Department of Physics, Chemistry and Biology

Bachelor’s Thesis

Sample preparation of 8-hydroxy-2’-deoxyguanosine with solidphase extraction methodology based on molecular imprinting

polymers and conventional silica based phases

Nina Bergman

June 14, 2011

LITH-IFM-G-SE-11/2468

Linkopings University Department of Physics, Chemistry and Biology581 83 Linkoping

Department of Physics, Chemistry and Biology

Sample preparation of 8-hydroxy-2’-deoxyguanosine with solidphase extraction methodology based on molecular imprinting

polymers and conventional silica based phases

Nina Bergman

Thesis work done at arbets- och miljomedicin, the University Hospital inLinkoping

June 14, 2011

Supervisor: Per Leanderson

Examiner: Stefan Svensson

Linkopings University Department of Physics, Chemistry and Biology581 83 Linkoping

Abstract

The aim of this study was to develop methods for sample preparation for 8-OHdG in bloodplasma samples with different solid phase extraction techniques using HPLC with an elec-trochemical detector. The solid phase extraction cartridges used were Chromabond® C18,Oasis® MAX, and three types of SupelMIP™ cartridges for chloramphenicol, riboflavin, andnitroimidazoles. The SupelMIP™ cartridges are based on molecularly imprinted polymers-technique. The separation of 8-OHdG in samples extracted from blood plasma was carriedout with a Thermo Quest Hypersil Division ODS column (250 mm × 4 mm, 3µm I.D.)and methanol:buffer (10:90, v/v) as mobile phase. Recovery and selectivity was studied forthe different solid phase extraction methods. The highest recovery was obtained using theChromabond C18 cartridge with a recovery of 92%, and CV coefficient 9.5% (n = 4). 8-OHdGcould not be extracted on MIP-cartridges for chloramphenicol or riboflavin, but was retainedon MIP columns for nitroimidazoles, and the highest recovery was 49%.

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Acknowledgments

I would like to thank my supervisor Per Leanderson for all my newfound skills that I obtainedduring the project, and also the rest of the employed staff at Arbets- och miljomedicin. I wouldalso like to thank my examiner Stefan Svensson who always took time to help and supportme during the whole project. Thank you for all the rewarding discussions about everything.Furthermore, I want to thank Roger Savenhed and Martin Josefsson at Linkopings Universitywho taught me so much about analytical chemistry. Your courses provided me with enoughknowledge to work on this project.

I would also like to thank Johan Thim and Kristin Bergman for their support, AleksandraKyslychenko for being a good friend, and Lorentz Larsson for helping me coming in contact withArbets- och miljomedicin. And last, but not least, Geertruida van Maldegem for introducingme to the world of chemistry.

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Abbrevations

8-OHdG 8-hydroxy-2�-deoxyguanosineCSSA citric acid, sodium acetate trihydrate, sodium hydroxide, acetic acidDNA deoxyribonucleic acidHDV hydrodynamic voltammogramHPLC high-performance liquid chromatographyMAX mixed-mode anion-exchangerMIP molecularly imprinted polymersROS reactive oxygen speciesSPE solid phase extraction

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Contents

Contents iv

1 Introduction 11.1 8-Hydroxy-2’-Deoxyguanosine . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Solid Phase Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Molecularly Imprinted Polymers . . . . . . . . . . . . . . . . . . . . . . . 31.4 High Performance Liquid Chromatography . . . . . . . . . . . . . . . . . 4

1.4.1 Stationary and Mobile Phases . . . . . . . . . . . . . . . . . . . . 51.4.2 Electrochemical Detector . . . . . . . . . . . . . . . . . . . . . . . 5

1.5 The Aim of This Project . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2 Materials and Methods 62.1 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.2 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.3 High Performance Liquid Chromatography . . . . . . . . . . . . . . . . . 7

2.3.1 The Electrochemical Detector . . . . . . . . . . . . . . . . . . . . 72.4 Sample Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.4.1 Aqueous Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.4.2 Blood Plasma Samples . . . . . . . . . . . . . . . . . . . . . . . . 7

2.5 Traditional Solid Phase Extraction Methods . . . . . . . . . . . . . . . . 72.5.1 Octadecyl Modified Silica Phase . . . . . . . . . . . . . . . . . . . 72.5.2 Mixed-Mode Anion-Exchanger . . . . . . . . . . . . . . . . . . . . 9

2.6 Commercial Molecularly Imprinted Polymers . . . . . . . . . . . . . . . . 9

3 Results 113.1 HPLC Method Development . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.1.1 Analytical Recovery . . . . . . . . . . . . . . . . . . . . . . . . . 123.2 Traditional Solid Phase Extraction . . . . . . . . . . . . . . . . . . . . . 13

3.2.1 Octadecyl Modified Silica Phase . . . . . . . . . . . . . . . . . . . 133.2.2 Mixed-Mode Anion-Exchanger . . . . . . . . . . . . . . . . . . . . 14

3.3 Commercial MIP Cartridges . . . . . . . . . . . . . . . . . . . . . . . . . 15

4 Discussion 174.1 HPLC Method Development . . . . . . . . . . . . . . . . . . . . . . . . . 174.2 Octadecyl Modified Silica Phase . . . . . . . . . . . . . . . . . . . . . . . 174.3 Mixed-Mode Anion-Exchanger . . . . . . . . . . . . . . . . . . . . . . . . 18

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4.4 Commercial Molecularly Imprinted Polymers . . . . . . . . . . . . . . . . 184.5 Conclusions and Future Work . . . . . . . . . . . . . . . . . . . . . . . . 19

Bibliography 21

Appendices 24

A Preparation of the CSSA Buffer 24

B Tables 25B.1 Volume Table for SPE with C18 . . . . . . . . . . . . . . . . . . . . . . . 25B.2 Repeated Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

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Chapter 1

Introduction

1.1 8-Hydroxy-2’-Deoxyguanosine

Reactive oxygen species (ROS) are formed in our cells all the time. This is a normal,important, and necessary process for the killing of invading microorganisms[1]. Normally,our bodies have a defense system (antioxidants) that keeps our levels of ROS balanced.However, carcinogenic substances like tobacco smoke, or ionisation radiation, amongstother things, can also produce ROS[2]. An overproduction can lead to a state of oxidativestress. This stress is linked to an elevated risk of oxidative damage to our DNA when thedefense system is out of balance. Oxidative damage to DNA can contribute to severaldiseases such as cancer. However, our bodies try to repair the damaged DNA by enzymereactions[3].

