Journal of Chromatography A, 796 (1998) 231-239.
8
10
1. Aim
The aim of the studies this thesis is based upon was to develop improved
methodology for the determination of selected biological markers of lipid
peroxidation using modern analytical instruments and microseparation tech-
niques. Such methods should ideally be fast and simple, keeping the num-
ber of steps in the analytical process at a minimum. Aspects of particular
concern in both the experimental work and the thesis include the effects of
sample treatments prior to analysis, evaluation of the developed methods
with respect to possible artefacts, the scope for results to be misinterpreted,
and other potential pitfalls when studying biological markers of lipid
peroxidation in biological samples.
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2. Introduction
Life processes, especially under stress conditions, generate a wide range of
potentially damaging by-products as well as useful metabolites. Oxidants pro-
duced in this way, often free radicals, can cause extensive damage to DNA,
proteins and lipids. Oxidative stress occurs when more oxidant by-products are
produced than the cell can defend itself against. Oxidative stress can, therefore,
occur when the production of free radicals increases, when quenching of free
radicals or repair of damaged macromolecules decreases, or when these changes
occur simultaneously. Sies similarly defined the process as “a disturbance in
the pro-oxidant-antioxidant balance in favour of the former, leading to poten-
tial damage” [1]. Processes associated with oxidative stress can result in an
elevated level of oxidatively modified or toxic molecules that can cause cellu-
lar malfunction and death. Oxidative stress and destruction caused by radicals
have been coupled to many diseases and also normal ageing.
Oxygen-dependent deterioration of fats and oils, also called rancidity, causes

problems when storing these substances and has been known since ancient
times. In more recent times, it has been concluded that lipid peroxidation is one
of the main processes induced by oxidative stress in vivo. Lipid peroxidation,
i.e. the oxidative destruction of polyunsaturated fatty acids (PUFAs), is an au-
tocatalytic, uncontrolled process leading to the formation of fatty acid hydrop-
eroxides. These primary lipid peroxidation products are unstable and trans-
form into more stable secondary products that can be used as biomarkers for
the process. Animal cell membranes are prone to lipid peroxidation since they
contain, among other constituents, the unsaturated fatty acids linoleic acid (18:2),
linolenic acid (18:3), arachidonic acid (20:4) and docohexaenoic acid (22:6).
Detailed definitions of key terms like oxidative stress, oxidative damage and
antioxidants can be found in a recent review by Halliwell and Whiteman [2].
The analytical challenges involved in determining minute concentrations of

analytes in complex biologically relevant samples are well known. During lipid
12
peroxidation a multitude of degradation products are formed and, as will be
discussed later, the samples are often very sensitive to harsh treatments. Mea-
suring lipid peroxidation in biological samples is, therefore, a delicate and dif-
ficult task. Improved analytical tools, enabling reliable determinations of
biomarkers associated with lipid peroxidation are urgently required in order to
investigate and elucidate the fundamental mechanisms underlying this process.
13
3. What is a biomarker?
The word biomarker is widely employed and quoting Naylor may give insight
into the extent of its usage; “[biomarker is]…an umbrella coalescence term
which covers the usage and development of tools and technologies, monitoring
drug discovery and development and understanding the prediction, causes, pro-
gression, regression, outcome, diagnosis and treatment of disease…“ [3].
A commonly used definition is the one proposed in 2001 by the Biomarkers
Definitions Working Group from the National Institute of Health and the
Food and Drug Administration (NIH/FDA) in the USA [4]: “A characteris-
tic that is objectively measured and evaluated as an indicator of normal
biological processes, pathogenic processes, or pharmacologic responses to
a therapeutic intervention”.
Broadly, there are three types of biomarkers: (1) disease biomarkers – used to
monitor and diagnose the progression of a disease; (2) drug efficacy/toxicity
biomarkers – used to monitor the efficacy or toxicity of a treatment regime;
and (3) pharmacodynamic markers for monitoring pharmacological responses.
A biomarker needs to be validated for sensitivity, specificity and reproducibil-
ity, and to be evaluated with respect to clinical endpoints, i.e. how patients feel,
function and/or survive when changes in the level of the biomarker occur [5].
In 2004 Halliwell and Whiteman presented a list of criteria (shown in Table 1)

that an ideal biomarker of oxidative damage should fulfil [2]. The same article
also gives a comprehensive survey of the various methods and molecules used
to measure and study oxidative damage to DNA, lipids and proteins.
14
Table 1 Criteria for an ideal biomarker of oxidative damage
A) Core criterion
The biomarker must be predictive for later development of disease.
B) Technical criteria
(i) The biomarker should detect a major part, or at least a fixed
percentage of total ongoing oxidative damage in vivo.
(ii) The coefficient of variation between different assays of the
same sample should be small in comparison with the difference
between subjects.
(iii) Its levels should not vary widely in the same subjects under the
same conditions at different times.
(iv) It must employ chemically robust measurement technology.
(v) It must not be confounded by diet.
(vi) It should ideally be stable during storage, i.e. neither lost nor
formed artifactually in stored samples.
15
4. Biological samples
This thesis deals with a variety of biological matrices: brain tissue from
rats and human plasma, cerebrospinal fluid (CSF) and urine. All types of
samples are associated with specific analytical challenges depending on
their origin and composition. A common feature of all the selected analytes
is that they can be considered as small molecules, having molecular weights
below 400 Da. The sample matrices studied in this work and some guide-
lines on storing samples to prevent further decomposition of lipid sample
constituents are described below.
4.1 Brain tissue

The total lipid content of the brain is approximately 10 %. The membranes in
the brain are rich in polyunsaturated, highly peroxidable fatty acids. Oxygen
consumption in the living brain is proportionally greater than in many other
organs. In addition, brain tissue contains only moderate levels of both enzy-
matic and non-enzymatic quenchers for the reactive oxygen species. Due to the
high lipid content in brain tissue, these samples must be stored at - 70 °C and an
antioxidant such as butylated hydroxytoluene (BHT) should be added to the
samples promptly.
4.2 Plasma
Blood sampling is one of the most convenient techniques for population screen-
ing purposes since it is minimally invasive. Whole blood is composed of two
fractions. The blood plasma accounts for 55 % of the volume, and formed ele-
ments, i.e. cells and cell fragments, comprise the remaining 45 %. When the
formed elements are removed by centrifugation the plasma remains. Plasma
consists of about 91.5 % water, 7 % proteins and 0.5-1 % lipids, the balance
consisting of other solutes such as electrolytes, nutrients, gases, waste prod-
ucts, regulatory substances and vitamins. The storage of plasma samples has
the same requirements as brain tissue samples.
16
4.3 Urine
Collection of urine samples is feasible for large clinical trials as it is noninvasive.
At a normal water intake, an adult person excretes 1-2 l of urine every day. The
major urine components are water, urea, creatinine and sodium-, chloride- and
potassium ions in the 50-250 mM range. Urine only contains minor amounts of
proteins and negligible amounts of lipids. Urine is the primary medium for
excretion of water-soluble waste products and other species that have been
made water-soluble by metabolism. Compared to plasma and CSF, urine pro-
vides an integrated index of analyte production over time. Since urine does not
contain any significant levels of lipids there is no risk of artefactual generation
of lipid peroxidation products by decomposition of sample constituents. It has
even been shown that urinary isoprostane (see section 5.2) levels remain un-
changed after a 5-day period at 37 °C [6]. The requirements for storing urine
samples are thus less strict and samples can be kept at - 20 °C.
4.4 Cerebrospinal fluid

Cerebrospinal fluid, CSF, is the clear fluid that continuously circulates in the
subarachnoid space (the space between the skull and the cortex), the ventricu-
lar system of the brain and the spinal cord. It gives mechanical and chemical
protection and is a medium for exchange of nutrients and waste products be-
tween the blood and the nervous tissue. The total amount of CSF is about 150 ml,
and around 500 ml is produced every day, which indicates its very active circu-
lation. CSF is in many ways similar to an ultrafiltrate of plasma and has a
protein content below 430 mg/l. The CSF is a very useful matrix for studies of
the central nervous system since the fluid reflects the metabolic state of the
brain under both healthy and disease conditions. Usually, it is obtained by a
procedure called lumbar puncture. CSF samples should be stored at - 70 °C.
17
5. Lipid peroxidation biomarkers
The targets of attack in lipid peroxidation are membrane lipids that surround
cells and cell organelles. Fig. 1 shows a schematic diagram of the phospholipid
bilayer of the outer membrane of a cell. When the lipids in the bilayer are
changed oxidatively, not only are potentially harmful molecular species formed,
but also the membrane fluidity is affected, which may lead to changes in or loss
of cell functions or even cell death.
Cell Phospholipid bilayer
O
Phospholipid
O
H2C O CH3
H3C O
H2C PO4 Base
Figure 1. The outer membrane of a cell, comprising a 60-100 Å thick

phospholipid bilayer.
5.1 Aldehydes
The general lipid peroxidation process is illustrated in Fig. 2. The initiation of
lipid peroxidation starts with a free radical attack. Hydrogen is abstracted from
the target fatty acid (LH) to form a fatty acid radical (L·). The carbon radical is
usually stabilized by a molecular rearrangement to form a conjugated diene.
Then, a peroxyl radical (LOO·) is produced by oxygen uptake. The peroxyl
radical can abstract a hydrogen atom from another PUFA, thus forming a fatty
acid hydroperoxide (LOOH). Hence, a new oxidation chain is initiated, in which
new fatty acid radicals are generated. This is called the propagation stage of
lipid peroxidation. The lipid hydroperoxides formed can then decompose to
form, among other species, a great variety of aldehydes. Aldehydes are rela-
18
Part of fatty acid with three double bonds
LH Hydrogen abstraction
H
C
Rearrangement to
conjugated diene
L C
O2 Oxygen uptake
LOO O The peroxyl radical abstracts

O H H from another PUFA causing an
autocatalytic chain reaction.
O Lipid hydroperoxide Fragmentation to

LOOH O aldehydes e.g.
H Cyclic peroxide malondialdehyde and
hydroxynonenal.
Cyclic endoperoxide
Figure 2. Representation of the initiation and propagation reactions of

