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DETERMINATION OF BIOMARKERS FOR

LIPID PEROXIDATION AND OXIDATIVE STRESS

− Development of analytical techniques and methods −

Kristina Claeson Bohnstedt

Doctoral ThesisDepartment of Analytical Chemistry

Stockholm UniversityStockholm, 2005

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Akademisk avhandling som för framläggande av filosofie doktorsexamen vidStockholms universitet offentligen försvaras i Magnélisalen, Kemiskaövningslaboratoriet, Svante Arrhenius väg 12, torsdagen den 27 januari 2005.

ISBN 91-7265-988-2© Kristina Claeson Bohnstedt, 2005Intellecta DocuSys AB, Sollentuna

Doctoral Dissertation, 2005Department of Analytical ChemistryStockholm University106 91 Stockholm

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Table of contents

Abstract

Preface - List of papers

Part One

1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 What is a biomarker? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Biological samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4.1 Brain tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164.2 Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164.3 Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174.4 Cerebrospinal fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5 Lipid peroxidation biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185.1 Aldehydes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5.1.1 MDA - formation, properties and analysis . . . . . . . . . . . . . . . 205.1.2 Hydroxynonenal - formation, properties and analysis . . . . . . 22

5.2 Isoprostanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235.2.1 Isoprostanes - formation, properties and analysis . . . . . . . . . . 23

Part Two

6 Preparation of biological samples for CE or LC . . . . . . . . . . . . . . . . . . 296.1 Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306.2 Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316.3 Liquid-liquid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326.4 Solid phase extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336.5 Column switching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336.6 Derivatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346.7 Other techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

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7 Capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367.1 Introduction to CE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367.2 Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397.3 In capillary sample concentration . . . . . . . . . . . . . . . . . . . . . . . . . 39

7.3.1 Field amplified stacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407.3.2 Isotachophoretic stacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

7.4 Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417.5 Detection in CE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437.6 Qualitative and quantitative aspects in CE . . . . . . . . . . . . . . . . . . 44

8 LC-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468.1 The effect of column diameter in LC . . . . . . . . . . . . . . . . . . . . . . 468.2 Porous graphitic carbon as a packing material in LC . . . . . . . . . 48

8.2.1 Manufacture and structure of PGC . . . . . . . . . . . . . . . . . . . . . 488.2.2 Performance of PGC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

8.3 ESI as an ionization technique for MS detection . . . . . . . . . . . . . 518.4 Ion supression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548.5 PGC and ESI-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558.6 Qualitative and quantitative aspects in ESI-MS . . . . . . . . . . . . . 56

Part Three

9 Summary of the papers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619.1 Paper I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619.2 Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639.3 Paper III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669.4 Paper IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689.5 Paper V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729.6 Unpublished results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

10 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7611 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8012 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.

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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.

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Preface

This thesis is divided into three parts. The first presents an introduction tooxidative stress, and then discusses types of biological samples and compoundsthat 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 partdiscusses 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 capillaryzone 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 ofmalondialdehyde.Journal of Chromatography B, 751 (2001) 315-323.

The author was responsible for the idea, the laboratory work (in collabo-ration with Gunnar Thorsén), data evaluation (except for the NMR andMS 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.Journal of Chromatography B, 763 (2001) 133-138.

The author and Gunnar Thorsén contributed equally to the laboratory work,data evaluation and writing of this paper.

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IV Kristina Claeson Bohnstedt, Bo Karlberg, Lars-Olof Wahlund,Maria Eriksdotter Jönhagen, Hans Basun, Staffan Schmidt.Determination of isoprostanes in urine samples from Alzheimer patientsusing porous graphitic carbon liquid chromatography-tandem mass spec-trometry.Journal of Chromatography B, 796 (2003) 11-19.

The author and Staffan Schmidt were jointly responsible for the idea, andthe author was responsible for the laboratory work, data evaluation andwriting of this paper.

V Kristina Claeson Bohnstedt, Bo Karlberg, Hans Basun,Staffan Schmidt.Porous graphitic carbon chromatography-tandem mass spectrometry forthe detection of isoprostanes in human cerebrospinal fluidJournal of Chromatography B, submitted.