One of the most reactive oxidants is the hydroxyl radical, and it can attack the nu-cleotides in our DNA strands, proteins, and lipids[2–4]. The hydroxyl radical can begenerated, e.g., after radiolysis of water caused by ionisation radiation, or after metal-driven dissociation of hydrogen peroxide. When the hydroxyl radical interacts with thenucleotide guanosine in the DNA, 8-hydroxy-2’-deoxyguanosine (8-OHdG) is formed[3].After DNA excision repair, 8-OHdG is transported out of the cell and into the circu-lation, where it can be used as a biomarker for oxidative stress and DNA damage[5].It can be measured in urine and blood plasma samples using high-performance liquidchromatography (HPLC)[6–8], gas-chromatography-mass spectrometry (GC-MS), liq-uid chromatography-mass spectrometry-mass spectrometry LC-MS/MS[6,9], and HPLCwith enzyme-linked immunosorbent assay (ELISA)[7]. The urinary concentrations inhealthy people has been estimated to 14.7 ± 4.6 (n = 15) nmol/l[9] and the concentra-tions in plasma to 1–70 pg/ml[10]. In cancer patients, the concentrations are significantlyhigher[9]. In Figure 1.1, the chemical structure of 8-OHdG is displayed.

1.2 Solid Phase Extraction

When the analyte (8-OHdG in this study) is present in a complex matrix, or in lowconcentrations for trace analysis, the sample preparation is important, and liquid-liquidextraction (LLE) and solid phase extraction (SPE) can be helpful tools to isolate the

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Figure 1.1: Chemical structure of 8-OHdG.

analyte from the matrix, concentrate, and simplify the chromatographic work[11]. TheSPE technique has many advantages, such as high reproducible recovery, concentrationof the analyte, requires less organic solvent than liquid-liquid extraction, and is a simpletechnique to use[12]. The SPE cartridge contains a reservoir where the sample is loaded,frits that function both as filters and to retain the sorbent[13], and a sorbent bed thatoften contains the same silica based material as the stationary phase in HPLC[14, pp.278–279]; see Figure 1.2.

Figure 1.2: This figure shows the SPE cartridge. The sample is loaded at the top of the cartridgeinto something called the reservoir, the frits function as filters, and the sorbent is where the sample isadsorbed.

The analyte in the sample is either adsorbed to the sorbent material, and can beremoved selectively after the interfering compounds have been washed away, or theanalyte could pass through the sorbent while the interfering compounds are adsorbedto the sorbent[12].

Because of the low concentration, and overlapping peaks due to the complex matrix,it is difficult to make a quantitative analysis of 8-OHdG today, so sample preparationtechniques such as SPE could be used to great advantage. For SPE analysis, C18 car-tridges, mixed mode anion exchange (MAX) cartridges[15], and strong cation exchange(SCX) cartridges[10], have been used to separate 8-OHdG from its matrix in previousstudies. To approach the problem with low concentrations using HPLC, an electrochem-ical detector is most often used for detection[15].

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SPE contains several steps: conditioning, loading of the samples, washing, andeluting[11, pp. 556–583][12][16, pp. 2–4].

The conditioning is made to activate the sorbent and to get the maximal phaseinterface between the sorbent and the sample[12]. Methanol is often used followed bywater or a buffer. The sample is then loaded and passed through the cartridge. For somecartridges, the sorbent is not allowed to run dry during these steps[16, pp. 2–4]. Theflow rate is another variable that is important for some cartridges[17]. The washing stepelutes weakly bonded compounds that can interfere in the chromatographic analysis, andthe eluting step is where the analyte is eluted through the cartridge and collected[12].

The sorbents can be polar, non-polar, have ion exchange functionalities, or be of amixed mode phase with several functionalities at the same time, which makes it possibleto obtain a greater selectivity[12]. The method development in SPE is usually based ontrial-and-error procedures[18].

The cartridges that are focused on in this study will be Chromabond® C18, Oasis®

MAX, and commercial MIP cartridges. The C18 cartridge, which has a nonpolar sor-bent, is of the same type that is often used in reversed phase HPLC columns, wherehydrophobic compounds will be adsorbed to the hydrophobic sorbent, and polar com-pounds will more easily pass through the cartridge. The Oasis® MAX cartridge containsa mixed mode of polymeric sorbents with reversed phase (C18) and anion functionalities.Quarternary amines provides the strong anion effect[7]. It is used to extract acidic com-pounds, with pKa 2–8[7, 8], from plasma, aqueous, or urine samples. Since 8-OHdG is aweak acid with its OH groups[15], and has pKa 6–8[19, 20], the Oasis® MAX cartridgemight be a good choice. According to a previous study, the Oasis® MAX cartridgesmade it possible to remove interfering peaks, but with lower recovery than cartridgescontaining only C18[6].

1.3 Molecularly Imprinted Polymers

A more recent sample preparation technique is molecularly imprinted polymers (MIP),which can provide higher selectivity and a high recovery because of the selective bindingsites that are imprinted in the polymer[21, 22]. This provides a lower background forcomplex matrices in HPLC analysis, which makes it possible to reach lower detectionlimits[22].

A template molecule, that resembles the target molecule as closely as possible, ismixed with functional monomers. By adding a crosslinking monomer and an initiator,a polymerization is made around the molecule. This polymerization forms a three-dimensional complex around the template molecule. When removing the templatemolecule, there is an imprinting of the molecule that is very specific to the target moleculeby size, shape, and to the chemical functionalities[22–24]. The process is described inFigure 1.3.

When choosing the functional monomers it is important to know what type of in-teraction that is desired because the monomers decide what type of interactions thereare between the target molecule and the monomers[24]. Two commonly used functionalmonomers are methacrylic acid (MAA) and 4-vinylpyridine (4-VP), both of which forms

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Figure 1.3: Construction of a MIP. The functional monomers are mixed with the template molecule. Acrosslinker and initiator are added to form a three-dimensional complex around the template molecule.The template molecule is then removed and leaves a molecular imprint.

non-covalent bondings to the template molecule[25]. Other types of monomers can alsobe used to form other interactions, such as hydrogen-bonding, Van deer Waals inter-actions, or ionic bonding, for example[23]. Common crosslinking agents are ethyleneglycol dimethacrylate (EDMA), divinylbenzene, or N,N’-methylene bisacrylamide. Acommon initiator for polymerisation is 2,2’-azobis(isobutyronitrile) (AIBN). The poly-mer can be packed into either ordinary SPE cartridges, or they can be connected directlyto the HPLC instrumentation as a pre-column[23], or packed in special syringes (calledMEPS), or in pipette tips[26]. Attempts to make a MIP for 8-OHdG in urine samples hasbeen published using HPLC with UV detection[9]. In their study, guanosine was used astemplate molecule, acrylamide and 4-Vinylpyridine was used as functional monomers,and N,N’-methylene bisacrylamide as crosslinking agent. The initiator used was AIBN,and dodecanol was used as solvent.

There are many commercial MIP cartridges for specific compounds, or groups ofcompounds, but no cartridge for just 8-OHdG seems to be available today. During theresearch for this project, no records has been found of investigations of commercial MIPcartridges designed for different compounds that has been tested with 8-OHdG.