lipid peroxidation.
tively stable, compared with free radicals and hydroperoxides. This allows them
to diffuse within or out of a cell, and to reach locations remote from the site
where the initial free radical attack occurred. The stability of the aldehydes,
and the fact that they are always produced when lipid hyderoperoxides break
down in biological systems, make their identification and measurement valu-
able. Aldehydes can thus work as biomarkers, providing an index of the extent
of lipid peroxidation, and enable the role of aldehydes in specific pathological
conditions to be examined.
Among the many different aldehydes that can be formed, the most intensively
studied are malondialdehyde and 4-hydroxy-2-trans-nonenal [7]. Schauenstein
and Esterbauer, in the 1960s, were among the first to recognize the importance
of these aldehydes [8].
19
5.1.1 Malondialdehyde – formation, properties and analysis
Malondialdehyde (MDA) is a volatile, 1,3-dicarbonyl compound of low mo-
lecular weight (72.07 g/mole). It is a weak acid, since the pKa value of the
enolic OH group is 4.5. Hence, in neutral and alkaline conditions the predomi-
nant form is the enolate ion (Fig. 3). The molecule absorbs light in the UV
region in both acidic (λmax = 245 nm, ε ≈ 13000) and neutral or basic solutions
(λmax = 267 nm, ε ≈ 31000). MDA is said to originate from the oxidative de-
composition of fatty acids containing three or more double bonds, such as lino-
lenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6) [9].
O O pKa H H
4.5
H H O O
H
Figure 3. Malondialdehyde, MDA.
Under physiological conditions MDA is moderately reactive and it can act both
as an electrophile and as a nucleophile. It reacts with biomolecules containing
primary amino groups, such as proteins, nucleic acids and amino phospholip-
ids [9]. The correlation between MDA and various diseases is discussed in [10]
and its potential as a biomarker of oxidative damage to lipids in [2,10,11],
amongst other texts.
Theories relating to MDA formation and methods for its quantification have
been extensively discussed [7,9,12]. The techniques used for the determination
of MDA can be divided into two classes: derivative and direct. The most com-
mon methods utilize the reaction (heated, in acidic conditions) between MDA
and two molecules of thiobarbituric acid (TBA), which generates a red, fluo-
20
rescent product. This product is then measured, either directly by UV/Vis spec-
troscopy or after liquid chromatography (LC) separation employing UV or fluo-
rescence detection. The approach has several drawbacks, including a lack of
selectivity and harsh derivatization conditions. As early as 1958 it was shown,
in studies of fish oil, that 98 % of the MDA that reacts in the TBA-test was not
originally present in the sample but was formed by decomposition of lipid per-
oxides in the sample during the acid heating stage of the TBA assay [13,14].
The commonly recognised lack of selectivity of this assay, i.e. the fact that
several compounds other than MDA yield products with similar absorption
wavelengths on heating with TBA, has led to use of the term TBARS
(thiobarbituric acid reactive species) [15]. This expression should be used when
discussing results generated by this methodology. Apart from the TBA-based
methods other derivative methods employ gas chromatography (GC) with elec-
tron capture (EC) [16-18] or mass spectrometric (MS) detection [19-23]. Gen-
erally, the derivative methods have low limits of detection (LODs) but they
have inherent problems, including the use of time-consuming sample treat-
ments and the risk of producing MDA from unstable sample constituents dur-
ing the derivatization step. As mentioned previously, MDA is known to bind to
proteins [15,24]. For that reason, it is desirable to know whether a given method
determines the free, bound or total MDA content. When a crude sample is
exposed to harsh assay conditions, such as elevated temperatures or the addi-
tion of solutions with extreme pH values, changes in the relative amounts of
free and bound forms of MDA can be expected. To avoid this, direct MDA
analysis, performed under mild treatment conditions, is preferable, since it limits
shifts in the free and bound equilibrium and minimizes the risk of MDA gen-
eration from sample constituents during analysis. Direct analytical methods
include LC [25-28] and capillary electrophoresis (CE) [29], both in conjunc-
tion with UV-absorbance detection of the native molecule.
Some researchers claim that malondialdehyde is less commonly monitored

than it used to be. However, well over a thousand papers were published in
2004 in which “malondialdehyde” was listed as one of the key words.
21
There is quite a large range in the levels of MDA reported in the literature.
Depending on methodology and sample type, the reported values range from
zero up to high micromolar concentrations. For example, reported plasma lev-
els range from low nM to around 40 µM depending on methodology. MDA
was the target molecule in studies I and II.
5.1.2 Hydroxynonenal – formation of, properties and analytical methods

Fig. 4 illustrates the structure of 4-hydroxy-2-trans-nonenal (HNE), a mol-
ecule with a formula weight of 156.2. It absorbs light in the UV region, with a
λmax at 221 nm (ε ≈ 14000). HNE is a major product of the peroxidative decom-
position of ω6 PUFAs, such as linoleic acid (18:2) and arachidonic acid (20:4).
H
OH
Figure 4. 4–hydroxy-2-trans-nonenal, HNE.
The HNE aldehyde group, the CC double bond and the hydroxy group are all
able to take part in the chemical modification of biomolecules. HNE can react
rapidly with thiol and amino groups at physiological pH levels [30]. This may
be a significant factor in the claim that HNE is one of the most toxic substances
produced during lipid peroxidation. It possesses cytotoxic, hepatotoxic, mu-
tagenic and genotoxic properties [31]. It has also been hypothesized that HNE
was one of the toxic agents in the “Spanish cooking oil syndrome” [32]. More
recently, it has been suggested that HNE may not be merely a toxic product of
lipid peroxidation, but may also function as a biological signal substance in
both pathological and physiological conditions [33,34].
22
Several authors have described methods for the determination of HNE. These
techniques are mainly derivative, but at high levels (more than 2 µM), LC-UV
can be used on the native molecule [7]. At lower levels, derivatization has so
far been necessary to enable detection. Various hydrazine reagents have been
used to facilitate its detection for LC-UV [7,35], LC with electrochemical de-
tection [36] and GC-MS (after silylation) [37,38]. Several other methods have
also been described [39,40]. Another relevant way to estimate HNE levels is to
measure adducts to biomolecules using GC-MS or LC-MS [41]. The journal
“Molecular aspects of medicine” recently dedicated an entire issue to HNE and
its role in lipid peroxidation [42].
As in MDA analysis, measures must be taken to minimize unintentional changes

in sample composition caused by handling when analysing HNE. The levels of
HNE in healthy tissues may be approximately 0.1 µM or lower, and the ratio
between free and bound forms has not been comprehensively established. Dur-
ing oxidative stress in vivo, increases to 1-20 µM, are possible [7]. HNE was
investigated in studies described in Paper III.
5.2 Isoprostanes
5.2.1 Isoprostanes – formation, properties and analysis
In 1990 Morrow et al. demonstrated the formation of a group of
prostaglandin F2-like lipid peroxidation products [6]. These substances
constitute a family of lipids formed in vivo by the enzyme-independent and
free radical catalyzed, peroxidation of arachidonic acid (AA) in membrane
phospholipids. Since the molecules are isomeric to the prostaglandin F2α
formed enzymatically by cyclooxygenase (COX), they have been called
F2-isoprostanes. Isoprostanes were discovered serendipitously during studies
of COX-derived prostaglandin D2. The researchers found, using GC-MS, that
plasma samples from healthy volunteers that were processed and analyzed
immediately contained peaks possessing characteristics of F-ring
prostaglandins. After storage of the plasma samples for several months at
23
- 20 °C identical peaks were found, but this time at 100-fold higher levels [43],
which had been formed from lipids in the plasma samples. The formation of
the F2-isoprostanes from AA is outlined in Fig. 5. Following the discovery of
the F2-isoprostanes, it has been shown that the isoprostane pathway can provide
a route for the generation of other classes of isoprostanes from unsaturated
fatty acids other than AA that have at least three double bounds. Examples are
the “neuroprostanes” originating from docohexaenoic acid (22:6) [44]. Two
structural features distinguish the F2-isoprostanes from their enzymatically
derived relatives: the hydroxyl groups on their prostane ring have a cis
orientation, and their side chains predominantly have a cis orientation in relation
to the prostane ring.
HO OH
HO
COOH
COOH
HO HO
OH
Class III FR + O2 Class IV
COOH
COX
PG
Arachidonic acid
OH
HO HO
FR + O2
COOH
COOH
HO HO
OH
Class VI
Class V
Figure 5. Free radical (FR) attack on arachidonic acid generates four dif-
ferent classes of stereoisomers of prostaglandin F2 α called F2-isoprostanes
(classes III-VI). Prostaglandins (PGs) are enzymatically formed from
arachidonic acid by the cyclooxygenase (COX) pathway.
24
The F2-isoprostanes can be divided into four subgroups of regioisomers, called
types III, IV, V and VI. Each regioisomer is comprised of eight racemic dias-
tereoisomers (see Fig. 5). All the F2-isoprostanes have a molecular mass of
354.5 and the pKa of the acidic group is approximately 5. The nomenclature
and abbreviations used for isoprostanes in general, and in the subgroup of F2-
isoprostanes, are often confusing as the two major research groups working with
isoprostanes (Morrow and FitzGerald, and their respective co-workers) use differ-
ent terminology [45,46]. The two research teams also propose two slightly dif-
ferent explanations for the formation of F2-isoprostanes from AA [47,48].
Elevated levels of F2-isoprostanes have been reported in various physiological

states associated with enhanced lipid peroxidation and oxidative stress, includ-
ing a range of cardiovascular [49-53] and neurological diseases [54-56]. Some
of the isoprostanes have proven to be biologically active, mediating vasocon-
striction [57]. A large amount of work has been devoted to the field of isoprostane
analysis since they were first described, and two main approaches have been
adopted for their quantification in various biological samples. The first is an
immunological approach involving radioimmunoassays (RIAs) and enzyme
immunoassays (EIAs) that in many cases are inexpensive and easy to perform
[58,59]. These methods are considered to give only a semi-quantitative esti-
mate of isoprostane levels, since the risk of cross reactivity is significant. The
second approach is based on chromatographic separation and detection by mass
spectrometry (MS). One of the most frequently used methods of this kind in-
volves gas chromatography-electron capture negative chemical ionization mass
spectrometry (GC-ECNI-MS) [48,60]. This technique has low detection limits
but is time consuming and labour intensive, requiring sample pre-treatment
steps such as thin layer chromatography, solid phase extraction and
derivatization. LC-MS has been used as an alternative to GC-ECNI-MS for the
determination of F2-isoprostanes in urine and plasma samples. Li and co-workers
have successfully separated F2-isoprostane isomers in each of the four classes
using a C18 stationary phase, but their results have been very difficult to repeat
[61]. Papers IV and V are assigned to F2-isoprostanes. The topic of isoprostanes
has recently been reviewed in [57,62].
25
6. Preparation of biological samples for
CE or LC
Table 2 presents a summary of the analytes, sample types, pre-treatments, sepa-
ration and detection procedures used in the work underlying this thesis. To suit
the demands of this type of sample matrices, the preparation procedures needed
to be kept as simple as possible while generating samples that could be injected
into CE systems (with UV or laser induced fluorescence detection instruments)
or analysed by LC-MS.
Table 2
Paper Analyte Sample type Pre-treatment Separation Detection
I MDA Rat brain tissue Precipitation/ CE UV

Ultrafiltration
II MDA Rat brain tissue Ultrafiltration CE/GC/LC UV/EC/MS
Unp.a MDA Human plasma Ultrafiltration CE/ITP UV
III HNE Rat brain tissue Derivatization MEKC LIF
IV IsoP Human urine Extraction PGC-LC ESI-MS
V IsoP Human CSF Ultrafiltration/ PGC-LC ESI-MS

Direct injection
a
Unpublished results
As a biological matrix contains a myriad of compounds of various types and

concentrations, the use of efficient separation steps such as CE or LC are very
helpful for separating target compounds from a sample. Depending on the prop-
erties of the analyte and the sample, some kind of sample preparation is almost
always needed before this final analysis step can be successfully performed.
29
The general reasons for subjecting biological samples to preparatory treatments
(for the determination of low molecular weight molecules) are similar for CE
and LC, and include;
{ Removal of unwanted compounds in the sample matrix, e.g. proteins

and salts.
{ Enrichment of the analyte.
{ Facilitation of detection, i.e derivatization.
The sample components that cause the most problems in both CE and LC are
often proteins, which readily adsorb onto the surface of the CE capillary walls,
thereby impairing or completely destroying the separation. The flow through
the capillary can even be irreversibly stopped. Another problem is when broad
protein peaks interfere with the detection of the desired analyte peaks in the
electropherogram. In LC, high protein contents in a sample can give rise to
clogging of the column or irreversibly affect the stationary phase. Studies on
deproteinization have been published for both CE [63] and LC applications
[64]. Precipitation and ultrafiltration are two commonly used modes of protein
removal and are discussed below.
6.1 Precipitation
Precipitation is a fast and simple means for protein removal and many samples
can be processed at the same time. It can be accomplished by the addition of
organic solvents, salts or acids to the sample followed by centrifugation of the
precipitate. The clear supernatant is then either injected directly, or subjected
to further preparation steps. Examples of precipitating agents used in literature
include acetonitrile, methanol, trichloroacetic acid and ammonium sulphate.
The effect of the media used for precipitation on the chromatographic or elec-
trophoretic system must be considered. One disadvantage with organic solvent
precipitation is the dilution of the sample. However, the organic solvent lowers
the sample’s conductivity, so the enhanced stacking of the charged sample com-
ponents in CE may compensate for some of the dilution. For a discussion about
30
stacking, see section 7.3. In reversed phase LC, organic solvent in the injected
sample, at a higher ratio than in the mobile phase, is detrimental to the separa-
tion. Concentration to dryness and reconstitution in an appropriate solvent can
alleviate this problem. This, however, adds an additional step and could de-
crease the total recovery as discussed later. The other two alternatives, precipi-
tation by salt or acid, are not advantageous in either CE or LC. In CE, conduc-
tivity should be kept low in the injected sample zone to allow satisfactory stack-
ing and low Joule heating. In LC, acid or salt can be detrimental to the column
packing material or harm separation. Further, strong acid can cause degrada-
tion of sample components including the analytes. One additional alternative is
to precipitate the proteins in a sample by heating. This treatment can be too
harsh for thermolabile analytes, and may lead to artefactual generation of
analytes from sample constituents, as is the case with lipid peroxidation mark-
ers. Precipitation by adding organic solvents (acetonitrile or methanol) was
used in studies I and III.
6.2 Ultrafiltration
One convenient way to remove proteins from samples without dilution is ultra-
filtration. Ultrafiltration involves filtration of the sample through a membrane
of specific pore size, using a centrifuge. Particles and molecules larger than the
threshold size (e.g. 30 kDa) are retained on the surface while smaller species
are allowed to pass through. This significantly reduces the amount of protein in
the sample [63,64]. Many samples can be run simultaneously and it is suitable
for a wide range of sample volumes since the ultrafiltration devices come in
different sizes. However, no sample enrichment is obtained with ultrafiltra-
tion. Furthermore, ultrafiltration allows only the free fraction of the analyte,
not the protein-bound forms (if any), to pass through the filter. This facilitates
studies of free versus protein-bound levels of the analyte. Consequently, it has
been employed frequently for protein binding studies of drugs [65-67]. A vital
aspect is to verify that the analytes of interest do not adsorb onto the membrane
or the polymeric material in the devices. Further, it is important to maintain
physiological settings regarding, for example, pH and temperature in free ver-
31
sus protein-bound form studies, since binding values can otherwise be affected.
Ultrafiltration was used in studies I, II and V.
If precipitation or ultrafiltration doesn’t generate sufficiently clean and