The author and Staffan Schmidt were jointly responsible for the idea, andthe author was responsible for the laboratory work, data evaluation andwriting of this paper.

Paper not included in this thesis

Joanna Olsson, Kristina Claeson, Bo Karlberg, Ann-CarolineNordström.Determination of indole-3-acetic acid and indole-3-acetylaspartic acid inpea plant with capillary electrophoresis and fluorescence detection.Journal of Chromatography A, 796 (1998) 231-239.

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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

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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.

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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.

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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.

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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.

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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.

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5. Lipid peroxidation biomarkers

The targets of attack in lipid peroxidation are membrane lipids that surroundcells and cell organelles. Fig. 1 shows a schematic diagram of the phospholipidbilayer of the outer membrane of a cell. When the lipids in the bilayer arechanged oxidatively, not only are potentially harmful molecular species formed,but also the membrane fluidity is affected, which may lead to changes in or lossof cell functions or even cell death.

5.1 AldehydesThe general lipid peroxidation process is illustrated in Fig. 2. The initiation oflipid peroxidation starts with a free radical attack. Hydrogen is abstracted fromthe target fatty acid (LH) to form a fatty acid radical (L·). The carbon radical isusually stabilized by a molecular rearrangement to form a conjugated diene.Then, a peroxyl radical (LOO·) is produced by oxygen uptake. The peroxylradical can abstract a hydrogen atom from another PUFA, thus forming a fattyacid hydroperoxide (LOOH). Hence, a new oxidation chain is initiated, in whichnew fatty acid radicals are generated. This is called the propagation stage oflipid peroxidation. The lipid hydroperoxides formed can then decompose toform, among other species, a great variety of aldehydes. Aldehydes are rela-

CellPhospholipid bilayer

Phospholipid

O

O

H2C

H2C

O

PO4

CH3

Base

CH3

O

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

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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].

O2

Rearrangement to

conjugated diene

Oxygen uptake

Hydrogen abstraction

The peroxyl radical abstracts

H from another PUFA causing an

autocatalytic chain reaction.

Lipid hydroperoxide

Cyclic peroxide

Cyclic endoperoxide

Fragmentation to

aldehydes e.g.

malondialdehyde and

hydroxynonenal.

L

LOOH

LOO

LH

C

C

O

O

O

O

H

H

H

Part of fatty acid with three double bonds

Figure 2. Representation of the initiation and propagation reactions oflipid peroxidation.

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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].

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-

O O

H H

H

H

H

OO

pKa4.5

Figure 3. Malondialdehyde, MDA.

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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.

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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).

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].

H

O

OH

Figure 4. 4–hydroxy-2-trans-nonenal, HNE.

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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 F2αformed 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

24

- 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 ofthe F2-isoprostanes from AA is outlined in Fig. 5. Following the discovery ofthe F2-isoprostanes, it has been shown that the isoprostane pathway can providea route for the generation of other classes of isoprostanes from unsaturatedfatty acids other than AA that have at least three double bounds. Examples arethe “neuroprostanes” originating from docohexaenoic acid (22:6) [44]. Twostructural features distinguish the F2-isoprostanes from their enzymaticallyderived relatives: the hydroxyl groups on their prostane ring have a cisorientation, and their side chains predominantly have a cis orientation in relationto the prostane ring.

COOH

HO

HO

COOH

OH

Class III

HO

HOOH

COOH

Class V

HO

HO

COOH

OH

Class VI

HO

HO

COOH

OH

Class IV

PGCOX

Arachidonic acid

FR + O2

FR + O2

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 fromarachidonic acid by the cyclooxygenase (COX) pathway.

25

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].

29

6. Preparation of biological samples forCE 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 UVUltrafiltration

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-MSDirect 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.

30

The general reasons for subjecting biological samples to preparatory treatments(for the determination of low molecular weight molecules) are similar for CEand LC, and include;

Removal of unwanted compounds in the sample matrix, e.g. proteinsand salts.Enrichment of the analyte.Facilitation of detection, i.e derivatization.