1.4 High Performance Liquid Chromatography

The HPLC system is most often used for less volatile compounds, and separates thecompounds depending on their polarity in relation to the mobile phase and stationaryphase used[11, pp. 556–583][14, pp. 313–329]. The HPLC system uses a pump systemto obtain enough pressure to make the mobile phase pass through the column at aspecific flow rate. The column contains a stationary phase of fine particles or polymersto provide a large surface area to get high-resolution separations[11, pp. 556–583]. Theanalyte is injected with a syringe into the injection valve and then introduced into thecolumn together with the mobile phase flow by the pressure from a pump. The analytesare separated on a column, and will pass through the column with the mobile phase andgive different retention times depending on the polarity for the analytes. The elutingcompounds are recognised by a detector which produce a signal that is converted bya chromatographic software to a chromatogram with peaks whose areas corresponds tothe amount of the eluting compound.

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1.4.1 Stationary and Mobile Phases

Reversed phase chromatography, where the mobile phase is more polar than the station-ary phase, is the most commonly used technique[11, 14]. The eluent strength becomeshigher the less polar the mobile phase is[14]. The stationary phase does normally containsilica particles bonded to carbon chains of different lengths, usually octadecyl (C18), anddifferent functional groups such as nitrile and phenyl, can also be used[27, pp. 177–140].The carbon chains makes the stationary phase more hydrophobic. Mobile phases of-ten used in reversed phase are water or buffers mixed with methanol, acetonitrile, ortetrahydrofurane (THF), in different ratios[27, pp. 174–177].

1.4.2 Electrochemical Detector

An electrochemical detector can be used for analytes that can be oxidized or reduced, andthis can be a very sensitive detection principle. Moreover, the detector is sensitive to bothtemperature and flow rate[11, pp. 556–583]. The sample that is eluted from the columnreaches the detector, where a potential is placed across two electrodes. Compounds thatare oxidable or reducible requires different potentials to cause a reaction. The minimalpotential that is necessary can be investigated in a hydrodynamic voltammogram (HDV).Normally, at the top of the HDV there is an optimum signal to noise ratio, and forpotentials near this optimum, the analyte of interest is fully oxidized (or reduced)[28].

1.5 The Aim of This Project

The aim of this project was to develop a method for sample preparation of 8-OHdG inblood plasma samples, using traditional solid phase extraction and molecular imprintingpolymers for 8-OHdG, followed by analysis with HPLC-ECD. The following questionswere considered:

• What recovery can be obtained from different SPE and MIP cartridges?

• Could a commercial MIP provide a higher selectivity than traditional SPE?

• Is it possible for us to make a molecularly imprinted polymer for 8-OHdG that canprovide a higher selectivity than traditional SPE or commercial MIP cartridges?

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Chapter 2

Materials and Methods

In this chapter, methods for sample preparation and concentration of 8-OHdG for twotraditional SPE cartridges, a C18 and a MAX cartridge, are considered. Furthermore,three different MIP cartridges, designed for chloramphenicol, riboflavin, or nitroimida-zoles, are also tested. To investigate what had an effect on the systems, the differentsteps used in SPE were varied and analysed. Aqueous samples spiked with 8-OHdGwere used in the beginning, and blood plasma samples were tested when the differentsteps seemed to work. A concentration of the sample was desired because of the lowamount found in blood plasma, so a concentration factor of ten was used in most of theanalysis.

2.1 Instrumentation

The chromatography system used was a HPLC-Jasco X-LC™ 3185 PU equipped withRheodyne 7125 (Cotati, California, USA) manual injector. The columns used were eitherGrace Smart RP 18 3u (100 mm × 4.6 mm I.D.) or Thermo Quest Hypersil DivisionODS (250 mm × 4 mm, 3µm I.D.) with an injection volume of 10 µl. The mobile phasewas methanol:CSSA buffer (10:90, v/v) with a flow rate of 1.0 ml/min using Grace Smartand 0.6 ml/min using Thermo Quest columns, respectively. The detection system usedwas an ESA Coulochem II electrochemical detector. The samples were evaporated in aSavant SpeedVac.

2.2 Chemicals

Ammonium acetate and sodium acetate trihydrate were purchased from Merck (Darm-stadt, Germany); sodium hydroxide and di-sodiumhydrogenphosphate from Riedel-deHaen (Seelze, Germany); 8-hydroxy-2’-deoxyguanosine (8-OHdG), methanol, hexane,and acetonitrile, from Sigma Aldrich (Spruce street, St.Louis, MO, USA); citric acid fromMay & Baker (Dagenham, England). Blood plasma was obtained from Linkopings Uni-versity Hospital, SPE cartridges used were Chromabond® C18 ec, 45 µm, 1ml/100mg,Oasis® MAX (186000367) 3cc/60 mg 30 µm, 0.25 meq/g. Commercial MIP cartridges

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were SupelMIP™ SPE cartridges made for chloramphenicol, 55 µm, 25 mg/10 ml (53240-U), riboflavin, 25mg/10 ml (53236-U) 58 µm, or nitroimidazoles 62 µm, 50 mg/3ml.

2.3 High Performance Liquid Chromatography

To obtain an adequate separation of the peaks in the plasma samples, several mobilephases were made with 6%, 8%, and 10% methanol/CSSA buffer, 2% acetonitrile/CSSAbuffer, 0.1% acetic acid and 6% methanol/CSSA buffer, and analysed under isocraticconditions. The specifics of how the CSSA buffer was made can be found in Appendix A.Two columns were tested: Grace Smart and Thermo Quest. Different flow rates werealso tested.

2.3.1 The Electrochemical Detector

To obtain a HDV for 8-OHdG, the voltage on the detector was set to different valuesbetween 100–600 mV, and samples with the same concentration were injected into theHPLC-system as the current (nA) and peak areas were registered.

2.4 Sample Preparations

2.4.1 Aqueous Samples

A stock solution of 8-OHdG with a concentration of 10 µmol/l was made in water. Fromthis solution, a working solution with the concentration of 100 nmol/l was prepared inmobile phase, or buffer.

2.4.2 Blood Plasma Samples

To precipitate the proteins, 110 µl of cold 5% trichloroacetic acid was added to 1000 µlof both a spiked plasma sample containing 100 nmol/l 8-OHdG and an unspiked plasmasample. The samples were vortexed for 15 seconds and centrifuged at 14100g for 2 min-utes. The supernatant was removed from the pellet and transferred to Eppendorf tubes.The supernatant was then mixed with an equal amount of 1 mol/l ammonium acetatewith pH 5.25.