concentrated samples, quite a wide range of alternative sample preparation
methods can be applied. Such methods can also provide more sophisti-
cated ways to remove proteins. Some examples of potentially useful pro-
cedures are described below.
6.3 Liquid–liquid extraction

Clean and concentrated samples can be obtained after applying liquid-liquid
extraction (LLE). LLE is a separation process that exploits differences in the
relative solubilities of the analytes in immiscible solvents. The compounds of
interest can be extracted from, for example, an aqueous sample, into a volatile
organic solvent such as pentane, hexane, diethyl ether or ethyl acetate. Impor-
tant variables to drive the extraction in the desired direction are pH and the
ionic strength of the aqueous solution. The organic solvents used for LLE can-
not be injected into CE or, in most cases, reversed phase LC systems. Thus,
after extraction, the organic phase is evaporated to dryness and the residue is
then reconstituted into a suitable solvent. Loss of analyte can occur during the
evaporation step, by adsorption to the equipment or incomplete reconstitution.
As LLE is not easily automated it can be a bottleneck in high-throughput analysis.
However, if a robot is used, many samples can be processed in parallel with
reduced manual handling, thereby saving a considerable amount of time. In
some cases, LLE can generate cleaner samples than solid phase extraction treat-
ment (SPE). For example, Bonfiglio showed that ion suppression effects in
electrospray ionization (see section 8.4) were reduced more by LLE than by
ACN precipitation or SPE [68]. So, if the recovery in the evaporation/reconsti-
tution step is high and a well-judged selection of solvents and pH is used, LLE
can give very clean extracts with satisfactory selectivity for the studied analyte.
A robot-run LLE extraction technique was used in the work associated with
Paper IV. Useful reviews on LLE of biological samples are presented in [69,70].
32
6.4 Solid phase extraction
Solid phase extraction (SPE) can be used to simultaneously enrich the analytes
in samples and remove salts, proteins and a wide variety of other potentially
interfering compounds. In SPE the sample is passed over a small tube filled
with porous solid particles such as silica-C18. Alternatively, a membrane disc
containing sorbent particles can be used. After preconditioning the sorbent, the
sample is applied. The sorbent is then selectively washed to remove unwanted
compounds without losing the analyte(s) of interest. Finally, the analytes are
eluted using a minimum of solvent. The solvent used should be compatible
with the next analytical step, otherwise it must be changed by evaporation/
reconstitution as in LLE. There are a great number of different, commercially
available SPE phases and the process can be coupled on-line in order to save
time and minimize the risk of sample loss. In recent years SPE has replaced
many LLE extractions, but in some cases SPE eluates are not as clean as ex-
tracts obtained after a judiciously performed LLE . Examples of reviews on SPE
of biological samples for CE and chromatographic applications are presented in
[71,72]. SPE was a minor part of the analytical procedures described in Paper IV.
6.5 Column switching

The term column switching has been widely used in the literature and has
been employed in applications ranging from on-line SPE to complex net-
works of columns. In a review, Campins-Falco et al. define the term as
encompassing “all techniques by which the direction of the flow of mobile
phase is changed by valves, so the effluent of a primary column is passed
to a secondary column for a defined period of time” [73]. Majors has de-
scribed some practical considerations regarding the technique [74]. Col-
umn switching can significantly facilitate analysis of biological samples
by providing on-line sample clean-up, and enrichment or by improving
resolution and selectivity through the use of different stationary phases.
To avoid band broadening the first column should have smaller, or at most,
the same dimensions as the second column. Further, again to minimize band
broadening, the retention capability of the first column should not exceed
33
that of the second column for the selected solvents, since efficient chroma-
tography is promoted if the sample is re-concentrated at the top of the sec-
ond column. An example of a column switching setup, described in paper V,
is illustrated in Fig. 6.
Pump I
Injector
Waste
ap
Tr
Analytical Column
MS-MS
Pump II
Figure 6. The column switching setup used in study V.
An increasingly popular way to facilitate the direct injection of biological flu-

ids in LC is to use restricted access materials (RAMs) in the pre-column. RAMs
are porous packing materials that prevent macromolecules, like proteins, from
penetrating the pores but allow free access of low molecular weight compounds.
The inert layer on the outside of the material does not interact with the sample
matrix while the porous inner surface of the material is covered with a phase,
for example C18, that retains analytes by reversed-phase interactions. Unretained
components, such as proteins, are washed out to waste before the retained frac-
tion is eluted onto the analytical column where the separation is performed.
Recent reviews on RAM have been presented by [75,76].
6.6 Derivatization
Derivatization is not a pure cleaning or concentration step in sample pre-treat-
ment. Instead, it can facilitate meaningful determinations of compounds by
increasing their volatility (prior to GC analysis), improving both the selectivity
34
(especially in chiral separations) and chromatographic efficiency, and enhanc-
ing detectability. The technique is often regarded as a necessary evil since it
adds an additional step to the sample preparation procedure and thus intro-
duces a further source of error in the analysis. The development of sensitive
and universal detection methods, such as MS, has reduced its importance in
many fields. Derivatization is used today, in most cases, prior to LC only if all
other alternatives have failed. In the field of CE and capillary
electrochromatography (CEC), where detection is often problematic due to the
low loads that can be accommodated, and the limited cross-column path lengths
for spectroscopic detection, derivatization can sometimes be beneficial.
Derivatization was used in study III to enable detection by laser-induced fluo-
rescence after a micellar electrokinetic chromatography (MEKC) separation
and in study II to increase the volatility and detectability of the target com-
pounds in GC-ECD and GC-MS. Recent books on the topic of derivatization
for chromatographic and electrophoresis based analyses include [77,78].
6.7 Other techniques

The sample preparation techniques mentioned above are some of the most
commonly used. However, there are of course a large number of other ways
to prepare a sample for analysis that have been described in the literature.
General reviews regarding sample pre-treatments of biological samples that
are suitable before analysis by CE, LC or both appear continuously and
helpful examples are [79-84]. Reviews concerning specific techniques such
as the use of molecular imprinted polymers (MIP) [85,86], capillary ultra-
filtration [87,88], solid phase micro extraction (SPME) [89], affinity tech-
niques [90], microdialysis [91,92], and membrane-based methods [93] have
also been published. The on-line sample concentration technique “stack-
ing” for CE is discussed in section 7.3.
35
7. Capillary electrophoresis
Electrophoresis in micrometer bore capillaries was first presented in the early
1980s [94], since then capillary electrophoresis has rapidly become a well-
known technique. Basic issues and applications of CE are covered in several
good books that are recommended for further reading [95-98]. This section
starts with a brief introduction to capillary electrophoresis, and is then dedi-
cated to a discussion of the analyte characteristics and conditions used to solve
problems associated with measuring lipid peroxidation in biological samples.
7.1 Introduction to CE
CE is performed in narrow tubes (20–100 µm i.d.) or on micro-fabricated chips.
The discussion here is focused on CE in fused silica capillaries, although most
of the basic elements are applicable to electrophoresis in general. The basic
instrumentation required in CE is inexpensive and simple, comprising the cap-
illary mentioned above, two buffer vials, a high voltage supply, a pair of elec-
trodes, a detector and a computer equipped with a data handling system. Fig. 7a
shows a schematic illustration of a CE system. Separation in electrophoresis
relies on the differences in the electrophoretic migration velocities of the sol-
utes in an electric field. The silanol groups at the inner surface of the capillary
have pKa values ranging from 2 to 5. When the capillary is filled with a back-
ground electrolyte (BGE) whose pH exceeds the pKa of these groups, a net
negative charge will be created on the wall surface. The negative charge at-
tracts layers of hydrated positively charged ions to the vicinity of the wall and
when an electric field is applied the entire bulk liquid moves towards the cath-
ode due to the viscosity of the solution. This phenomenon is called electroos-
motic flow (EOF). Since the driving force for the EOF is evenly distributed along
the capillary walls a flat velocity profile is obtained, compared to the parabolic
flow arising from mechanical pumping. The flat profile eliminates a large contri-
bution to bandbroadening, thereby increasing the theoretical platenumbers.
When the capillary is filled with a BGE, and small amount of sample solution
36
Figure 7. Schematic representation of a CE system. (a) EOF in the nor-
mal direction. (b) Reversed EOF.
37
is introduced into one end and an electric field is applied, sample molecules are
transported by the joint action of electrophoretic migration and EOF. Some of
the fundamental formulas applying to electrophoresis are shown below.
The velocity of an ion in an electric field is given by
v = µe E (1)
where v = ion velocity, µe = electrophoretic mobility and E = applied electric field.
The mobility µe of a given ion in a given medium is a constant which is charac-

teristic of that ion. In an electric field a charged analyte is acted on by an elec-
tric force FE that is given by
FE = q E (2)
and a frictional force FF that is given (for a spherical hydrated ion) by
FF = -6 π η r v (3)
where q = ion charge, η = solution viscosity, r = ion radius and

v = ion velocity
During steady state electrophoresis these forces balance each other and are
equal but opposite
q E = 6 πηrv (4)
Solving for velocity using the above formulas gives an equation that describes
the electrophoretic mobility of an ion in physical parameters
µe = q / 6 π η r (5)
38
7.2 Injection
The two general methods that can be applied for injection in CE are hydrody-
namic injection and electrokinetic injection. In the first case, a pressure differ-
ence is created across the inlet and outlet vials, forcing the sample solution into
the capillary inlet end. The volume of the sample injected depends on the mag-
nitude and duration of the pressure applied, the BGE viscosity, and the capil-
lary dimensions. In the second case a voltage is applied which causes the sample
ions to migrate into the capillary as a result of electroosmosis and electro-
phoretic mobility. The amount injected depends on the electrophoretic mobil-
ity of the solutes, the electroosmotic flow rate, the applied voltage, the capil-
lary dimensions, the solute concentration and the duration of the injection.
Hydrodynamic injection is, conceptually, the simplest method. It is reproduc-
ible, since the sample matrix has virtually no effect on the injected amount. In
electrokinetic injection, variations in sample conductivity, which may arise
due to matrix effects, influence the quantity loaded. This often affects repro-
ducibility in a negative way. In all the work presented here involving CE, hy-
drodynamic injections were used.
7.3 In-capillary sample concentration