The sample components that cause the most problems in both CE and LC areoften proteins, which readily adsorb onto the surface of the CE capillary walls,thereby impairing or completely destroying the separation. The flow throughthe capillary can even be irreversibly stopped. Another problem is when broadprotein peaks interfere with the detection of the desired analyte peaks in theelectropherogram. In LC, high protein contents in a sample can give rise toclogging of the column or irreversibly affect the stationary phase. Studies ondeproteinization have been published for both CE [63] and LC applications[64]. Precipitation and ultrafiltration are two commonly used modes of proteinremoval and are discussed below.

6.1 PrecipitationPrecipitation is a fast and simple means for protein removal and many samplescan be processed at the same time. It can be accomplished by the addition oforganic solvents, salts or acids to the sample followed by centrifugation of theprecipitate. The clear supernatant is then either injected directly, or subjectedto further preparation steps. Examples of precipitating agents used in literatureinclude 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 solventprecipitation is the dilution of the sample. However, the organic solvent lowersthe 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

31

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-

32

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].

33

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

34

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.

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

Injector

Pump I

Trap

Waste

Pump IIMS-MS

Analytical Column

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

35

(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.

36

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

37

Figure 7. Schematic representation of a CE system. (a) EOF in the nor-mal direction. (b) Reversed EOF.

38

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 π η r v (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)

39

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.

40

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

41

thus develops. When an ion diffuses into a neighbouring zone, its velocitychanges and it returns to its original zone. Since the current is constant there isa constant ratio between the concentration and the mobility in each zone. Theconcentration in each zone is determined by the concentration of the leadingelectrolyte. 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 iscalled transient ITP. If a sample contains high concentrations of a species thatcan serve as a leading electrolyte, ITP effects can occur. This state can eitherbe 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 inRefs. [111,112]. An ITP state was probably induced during the analysis ofthe plasma samples as discussed in section 9.6. In this case, the analytepeak was equivalent to nearly 3×106 theoretical plates, which is signifi-cantly more than can be explained by conventional CE stacking theory insuch a high ionic strength sample.

7.4 SeparationCE 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 ofinterest are small and/or highly charged a faster separation can be achieved byreversing the EOF, as shown in Fig. 7b. In this mode the directional vectors ofelectrophoretic migration and EOF combine, and thus increase the speed of theanalysis. Reversal of EOF was demonstrated by Zare and co-workers for deter-mination of low molecular weight carboxylic acids [113] and was also earlierpresented by Terabe [114] and Tsuda [115]. Reversal of the EOF can be ac-complished by adding quaternary amines such as the cationic surfactant cetyltrimethyl ammonium bromide (CTAB) to the BGE. CTAB monomers adhere

42

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.

micelle

analyte

EOF

K analyte µ

µ

Figure 8. The different transport mechanisms involved in MEKC(anionic surfactant).

43

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

44

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

45

area. Dividing the integrated peak area by migration time corrects for this, andwas 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 theareas of the analyte peaks. Probably the most reliable method of calibrationis 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. Internalstandard methodology was used in the research associated with Paper II. In[98] the authors define the following criteria for the selection of a suitableinternal 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 theelectropherogram.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 andany components in the samples.It should have a detector response similar to that of the solutes ofinterest.It should be chemically similar to the solutes of interest if the samplesare 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 internalstandard height or area ratio of approximately unity.

46

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 LC1.5 – 3.2 mm 100 – 500 µl/min microbore LC0.5 – 1.5 mm 10 – 100 µl/min micro LC150 – 500 µm 1 – 10 µl/min capillary LC10 – 150 µm 10 – 100 nl/min nano LC

47

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/(d

2)2, where d

1

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

48

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.

49

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

OH

OH

PGC Silica-C18

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

50

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-Dspatial arrangement. Early work on the retention by PGC of simple aliphaticand 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 polaranalyte with the polarizable surface of graphite. It is this second mechanismthat 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 smallaromatic compounds and found that retention was increased when one hydro-gen was substituted by a polar functional group [148]. Several other researchgroups have also studied the retention of polar analytes on PGC [149]. PGChas layers of hexagonally arranged carbon atoms in a two-dimensional struc-ture, where the atoms are in sp2-hybrid states. The delocalized π-electrons andthe high polarizability of the large layers of carbon are features that govern thesecond retention mechanism. In Fig. 10 the charge-induced dipole that appearswhen a polar analyte approaches the surface of the graphite is shown.