2.5 Traditional Solid Phase Extraction Methods

2.5.1 Octadecyl Modified Silica Phase

Several measurements with different conditions were made to develop a sample prepa-ration method with C18 cartridges to see which conditions were the most suitable. Thiswas accomplished by trying to separate the peaks and calculating the recovery for 8-OHdG in aqueous and blood plasma samples. The conditions investigated were differentSPE cartridges, washing, and eluents. According to a previous study, 8-OHdG in urine

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samples has been successfully extracted using C18 cartridges[29]. Using their proposedmethod as a starting point, several tests were carried out with different variables; seeTable B.1 in the Appendix for specifics. Tests were made to see if acetonitrile or hexanecould work as washing solution, and if acetonitrile could be used as eluent.

The final method obtained was the following: The C18 cartridges were first pre-conditioned with 1000 µl methanol followed with 1000 µl 25 mmol/l phosphate bufferwith pH 5.5. The sample, 1000 µl for aqueous samples and approximately 700 µl forblood plasma samples, was then loaded into the cartridge and washed with 300 µl ofthe 25 mmol/l phosphate buffer and 300 µl water. The cartridges were dried withnitrogen gas and placed in a SpeedVac at 40°C for 15 minutes. Elution was performedwith 2 × 300 µl methanol. The samples were dried in a SpeedVac at 40°C for 1 hour,resuspended in 100 µl mobile phase, shaken at 2400 shakes/minute for 2 minutes, andcentrifuged at 14100g for one minute. The evaporation step is made to concentrate theamount of 8-OHdG by a factor ten in the samples. See Figure 2.1 for a sketch of theproposed SPE method.

Figure 2.1: The proposed SPE method using C18 cartridges. First the cartridge is conditioned withmethanol and phosphate buffer. The sample is then loaded into the cartridge. The cartridge is washedwith phosphate buffer and water. The 8-OHdG is then eluted with methanol.

Blood plasma samples were tested with the final method for C18. Hexane was used asa third washing step to try to dispose of peaks that elute late, and to get a shorter analysistime. Repeated extractions were made with five blood plasma samples spiked with 100nmol/l 8-OHdG to investigate the variation between the samples. These samples wereprepared using 250 µl plasma for each sample, the remaining chemicals were rescaled tomatch the amount of blood plasma.

Another extraction, using 500 µl spiked blood plasma and chemicals rescaled tomatch this volume, was injected and analysed four times, to investigate the variationwithin the samples.

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2.5.2 Mixed-Mode Anion-Exchanger

In a previous study[6], an Oasis® MAX cartridge has been used successfully to removeinterfering peaks. Different pH values of the samples and washing solutions were testedto see if pH has an effect on the loading and washing systems. Aqueous samples weremade with 100 nmol/l 8-OHdG in 25 mmol/l phosphate buffers with pH 2, 4, 5, 7, and 9.For each sample, 1000 µl was passed through one of five different MAX cartridges. Eachcartridge was then washed with 1000 µl of the 25 mmol/l phosphate buffer which hasthe corresponding pH. Tests using hexane and methylene chloride as washing solutions,and methanol:acetic acid (98:2, v/v) and pure methanol as eluents, were made.

To see if the speed when the sample is loaded and passed through the cartridge thatprovides the first fraction is important, one sample was forced through the cartridge at ahigh speed, and one was allowed to pass through the cartridge slowly. The first fractionswere collected and analysed. Investigations were also made to see if the concentration ofthe buffers had any effect. This was accomplished by making new samples of 100 nmol/l8-OHdG: one in 1 mmol/l ammonium acetate buffer and one in 25 mmol/l phosphatebuffer.

The final method obtained was the following: The cartridges were conditioned with2000 µl methanol and 2000 µl Milli-Q water. The blood plasma samples were loaded intothe cartridge and washed respectively with 1000 µl phosphate buffer, pH 5, and 1000 µlmethylene chloride. The samples were then eluted with 1000 µl of methanol:acetic acid(98:2, v/v). The samples were evaporated in a SpeedVac at 40°C for 1 hour, and thenresuspended in 100 µl mobile phase consisting of methanol:CSSA buffer (8:92, v/v),shaken at 2400 shakes/minute for 2 minutes, and centrifuged at 14100g for one minute.

2.6 Commercial Molecularly Imprinted Polymers

To see if a greater selectivity could be obtained for 8-OHdG, three different MIP car-tridges were tested: SupelMIP™ SPE cartridges for chloramphenicol, riboflavin, ornitroimidazoles. Gravity flow was used during all sample loading on these cartridges.

As a first step, aqueous solutions spiked with 100 nmol/l 8-OHdG were tested, andlater blood plasma samples were tested on the nitroimidazole cartridge. The stepsbelow follows those proposed in the data sheets for the respective cartridge; see [30–32],respectively.

SupelMIP™ SPE Chloramphenicol. The cartridges were conditioned with 1000 µlmethanol, 1000 µl milli-Q water, and the samples were loaded into the cartridges. Duringthese steps, the cartridges were not allowed to dry. 8-OHdG was eluted with 2×1000 µlmethanol:acetic acid:water (89:1:10, v/v/v) and the first fraction for each sample wasanalysed on the HPLC to see which amount of 8-OHdG that was retained in the car-tridge.

SupelMIP™ SPE Riboflavin. The cartridges were conditioned with 1000 µlmethanol, 1000 µl milli-Q water, and the cartridges were not allowed to dry duringthese steps. 8-OHdG was then eluted with 3 × 1000 µl acetonitrile:water(70:30,v/v).The first fractions were collected and analysed on the HPLC.

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SupelMIP™ SPE Nitroimidazoles. The cartridges were conditioned with 1000 µltoluene, 1000 µl acetonitrile, and 1000 µl 10 mmol/l ammonium acetate with pH 6. Thesamples, 1000 µl 100 nmol/l 8-OHdG in 10 mmol/l ammonium acetate buffer at pH 6,were then loaded into the cartridge. The cartridges were washed with 1000 µl milli-Qwater, 2×1000 µl hexane, and 1000 µl heptane:toluene (3:1, v/v). Between each washingstep, a vacuum was applied. 8-OHdG was then eluted with 2×1000 µl acetonitrile:water(60:40 v/v) with 0.5% acetic acid. The samples were evaporated in a SpeedVac at 40°Cover the night, and resuspended in 100 µl mobile phase consisting of methanol:CSSAbuffer (10:90, v/v).

Tests were made with different conditioning solvents, similar to those used for thenitroimidazole cartridge, on the riboflavin and chloramphenicol cartridges, and attemptsto elute 8-OHdG with methanol, or acetonitrile, with 0.5% acetic acid were made onthe nitroimidazole cartridge to see if the evaporation time in the SpeedVac could bereduced with a different eluent. To see if the sample loading volume was important, oneblood plasma sample was diluted with 100 µl 1 mmol/l ammonium acetate buffer andone with 500 µl 1 mmol/l ammonium acetate buffer.

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Chapter 3

Results

3.1 HPLC Method Development

In the first part of the study, a HPLC method for analysis of 8-OHdG was set up.Different variables such as potential, mobile phases, different columns, and flow rateswere investigated. The potentials were plotted against the noted peak areas in an HDVgraph; see Figure 3.1. The information from the HDV shows that the optimal signal tonoise ratio is near 300 mV. This potential gives a complete oxidation of 8-OHdG andwas therefore used in all further experiments unless otherwise noted.