The term “sample stacking” was first used by Ornstein [99] to describe the
stacking of proteins according to their mobilities in disc electrophoresis. An
up-to-date and broad definition of stacking is that it covers “all on-line sample
concentration techniques in electrophoresis” [100]. A number of techniques
have been developed that concentrate samples on standard CE equipment. These
techniques can be broadly divided into two classes: those based on electric
field amplification and those based on isotachophoresis. When the simple term
“stacking” is used in the literature, it usually refers to field amplified stacking.
Refs [100-102] are recent review articles on stacking in CE. In studies I
and II field amplified stacking was used and in studies outlined in the “Un-
published results” chapter (see section 9.6), an isotachophoretic stacking
state was induced.
39
7.3.1 Field amplified stacking
Mikkers et al. described on-capillary sample concentration, obtained through
field strength differences between the sample zone and the BGE [103]. Chien
and Burgi later continued the work [104-106]. The basis of stacking is to pro-
vide an electric field of high strength across the injection zone. When the con-
ductivity of the injected sample zone is lower than that of the background elec-
trolyte, the field strength increases. A lower conductivity is usually achieved
by using dilute samples or by the addition of organic solvents that reduces the
conductivity in the samples. Electrophoretic velocity is proportional to the elec-
tric field, and after injection the solute ions rapidly migrate through the dilute
sample zone until they reach the concentration boundary between the sample
zone and the run BGE. The solutes then encounter a lower electric field which
makes them slow down, and a narrow stacked zone is formed. In the work
presented here, efforts were made to obtain a good stacking effect by keeping
the influencing variables, such as injected sample zone conductivity, BGE con-
centration and mobility at appropriate levels.
7.3.2 Isotachophoretic stacking

Everaerts et al. presented another major advance in sample concentration in
CE, namely isotachophoretic stacking (ITP) [107-109]. ITP is a variation of
electrophoresis that can also be used to increase sample concentration prior to
ordinary CE. It is based on equation 1, which shows that the velocity of an ion
in an electric field of strength E is dependent on the mobility (µe) of the ion.
The molecules to be separated are sandwiched between a leading electrolyte

with a high-mobility ion and a terminating electrolyte with a low-mobility ion.
When the electric field is applied, the sample components begin to arrange
themselves into zones, according to their mobility. The ions with highest mo-
bility give rise to the highest conductivity, so the field strength is lowest across
the zone that contains them. Conversely, the least mobile ions generate the
highest field strength and their velocity is increased. A steady state velocity
40
thus develops. When an ion diffuses into a neighbouring zone, its velocity
changes and it returns to its original zone. Since the current is constant there is
a constant ratio between the concentration and the mobility in each zone. The
concentration in each zone is determined by the concentration of the leading
electrolyte. Zones that are less concentrated than the leading electrolyte be-
come compressed, so that an appropriate concentration is achieved. The sim-
plest way to create an ITP system is to dissolve the sample in a buffer that,
together with the background electrolyte, will create ITP conditions. This is
called transient ITP. If a sample contains high concentrations of a species that
can serve as a leading electrolyte, ITP effects can occur. This state can either
be induced by the addition of a highly mobile ion such as chloride, as sug-
gested by Foret [96,110] or be self-generated by the sample constituents.
The latter is especially likely when complex samples are being processed.
The phenomenon is called “self-stacking” and is further discussed in
Refs. [111,112]. An ITP state was probably induced during the analysis of
the plasma samples as discussed in section 9.6. In this case, the analyte
peak was equivalent to nearly 3×106 theoretical plates, which is signifi-
cantly more than can be explained by conventional CE stacking theory in
such a high ionic strength sample.
7.4 Separation
CE for anionic species is most commonly run with the electrophoretic migra-
tion and EOF flowing in opposite directions, as in Fig. 7a. If the analytes of
interest are small and/or highly charged a faster separation can be achieved by
reversing the EOF, as shown in Fig. 7b. In this mode the directional vectors of
electrophoretic migration and EOF combine, and thus increase the speed of the
analysis. Reversal of EOF was demonstrated by Zare and co-workers for deter-
mination of low molecular weight carboxylic acids [113] and was also earlier
presented by Terabe [114] and Tsuda [115]. Reversal of the EOF can be ac-
complished by adding quaternary amines such as the cationic surfactant cetyl
trimethyl ammonium bromide (CTAB) to the BGE. CTAB monomers adhere
41
to the capillary wall by ionic interactions. The hydrophobic ends of the at-
tached molecules associate with the hydrophobic ends of the CTAB molecules
in solution and their exposed positive charges attract negatively charged an-
ions from the BGE. Upon application of an electric field the solvated anions
migrate toward the anode, dragging the bulk solution with them and thus re-
verse the EOF. This kind of dynamic modification of the capillary wall is also
known to reduce the detrimental adsorption of sample constituents, such as
proteins, onto the capillary surface [116]. Reversed flow using CTAB as a
modifier, was employed in the research associated with Papers I and II. When
the amount of surfactant is increased above its “critical micelle concentration”
(CMC), micelles are formed in the separation system. Micellar electrokinetic
chromatography (MEKC), first demonstrated by Terabe and co-workers [117],
is a type of CE that allows the separation of both charged and neutral solutes.
The micelles in the BGE act as a pseudo stationary phase and separation is
achieved by the solutes’ differential partitioning between the micelles and the
surrounding electrolyte. Fig. 8 shows a schematic representation of the differ-
ent transport mechanisms in MEKC. MEKC analyses, with direct injection of
body fluids have been presented [118,119], thereby displaying the robustness
of the technique for handling crude samples. In study III, MEKC was used
for the separation of complex samples, and in study II as part of an analyte
purity test, since the technique’s separation mechanism is different to that
of ordinary CE.
µanalyte
K analyte
µ micelle
EOF
Figure 8. The different transport mechanisms involved in MEKC

(anionic surfactant).
42
7.5 Detection in CE
The mode of detection should be selected to complement the properties of the
analyte, and parameters such as sensitivity, limit of detection, selectivity, lin-
ear range and noise should then be considered. Generally, all the detection
techniques used in miniaturized LC are also relevant to CE. The very small
sample volumes used in CE are often considered to be an advantage, in com-
parison with many other separation techniques, but it also makes detection
more challenging. One of the most commonly used detection techniques is UV
spectroscopy. However, although this approach is versatile it has poor concen-
tration detection limits due to the short pathlength of the fused silica capillar-
ies. CE-UV was used in studies I and II. MS detection has lower detection
limits than UV and provides structural information. Even if CE-MS is a grow-
ing field it is still not as widely available as UV detection, mainly due to its
high costs. Electrochemical detection (EC) often has better detection limits
and is more selective than UV detection. Further, the selectivity can be tuned
by varying the applied potential. However, EC detection for CE is somewhat
more difficult to use and requires a practician skilled in electrochemistry. La-
ser-induced fluorescence (LIF) is the most sensitive type of detection currently
available for CE and early designs and applications of LIF detection systems
have been covered in a review by Dovichi [120]. Tao and Kennedy review
more recent advances [121]. The very low detection limits of LIF can be attrib-
uted to the nature of the laser radiation. The high powered, unidirectional and
monochromatic light of a laser can be focused onto a very small volume, if
compared to other types of irradiation sources, such as for example, Xe-lamp
based fluorescence. Since most molecules lack native fluorescence,
derivatization is often necessary. Many of the CE-LIF detections performed in
the yocto to zepto mole range have been achieved by the derivatization of
analytes with excellent fluorophores. In [121] the following criteria for an ideal
derivatization reagent for CE-LIF, are listed; (1) fast reaction rates with the
analytes, (2) formation of strongly fluorescent compounds with the analytes
providing excitation maxima that match the wavelengths of convenient lasers,
and (3) good chemical and photo stability. LIF detection, utilizing an argon ion
43
laser, was applied in the research associated with Paper III. A picture of the
laser setup used is presented in section 9.3. Strategies to improve sensitivity
and detection limits in capillary electrophoresis of biological fluids are reviewed
by Hempel [122], and Swinney reviews CE detection methodologies in [123].
7.6 Qualitative and quantitative aspects in CE

In CE, the sample matrix can influence migration times. This makes peak iden-
tification on the basis of migration times somewhat unreliable. Consequently,
multi-wavelength absorption detection, in which every peak can be represented
by absorption spectra, improves identification and may provide valuable struc-
tural information. Diode array detection (DAD) was used in the research asso-
ciated with Papers I and II. Other examples of detection techniques that may
provide structural information include MS [124,125] and mid-IR analysis [126].
Spiking the sample with a standard containing the substance of interest can
strengthen identification of a peak in an electropherogram. Despite the high
separation efficiencies of CE, there may still be uncertainties regarding the
purity of a peak, and whether or not it contains unknown compounds with simi-
lar migration velocities. This is especially problematic with heterogeneous
samples. There are several possible solutions to this problem: the use of very
selective detectors, such as LIF or EC, that enhance the reliability of peak iden-
tification, or as mentioned above, the use of a detector that generates structural
information concerning the compounds represented by the peaks, enabling com-
parison of the peak spectra obtained with those of the standards. An alternative
is to change the selectivity of the entire separation system, thereby elucidating
peak purity.
Quantitative analysis in CE is similar to that of LC and is commonly performed

using peak area, rather than height, since calibration plots based on area are
more linear and display linearity over a wider concentration range [95]. In
contrast to LC, the migration velocities of the solutes in CE affect the peak
areas. Analytes of low mobility remain in the detection window for a longer
time than those of higher mobility, and will therefore have an increased peak
44
area. Dividing the integrated peak area by migration time corrects for this, and
was used in both studies I and II. For calibration, conventional external stan-
dardization can be used. An alternative is to apply a standard addition tech-
nique, which was used in study I, as the sample matrix can putatively affect the
areas of the analyte peaks. Probably the most reliable method of calibration
is to use internal standards. Adding an internal standard to the sample cor-
rects for quantitative losses during clean up, as well as instrumental impre-
cision, which is primarily caused by variations in injection parameters.
Internal standards also help to correct for migration time variations. Internal
standard methodology was used in the research associated with Paper II. In
[98] the authors define the following criteria for the selection of a suitable
internal standard in CE:
{ It must be well separated from solutes and interferences in the samples.

{ It should elute close to the compounds of interest, ideally at a gap in the
electropherogram.
{ It must not be present in any of the samples.
{ It should be commercially available at high purity.
{ It must be stable and non-reactive with the capillary, the run BGE and
any components in the samples.
{ It should have a detector response similar to that of the solutes of
interest.
{ It should be chemically similar to the solutes of interest if the samples
are to be derivatized or pre-treated.
{ It should have an electrophoretic mobility that is similar to the run BGE,
to ensure good peak symmetry.
{ It should be added at a concentration that produces a solute to internal
standard height or area ratio of approximately unity.
45
8. LC-MS
Reversed phase liquid chromatography (LC) coupled with mass spectrometry
(MS) detection is one of the most widely applied techniques in the field of
analytical chemistry today, and has become an essential part of everyday work
in any analytical laboratory. General LC theory is covered in several good books
such as [127,128] as is the theory regarding electrospray-MS in [129,130]. The
following section discusses specific aspects of LC-MS that have been given
close attention in order to solve the problems involved in the separation and
detection of minute amounts of polar and structurally closely related solutes in
complex biological matrixes.
8.1 The effect of column diameter in LC

As interest in analyzing very small volumes of biological samples has grown
there has been a rapid development of micro-separation techniques such as
micro-LC, capillary-LC and CE. The terminology used in this area is some-
times confusing and Chervet et al. suggested a nomenclature, presented in Table
3 below, based on the flow rates in packed micro-columns [131]. According to
their nomenclature, a 1 mm i.d. column used with a flow rate of 50 µl/min, as in
the system described in Papers IV and V, is classified as a micro–LC column.
Reduction in column diameter in LC can be motivated both economically and
environmentally since, for instance, when the i.d. of the column is reduced
Table 3 Names and definitions for LC techniques