As PGC is inert to pH extremes and solvents, method development on PGC canbe 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-

Figure 10. Induced dipole when a point charge approaches a PGC surface.

51

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

52

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”.

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-

To MS analyzer

Taylor cone

Flow of liquid

Atmospheric pressure

Oxidation

Reduction

Electrons

Electrons

10-5 - 10-6 torr

Figure 11. Schematic diagram of the electrospray process in positiveion mode.

53

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-

54

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

55

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

56

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

57

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.

61

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.

62

B. Lipid peroxidation was stimulated in a second aliquot by the addition ofiron.

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 waspresent in the control samples (A). In the fractions where oxidation was notprevented (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 showingthese results are presented in Figs. 12 and 13.

min0.5 1 1.5 2 2.5 3

mAU

0

1

2

3

4

DAD1 C, Sig=267,10 Ref=off (STINA\ANA00088.D)

MDA

min0.5 1 1.5 2 2.5 3

mAU

0

1

2

3

4

DAD1 C, Sig=267,10 Ref=off (STINA\ANA00090.D)

MDA

a

b

0.5 1 1.5 2 2.5 3

0.5 1 1.5 2 2.5 3

min

0

1

2

3

4

0

1

2

3

4

mAU

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 BHTadded (fraction D).

63

Ultrafiltration is assumed to remove all protein-bound forms of MDA, leavingonly the free fraction for analysis. Since the precipitation procedure generatedthe same results, it clearly also leaves only the free MDA. The total analyticalprocesses, including sample homogenization, the addition of antioxidant, pro-tein removal, separation and detection within the CE system, took less than20 minutes.

9.2 Paper IIThe title of paper II is “Methyl malondialdehyde as an internal standard for thedetermination of malondialdehyde”.

During the work described in Paper I, a standard addition technique was usedfor 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 thepossibilities for creating an internal standard that fulfilled the criteria listed insection 7.6.

min0.5 1 1.5 2 2.5 3

mAU

0

5

10

15

20

25

30

35

DAD1 C, Sig=267,10 Ref=off (STINA\ANA00081.D)

MDA

0.5 1 1.5 2 2.5 3 min

0

5

10

15

20

25

30

35

mAU

Figure 13. Electropherogram of rat brain homogenate where lipidperoxidation has been induced by the addition of iron (fraction B).

64

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.

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.

O O

HH

Figure 14. Methyl malondialdehyde, MeMDA.

65

min0.5 1 1.5 2 2.5

mAU

0

1

2

3

4

5

6

7

8

DAD1 C, Sig=267,10 Ref=off (STINA\IS000030.D)

0

2

4

6

8

mAU

MDA

Me-MDA

1 0.5 1.5 2 2.5 min

mAU

Figure 15. Electropherogram obtained from a standard solution contain-ing 10 µM MDA and 12 µM Me-MDA.

min0.5 1 1.5 2 2.5 3 3.5

mAU

0

2

4

6

8

min0.5 1 1.5 2 2.5 3 3.5

mAU

0

2

4

6

8

min0.5 1 1.5 2 2.5 3 3.5

mAU

0

2

4

6

8

0.5 1 1.5 2 2.5 min

0

2

4

6

8

0

2

4

6

8

0

2

4

6

8

mAU

MDA

MDA

MDA

Me-MDA

a

b

c

3.53

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 theinternal standard Me-MDA added.

66

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

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

S OO

NHNH

2

NCH

3CH

3

S OO

NH

NCH

3CH

3

N

H

+O

H

OH

H+

OH

Figure 17. Reaction between HNE (the analyte) and the derivatizationreagent DNSH.

67

Optical bench

Full frame argon ion laser

CE

Equipment

CE capillaryFocusing lens

Shortpass filter

Longpass filters

Outlet vial

Microscope

lens

Photomultiplier tube

To data handling system

Direction of light

Detection window

Figure 18. The MEKC-LIF setup used in study III.