Figure 3.1: An HDV for 8-OHdG. The potentials were set between 100-600 mV and plotted against thepeak areas. The optimum signal to noise ratio is near 300 mV.

Using a mobile phase consisting of methanol:CSSA buffer (6:94, v/v) on the GraceSmart column, and the flow rate set to 1 ml/min, resulted in a retention time of 4.6 min-utes. However, when blood plasma samples were used, there was an interfering peakin the chromatogram at almost the same retention time as 8-OHdG. Attempts to ob-tain a better separation of the peaks by changing the flow rate, and changing the mobile

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phase to either acetonitrile:CSSA buffer (2:98, v/v), or acetic acid:methanol:CSSA buffer(0.1:5:94.9, v/v/v), had almost no effect on the Grace Smart column. A mobile phasewith acetonitrile:CSSA buffer (2:98, v/v) resulted in a retention time of 5.59 minutesand no improvement of the separation compared with 6% methanol. When changingthe mobile phase to methanol:CSSA buffer (10:90, v/v), using Thermo Quest HypersilDivision ODS, instead with a flow rate of 0.5 ml/min, the 8-OHdG peak was separatedfrom the interfering peak, although the retention time of 8-OHdG was 18.2 minutes.These chromatographic conditions were therefore used in all further analysis of bloodplasma samples. A Chromatogram of 8-OHdG, separated on the Thermo Quest columnusing C18 SPE, is shown in Figure 3.2.

Figure 3.2: Chromatogram of one blood plasma sample, spiked with 100 nmol/l 8-OHdG. The peaksare separated on the Thermo Quest Hypersil Division ODS (250 mm × 4 mm, 3µm I.D.) column, at aflow rate of 0.5 ml/min, and mobile phase consisting of methanol:CSSA buffer (10:90, v/v).

Another attempt that was made, was to change the potential by changing cell E1to 150 mV. This had no significant effect on which compounds, interfering or not, thatwere detected.

3.1.1 Analytical Recovery

In the absence of an internal standard, the recovery of 8-OHdG is calculated usingthe peak area ratio of the sample using the SPE method of interest, to one knownreference sample spiked with 100 nmol/l 8-OHdG in buffer. Since the evaporation andresuspendation enrich the samples, the peak area of the reference samples were multipliedby the corresponding concentration factor. The analysis of the aqueous reference sampleswere carried out on the same day, and under the same conditions, as for the blood plasmasamples.

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3.2 Traditional Solid Phase Extraction

3.2.1 Octadecyl Modified Silica Phase

The C18 method provided a high recovery, and 8-OHdG did not pass through in thefirst fraction or with the first washing step of the cartridges. Washing with hexane gaveno loss in recovery in aqueous samples, but tests with blood plasma did not show anyimprovement of washing away late eluting peaks in the chromatogram. Washing withacetonitrile resulted in a poor recovery and could perhaps be used as eluent instead.In plasma samples, using a C18 cartridge not only gave a high recovery, but also re-moved a considerable amount of background in the latter part of the chromatogram; seeFigure 3.3.

Figure 3.3: Chromatogram of one spiked blood plasma sample (100 nmol/l 8-OHdG) using solid phaseextraction on a C18 SPE cartridge, and one diluted blood plasma sample (blood plasma:1 mol/l ammo-nium acetate buffer, 1:1, v/v) without SPE. The black line shows the spiked blood plasma sample, usingSPE with a C18 cartridge, and the purple line shows the blood plasma without SPE. The retentiontime for 8-OHdG in the spiked blood plasma sample was 4.7 min. The HPLC column used was GraceSmart RP 18 3u (100 mm × 4.6 mm I.D.) with a flow rate of 1.0 ml/min, and mobile phase consist-ing of methanol:CSSA buffer (8:92, v/v). The peaks are not separated under these chromatographicconditions.

In Table 3.1, the peak areas for each step of the extraction and calculated recovery foraqueous samples spiked with 100 nmol/l 8-OHdG is shown; see Table B.1 in Appendix forspecifics of the different tests. In blood plasma samples, hexane was tested as a washingstep to see if the peaks that elute late could be avoided so a shorter analysis time couldbe obtained, but the results showed no significant difference to chromatograms withouthexane. The results of the analysis of the blood plasma samples are described below.

13

Table 3.1: Areas in mVs and recovery for aqueous solutions of 100 nmol/l 8-OHdG using SPE with a C18cartridge. The volume of the spiked aqueous sample used in these tests is 900µl, so the concentrationfactor is nine. The table shows the peak areas of the reference sample with 100 nmol/l 8-OHdG beforeextraction; in the first fraction; in wash 1–3; in the eluent after extraction, evaporation, and redilutionof the sample in 100 µl mobile phases; and the calculated recovery for each test.

Test Ref. sample Frac 1 Wash 1 Wash 2 Wash 3 Elution Recovery (%)1a 150 - - - 833 166 121b 150 - - - - 1274 942a 156 - - 2 - 2305 1642b 156 - - - - 2000 1422c 156 - - - - 1536 1093a 163 - - - - 1631 1113b 163 - - - - 1742 1193c 163 - - - - 1648 1123d 207 - - - - 2191 118

To examine the variation in measurements, five blood plasma samples were preparedwith concentration 100 nmol/l 8-OHdG. Assuming that the measurements follow a nor-mal distribution, the confidence interval

64.9 < µ < 75.0 (%)

was obtained for the recovery µ using a confidence degree of 95%. See Table B.2ain Appendix. In this case, the variational coefficient CV was calculated to be 5.8%.Similarly, the same sample with the concentration 100 nmol/l was injected four timesto investigate how much the measurements varies within the same sample. Assuming anormal distribution again, a 95% confidence interval for the recovery µ was calculatedto be

70.8 < µ < 95.9 (%)

The variational coefficient obtained was 9.5%. See Table B.2b in Appendix.

3.2.2 Mixed-Mode Anion-Exchanger

The results from the analysis of the first fractions of the aqueous samples at different pHvalues, was that everything was trapped in the cartridges independently of pH value. Infurther aqueous solutions, pH 5 was chosen. When using a syringe to force the samplethrough the cartridge, 17% passes through without bonding to the cartridge, and can beseen in the first fraction. The concentration of the buffer had no detectable effect on thesample loading. In one of the tests, where hexane was used in the washing step, there wasa loss of 7% 8-OHdG that passed through with the hexane. Using methylene chlorideas the washing solvent instead caused no loss, so this was used in further analysis asthe washing step. Eluting one aqueous sample with methanol and 0.5% acetic acid gavethe highest recovery (109%). The sample eluted with pure methanol resulted in 85%recovery in an aqueous sample. Using the final method for blood plasma samples, the

14

recovery obtained was 65%. A chromatogram of a spiked blood plasma sample extractedwith a MAX cartridge is shown in Figure 3.4.