Column i.d. Flow rate Name
3.2 – 4.6 mm 0.5 – 2.0 ml/min conventional LC

1.5 – 3.2 mm 100 – 500 µl/min microbore LC
0.5 – 1.5 mm 10 – 100 µl/min micro LC
150 – 500 µm 1 – 10 µl/min capillary LC
10 – 150 µm 10 – 100 nl/min nano LC
46
from 4.6 to 1 mm (maintaining linear flow velocity) solvent requirements are
reduced by 95 %. A small diameter and hence a low flow rate of mobile phase
can also increase the system’s compatibility with certain detectors such as
electrospray-MS (ESI-MS) instruments [130]. Many modern LC-ESI-MS in-
terfaces can handle LC-flows of 1 ml/min or even more. However, most inter-
faces can benefit from lower flow rates that relax the requirements on the vacuum
pumping system. Abian et al [132] discusses some further aspects on column
dimensions and hence flow rates in LC-ESI-MS in a recent review. Reducing
the column diameter can also affect sensitivity and detection limits since a
higher sample peak concentration is produced in the detector, given that the
other parameters affecting efficiency of the LC system are constant. A minia-
turized column thus gives the lowest detection limit when true concentration
sensitive detectors such as UV and refractive index are used. This is also the
case in ESI-MS, since it behaves as a concentration sensitive detector [133-135].
The theoretical gain in mass sensitivity can be estimated as (d1)2/(d2)2, where d1
and d2 are columns of different internal diameter and d1>d2. The predicted
increase in mass sensitivity, when a 1 mm i.d column is used instead of a con-
ventional 4.6 mm column, is thus a factor of 21.Therefore, in situations where
sample amounts are limited, small bore columns can provide better detection
limits than conventional columns. When downscaling LC the factors generat-
ing band broadening effects become increasingly important to control. Band
broadening brings loss of efficiency and hence reductions in resolution and
sensitivity. The total band broadening reflects the sum of the contributory fac-
tors from the components in the chromatographic system (injection, column,
detection and connecting fittings and tubing). Therefore, the requirements for
appropriate systems, including those for injecting the samples, delivering the
solvent gradient, and the connections between the various components, need to
be carefully addressed. In ref [136] the maximum volume that can be injected
into a 1 mm i.d. column while maintaining column efficiency is calculated to
be approximately 500 nl. To alleviate the problems associated with small in-
jection volumes, on-column focusing (where the sample is dissolved in a non-
eluting medium) or micro pre-columns combined with column switching tech-
niques can be used (as in study V), thereby allowing much larger injections to
47
be made [137]. As for the mobile phase delivery system, the gradient delay
needs to be minimised, the proportioning needs to be accurate and homog-
enous mixing is required. Instrumental requirements in micro column LC are
further discussed in refs [131,136].
8.2 Porous graphitic carbon as packing material in LC

8.2.1 Manufacture and structure of PGC
The development of porous graphitic carbon (PGC) was motivated by a desire
to overcome problems associated with reversed phase silica gels such as their
limited hydrolytic stability and underivatized silanol groups causing second-
ary chromatographic interactions. In the past, use of carbon as a packing mate-
rial in chromatography has been hampered by problems with particle fragility,
retention capacity and poor mass transfer. Various attempts to use carbon-based
separation in chromatography are reviewed in [138,139]. In 1982 Knox and
Gilbert presented a new methodology for the production of a glassy carbon
using silica gel as template [140]. This material had the desired properties with
respect to surface area and mechanical strength. The porosity of the PGC was
accomplished by impregnating porous silica with a phenol-formaldehyde mix-
ture, whereby a resin was formed within the pores of the silica gel after heat-
ing. The resin was then carbonized by raising the temperature to 1000 °C. After
dissolution of the silica template with alkali, graphitization was performed at
temperatures above 2000 °C. This removed micropores and produced a crys-
talline product with 7 µm particle size. In 1988, porous graphitic carbon be-
came commercially available under the name Hypercarb from Shandon/Hypersil
and the company further improved the PGC performance by introducing a
material with 5 µm particle size in 1994. Very recently, PGC has also become
available in 3 µm particle size [141]. The structure of PGC is composed of flat
sheets of hexagonally arranged carbon atoms with a satisfied valence and is thus
completely different from traditional silica-bonded phases, as depicted in Fig 9.
The material is geometrically stable, it does not swell or shrink, it is inert to 100 %
water and organic solvents and it is stable over the entire pH range.
48
OH
OH
PGC Silica-C18
Figure 9. PGC structure in comparison with traditional silica-based C18.
8.2.2 Performance of PGC

Porous graphitic carbon has been successfully employed in various research
areas and has proven to be very useful in the separation of polar analytes and
structurally closely related compounds. Retention mechanisms on PGC are dif-
ferent from those on silica-based bonded phases and have not yet been fully
elucidated. Some aspects of these mechanisms are discussed below. In 1997
Knox and Ross published comprehensive reviews on the performance and ap-
plications of separations on carbon-based materials [142,143].
Two mechanisms are responsible for retention on PGC. First, the dispersive
interactions that are stronger with 100 % carbon compared to C18. This results
in PGC behaving as a strong reversed-phase stationary phase and the retention
increases as the hydrophobicity of the analyte increases. Compared to C18
materials, a higher concentration of organic modifier is usually required to
elute a hydrophobic solute from a PGC column. The selectivity for closely
related compounds, for example stereoisomers, is often improved on PGC com-
pared to C18 and similar materials. An explanation for this is that the strength
of analyte interactions with PGC material is largely dependent on the molecu-
lar area in contact with the flat graphite surface, and also on the type and posi-
tioning of the functional groups. A flexible molecule cannot align itself as closely
49
as a rigid planar molecule to the PGC surface. So, retention of a planar mol-
ecule is often stronger since it has more interactions than a molecule with a 3-D
spatial arrangement. Early work on the retention by PGC of simple aliphatic
and aromatic compounds was carried out by Möckel [144], Kriz [145], Tanaka
[146] and Wan [147], inter alia.
The second mechanism is related to charge-induced interactions of a polar

analyte with the polarizable surface of graphite. It is this second mechanism
that accounts for the expression “polar retention effect on graphite” (PREG),
describing the increase in retention observed as the polarity of the analyte in-
creases. As early as 1989 Bassler et al. studied the retention on PGC of small
aromatic compounds and found that retention was increased when one hydro-
gen was substituted by a polar functional group [148]. Several other research
groups have also studied the retention of polar analytes on PGC [149]. PGC
has layers of hexagonally arranged carbon atoms in a two-dimensional struc-
ture, where the atoms are in sp2-hybrid states. The delocalized π-electrons and
the high polarizability of the large layers of carbon are features that govern the
second retention mechanism. In Fig. 10 the charge-induced dipole that appears
when a polar analyte approaches the surface of the graphite is shown.
Figure 10. Induced dipole when a point charge approaches a PGC surface.
As PGC is inert to pH extremes and solvents, method development on PGC can

be very flexible and more consideration can often be paid to matching the re-
quirements of the subsequent detection step than is the case with more tradi-
tional modes of separation for polar analytes. Further, harsh regeneration/reju-
50
venation regimes can be used without destroying the stationary phase or sepa-
ration efficiency. This facilitates the analysis of biological samples, especially
during the method development phase, since samples that have not been highly
purified may be injected into the system. PGC was used in studies IV and V for
the successful separation of the polar and stereoisomeric F2-isoprostanes.
8.3 ESI as an ionization technique for MS detection

A mass spectrometer is an instrument designed to separate and detect charged
species according to their mass to charge (m/z) ratio. Its essential components
include a sample inlet, ion source, mass analyzer, detector and data handling
system. In the case of MS, when used as a detection technique for LC, the ion
source must transform analytes in solution to gas-phase ions before they reach
the mass analyzer, which works under very low pressure. The development of
atmospheric pressure ionization (API) techniques has led to a solution to this
problem. The API techniques, of which electrospray ionization (ESI) is the
most popular, are generally regarded as soft modes of ionization in the sense
that the labile molecules are transformed into ions without causing extensive
fragmentation. ESI often produce multiple charged ions from large mol-
ecules such as proteins and peptides, this facilitate their analysis since they
then fit in the m/z working range of the mass analyzer. Dole and coworkers
reported generation of gas-phase ions by spraying a solution from the tip
of an electrically charged capillary as early as 1968 [150]. Fenn and co-
workers adapted this idea of Dole, and developed electrospray as an ionization
technique for MS in 1984 [151]. The tremendous success of the combination of
LC and API techniques is mainly due to the facts that API systems are robust
and relatively easy to use, the system can manage the liquid volumes typically
handled in LC and that the typical LC analytes (nonvolatile, polar, and ther-
mally unstable) are suited for API.
In electrospray ionization the analyte solution is passed via a capillary through

a small diameter tip held at a high potential (in the range of 1-5 kV) and at
atmospheric pressure. As the solution emerges from the tip the electric field
51
generates a spray of highly charged droplets. The theoretical explanation for
these phenomena, in positive mode ESI, is that when a positive potential is
created between the spray tip and the MS entrance, positive ions are drawn out,
by electrostatic attraction, causing the liquid to protrude and thus form a Tay-
lor cone [152], as depicted in Fig. 11. This initiates charged droplet formation
when the electrostatic force exceeds the surface tension at the tip of the Taylor
cone. If the polarity of the spray tip and the counter electrode (MS-entrance) is
reversed the opposite will occur, i.e. negative ions will protrude in the Taylor
cone, negatively charged droplets will be formed and so forth. That is called
“negative mode ESI”.
Taylor cone Reduction
Atmospheric pressure
Flow of liquid To MS analyzer
Oxidation
10-5 - 10-6 torr
Electrons
Electrons
Figure 11. Schematic diagram of the electrospray process in positive

ion mode.
Two models, which on first consideration may seem contradictory, are com-
monly used to explain the process whereby the gaseous ions are formed from
the charged droplets. Dole used the “charge residue model, CRM” [150]. Here,
the evaporation of the solvent from the initially formed drops leads to a reduc-
tion in their diameter, and as the surface charge increases they reach the Rayleigh
instability limit. Then a coulomb explosion occurs at the point where the repul-
sion of charge is greater than the surface tension holding the droplet together,
generating even smaller charged droplets. This process of evaporation and cou-
52
lomb explosions continues until the last solvent evaporates and leaves the ion.
Iribarne and Thompson proposed another model (the ion evaporation model,
IEM) [153] in which they assume that ion emission to the gas phase occurs
directly from the small offspring droplets. Recently, the possibility has been
discussed that the two models may not be mutually contradictory but, instead,
may explain processes involving different kinds of molecules. The IEM seems
more valid for small molecules while the CRM is applicable for larger mol-
ecules, such as proteins.
The ideal flow rates for pure ESI are below 1 µl/min. In most commercial
instruments available today, a nebulizer gas pneumatically assists the forma-
tion of a spray. This is called ionspray and was introduced by Bruins [154].
Here, higher flows can be used than are possible with pure electrospray. In
addition, most instruments also have systems for heating the areas around the
sprayer tip to further aid solvent evaporation. The term ESI is commonly used,
even if the spray is pneumatically assisted. The success of ESI-MS detection
for LC is dependent on several factors, including analyte characteristics, in-
strumental parameters and solution characteristics. Generally, it can be said
that pneumatically assisted ESI is more robust than pure ESI with respect to
deviations from ideal conditions.
Important analyte characteristics include their readiness to become charged,

and ESI is most suited for compounds that readily form ions in solution or
can be ionized by adjusting the pH. An alternative way to obtain charged
analytes is by adduct formation with small ions like Na+. ESI of acidic com-
pounds are most often performed in negative mode, utilizing high pH buffers,
while positive mode and low pH buffers are normally chosen for basic com-
pounds. However, the acid/base chemistry in solution cannot be directly trans-
posed to the reactions occurring in ESI, and charging can occur contrary to
expectations based solely on pKa values. Ions can be protonated/deprotonated
during the desorption process or in the gas phase, depending on the gas phase
proton affinities of the analytes. Further, ionization through electrochemical
oxidation or reduction may occur in the ES ion source. Other analyte character-
53
istics, such as lipophilicity may also influence the ionization efficiency. For
instance, a high affinity for the droplet surface is beneficial for a high ESI
response [155].
Instrumental parameters influence the ability to obtain a stable and effective

spray. Examples of important variables are: the applied voltage, the nebulizing
gas flow, the position and distance between the spray capillary and the counter
electrode, the solution flow rate and the shape and diameter of the spray tip. A
lower flow rate generally results in a smaller initial size of the droplet pro-
duced in the ESI interface. This is beneficial since, amongst other things, smaller
droplets have a higher surface to volume ratio that makes a larger proportion of
the analyte molecules available for desorption.
Solution characteristics such as pH, volatility, viscosity, dielectric constant,

surface tension, and conductivity, affect the ESI process and the ideal solvent
composition varies with the application. Solutions with too high surface ten-
sion, such as pure water, are difficult to electrospray, but it is also difficult to
obtain a stable spray from highly nonpolar liquids such as hexane due to their
high volatility, very low surface tension and low dielectric constant. When LC
separation systems are interfaced with ESI-MS further demands are put on the
properties of the solution, in order to obtain sufficient chromatographic separa-
tion. Deviations from the ideal solvent composition and flow rate may be in-
evitable, and may result in significant impairments in ESI performance. Non-
volatile buffers and ion-pairing reagents must be avoided since they can cause
background interference and signal suppression, as discussed in next section.
8.4 Ion suppression