0

30000

60000

90000

17 18 19 20 21 22 23

min

Re

l. F

luo

resce

nce

0

30000

60000

90000

17 18 19 20 21 22 23

min

Re

l. F

luo

resce

nce

0

30000

60000

90000

17 18 19 20 21 22 23

min

Re

l. F

lou

resce

nce

HNE

HNE

a

b

c

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.

68

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.

69

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.

70

5.00 10.00 15.00 20.00 25.00Time0

100

%

0

100

%

353 > 193

Class III

357 > 197

HO

CO

OHCH

3

OH

HO

HO

HO

OH

CH

3

CO

OH

HO

HO

CO

OHCH

3

OH

HO

HO

CO

OH

OH

CH

3

DDD

D

HO

HO

CO

OH

OH

CH

3

(a)

(b)

(c)

(d)

(e)

Figure 20. Separation of the available F2-isoprostane standards, spikedin a urine sample, on PGC with MS/MS detection. (a) Isoprostane 15(R)F2α, (b) Isoprostane F2α, (c) Isoprostane F2β, (d) Prostaglandin F2αand (e) Isoprostane F2α-d4.

71

10. 00 20. 00Time0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

357 > 197IS

353 > 193

Class III

353 > 151

Class V

353 > 127

Class IV

353 > 115

Class VI

iP15(R) F2α

iPF2αPGF2α

Figure 21. Four classes of F2-isoprostanes in a urine sample from anAlzheimer patient. Separation on PGC with MS/MS detection.

72

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.

73

10.00 20.00Time0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

ab

c

d, IS

357 > 197

353 > 193Class III

353 > 151Class V

353 > 127Class IV

353 > 115Class VI

Figure 22. CSF sample spiked withisoprostane standard. (a) 8-iso-15(R)-Prostaglandin F2α, (b) 8-iso Prostaglan-din F2α, (c) 8-iso Prostaglandin F2β and(d) 8-iso Prostaglandin F2α-d4 (IS). The8-iso Prostaglandin F2α peak correspondsto 32 pg/ml. Separation on PGC with MS/MS detection.

74

9.6 Unpublished resultsEven though brain tissue is a very interesting material for studying oxidativestress, it is impossible to use for monitoring oxidative status in living humans.In contrast, blood plasma is easily collected. A typical finger prick yields asample amount in the µL range, which is a sufficient volume for CE analysis.For this reason, experiments using the separation system described in Papers Iand II, were conducted with plasma samples. A portion of 50 µL of plasmafrom donor bags was treated with either BHT to prevent oxidation or iron topromote oxidation. The samples were injected onto the CE system after 5-minute ultrafiltration through filters with a molecular cutoff at 30 kDa. Fig. 23shows electropherograms of (a) BHT-plasma, (b) oxidized plasma and (c) spikedplasma. The MDA peaks are extremely narrow (0.3 s at half peak width), withplatenumbers around 3×106, presumably due to transient ITP generated by thesample components. The high ionic strength of undiluted plasma should resultin peak broadening, due to the relatively low electric field across the injectedsample zone, but the induced ITP state clearly creates the opposite effect. The

mAU

-0.5

0

0.5

1

1.5

2

2.5

mAU

-0.5

0

0.5

1

1.5

2

2.5

mAU

-0.5

0

0.5

1

1.5

2

2.5

0

1

2

3

0

1

2

3

0

1

2

3

mAU

MDA

MDA

MDA

a

b

c

0.5 1 1.5 2 2.5 3 3.5 4 min

0.5 1 1.5 2 2.5 3 3.5 4

0.5 1 1.5 2 2.5 3 3.5 4

Figure 23. Electropherograms of ultrafiltered plasma samples. (a) BHT-treated sample. (b) Oxidized sample. (c) The same sample as in (a) butspiked to 1 mM with MDA standard solution, the entire peak is not shownsince it reached 13 mAU.

75

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.

76

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

77

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

78

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

79

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.

80

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.

81

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 !

82

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