Figure 3.4: Chromatogram of one spiked blood plasma sample with 100 nmol/l 8-OHdG using solidphase extraction on a MAX cartridge. The HPLC column used was Thermo Quest Hypersil DivisionODS (250 mm × 4 mm, 3µm I.D.) with a flow rate of 0.6 ml/min, and mobile phase consisting ofmethanol:CSSA buffer (8:92, v/v). There is a ridge at the end of the chromatogram, which indicatesthat not all of the nonpolar compounds had been removed during the SPE.

3.3 Commercial MIP Cartridges

At first, aqueous solutions spiked with 100 nmol/l 8-OHdG were tested. To the riboflavincartridge, 62% 8-OHdG was retained. and to the chloramphenicol cartridge, 50% wasretained. However, when the cartridges for nitroimidazoles were used, 100% of the 8-OHdG was retained. To see if the riboflavin and chloramphenicol cartridges could workwith conditions similar to those used with the nitroamidozole cartridge, the same pre-conditioning procedure was used for those cartridges. On the chloramphenicol cartridge,only 29% was retained, and on the riboflavin cartridge, 45% was retained. Since it didnot work sufficiently well, it was decided to do further analysis only on the nitroimidazolecartridge. Tests to elute 8-OHdG with acetonitrile:water (60:40, v/v) and 0.5% aceticacid, methanol with 0.5% acetic acid, and acetonitrile with 0.5% acetic acid, gave therecoveries 44%, 86%, and 47%, respectively; see Table 3.2.

The use of methanol with 0.5% acetic acid also reduced the evaporation time fromone night to one hour, so all further elutions were made with methanol and 0.5% aceticacid since it also provided the highest recovery in the aqueous samples. After the firstwashing step with water, there was a small loss of 8-OHdG where 17% seemed to follow

15

Table 3.2: Areas in mVs and recovery for aqueous samples with the concentration 100 nmol/l 8-OHdGextracted on the nitroimidazole MIP cartridge. Peak areas of the reference sample before the extraction;in the first fraction; in wash 1–3; in the eluent after extraction, evaporation, and redilution of the samplein 100 µl mobile phase; and the calculated recovery. In test A, acetonitrile:water (60:40,v/v) with 0.5%acetic acid was used to elute 8-OHdG. In test B and C, methanol and 0.5% acetic acid, and acetonitrileand 0.5% acetic acid, respectively, were tested.

Test Ref. sample Frac 1 Wash 1 Wash 2 Wash 3 Elution RecoveryA 152 - 26 209 6 668 44%B 152 - - 89 14 1310 86%C 152 - - 196 6 708 47%

the water through the cartridge in one of the samples. In the second and third washingstep, there is too much loss, so these washing steps are eliminated. In blood plasmasamples, the samples that were diluted with 100 µl and 500 µl 1 mmol/l ammoniumacetate buffer gave the recoveries 13% and 49%, respectively. A chromatogram usingnitroimidazole cartridge can be found in Figure 3.5

Figure 3.5: Chromatogram of one blood plasma sample spiked with 100 nmol/l 8-OHdG using solidphase extraction with MIP nitroimidazole cartridges. The column used was Thermo Quest HypersilDivision ODS (250 mm × 4 mm, 3µm I.D.) with at a flow rate of 0.5 ml/min, and mobile phaseconsisting of methanol:CSSA buffer (10:90, v/v).

16

Chapter 4

Discussion

4.1 HPLC Method Development

In the beginning of the project, the HPLC column used was Grace Smart RP 18 3u(100 mm × 4.6 mm I.D.). Different mobile phases and flow rates were tested to see ifsufficient separation of the peaks could be obtained in blood plasma samples. This wasnot successful due to the complex matrix. Many of these tests are left out of the reportsince they did not contribute to any important progress.

According to a previous study[10], 8-OHdG has been separated from plasma samplesusing a Develosil C30 column. There was no access to any type of HPLC column witha C30 stationary phase, so other columns were tested instead. One such column wasthe Thermo Quest Hypersil Division ODS (250 mm × 4 mm, 3µm I.D.), which made itpossible to separate the peaks with an approximate retention time of 18 minutes usinga flow rate of 0.5 ml/min. The time of analysis was quite long for the blood plasmasamples, about 35 minutes per analysis. One possible solution could be to use gradientseparation, and increase the concentration of methanol later in the chromatogram. Thiscould decrease the time of analysis. However, this method will also require some timefor the mobile phase system to stabilise between each new analysis.

4.2 Octadecyl Modified Silica Phase

The C18 cartridge gave a quite high recovery, about 70%, but the results show a vari-ation, even within the same sample. The recoveries in the aqueous samples are a lothigher than 100% in some cases, which could be an effect from the variation. To providean accurate analysis in future work, an internal standard is needed. One possible reasonfor the variation is the manual injection, so an auto injector should be able to provide alower variation. Another reason can be because of the low amount of blood plasma thatwas used in the tests, but since there was a lack of blood plasma during this analysisthere was no possibility to use larger amounts for each extraction. The pipettes mayalso have contributed to some quantity of the variation.

Since there was no access to a suitable internal standard during the analysis in thisproject, it is difficult to make any quantitative analysis before this problem with the

17

variation is solved. The chromatograms using SPE with C18 in blood plasma, showsthat a large portion of the interfering matrix has been removed; see Figure 3.3.

4.3 Mixed-Mode Anion-Exchanger

When sample preparation was made with the Oasis® MAX cartridge, the pH values werevaried, but it was found that pH had no effect on the bonding of 8-OHdG in aqueoussolutions. All 8-OHdG was retained in the sorbent in the cartridge independently of pHvalue. This indicates that only the reversed phase effect is active. A better result couldpossibly had been obtained in plasma samples if also the anion effect had been activatedby treatment of the cartridge and sample with an alkaline solution. This was not tested.If a lower pH, less than 2.5, had been used, some of the interactions to the proteins inthe matrix could perhaps had been broken. In the analysis, approximately pH 5 wasused. Hence, only the reversed phase effect was active. A combined effect, from bothreversed phase and anion exchanger effects, should probably provide a higher recovery.

In the chromatograms for the MAX cartridge, there is a ridge at the end; see, e.g.,Figure 3.4. This indicates that the SPE with the MAX cartridge does not work suf-ficiently well to remove the nonpolar compounds. This causes the analysis to takeapproximately 10 minutes longer compared to SPE made with the C18 cartridge, sincethe base line takes longer to stabilise between the different analyses. The recovery is lessthan that obtained for C18, but the peaks are still separated and there are no interferingpeaks at the same retention time as 8-OHdG.

To summarise, the MAX cartridge provided 65% recovery for blood plasma samples,and the time of analysis was longer compared with that after C18 cartridges due to thetime it takes for the baseline to stabilise after each analysis.