An often-heard assertion is that utilization of LC-MS-MS practically guaran-
tees selectivity to the extent that sample preparation may be highly simplified
or even eliminated. Contrary to this belief, several cases in which there is a
need for efficient extraction from biological materials and/or chromatographic
separation have been described by Matuszewski et al. [156] and Bonfiglio
54
et al. [68], amongst others. When ion suppression occurs the response observed
for a given concentration of analyte is different in the presence of components
from the sample or the mobile phase. A possible explanation of ion suppres-
sion is that there may be competition for a limited resource. For instance, it has
been suggested that limited space on the droplets’ surfaces can cause suppres-
sion of the analyte signal, and that surface-active analytes are less prone to ion
suppression than more polar ones that preferentially remain in the interior of
the charged droplets [155]. The charge-inducing factors that form the ions can
also be limited resources and when the sample or the LC-mobile phase has
constituents that are more readily charged than the analyte(s) the signal can be
suppressed. Alternatively, ion suppression can arise when formed ion pairs are
not broken apart by the ESI process. Therefore, the addition of ion-pairing
agents can promote ion suppression in LC-ESI-MS and should thus be avoided.
Ion suppression effects caused by compounds in the sample matrix or the mo-
bile phase can be reduced if lower flow rates are used, this is for example
described by Wilm and Mann [157].
8.5 PGC and ESI-MS

Ionic and very polar analytes are traditionally separated by ion-exchange chro-
matography or reversed phase chromatography with an ion-pairing agent added
to the mobile phase. When MS is used for detection the mobile phases used in
these chromatographic modes are not at all suitable, due to their involatility
and tendency to raise detection limits by ion suppression. Typical reversed
phase stationary phases such as C18 do not provide sufficient retention of the
ionic or very polar analytes when using MS-friendly mobile phases. Given the
points discussed above, it should be evident that PGC is a very good stationary
phase in conjunction with MS for the determination of polar and structurally
closely related analytes. Polar compounds are well retained on the material and
it is therefore possible to elute them with a high percentage of organic modi-
fier, which facilitates nebulization in ESI and is thus beneficial for the analy-
sis. Further, pH can often be adjusted to optimise detection responses without
compromising phase stability or impairing separation. In studies IV and V strong
55
retention was obtained for the negatively charged analytes at pH 9 with a high
content of organic modifier in the mobile phase. The ability of PGC to separate
so closely related molecules as stereoisomers is essential in the study of the
isoprostanes. Here, separation prior to detection is required as the MS is unable
to distinguish between those molecules as they have exactly the same m/z and
will fragment in the same manner.
8.6 Qualitative and quantitative aspects in ESI-MS

Qualitative and quantitative aspects of ESI-MS are briefly discussed be-
low, and more comprehensive discussions of the topic can be found else-
where [130,158].
A number of mass analyzers are available for LC-MS e.g. single or triple qua-
drupoles, ion trap, time of flight and sector instruments. Apart from separating
charged species according to their mass to charge ratio (m/z), these analyzers
can also be used to fragment analytes. In fragmentation, collision induced dis-
sociation (CID) is accomplished by the impact of accelerated charged species
and surrounding gas molecules. In MS/MS experiments (also called tandem
MS), parent ions, collision energies and collision gas pressure are selected in a
controlled fashion so that CID gives desired information concerning the analytes.
In the work underlying this thesis triple quadrupole instruments were used.
The detection limits, sensitivity and selectivity of a MS analysis performed by
a scanning instrument such as a quadrupole are affected by which ion monitor-
ing mode is used. The selectivity can be largely improved by running the in-
strument in selected ion monitoring mode (SIM), where the scan range is lim-
ited to just one or a few m/z. SIM lowers both the chemical noise and the
detection limit. Selected reaction monitoring (SRM), means that both specific
precursor and product ions are selected (which requires tandem-MS), this pro-
vides even higher selectivity than SIM and often also lower detection limits.
The application of stable isotope-labelled internal standards (IS) that coelute

with the analytes is the most reliable quantification mode in LC-MS since it is
56
unaffected by ion suppression. As labelled standards are sometimes unavail-
able a structural analogue to the analyte can be used instead. In studies IV and
V the IS used was a deuterated analogue to one of the analytes that was also a
structural analogue to the rest of the analytes. If no suitable IS can be found
calibration can also be performed using conventional external calibration, stan-
dard addition or multivariate regression methods.
A general issue to consider in any quantitative analysis is the limit of de-

tection (LOD). The term “sensitive” is sometimes used, maybe loosely, as
meaning “having low detection limits”. However, the term “sensitivity” is
defined as the slope of the calibration curve, and is synonymous with the
ability to discriminate between small differences in concentration. LOD is
the lowest measurable concentration, which is easy to agree on, but defin-
ing it is more complicated. Currie comprehensively discusses the topic in a
recent review [159]. However, in this thesis the simple ratio of 3S/N has
been used since it can be regarded as a sufficient measure of a method’s
capability to detect specific analytes.
57
9. Summary of the papers
9.1 Paper I
The title of Paper I is “Free malondialdehyde determination in rat brain
tissue by capillary zone electrophoresis: Evaluation of two protein removal
procedures”
Paper I is a continuation of a study instigated by Olsson et al. [29]. Several

questions prompted the investigations described in it:
{ Is it possible to simplify sample pre-treatment, and if so, how does this

affect the outcome of the analysis?
{ How quickly can the whole process, from sample collection to final analy-
sis, be performed?
{ What steps in the process are the most important?
{ How easily is artifactual MDA generated from sample constituents?
{ How much MDA can be generated in frozen samples, or when oxidative
stress is induced?
Two protein removal procedures were developed to allow determination of

malondialdehyde in rat brain tissue: one based on ultrafiltration and the other
on precipitation by the addition of acetonitrile. The methods generated similar
results and both were fast and easy to perform. The CE analysis was also very
rapid, since a cationic surfactant, CTAB, was used to reverse the EOF in the
system. MDA migrated as one of the first peaks after only 3 min. The LOD of
MDA was 0.2 µM.
Brain homogenates from three rats were used in the study. Prior to analysis,
four samples from each rat were prepared as follows.
A. In order to prevent oxidation, 0.075 % BHT was added to one aliquot

from each rat. This provided a control brain sample.
61
B. Lipid peroxidation was stimulated in a second aliquot by the addition of
iron.
C. The third brain portion was spiked with an MDA standard to a final concentra-
tion of 5 µM. BHT (0.075 %) was added to prevent oxidation.
D. The fourth aliquot of each set was left untreated.
Both protein removal methods demonstrated that a small amount of MDA was
present in the control samples (A). In the fractions where oxidation was not
prevented (D) there was an approximately five-fold increase in MDA, pro-
duced as an artifact following sampling. The capacity of the samples to gener-
ate MDA was demonstrated in fraction (B) where oxidative stress was induced.
Here, there was a thirty-fold increase in MDA. Electropherograms showing
these results are presented in Figs. 12 and 13.
mAU
DAD1 C, Sig=267,10 Ref=off (STINA\ANA00088.D)
mAU
44
a
33 MDA
22
11
00
0.5 1 1.5
0.5 1
DAD1 C, Sig=267,10 Ref=off (STINA\ANA00090.D) 1.5 22 2.5 2.5
33 min
mAU
44 MDA
b
33
22
11
00
0.5
0.5 1 1 1.5
1.5 22 2.5 2.5
33 min min
Figure 12. Electropherograms of a rat brain homogenate generated from

(a) a sample in which oxidation was prevented by the addition of BHT
(fraction A), (b) the same rat brain homogenate as in (a) but with no BHT
added (fraction D).
62
mAU
DAD1 C, Sig=267,10 Ref=off (STINA\ANA00081.D)
mAU
3535 MDA
3030
2525
2020
1515
1010
55
00
0.5 1 1.5 2 2.5 3 min
0.5 1 1.5 2 2.5 3 min
Figure 13. Electropherogram of rat brain homogenate where lipid

peroxidation has been induced by the addition of iron (fraction B).
Ultrafiltration is assumed to remove all protein-bound forms of MDA, leaving

only the free fraction for analysis. Since the precipitation procedure generated
the same results, it clearly also leaves only the free MDA. The total analytical
processes, including sample homogenization, the addition of antioxidant, pro-
tein removal, separation and detection within the CE system, took less than
20 minutes.
9.2 Paper II
The title of paper II is “Methyl malondialdehyde as an internal standard for the
determination of malondialdehyde”.
During the work described in Paper I, a standard addition technique was used
for the quantitative analysis. Although this worked well, it highlighted the ben-
efits of using an internal standard. The aim of this study was to explore the
possibilities for creating an internal standard that fulfilled the criteria listed in
section 7.6.
63
The paper includes a detailed description of the synthesis of the sodium salt of
methyl-MDA (Me-MDA). The basic procedure involved mixing the commer-
cially available starting material and sodium hydroxide with water in a flask,
heating the mixture in a water bath, then evaporating the resulting solution at
reduced pressure until crystals formed. The crystals (NaMe-MDA) were then
washed and dried. The identity and purity of the synthesis product were tested
using various techniques, including carbon and proton NMR and UV absorp-
tion spectroscopy. Its purity was further verified using systems with different
separation mechanisms such as CE-UV, MEKC-UV, LC-UV and GC-MS. The
sodium salt of Me-MDA appeared to be stable for years when stored in a dry
atmosphere, at room temperature. This was confirmed using UV spectroscopy.
O O
H H
Figure 14. Methyl malondialdehyde, MeMDA.
Me-MDA (Fig. 14.) was assessed as an internal standard for the determination
of MDA in rat brain tissue. The research focused on the use of capillary elec-
trophoresis, using the same system as described in Paper I. The quantification
of MDA in rat brain tissues, using Me-MDA, generated results that were in
accordance with those described in Paper I. Figs. 15 and 16 show typical elec-
tropherograms obtained. To investigate further the versatility of Me-MDA as
an internal standard, an LC-UV method was developed. GC-ECD and GC-MS
were also shown to be possible techniques for analysing MDA, when using
Me-MDA as an internal standard. Pentafluorophenylhydrazine was used for
the derivatization of MDA and Me-MDA prior to GC analysis. Since this
paper was first published, the use of Me-MDA as an internal standard has
been further evaluated and validated by other researchers, who have inves-
tigated its applicability in LC [160,161], GC [162-164], and CE [165] de-
terminations of MDA.
64
mAU
mAU
DAD1 C, Sig=267,10 Ref=off (STINA\IS000030.D)
mAU
88
MDA
7
66 Me-MDA
5
44
22
00
0.5 1 1.5
0.5 1 1.5 22 2.5
2.5 min
min
Figure 15. Electropherogram obtained from a standard solution contain-

ing 10 µM MDA and 12 µM Me-MDA.
mAU
mAU
88
66 a
44 MDA
22
00
0.5 1 1.5 2 2.5 3 3.5 min
mAU
88
MDA
66 b
44
22
00
0.5 1 1.5 2 2.5 3 3.5 min
mAU Me-MDA
88
66 c MDA
44
22
00
0.5
0.5 11 1.5 1.5
22 2.5
2.5 3
3
3.5
3.5 min
min
Figure 16. Electropherograms obtained from ultrafiltered rat brain ho-

mogenate samples: (a) treated with BHT to prevent oxidation, (b) oxi-
dized by the addition of iron, (c) the same sample as in (b), but with the
internal standard Me-MDA added.
65
9.3 Paper III
The title of paper III is “Micellar electrokinetic chromatography separation
and laser induced fluorescence detection of the lipid peroxidation product
4-hydroxynonenal.”
MDA is not the only aldehydic lipid peroxidation product that can act as a
biomarker for oxidative stress and injury. In Paper III, we considered the use of
HNE as a biomarker. This molecule is claimed to be one of the most cytotoxic
species produced during lipid peroxidation in vivo. It has recently been as-
serted to also work as a biological signal in both pathological and physiologi-
cal conditions. The aim of this project was to develop a method for determining
HNE in biological matrices. The inherent properties and low levels of HNE in
samples make it difficult to detect without derivatization. MEKC in combina-
tion with LIF is known to have extraordinarily low detection limits and to be
robust towards biological samples. The derivatization of HNE, using the fluo-
rescent reagent DNSH, was tested and screened for important factors using a
reduced factorial design, and a MEKC method was developed. The reaction
between HNE and DNSH is presented in Fig. 17. The significant factors for
H3C CH3
N
H3C CH3
N
O +
+ H
O S O
H
NH
O S O OH N
NH
H2N H
OH
Figure 17. Reaction between HNE (the analyte) and the derivatization
reagent DNSH.
derivatization were found to be the reaction temperature, the volume fraction

of organic solvent and the amount of acid present. The reaction time and the
amount of excess reagent were factors of lesser importance. Following separa-
tion in the MEKC system, the DNSH-HNE derivative was detected by using
66
Optical bench Direction of light
Full frame argon ion laser

Shortpass filter
CE capillary
Focusing lens To data handling system
Microscope
lens
CE
Equipment
Outlet vial
Detection window
Photomultiplier tube
Longpass filters
Figure 18. The MEKC-LIF setup used in study III.
90000
a
Rel. Fluorescence
60000
30000
min
0
17 18 19 20 21 22 23
90000
b
Rel. Fluorescence
HNE
60000
30000
min
0
17 18 19 20 21 22 23
90000 c
Rel. Flourescence
60000 HNE
30000
min
0
17 18 19 20 21 22 23
Figure 19. Separation of DNSH-derivatized samples. (a) Rat brain homoge-

nate prevented from oxidation by the addition of BHT. (b) Rat brain homo-
genate where lipid peroxidation has been induced. (c) Standard HNE sample.
67
LIF, with the 351-nm line from an argon ion laser. A schematic diagram of the
MEKC-LIF setup is presented in Fig. 18. Samples similar to those described in
Paper I were analysed, i.e. rat brain homogenates where lipid peroxidation was
either prevented or induced. The results, shown in Fig. 19, illustrate that HNE
was identified in the samples subjected to lipid peroxidation but not in the
control samples treated with BHT. Some side reactions of the reagent generate
additional peaks in the chromatogram, but they do not interfere with the HNE
peak. The LOD of the injected sample was 30 nM, corresponding to 0.3 fmol.
Additional work would improve the presented method, including determining
how the derivatization process influences the balance between bound and free
forms of HNE and establishing a method for proper calibration.
9.4 Paper IV
The title of paper IV is “Determination of isoprostanes from Alzheimer
patients using porous graphitic carbon liquid chromatography-tandem mass
spectrometry”
As mentioned earlier aldehydes are not the only products of lipid peroxidation.
Isoprostanes are attracting increasing interest as probably the best available
biomarkers of oxidative damage to lipids. Previous methods for isoprostane
determination were mainly based on GC-MS and were very tedious and manual
labour intensive. Further, there was a disagreement within the literature about
whether or not the urinary F2-isoprostane levels were elevated in Alzheimer’s
disease (AD). The aim of this study was to develop a sensitive, simple and fast
method for the determination of all four classes of F2-isoprostanes in urine
samples from AD patients. As CE is not well suited for this analysis PGC-LC
was used instead.
In the study associated with this paper we had the advantage of being able to
work with samples from thoroughly diagnosed AD patients. The sample pre-
treatment developed was a robotized LLE procedure for extracting the
isoprostanes from urine samples, giving acceptable recoveries in a short time.
68
Further, the F2-isoprostanes could be separated, even though they only differ,
within classes, in the orientation of their –OH groups, by the use of a PGC
column. Separation of the available standards is shown in Fig. 20. During the
development of the chromatography step of the analysis a great difference in
the ability of ACN and methanol to elute isoprostanes retained on PGC was
discovered. For detection, MS-MS was used and class-specific product ions
were selected to discriminate between the four classes. Urinary levels of all
four classes of F2-isoprostanes were examined in both control and AD patient
samples. Figure 21 shows results obtained from a urine sample of an AD pa-
tient. No statistically significant difference could be observed, regarding F2-
isoprostane levels, between control and AD patients, either when one analyte
at a time was compared or when principal component analysis (PCA) was
performed on all of the major peaks in the chromatograms. The limit of detec-
tion (calculated as 3S/N) for the developed system was 0.7 pg injected, corre-
sponding to 35 pg/ml, which was sufficient for detecting the expected levels in
the real samples.
69
HO
HO
Class III
(b) 353 > 193

OH
100 COOH
HO
CH3
HO
HO
HO
HO
HO
(c)
OH
(d)
%
OH
OH
COOH
CH3
COOH
CH3
COOH
CH3
(a)
0
HO
HO
100
357 > 197
(e)
OH
D
D
D
D
COOH
CH3
0 Time
5.00 10.00 15.00 20.00 25.00
Figure 20. Separation of the available F2-isoprostane standards, spiked

in a urine sample, on PGC with MS/MS detection. (a) Isoprostane 15(R)
F2α, (b) Isoprostane F2α, (c) Isoprostane F2β, (d) Prostaglandin F2α
and (e) Isoprostane F2α-d4.
70
100 IS
357 > 197
100 iPF2α 353 > 193

PGF2α
iP15(R) F2α
Class III
%
100 353 > 151
Class V
%
100 353 > 127
Class IV
%
100 353 > 115
Class VI
%
0 Time
10. 0 0 2 0. 00
Figure 21. Four classes of F2-isoprostanes in a urine sample from an

Alzheimer patient. Separation on PGC with MS/MS detection.
71
9.5 Paper V
The title of paper V is “Porous graphitic carbon chromatography-tandem
mass spectrometry for the determination of isoprostanes in human cere-
brospinal fluid”
During the work leading to Paper IV it became evident that urine levels of
isoprostanes were not increased in AD. Most previously published results sug-
gest that cerebrospinal fluid isoprostane levels tend to be elevated in AD. How-
ever, all of the earlier published results were obtained using the time- and
labour intensive CG-MS approach mentioned above. The aims of this investi-
gation were not only to use the developed PGC-MS-MS methodology for CSF
samples, but also to simplify the sample pre-treatment and to be able to inject
an entire 300 µl CSF sample onto a microbore PGC column (1 mm i.d.) with-
out destroying the separation or the column. This was accomplished by develop-
ing a column switching method in which the entire sample was loaded onto a short
PGC column working as a trap for the analytes. The sample was thereby concen-
trated, desalted and to some extent purified before it was switched onto the analyti-
cal column. To further improve the robustness of the developed method the
CSF samples were ultrafiltered before injection. The very low levels of
isoprostanes could be detected in the CSF samples, but the isoprostanes for
which standards were available could not be found in the chromatograms. Fig-
ure 22 shows a chromatogram of a CSF sample to which standard isoprostanes
have been added. The F2α peak and the unknown isoprostane eluting shortly
after it correspond to 32 and 25 pg/ml, respectively. The LOD for the devel-
oped procedure was 14 pg/ml.
The column switching setup used in this work is presented in Fig. 6 in

section 6.5.
72
Figure 22. CSF sample spiked with 100 d, IS
isoprostane standard. (a) 8-iso-15(R)-
Prostaglandin F2α, (b) 8-iso Prostaglan- 357 > 197
din F2α, (c) 8-iso Prostaglandin F2β and
%
(d) 8-iso Prostaglandin F2α-d4 (IS). The
8-iso Prostaglandin F2α peak corresponds
to 32 pg/ml. Separation on PGC with MS/
MS detection. 0
b
100 a
353 > 193
c Class III
%
100
353 > 151
Class V
100
353 > 127
Class IV
100
353 > 115
Class VI
0 Time
10.00 20.00
73
9.6 Unpublished results
Even though brain tissue is a very interesting material for studying oxidative
stress, it is impossible to use for monitoring oxidative status in living humans.
In contrast, blood plasma is easily collected. A typical finger prick yields a
sample amount in the µL range, which is a sufficient volume for CE analysis.
For this reason, experiments using the separation system described in Papers I
and II, were conducted with plasma samples. A portion of 50 µL of plasma
from donor bags was treated with either BHT to prevent oxidation or iron to
promote oxidation. The samples were injected onto the CE system after 5-
minute ultrafiltration through filters with a molecular cutoff at 30 kDa. Fig. 23
shows electropherograms of (a) BHT-plasma, (b) oxidized plasma and (c) spiked
plasma. The MDA peaks are extremely narrow (0.3 s at half peak width), with
platenumbers around 3×106, presumably due to transient ITP generated by the
sample components. The high ionic strength of undiluted plasma should result
in peak broadening, due to the relatively low electric field across the injected
sample zone, but the induced ITP state clearly creates the opposite effect. The
mAU
3
mAU
2.5 a
22
1.5
11 MDA
0.5
00
-0.5
3
mAU 0.5 1 1.5 2 2.5 3 3.5 4
2.5
b MDA
22
1.5
11
0.5
00
-0.5
3
mAU 0.5 1 1.5 2 2.5 3 3.5 4
2.5
c MDA
22
1.5
11
0.5
00
-0.5
0.5 1 1.5 2 2.5 3 3.5 4 min
Figure 23. Electropherograms of ultrafiltered plasma samples. (a) BHT-

treated sample. (b) Oxidized sample. (c) The same sample as in (a) but
spiked to 1 mM with MDA standard solution, the entire peak is not shown
since it reached 13 mAU.
74
chloride content of plasma is, for example, approximately 100 mM, and this
ion may serve as a leading ion generating the noted ITP effects, as discussed in
section 7.3. A rough quantitative analysis suggests a LOD of about 60 nM and
MDA levels in the samples treated with BHT of approximately 0.15 µM. Un-
fortunately, no definite conclusions could be drawn from the MDA levels found
in these donor bag plasma samples. The antioxidant, BHT, was not added until
immediately prior to analysis, so the MDA measured may have been produced
during storage of the bags and, consequently, may not have been endogenous.
The system was unfortunately not as robust as desired. The peak shapes were
affected by unknown sample constituents and the migration times were too
inconsistent. Thus, additional work is required before the method can be used
for reliably monitoring oxidative stress using plasma samples.
75
10. Discussion
The major foci of this thesis and the work underlying it are methods for analysing
biomarkers of oxidative stress and the issues that have to be considered when
measuring them. The development of analytical techniques and methods for
studying biological markers of lipid peroxidation in animals and humans is a
difficult and very interesting task. The suitability of the selected analyte(s) for
the problem addressed always has to be carefully considered. Further, since the
analytes are endogenous, ubiquitous and can be increased by the transforma-
tion of various sample components, samples must also be handled in an appro-
priate way to minimise their artefactual formation. No real “blank” biological
samples are available as they are in, for example, analysis of pharmaceutical
drugs. However, even if the research area is sometimes frustrating there is no
doubt that research into oxidative stress and lipid peroxidation is relevant and
of great interest.
As estimates of oxidative stress in vivo are necessarily indirect, because of the