4.4 Commercial Molecularly Imprinted Polymers

The surprising result when testing the commercial MIP columns, was that one of themactually worked for 8-OHdG. Since MIP cartridges are tailor-made to match a specificcompound, or a group of similar compounds, it is surprising that recovery of 8-OHdG inplasma samples was high when using the nitroimidazole cartridge. The discovery thatelution with methanol and 0.5% acetic acid could be made instead of using the proposedacetonitrile:water (60:40, v/v) solution is very useful since it reduces the evaporationtime from one night to one hour. The water does not seem to have a great effect onthe elution process, but this does not explain why the tests with acetonitrile and 0.5%acetic acid did not work as well as an eluent.

The other two cartridges tested (riboflavin and chloramphenicol) did not work suffi-ciently well, even for aqueous samples. The reason for this might be that the moleculesare so different, both in size and other properties, that 8-OHdG does not bind to thesorbent in these cartridges. Therefore, these cartridges were not used for blood plasmasamples.

18

Since the knowledge of how the cartridges work and what they are made of is lacking,it is very difficult to proceed with developing a method that works better than the onesuggested previously. The loading and washing step had a recommended flow rate ofless than 0.5 ml/min, or to use gravity flow if possible. In this study, gravity flowwas applied. This process was time consuming and complicated due to the fact thatthe sorbent was not allowed to dry during the conditioning of cartridge. It is also notclear if everything was eluted when using this eluent, so further analysis is necessaryto ensure that everything is extracted from the cartridge so following samples are notcontaminated by leftover substances if the cartridges are used more than once.

Another phenomenon that was somewhat surprising was the fact that blood plasmasamples that were diluted provided a higher recovery than the corresponding undilutedones. One possible explanation could be that in the undiluted sample, proteins and othercompounds interfere more when 8-OHdG is attached to binding sites in the cartridge.

The chromatograms for blood plasma samples using the nitroimidazole cartridgeshows that all of the interfering compounds have been removed; see Figure 3.5. Com-pared with the chromatogram where the MAX cartridge was used, this one is better dueto the lack of the ridge at the end, and therefore has a shorter time of analysis. Butstill, the C18 method is the best choice since it provides the highest recovery.

4.5 Conclusions and Future Work

The recovery for all methods were lower in blood plasma samples than in the aqueoussamples, which was expected. One possible reason for this is that the complex matrixprobably takes binding sites from 8-OHdG in the cartridges. Another reason for the lossof recovery in blood plasma samples is that 8-OHdG could be affected by the proteinprecipitation when trichloroacetic acid was used. One test was made to try to ultra-filtrate the blood plasma instead, using a membrane with a molecular weight cut offat 10000 dalton, but the filter broke because of the centrifugal forces. A different typeof filter is necessary, but was not available at the time. There should be more analysismade with filtration, or alternative precipitation techniques, to see if this has an effecton the recovery.

The C18 cartridge was the most straight-forward sample preparation method to workwith. The flow rate had no significant effect and the cartridge was allowed to run drybetween the steps, so the laboratory work was less demanding. The MAX cartridgewas more time consuming since the flow rate of the sample loading had an effect on therecovery. Furthermore, the method requires several chemicals that are toxic and badfor the environment, and it was necessary to carry out the extractions under a fumehood. The nitroimidazole MIP cartridge was the most difficult to work with for severalreasons. The cartridge was not allowed to run dry between steps, and for some partsof the extraction it was necessary to apply a vacuum. Since the flow rate was very low,using only the gravity, the process also took a longer time.

If there would have been time to produce an in-house MIP specifically designed for8-OHdG, it should have been possible to achieve a higher selectivity for 8-OHdG and toobtain a greater recovery.

19

Since there was variation, an internal standard, and an auto injector, is probablyneeded in further analysis. The variation could also have been affected by the lowvolumes of blood plasma used during those tests. Investigations using a larger amountof blood plasma may reduce the variation caused by the the pipettes. An automation ofthe SPE method is another alternative to reduce the variation between the samples bylowering the operator errors[16, pp. 243–244]. The fact that the variation within the samesample was higher than the variation between the samples remains unexplained. All ofthe repeated tests were carried out during the same day and with the same procedure.

The recovery calculation is also uncertain since the peak areas of the reference solu-tions are multiplied by the concentration factor. One should probably use a referencesolution of the same concentration as the enriched sample instead of using a multiplica-tion factor. By doing this, it is possible to avoid extrapolating information.

The contents in blood plasma also varies from person to person, so there is a pos-sibility that the method may vary between different people. To be able to make aquantitative analysis in patients, the limit of quantification has to be determined tosee if the proposed method with C18 could work with the chromatographic conditionsobtained. A calibration curve should be derived for spiked blood plasma samples, sothat the concentration can be estimated in unknown blood plasma samples. If it turnsout not to be possible to detect low enough concentrations using SPE with C18 in bloodplasma samples, one should look into constructing a MIP to remove more of the matrixand reach a lower detection limit.

To successfully construct a MIP for 8-OHdG requires a lot of time and planning.The first step is to decide what chemicals that should be used. A larger study of thecurrent literature is needed to see what type of crosslinking agent, functional monomers,initiator, solvents, and template molecule, that can be used. A template molecule mustbe chosen to be as similar as possible to the target molecule, which in this case is8-OHdG. Possibly one can use guanosine, which was suggested in a previous study[9],since 8-OHdG is too expensive. The functional monomer and crosslinking agent must bepicked carefully. One must consider what types of interactions that are desirable is thiscase, to obtain a high selectivity for 8-OHdG in blood plasma samples. When it is decidedwhat chemicals to use, a suitable cartridge, pipette, pre-column, or similar object, hasto be chosen to contain the molecular imprinted polymer. After this, it is possible tocalculate the amounts of chemicals that are needed. When the MIP is completed, onecan proceed with method development, e.g., testing different conditioning steps, washingsteps, and eluents.

20

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[3] Valavanidis, A. et al, 8-hydroxy-2�-deoxyguanosine(8OHdG): A Critical Biomarkerof Oxidative Stress and Carcinogenesis, Journal of environmental science and health.Part C, Environmental carcinogenesis & ecotoxicology reviews 27 (2009), no. 2,120–139

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[5] Kimura, S. et al, Evaluation of Urinary 8-Hydroxydeoxyguanine in Healthy JapanesePeople, Basic and Clinical Pharmacology and Toxicology 98 (2006), no. 5, 496–502.

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[7] Oasis® technical note, Oasis® MAX product and generic method information, Wa-ters Corporation 2003, http://www.younglin.com/brochure_pdf/waters/MAX.pdf; verified 2011-05-22.

[8] Oasis® care and use manual, Oasis mixed-mode ion-exchange cartridges and 96-well plates, Waters Corporation 2010, http://www.waters.com/waters/support.htm?locale=en_us&lid=10076790&cid=511442&type=USRM; verified 2011-05-22.

[9] Zhang, S.W. et al, Molecularly imprinted monolith in-tube solid-phase microex-traction coupled with HPLC/UV detection for determination of 8-hydroxy-2’-deoxyguanosine in urine, Analytical and Bioanalytical Chemistry 395 (2009), no. 2,479–487.

[10] Koide, S. et al, Determination of human serum 8-hydroxy-2’-deoxyguanosine(8OHdG ) by HPLC-ECD combined with solid phase extraction (SPE ), Journal ofChromatography B 878 (2010), no. 23, 2163–2167.

[11] Harris, D.C., Quantitative Chemical Analysis, 7th ed., W.H. Freeman, NewYork 2007.

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[12] Biotage®, QuickStart guide to SPE, Biotage 2011, http://www.biotage.com/DynPage.aspx?id=49597; verified 2011-05-22.

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[14] Simonsen, F., Analysteknik: instrument och metoder, Studentlitteratur, Lund 2005.

[15] Zhang, S.W. et al, Determination of urinary 8-hydroxy-2’-deoxyguanosine by cap-illary electrophoresis with molecularly imprinted monolith in-tube solid phase mi-croextraction, Chinese Chemical Letters 21 (2010), no. 1, 85–88.

[16] Thurman, E.M. et al, Solid-Phase Extraction: Principles and Practice, John Wiley& Sons, New York 1998

[17] Argonaut technical note, Method Development in Solid Phase Extraction using ISO-LUTE SCX and SCX-3 SPE Columns for the extraction of Aqueous Samples, Dr.Weber Consulting Kft. 2001, http://www.weber.hu/PDFs/SPE/Tn106_SCX_SPE.pdf; verified 2011-05-22.

[18] Poole, C.F. et al, Contributions of theory to method development in solid-phaseextraction, Journal of Chromatography A 885 (2000), no 1–2, 17–39.

[19] Arnett, S.D. et al, Enhanced pH-mediated stacking of anions for CE incorporatinga dynamic pH junction, Electrophoresis 28 (2007), no. 20, 3786–3793.

[20] Qian, K. et al, Preparation and application of a molecularly imprinted polymer forthe determination of trace metolcarb in food matrices by high performance liquidchromatography, Journal of Separation Science 33 (2010), no. 14, 2079–2085.

[21] Komiyama, M. et al, Molecular Imprinting: From Fundamentals to Applications,Wiley-VCH Verlag, 2003 Weinheim.

[22] SupelMIP data sheet 407075, Molecularly imprinted polymers for thehighly selective extraction of trace analytes from complex matrices, SupelcoAnalytical 2009, http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/General_Information/t407075.pdf; verified 2011-05-23.

[23] Lasakova, M. et al, Molecularly imprinted polymers and their application in solidphase extraction, Journal of Separation Science 32 (2009), no. 5–6, 799–812.

[24] Turiel, E. et al, Molecularly imprinted polymers for sample preparation: A review,Analytica Chimica Acta 668 (2010), no. 2, 87–99.

[25] Hwang, C.C. et al, Chromatographic characteristics of cholesterol-imprinted poly-mers prepared by covalent and non-covalent imprinting methods, Journal of Chro-matography A 962 (2002), no. 1–2, 69–78.

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[26] Altun, Z. et al, Increasing sample preparation throughput using monolithicmethacrylate polymer as packing material for 96-tip robotic device, Journal of LiquidChromatography and Related Technologies 29 (2006), no. 10, 1477–1489.

[27] Meyer, V.R., Practical High-Performance Liquid Chromatography, 5th ed., JohnWiley & Sons, West Sussex 2010.

[28] ESA technical note 70-6348, Hydrodynamic Voltammograms: Generation, Expla-nation, and Optimization of Applied Potentials, ESA inc., http://www.esainc.com/download/?id=131; verified 2011-05-22.

[29] Sabatini, L. et al, A method for routine quantitation of urinary 8-hydroxy-2-deoxyguanosine based on solid-phase extraction and micro-high-performance liquidchromatography/electrospray ionization tandem mass spectrometry, Rapid Commu-nications in Mass Spectrometry 19 (2004), no. 2, 147–152.

[30] Supelco instruction set (data sheet), SupelMIP™ SPE - Chloramphenicol, Sigma-Aldrich Co. 2008. http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/Product_Information_Sheet/t706024.pdf; verified 2011-05-22.

[31] Supelco instruction set (data sheet), SupelMIP™ SPE - Riboflavin (Vitamin B2),Sigma-Aldrich Co. 2007. http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/Product_Information_Sheet/t706022.pdf; verified 2011-05-22.

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Appendix A

Preparation of the CSSA Buffer

Total volume is 1l.Citric acid:

C = 12.5 mmol/l, M = 210.14 g/mol, m = 2.62 g, and n = 12.47 mmol.Sodium acetate trihydrate:

C = 25 mmol/l, M = 136.08 g/mol, m = 3.4 g, and n = 24.99 mmol.Sodium hydroxide:

C = 30 mmol/l, M = 40.00 g/mol, m = 1.2 g, and n = 30.00 mmol.Acetic acid:

C = 10 mmol/l, M = 60.05 g/mol, m ≈ 0.6 g, and n ≈ 10 mmol.

24

Appendix B

Tables

B.1 Volume Table for SPE with C18

Table B.1: Solvent and volume table for different experiments with SPE. The variables were tried out toget the best conditions and recovery of 8-OHdG in aqueous samples for the C18 cartridge. A is 900 µlmethanol, B is 300 µl water, C is 300 µl 25 mM phosphate buffer with pH 5.5, D is 100 µl mobile phasewith methanol:CSSA buffer (8:92, v/v), E is 900 µl 25 mM phosphate buffer with pH 5.5, F is 300 µlacetonitrile, G is 300 µl hexane, and H is 300 µl methanol.

Test Conditioning Wash 1 Wash 2 Drying Wash 3 Elution Resuspendation1a A + E C B Yes F 2× H D1b A + E C B Yes - 2× H D2a A + E C B Yes G 2× H D2b A + E C B Yes - 2× H D2c A + E C B Yes - 2× F D3a A + E C B Yes G 2× H D3b A + E C B Yes G 2× H D3c A + E C B Yes - 2× H D3d A + E C B Yes - 2× H D

25

B.2 Repeated Tests

Table B.2: Repeated tests for the C18 cartridge with 100 nmol/l 8-OHdG in blood plasma samples.

Between samplesPeak area Recovery (%)

930 68967 70896 651045 76971 71

(a) Analysis of five different bloodplasma samples with the concen-tration 100 nmol/l.

Within samplesPeak area Recovery (%)

1200 871270 921090 791027 75

(b) Analysis of the same bloodplasma sample four times. Concen-tration 100 nmol/l.

26