short-lived nature of free radicals, the search for good biomarkers is important.
The relative stability of aldehydic lipid peroxidation products suggested, at an
early stage, their potential as biomarkers. A wide range of aldehydes can be
formed in biological samples and their concentrations are usually very low,
making analysis intricate. The complexity of the samples and the difficulty in
discriminating between the free and bound forms of the aldehydes make their
analysis still more challenging. Consequently, there is often disagreement within
the scientific literature regarding the relevance or interpretation of these oxida-
tive stress markers. This is typically true for MDA. In general, there is an agree-
ment that MDA is formed in vivo when cell membranes are injured by free
radicals. However, its use as a biomarker for oxidative stress has been ham-
pered by the extensive problems associated with the existing analytical meth-
odology. The discrepancies between the published results are often due to arti-
ficial formation during sample preparation and storage, or to unselective sepa-
ration or detection. In the research associated with Papers I and II, efforts were
76
made to study the factors influencing the analysis of MDA and to determine
how it can be measured while minimizing the risk of producing artifacts. The
properties of CE make this technique well suited for this molecule and its ma-
trices, allowing minimal sample pre-treatment and rapid analysis. In addition,
the internal standard described in Paper II increases the potential for accurate
quantification and, in this case also validation, since the same sample can be
analyzed using a number of other techniques without changing the internal
standard. Me-MDA fulfils all the internal standard criteria outlined in section
7.6 except for its commercial availability. However, the synthesis procedure
described uses inexpensive starting materials, it is simple, fast and results in a
stable product. To monitor oxidative stress in living humans simple sampling
techniques must be employed. One possibility is to measure MDA in blood
plasma. In this case, the small sample volume requirements of CE might allow
the use of finger prick samples. In summary, I would claim that MDA is a good
biomarker for free radical damage to cell membranes, if the analysis is per-
formed judiciously and with suitable methodology.
As for MDA, the determination of HNE is always followed by questions whether

the results represent the “true” levels within the sample. Metabolism and reac-
tions generated by the sample itself, or by the sample handling procedures, can
give rise to erroneous results. However, HNE is a very interesting molecule,
having both inherently toxic effects and the ability to serve as a biomarker. The
development of the new methodology presented here could help avoid the prob-
lems mentioned above.
Isoprostanes are probably the best biomarkers for lipid peroxidation discov-
ered so far, even though they are not completely “ideal” according to the crite-
ria listed in Table 1. The available methodology for their determination is ei-
ther too unspecific or too labour intensive, so to speed up progress in this field,
and further clarify their qualities, there is a need for improved methodology.
The properties of isoprostanes make it evident that CE is not a suitable alterna-
tive for their determination. Narrow-bore LC using porous graphitic carbon as
stationary phase in combination with tandem MS is a much more attractive
77
alternative. With the methodology developed in the studies associated with
this thesis the analysis of biological fluids such as urine and CSF for the deter-
mination of F2-isoprostanes can be accomplished in a simple and fast manner
without forfeiting the quality of the analysis.
It has been repeatedly pointed out in this thesis that measurement of lipid
peroxidation is a difficult task. Probably there is no perfect, ideal marker that is
satisfactory in all respects, nor will a perfect analytical method for its determi-
nation ever be developed. However, the analytical tools can always be enhanced
and the usage as well as the interpretation of the biological markers can be
further improved. Capillary electrophoresis requires only very simple sample
purification. When the sample amount is limited, as in the case of brain samples
or small blood samples, the minute injection volumes required for CE is an
asset. Analysis speed can be very fast if the systems are used optimally. If CE
fails as a suitable analytical technique, LC in combination with tandem MS
may offer a very powerful alternative. If the analytes are polar and/or structur-
ally closely related, as is the case with isoprostanes, PGC can provide excellent
retention and separation properties. However, it should be noted that method
development on PGC is not as straightforward as it is, for example, when using
C18. My impression of PGC is that it sometimes leaves you confused but that
it can be a very useful and fascinating tool to solve problematic separations.
An interesting question is whether free radicals and the degradation products

formed are the cause of a disease or merely symptoms. Most of the methods
developed, including those presented here, can only demonstrate that free radi-
cal damage is occurring, but not whether it is a cause or a consequence of
disease. The biomarkers discussed in this thesis cannot be used as sole markers
of a certain disease. Instead, they can be used to study if lipid peroxidation is
associated with a disease, progression of disease or to follow the effects of a
certain treatment.
Attempts to find new and better biomarkers for oxidative stress and lipid
peroxidation will continue. Two approaches can be used to discover putative
78
biomarker candidates. The first is the traditional way, in which collected expe-
rience and knowledge concerning the pathways and chemistry of lipid
peroxidation in vivo are used to select new biomarker candidates. Secondly, a
multivariate approach, where total compositions of metabolites in biological
fluids are screened for divergence in a less prejudiced manner, can be used to
find potential markers. After the hypothetical markers have been selected by
either of the two approaches, more in-depth studies are needed to further evalu-
ate candidate substances. I believe that the fastest way to progress in the field
of novel biomarkers is by collaboration between people skilled in lipid
peroxidation, traditional analytical chemistry and chemometrics.
Finally, as Halliwell, one of the main authorities in the field of lipid peroxidation,
points out; “think how, what and how much” [2]. Whatever method is used to
measure oxidative damage, it is necessary to think carefully about how it works,
factors that are likely to confound the results and how quantitative it can be. If
attention is paid to those issues and suitable techniques and methods are used,
the risks of erroneous results and interpretations are minimized.
79
11. Acknowledgements
När det är dags att skriva sin “acknowledgement” så inser man verkligen vilka
hjälpsamma, roliga och smarta människor man har haft runt sig under sina år
som doktorand. Det är många personer som jag stött på under arbetets gång
som förtjänar ett stort tack.
Bo Karlberg min handledare, tack Bosse för att du ville ha mig som doktorand
– jag visste inte ens själv att jag ville doktorera innan du frågade. Jag uppskattar
verkligen ditt stöd och din positiva anda.
Alla er som jag har samarbetat med under åren; Joanna Oreskär (Olsson), Ann-
Caroline Nordström, Fredrik Åberg, Gunnar Thorsén, Hans Basun och Staffan
Schmidt – tack för gott samarbete och för att ni har lärt mig så mycket. Ett
särskilt tack till Staffan för att jag fick komma och arbeta på Astrazeneca i
Södertälje och för att du gjorde tiden där så rolig.
Mina rumskamrater, Malin, Jenny, Magnus och utflugna Karin, jag kan inte
tänka mig bättre personer att dela rum med. Det är alltid någon som har tid att
lyssna, förklara, skämta, fika, osv.
Alla ni som var doktorander på institutionen när jag började läsa kurser inom
analytisk kemi bl.a. Anders Ch, Elisabeth, Gunnar, Joanna, Jonas B, Ludvig,
Magnus A, Petr och Åsa, – det var ni som fick mig att trivas och vilja stanna
kvar.
Ni som är/har varit doktorander samtidigt som mig, Bodil, Caroline, Christoffer,
Cristina, Helena I, Helena H, Johanna, Kent, Leila, Magnus E, Nana, Ove,
Petter, Ragnar, Sindra, Stina M, Thorvald och Yvonne - det är mycket tack
vare er som det har varit riktigt kul att gå till jobbet varje dag.
80
All nuvarande och före detta personal på institutionen, Anders C, Anita,
AnneMarie, BengtOve, Björn, Carlo, Conny, Eva, Håkan, Lena, Ralf, Ramon,
Roger, Rolf, Sven, Ulla och Ulrika – tack för all hjälp och för att ni bidrar till
vår trevliga atmosfär.
Ett särskilt tack till Gunnar, Jonas B, Malin, Ralf och Ulrika för att ni läst och
gett mig värdefulla kommentarer på manuskriptet till avhandlingen.
Jonas R – utan din hjälp så skulle säkerligen min dator havererat och min
avhandling skulle se tokig ut.
Alla kompisar som inte bryr sig om analytisk kemi – ni gör min värld lite
större.
Mamma Sonja och pappa Tommy, tack för att ni alltid ställer upp för mig i alla
lägen. Tack för bilskjuts till alla möjliga och omöjliga ställen, för all god mat
och för alla goda råd. Svärmor Anna, svärfar Lennart och svägerska Paulina,
tack för att ni är så gulliga och tar hand om mig och min lilla familj när det
behövs.
Sist men viktigast - mina alldeles, alldeles underbara killar, Fredrik och lille
Alvin – ni är bäst !
81
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DETERMINATION OF BIOMARKERS FOR

LIPID PEROXIDATION AND OXIDATIVE STRESS

− Development of analytical techniques and methods −

Kristina Claeson Bohnstedt

Akademisk avhandling som för framläggande av filosofie doktorsexamen vid

The measurement of low molecular weight biomarkers of lipid peroxidation

Paper I focuses on malondialdehyde, describing and evaluating two new sim-

Paper II describes the synthesis, characterization and implementation of a new

Paper IV is focused on the development of a new methodology for determin-

Paper V describes porous graphitic carbon chromatography-tandem mass spec-

This thesis is based upon the following publications

I Kristina Claeson, Fredrik Åberg, Bo Karlberg.

II Kristina Claeson, Gunnar Thorsén, Bo Karlberg.

III Kristina Claeson, Gunnar Thorsén, Bo Karlberg.

V Kristina Claeson Bohnstedt, Bo Karlberg, Hans Basun,

Paper not included in this thesis

Joanna Olsson, Kristina Claeson, Bo Karlberg, Ann-Caroline

Oxygen-dependent deterioration of fats and oils, also called rancidity, causes

The analytical challenges involved in determining minute concentrations of

In 2004 Halliwell and Whiteman presented a list of criteria (shown in Table 1)

4.1 Brain tissue

4.4 Cerebrospinal fluid

Cell Phospholipid bilayer

Figure 1. The outer membrane of a cell, comprising a 60-100 Å thick

LOO O The peroxyl radical abstracts

O Lipid hydroperoxide Fragmentation to

Figure 2. Representation of the initiation and propagation reactions of

Some researchers claim that malondialdehyde is less commonly monitored

5.1.2 Hydroxynonenal – formation of, properties and analytical methods

As in MDA analysis, measures must be taken to minimize unintentional changes

Elevated levels of F2-isoprostanes have been reported in various physiological

Paper Analyte Sample type Pre-treatment Separation Detection

I MDA Rat brain tissue Precipitation/ CE UV

II MDA Rat brain tissue Ultrafiltration CE/GC/LC UV/EC/MS

Unp.a MDA Human plasma Ultrafiltration CE/ITP UV

III HNE Rat brain tissue Derivatization MEKC LIF

IV IsoP Human urine Extraction PGC-LC ESI-MS

V IsoP Human CSF Ultrafiltration/ PGC-LC ESI-MS

As a biological matrix contains a myriad of compounds of various types and

{ Removal of unwanted compounds in the sample matrix, e.g. proteins

If precipitation or ultrafiltration doesn’t generate sufficiently clean and

6.3 Liquid–liquid extraction

6.5 Column switching

Figure 6. The column switching setup used in study V.

An increasingly popular way to facilitate the direct injection of biological flu-

6.7 Other techniques

The velocity of an ion in an electric field is given by

where v = ion velocity, µe = electrophoretic mobility and E = applied electric field.

The mobility µe of a given ion in a given medium is a constant which is charac-

and a frictional force FF that is given (for a spherical hydrated ion) by

where q = ion charge, η = solution viscosity, r = ion radius and

7.3 In-capillary sample concentration

7.3.2 Isotachophoretic stacking

The molecules to be separated are sandwiched between a leading electrolyte

Figure 8. The different transport mechanisms involved in MEKC

7.6 Qualitative and quantitative aspects in CE

Quantitative analysis in CE is similar to that of LC and is commonly performed

{ It must be well separated from solutes and interferences in the samples.

8.1 The effect of column diameter in LC

Table 3 Names and definitions for LC techniques

3.2 – 4.6 mm 0.5 – 2.0 ml/min conventional LC

8.2 Porous graphitic carbon as packing material in LC

Figure 9. PGC structure in comparison with traditional silica-based C18.

8.2.2 Performance of PGC